thioinosine and 4-nitrobenzylthioinosine

The chicken erythroblast cell line HD3 is transformed by a temperature-sensitive mutant of avian erythroleukemia virus. Upon shift to the non-permissive temperature in the presence of inducers (hemin and butyric acid), HD3 cells differentiate to an erythrocyte phenotype and provide a model system for analyzing events associated with this process. Expression of some cell surface proteins undergoes drastic changes as cells mature to the erythrocyte stage with a selective loss of membrane proteins that appears to be species-specific. Specific changes also occur in the expression and activities of cytosolic enzymes reflecting alterations of metabolism. HD3 differentiation is characterized by increased transferrin receptor (TFR) expression and increased hemoglobin (Hb) synthesis, a marker for the erythrocyte. In parallel, there is a decrease in glucose transport and an increase in nucleoside transport signifying a switch from glycolytic hexose metabolism to metabolism of pentose from nucleoside. Likewise the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD) declines while glucose-6-phosphate dehydrogenase (G6PDH) activity remains constant. Commitment to the erythrocyte lineage alters expression of specific genes: TFR mRNA level increases while expression decreases for GLUT1 and GLUT3 glucose transporter mRNAs and GAD mRNA. However, the relationship between GAD activity and GAD mRNA was complex indicating modulation of GAD mRNA and protein half-lives. Serine/threonine and tyrosine phosphorylation and cAMP levels were shown to regulate the level of these messages. In this review, we describe how HD3 differentiation involves changes in plasma membrane composition, metabolism and gene expression that are orchestrated at different levels of control by multiple signaling modalities.

Topics: Animals; Biological Transport; Blood Glucose; Cell Differentiation; Cell Line; Chickens; Erythroblasts; Glucose Transporter Type 1; Glucose Transporter Type 2; Glyceraldehyde-3-Phosphate Dehydrogenases; Hemoglobins; Iron; Monosaccharide Transport Proteins; Receptors, Transferrin; Thioinosine

The nucleoside transport system (NTS), an integral part of the adenosine inactivation mechanism, is physiologically significant in adenosine-mediated autoregulation of cardiovascular function. Radioreceptor approaches have been employed in the characterization of nucleoside transporters as potential targets in the therapy of cardiovascular diseases. Unanswered questions that are being addressed relate to their pharmacological regulation and their involvement in the pathogenesis of disease states. A brief overview of the characterization of nucleoside transporters in the cardiovascular system is presented and discussed in terms of their relevance to cardiovascular function.

Topics: Animals; Cardiovascular Physiological Phenomena; Carrier Proteins; Hypertension; Membrane Proteins; Nucleoside Transport Proteins; Radioligand Assay; Thioinosine; Tritium

The permeation of nucleosides across the plasma membrane of mammalian cells is complex and mediated by at least five distinct transporters that differ in their sensitivity to inhibitors and in their specificity for nucleosides. The basic properties and permeant specificity of these transporters are summarized in Table 3. It appears that there may be differences in the distribution of these transporters in tumors and normal tissues that might be exploited for chemotherapeutic purposes. The human tumor cell lines examined express predominantly the NBMPR-sensitive equilibrative transporter es which can be blocked by low concentrations of NBMPR and dipyridamole. It is reasonable to expect that tumors with transport properties similar to the CCRF-CEM and Rh28 cell lines (Table 1) that have no detectable NBMPR-insensitive transport activity will be highly susceptible to the therapeutic approach of combining a transport inhibitor such as dipyridamole or NBMPR with an inhibitor of de novo pyrimidine biosynthesis. On the other hand, this approach to therapy is unlikely to succeed against tumors with transport phenotypes similar to the WI-L2 cell line that may permit the salvage nucleosides in the presence of these inhibitors. The majority of tumor cells examined, however, fall between these extremes, and it is not yet known what level of NBMPR-insensitive transport activity can be tolerated without seriously compromising this therapeutic approach. With respect to normal tissues, the mature absorptive cells of the intestine have predominantly Na(+)-dependent nucleoside transporters that are insensitive to NBMPR and dipyridamole. The proliferating crypt cells also appear to have Na(+)-dependent nucleoside transport, although they may also have an NBMPR-sensitive component of transport (Belt, unpublished data). Bone marrow granulocyte-macrophage progenitor cells also appear to have one or more concentrative nucleoside transporters. Thus these tissues, which are most vulnerable to the toxicity of antimetabolites, may be able to salvage nucleosides in the presence of inhibitors of equilibrative transport and be protected from the toxicity of de novo synthesis inhibitors. It is likely, however, that a successful application of this therapeutic approach will require the analysis of the nucleoside transport phenotype of individual tumors in order to identify those patients that may benefit from such therapy. Since the development of antibodies and cDNA probes for the vari

Topics: Animals; Biological Transport, Active; Carrier Proteins; Cell Line; Hematopoietic Stem Cells; Humans; Intestinal Mucosa; Membrane Proteins; Mice; Nucleoside Transport Proteins; Nucleosides; Sodium; Thioinosine; Tumor Cells, Cultured

Topics: Adenine; Algorithms; Animals; Biological Transport; Dilazep; Dipyridamole; Hypoxanthine; Hypoxanthines; Kinetics; Lidoflazine; Monosaccharide Transport Proteins; Nucleosides; Temperature; Thioinosine; Thionucleosides

Inhibitors of adenosine uptake or transport have been used clinically for some time in certain cardiovascular diseases. More recently, some of them have also been investigated for possible clinical use in combination with antimetabolites based on the observed heterogeneity of nucleoside transport in mammalian tumor cells. Such a heterogeneity of adenosine uptake and uptake sites has now also been suggested in the mammalian CNS. The aim of this article is, therefore, to review the present status of our knowledge of adenosine uptake in the mammalian CNS, compare it with our far more advanced knowledge of nucleoside transport in other mammalian cells and suggest direction of future research. The possible implications for the development of adenosine uptake inhibitors as adenosinergic neuropharmaceuticals will be discussed based on our knowledge of the physiological function of adenosine in the CNS.

Topics: Adenosine; Animals; Biological Transport; Brain; Dipyridamole; Erythrocytes; Nervous System; Thioinosine; Tumor Cells, Cultured

Topics: Affinity Labels; Carrier Proteins; Humans; Inosine; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Photochemistry; Thioinosine

The hypothesis that inhibition of adenosine uptake may play an important role in the central actions of the benzodiazepines is presented. The evidence supporting this hypothesis is discussed. Brain concentrations of the benzodiazepines are adequate for inhibition of adenosine uptake. Benzodiazepines, such as RO15-1788 and RO5-4864, which do not enhance gamma-aminobutyric acid binding, may exert some of their central effects by inhibiting the uptake of adenosine.

Topics: Adenosine; Animals; Anti-Anxiety Agents; Anticonvulsants; Benzodiazepinones; Brain; Cats; Cell Line; Cerebral Cortex; Diazepam; Flumazenil; Rats; Synaptosomes; Thioinosine

Topics: Animals; Biological Transport, Active; Diazepam; Dilazep; Dipyridamole; Erythrocyte Membrane; Erythrocytes; Humans; Inosine; Kinetics; Membrane Proteins; Models, Molecular; Nucleosides; Papaverine; Species Specificity; Thioinosine; Uridine

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 μg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 μg/mL).

Topics: Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Microbial Sensitivity Tests; Staphylococcus aureus; Structure-Activity Relationship; Thioinosine; Topoisomerase II Inhibitors; Topoisomerase Inhibitors

The present study was performed to investigate the capability of gemcitabine and pemetrexed to synergistically interact with respect to cytotoxicity and apoptosis in T24 and J82 bladder cancer cells, and to establish a correlation between drug activity and gene expression of selected genes in tumour samples. The interaction between gemcitabine and pemetrexed was synergistic; indeed, pemetrexed favoured gemcitabine cytotoxicity by increasing cellular population in S-phase, reducing Akt phosphorylation as well as by inducing the expression of a major gemcitabine uptake system, the human equilibrative nucleoside transporter-1 (hENT1), and the key activating enzyme deoxycytidine kinase (dCK) in both cell lines. Bladder tumour specimens showed an heterogeneous gene expression pattern and patients with higher levels of dCK and hENT1 had better response. Moreover, human nucleoside concentrative transporter-1 was detectable only in 3/12 patients, two of whom presented a complete response to gemcitabine. These data provide evidence that the chemotherapeutic activity of the combination of gemcitabine and pemetrexed is synergistic against bladder cancer cells in vitro and that the assessment of the expression of genes involved in gemcitabine uptake and activation might be a possible determinant of bladder cancer response and may represent a new tool for treatment optimization.

Topics: Administration, Intravesical; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Deoxycytidine; Deoxycytidine Kinase; Dipyridamole; Dose-Response Relationship, Drug; Drug Synergism; Equilibrative Nucleoside Transporter 1; Excitatory Amino Acid Antagonists; Female; Gemcitabine; Gene Expression Regulation, Neoplastic; Glutamates; Guanine; Humans; Male; Middle Aged; Neoplasm Staging; Pemetrexed; Phosphoribosylglycinamide Formyltransferase; Phosphorylation; Proto-Oncogene Proteins c-akt; S Phase; Thioinosine; Thymidylate Synthase; Treatment Outcome; Urinary Bladder Neoplasms

We report on the characterization of NbTi films at [Formula: see text] 11 GHz and in DC magnetic fields up to 4 T, performed by means of the coplanar waveguide resonator technique, providing quantitative information about the penetration depth, the complex impedance, and the vortex-motion-induced complex resistivity. This kind of characterization is essential for the development of radiofrequency cavity technology. To access the vortex-pinning parameters, the complex impedance was analyzed within the formalism of the Campbell penetration depth. Measurements in this frequency range allowed us to determine the complete set of vortex-pinning parameters and the flux flow resistivity, both analyzed and discussed in the framework of high-frequency vortex dynamics models. The analysis also benefits from the comparison with results obtained by a dielectric-loaded resonator technique on similar samples and by other ancillary structural and electromagnetic characterization techniques that provide us with a comprehensive picture of the material. It turns out that the normalized flux flow resistivity follows remarkably well the trend predicted by the time dependent Ginzburg-Landau theory, while the pinning constant exhibits a decreasing trend with the field which points to a collective pinning regime.

Topics: Electric Impedance; Magnetic Fields; Motion Pictures; Thioinosine

Novel bacterial topoisomerase inhibitors (NBTIs) are an important new class of antibacterials targeting bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Notwithstanding their potent antibacterial activity, they suffer from a detrimental class-related hERG blockage. In this study, we designed and synthesized an optimized library of NBTIs comprising different linker moieties that exhibit reduced hERG inhibition and retain inhibitory potencies on DNA gyrase and topoisomerase IV of Staphylococcus aureus and Escherichia coli, respectively, as well as potent antibacterial activities. Substitution of the linker's tertiary amine with polar groups outcome in diminished hERG inhibition. Compound 17 expresses nanomolar enzyme inhibitory potency and antibacterial activity against both Gram-positive and Gram-negative bacteria as well as reduced hERG inhibition relative to our previously published NBTI analogs. Here, we point to some important NBTI's structural features that influence their hERG inhibitory activity.

Topics: Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Escherichia coli; Gram-Negative Bacteria; Gram-Positive Bacteria; Microbial Sensitivity Tests; Naphthyridines; Structure-Activity Relationship; Thioinosine; Topoisomerase II Inhibitors

Topics: Animals; Mice; Microbial Sensitivity Tests; Mycobacterium abscessus; Mycobacterium Infections, Nontuberculous; Mycobacterium tuberculosis; Nontuberculous Mycobacteria; Thioinosine; Topoisomerase II Inhibitors

Neuroinflammation and apoptosis are two key factors contributing to early brain injury (EBI) after subarachnoid hemorrhage (SAH) and are strongly associated with a poor prognosis. Recently, equilibrative nucleoside transporter 1 (ENT1) was emerged to accelerate the severity of inflammation and cell apoptosis in several nervous system diseases, including cerebral ischemia, neurodegeneration and epilepsy. However, no study has yet elaborated the expression levels and effects of ENT1 in EBI after SAH.. Sprague-Dawley rats were subjected to SAH by endovascular perforation. Nitrobenzylthioinosine (NBTI) was intranasally administered at 0.5 h after SAH. The protein expression levels of ENT1, NLRP3, Bcl2, Bax, ACS, Caspase-1, IL-1 were detected by western blot. The modified Garcia score and beam balance score were employed to evaluate the neurologic function of rats following SAH. In addition, hematoxylin-eosin, fluoro-jade C and TdT-mediated dUTP nick-end labeling staining were then used to evaluate brain tissue damage and neuronal apoptosis.. Analysis indicated that endogenous levels of ENT1 were significantly upregulated at 24-hour post-SAH, accompanied by NLRP3 inflammasome activation and Bcl2 decline. The administration of NBTI, an inhibitor of ENT1, at a dose of 15 mg/kg, ameliorated neurologic deficits and morphologic lesions at both 24 and 72 h after SAH. Moreover, ENT1 inhibition efficiently mitigated neuronal degeneration and cell apoptosis. In addition, NBTI at 15 mg/kg observably increased Bcl2 content and decreased Bax level. Furthermore, suppression of ENT1 notably reduced the expression levels of NLRP3, apoptosis associated speck like protein containing CARD, caspase-1 and IL-1β.. NBTI relieved SAH-induced EBI partly through ENT1/NLRP3/Bcl2 pathway.

Topics: Animals; Apoptosis; Disease Models, Animal; Equilibrative Nucleoside Transporter 1; Inflammasomes; Neuroinflammatory Diseases; NLR Family, Pyrin Domain-Containing 3 Protein; Proto-Oncogene Proteins c-bcl-2; Rats; Subarachnoid Hemorrhage; Thioinosine

Equilibrativenucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors using K

Topics: Darunavir; Drug Interactions; Equilibrative Nucleoside Transporter 1; HeLa Cells; Humans; Nucleosides; Ribavirin; Ribonucleosides; Thioinosine

We performed accumulation assays with Hoechst 33342 (a model dual substrate of ABCB1 and ABCG2) and bi-directional transport studies with the ABCG2 substrate [. NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC. NBMPR at a concentration of 0.10 mM abolishes ABCG2 activity. Researchers using NBMPR to evaluate the effect of ENTs on pharmacokinetics must therefore interpret their results carefully if studying compounds that are substrates of both ENTs and ABCG2.

Topics: Animals; Antiviral Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport; Cell Line; Dogs; Female; Humans; Madin Darby Canine Kidney Cells; Neoplasm Proteins; Placenta; Pregnancy; Rats; Rats, Wistar; Thioinosine

Cytarabine (Ara-C) is a nucleoside analog used in the treatment of acute myeloid leukemia (AML). Despite the many years of clinical use, the identity of the transporter(s) involved in the disposition of Ara-C remains poorly studied. Previous work demonstrated that concurrent administration of Ara-C with nitrobenzylmercaptopurine ribonucleoside (NBMPR) causes an increase in Ara-C plasma levels, suggesting involvement of one or more nucleoside transporters. Here, we confirmed the presence of an NMBPR-mediated interaction with Ara-C resulting in a 2.5-fold increased exposure. The interaction was unrelated to altered blood cell distribution, and subsequent studies indicated that the disposition of Ara-C was unaffected in mice with a deficiency of postulated candidate transporters, including ENT1, OCTN1, OATP1B2, and MATE1. These studies indicate the involvement of an unknown NBMPR-sensitive Ara-C transporter that impacts the pharmacokinetic properties of this clinically important agent.

Topics: Animals; Antimetabolites, Antineoplastic; Cytarabine; Drug Interactions; Equilibrative Nucleoside Transporter 1; Female; Humans; Leukemia, Myeloid, Acute; Liver-Specific Organic Anion Transporter 1; Mice; Mice, Knockout; Organic Cation Transport Proteins; Symporters; Thioinosine

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.

Topics: Animals; Antiviral Agents; Cell Line; Guanosine; Macaca mulatta; Mycophenolic Acid; Parainfluenza Virus 2, Human; Ribavirin; RNA, Viral; Thioinosine; Virus Internalization; Virus Replication

Entecavir (ETV), a first-line antiviral drug against hepatitis B virus (HBV), has the possibility to be used to prevent mother-to-child transmission. The aim of present study was to clarify the mechanism of ETV uptake into hepatocytes and evaluate the alteration of ETV's hepatic distribution during pregnancy.. The roles of equilibrative nucleotide transporter (ENT) 1 and organic anion transporter (OAT) 2 in ETV accumulation and anti-HBV efficacy were studied in human ENT1 or OAT2 overexpressed cell models and HepG2.2.15 cells, respectively; meanwhile, the liver-to-plasma ETV concentration ratios in non-pregnant and pregnant mice were measured to evaluate the effect of pregnancy on ETV hepatic distribution.. ETV was shown to be a substrate of ENT1 and OAT2. An ENT1 inhibitor significantly decreased the efficacy of ETV in HepG2.2.15 cells, while overexpression of OAT2 increased susceptibility of HBV to ETV. The liver-to-plasma ETV concentration ratios in pregnant mice were sharply reduced; whereas, the absolute concentration of ETV in the liver did not obviously alter in pregnancy. Although oestradiol and progesterone showed a concentration-dependent inhibition on ETV accumulation both in hepatic cell lines and in primary human hepatocytes, a physiologically relevant concentration of oestradiol and progesterone did not affect antiviral activity of ETV.. OAT2 and ENT1 are the main transporters involved in the hepatic uptake and anti-HBV efficacy of ETV. The concentration of ETV in the liver was not obviously altered during pregnancy, which indicates that dosage adjustment in pregnancy is not necessary.

Topics: Animals; Antiviral Agents; Cell Proliferation; Cells, Cultured; Dogs; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Female; Guanine; HEK293 Cells; Hepatitis B virus; Hepatocytes; Humans; Indomethacin; Mice; Mice, Inbred ICR; Organic Anion Transporters, Sodium-Independent; Pregnancy; Structure-Activity Relationship; Thioinosine

The human equilibrative nucleoside transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in humans. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are used clinically. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited, owing to the absence of structural information on hENT1. Here, we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1 and provide insight into adenosine recognition and transport. Our studies provide a platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.

Topics: Adenosine; Crystallography, X-Ray; Dilazep; Equilibrative Nucleoside Transporter 1; Humans; Models, Molecular; Protein Conformation; Thioinosine

Trifluridine (FTD) exhibits anticancer activities after its oral administration despite its hydrophilic nature. It was previously reported that concentrative nucleoside transporter (CNT) 1 mediates the apical uptake of FTD in human small intestinal epithelial cells (HIECs). In the present study, FTD was also identified as a substrate for equilibrative nucleoside transporter (ENT) 1 and ENT2 in transporter gene-transfected cells. An immunocytochemical analysis revealed that ENT1 was expressed at the basolateral and apical membranes of HIECs. Cellular accumulation increased in the presence of S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT selective inhibitor. Cytotoxicity in HIEC monolayers at low FTD concentrations was increased by NBMPR, and this may have been due to inhibition of the ENT-mediated basolateral transport of FTD by NBMPR. These results suggest that ENTs reduce the intestinal cytotoxicity of FTD by facilitating its basolateral efflux. On the other hand, the intracellular accumulation and cytotoxicity of FTD in HIECs were decreased at higher concentrations of FTD by NBMPR, and this may have been due to the NBMPR inhibition of the apical uptake of FTD, which has been suggested to be mediated by CNTs and ENTs. In conclusion, ENTs were responsible for intestinal transepithelial permeation by mediating the basolateral efflux of FTD after its uptake by CNT1 from the apical side, resulting in decreases in its intracellular accumulation and intestinal toxicity in humans. Equilibrative nucleoside transporters may also partially contribute to the low-affinity uptake of FTD across the apical membrane along with high-affinity CNT1.

Topics: Biological Transport; Cell Survival; Cells, Cultured; Drug Interactions; Epithelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Intestine, Small; Thioinosine; Trifluridine

Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that, in addition to activating protein kinase A, 8CPT-modified cAMP analogues suppress the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver-specific glucose-lowering properties of AICAR and metformin.. Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP) and AMPK signalling were measured, and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells.. AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not by metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR, at least in part, by impeding its uptake into hepatocytes.. We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and the effects of AICAR, but not metformin, on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Biological Transport; Equilibrative Nucleoside Transporter 1; Female; Glucose; Hepatocytes; Hypoglycemic Agents; Liver; Metformin; Mice; Phosphorylation; Ribonucleotides; Signal Transduction; Thioinosine

Nitrobenzylthioinosine (NBTI), a specific inhibitor of type 1 equilibrative nucleoside transporter, could regulate the extracellular adenosine concentration and have protective roles in seizures. However, the protection mechanism of NBTI in seizures remains poorly understood. Here, the expression pattern and subcellular distribution of adenosine A1 receptor were detected by Western blot analysis and double-labeling immunofluorescence staining in Lithium Chloride-Pilocarpine induced epileptic rat model. At 24 h after pilocarpine induced rat seizures, hippocampal slices were prepared and the evoked excitatory postsynaptic currents (eEPSCs) amplitude of pyramidal neurons in hippocampus CA1 region was recorded using whole-cell patch clamp. In vivo, compared to control group, Western blotting analysis showed that the expression of adenosine A1 receptor protein was increased at 24 h and 72 h after seizure, didn't change at 0 min and 1 w, and decreased at 2 w. Double-label immunofluorescence revealed that adenosine A1 receptor was mainly expressed in the membrane and cytoplasm of neurons. In Vitro, adenosine decreased the eEPSCs amplitude of pyramidal neurons in hippocampus CA1 region, NBTI also had the same effect. Meantime, NBTI could further inhibit eEPSCs amplitude on the basis of lower concentration adenosine (50µM), and adenosine A1 receptor inhibitor DPCPX partially reversed this effect. Taken together, we confirmed that the expression of adenosine A1 receptor protein was increased in the early seizures and decreased in the late seizures. At the same time, NBTI mimics adenosine to attenuate the epileptiform discharge through adenosine A1 receptor, which might provide a novel therapeutic approach toward the control of epilepsy.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Animals; Disease Models, Animal; Epilepsy; Gene Expression; Hippocampus; Male; Patch-Clamp Techniques; Protein Transport; Pyramidal Cells; Rats; Receptor, Adenosine A1; Thioinosine

The term receptor reserve, first introduced and used in the traditional receptor theory, is an integrative measure of response-inducing ability of the interaction between an agonist and a receptor system (consisting of a receptor and its downstream signaling). The underlying phenomenon, i.e., stimulation of a submaximal fraction of receptors can apparently elicit the maximal effect (in certain cases), provides an opportunity to assess the receptor reserve. However, determining receptor reserve is challenging for agonists with short half-lives, such as adenosine. Although adenosine metabolism can be inhibited several ways (in order to prevent the rapid elimination of adenosine administered to construct concentration-effect (E/c) curves for the determination), the consequent accumulation of endogenous adenosine biases the results. To address this problem, we previously proposed a method, by means of which this bias can be mathematically corrected (utilizing a traditional receptor theory-independent approach). In the present investigation, we have offered in silico validation of this method by simulating E/c curves with the use of the operational model of agonism and then by evaluating them using our method. We have found that our method is suitable to reliably assess the receptor reserve for adenosine in our recently published experimental setting, suggesting that it may be capable for a qualitative determination of receptor reserve for rapidly eliminating agonists in general. In addition, we have disclosed a possible interference between FSCPX (8-cyclopentyl-

Topics: Adenosine; Animals; Biological Transport; Computer Simulation; Equilibrative Nucleoside Transporter 1; Guinea Pigs; Half-Life; Kinetics; Models, Statistical; Myocardium; Myocytes, Cardiac; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A1; Thioinosine; Xanthines

The main roles of equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) are to transfer single nucleosides and analogues for the nucleic acid salvage pathway. Oligodeoxyribonucleotides (ODNs) can be transported into the cytoplasm or nucleus of cells under certain conditions. Among ODNs composed of a single type of nucleotide, the transport efficiency differs with the length and nucleotide composition of the ODNs and varies in different types of leukaemia cells; among the 5 tested random sequence ODNs and 3 aptamers with varying sequences, the data showed that some sequences were associated with significantly higher transport efficiency than others. The transport of ODNs was sodium, energy, and pH-independent, membrane protein-dependent, substrate nonspecific for ODNs and 4-nitrobenzylthioinosine (NBMPR)-insensitive, but it showed a low sensitivity to dipyridamole (IC50 = 35.44 µmol/L), distinguishing it from ENT1-4 and CNTs. The delivery efficiency of ODNs was superior to that of Lipofection and Nucleofection, demonstrating its potential applications in research or therapeutics. Moreover, this process was associated with p38 mitogen activated protein kinase (p38MAPK) instead of c-Jun N-terminal kinase (JNK) signalling pathways. We have denoted ODN transmembrane transport as equilibrative nucleic acid transport (ENAT). Overall, these findings indicate a new approach and mechanism for transmembrane transport of ODNs.

Topics: Adolescent; Adult; Aged; Biological Transport; Child; Child, Preschool; Cytoplasm; Dipyridamole; Female; Humans; Infant; K562 Cells; Leukemia; Male; Middle Aged; Oligodeoxyribonucleotides; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Thioinosine

To evaluate the role of nucleoside transporters in the nose-to-brain uptake of [

Topics: Animals; Area Under Curve; Dideoxynucleosides; Nasal Mucosa; Nucleoside Transport Proteins; Olfactory Bulb; Positron-Emission Tomography; Rats; Rats, Sprague-Dawley; Thioinosine; Tissue Distribution

Adenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi.. Overall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 μmol/L, but abolished by 10 μmol/L NBTI in cells non-treated or treated with NH. hENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus.

Topics: Adenosine; Biological Transport; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Female; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen-Ion Concentration; Pregnancy; Thioinosine

The neuroprotective role of carbon monoxide (CO) has been studied in a cell-autonomous mode. Herein, a new concept is disclosed - CO affects astrocyte-neuron communication in a paracrine manner to promote neuroprotection. Neuronal survival was assessed when co-cultured with astrocytes that had been pre-treated or not with CO. The CO-pre-treated astrocytes reduced neuronal cell death, and the cellular mechanisms were investigated, focusing on purinergic signaling. CO modulates astrocytic metabolism and extracellular ATP content in the co-culture medium. Moreover, several antagonists of P1 adenosine and P2 ATP receptors partially reverted CO-induced neuroprotection through astrocytes. Likewise, knocking down expression of the neuronal P1 adenosine receptor A2A-R (encoded by Adora2a) reverted the neuroprotective effects of CO-exposed astrocytes. The neuroprotection of CO-treated astrocytes also decreased following prevention of ATP or adenosine release from astrocytic cells and inhibition of extracellular ATP metabolism into adenosine. Finally, the neuronal downstream event involves TrkB (also known as NTRK2) receptors and BDNF. Pharmacological and genetic inhibition of TrkB receptors reverts neuroprotection triggered by CO-treated astrocytes. Furthermore, the neuronal ratio of BDNF to pro-BDNF increased in the presence of CO-treated astrocytes and decreased whenever A2A-R expression was silenced. In summary, CO prevents neuronal cell death in a paracrine manner by targeting astrocytic metabolism through purinergic signaling.

Topics: Adenosine; Adenosine Triphosphate; Animals; Apoptosis; Astrocytes; Carbon Monoxide; Coculture Techniques; Cysteine; Extracellular Space; Gene Silencing; Glycyrrhetinic Acid; Mice, Inbred C57BL; Models, Biological; Neurons; Neuroprotection; Paracrine Communication; Pyrimidines; Receptor, trkB; Receptors, Adenosine A2; Receptors, Purinergic; Serine; Signal Transduction; Suramin; Thioinosine; Triazoles; Xanthines

Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-cancer, anti-parasitic and anti-viral drugs. Previous work, and in silico prediction, suggest that hENT1 is glycosylated at Asn(48) in the first extracellular loop of the protein and that glycosylation plays a role in correct localization and function of hENT1. Site-directed mutagenesis of wild-type (wt) hENT1 removed potential glycosylation sites. Constructs (wt 3xFLAG-hENT1, N48Q-3xFLAG-hENT1 or N288Q-3xFLAG-hENT2) were transiently transfected into HEK293 cells and cell lysates were treated with or without peptide-N-glycosidase F (PNGase-F), followed by immunoblotting analysis. Substitution of N48 prevents hENT1 glycosylation, confirming a single N-linked glycosylation site. N48Q-hENT1 protein is found at the plasma membrane in HEK293 cells but at lower levels compared with wt hENT1 based on S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding analysis (wt 3xFLAG-ENT1 Bmax, 41.5±2.9 pmol/mg protein; N48Q-3xFLAG-ENT1 Bmax, 13.5±0.45 pmol/mg protein) and immunofluorescence microscopy. Although present at the membrane, chloroadenosine transport assays suggest that N48Q-hENT1 is non-functional (wt 3xFLAG-ENT1, 170.80±44.01 pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06 pmol/mg protein; mock-transfected 74.31±19.65 pmol/mg protein). Co-immunoprecipitation analyses suggest that N48Q ENT1 is unable to interact with self or with wt hENT1. Based on these data we propose that glycosylation at N48 is critical for the localization, function and oligomerization of hENT1.

Topics: Biological Transport; Cell Line; Equilibrative Nucleoside Transporter 1; Glycosylation; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Thioinosine

Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 μM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.

Topics: Antimetabolites, Antineoplastic; Binding Sites; Cytidine; Deoxycytidine; Equilibrative Nucleoside Transporter 1; Gemcitabine; HEK293 Cells; Humans; Protein Transport; Thioinosine

Type 1 equilibrative nucleoside transporter (ENT1) promotes glutamate release by inhibition of adenosine signaling. However, whether ENT1 plays a role in epileptic seizure that involves elevated glutamatergic neurotransmission is unknown. Here, we report that both seizure rats and patients show increased expression of ENT1. Intrahippocampal injection of a specific inhibitor of ENT1, nitrobenzylthioinosine (NBTI), attenuates seizure severity and prolongs onset latency. In order to examine whether NBTI would be effective as antiepileptic after peripheral application, we injected NBTI intraperitoneally, and the results were similar to those obtained after intrahippocampal injection. NBTI administration leads to suppressed neuronal firing in seizure rats. In addition, increased mEPSC in seizure are inhibited by NBTI. Finally, NBTI results in deactivation of phosphorylated cAMP-response element-binding protein in the seizure rats. These results indicate that ENT1 plays an important role in the development of seizure. Inhibition of ENT1 might provide a novel therapeutic approach toward the control of epileptic seizure.

Topics: Action Potentials; Adenosine; Adolescent; Adult; Animals; Anterior Temporal Lobectomy; Anticonvulsants; CA1 Region, Hippocampal; Carrier Proteins; Child, Preschool; Convulsants; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Drug Resistance; Epilepsy, Temporal Lobe; Equilibrative Nucleoside Transporter 1; Glutamates; Humans; Male; Middle Aged; Nerve Tissue Proteins; Phosphorylation; Pilocarpine; Protein Processing, Post-Translational; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Seizures; Thioinosine; Young Adult

Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5'nucleotidase inhibitor adenosine 5'-(α,β-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5'-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish.

Topics: Adenine; Adenosine; Adenosine A1 Receptor Antagonists; Adenosine Diphosphate; Animals; Benzyl Compounds; Convulsants; Dipyridamole; Gene Expression Regulation; Genes, fos; Pentylenetetrazole; Phenethylamines; Phosphodiesterase Inhibitors; Seizures; Signal Transduction; Thioinosine; Xanthines; Zebrafish

Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events.

Topics: Animals; Binding Sites; Biological Transport; Carrier Proteins; Cell Membrane; Deoxycytidine; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Female; Gemcitabine; Hepatic Artery; Hepatocytes; Humans; Infusion Pumps; Male; Membrane Proteins; Middle Aged; Rats; Rats, Wistar; Thioinosine

DNA hypermethylation, an epigenetic change that silences gene expression without altering nucleotide sequences, plays a critical role in the formation and progression of colorectal cancers as well as in the acquisition of drug resistance. Decitabine (DAC), a DNA methyltransferase 1 inhibitor of nucleoside analogues, has been shown to restore gene expression silenced by hypermethylation. In the present study, the mechanisms underlying both uridine and DAC uptake were examined in the human colon cancer cell line HCT116. Real-time polymerase chain reaction analysis revealed that ENT1 mRNA was the most abundant among the nucleoside transporters examined in HCT116 cells. The ENT1 protein was detected in the membrane fraction, as determined by Western blotting. The uptake of uridine or DAC was time- and concentration-dependent, but also Na(+)-independent. The uptake of these agents was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENTs), and was also decreased in cells treated with ENT1 small interfering RNA. The uptake of both uridine and DAC was inhibited by uridine, cytidine, adenosine, or inosine, while that of DAC was also inhibited by thymidine. The expression of MAGEA1 mRNA, the DNA of which was methylated in HCT116 cells, was increased by DAC treatment, and this increment was attenuated by concomitant treatment with NBMPR. The IC50 value of DAC was also increased in the presence of NBMPR. These results suggest that DAC is mainly taken up by ENT1 and that this uptake is one of the key determinants of the activity of DAC in HCT116 cells.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Carrier Proteins; Colorectal Neoplasms; Decitabine; DNA Methylation; Equilibrative Nucleoside Transporter 1; Gene Expression; HCT116 Cells; Humans; Melanoma-Specific Antigens; Real-Time Polymerase Chain Reaction; RNA, Messenger; Thioinosine; Uridine

Sleep impairments are comorbid with a variety of neurological and psychiatric disorders including depression, epilepsy, and alcohol abuse. Despite the prevalence of these disorders, the cellular mechanisms underlying the interaction between sleep disruption and behavior remain poorly understood. In this study, the impact of chronic sleep loss on sleep homeostasis was examined in C57BL/6J mice following 3 d of sleep restriction. The electroencephalographic power of slow-wave activity (SWA; 0.5-4 Hz) in nonrapid eye movement (NREM) sleep and adenosine tone were measured during and after sleep restriction, and following subsequent acute sleep deprivation. During the first day of sleep restriction, SWA and adenosine tone increased, indicating a homeostatic response to sleep loss. On subsequent days, SWA declined, and this was accompanied by a corresponding reduction in adenosine tone caused by a loss of one source of extracellular adenosine. Furthermore, the response to acute sleep deprivation (6 h) was significantly attenuated in sleep-restricted mice. These effects were long-lasting with reduced SWA and adenosine tone persisting for at least 2 weeks. To investigate the behavioral consequences of chronic sleep restriction, sensitivity to the motor-impairing effects of alcohol was also examined. Sleep-restricted mice were significantly less sensitive to alcohol when tested 24 h after sleep restriction, an effect that persisted for 4 weeks. Intracerebroventricular infusion of an adenosine A1 receptor antagonist produced a similar decrease in sensitivity to alcohol. These results suggest that chronic sleep restriction induces a sustained impairment in adenosine-regulated sleep homeostasis and consequentially impacts the response to alcohol.

Topics: Action Potentials; Adenosine; Alcohols; Animals; Brain Waves; Electroencephalography; Excitatory Postsynaptic Potentials; Extracellular Fluid; Hippocampus; Homeostasis; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Movement Disorders; Neurons; Purinergic P1 Receptor Antagonists; Sleep Deprivation; Sleep Stages; Theophylline; Thioinosine; Time Factors; Wakefulness

Simultaneous inhibition of the cardiac equilibrative-p-nitrobenzylthioinosine (NBMPR)-sensitive (es) type of the equilibrative nucleoside transport 1 (ENT1) nucleoside transporter, with NBMPR, and adenosine deaminase, with erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), prevents release of myocardial purines and attenuates myocardial stunning and fibrillation in canine models of warm ischemia and reperfusion. It is not known whether prolonged administration of hypothermic cardioplegia influences purine release and EHNA/NBMPR-mediated cardioprotection in acutely ischemic hearts.. Anesthetized dogs (n = 46), which underwent normothermic aortic crossclamping for 20 minutes on-pump, were divided to determine (1) purine release with induction of intermittent antegrade or continuous retrograde hypothermic cardioplegia and reperfusion, (2) the effects of postischemic treatment with 100 μM EHNA and 25 μM NBMPR on purine release and global functional recovery, and (3) whether a hot shot and reperfusion with EHNA/NBMPR inhibits purine release and attenuates ventricular dysfunction of ischemic hearts. Myocardial biopsies and coronary sinus effluents were obtained and analyzed using high-performance liquid chromatography.. Warm ischemia depleted myocardial adenosine triphosphate and elevated purines (ie, inosine > adenosine) as markers of ischemia. Induction of intermittent antegrade or continuous retrograde hypothermic (4°C) cardioplegia releases purines until the heart becomes cold (<20°C). During reperfusion, the levels of hypoxanthine and xanthine (free radical substrates) were >90% of purines in coronary sinus effluent. Reperfusion with EHNA/NBMPR abolished ventricular dysfunction in acutely ischemic hearts with and without a hot shot and hypothermic cardioplegic arrest.. Induction of hypothermic cardioplegia releases purines from ischemic hearts until they become cold, whereas reperfusion induces massive purine release and myocardial stunning. Inhibition of cardiac es-ENT1 nucleoside transporter abolishes postischemic reperfusion injury in warm and cold cardiac surgery.

Topics: Adenine; Adenosine Triphosphate; Animals; Cold Ischemia; Disease Models, Animal; Dogs; Equilibrative Nucleoside Transporter 1; Female; Heart Arrest, Induced; Hypothermia, Induced; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardial Stunning; Myocardium; Recovery of Function; Thioinosine; Time Factors; Ventricular Function, Left; Warm Ischemia

Although the A1 adenosine receptor (A1 receptor), the main adenosine receptor type in cardiac muscle, is involved in powerful cardioprotective processes such as ischemic preconditioning, the atrial A1 receptor reserve has not yet been quantified for the direct negative inotropic effect of adenosine. In the present study, adenosine concentration-effect (E/c) curves were constructed before and after pretreatment with FSCPX (8-cyclopentyl-N3-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-N1-propylxanthine), an irreversible A1 receptor antagonist, in isolated guinea pig atria. To prevent the intracellular elimination of the administered adenosine, NBTI (S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine), a nucleoside transport inhibitor, was used. As expected, NBTI alone and FSCPX-pretreatment alone shifted the adenosine E/c curve to the left and right, respectively. However, in the presence of NBTI, FSCPX-pretreatment appeared to increase the maximal response to adenosine. By means of the receptorial responsiveness method (RRM), our recently developed procedure, adenosine E/c curves generated in the presence of NBTI were corrected for the bias caused by the endogenous adenosine accumulated by NBTI. The corrected curves indicate a substantial A1 receptor reserve for the direct negative inotropy evoked by adenosine. In addition, our results suggest that accumulation of an endogenous agonist may bias the E/c curve constructed with the same or similar agonist that can lead to seemingly paradoxical results.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Animals; Atrial Function; Dose-Response Relationship, Drug; Guinea Pigs; Heart Atria; In Vitro Techniques; Male; Myocardial Contraction; Receptor, Adenosine A1; Thioinosine; Xanthines

The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers.. HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry.. DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations > 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs.. Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

Topics: Anthracyclines; Antibiotics, Antineoplastic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Dipyridamole; HL-60 Cells; Humans; Leukemia; MCF-7 Cells; Nucleoside Transport Proteins; Nucleosides; Purinergic Antagonists; Suramin; Temperature; Thioinosine; Verapamil

To elucidate the mechanism(s) of hypoxanthine production in Müller cells and the elimination of hypoxanthine across the inner blood-retinal barrier (BRB).. The hypoxanthine biosynthesis and adenosine transport in Müller cells were investigated using a conditionally immortalized rat Müller cell line, TR-MUL5 cells. The elimination of hypoxanthine across the inner BRB was assessed by an in vivo microdialysis method and an in vitro transport study using a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells.. [(3)H]Hypoxanthine was detected in TR-MUL5 cells and TR-MUL5 cell-cultured medium 3 hours after [(3)H]adenosine incubation, indicating that the hypoxanthine is produced in TR-MUL5 cells. [(3)H]Adenosine was taken up into TR-MUL5 cells, which express mRNAs of nucleoside transporters (ENT1-2 and CNT1-2), in an Na(+)-independent and concentration-dependent manner (Km = 20 μM). Moreover, 100 μM nitrobenzylmercaptopurine riboside (NBMPR) and azidothymidine, which are inhibitors of ENT2, inhibited [(3)H]adenosine uptake, suggesting that ENT2 is a major contributor to adenosine transport in Müller cells. [(3)H]Hypoxanthine was eliminated from the rat vitreous humor and this process was inhibited in the presence of NBMPR. [(3)H]Hypoxanthine uptake by TR-iBRB2 cells took place in an Na(+)-independent and concentration-dependent manner with Km values of 4.3 μM and 2.9 mM, and was inhibited by 100 μM NBMPR.. Our findings suggest that hypoxanthine is produced from adenosine in Müller cells and ENT2 plays a major role in adenosine uptake in Müller cells. Hypoxanthine in the retina is eliminated via Na(+)-independent equilibrative nucleoside transporters.

Topics: Adenosine; Animals; Blood-Retinal Barrier; Cell Line; Chromatography, High Pressure Liquid; DNA Primers; Dose-Response Relationship, Drug; Endothelium, Vascular; Equilibrative Nucleoside Transport Proteins; Hypoxanthine; Male; Membrane Transport Proteins; Microdialysis; Neuroglia; Nucleoside Transport Proteins; Rats; Rats, Wistar; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thioinosine; Zidovudine

The high density of A1 adenosine receptors in the brain results in significant potential for central nervous system (CNS)-related adverse effects with A1 agonists. Tecadenoson is a selective A1 adenosine receptor agonist with close similarity to adenosine. We studied the binding and transmembrane transport of tecadenoson by recombinant human equilibrative nucleoside transporters (hENTs) hENT1 and hENT2, and human concentrative nucleoside transporters (hCNTs) hCNT1, hCNT2, and hCNT3 in vitro and by mouse mENT1 in vivo. Binding affinities of the five recombinant human nucleoside transporters for tecadenoson differed (hENT1 > hCNT1 > hCNT3 > hENT2 > hCNT2), and tecadenoson was transported largely by hENT1. Pretreatment of mice with a phosphorylated prodrug of nitrobenzylmercaptopurine riboside, an inhibitor of mENT1, significantly decreased brain exposure to tecadenoson compared with that of the untreated (control) group, suggesting involvement of mENT1 in transport of tecadenoson across the blood-brain barrier (BBB). In summary, ENT1 was shown to mediate the transport of tecadenoson in vitro with recombinant and native human protein and in vivo with mice. The micromolar apparent Km value of tecadenoson for transport by native hENT1 in cultured cells suggests that hENT1 will not be saturated at clinically relevant (i.e., nanomolar) concentrations of tecadenoson, and that hENT1-mediated passage across the BBB may contribute to the adverse CNS effects observed in clinical trials. In contrast, in cases in which a CNS effect is desired, the present results illustrate that synthetic A1 agonists that are transported by hENT1 could be used to target CNS disorders because of enhanced delivery to the brain.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Affinity Labels; Animals; Biological Transport; Blood-Brain Barrier; Brain; Cells, Cultured; Equilibrative Nucleoside Transporter 1; Furans; Humans; Membrane Transport Modulators; Mice; Nucleoside Transport Proteins; Prodrugs; Thioinosine

Topics: Microwaves; Molecular Structure; Stereoisomerism; Thioinosine; Thiourea

We investigated the contribution of endogenous adenosine to amitriptyline-induced cardiovascular toxicity in rats. A control group of rats was pretreated with intraperitoneal (i.p.) 5% dextrose and received intravenous 0.94 mg/kg/min of amitriptyline for 60 minutes. The second and third groups of rats pretreated with i.p. 10 mg/kg of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor, and i.p. 1 mg/kg of S-(4-nitrobenzyl)-6-thioinosine (NBTI), a facilitated adenosine transport inhibitor, received 5% dextrose and amitriptyline infusion, respectively. Outcome parameters were mean arterial pressure (MAP), heart rate (HR), QT and QRS durations, and plasma adenosine concentrations. Plasma adenosine concentrations were increased in all groups. In the control group, amitriptyline decreased MAP and HR and prolonged QT and QRS durations after 10 minutes of infusion. In EHNA/NBTI-pretreated rats, amitriptyline prolonged QRS duration at 10 and 20 minutes. In EHNA/NBTI pretreated rats, amitriptyline-induced MAP, HR reductions, and QRS prolongations were more significant than that of dextrose-infusion-induced changes. Our results indicate that amitriptyline augmented the cardiovascular effects of endogen adenosine by increasing plasma levels of adenosine in rats.

Topics: Adenine; Adenosine; Amitriptyline; Animals; Antidepressive Agents, Tricyclic; Arterial Pressure; Glucose; Heart Rate; Infusions, Intravenous; Long QT Syndrome; Male; Rats; Rats, Wistar; Thioinosine; Time Factors

We previously showed that equilibrative nucleoside transporter 1 (ENT1) is a primary ribavirin transporter in human hepatocytes. However, because the role of this transporter in the antiviral mechanism of the drug remains unclear, the present study aimed to elucidate the role of ENT1 in ribavirin antiviral action. OR6 cells, a hepatitis C virus (HCV) replication system, were used to evaluate both ribavirin uptake and efficacy. The ribavirin transporter in OR6 cells was identified by mRNA expression analyses and transport assays. Nitrobenzylmercaptopurine riboside (NBMPR) and micro-RNA targeted to ENT1 mRNA (miR-ENT1) were used to reduce the ribavirin uptake level in OR6 cells. Our results showed that ribavirin antiviral activity was associated with its accumulation in OR6 cells, which was also closely associated with the uptake of the drug. It was found that the primary ribavirin transporter in OR6 cells was ENT1 and that inhibition of ENT1-mediated ribavirin uptake by NBMPR significantly attenuated the antiviral activity of the drug as well as its accumulation in OR6 cells. The results also showed that even a small reduction in the ENT1-mediated ribavirin uptake, achieved in this case using miR-ENT1, caused a significant decrease in its antiviral activity, thus indicating that the ENT1-mediated ribavirin uptake level determined its antiviral activity level in OR6 cells. In conclusion, our results show that by facilitating its uptake and accumulation in OR6 cells, ENT1 plays a pivotal role in the antiviral effectiveness of ribavirin and therefore provides an important insight into the efficacy of the drug in anti-HCV therapy.

Topics: Antiviral Agents; Biological Transport; Blotting, Western; Cell Line, Tumor; Equilibrative Nucleoside Transporter 1; Genes, Reporter; Hepacivirus; Hepatocytes; Humans; Luciferases; MicroRNAs; Ribavirin; RNA, Messenger; Thioinosine

The inhibition of adenosine deaminase with erythro-9 (2-hydroxy-3-nonyl)-adenine (EHNA) and the es-ENT1 transporter with p-nitro-benzylthioinosine (NBMPR), entraps myocardial intracellular adenosine during on-pump warm aortic crossclamping, leading to a complete recovery of cardiac function and adenosine triphosphate (ATP) during reperfusion. The differential role of entrapped intracellular and circulating adenosine in EHNA/NBMPR-mediated protection is unknown. Selective (8-cyclopentyl-1,3-dipropyl-xanthine) or nonselective [8-(p-sulfophenyl)theophyline] A1 receptor antagonists were used to block adenosine A1-receptor contribution in EHNA/NBMPR-mediated cardiac recovery.. Anesthetized dogs (n = 45), instrumented to measure heart performance using sonomicrometry, were subjected to 30 minutes of warm aortic crossclamping and 60 minutes of reperfusion. Three boluses of the vehicle (series A) or 100 μM EHNA and 25 μM NBMPR (series B) were infused into the pump at baseline, before ischemia and before reperfusion. 8-Cyclopentyl-1,3-dipropyl-xanthine (10 μM) or 8-(p-sulfophenyl)theophyline (100 μM) was intra-aortically infused immediately after aortic crossclamping distal to the clamp in series A and series B. The ATP pool and nicotinamide adenine dinucleotide was determined using high-performance liquid chromatography.. Ischemia depleted ATP in all groups by 50%. The adenosine/inosine ratios were more than 10-fold greater in series B than in series A (P < .001). ATP and function recovered in the EHNA/NBMPR-treated group (P < .05 vs control group). 8-Cyclopentyl-1,3-dipropyl-xanthine and 8-(p-sulfophenyl)theophyline partially reduced cardiac function in series A and B to the same degree but did not abolish the EHNA/NBMPR-mediated protection in series B.. In addition to the cardioprotection mediated by activation of the adenosine receptors by extracellular adenosine, EHNA/NBMPR entrapment of intracellular adenosine provided a significant component of myocardial protection despite adenosine A1 receptor blockade.

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Chromatography, High Pressure Liquid; Constriction; Disease Models, Animal; Dogs; Ischemic Preconditioning; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardial Stunning; Nucleoside Transport Proteins; Receptor, Adenosine A1; Theophylline; Thioinosine; Xanthines

To determine the role of the p-nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter 1 (es-ENT1) in postmyocardial infarction reperfusion injury-mediated ventricular fibrillation and regional dysfunction. We used erythro-9 (2-hydroxy-3-nonyl)-adenine and p-nitrobenzylthioinosine to inhibit both adenosine deamination and transport in a canine model of off pump acute myocardial infarction.. Anesthetized adult dogs (n = 37), instrumented to monitor the percentage of systolic segmental shortening and wall thickening using sonomicrometry, underwent 90 minutes of left anterior descending coronary artery occlusion and 120 minutes of reperfusion. Myocardial coronary blood flow, adenosine triphosphate pool, infarct size, and the incident of ventricular fibrillation and cardioversion were also measured. The dogs received an intravenous infusion of the vehicle (control) or 100 μM of erythro-9 (2-hydroxy-3-nonyl)-adenine and 25 μM p-nitrobenzylthioinosine before ischemia (preconditioning group) or just before reperfusion (postconditioning group).. In the control group, adenosine triphosphate depletion was associated with the accumulation of more inosine than adenosine during ischemia and washed out during reperfusion. Myocardial adenosine and inosine were the major nucleosides in the pre- and postconditioning groups during ischemia and remained detectable during reperfusion. In both groups, recovery of systolic segmental shortening and wall thickening and a reduction in the incidence of ventricular fibrillation (P < .05 vs the control group) coincided with retention of myocardial nucleosides. The infarct size in the 3 groups was not significantly different, independent of myocardial blood flow during ischemia.. Preconditioning or postconditioning with erythro-9 (2-hydroxy-3-nonyl)-adenine/p-nitrobenzylthioinosine significantly reduced the incidence of ventricular fibrillation and cardioversion and attenuated regional contractile dysfunction mediated by postmyocardial infarction reperfusion injury. It is concluded that p-nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter 1 played a major role in these events.

Topics: Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Analysis of Variance; Animals; Coronary Circulation; Dogs; Equilibrative Nucleoside Transporter 1; Ischemic Preconditioning; Least-Squares Analysis; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardial Stunning; Thioinosine; Ventricular Fibrillation

The purpose of this study was to clarify the transport characteristics of nucleosides in rat placenta and the changes of functional expression of nucleoside transporters in rat placenta with experimental diabetes mellitus. Placental uptake clearances of [(3)H]adenosine and [(3)H]zidovudine from maternal blood was much higher than that of [(14)C]mannitol. Xenopus oocytes injected with rat ENT1 and ENT2 cRNA took up [(3)H]adenosine with K(m) values of 6.1 and 26 µM, respectively. [(3)H]Adenosine transport by rat placental brush-border membrane vesicles (BBMV) was saturable and was inhibited by nitrobenzylthioinosine (NBMPR), a specific ENT inhibitor, in a manner consistent with involvement of both rat ENT1 and ENT2. [(3)H]Didanosine was modestly taken up by placenta, and the inhibitory effect of 100 µM NBMPR on [(3)H]ddI uptake by BBMV suggested a role of ENT2-mediated transport. Expression of ENT1, ENT2, ENT3, CNT2, and CNT3 mRNAs was detected in placenta of control and streptozotocin (STZ)-induced diabetic pregnant rats, and CNT2 (SLC28A2) expression was significantly increased in STZ-induced diabetic rats. Consistently, Na(+)-dependent adenosine uptake by BBMV from STZ-induced diabetic pregnant rats was higher than that from control rats. These results suggest the involvement of placental ENT2 as well as ENT1 in nucleoside uptake from maternal blood, and the induction of CNT2 in experimental diabetes mellitus.

Topics: Adenosine; Animals; Biological Transport; Carrier Proteins; Diabetes Mellitus, Experimental; Diabetes, Gestational; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Female; Maternal-Fetal Exchange; Membrane Transport Proteins; Microvilli; Nucleosides; Oocytes; Placenta; Pregnancy; Rats; Rats, Wistar; RNA, Messenger; Thioinosine; Trophoblasts; Up-Regulation; Xenopus laevis; Zidovudine

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. A previous study from our laboratory established Cys222 in transmembrane (TM) 6 as the residue responsible for methyl methanethiosulfonate (a membrane-permeable sulfhydryl modifier)-mediated enhancement of the binding of the ENT1 inhibitor nitrobenzylmercaptopurine riboside (NBMPR) in intact cells. However, the capacity of charged sulfhydryl reagents to inhibit the binding of NBMPR in broken cell preparations (allowing cytoplasmic access) was not affected by mutation of any of the cysteines (Cys87, 193, 213, or 222) in the N-terminal half of the protein. We thus hypothesized that the inhibitory effects of the modifiers were due to the one or more of the six cysteine residues in the C-terminal half of ENT1, particularly one or both of those in the fifth intracellular loop (Cys414 and Cys416). Each of the cysteines were mutated to serine or alanine and expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate sulfhydryl-modifying reagents. Transporter function was assessed by the site-specific binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. These studies established that Cys378 is an extracellular-located residue modified by [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET) to inhibit the binding of NBMPR to intact cells. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding, and this enhancement was eliminated by the comutation of Cys378, indicating that disruption of the fifth intracellular loop modifies the conformation of TM10 and its extracellular extension. Mutation of Cys416 led to the loss of the ability of the charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes and also led to the loss of transport function. This finding further supports an allosteric interaction between the fifth intracellular loop and the extracellular NBMPR binding domain and implicates this region in the translocation function of human ENT1.

Topics: 2-Chloroadenosine; Allosteric Regulation; Cell Line; Cysteine; Equilibrative Nucleoside Transporter 1; Humans; Mesylates; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Transport; Thioinosine; Transfection

Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A(2A) adenosine receptors (A(2A)AR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A(2A)AR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2A)AR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K(m) for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606) or pGL3-hCAT-1(-650) constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606), and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2A)AR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.

Topics: Adenosine; Adolescent; Adult; Amino Acid Transport System y+; Arginine; Biological Transport; Cationic Amino Acid Transporter 1; Extracellular Space; Female; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Insulin; Kinetics; Male; Promoter Regions, Genetic; Receptor, Adenosine A2A; Thioinosine; Transcription, Genetic; Young Adult

Tetrahydrobiopterin (BH(4)) is synthesized de novo in particular cells, but in the case of a systemic or local BH(4) deficiency, BH(4) supplementation therapy is applied. BH(4)-responsive PKU has also been effectively treated with BH(4) supplementation. However, the rapid clearance of the supplemented BH(4) has prevented the therapy from being widely accepted. Deposition of BH(4) after supplementation involves oxidation of BH(4) to dihydrobiopterin (BH(2)) and subsequent conversion to BH(4) by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH(4) across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH(2) or BH(4) using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP>BH(2)>BH(4). hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH(4) via uptake that is likely mediated by ENT2 (SP>BH(2)>BH(4)). When exogenous BH(2) was given to mice, it was efficiently converted to BH(4) and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH(4) deposition after BH(2) administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH(2) was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH(2), and BH(4) across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH(4) biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH(2) uptake for BH(4) accumulation and the release for modifying the intracellular BH(2)/BH(4) ratio.

Topics: Animals; Biopterins; Endothelial Cells; Endothelium, Vascular; Equilibrative Nucleoside Transport Proteins; Female; HeLa Cells; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Oocytes; Pterins; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thioinosine; Xenopus laevis

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 μM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 μM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 μM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.

Topics: Action Potentials; Adenosine; Adenosine A1 Receptor Antagonists; Animals; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Female; Glucose; Glutathione; Hippocampus; Humans; Hypoxia; In Vitro Techniques; Mice; Mice, Transgenic; Neurons; Patch-Clamp Techniques; Protein Binding; Purinergic P1 Receptor Agonists; Statistics, Nonparametric; Synaptic Transmission; Thioinosine; Tritium; Xanthines

To adopt an individualized approach to assess cytarabine (ara-C) hematotoxicity, we studied the relationship between pharmacogenetic variability in the cytidine deaminase gene (CDA) and ara-C toxicity in native peripheral blood mononuclear cells from 100 healthy volunteers.. Peripheral blood mononuclear cells were incubated for 48 h with 3 µM ara-C, and cell viability was analyzed by flow cytometry with and without the addition of an equilibrative nucleoside transporter transport inhibitor. CDA promoter and exonic variants were genotyped to derive haplotypes for the CDA gene.. Significant between-subject variability was observed in ara-C toxicity (21-fold with 40.1% coefficient of variation compared with 1.2-fold within-subject variability [9.6% coefficient of variation]). Inhibition of hENT1 reversed ara-C cytotoxicity. The linked CDA promoter variants -451C>T, -92A>G, -31Del and the exonic 79A>C variant were associated with ara-C toxicity (p < 0.05). CDA*2A haplotype was associated with ara-C toxicity (p = 0.03).. Genetic polymorphisms within CDA may be risk factors for ara-C-induced hematotoxicity. Original submitted 6 October 2010; Revision submitted 29 November 2010.

Topics: Adult; Antimetabolites, Antineoplastic; Cell Survival; Cells, Cultured; Cytarabine; Cytidine Deaminase; Drug-Related Side Effects and Adverse Reactions; Equilibrative Nucleoside Transporter 1; Exons; Female; Genotype; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Risk Factors; Thioinosine; Young Adult

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.. RT-PCR was used to investigate the expression of mRNA encoding for A(1), A(2A), A(2B) and A(3) receptors. Contractile activity was examined in vitro as changes in isometric tension.. In mouse duodenum, all four classes of adenosine receptors were expressed, with the A(2B) receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A(1) receptor antagonist and mimicked by N(6) -cyclopentyladenosine (CPA), selective A(1) agonist. The relaxation induced by A(1) receptor activation was insensitive to tetrodotoxin (TTX) or N(ω) -nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A(1) receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A(2A) receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A(2A) receptor antagonist or l-NAME.. Adenosine can negatively regulate mouse duodenal motility either by activating A(1) inhibitory receptors located post-junctionally or controlling neurotransmitter release via A(1) or A(2A) receptors. Both receptors are available for pharmacological recruitment, even if only A(2A) receptors appear to be preferentially stimulated by endogenous adenosine.

Topics: Adenosine; Animals; Duodenum; Electric Stimulation; Gastrointestinal Motility; In Vitro Techniques; Isometric Contraction; Male; Mice; Mice, Inbred C57BL; Nucleoside Transport Proteins; Purinergic Antagonists; Purinergic P1 Receptor Agonists; Receptors, Purinergic P1; RNA, Messenger; Thioinosine

Adenosine concentrations are regulated by purinergic enzymes and nucleoside transporters. Transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1) have been generated (Parkinson et al., 2009 [7]). The present study tested the hypothesis that mice homozygous and heterozygous for the transgene exhibit differences in hENT1 mRNA and protein expression, and in behavioral responses to caffeine and ethanol, two drugs with adenosine-dependent actions. Real time polymerase chain reaction (PCR) was used to identify mice heterozygous and homozygous for the transgene. Gene expression, determined by real time PCR of cDNA reverse transcribed from cerebral cortex RNA, was 3.8-fold greater in homozygous mice. Protein abundance, determined by radioligand binding assays using 0.14nM [(3)H]S-(4-nitrobenzyl)-6-thioinosine ([(3)H]NBTI), was up to 84% greater in cortex synaptosome membranes from homozygous than from heterozygous mice. In western blots with an antibody specific for hENT1, a protein of approximately 40kDa was strongly labelled in cortex samples from homozygous mice, weakly labelled in samples from heterozygous mice and absent from samples from wild type mice. In behavioral assays, transgenic mice showed a greater response to ethanol and a reduced response to caffeine than wild type littermates; however, no significant differences between heterozygous and homozygous mice were detected. These data indicate that the difference in ENT1 function between wild type and heterozygous mice was greater than that between heterozygous and homozygous mice. Therefore, either heterozygous or homozygous hENT1 transgenic mice can be used in studies of ENT1 regulation of adenosine levels and adenosine dependent behaviors.

Topics: Adenosine Kinase; Analysis of Variance; Animals; Behavior, Animal; Caffeine; Cerebral Cortex; Equilibrative Nucleoside Transporter 1; Ethanol; Gene Expression Regulation; Humans; Mice; Mice, Transgenic; Motor Activity; Protein Binding; Thioinosine; Tritium

The evaluation of the mutagenic and antimutagenic actions of extracts obtained from aerial part of Teucrium ramosissimum was assayed using the Salmonella typhimurium assay system. The effect of the same extracts on genotoxicity and SOS response induced by aflatoxin B(1) as well as nitrofurantoin was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The different extracts showed no mutagenicity when tested with Salmonella typhimurium strains TA100, TA98 and TA1535 either with or without S9 mix. In contrast, our results prove that T. ramosissimum extracts possess antimutagenic effects against sodium azide, aflatoxin B1, benzo[a]pyrene and 4-nitro-o-phenylenediamine. Moreover, the T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixtures. However, all the extracts significantly decreased the genotoxicity induced by aflatoxin B(1) and nitrofurantoin. The result obtained by the Ames test confirms those of SOS chromotest. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) and the non enzymatic (NBT/riboflavine, DPPH and ABTS assays) systems. All extracts exhibited high antioxidant activity except the chloroform and the methanol extracts in DPPH and NBT/riboflavine assays respectively. Our results underline the potential of T. ramosissimum to avoid mutations and also its antioxidant potential.

Topics: Affinity Labels; Antimutagenic Agents; Antioxidants; Bacteria; Benzothiazoles; Biphenyl Compounds; DNA Repair; Dose-Response Relationship, Drug; Flavonoids; Free Radical Scavengers; Microbial Sensitivity Tests; Mutagenicity Tests; Mutagens; Phenols; Picrates; Plant Extracts; Plant Leaves; Salmonella typhimurium; Sterols; Sulfonic Acids; Tannins; Teucrium; Thioinosine; Xanthine Oxidase

The objective of this study was to evaluate the immunomodulatory effects of proanthocyanidin A-1 (PAA-1) from leaves of Rhododendron spiciferum (Ericaceae). In vitro tests showed that PAA-1 stimulated cell proliferation of splenocytes and peritoneal macrophages significantly enhanced the cytotoxicity of natural killer (NK) cells and increased CD4(+) and CD8(+) cell populations. PAA-1 also regulated the expression of Th1- and Th2-related cytokines. Moreover, this study showed that PAA-1 exhibited a significant effect on NBT dye reduction and lysosomal enzyme activity responses in macrophages, indicating effective phagocytic activation. These results revealed that PAA-1 exhibits immunomodulatory activity without a clear dose response.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Immunologic Factors; Killer Cells, Natural; Lysosomes; Macrophages, Peritoneal; Male; Mice; Mice, Inbred BALB C; Phagocytes; Plant Leaves; Proanthocyanidins; Rhododendron; Spleen; Thioinosine

Transport across the cell membrane is crucial in drug delivery. However, the process is complicated because nucleoside derivatives that are commonly used as anti-viral drugs are transported through two different types of specific transporters: concentrative transporters and equilibrative transporters. Cross-disciplinary approaches involving both biological experiments and theoretical considerations are therefore necessary to study the transport of nucleoside analogues such as ribavirin. Here we constructed an experimental model system using the Xenopus laevis oocyte that expressed examples of both types of transporters: human concentrative nucleoside transporter 3 and human equilibrative transporter 1. We also performed a kinetic study. Experimental results showed that the transport of ribavirin could be reduced by inhibiting one of the two types of transporters, which seems to be counterintuitive. We therefore designed a simple mathematical model of the dynamics of ribavirin uptake and analyzed the model behaviors using a numerical simulation. The theoretical results reproduced the experimentally observed phenomena and suggested a possible mechanism for the process. Based on this mechanism, we predicted some potential methods for the effective uptake of ribavirin from a dynamics point of view.

Topics: Animals; Equilibrative Nucleoside Transporter 1; Kinetics; Models, Biological; Nucleoside Transport Proteins; Oocytes; Ribavirin; Sodium Chloride; Thioinosine; Xenopus laevis

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.

Topics: Animals; Chlorocebus aethiops; COS Cells; Equilibrative Nucleoside Transport Proteins; Mice; Oligopeptides; Peptide Fragments; Peptides; Recombinant Fusion Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thioinosine; Trypsin

Exogenous adenosine produces potent synaptic inhibition in spinal substantia gelatinosa (SG), a region involved in nociceptive and thermoreceptive mechanisms. To examine the possibility that endogenous adenosine tonically modulates excitatory synaptic transmission in spinal SG, whole-cell, voltage-clamp recordings were made from SG neurons in adult rat spinal cord slices. In all SG neurons sensitive to exogenous adenosine, the adenosine uptake inhibitor, NBTI, mimics adenosine's inhibitory actions on dorsal root evoked EPSCs (eEPSCs) and miniature spontaneous EPSCs (mEPSCs). These inhibitory effects were antagonized by A1 adenosine receptor antagonist, DPCPX. DPCPX also potentates eEPSCs in those SG neurons in which adenosine or adenosine A1 receptor agonists (CHA, CCPA) suppressed eEPSCs. DPCPX often increases mEPSC frequency without altering mEPSC amplitude, suggesting presynaptic action on adenosine A1 receptors. Selective A2 (DMPX) and A2a (ZM 241385) adenosine receptor antagonists had no or minimal effects upon either eEPSCs or mEPSCs. The adenosine degrading enzyme, adenosine deaminase, mimicked the effects of DPCPX on the mEPSC frequency. We conclude that the excitatory synaptic transmission in the spinal SG is under an inhibitory tone of endogenous adenosine through the activation of A1 receptors. The present results suggested that the background activity of A1 receptors in the spinal SG might be contributed to setting the physiological "noceceptive thresholds".

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Adenosine Deaminase; Animals; Electric Stimulation; Excitatory Postsynaptic Potentials; Miniature Postsynaptic Potentials; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Substantia Gelatinosa; Synaptic Transmission; Thioinosine; Xanthines

Very limited information exists on transformation processes of carbon nanotubes in the natural aquatic environment. Because the conjugated pi-bond structure of these materials is efficient in absorbing sunlight, photochemical transformations are a potential fate process with reactivity predicted to vary with their diameter, chirality, number and type of defects, functionalization, residual metal catalyst and amorphous carbon content, and with the composition of the water, including the type and composition of materials that act to disperse them into the aqueous environment. In this study, the photochemical reactions involving colloidal dispersions of carboxylated single-walled carbon nanotubes (SWNT-COOH) in sunlight were examined. Production of reactive oxygen species (ROS) during irradiation occurs and is evidence for potential further phototransformation and may be significant in assessing their overall environmental impacts. In aerated samples exposed to sunlight or to lamps that emit light only within the solar spectrum, the probe compounds, furfuryl alcohol (FFA), tetrazolium salts (NBT2+ and XTT), and p-chlorobenzoic acid (pCBA), were used to indicate production of 1O2, O2.-, and .OH, respectively. All three ROS were produced in the presence of SWNT-COOH and molecular oxygen (3O2). 1O2 production was confirmed by observing enhanced FFA decay in deuterium oxide, attenuated decay of FFA in the presence of azide ion, and the lack of decay of FFA in deoxygenated solutions. Photogeneration of O2.- and .OH was confirmed by applying superoxide dismutase (SOD) and tert-butanol assays, respectively. In air-equilibrated suspensions, the loss of 0.2 mM FFA in 10 mg/L SWNT-COOH was approximately 85% after 74 h. Production of 1O2 was not dependent on pH from 7 to 11; however photoinduced aggregation was observed at pH 3.

Topics: Carboxylic Acids; Chlorobenzoates; Furans; Hydrogen-Ion Concentration; Nanotubes, Carbon; Reactive Oxygen Species; Singlet Oxygen; Sunlight; Tetrazolium Salts; Thioinosine; Water

5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), 5'-S-(2-acetamidoethyl)-6-N-[(4-substituted)benzyl]-5'-thioadenosine analogues, 5'-S-[2-(6-aminohexanamido)]ethyl-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAHENTA), and related compounds were synthesized by S(N)Ar displacement of fluoride from 6-fluoropurine intermediates with 4-(substituted)benzylamines. Conjugation of the pendant amino groups of SAENTA and SAHENTA with fluorescein-5-yl isothiocyanate (FITC) gave fluorescent probes that bound at nanomolar concentrations specifically to human equilibrative nucleoside transporter 1 (hENT1) produced in recombinant form in model expression systems and in native form in cancer cell lines. Transporter binding effects were studied and the ability of the probes to predict the potential antitumor efficacy of 2'-deoxy-2',2'-difluorocytidine (gemcitabine) was demonstrated.

Topics: Adenosine; Animals; Antineoplastic Agents; Cell Line, Tumor; Deoxycytidine; Equilibrative Nucleoside Transporter 1; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gemcitabine; Humans; Mice; Oocytes; Protein Binding; Stereoisomerism; Structure-Activity Relationship; Thioinosine; Thionucleosides; Xenopus; Yeasts

(18)F-3'-Deoxy-3'-fluorothymidine ((18)F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for (18)F-FLT uptake in normal tissues and tumors.. ENT1-knockout (ENT1(-/-)) mice were generated and compared with wild-type (ENT1(+/+)) mice using small-animal (18)F-FLT PET. In addition, ENT1(+/+) mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by (18)F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and (18)F-FLT uptake using real-time polymerase chain reaction and (3)H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by (18)F-FLT small-animal PET.. Compared with noninjected ENT1(+/+) mice, ENT1(+/+) mice injected with NBMPR-P and ENT1(-/-) mice displayed a reduced percentage injected dose per gram (%ID/g) for (18)F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for (18)F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1(-/-) mice displayed 1.65-fold greater plasma thymidine levels than did ENT1(+/+) mice. Spleen tissue from ENT1(+/+) and ENT1(-/-) mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold (3)H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection.. Loss of ENT1 activity significantly affected (18)F-FLT biodistribution in mice and (18)F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of (18)F-FLT uptake in normal and transformed cells.

Topics: Animals; Biological Transport; Cell Line, Tumor; Cell Transformation, Neoplastic; Dideoxynucleosides; Equilibrative Nucleoside Transporter 1; Female; Gene Knockdown Techniques; Gene Knockout Techniques; Humans; Mice; Mice, Knockout; Neoplasms; Positron-Emission Tomography; RNA, Messenger; RNA, Small Interfering; Spleen; Thioinosine; Thymidine; Thymidine Kinase; Transfection

Extracellular adenosine removal is via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in the endothelium, thus regulating adenosine-induced revascularization and angiogenesis. Since human endothelial progenitor cells (hEPCs) promote revascularization, we hypothesize differential expression of nucleoside transporters in hEPCs. hEPCs were cultured 3 (hEPC-3d) or 14 (hEPC-14d) days. RT-PCR for prominin 1, CD34, octamer-4, kinase insert domain receptor, oxidized low-density lipoprotein (lectin-like) receptor 1 and tyrosine endothelial kinase was used to evaluate phenotypic differentiation. Flow cytometry was used to estimate CD34(+)/KDR(-) (non-differentiated), CD34(-)/KDR(+) (differentiated) or CD34(+)/KDR(+) (mixed) cell populations. Adenosine transport was measured in absence or presence of sodium, S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1-10 μM), inosine, hypoxanthine or guanine (0.1-5 mM), hENTs protein abundance by western blot, and hENTs, hCNT1, hCNT2 and hCNT3 mRNA expression by real time RT-PCR. hEPC-3d cells were CD34(+)/KDR(-) compared with hEPC-14d cells that were CD34(-)/KDR(+). hEPC-3d cells exhibit hENT1-like adenosine transport (NBTI-sensitive, Na(+)-independent), which is absent in hEPC-14d cells. hEPC-14d cells exhibit two transport components: component 1 (NBTI insensitive, Na(+)-independent) and component 2 (NBTI insensitive, Na(+)-dependent, Hill coefficient ∼1.8), the latter resembling CNT3-like transport. hEPC-3d cells express hENT1 protein and mRNA, which is reduced (∼90%) in hEPC-14d cells, but instead only hCNT3 mRNA is expressed in this cell type. hENT2, hCNT1 and hCNT2 were undetectable in hEPCs. Thus, hEPCs exhibit a differential expression of hENT1 and hCNT3 functional nucleoside transporters, which could be related with its differentiation stage.

Topics: Adenosine; Biological Transport; Blotting, Western; Cell Differentiation; Endothelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Kinetics; Reverse Transcriptase Polymerase Chain Reaction; RNA; Stem Cells; Thioinosine

Adenosine is a candidate modulator of sperm motility in the female reproductive tract that increases sperm flagellar beat frequency in vitro. Past work suggested that this acceleration may involve equilibrative (ENT) and concentrative (CNT) nucleoside transporters. Here we show that Slc29a1 (ENT-1) is the predominant nucleoside transporter expressed in the mouse testis. Unexpectedly, the beat of Slc29a1-null sperm still accelerates in response to 2-chloro-2'-deoxyadenosine (Cl-dAdo). Moreover, in wild-type sperm neither blockade of CNTs by removal of external Na(+), nor inhibition of ENTs with nitrobenzylthioionosine, prevents acceleration of the sperm beat by Cl-dAdo. In contrast, pertussis toxin produces strong blockade, indicating involvement of a Gα(i/o)-coupled adenosine receptor. Although agonists selective for adenosine receptors A1R, A2aR, and A2bR are ineffective, A3R-selective agonists Cl-IB-MECA and IB-MECA do accelerate the beat. Consistent with this pharmacological profile, the predominant Adora transcripts in the testis are products of the nested Adora3i1 and Adora3i2 genes. Surprisingly, Cl-IB-MECA and Cl-dAdo still accelerate the beat of Adora3i1-null sperm indicating that the remaining Adora3i2 transcript produces an A3R that functions in sperm. When cloned Adora3i2 is heterologously expressed in tsA-201 cells, Cl-dAdo decreases forskolin-evoked accumulation of cAMP, indicating that Adora3i2 specifies a functional A3Ri2 adenosine receptor that couples through Gα(i). Database mining reveals that mouse Adora3i2 is expressed primarily in testis, almost exclusively in spermatids. Expression of the orthologous ADORA3i3 transcript also is most prominent in human testis; presumably producing an A3Ri3 receptor that is functional in sperm and that may be a target for development of male-directed contraceptives.

Topics: Animals; Colforsin; Cyclic AMP; Humans; Male; Mice; Mice, Knockout; Nucleosides; Pertussis Toxin; Receptor, Adenosine A3; Spermatids; Spermatozoa; Testis; Thioinosine

The functionality of human erythrocyte nucleoside transporter ENT1 was examined at ice-cold temperatures (ICT; measured temperature, 0.5-0.7 degrees C) using rightside-out membrane vesicles (ROVs). The uptake of uridine, an ENT1 substrate, showed saturation kinetics and was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), a specific ENT1 inhibitor, at both 23 degrees C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabeled uridine or ribavirin, another ENT1 substrate, and the overshoot phenomenon was observed at ICT. Similarly, [3H]ribavirin uptake was markedly trans-stimulated by unlabeled ribavirin or uridine at ICT. The trans-stimulated uptake of [3H]uridine at ICT was inhibited by ENT1 inhibitors/substrates such as NBMPR, dipyridamole, adenosine, and ribavirin in a concentration-dependent manner. The inhibition of [3H]uridine uptake by NBMPR and dipyridamole at ICT was also observed in intact red blood cells. Like uridine uptake, [3H]D-glucose uptake by ROVs, which is mediated by facilitative glucose transporter GLUT1, was trans-stimulated by unlabeled D-glucose at ICT, and the overshoot phenomenon was observed. In contrast, the ability of ATP-dependent transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein via multidrug resistance-associated protein 5 in inside-out membrane vesicles disappeared at ICT. These results clearly indicate that human erythrocyte transporters such as ENT1 function even at very low temperatures near 0 degrees C. The significance of these findings in transporter research is discussed.

Topics: Cold Temperature; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Erythrocyte Membrane; Glucose Transporter Type 1; Humans; Ribavirin; Thioinosine; Uridine

Adenosine serves as a homeostatic factor, regulating hippocampal activity through A(1) receptor-mediated inhibition. Gamma frequency oscillations, associated with cognitive functions, emerge from increased network activity. Here we test the hypothesis that hippocampal gamma oscillations are modulated by ambient adenosine levels. In mouse hippocampal slices exogenous adenosine suppressed the power of both kainate-induced gamma oscillations and spontaneous gamma oscillations, observed in a subset of slices in normal aCSF. Kainate-induced gamma oscillation power was suppressed by the A(1) receptor agonist PIA and potentiated by the A(1) receptor antagonist 8-CPT to three times matched control values with an EC(50) of 1.1microM. 8-CPT also potentiated spontaneous gamma oscillation power to five times control values. The A(2A) receptor agonist CGS21680 potentiated kainate-induced gamma power to two times control values (EC(50) 0.3nM), but this effect was halved in the presence of 8-CPT. The A(2A) receptor antagonist ZM241385 suppressed kainate-induced gamma power. The non-selective adenosine receptor antagonist caffeine induced gamma oscillations in slices in control aCSF and potentiated both kainate-induced gamma and spontaneous gamma oscillations to three times control values (EC(50) 28muM). Decreasing endogenous adenosine levels with adenosine deaminase increased gamma oscillations. Increasing endogenous adenosine levels with the adenosine kinase inhibitor 5-iodotubericidin suppressed gamma oscillations. Partial hypoxia-induced suppression of gamma oscillations could be prevented by 8-CPT. These observations indicate that gamma oscillation strength is powerfully modulated by ambient levels of adenosine through A(1) receptors, opposed by A(2A) receptors. Increased gamma oscillation strength is likely to contribute to the beneficial cognitive effects of caffeine.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Analysis of Variance; Animals; Biological Clocks; Dose-Response Relationship, Drug; Evoked Potentials; Excitatory Amino Acid Agonists; Fourier Analysis; Hippocampus; Hypoxia; In Vitro Techniques; Kainic Acid; Male; Mice; Mice, Inbred C57BL; Phenethylamines; Receptor, Adenosine A1; Receptor, Adenosine A2A; Theophylline; Thioinosine; Time Factors; Triazines; Triazoles

3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying K(i) values ranging from 10,000 to 0.7nM. Both CoMFA and CoMSIA analysis gave reliable models with q2 values >0.50 and r2 values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r2 values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.

Topics: Equilibrative Nucleoside Transporter 1; Humans; Models, Molecular; Quantitative Structure-Activity Relationship; Thioinosine

The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.

Topics: Amino Acid Sequence; Animals; Antimetabolites, Antineoplastic; Cell Line, Tumor; Cell Membrane; Cytarabine; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Glycine; Humans; Leukemia, T-Cell; Molecular Sequence Data; Point Mutation; Protein Structure, Tertiary; RNA, Messenger; Thioinosine

The polar nucleoside drug ribavirin is front-line treatment for chronic hepatitis C virus infection. The human equilibrative nucleoside transporter (ENT) 1 transports ribavirin into erythrocytes where it is phosphorylated. These phosphorylated metabolites accumulate in the erythrocytes and produce dose-limiting hemolytic anemia. Here, we examined the in vitro and ex vivo transport and metabolism of ribavirin by erythrocytes isolated from humans and Ent1-null mice. Ribavirin (2.4 microM) uptake was significantly higher (1044 +/- 255 amol/microg/10 s) into erythrocytes from Ent1(+/+) mice compared with that from Ent1(-/-) mice (76.48 +/- 11.20 amol/microg/10 s). Our results showed a saturable (K(m) of 382 +/- 75.1 microM) transport of [(3)H]ribavirin into erythrocytes from Ent1(+/+) mice. We found that ribavirin concentration rapidly (within 60 s) reached equilibrium in erythrocytes using a time course of [(3)H]ribavirin transport (2.5 microM) and metabolism in mouse and human erythrocytes for 8 h. However, total radioactivity of ribavirin was predominantly attributed to the phosphorylated metabolites ribavirin monophosphate and ribavirin triphosphate. Our findings allow us to estimate ribavirin transport, diffusion, and metabolic clearance and to predict in vivo accumulation of ribavirin phosphates in erythrocytes of both mice and humans. Our modeling of ribavirin in erythrocytes on long-term administration of ribavirin suggests that the accumulation of ribavirin inside the cells is dependent on ENT1/Ent1 transport and the rates of intracellular phosphorylation and the degradation of the phosphorylated metabolites. We predict that Ent1(+/+) and Ent1(-/-) mice will serve as excellent models to investigate the contribution of Ent1 to the pharmacokinetics and toxicity of ribavirin in vivo.

Topics: Algorithms; Animals; Cell Line; Chromatography, High Pressure Liquid; Data Interpretation, Statistical; DNA, Complementary; Dogs; Equilibrative Nucleoside Transporter 1; Erythrocytes; Humans; Kinetics; Mice; Mice, Knockout; Models, Statistical; Ribavirin; Thioinosine; Transduction, Genetic

Metformin improves cardiovascular outcomes in patients with type 2 diabetes compared with other glucose-lowering drugs. Experimental studies have shown that metformin can increase the intracellular concentration of adenosine monophosphate, which is a major determinant of the intracellular formation of adenosine. We hypothesize that metformin, given at reperfusion, can limit myocardial infarct size due to increased adenosine receptor stimulation. Isolated perfused hearts from Sprague-Dawley rats were subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion. Perfusion with metformin (50 microM) for the first 15 minutes of reperfusion reduced infarct size (percent area at risk) from 42% +/- 2% to 19% +/- 4% (n >or= 6; P < 0.01), which was blocked by a concomitant perfusion with the adenosine receptor antagonist 8-p-sulfophenyltheophylline (100 microM; 43% +/- 3%) or nitrobenzylthioinosine (a blocker of transmembranous adenosine transport; 1 microM; 45% +/- 5%). In addition, intravenous administration of metformin (5 mg/kg) reduced infarct size in a rat in situ model of myocardial infarction (34% +/- 6% vs. 62% +/- 5%; P < 0.01), which was completely abolished by 8-p-sulfophenyltheophylline (61% +/- 3%). We conclude that metformin, given at reperfusion, reduces infarct size in a rat model of myocardial infarction, which is critically dependent on adenosine receptor stimulation, probably via increased intracellular formation of adenosine.

Topics: Adenosine; Animals; Biological Transport; Hypoglycemic Agents; In Vitro Techniques; Male; Metformin; Myocardial Infarction; Myocardial Reperfusion Injury; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Theophylline; Thioinosine

It is generally accepted that the clinical efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) arises mainly from the inhibition of cyclooxygenase (COX). However, more evidence has suggested that certain pharmacological actions of NSAIDs may be mediated by COX-independent mechanisms. The present study investigated the effects of NSAIDs on adenosine uptake in human aortic smooth muscle cells (HASMCs). Among the NSAIDs tested (all at 100 microM), aspirin, ibuprofen and naproxen had no effect on [(3)H]adenosine uptake. Piroxicam inhibited [(3)H]adenosine uptake by 30%, while etodolac, indomethacin, ketoprofen, mefenamic acid and sulindac inhibited [(3)H]adenosine by 13-18%. Sulindac sulfide, an active metabolite of sulindac, inhibited [(3)H]adenosine uptake and [(3)H]nitrobenzylmercaptopurine ribonucleoside (NBMPR) binding of HASMCs with IC(50) values of 40.67+/-4.82 and 24.19+/-3.76 muM, respectively. Kinetic studies revealed that sulindac sulfide was a competitive inhibitor of adenosine uptake. Using the nucleoside-transporter-deficient PK15NTD cells that stably express equilibrative nucleoside transport (ENT) 1 and ENT2, it was found that the inhibitory effect of sulindac sulfide on ENT1 was greater than that on ENT2. Sulindac sulfide increased the extracellular adenosine level. In addition, it inhibited the proliferation of HASMCs and this anti-proliferative effect could be abolished by adenosine A(2B) receptor antagonist. Our results suggest that sulindac sulfide may exert pharmacological effects through the inhibition of adenosine uptake, which modulates the availability of adenosine in the vicinity of adenosine receptors.

Topics: Adenosine; Anti-Inflammatory Agents, Non-Steroidal; Aorta; Cell Proliferation; Cells, Cultured; Coloring Agents; Dose-Response Relationship, Drug; Etodolac; Humans; Indomethacin; Inhibitory Concentration 50; Ketoprofen; Kinetics; Mefenamic Acid; Muscle, Smooth, Vascular; Piroxicam; Sulindac; Temperature; Tetrazolium Salts; Thiazoles; Thioinosine

At the rat motor nerve terminals, activation of muscarinic M(1) receptors negatively modulates the activity of inhibitory muscarinic M(2) receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involved endogenous adenosine tonically activating A(1) receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [(3)H]-choline (2.5 microCi/ml). Selective activation of muscarinic M(1) and adenosine A(1) receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 microM) and R-N(6)-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [(3)H]-ACh release induced by muscarinic M(2) receptor activation with oxotremorine (10 microM). Attenuation of the inhibitory effect of oxotremorine (10 microM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M(1) autoreceptors, suggesting that suppression of M(2)-inhibiton by A(1) receptor activation is independent on muscarinic M(1) receptor activity. Conversely, the negative crosstalk between M(1) and M(2) autoreceptors seems to involve endogenous adenosine tonically activating A(1) receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 microM) by McN-A-343 (3 microM) was suppressed by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 microM). The results suggest that the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A(1) receptors at the rat motor endplate.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Adenosine; Adenosine A1 Receptor Agonists; Adenosine Deaminase; Animals; Female; Male; Motor Endplate; Neurotransmitter Agents; Nucleoside Transport Proteins; Oxotremorine; Phrenic Nerve; Pirenzepine; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Thioinosine; Tritium; Xanthines

The low efficiency of islet transplantation necessitating sequential transplantations with the use of 2 to 3 donors for a recipient has been a major obstacle facing clinical islet transplantation. We determined whether adenosine has any beneficial effects on preventing early loss of transplanted islets in the liver, thereby facilitating successful islet transplantation from one donor to one recipient in mice.. Two hundred islets, the number of islets from a single mouse pancreas, were grafted into the liver of streptozotocin-induced diabetic C57BL/6 mice. Adenosine was administered once at the time of islet transplantation. Mononuclear cells in the liver of mice receiving islets were isolated and examined by flow cytometry.. A single injection of adenosine at the time of transplantation ameliorated hyperglycemia of diabetic mice receiving 200 syngenic islets with suppression of interferon (IFN)-gamma production of hepatic NKT cells and neutrophils, while that of control did not. The IFN-gamma production of NKT cells and neutrophils in the liver of mice treated with alpha-galactosylceramide, a synthetic ligand of NKT cells was suppressed by adenosine. The beneficial effect of adenosine was also observed for BALB/c islet allografts when alloimmune rejection was prevented by anti-CD4 antibody.. Adenosine suppresses the NKT cell-mediated IFN-gamma production of neutrophils in the liver of mice receiving islets, thus leading to prevention of early loss of transplanted syngenic and allogenic islets. The findings indicate that adenosine may improve efficiency of clinical islet transplantation.

Topics: Adenosine; Animals; Antibodies; Blood Glucose; CD11b Antigen; CD4 Antigens; Diabetes Mellitus, Experimental; Dipyridamole; Dose-Response Relationship, Drug; Galactosylceramides; Graft Rejection; Graft Survival; Immunosuppressive Agents; Injections, Intraperitoneal; Interferon-gamma; Islets of Langerhans Transplantation; Liver; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Natural Killer T-Cells; Neutrophils; Nucleoside Transport Proteins; Receptors, Chemokine; Thioinosine; Time Factors; Transplantation Tolerance; Transplantation, Homologous

Malignant gliomas are the most common and devastating primary tumors in the brain and, despite treatment, patients with these tumors have a poor prognosis. The participation of ecto-5'-NT/CD73 per se as a proliferative factor, being involved in the control of cell growth, differentiation, invasion, migration and metastasis processes has been previously proposed. In the present study, we evaluated the activity and functions of ecto-5'-NT/CD73 during the proliferation process of rat C6 and human U138MG glioma cell lines. Increasing confluences and culture times led to an increase in ecto-5'-NT/CD73 activity in both C6 and U138MG glioma cells. RT-PCR analysis and flow cytometry analysis showed a significant increase in ecto-5'-NT/CD73 mRNA and protein levels, respectively, comparing confluent with sub-confluent cultures in human U138MG glioma cells. Ecto-5'-nucleotidase/CD73 may regulate the extracellular adenosine 5'-monophosphate (AMP) and adenosine levels. Treatment with 1 microM APCP, a competitive ecto-5'-NT/CD73 inhibitor, caused a significant reduction of 30% in glioma cell proliferation. In addition, 100 microM adenosine increases cell proliferation by 36%, and the treatment with adenosine plus NBTI and dipyridamole, produced an additional and significant increase of on cell proliferation. The inhibitory effect on cell proliferation caused by APCP was reverted by co-treatment with NBTI and dipyridamole. AMP (1 mM and 3 mM) decreased U138MG glioma cell proliferation by 29% and 42%, respectively. Taken together, these results suggest the participation of ecto-5'-NT/CD73 in cell proliferation and that this process is dependent upon the enzyme's production of adenosine, a proliferative factor, and removal of AMP, a toxic molecule for gliomas.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Monophosphate; Affinity Labels; Animals; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioma; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Rats; Thioinosine

The aim of the present study was to characterize the intestinal absorption of ribavirin (1-beta-d-ribofuranosyl-1, 2, 4-trizole-3-carboxamide). We evaluated the contribution of Na(+)-dependent and -independent transport to ribavirin absorption in the rat intestine using an in situ closed loop method. In addition, we performed pharmacokinetic analysis of the uptake of ribavirin in human intestinal epithelial LS180 cells, and also evaluated the effect of extracellular Na(+) concentration and an inhibitor of the Na(+)-independent equilibrative nucleoside transporter, nitrobenzylmercaptopurine ribonucleoside (NBMPR), on the uptake of ribavirin in the cells. In the presence and also absence of Na(+) in rat intestinal loops, more than 80% of the administered dose (50 microg at a concentration of 100 microg/ml=409 microM) of ribavirin was absorbed in 40 min. The absorption of ribavirin in the rat intestine was significantly reduced by coadministration of 10 mg/ml (=37.3 mM) inosine. In LS180 cells, 100 microM ribavirin was taken up time-dependently, and the influx clearance of the drug was similar to the efflux clearance. Five mM inosine and mizoribine reduced the uptake of 100 microM ribavirin in LS180 cells. The absence of extracellular Na(+) decreased the uptake of 100 microM ribavirin only weakly in the cells, whereas the uptake of 100 microM-2 mM ribavirin was markedly decreased by 100 microM NBMPR. These findings suggested that Na(+)-independent nucleoside transport contributes significantly to intestinal absorption of ribavirin at relatively high concentrations (>or=100 microM).

Topics: Animals; Antiviral Agents; Biological Transport; Cell Line; Dose-Response Relationship, Drug; Humans; Intestinal Absorption; Intestinal Mucosa; Male; Nucleoside Transport Proteins; Rats; Rats, Wistar; Ribavirin; Sodium; Thioinosine; Time Factors

Experience with unsignaled, inescapable shock represents a profound challenge to brain metabolic function and physiology. The authors have argued that behavioral impairment following this traumatic stress is a consequence of enhanced brain adenosine signaling, which promotes metabolic recovery by profoundly inhibiting neural activation. The authors tested this hypothesis by artificially increasing extracellular brain adenosine concentration by blocking uptake transport with NBTI in rats given only restraint stress in five experiments. NBTI impaired shuttle-escape performance in the manner of inescapable shock in a dose-dependent manner and acted synergistically with an ineffective number of inescapable shocks to maximally impair test performance. These deficits produced by inescapable shock and NBTI were reversed by the nonselective adenosine receptor antagonist caffeine, and the highly selective A-sub(2A) receptor antagonist CSC (8-(3-chloro-styrl)caffeine). The highly selective A-sub-1 receptor antagonist DPCPX (8-Cyclopentyl-1,3-Dipropylxanthine) failed to improve performance in rats preexposed to inescapable shock or pretreated with NBTI. These data suggest that enhanced adenosine signaling at a brain A-sub(2A) receptor impairs escape performance following inescapable shock in the learned helplessness paradigm.

Topics: Adenosine; Analysis of Variance; Animals; Behavior, Animal; Brain; Caffeine; Electroshock; Escape Reaction; Freezing Reaction, Cataleptic; Male; Nucleoside Transport Proteins; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Reaction Time; Signal Transduction; Thioinosine; Xanthines

3H-1,2-Dithiole-3-thione (D3T), a potent member of dithiolethiones, induces phase 2 enzymes by activating an Nrf2/Keap1-dependent signaling pathway. It was proposed that interaction between D3T and two adjacent sulfhydryl groups of Keap1 might cause dissociation of Keap1 from Nrf2, leading to Nrf2 activation. This study was undertaken to investigate the reactions between D3T and thiols, including the dithiol compound, dithiothreitol (DTT), and the monothiol, glutathione (GSH). We reported here that under physiologically relevant conditions incubation of D3T with DTT caused remarkable oxygen consumption, indicating a redox reaction between D3T and the dithiol molecule. Incubation of D3T with GSH also led to oxygen consumption, but to a less extent. Electron paramagnetic resonance (EPR) studies showed that the redox reaction between D3T and DTT generated superoxide. Superoxide was also formed from the redox reaction of D3T with GSH. These findings demonstrate that D3T reacts with thiols, particularly a dithiol, generating superoxide, which may provide a mechanistic explanation for induction of Nrf2-dependent phase 2 enzymes by D3T.

Topics: Antineoplastic Agents; Chemoprevention; Dithiothreitol; Dose-Response Relationship, Drug; Glutathione; Oxidation-Reduction; Oxygen Consumption; Pyrroles; Reactive Oxygen Species; Spin Trapping; Sulfhydryl Compounds; Superoxide Dismutase; Superoxides; Thioinosine; Thiones; Thiophenes

We investigated the potential drug-drug interaction between imatinib and fludarabine, which may be concomitantly administered in chronic myeloid leukemia (CML) patients receiving fludarabine-based conditioning for allogeneic hematopoietic cell transplantation (HCT). Imatinib is an inhibitor of human equilibrative transporters (hENTs), which are responsible for the intracellular uptake of fludarabine.. Intracellular accumulation of fludarabine triphosphate (F-ara-ATP), the active metabolite of fludarabine, was measured in CD4(+) and CD8(+) T-lymphocytes isolated from healthy volunteers, which were treated in vitro with fludarabine alone, and in the presence of either imatinib or NBMPR, a known hENT inhibitor.. Imatinib significantly inhibited F-ara-ATP accumulation in CD4(+) and CD8(+) T-lymphocytes in a concentration-dependent manner. The observed imatinib inhibition was comparable to inhibition observed with NBMPR. The inhibition of F-ara-ATP by imatinib is likely due to inhibition of nucleoside transporters hENT1 and hENT2.. There is significant in vitro drug interaction between imatinib and fludarabine. This effect may be of important consideration in patients receiving fludarabine-based conditioning prior to HCT.

Topics: Antineoplastic Agents; Benzamides; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dose-Response Relationship, Drug; Drug Interactions; Humans; Imatinib Mesylate; In Vitro Techniques; Piperazines; Pyrimidines; Thioinosine; Vidarabine

Sheep choroid plexus epithelium expresses equilibrative nucleoside transporters (ENT) 1 and 2 and concentrative nucleoside transporter 2 at the transcript level. This study aimed to explore the kinetics and functional role of these transporters at the basolateral side of the sheep choroid plexus epithelium perfused in situ. The cellular uptake of [(3)H]adenosine and [(3)H]uridine was insensitive to 1 microm nitrobenzylthioinosine (NBTI), and the uptake of [(3)H]adenosine was reduced significantly when 10 microm NBTI was present in low-Na(+) Ringer solution. This might suggest that ENT2, a transporter sensitive to micromolar NBTI, is functionally active at the basolateral side of the choroid plexus epithelium while ENT1, a transporter sensitive to nanomolar NBTI, is not active. When low-Na(+) Ringer solution was used for the in situ perfusion, the Na(+) concentration in the venous effluent decreased to 14 mm; under these conditions the maximal uptake (U(max)) of [(3)H]adenosine and [(3)H]uridine did not change significantly when compared with the U(max) obtained when Ringer solution that contained 145 mm Na(+) was used. Kinetic analysis revealed apparent Michaelis-Menten constants (K(m,app)) for cellular uptake of [(3)H]adenosine, [(3)H]inosine and [(3)H]thymidine of 1.2 +/- 0.2, 15.7 +/- 2.6 and 3.8 +/- 0.9 microm, respectively. The HPLC and HPLC-fluorometric analysis of the sheep plasma and cerebrospinal fluid revealed nanomolar concentrations of adenosine and thymidine and micromolar levels of inosine and nucleobases. Considering the estimated K(m,app) values, it appears that under normal conditions inosine is the more important nucleoside substrate for uptake by the basolateral membrane of the choroid plexus epithelium than other nucleosides.

Topics: Adenosine; Animals; Cell Polarity; Cell Survival; Choroid Plexus; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Epithelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Inosine; Kinetics; Membrane Transport Proteins; Models, Biological; Nucleoside Transport Proteins; Nucleosides; Perfusion; Sheep; Sodium; Thioinosine; Thymidine; Uridine

Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors. In our previous study [J. Med. Chem. 2003, 46, 831-837], novel regioisomeric nitro-1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of NBMPR were synthesized and evaluated as ENT1 ligands. 7-NO(2)-1,2,3,4-Tetrahydroisoquino-2-yl purine riboside was identified as the analogue with the nitro group in the best orientation at the NBMPR binding site of ENT1. In the present study, further conformational constraining was introduced by synthesizing 5'-O,8-cyclo derivatives. The flow cytometrically determined binding affinities indicated that the additional 5'-O,8-cyclo constraining was unfavorable for binding to the ENT1 transporter. The structure-activity relationship (SAR) acquired was applied to pharmacophore mapping using the PHASE program. The best pharmacophore hypothesis obtained embodied an anti-conformation with three hydrogen-bond acceptors, one hydrophobic center, and two aromatic rings involving the 3'-OH, 4'-oxygen, the NO(2) group, the benzyl phenyl and the imidazole and pyrimidine portions of the purine ring, respectively. A PHASE 3D-QSAR model derived with this pharmacophore yielded an r(2) of 0.916 for four (4) PLS components, and an excellent external test set predictive r(2) of 0.78 for 39 compounds. This pharmacophore was used for molecular alignment in a comparative molecular field analysis (CoMFA) 3D-QSAR study that also afforded a predictive model with external test set validation predictive r(2) of 0.73. Thus, although limited, this study suggests that the bioactive conformation for NBMPR at the ENT1 transporter could be anti. The study has also suggested an ENT1 inhibitory pharmacophore, and established a predictive CoMFA 3D-QSAR model that might be useful for novel ENT1 inhibitor discovery and optimization.

Topics: Drug Design; Equilibrative Nucleoside Transporter 1; Glycosides; Humans; K562 Cells; Models, Biological; Models, Molecular; Molecular Structure; Quantitative Structure-Activity Relationship; Static Electricity; Thioinosine

The placenta requires nucleosides as nutrients for fetal growth, so it is important to examine potential interactions between placental transports of nucleosides and drugs to ensure the safety of pharmacotherapy during pregnancy. The purposes of this study are to clarify the uptake mechanisms of nucleosides from the maternal side of the syncytiotrophoblast and to investigate the inhibitory effect of various drugs on nucleoside uptake, using the rat syncytiotrophoblast cell line TR-TBT 18d-1, which shows syncytial-like morphology and functional expression of several transporters. Initial uptake of [(3)H]uridine or [(3)H]adenosine from the apical side of TR-TBT 18d-1 was markedly reduced by an excess of the respective unlabelled compound, and was slightly reduced by replacement of Na(+) with N-methyl-d-glucamine, indicating that both uptakes were Na(+)-independent. [(3)H]Uridine and [(3)H]adenosine uptakes in the absence of Na(+) were significantly and concentration-dependently inhibited by both 0.1 microM and 100 microM nitrobenzylthioinosine, suggesting the involvement of equilibrative nucleoside transporters (ENTs, SLC29). Kinetic analysis of adenosine uptake yielded a K(m) value of approximately 17 microM. These results are consistent with the reported uptake characteristics of uridine and adenosine by ENT1 and ENT2. The uptakes were significantly reduced by high concentrations of several nucleoside drugs, including cytarabine, vidarabine, zidovudine, mizoribine, caffeine and amitriptyline, but the effects were small within the therapeutic concentration ranges. In summary, our results suggest that ENTs are involved in apical uptake of uridine and adenosine in the syncytiotrophoblast. However, therapeutic concentrations of the drugs tested in this study might have little influence on maternal-to-fetal nucleoside transfer.

Topics: Animals; Antifungal Agents; Antineoplastic Agents; Antiviral Agents; Biological Transport; Cell Line; Dose-Response Relationship, Drug; Equilibrative-Nucleoside Transporter 2; Immunosuppressive Agents; Nucleosides; Rats; Thioinosine; Tritium; Trophoblasts

This study was designed to evaluate the cytotoxic activity of several nucleoside and nucleobase analog drugs as possible new agents for treatment of malignant mesothelioma and to identify factors responsible for the clinical variation of nucleoside analog drug response in chemotherapy of mesothelioma. Three human mesothelioma cell lines (MSTO-211H, H2452 and H2052) were tested for gemcitabine sensitivity and nucleoside transport activity. MSTO-211H, H2452 and H2052 exhibited differences in sensitivity to gemcitabine, nucleoside transport rates and hENT1 site densities. In H2052 cells, gemcitabine, 5-fluoro-2'-deoxyuridine, clofarabine and cladribine were most active with IC(50) values of 46, 43, 240 and 490 nM, respectively, whereas 5-fluorouracil was the least cytotoxic drug tested. In H2052 cells, the combination of gemcitabine and fludarabine or cladribine resulted in synergistic cytotoxic response. In nucleobase transport studies, hypoxanthine and 6-mercaptopurine but not 5-fluorouracil was transported into H2052 cells by a novel purine-specific, sodium-independent nucleobase transport activity. In summary differences in nucleoside analog drug transport activities are likely to contribute to the observed clinical variation in nucleoside analog response in patients and for the first time a correlation between nucleobase drug sensitivities and transport activities was shown. A novel combination of gemcitabine and fludarabine or cladribine had synergistic cytotoxic activity against the least sensitive mesothelioma cell line. These drug combinations merit further evaluation as effective therapeutic regimens in patients with aggressive mesothelioma.

Topics: Antineoplastic Agents; Biological Transport; Cell Line, Tumor; Cell Survival; Dipyridamole; Drug Synergism; Equilibrative Nucleoside Transporter 1; Humans; Mesothelioma; Nucleosides; Papaverine; Purines; Pyrimidines; Thioinosine

The purine nucleoside adenosine is a physiologically important molecule in the heart. Brief exposure of cardiomyocytes to hypoxic challenge results in the production of extracellular adenosine, which then interacts with adenosine receptors to activate compensatory signaling pathways that lead to cellular resistance to subsequence hypoxic challenge. This phenomenon is known as preconditioning (PC), and, while adenosine is clearly involved, other components of the response are less well understood. Flux of nucleosides, such as adenosine and inosine, across cardiomyocyte membranes is dependent on equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2). We have previously shown in the murine cardiomyocyte HL-1 cell line that hypoxic challenge leads to an increase in intracellular adenosine, which exits the cell via ENT1 and preconditions via A1 and A3 adenosine receptor-dependent mechanisms. However, the role and contribution of inosine and ENT2 are unclear. In this study, we confirmed that ENT1 and ENT2 are both capable of transporting inosine. Moreover, we found that hypoxic challenge leads to a significant increase in levels of intracellular inosine, which exits the cell via both ENT1 and ENT2. Exogenously added inosine (5 microM) preconditions cardiomyocytes in an A1 adenosine receptor-dependent manner since preconditioning can be blocked by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) but not the A3 adenosine receptor antagonist MRS-1220 (200 nM). These data suggest that cardiomyocyte responses to hypoxic PC are more complex than previously thought, involving both adenosine and inosine and differing, but overlapping, contributions of the two ENT isoforms.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine A3 Receptor Antagonists; Animals; Cell Hypoxia; Cell Line; Cell Survival; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Inosine; Mice; Myocytes, Cardiac; Nucleoside Transport Proteins; Quinazolines; Receptor, Adenosine A1; Receptor, Adenosine A3; Thioinosine; Triazoles; Up-Regulation; Xanthines

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.

Topics: Adenosine; Biological Transport; Dilazep; Dipyridamole; Enzyme Activation; Equilibrative Nucleoside Transporter 1; Humans; Kinetics; Models, Biological; Mutation; Nucleosides; Piperazines; Protein Binding; Thioinosine; Tryptophan; Vasodilator Agents

NB4 cells express multiple nucleoside transporters (NTs), including: hENT1 (es), and hENT2 (ei), and the CNT subtype referred to as, csg; a concentrative sensitive guanosine specific transporter. csg activity is a distinguishing feature of the NB4 cell line and its presence suggests a particular requirement of these cells for guanosine salvage. Proliferation and differentiation pathways determine, in part, the number of NTs in cells and tissues. In this study, all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of NB4 cells resulted in biphasic changes in guanosine transport. Transient increases in csg and es activity, the result of an increase in V(max) (pmol/muls) of both transporter systems, served as early markers of differentiation while expression of a fully differentiated phenotype was accompanied by a selective loss of csg activity and the return of es activity to that of proliferating cells. Intracellular incorporation of [(3)H]-guanosine decreased as cells matured despite increased transport rates and suggested a reduced intracellular requirement of NB4-granulocytes compared to their proliferating counterparts. Whether a loss of csg activity could serve to assess clinical response to differentiation therapies is not known. Nitrobenzylthioinosine (NBMPR) binding sites within nuclear membrane (NM) preparations, suggested the presence of functional intracellular NTs. An increase in plasma membrane (PM) associated transporters coincided with the early increase in guanosine transport and a decrease in NBMPR binding to NM fractions and suggests that intracellular NTs may serve as a reserve pool for translocation to the (PM) when additional transport capacity is required. The modulation of transporters during differentiation could potentially regulate drug bioavailability and cytotoxicity and should be evaluated prior to combining differentiating agents with traditional nucleoside analogs in the treatment of APL.

Topics: Affinity Labels; Biological Transport; Cell Differentiation; Cell Membrane; Granulocytes; Guanosine; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Neutrophils; Nucleoside Transport Proteins; Subcellular Fractions; Thioinosine; Tretinoin; Tumor Cells, Cultured; Uridine

Cytosine arabinoside (1-beta-D-arabinofuranosylcytosine;Ara-C) is the most important antimetabolite used for acute leukemia. We established Ara-C (0.003-1 micromol/l)-resistant NALM-6 leukemia cells, and attempted the characterization of their resistance.. The Ara-C-resistant cell lines were developed by stepwise increases in the drug. The mRNA expressions were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The uptake of Ara-C, deoxycytidine kinase (dCK) activity and cytidine deaminase (CDA) activity were measured using radioisotope methods. Cytotoxicity was evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay.. The mRNA expression of human equilibrative nucleoside transporter-1 (hENT-1), which is an uptake transporter of Ara-C, was initially decreased during the acquisition of resistance to Ara-C. The expression of dCK, an activation enzyme, and of CDA, an inactivation enzyme, was decreased and increased in the late phase, respectively. The cytotoxic effect of Ara-C on parental NALM-6 cells was ameliorated by hENT-1 inhibitors. There were no differences in the cytotoxic effect of other anticancer drugs, but there was similar resistance to nucleoside analogues via hENT-1 between the parental and resistant cells.. Decreased hENT-1 expression and function is causatively responsible for the acquisition of Ara-C resistance and alterations in dCK and CDA contribute to the higher concentration range.

Topics: Cell Line, Tumor; Cell Survival; Cytarabine; Cytidine Deaminase; Cytoprotection; Deoxycytidine Kinase; Dipyridamole; Drug Resistance, Neoplasm; Gene Expression; Humans; Leukemia; RNA, Messenger; Thioinosine

We have investigated the cardioprotective effects of novel tetrahydroisoquinoline nitrobenzylmercaptopurine riboside (NBMPR) analog nucleoside transport (NT) inhibitors, compounds 2 and 4, in isolated perfused rat hearts. Langendorff-perfused heart preparations were subjected to 10 min of treatment with compound 2, compound 4, or vehicle (control) followed by 30 min of global ischemia and 120 min of reperfusion. For determination of infarct size, reperfusion time was 180 min. At 1 microM, compounds 2 and 4 provided excellent cardioprotection, with left ventricular developed pressure (LVDP) recovery and end-diastolic pressure (EDP) increase of 82.9 +/- 4.0% (P<0.001) and 14.1 +/- 2.0 mmHg (P<0.03) for compound 2-treated hearts and 79.2 +/- 5.9% (P<0.002) and 7.5 +/- 2.7 mmHg (P<0.01) for compound 4-treated hearts compared with 41.6 +/- 5.2% and 42.5 +/- 6.5 mmHg for control hearts. LVDP recovery and EDP increase were 64.1 +/- 4.2% and 29.1 +/- 2.5 mmHg for hearts treated with 1 microM NBMPR. Compound 4 was the best cardioprotective agent, affording significant cardioprotection, even at 0.1 microM, with LVDP recovery and EDP increase of 76.0 +/- 4.9% (P<0.003) and 14.1 +/- 1.0 mmHg (P<0.03). At 1 microM, compound 4 and NBMPR reduced infarct size, with infarct area-to-total risk area ratios of 29.13 +/- 3.17 (P<0.001) for compound 4 and 37.5 +/- 3.42 (P<0.01) for NBMPR vs. 51.08 +/- 5.06% for control hearts. Infarct size was more effectively reduced by compound 4 than by NBMPR (P<0.02). These new tetrahydroisoquinoline NBMPR analogs are not only potent cardioprotective agents but are, also, more effective than NBMPR in this model.

Topics: Animals; Cardiotonic Agents; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Female; Heart; In Vitro Techniques; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Perfusion; Rats; Rats, Wistar; Tetrahydroisoquinolines; Thioinosine; Ventricular Function, Left; Ventricular Pressure

Even though extracellular adenosine plays multiple roles in the cochlea, the mechanisms that control extracellular adenosine concentrations in this organ are unclear. This study investigated the expression of nucleoside transporters and adenosine uptake in the rat cochlea. Reverse transcription-polymerase chain reaction revealed the expression of mRNA transcripts for two equilibrative (ENT1 and ENT2) and two concentrative (CNT1 and CNT2) nucleoside transporters. Exogenous adenosine perfused through the cochlear perilymphatic compartment was taken up by cells lining the compartment. Adenosine uptake was sensitive to changes in extracellular Na concentrations and inhibited by nitrobenzylthioinosine (an adenosine uptake blocker). The study suggests that the bi-directional nucleoside transport supports the uptake and recycling of purines and regulates the activation of adenosine receptors by altering adenosine concentrations in cochlear fluid spaces.

Topics: Adenosine; Animals; Carrier Proteins; Cochlea; Equilibrative Nucleoside Transporter 1; Female; Gene Expression Regulation; Hair Cells, Auditory; Male; Membrane Transport Proteins; Nucleoside Transport Proteins; Perilymph; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Sodium; Sodium Channels; Thioinosine

The aim of the present study was to investigate the effect of hyperthyroidism on the trans-sarcolemmal adenosine (Ado) flux via equilibrative and nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporters (ENT1) in guinea pig atria, by assessing the change in the Ado concentration of the interstitial fluid ([Ado]ISF) under nucleoside transport blockade with NBTI. For the assessment, we applied our novel method, which estimates the change in [Ado]ISF utilizing the altered inotropic response to N6-cyclopentyladenosine (CPA), a relative stable selective agonist of A1 Ado receptors, by providing a relative index, the equivalent concentration of CPA. Our results show an interstitial Ado accumulation upon ENT1 blockade, which was more extensive in the hyperthyroid samples (CPA concentrations equieffective with the surplus [Ado]ISF were two to three times higher in hyperthyroid atria than in euthyroid ones, with regard to the negative inotropic effect of CPA and Ado). This suggests an enhanced Ado influx via ENT1 in hyperthyroid atria. It is concluded that hyperthyroidism does not alter the prevailing direction of the Ado transport, moreover intensifies the Ado influx in the guinea pig atrium.

Topics: Adenosine; Adenosine Deaminase; Animals; Biological Transport; Cyclopentanes; Guinea Pigs; Heart Atria; Hyperthyroidism; Male; Receptors, Purinergic P1; Solvents; Thioinosine; Thyroid Gland; Thyroxine

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays. hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [(3)H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [(3)H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs.

Topics: Antibodies, Monoclonal; Antibody Specificity; Biomarkers; Blotting, Western; Cells, Cultured; Equilibrative Nucleoside Transporter 1; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Kidney; Kidney Tubules, Collecting; Kidney Tubules, Proximal; Kinetics; Loop of Henle; Membrane Transport Proteins; Membranes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thioinosine; Uridine

In C6 glioma cells, adenine nucleotides, especially AMP, and adenosine inhibited cell proliferation in time- and concentration-dependent manners. alpha,beta-methylene-ADP, an ecto-5'-nucleotidase inhibitor, suppressed the hydrolysis of AMP and reversed the inhibition of cell growth induced by AMP but not by adenosine. Adenosine deaminase eliminated both AMP- and adenosine-mediated growth inhibitions. 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, had little effect on the cell growth. Equilibrative nucleoside transporters, ENT-1 and ENT-2, were expressed in C6 cells by determining their mRNAs. ENT inhibitors, nitrobenzylthioinosine and dipyridamole, suppressed the uptake of [(3)H]adenosine into C6 cells, and attenuated AMP- or adenosine-mediated growth inhibition. Furthermore, an adenosine kinase inhibitor 5-iodotubercidin reversed the growth inhibition induced by AMP and adenosine. When uridine was added in the extracellular space, AMP- or adenosine-induced cell growth inhibition was completely reversed, suggesting that intracellular pyrimidine starvation would be involved in their cytostatic effects. These results indicate that extracellular adenine nucleotides inhibit C6 cell growth via adenosine, which is produced by ecto-nucleotidases including CD73 at the extracellular space and then incorporated into cells by ENT2. Intracellular AMP accumulation by adenosine kinase after adenosine uptake would induce C6 cell growth inhibition through pyrimidine starvation.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adenosine; Adenosine Deaminase; Adenosine Diphosphate; Adenosine Kinase; Adenosine Monophosphate; Animals; Brain Neoplasms; Cell Count; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Glioma; Hydrolysis; Rats; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Thioinosine; Uridine

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S(6)-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a K(d) of 0.1-1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C(2)-purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C(2)-purine position of NBMPR. Structure-activity relationships (SAR) indicate that increasing the steric bulk at the C(2)-purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl mercaptopurine riboside (7), exhibiting a K(i) of 2.1 nM. This information, when coupled with the information obtained from other structure-activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.

Topics: Cell Line; Drug Evaluation, Preclinical; Equilibrative Nucleoside Transporter 1; Flow Cytometry; Humans; Molecular Structure; Purines; Structure-Activity Relationship; Thioinosine

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

Topics: 2-Chloroadenosine; Animals; Cell Culture Techniques; Cell Line; Cells, Cultured; Child, Preschool; Coronary Vessels; Dilazep; Dipyridamole; Dogs; Endothelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Female; Humans; Hypoxanthine; Kinetics; Microcirculation; Nucleobase Transport Proteins; Piperazines; Protein Binding; Purines; Rats; Swine; Thioinosine; Transfection; Tritium

Our objectives were to study the biological activity of a novel gemcitabine-cardiolipin conjugate (NEO6002) and compare that with gemcitabine. Cytotoxicity in vitro was determined against several gemcitabine-sensitive parental and gemcitabine-resistant cancer cell lines using the sulforhodamine B assay. The in vivo toxicity was examined by changes in body weight and hematologic indices of conventional mice. Immunodeficient SCID mice bearing P388 and BxPC-3 tumor xenografts were used to evaluate the in-vivo therapeutic efficacy. Both NEO6002 and gemcitabine showed pro-apoptotic and cytotoxic effects against all gemcitabine-sensitive cell lines tested. Unlike gemcitabine, the cytotoxicity of NEO6002 was independent of nucleoside transporter (NT) inhibitors, indicating a different internalization route of NEO6002. The conjugate demonstrated a favorable activity not only in ARAC-8C, a NT-deficient gemcitabine-resistant human leukemia cell line, but also in several other gemcitabine-resistant cell lines. At the in-vivo level, a comparative toxicity study showed a significant body weight loss and a decrease in white blood cell counts in gemcitabine-treated mice, whereas the influence of NEO6002 was mild. Treatment of NEO6002 at 27 micromol/kg increased the median survival of CD2F1 mice bearing P388 cells by up to 73%, while at the same doses and schedule of gemcitabine resulted in toxic deaths of all treated mice. At a dose of 18 micromol/kg, NEO6002 inhibited the growth of BxPC-3 xenografts by 52%, while only 32% of tumor inhibition was achieved with gemcitabine. We conclude that NEO6002 may be an effective chemotherapeutic agent with improved tolerability and can potentially circumvent NT-deficient, gemcitabine-resistant tumors.

Topics: Animals; Antineoplastic Agents; Body Weight; Cardiolipins; Cell Proliferation; Deoxycytidine; Dipyridamole; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gemcitabine; HT29 Cells; Humans; Leukemia P388; Leukocyte Count; Mice; Mice, SCID; Neutropenia; Nucleoside Transport Proteins; Pancreatic Neoplasms; Thioinosine; Xenograft Model Antitumor Assays

In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([3H]guanine) and pyrimidine ([3H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time and concentration dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single Na+-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells.

Topics: Adenine; Animals; Biological Transport; Blood-Testis Barrier; Cytosine; Guanine; Hypoxanthine; Kinetics; Male; Meglumine; Nucleobase Transport Proteins; Nucleoside Transport Proteins; Purine Nucleosides; Pyrimidine Nucleosides; Rats; Rats, Inbred Strains; Sertoli Cells; Sodium; Thioinosine; Thymine; Uracil; Uridine

Several accepted methods are available to estimate the adenosine (Ado) concentration of interstitial fluid ([Ado]ISF) in functioning heart, providing results spanning over nano- to micromolar concentrations. This extremely large range points to the necessity of novel approaches for estimating [Ado]ISF or at least the alteration from basal [Ado]ISF. In the present study, the change in [Ado]ISF was characterized following nucleoside transport (NT) blockade elicited by 10 micromol/L dipyridamole or 10 micromol/L nitrobenzylthioinosine in isolated guinea pig atria, by means of our novel procedure referred to as receptorial responsiveness method (RRM). The RRM provided an index of the change in [Ado]ISF under NT blockade, namely the concentration of N-cyclopentyladenosine (CPA; a relatively stable A1 Ado receptor agonist), which is equieffective with the change in [Ado]ISF regarding the contractility. Our results show that dipyridamole or nitrobenzylthioinosine produced an elevation in [Ado]ISF at the cardiomyocyte A1 Ado receptors equivalent to about 16 or 20 nmol/l CPA, respectively. In addition, nitrobenzylthioinosine was found more appropriate for selective NT blockade than dipyridamole.

Topics: Adenosine; Animals; Biological Transport; Dipyridamole; Extracellular Fluid; Guinea Pigs; Heart Atria; Male; Myocardial Contraction; Nucleosides; Thioinosine

To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.

Topics: Animals; Binding Sites; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Female; Fluorescent Dyes; HeLa Cells; Humans; Membrane Transport Proteins; Microscopy, Confocal; Nucleoside Transport Proteins; Oocytes; Photobleaching; Pyrimidine Nucleosides; Recombinant Proteins; Saccharomyces cerevisiae; Thioinosine; Time Factors; Tumor Cells, Cultured; Xenopus laevis

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

Topics: Affinity Labels; Animals; Autoradiography; Brain; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; In Situ Hybridization; Mazindol; Mice; Mice, Inbred C57BL; Mice, Knockout; Nucleoside Transport Proteins; Protein Binding; Receptor, Adenosine A2A; Receptors, Dopamine D1; Receptors, Dopamine D3; RNA, Messenger; Thioinosine; Tritium

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

Topics: Animals; Arabinonucleosides; Biological Transport; Cell Line, Tumor; Cell Survival; Cytarabine; Cytidine; Dose-Response Relationship, Drug; Equilibrative-Nucleoside Transporter 2; Female; HeLa Cells; Humans; Membrane Potentials; Nucleoside Transport Proteins; Oocytes; Saccharomyces cerevisiae; Thioinosine; Transfection; Tritium; Uridine; Xenopus laevis

L-Arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D-glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D-glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D-glucose. Insulin increased hENT2 protein abundance in normal or high D-glucose, but reduced hENT1 protein abundance in normal D-glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D-glucose. Insulin effect on hENT1 mRNA expression in normal D-glucose was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development.

Topics: Adenosine; Affinity Labels; Biological Transport; Cells, Cultured; Citrulline; Endothelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Glucose; Humans; Insulin; Nitric Oxide Synthase Type III; Protein Isoforms; Thioinosine; Umbilical Veins

Derivatives of 2'-deoxyuridine that contain fluoroalkyl groups at the C5 position and derivatives of thymidine that contain fluoroalkyl groups at the N3 position were synthesized and examined in three in vitro assays designed to evaluate their potential as radiopharmaceuticals for imaging cellular proliferation. Three of the former nucleosides and five of the latter were synthesized. The three assays were as follows: (a) phosphoryl transfer assay, which showed that all three of the former nucleosides and four of the latter ones were phosphorylated by recombinant human thymidine kinase 1 (TK1) and that N(3)-(2-fluoroethyl)-thymidine (NFT202) was the most potent substrate of the eight nucleosides studied; (b) transport assay, which indicated that all eight nucleosides had good affinity for an 6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosylpurine-sensitive mouse erythrocyte nucleoside transporter, with inhibition constants in the range of 0.02-0.55 mM; and (c) degradation assay, which showed that all but one of the former nucleosides and none of the latter were degraded by recombinant Escherichia coli thymidine phosphorylase (an enzyme that catalyzes the glycosidic bond of thymidine and 2'-deoxyuridine derivatives). From these in vitro screening assays, we selected NFT202 as a candidate for subsequent in vivo evaluation because this compound met the three minimum requirements of the in vitro screening assays and had the most potent phosphorylation activity as a substrate for recombinant human TK1.

Topics: Animals; Cell Proliferation; Drug Design; Fluorine; Humans; Mice; Phosphorylation; Radiopharmaceuticals; Structure-Activity Relationship; Thioinosine; Thymidine; Thymidine Kinase; Thymidine Phosphorylase

We previously demonstrated that insulin has a neuroprotective role against oxidative stress, a deleterious condition associated with diabetes, ischemia, and age-related neurodegenerative diseases. In this study, we investigated the effect of insulin on neuronal glucose uptake and metabolism after oxidative stress in rat primary cortical neurons. On oxidative stress, insulin stimulates neuronal glucose uptake and subsequent metabolism into pyruvate, restoring intracellular ATP and phosphocreatine. Insulin also increases intracellular and decreases extracellular adenosine, counteracting the effect of oxidative stress. Insulin effects are apparently mediated by phosphatidylinositol 3-K and extracellular signal-regulated kinase signaling pathways. Extracellular adenosine under oxidative stress is largely inhibited after blockade of ecto-5'-nucleotidase, suggesting that extracellular adenosine results preferentially from ATP release and catabolism. Moreover, insulin appears to interfere with the ATP release induced by oxidative stress, regulating extracellular adenosine levels. In conclusion, insulin neuroprotection against oxidative stress-mediated damage involves 1) stimulation of glucose uptake and metabolism, increasing energy levels and intracellular adenosine and, ultimately, uric acid formation and 2) a decrease in extracellular adenosine, which may reduce the facilitatory activity of adenosine receptors.

Topics: Adenine Nucleotides; Adenosine; Androstadienes; Animals; Cells, Cultured; Cerebral Cortex; Chromones; Flavonoids; Glucose; Insulin; Lactic Acid; Morpholines; Neurons; Oxidative Stress; Phosphocreatine; Pyruvic Acid; Rats; Rats, Wistar; Thioinosine; Wortmannin

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

Topics: 2-Chloroadenosine; Bone Neoplasms; Casein Kinase II; Catalytic Domain; Cell Line, Tumor; Computer Systems; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Formycins; Gene Expression Regulation, Neoplastic; Humans; Nucleosides; Osteosarcoma; Polymerase Chain Reaction; Substrate Specificity; Thioinosine; Transfection; Tritium

Nucleosides are essential for nucleotide synthesis in testicular spermatogenesis. In the present study, the mechanism of the supply of nucleosides to the testicular system across the blood-testis barrier was studied using primary-cultured Sertoli cells from rats and TM4 cells from mice. Uptake of uridine by these cells was time- and concentration-dependent. Uridine uptake was decreased under Na(+)-free conditions, and the system was presumed to be high affinity, indicating an Na(+)-dependent concentrative nucleoside transporter (CNT) is involved. On the other hand, nitrobenzylthioinosine, a potent inhibitor of Na(+)-independent equilibrative nucleoside transporters (ENTs), inhibited uridine uptake by the Sertoli cells in a concentration-dependent manner. Expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 was detected in Sertoli cells by reverse transcriptase-polymerase chain reaction analysis. Inhibition studies of the uptake of uridine by various nucleosides both in the presence and absence of Na(+) indicated that the most of those expressed nucleoside transporters, ENTs and CNTs, are involved functionally. These results demonstrated that Sertoli cells are equipped with multiple nucleoside transport systems, including ENT1, ENT2, and CNTs, to provide nucleosides for spermatogenesis.

Topics: Algorithms; Animals; Biological Transport, Active; Blood-Testis Barrier; Cell Line; Cells, Cultured; Kinetics; Male; Mice; Nucleosides; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sertoli Cells; Sodium; Thioinosine; Uridine

The human equilibrative nucleoside transporter 1 (hENT1) is an important modulator of the physiological action of adenosine. We identified amino acid residues involved in adenosine transport using a yeast-based assay to rapidly screen and identify randomly generated hENT1 mutants that exhibited decreased sensitivity to inhibition of adenosine transport by various hENT1 competitive inhibitors. We identified Met89 and Ser160 as important in the affinity of hENT1 for various substrates and inhibitors. Mutation to Met89Cys or Ser160Cys significantly (p < 0.05) increased the S6-(4-nitrobenzyl)-mercaptopurine riboside (NBMPR) IC50 values by approximately 4- and 6-fold, respectively (42 +/- 13 and 65 +/- 1.6 nM) compared with the wild-type transporter (11 +/- 0.7 nM). The double mutant Met89Cys/Ser160Cys synergistically increased the NBMPR IC50 value to approximately 19-fold of that of the wild-type transporter. In contrast, compared with wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p < 0.05) increased by only the Ser160Cys (approximately 2.6-fold) or the double mutant Met89Cys/Ser160Cys (approximately 4.7-fold) but not by the Met89Cys mutant. Mutation to Met89Cys or Ser160Cys increased the Km of adenosine (approximately 8- and 3-fold) and the Ki of guanosine (approximately 6- and 2-fold). The double mutant increased both the Km value of adenosine and the Ki value of guanosine by approximately 8-fold and seemed to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone. Together, these data indicate that transmembrane domains (TMDs) 2 (Met89) and 4 (Ser160) of hENT1 interact and are important in conferring sensitivity to NBMPR. In contrast, Ser160 and Met89 of hENT1, respectively, play a dominant role in conferring sensitivity to dipyridamole and adenosine/guanosine affinity.

Topics: Adenosine; Amino Acid Sequence; Biological Transport; Dipyridamole; Equilibrative Nucleoside Transporter 1; Genetic Complementation Test; Guanosine; Humans; Kinetics; Methionine; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Serine; Thioinosine

Previously, we have shown that hydrogen peroxide (H2O2) and glucose deprivation (GD) induced ATP loss and cell death in astrocytes. Here, we reported that adenosine and related purine nucleos(t)ides recovered cellular ATP level and completely prevented the cell death in rat primary astrocytes co-treated with H2O2 and glucose deprivation. Time- and concentration-dependently, H2O2 induced cell death and ATP loss in glucose-deprived astrocytes. Adenosine or ATP prevented both astrocytic death and ATP loss caused by H2O2/GD in dose-dependent manner. Further, inhibition of adenosine deamination or transport with erythro-9-(-hydroxy-3-nonyl)adenosine or S-(4-nitrobenzyl)-6-thioinosine largely attenuated the protective effect of adenosine. Other purine nucleos(t)ides such as inosine, guanosine, ADP, AMP, ITP and GTP also showed similar protective effects. Adenosine or ATP also blocked the mitochondrial dysfunction and glutathione (GSH) depletion in H2O2-treated glucose-deprived astrocytes. The present results suggest that adenosine and related purine nucleos(t)ides may protect astrocytes from H2O2 and glucose deprivation induced the potentiated death by restoration of cellular ATP level.

Topics: Adenine; Adenosine; Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Benzimidazoles; Carbocyanines; Cell Death; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Glucose; Hydrogen Peroxide; In Vitro Techniques; L-Lactate Dehydrogenase; Membrane Potentials; Mitochondria; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Purines; Rats; Rats, Sprague-Dawley; Theobromine; Thioinosine; Time Factors; Xanthines

The blood-brain barrier (BBB) transport of synthetic A(1) receptor agonists was studied in an in situ brain perfusion model in the presence and absence of the selective nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI). For 8-methylamino-N(6)cyclopentyladenosine (MCPA), N(6)-cyclopentyladenosine (CPA), 2'deoxy-N(6)-cyclopentyladenosine (2'dCPA) and 5'deoxy-N(6)-cyclopentyl adenosine (5'dCPA) the brain uptake clearance was low with values of 0.0045+/-0.0012, 0.018+/-0.0020, 0.022+/-0.0028 and 0.12+/-0.054 ml min(-1)g(-1), respectively. In the presence of an average NBTI plasma concentration of 2.6+/-0.3 microg ml(-1) (NBTI dose: 3 mg kg(-1) i.v.) the values of the brain uptake clearance were 0.0062+/-0.0012, 0.013+/-0.0017, 0.014+/-0.0030 and 0.13+/-0.066 ml min(-1)g(-1), respectively and not significantly different from the values in the absence of NBTI. In a separate experiment the brain uptake of MCPA from phosphate buffered saline (PBS) and whole blood were compared. The brain uptake clearance from whole blood (0.0012+/-0.001 ml min(-1)g(-1)) was significantly lower than from PBS (0.0045+/-0.0012 ml min(-1)g(-1)). The results of these studies show that the rENT1 nucleoside transporter does not contribute significantly to the transport of synthetic A(1) receptor agonists across the BBB and that binding to blood constituents restricts the brain uptake.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Brain; Equilibrative Nucleoside Transporter 1; Male; Permeability; Protein Binding; Rats; Rats, Wistar; Receptor, Adenosine A1; Thioinosine

4-nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. Here we report on the synthesis and the biological evaluation of compounds that are less polar than NBTI. Compound screening in our laboratory indicated that introduction of an alkylamine substituent at the C(8)-position of N(6)-cyclopentyladenosine (CPA, 2) led to an increment in affinity for the transport protein. It was investigated whether this would also apply for NBTI derivatives. Two series of C(8)-alkylamine-substituted compounds were prepared, one in which the nitro group was absent (46-58) and another in which the ribose moiety was replaced by a benzyl group (72-75). Comparison of the biological data of these compounds with 6-benzylthioinosine (4, K(i) = 53 nM) and 9-benzyl-6-(4-nitrobenzylsulfanyl)purine (59, K(i) = 135 nM) confirmed the hypothesis. The K(i) values improved upon elongation of the alkylamine chain from methylamine to n-hexylamine with an optimum for n-pentylamine (50, K(i) = 2.3 nM). Substitution with 2-methylbutylamine (52), cyclopropylamine (53), cyclopentylamine (54, 72), and cyclohexylamine (55, 73) revealed that the presence of a bulky group enhanced the affinity. The presence of tertiary amines obtained by substitution with pyrrolidine, piperidine, and morpholine gave only poor results. For both series substitution with cyclopentylamine was most effective. Compound 54 (LUF5942) proved the most active, showing a comparable affinity (K(i) = 0.64 nM) to NBTI but a significantly lower polar surface area.

Topics: Amines; Animals; Biochemistry; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Drug Evaluation, Preclinical; Equilibrative Nucleoside Transporter 1; Erythrocyte Membrane; Humans; Nucleoside Transport Proteins; Purines; Receptor, Adenosine A1; Structure-Activity Relationship; Thioinosine

Quantitative autoradiography has been used to assess whether [3H]paracetamol (3 microM) binds specifically to any area of the murine brain and spinal cord and to investigate whether paracetamol (1-100 microM) competes for binding to the nociceptin opioid peptide (NOP) receptor or to the nitrobenzylthioinosine (NBTI)-sensitive adenosine transporter in the brains of mice. [3H]paracetamol binding was homogenous and, although there was some indication of specific binding overall, this binding in most individual regions failed to reach statistical significance. However, thoracic segments of the spinal cord were found to have significantly higher specific binding than cervical and lumbar regions. Paracetamol did not significantly compete for binding to the NOP receptor or to the NBTI-sensitive adenosine transporter, showing that it does not mediate its effect via these sites. Although paracetamol did bind specifically to the murine brain and spinal cord, the binding was not region-specific, suggesting binding is not related to any particular neurotransmitter system.

Topics: Acetaminophen; Analysis of Variance; Animals; Autoradiography; Binding Sites; Binding, Competitive; Brain; Male; Membrane Transport Proteins; Mice; Nociceptin; Nociceptin Receptor; Nucleoside Transport Proteins; Opioid Peptides; Receptors, Opioid; Spinal Cord; Thioinosine; Tritium

The etiology of the atherosclerosis that occurs in diabetes mellitus is unclear. Adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM d-glucose, which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT-1) and ENT-2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT-1 and inhibited by nitrobenzylmercaptopurine riboside (NBMPR, IC(50) = 0.69 +/- 0.05 nM). Adenosine transport in HASMCs was increased by >30% after treatment for 48 h with 25 mM d-glucose, but not with equimolar d-mannitol and l-glucose. Kinetic studies showed that d-glucose increased V(max) of adenosine transport without affecting K(m). Similarly, d-glucose increased B(max) of high-affinity [(3)H]NBMPR binding, while the dissociation constant (K(d)) was not changed. Consistent with these observations, 25 mM d-glucose increased mRNA and protein expression of ENT-1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD-98059 (40 microM) and U-0126 (10 microM), abolished the effect of d-glucose on ENT-1. We conclude that d-glucose upregulates the protein and message expression and functional activity of ENT-1 in HASMCs, possibly via MAPK/ERK-dependent pathways. Pathologically, the increase in ENT-1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and, thus, alter vascular functions in diabetes.

Topics: Adenosine; Aorta; Base Sequence; Biological Transport; Cells, Cultured; DNA Primers; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Glucose; Humans; Kinetics; Muscle, Smooth, Vascular; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thioinosine

Adenosine is an endogenous nucleoside that signals through G-protein coupled receptors. Extracellular adenosine is required for receptor activation and two pathways have been identified for formation and cellular release of adenosine. The CLASSICAL pathway relies on intracellular formation of adenosine from adenine nucleotides and cellular efflux of adenosine via equilibrative nucleoside transporters (ENTs). The ALTERNATE pathway involves cellular release of adenine nucleotides, hydrolysis via ecto-5'-nucleotidases and extracellular formation of adenosine.. A rat model of cerebral ischemia and primary cultures of rat forebrain astrocytes and neurons were used.. Using a rat model of cerebral ischemia, the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) significantly increased post-ischemic forebrain adenosine levels and significantly decreased hippocampal neuron injury relative to saline-treatment. NBMPR-induced increases in adenosine receptor activation were not detected, suggesting that altering the intracellular:extracellular distribution of adenosine can affect ischemic outcome. Using primary cultures of rat forebrain astrocytes and neurons, adenosine release was evoked by ischemic-like conditions. Dipyridamole, an inhibitor of ENTs, was more effective at inhibiting adenosine release from neurons than from astrocytes. In contrast, alpha , beta-methylene ADP, an inhibitor of ecto-5'-nucleotidase, was effective at inhibiting adenosine release from astrocytes, but not from neurons. Thus, during ischemic-like conditions, neurons released adenosine via the CLASSICAL pathway, while astrocytes released adenosine via the ALTERNATE pathway.. These cell type differences in pathways for adenosine formation during ischemia may allow transport inhibitors to block simultaneously adenosine release from neurons and adenosine uptake into astrocytes. In principle, this could improve neuronal ATP levels without decreasing adenosine receptor activation.

Topics: Adenosine; Affinity Labels; Animals; Astrocytes; Brain Ischemia; Cells, Cultured; Deoxyglucose; Dipyridamole; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Glucose; Hypoxia; Inosine; Models, Biological; Neurons; Oligomycins; Phosphodiesterase Inhibitors; Prosencephalon; Purines; Rats; Thioinosine; Tritium

Reduced oxygen level (hypoxia) induces endothelial dysfunction and release of the endogenous nucleoside adenosine. Human umbilical vein endothelium (HUVEC) function in an environment with 3% to 5% O2 and exhibit efficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whether adenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O2) reduced maximal hENT1-adenosine transport velocity (V(max)) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reduced hENT1 protein and mRNA levels, effects unaltered by N(omega)-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase [NOS] inhibitor) or PD-98059 (inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxia reduced endothelial NOS (eNOS) activity and eNOS phosphorylation at Ser(1177), but increased eNOS protein level. Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44(mapk) phosphorylation. Thus, hypoxia-increased extracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not to involve NO, but p42/44(mapk) may be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results could be important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterine growth restriction, or where adenosine transport is altered, such as gestational diabetes.

Topics: Adenosine; Biological Transport; Cell Hypoxia; Cells, Cultured; Down-Regulation; Endothelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Fetal Growth Retardation; Gene Expression Regulation; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Phosphorylation; RNA, Messenger; Thioinosine; Umbilical Veins

Adenosine is a widespread neuromodulator that can be directly released in the extracellular space during sustained network activity or can be generated as the breakdown product of adenosine triphosphate (ATP). Whole cell patch-clamp recordings were performed from CA3 principal cells and interneurons in hippocampal slices obtained from P2-P7 neonatal rats to study the modulatory effects of adenosine on giant depolarizing potentials (GDPs) that constitute the hallmark of developmental networks. We found that GDPs were extremely sensitive to the inhibitory action of adenosine (IC(50) = 0.52 microM). Adenosine also contributed to the depressant effect of ATP as indicated by DPCPX-sensitive changes of ATP-induced reduction of GDP frequency. Similarly, adenosine exerted a strong inhibitory action on spontaneous glutamatergic synaptic events recorded from GABAergic interneurons and on interictal bursts that developed in CA3 principal cells after blockade of gamma-aminobutyric acid type A (GABA(A)) receptors with bicuculline. All these effects were prevented by DPCPX, indicating the involvement of inhibitory A1 receptors. In contrast, GABAergic synaptic events were not changed by adenosine. Consistent with the endogenous role of adenosine on network activity, DPCPX per se increased the frequency of GDPs, interictal bursts, and spontaneous glutamatergic synaptic events recorded from GABAergic interneurons. Moreover, the adenosine transport inhibitor NBTI and the adenosine deaminase blocker EHNA decreased the frequency of GDPs, thus providing further evidence that endogenous adenosine exerts a powerful control on GDP generation. We conclude that, in the neonatal rat hippocampus, the inhibitory action of adenosine on GDPs arises from the negative control of glutamatergic, but not GABAergic, inputs.

Topics: Action Potentials; Adenine; Adenosine; Adenosine Triphosphate; Analgesics; Animals; Animals, Newborn; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutamic Acid; Hippocampus; In Vitro Techniques; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Thioinosine; Xanthines

Evidence suggests that adenosine (AD) is an endogenous sleep factor. The hypnogenic action of AD is mediated through its inhibitory A1 and excitatory A2A receptors. Although AD is thought to be predominantly active in the wake-active region of the basal forebrain (BF), a hypnogenic action of AD has been demonstrated in several other brain areas, including the preoptic area. We hypothesized that in lateral preoptic area (LPOA), a region with an abundance of sleep-active neurons, AD acting via A1 receptors would induce waking by inhibition of sleep-active neurons and that AD acting via A2A receptors would promote sleep by stimulating the sleep-active neurons. To this end, we studied the effects on sleep of an AD transport inhibitor, nitrobenzyl-thio-inosine (NBTI) and A1 and A2A receptor agonists/antagonists by microdialyzing them into the LPOA. The results showed that, in the sleep-promoting area of LPOA: 1) A1 receptor stimulation or inhibition of AD transport by NBTI induced waking and 2) A2A receptor stimulation induced sleep. We also confirmed that NBTI administration in the wake promoting area of the BF increased sleep. The effects of AD could be mediated either directly or indirectly via interaction with other neurotransmitter systems. These observations support a hypothesis that AD mediated effects on sleep-wake cycles are site and receptor dependent.

Topics: Adenosine; Animals; Male; Microdialysis; Preoptic Area; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptor, Adenosine A2A; Sleep; Thioinosine; Wakefulness

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Topics: Adenine; Adenosine; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; Deoxyadenosines; Dose-Response Relationship, Drug; Humans; Receptors, Estrogen; Thioinosine; Time Factors

ENT1 is an equilibrative nucleoside transporter that enables trans-membrane bi-directional diffusion of biologically active purines such as adenosine. In spinal cord dorsal horn and in sensory afferent neurons, adenosine acts as a neuromodulator with complex pro- and anti-nociceptive actions. Although uptake and release mechanisms for adenosine are believed to exist in both the dorsal horn and sensory afferent neurons, the expression profile of specific nucleoside transporter subtypes such as ENT1 is not established. In this study, immunoblot analysis with specific ENT1 antibodies (anti-rENT1(227-290) or anti-hENT1(227-290)) was used to reveal the expression of ENT1 protein in tissue homogenates of either adult rat dorsal horn or dorsal root ganglia (DRG). Immunoperoxidase labeling with ENT1 antibodies produced specific staining in dorsal horn which was concentrated over superficial laminae, especially the substantia gelatinosa (lamina II). Immunofluorescence double-labeling revealed a punctate pattern for ENT1 closely associated, in some instances, with cell bodies of either neurons (confirmed with NeuN) or glia (confirmed with CNPase). Electron microscopy analysis of ENT1 expression in lamina II indicated its presence within pre- and post-synaptic elements, although a number of other structures, including myelinated and unmyelinated, axons were also labeled. In sensory ganglia, ENT1 was localized to a high proportion of cell bodies of all sizes that co-expressed substance P, IB4 or NF, although ENT1 was most highly expressed in the peptidergic population. These data provide the first detailed account of the expression and cellular distribution of ENT1 in rat dorsal horn and sensory ganglia. The functional significance of ENT1 expression with regard to the homeostatic regulation of adenosine at synapses remains to be established.

Topics: Animals; Carrier Proteins; Equilibrative Nucleoside Transporter 1; Ganglia, Sensory; Ganglia, Spinal; Immunohistochemistry; Lumbar Vertebrae; Neuroglia; Neurons; Nucleoside Transport Proteins; Posterior Horn Cells; Rats; Rats, Wistar; Spinal Cord; Thioinosine; Tissue Distribution

Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3-300 microM), dilazep (1-100 microM), NBTI (10-500 nM), and adenosine (50-250 microM) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 microM). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a dose-dependent inhibition of glucose transport by dipyridamole (basal, IC(50) = 12.2 microM, insulin stimulated, IC(50) = 13.09 microM) and dilazep (basal, IC(50) = 5.7 microM, insulin stimulated, IC(50) = 19 microM) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses.

Topics: Adenosine; Animals; Biological Transport; Cardiotonic Agents; Cell Line; Deoxyglucose; Dilazep; Dipyridamole; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Kinetics; Mice; Monosaccharide Transport Proteins; Muscle Proteins; Myocytes, Cardiac; Thioinosine

We postulated that adenosine function could be related to some of the neurological features of Lesch-Nyhan syndrome and therefore characterized adenosine transport in PBLs (peripheral blood lymphocytes) obtained from Lesch-Nyhan patients (PBL(LN)) and from controls (PBL(C)). Adenosine transport was significantly lower in PBL(LN) when compared with that in PBL(C) and a significantly lower number of high affinity sites for [(3)H]nitrobenzylthioinosine binding were quantified per cell ( B (max)) in PBL(LN) when compared with that in PBL(C). After incubation with 25 microM hypoxanthine, adenosine transport was significantly decreased in PBL(LN) with respect to PBL(C). Hypoxanthine incubation lowers [(3)H]nitrobenzylthioinosine binding in PBL(C), with respect to basal conditions, but does not affect it in PBL(LN). This indicates that hypoxanthine affects adenosine transport in control and hypoxanthine-guanine phosphoribosyltransferase-deficient cells by different mechanisms.

Topics: Adenosine; Biological Transport; Cells, Cultured; Cyclic AMP; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Lymphocytes; Thioinosine

Nucleosides such as adenosine, as well as many nucleoside-based drugs, permeate cell membranes via a family of equilibrative nucleoside transporters (ENTs). We assessed the effects of (3-[1-(6,7-diethoxy-2-morpholino-quinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), a novel anti-inflammatory agent that potentiates the actions of adenosine, on the es (inhibitor-sensitive) and ei (inhibitor-resistant) subtypes of ENTs in human, mouse, and rat cells. KF24345 was similar to the prototypical high-affinity inhibitor nitrobenzylthioinosine (NBMPR) for blocking the human es transporter (K(I) of approximately 0.4 nM), but was 50-fold more effective than NBMPR at blocking the human ei transporter (K(I) of approximately 100 nM). KF24345 displayed significantly less species heterogeneity in its affinity for the es transporter than did dipyridamole, a widely used inhibitor of nucleoside transport; KF24345 may thus prove useful as an inhibitor for studies of nucleoside metabolism in a range of animal models. Furthermore, KF24345 seemed to act as a noncompetitive inhibitor of both [(3)H]NBMPR binding and [(3)H]nucleoside uptake by human es transporters, and these kinetics were consistent with an observed slow dissociation of KF24345 from the inhibitor binding site. KF24345 also exhibited unusual biphasic profiles for inhibition of [(3)H]NBMPR binding to membranes prepared from a recombinant human es transporter model (PK15-hENT1), suggesting the presence of multiple populations of NBMPR binding proteins in these membranes. The atypical tight binding interaction of KF24345 with the es transporter may prove useful for the molecular delineation of inhibitor binding domains and will facilitate its use as an in vivo inhibitor of nucleoside transport in studies focused on the biological effects of adenosine.

Topics: Adenosine; Allosteric Regulation; Animals; Binding Sites; Biological Transport; Cells, Cultured; Formycins; Humans; Mice; Pyrimidinones; Quinazolines; Rats; Thioinosine; Tritium; Tumor Cells, Cultured

We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 [e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole] were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92-->Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260+/-16 mM) when compared with the wild-type transporter (65+/-7.8 mM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.

Topics: Adenosine; Amino Acid Substitution; Biological Transport; Dilazep; Dipyridamole; Drug Resistance; Equilibrative Nucleoside Transporter 1; Guanosine; Humans; Inhibitory Concentration 50; Inosine; Leucine; Mutagenesis; Mutation, Missense; Point Mutation; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Structure-Activity Relationship; Thioinosine

hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family. hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine. hENT2 is expressed in only a few tissues (e.g. muscle). hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR). hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine. hENT1 is also more sensitive to NBMPR inhibition (IC50 approximately 0.4-8 nM) when compared with hENT2 (IC50 approximately 2.8 microM). This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine). Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ). Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2. For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine. However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine. Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.

Topics: Biological Transport; Dilazep; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Glycine; Humans; Nucleosides; Thioinosine; Vasodilator Agents

Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A1 receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H2O2, suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H2O2-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H2O2 exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage.

Topics: Adenine; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Cell Survival; Cells, Cultured; Dipyridamole; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen Peroxide; Inosine; Lipid Peroxidation; Male; Niacinamide; Oxidants; Oxidative Stress; Rats; Sertoli Cells; Thioinosine

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel halogenated nitrobenzylthioinosine analogs at the human es nucleoside transporter. Structure-activity relationships indicate the importance of hydrophobicity and electron withdrawing capacity of substituents at the para-position of the 6-position benzyl substituent. All of the compounds showed high binding affinity as shown by their ability to displace the fluorescent es transporter ligand, SAENTA-X8-fluorescein. Compound 16 (6-S-(para-iodobenzyl)-6-thioinosine) was the most tightly bound within the series with a K(i) of 3.88 nM (NBMPR exhibited a K(i) of 0.70 nM). This compound has higher affinity than the widely used nonnucleoside, nucleoside transport inhibitor, dipyridamole (K(i) = 8.79 nM), and may serve as a new lead compound.

Topics: Flow Cytometry; Membrane Transport Modulators; Membrane Transport Proteins; Nucleosides; Thioinosine

We studied the binding of [3H]nitrobenzylthioinosine (NBMPR) and the uptake of [3H]formycin B by the es (equilibrative inhibitor-sensitive) nucleoside transporter of Madin Darby Canine Kidney (MDCK) cells. NBMPR inhibited [3H]formycin B uptake with a Ki of 2.7+/-0.6 nM, and [3H]NBMPR had a KD of 1.3+/-0.3 nM for binding to these cells; these values are significantly higher than those obtained in human and mouse cell models. In contrast, other recognized es inhibitors, such as dipyridamole, were significantly more effective as inhibitors of [3H]NBMPR binding and [3H]formycin B uptake by MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function by stable expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhibitor sensitivities that were comparable to those obtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es/ENT1 transporter has distinctive inhibitor binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Carrier Proteins; Cell Line; Cloning, Molecular; Dilazep; Dipyridamole; DNA, Complementary; Dogs; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Formycins; Kinetics; Molecular Sequence Data; Piperazines; Protein Binding; Protein Conformation; Radioligand Assay; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thioinosine; Tritium

The effect of hypoxia on the release of adenosine was studied in vitro in the rat whole carotid body (CB) and compared with the effect of hypoxia (2%, 5% and 10% O(2)) on adenosine concentrations in superior cervical ganglia (SCG) and carotid arteries. Moderate hypoxia (10% O(2)) increased adenosine concentrations released from the CBs by 44%, but was not a strong enough stimulus to evoke adenosine release from SCG and arterial tissue. The extracellular pathways of adenosine production in rat CBs in normoxia and hypoxia were also investigated. S-(p-nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole were used as pharmacological tools to inhibit adenosine equilibrative transporters (ENT) and alpha,beta-methylene ADP (AOPCP) to inhibit ecto-5'-nucleotidase. Approximately 40% of extracellular adenosine in the CB came from the extracellular catabolism of ATP, under both normoxic and hypoxic conditions. Low pO(2) triggers adenosine efflux through activation of NBTI-sensitive ENT. This effect was only apparent in hypoxia and when adenosine extracellular concentrations were reduced by the blockade of ecto-5'-nucleotidase. We concluded that CB chemoreceptor sensitivity could be related to its low threshold for the release of adenosine in response to hypoxia here quantified for the first time.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Diphosphate; Animals; Carotid Body; Carrier Proteins; Chemoreceptor Cells; Dipyridamole; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Membrane Transport Proteins; Nucleoside Transport Proteins; Rats; Rats, Wistar; Thioinosine

To investigate the effects of potential inhibitors of membrane transport on the tubular secretion of AM188, an antiviral guanosine analog, in the isolated perfused rat kidney (IPK).. AM188 was administered to the IPK perfusate as a bolus/infusion regimen. In inhibitor groups, probenecid, p-aminohippuric acid (PAH), cimetidine, or nitrobenzylthioinosine was added to the perfusing medium.. In control IPKs, the ratio of renal clearance of AM188 (CLR) to GFR was 7.7 +/- 0.51 (mean +/- SD). The CL(R)/GFR ratio for AM188 was 6.20 +/- 0.41*, 2.85 +/- 0.20*, 1.45 +/- 0.07*, and 0.80 +/- 0.01* when the concentration of probenecid in perfusate was 10, 50, 100, and 1000 microM, respectively (*p < 0.05 compared to control group); the ratio was 7.71 +/- 0.38, 6.02 +/- 0.42*, 1.71 +/- 0.15*, and 0.91 +/- 0.07* for the PAH group and 6.42 +/- 1.70*, 5.33 +/- 1.53*, 3.16 +/- 0.81*, and 1.21 +/- 0.20* for the cimetidine group when the concentrations were 10, 100, 1000 and 10,000 microM, respectively; and the ratio was 5.33 +/- 0.21* when the concentration of nitrobenzylthioinosine was 5 microM.. These results suggest that renal tubular secretion of AM188 involves organic anion and cation transport systems.

Topics: Animals; Antiviral Agents; Cimetidine; Glomerular Filtration Rate; Guanosine; Male; Metabolic Clearance Rate; Nephrons; p-Aminohippuric Acid; Perfusion; Probenecid; Rats; Thioinosine; Tissue Distribution

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.

Topics: Animals; Capillaries; Cell Separation; Cells, Cultured; Dilazep; Dipyridamole; DNA Primers; Endothelial Cells; Formycins; Muscle, Skeletal; Nucleoside Transport Proteins; Piperazines; Radioligand Assay; Rats; Reverse Transcriptase Polymerase Chain Reaction; Thioinosine; Vasodilator Agents

Cyclopentenyl cytosine (CPEC) is a carbocyclic cytidine analog inhibitor of CTP synthetase and experimental drug for combination chemotherapy. CPEC treatment (50 nM) depleted intracellular CTP and induced a specific S-phase arrest and erythroid differentiation of human erythroleukemia K562 cells. The equilibrative nucleoside transporters (ENT1, 2) facilitated uptake of CPEC into K562 cells as evidenced by both NBMPR and dipyridamole inhibition of CPEC-mediated CTP depletion and erythroid differentiation. Incubation with the pyridinylimidazole p38 MAPK inhibitors, SB203580 or SB220025, suppressed both the CPEC-induced cell cycle arrest and differentiation of K562 cells. SB203580 also prevented the cell cycle arrest and erythroid differentiation of K562 cells induced by Leflunomide (LEF), a non-nucleoside inhibitor of the de novo pyrimidine pathway, without affecting LEF-induced depletion of pyrimidine pools. Finally, selective knockdown of p38 MAPK by using Smart Pooltrade mark siRNA to p38 MAPK significantly decreased the CPEC-induced differentiation of K562 cells. These results suggest that endogenous activity of p38 MAP kinases may be required for committing K562 cells to cell cycle arrest and erythroid differentiation under conditions of CTP depletion.

Topics: Affinity Labels; Carbon-Nitrogen Ligases; Cell Differentiation; Cytidine; Cytidine Triphosphate; Dipyridamole; Enzyme Inhibitors; Equilibrative Nucleoside Transporter 1; Erythroid Precursor Cells; Humans; Isoxazoles; K562 Cells; Leflunomide; Leukemia, Myeloid; p38 Mitogen-Activated Protein Kinases; Phosphodiesterase Inhibitors; Pyrimidines; RNA, Small Interfering; S Phase; Thioinosine

There is a large body of evidence indicating important interactions between the adenosine and the opioid systems in regulating pain, opioid dependence and withdrawal. Mice lacking the proenkephalin gene and therefore lacking the endogenous enkephalin peptides have been successfully developed and exhibit decreased locomotor activity, are hyperalgesic and show enhanced anxiety and aggression. In addition, an upregulation of mu and delta receptors was also observed in the brains of knockout mice. To investigate if there are any compensatory alterations in adenosine systems in the brains of mutant mice, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI)-sensitive adenosine transporters in the brains of wild-type and homozygous enkephalin knockout mice. Adjacent coronal brain sections were cut from brains of +/+ and -/- mice for the determination of binding of [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H]DPCPX), [(3)H]2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H]CGS21680) or [(3)H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant increase in [(3)H]DPCPX and [(3)H]NBTI binding but no significant change in [(3)H]CGS21680 binding was detected in enkephalin knockout brains. The results provide further evidence of functional interactions in the brain between opioid receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters but not A(2A) receptors.

Topics: Animals; Autoradiography; Brain; Enkephalins; Male; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nucleoside Transport Proteins; Protein Binding; Protein Precursors; Receptors, Purinergic P1; Thioinosine; Xanthines

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT-PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non-small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r =-0.6769, P = 0.0005), whereas dCK expression levels were not. In a highly gemcitabine-sensitive cell line, NCI-H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI-H23 cells to gemcitabine and subsequently established the drug-resistant clone H23/GEM-R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.

Topics: Affinity Labels; Antimetabolites, Antineoplastic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Division; Deoxycytidine; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Gemcitabine; Humans; Lung Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Thioinosine; Tumor Cells, Cultured

5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.

Topics: Adenosine; Affinity Labels; Amino Acid Chloromethyl Ketones; Aminoimidazole Carboxamide; Apoptosis; Caspases; Cyclic AMP-Dependent Protein Kinases; Cytochrome c Group; Enzyme Inhibitors; Humans; Jurkat Cells; Phosphorylation; Ribonucleosides; Thioinosine

Novel regioisomers of conformationally constrained analogues of the potent es nucleoside transporter ligand, nitrobenzylmercaptopurine riboside (NBMPR), designed for probing its bound (bioactive) conformation, were synthesized and evaluated as es transporter ligands by flow cytometry. Purine 6-position 5, 6, 7, or 8-nitro-1,2,3,4-tetrahydroisoquinolylpurine ribosides, in which the nitrobenzyl moiety in NBMPR has been locked into the nitro-1,2,3,4-tetrahydroisoquinoline system, were synthesized by reaction of the appropriate nitro-1,2,3,4-tetrahydroisoquinoline with 6-chloropurine riboside. Flow cytometry was performed using 5-(SAENTA)-X8-fluorescein as the competitive ligand. A high degree of variation in the es transporter binding capacity of the target compounds was observed, with the K(i) values ranging from 0.45 nM for the most tightly bound compound (4) to 300 nM for the least tightly bound compound (5). The K(i) of NBMPR was 0.70 nM, a little higher than that of compound 4. Compound 4 is the isomer that has the nitro group in the best orientation at the es transporter binding site compared to the other three compounds, 2, 3, and 5.

Topics: Equilibrative Nucleoside Transporter 1; Flow Cytometry; Humans; Isoquinolines; Nucleic Acid Conformation; Protein Binding; Structure-Activity Relationship; Thioinosine; Tumor Cells, Cultured

Adenosine modulates nociceptive processing in the superficial dorsal horn of the spinal cord. In other tissues, membrane transporters influence profoundly the extracellular levels of adenosine. To investigate the putative role of nucleoside transporters in the regulation of excitatory synaptic transmission in the dorsal horn, we employed immunohistochemistry and whole-cell patch-clamp recording of substantia gelatinosa neurons in slices of rat spinal cord in vitro. The rat equilibrative nucleoside transporter (rENT1) was revealed by antibody staining to be abundant in neonatal and mature dorsal horn, especially within laminae I-III. This was confirmed by immunoblots of dorsal horn homogenate. Nitrobenzylthioinosine (NBMPR), a potent non-transportable inhibitor of rENT1, attenuated synaptically evoked EPSCs onto lamina II neurons in a concentration-dependent manner. Application of an adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine produced a parallel rightward shift in the NBMPR concentration-effect curve. The effects of NBMPR were partially reversed by adenosine deaminase, which facilitates the metabolic degradation of adenosine. The modulation by NBMPR of evoked EPSCs was mimicked by exogenous adenosine or the selective A1 receptor agonist, 2-chloro-N6-cyclopentyl adenosine. NBMPR reduced the frequency but not the amplitude of spontaneous miniature EPSCs and increased the paired-pulse ratio of evoked currents, an effect that is consistent with presynaptic modulation. These data provide the first direct evidence that nucleoside transporters are able to critically modulate glutamatergic synaptic transmission.

Topics: Animals; Dose-Response Relationship, Drug; Electrophysiology; Equilibrative Nucleoside Transporter 1; Excitatory Postsynaptic Potentials; Female; Glutamic Acid; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Membrane Potentials; Nucleoside Transport Proteins; Patch-Clamp Techniques; Posterior Horn Cells; Rats; Rats, Wistar; Receptor, Adenosine A1; Recombinant Proteins; Synapses; Synaptic Transmission; Thioinosine; Xanthines

Nitrobenzylthioinosine (NBTI, 1) was systematically modified by attachment of substituents at positions C6 and N9, and also by substitution of N1 with C. These modifications were chosen to reduce the polarity of the new compounds. Incorporation of the nitro functionality into a benzoxadiazole ring system was considered first. These new nucleosides showed high affinity (1.5-10nM) towards the nucleoside transport protein as present on human erythrocyte ghosts. Next, modification of this benzoxadiazole ring system with C, S and O in different positions produced a number of less polar nucleosides with affinity in the higher nanomolar range. Modification of N9 was achieved with different alkyl and alcohol substituents. An n-butyl substituent proved best, although all variations yielded substantial decreases in affinity. Replacement of N1 by a carbon atom in combination with a 2-Cl substituent also resulted in a relatively potent NBTI derivative (47 nM).

Topics: Alkylation; Biological Transport, Active; Erythrocyte Membrane; Humans; In Vitro Techniques; Indicators and Reagents; Nucleosides; Radioligand Assay; Structure-Activity Relationship; Thioinosine

Arylalkylamine N-acetyltransferase (AANAT) is a key regulatory enzyme in the synthesis of melatonin, which displays daily fluctuations in chicken retinal photoreceptors in vivo. The purpose of the present study was to determine if cultures of embryonic neural retina cells express diurnal rhythms of AANAT activity.. Cell cultures were prepared from chick embryonic day 6 neural retina and incubated for 4 to 8 days in vitro (DIV). Cells were incubated under a daily light-dark (LD) cycle and were harvested day and night. Culture conditions were modified to test the effects of cell density, serum concentration, incubation temperature, S-(4-nitrobenzyl)-6-thioinosine (NBTI), and taurine on AANAT activity. AANAT activity was assayed in cell homogenates by measuring the catalytic formation of N-acetyltryptamine from tryptamine and acetyl coenzyme A.. Cells cultured in medium containing 10% fetal bovine serum (FBS) failed to show any diurnal fluctuation in AANAT activity on DIV 5 and 6. However, if the culture medium was replaced on DIV 4 with one containing 1% FBS, and 5 microM NBTI or 5 mM taurine, the cells expressed significant diurnal rhythms of enzyme activity. NBTI was more potent and effective than taurine. Culture conditions were optimized with respect to cell density, serum concentration, incubation temperature, and NBTI concentration. Under optimized conditions, overall cell survival and the density of photoreceptor cells were increased relative to that with the other culture conditions tested.. The results indicate that diurnal rhythms of AANAT activity are expressed in embryonic retinal cells incubated under particular culture conditions. The results show that the mechanisms regulating melatonin synthesis in chicken retinal cells are established during early embryonic life. This culture preparation will be useful in elucidating the photic control mechanisms involved in regulation of melatonin biosynthesis in photoreceptor cells.

Topics: Animals; Arylamine N-Acetyltransferase; Cell Count; Cell Survival; Cells, Cultured; Chick Embryo; Circadian Rhythm; Hot Temperature; Retina; Taurine; Thioinosine; Tryptamines

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.

Topics: 5'-Nucleotidase; Adenine; Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; Animals; Cells, Cultured; Dipyridamole; Enzyme Inhibitors; Extracellular Space; Germ Cells; Kinetics; Male; Paracrine Communication; Phosphodiesterase Inhibitors; Purines; Rats; Rats, Wistar; Receptors, Purinergic; Seminiferous Tubules; Sertoli Cells; Thioinosine

The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410-425) were used to study uptake of [3H]thymidine, [3H]adenine, and [3H]ganciclovir. Transport of [3H]adenine and [3H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34 degrees C. Thymidine uptake by SIRC displayed saturable kinetics (K(m) = 595.9 +/- 80.4 microM, and V(max) = 289.5 +/- 17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [3H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (K(m) = 14.4 +/- 2.3 microM, V(max) = 0.4 +/- 0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [3H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [3H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.

Topics: Adenine; Animals; Biological Transport; Cornea; Epithelium, Corneal; Ganciclovir; Male; Membrane Transport Proteins; Nucleosides; Nucleotide Transport Proteins; Rabbits; Sodium; Thioinosine; Thymidine; Tritium

Chronic treatment with opioids induces adaptations in neurons leading to tolerance and dependence. Studies have implicated the midbrain periaqueductal gray (PAG) in the expression of many signs of withdrawal. Patch-clamp recording techniques were used to examine whether augmentation of adenylyl cyclase signalling produces hyperexcitation in GABAergic nerve terminals within the mouse PAG. Both the rate of mIPSCs and the amplitude of evoked IPSCs during naloxone-precipitated withdrawal was profoundly enhanced in chronically morphine treated mice, compared to vehicle treated controls, in the presence but not the absence an adenosine A(1) receptor antagonist DPCPX. Enhanced GABAergic transmission in the presence of DPCPX was abolished by blocking protein kinase A. Inhibitors of cAMP transport, phosphodiesterase and nucleotide transport mimicked the effect of DPCPX. Coupling efficacy of micro-receptors to presynaptic inhibition of GABA release was increased in dependent mice in the presence of DPCPX. The increased coupling efficacy was abolished by blocking protein kinase A, which unmasked an underlying micro-receptor tolerance. These findings indicate that enhanced adenylyl cyclase signalling following chronic morphine treatment produces (1) GABAergic terminal hyperexcitability during withdrawal that is retarded by a concomitant increase in endogenous adenosine, and (2) enhanced micro-receptor coupling to presynaptic inhibition that overcomes an underlying tolerance.

Topics: Action Potentials; Adenosine; Affinity Labels; Animals; Colforsin; Cyclic AMP; Dipyridamole; Dose-Response Relationship, Drug; Drug Interactions; Enkephalins; Enzyme Inhibitors; gamma-Aminobutyric Acid; In Vitro Techniques; Isoquinolines; Male; Mesencephalon; Mice; Mice, Inbred C57BL; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Neural Inhibition; Neurons; Patch-Clamp Techniques; Periaqueductal Gray; Probenecid; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Substance Withdrawal Syndrome; Sulfonamides; Synaptic Transmission; Thioinosine; Time Factors; Uricosuric Agents; Vasodilator Agents; Xanthines

Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine [NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-beta-D-ribofuranosylpurine], an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological beta-L-enantiomers of purine nucleosides, beta-L-adenosine, beta-L-deoxyadenosine, and beta-L-guanosine. The beta-L-pyrimidine nucleosides, beta-L-uridine, beta-L-cytidine, and beta-L-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside beta-L-enantiomers by the intracellular parasites also implies that Toxoplasma-infected cells can transport these nucleosides. In sharp contrast, under the same conditions, uninfected fibroblast cells did not transport NBMPR or any of the unnatural beta-L-nucleosides. beta-D-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and beta-L-stereoisomers of the purine nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. Furthermore, infection with a Toxoplasma mutant deficient in parasite adenosine/purine nucleoside transport reduced or abolished the uptake of beta-D-adenosine, NBMPR, and purine beta-L-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine beta-L-nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. These results also demonstrate that, in contrast to the mammalian nucleoside transporters, the Toxoplasma adenosine/purine nucleoside transporter(s) lacks stereospecificity and substrate specificity in the transport of purine nucleosides. In addition, infection with T. gondii confers the properties of the parasite's purine nucleoside transport on the parasitized host cells and enables the infected cells to transport purine nucleosides that were not transported by uninfected cells. These unique characteristics of purine nucleoside transport in T. gondii may aid in the identification of new promising antitoxoplasmic drugs.

Topics: Animals; Biological Transport; Cells, Cultured; Dipyridamole; Fibroblasts; Humans; Hypoxanthine; Mice; Mice, Inbred Strains; Nucleoside Transport Proteins; Purine Nucleosides; Stereoisomerism; Thioinosine; Toxoplasma

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence 22Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1-5 U/ml) caused an excitatory effect on 22Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25 microM). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1-10 microM) and the adenosine deaminase inhibitor EHNA (10-25 microM) inhibited 22Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.

Topics: Adenine; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Brain; In Vitro Techniques; Rats; Rats, Wistar; Sodium; Sodium Radioisotopes; Synaptosomes; Thioinosine

We have previously shown that extracellular adenosine levels increase locally in the basal forebrain (BF) during prolonged wakefulness, yet the cellular mechanisms of this local accumulation have remained unknown. The extracellular adenosine levels are strictly regulated by adenosine metabolism and its transport through cell membrane by the nucleoside transporters. As we previously showed that the key adenosine metabolizing enzymes were not affected by prolonged wakefulness, we now focussed on potential changes in the nucleoside transporters. In the present study, we measured the binding of nitrobenzylthioinosine (NBMPR), an ENT1 transporter inhibitor, and the ENT1 transporter mRNA after prolonged wakefulness and recovery sleep. Rats were sleep-deprived for 3 or 6 h using gentle handling. After 6 h one group was allowed to sleep for 2 h. NBMPR binding was determined from BF and cortex by incubating tissue extracts with [3H] NBMPR. The in situ hybridization was carried out on 20 microm cryosections using [35S]dATP-labelled oligonucleotide probe for ENT1 mRNA. The NBMPR binding was significantly decreased in the BF, but not in the cortex, after 6 h sleep deprivation when compared with the time-matched controls, suggesting a decline in adenosine transport. The expression of ENT1 mRNA did not change during prolonged wakefulness or recovery sleep in either cortex or the BF, although circadian variations were measured in both areas. We conclude that the regional decrease in adenosine transport could contribute to the gradual accumulation of extracellular adenosine in the basal forebrain during prolonged wakefulness.

Topics: Adenosine; Animals; Binding Sites; Cerebral Cortex; Circadian Rhythm; Culture Techniques; Equilibrative Nucleoside Transporter 1; In Situ Hybridization; Male; Prosencephalon; Rats; Rats, Wistar; Recovery of Function; RNA, Messenger; Sleep Deprivation; Thioinosine; Wakefulness

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Adhesion; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Deoxyadenosines; Enzyme Precursors; Flow Cytometry; Humans; Microscopy, Fluorescence; Pentostatin; Thioinosine

The significance of PDE2 on the atrial inotropy was studied in eu- and hyperthyroidism. The contractile force was measured and negative inotropic capacity of N6-cyclopentyladenosine (CPA) was determined on left atria isolated from 8-day thyroxine- or solvent-treated guinea pigs, in the presence or absence of EHNA (adenosine deaminase and PDE2 inhibitor) or NBTI (nucleoside transporter inhibitor). EHNA was administered to inhibit PDE2, while NBTI was used to model the accumulation of endogenous adenosine. The reduction of the contractile force caused by EHNA was smaller in the thyroxine-treated atria than in the solvent-treated samples. Contrary, NBTI induced a decrease in the contractile force without significant difference between the two groups. In addition, EHNA enhanced the efficiency of CPA in thyroxine-treated atria and did not affect it in solvent-treated samples, while the response to CPA was decreased by NBTI in all atria, especially in hyperthyroidism. On the basis of greater retention of the contractile force and sustained/enhanced responsiveness to CPA in the presence of EHNA we conclude that PDE2's inhibition has a significant positive inotropic effect in guinea pig atria and this effect is proven to be augmented in hyperthyroidism.

Topics: Adenine; Adenosine; Animals; Cardiotonic Agents; Culture Techniques; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Guinea Pigs; Heart Atria; Hyperthyroidism; Male; Myocardial Contraction; Phosphoric Diester Hydrolases; Thioinosine

Adenosine is a physiologically important nucleoside in the cardiovascular system where it can act as a cardioprotectant and modulator of energy usage. Adenosine transporters (ATs) modulate cellular adenosine levels, which, in turn, can affect a number of processes such as receptor activation and glucose uptake, but their role in cardiac physiology is poorly understood. Therefore, we have developed a new cell model by determining various adenosine-related characteristics of HL-1, an immortalized atrial cardiomyocyte murine cell line. Adenosine uptake in HL-1 cells is sodium independent, saturable, and inhibitable by nucleoside transport inhibitors (nitrobenzylthioinosine (NBTI), dipyridamole, dilazep). Reverse transcription--polymerase chain reaction analysis confirmed that HL-1 cells possess mouse equilibrative nucleoside transporters 1 and 2 (mENT1, mENT2) and kinetic analyses indicate moderate-affinity (Km = 51.3 +/- 12.9 microM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (B(max) = 520 +/- 10 fmol/mg protein, Kd = 0.11 +/- 0.04 nM, 1.6 x 10(5) NBTI-binding sites/cell). HL-1 cells possess adenosine receptor, metabolic enzyme, protein kinase C isoform, and insulin-stimulated glucose transport profiles that match normal mouse heart. Therefore, HL-1 is an excellent model to study ATs within cardiomyocytes and the first model for evaluating in detail the role of the ATs in modulating effects of adenosine.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Affinity Labels; Animals; Biological Transport; Blotting, Northern; Carrier Proteins; Cells, Cultured; DNA Primers; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Glucose; Insulin; Membrane Transport Proteins; Mice; Models, Biological; Myocytes, Cardiac; Nucleoside Transport Proteins; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thioinosine

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.

Topics: Amino Acid Sequence; Animals; Biological Transport; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Membrane Transport Proteins; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Rats; Recombinant Proteins; Structure-Activity Relationship; Thioinosine; Uridine; Xenopus laevis

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63+/-3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.

Topics: Adenine; Allopurinol; Animals; Binding, Competitive; Biological Transport; Carbon Radioisotopes; Choroid Plexus; Dose-Response Relationship, Drug; Hypoxanthine; In Vitro Techniques; Mannitol; Perfusion; Sheep; Sodium; Thioinosine; Thymine; Tritium; Uracil; Xanthine Oxidase

Gemcitabine and capecitabine are nucleoside analogues used in chemotherapy strategies for the treatment of breast cancer. We previously demonstrated that deficiency in hENT1, the most abundant and widely distributed plasma membrane nucleoside transporter in human cells, confers high-level resistance to gemcitabine toxicity in vitro, whereas the relationship between hENT1 activity and capecitabine toxicity is unknown. To determine the relationship between capecitabine cytotoxicity and hENT1 abundance, cultured MDA-MB-435s human mammary carcinoma cells were exposed to graded concentrations of the capecitabine metabolites, 5'-deoxy-5-fluorouridine or 5-fluorouracil, in the presence and absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a tight-binding inhibitor of hENT1. The presence of NBMPR reduced the cytotoxic effects of 5'-deoxy-5-fluorouridine, indicating that hENT1 also enabled cellular uptake of this capecitabine metabolite by breast cancer cells. We report here the development of an immunohistochemical method to assess the hENT1 abundance of malignant cells in solid tumors. Frozen sections of 33 primary breast cancers were stained with monoclonal antibodies raised against a synthetic peptide derived from the large intracellular loop of hENT1, and staining intensity was scored on a 0-4+ scale. hENT1 staining intensity varied markedly among breast samples (4 with score 0, 5 with score 1+, 7 with score 2+, 14 with score 3+, 3 with score 4+), suggesting that at least 9 of the tumors were hENT1 deficient. We conclude that because hENT1 deficiency has previously been associated with nucleoside drug resistance, immunohistochemical staining of hENT1 warrants further study as a predictive tool for guiding the appropriate use of gemcitabine and capecitabine in the treatment of breast cancer.

Topics: Affinity Labels; Animals; Antibodies, Monoclonal; Antibody Formation; Antimetabolites, Antineoplastic; Breast Neoplasms; Capecitabine; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Deoxycytidine; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Female; Floxuridine; Fluorouracil; Humans; Immunoenzyme Techniques; Membrane Transport Proteins; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Staging; Thioinosine

Chronic incubation with elevated D-glucose reduces adenosine transport in endothelial cells. In this study, exposure of human umbilical vein endothelial cells to 25 mmol/L D-glucose or 100 micromol/L ATP, ATP-gamma-S, or UTP, but not ADP or alpha,beta-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport by D-glucose, ATP, and ATP-gamma-S was associated with reduced maximal binding, with no changes in the apparent dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (approximately 60+/-10%, P<0.05; n=6) in the number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8+/-0.1x10(6) in 5 mmol/L D-glucose) and in hENT1 mRNA levels was observed in cells exposed to D-glucose or ATP-gamma-S. Incubation with elevated D-glucose, but not with D-mannitol, increased the ATP release by 3+/-0.2-fold. The effects of D-glucose and nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P2Y purinoceptor antagonist), suramin (Galpha(s) protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (nonselective P2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine transport via hENT1 in endothelial cells cultured in 25 mmol/L D-glucose could be due to stimulation of P2Y2 purinoceptors by ATP, which is released from these cells in response to D-glucose. This could be a mechanism to explain in part the vasodilatation observed in the early stages of diabetes mellitus or in response to D-glucose infusion.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Binding, Competitive; Biological Transport; Cells, Cultured; Endothelium, Vascular; Equilibrative Nucleoside Transporter 1; Glucose; Humans; Membrane Transport Proteins; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; RNA, Messenger; Thioinosine; Umbilical Veins; Uridine Triphosphate

NB1011, a novel anticancer agent, targets tumor cells expressing high levels of thymidylate synthase (TS). NB1011 is converted intracellularly to bromovinyldeoxyuridine monophosphate (BVdUMP) which competes with the natural substrate, deoxyuridine monophosphate, for binding to TS. Unlike inhibitors, NB1011 becomes a reversible substrate for TS catalysis. Thus, TS retains activity and converts BVdUMP into cytotoxic product(s). In vitro cytotoxicity studies demonstrate NB1011's preferential activity against tumor cells expressing elevated TS protein levels. Additionally, NB1011 has antitumor activity in vivo. To identify drugs which interact synergistically with NB1011, we screened 13 combinations of chemotherapeutic agents with NB1011 in human tumor and normal cells. Dipyridamole and p-nitrobenzylthioinosine (NBMPR), potent inhibitors of equilibrative nucleoside transport, synergized with NB1011 selectively against 5-fluorouracil (5-FU)-resistant H630R10 colon carcinoma cells [combination index (CI)=0.75 and 0.35] and Tomudex-resistant MCF7TDX breast carcinoma cells (CI=0.51 and 0.57), both TS overexpressing cell lines. These agents produced no synergy with NB1011 in Det551 and CCD18co normal cells (CI > 1.1) lacking TS overexpression. Dipyridamole potentiated NB1011's cytotoxicity in medium lacking nucleosides and bases, suggesting a non-salvage-dependent mechanism. We demonstrate that nucleoside transport inhibitors, dipyridamole and NBMPR, show promise for clinically efficacious combination with NB1011.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Breast Neoplasms; Bromodeoxyuridine; Carrier Proteins; Cell Survival; Colonic Neoplasms; Dipyridamole; Drug Resistance, Neoplasm; Drug Synergism; Fluorouracil; Humans; Membrane Proteins; Nucleoside Transport Proteins; Quinazolines; Thioinosine; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

Nitrobenzylthioinosine (NBTI), a nucleoside-transport inhibitor, has been found to possess the ability to prevent the cytotoxic action of Shiga toxin (Stx) 1 in human renal cortical epithelial cells (HRCECs), thereby protecting HRCECs from cell death. Further examination revealed that NBTI does not affect either the binding or the endocytosis of Stx1 but alters the intracellular transport of Stx1. Generally, endocytosed Stx1 is thought to be transported from endosomes to the endoplasmic reticulum. In NBTI-treated cells, however, the endocytosed Stx1 is delivered to an early endosome, but no further transportation occurs. Moreover, Stx1 is rapidly excreted from NBTI-treated HRCECs, preventing the accumulation of Stx1. Investigation of the NBTI-mediated protection mechanism against Stx cytotoxicity may provide insights into the analysis of Stx-mediated cell damage and lead to improvements in therapeutic approaches for diseases caused by Stx.

Topics: Adenosine Triphosphate; Biological Transport; Cells, Cultured; Deoxyglucose; Epithelial Cells; Golgi Apparatus; Humans; Kidney Cortex; Shiga Toxin 1; Thioinosine

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate (pCMBS) were tested for their effects on ligand interactions with the [(3)H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no "reversal" in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane; Detergents; Dithiothreitol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Equilibrative Nucleoside Transporter 1; Erythrocytes; Ethylmaleimide; Humans; Kinetics; Ligands; Mice; Nucleoside Transport Proteins; Protein Binding; Protein Structure, Secondary; Protein Transport; Sulfhydryl Reagents; Thioinosine; Time Factors; Uridine

There is a large body of evidence indicating important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed and the animals show complete loss of analgesic responses to morphine as well as differences in pain sensitivity. To investigate if there are any compensatory alterations in adenosine systems in mutant animals, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI) sensitive adenosine transporters in the brains and spinal cords of wild type, heterozygous and homozygous mu-opioid receptor knockout mice. Adjacent coronal sections were cut from the brains and spinal cords of +/+, +/- and -/- mice for the determination of binding of [3H]DPCPX, [3H]CGS21680 or [3H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant reduction in [3H]DPCPX and [3H]NBTI binding was detected in mutant mice brains but not in spinal cords. No significant change in A(2A) binding was detected in mu-opioid receptor knockout brains. The results suggest there may be functional interactions between mu-receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters in the brain but not in the spinal cord.

Topics: Animals; Autoradiography; Brain; Carrier Proteins; Female; Male; Membrane Transport Proteins; Mice; Mice, Knockout; Nucleoside Transport Proteins; Receptor, Adenosine A2A; Receptors, Opioid, mu; Receptors, Purinergic P1; Spinal Cord; Thioinosine

The human concentrative (Na(+)-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na(+)-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na(+)-dependent cib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na(+) concentration indicated a Na(+):uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element.

Topics: Amino Acid Sequence; Animals; Biological Transport; Carrier Proteins; Cell Differentiation; Dilazep; Dipyridamole; Electric Conductivity; Evolution, Molecular; HL-60 Cells; Humans; Membrane Transport Proteins; Mice; Models, Molecular; Molecular Sequence Data; Purine Nucleosides; Pyrimidine Nucleosides; Recombinant Proteins; Sequence Homology, Amino Acid; Sodium; Substrate Specificity; Symporters; Thioinosine; Uridine

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys(140)) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys(140) to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys(140) as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys(140) in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4 has been shown previously to be within a structural domain (TMs 3-6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Cysteine; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Kinetics; Molecular Sequence Data; Nucleoside Transport Proteins; Oocytes; Rats; Sequence Alignment; Thioinosine; Transcription, Genetic; Uridine; Xenopus

The objectives of our study were to identify the types of nucleoside transporters present in the human fetal small intestine and to characterize their developmental activity, longitudinal distribution, and transport kinetics compared with those present in the adult intestine. Nucleoside uptake by intestinal brush-border membrane vesicles was measured by an inhibitor-stop rapid filtration technique. Only the purine-specific (N1; hCNT2) and the pyrimidine-specific (N2; hCNT1) Na(+)-dependent nucleoside transporters were found to be present on the brush-border membranes of the enterocytes along the entire length of the fetal and adult small intestines. The activity of these transporters was higher in the proximal than in the distal small intestine. Both the N1 and N2 transporters found in the fetal intestine shared similar kinetic properties (Michaelis-Menten constant and Na(+)-nucleoside stoichiometry) to those in the adult intestine. During the period of rapid morphogenesis (11-15 wk gestation), no temporal differences were apparent in the activity of the N1 and N2 transporters in the fetal small intestine. These findings have implications for the absorption of drugs from the amniotic fluid by the fetus after maternal drug administration of nucleoside drugs such as the antivirals zidovudine and didanosine.

Topics: Adult; Biological Transport; Carrier Proteins; Cell Membrane; Cytidine; Cytoplasmic Vesicles; Drug Design; Female; Fetus; Gene Expression Regulation, Developmental; Guanosine; Humans; Inosine; Intestine, Small; Male; Membrane Transport Proteins; Microvilli; Organ Specificity; Protein Isoforms; Sodium; Thioinosine; Uridine

[reaction: see text] A new synthetic route to ring-constrained (N)-methanocarba nucleosides and nucleotides is presented. Ring closure of a diene intermediate using Grubbs catalyst provides a new avenue for the preparation of the cyclopentenone derivative 6, which is a versatile intermediate for various carbocycles. The product was almost as potent an inhibitor of es-mediated nucleoside transport as the parent compound, inhibiting initial rates of uptake of uridine into cultured CCRF-CEM cells by 50% at approximately 30-50 nM.

Topics: Biological Transport; Catalysis; Cells, Cultured; Cyclization; Cyclopentanes; Humans; Molecular Structure; Nucleotides; Structure-Activity Relationship; Thioinosine

We have isolated a mouse cDNA clone corresponding to a novel isoform of the NBMPR-sensitive equilibrative nucleoside transporter (ENT1). The cDNA contains a 6 bp deletion in the open reading frame that changes the amino acid composition in a consensus casein kinase II (CKII) phosphorylation site at Ser-254. The clone containing Ser-254 is termed mENT1.1 and the clone lacking the serine termed mENT1.2. The deduced amino acid sequence of mENT1.1 corresponds to the previously cloned human and rat ENT1 proteins at Ser-254. Tissue distribution studies show that mRNA for both ENT1 isoforms are ubiquitously co-expressed in mouse. Analysis of genomic DNA corresponding to mouse ENT1 indicates the isoforms can be produced by alternative splicing at the end of exon 7. CEM/C19 cells stably expressing mENT1.1 and mENT1.2 show similar dose response curves for NBMPR and dipyridamole inhibition of [(3)H]adenosine uptake as well as exhibiting comparable selectivity for both purine and pyrimidine nucleosides but not the corresponding nucleobases.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Casein Kinase II; Cloning, Molecular; Dipyridamole; Equilibrative Nucleoside Transporter 1; Gene Expression Regulation; Humans; Inhibitory Concentration 50; Leukemia, T-Cell; Membrane Proteins; Mice; Molecular Sequence Data; Nucleoside Transport Proteins; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Rats; Sequence Homology, Amino Acid; Thioinosine; Tumor Cells, Cultured

Hypoxanthine (HPX) uptake was investigated in four human tumour cell lines previously characterised as being sensitive (ds: A549 and MCF7) or insensitive (di: COR-L23 and T-47D) to dipyridamole (DP)-induced inhibition of HPX rescue from antipurine antifolate-induced growth inhibition. The aim of the study was to determine the mechanism underlying the differential sensitivity of HPX rescue to DP. The time-course of HPX uptake in the two ds cell lines was different in comparison to the two di cell lines. The initial rate of HPX uptake in the di cell lines was more rapid than in the ds cell lines such that at 60 sec the amount of HPX taken up by the former was 2-6 times higher than that taken up by the later. The K(t) and T(max) for HPX transport in di COR-L23 cells were 870 microM and 4.75 microM/10(6) cells/min and 1390 microM and 1.78 microM/10(6) cells/min in ds A549 cells. HPX transport was not sodium-dependent in these cells. Equilibrative nucleoside transporter 2 (ENT2)-mediated thymidine transport was also higher in di cells. DP inhibited HPX uptake into ds cell lines by > or =48% and by < or =20% in the di cell lines. Competition studies with HPX and thymidine transport via ENT2 indicated an overlap between nucleoside and nucleobase transport transporters in the breast cancer cell lines (MCF7 and T-47D). These studies showed that more rapid and extensive HPX uptake, as well as reduced sensitivity to DP inhibition, is associated with the inability of DP to prevent HPX rescue from antipurine antifolate-induced growth inhibition in certain human tumour cell lines.

Topics: Affinity Labels; Binding, Competitive; Biological Transport; Carrier Proteins; Cell Division; Chromatography, High Pressure Liquid; Dipyridamole; Dose-Response Relationship, Drug; Drug Interactions; Equilibrative Nucleoside Transport Proteins; Folic Acid Antagonists; Humans; Hypoxanthine; Kinetics; Phosphodiesterase Inhibitors; Sodium; Thioinosine; Thymidine; Tumor Cells, Cultured

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

Topics: Animals; Biological Transport; Carrier Proteins; Cells, Cultured; Epididymis; Epithelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Guanosine; Male; Membrane Proteins; Membrane Transport Proteins; Nucleosides; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Sodium; Thioinosine; Thymidine; Tritium; Uridine

Previous work showed the presence of adenosine receptors as well as adenosine uptake and release mechanisms in developing chick retinal neurons in culture. In the present work we show that exogenous glutamate or kainate promotes extensive cell death in these cultures which is blocked when the cultures are previously incubated with adenosine. Addition of glutamate or kainate to purified cultures of retinal neurons and photoreceptors induced massive death of cultured cells which was inhibited in both cases by preincubation with MK801, an NMDA antagonist, or DNQX, an AMPA/kainate antagonist. Cell death was also greatly attenuated by preincubation with adenosine plus EHNA, an adenosine deaminase inhibitor, NBI, an adenosine uptake blocker, the permeable cAMP analogs 8-Br cAMP and Sp cAMP and the A(2a) agonists CGS 21680 and DPMA, but not with the A(1) receptor agonist CHA. Kinetic studies performed determining the intracellular LDH activity showed that maximal death was observed after 8 h and in concentrations of glutamate as low as 50 microM. We also observed a time-dependent protective effect of adenosine beginning after 1 h of preincubation and reaching a maximal effect after 24 h, indicating its association with changes in cellular metabolism induced by long-term exposure of cells to the nucleoside. The results show that adenosine inhibits glutamate toxicity in retinal neurons through a long-term activation of A(2a) receptors and elevation of intracellular cyclic AMP levels.

Topics: Adenosine; Animals; Cell Survival; Cells, Cultured; Chick Embryo; Cyclic AMP; Drug Synergism; Glutamic Acid; L-Lactate Dehydrogenase; Neurons; Neurotoxins; Purinergic P1 Receptor Agonists; Receptor, Adenosine A2A; Receptors, Purinergic P1; Retina; Thioinosine; Time Factors

The rat equilibrative nucleoside transporters rENT1 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR). Structurally, the proteins have a large glycosylated extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, we have generated chimeras between NBMPR-sensitive rENT1 and NBMPR-insensitive rENT2, using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) to identify structural domains of rENT1 responsible for transport inhibition by NBMPR. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 NBMPR-insensitive. Domain swaps within the amino-terminal halves of rENT1 and rENT2 identified two contiguous regions, TMs 3-4 (rENT1 residues 100-171) and TMs 5-6 (rENT1 residues 172-231), as the major sites of NBMPR interaction. Since NBMPR is a nucleoside analogue and functions as a competitive inhibitor of zero-trans nucleoside influx, TMs 3-6 are likely to form parts of the substrate translocation channel.

Topics: Affinity Labels; Animals; Biological Transport; Carrier Proteins; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Oocytes; Peptide Mapping; Purine Nucleosides; Rats; Recombinant Fusion Proteins; Thioinosine; Uridine; Xenopus

Adenosine plays a significant role in various physiological and regulatory processes including coronary vasodilatation. In the current study, a high-affinity adenosine transporter in freshly dissociated porcine coronary smooth muscle (PCSM) cells and cultured human coronary smooth muscle (HCSM) cells was characterized. Kinetic analysis of the transport process revealed a V(max) of 82+/-17 pm/mg protein/min and a K(m) of 4.3+/-2.1 microm for PCSM cells, whereas a K(m) of 4.8 microm and V(max) of 254 pm/mg/min was observed for cultured HCSM. Concentration-dependent inhibition of adenosine uptake by S-(4-nitrobenzyl)-6-thioinosine (NBTI) was observed in both PCSM (IC(50), 0.08 microm) and HCSM (0.1 microm) cells. Both cell types also demonstrate a high-affinity, single binding site for NBTI (PCSM, B(max) 144.8+/-23 fmol/mg protein and K(d) 1.1+/-0.35 nm; HCSM, B(max) 672+/-62 fmol/mg protein and K(d) 0.45+/-0.14 nm). Adenosine uptake in these cells was not affected by extracellular sodium concentration. RT-PCR analysis of mRNA from individually selected PCSM and HCSM cells demonstrated expression of an NBTI-sensitive equilibrative transporter. Smooth muscle cells isolated from porcine brachial and femoral arteries also transported adenosine at levels similar to that of coronaries. These data demonstrate that vascular coronary smooth muscle possess an NBTI-sensitive equilibrative transporter for adenosine which could function in regulation of vasodilation.

Topics: Adenosine; Animals; Carrier Proteins; Cells, Cultured; Coronary Vessels; Female; Humans; Muscle, Smooth, Vascular; Sodium; Swine; Thioinosine; Tumor Cells, Cultured; Vasodilator Agents

The pattern of nucleoside transporter expression in hepatocytes was studied in the developing rat liver.. Hepatocytes isolated from fetuses, neonates and adult rats were used for uridine uptake measurements and concentrative nucleoside transporter (CNT) expression.. Adult hepatocytes showed the highest Na-dependent uridine uptake, but fetal hepatocytes exhibited a significant NBTI-sensitive component of equilibrative Na+-independent transport, which was either negligible or absent in neonatal and adult rat hepatocytes. Low Na+-dependent uridine uptake was associated with low amounts of CNT1 and CNT2 transporter proteins, both with apparent Km values in the low micromolar range. Hepatocyte primary cultures from 20-day-old fetuses showed very low amounts of CNT2 mRNA, and expressed both carrier proteins. Incubation of fetal hepatocytes with dexamethasone and T3 resulted in a significant increase in Na+-dependent uridine uptake and an accumulation of the CNT2 protein and mRNA.. The expression of concentrative nucleoside carriers in hepatocytes from developing rat liver is developmentally regulated. Addition of endocrine factors known to induce differentiation of fetal hepatocytes results in selective up-regulation of CNT2 expression.

Topics: Animals; Animals, Newborn; Biological Transport, Active; Cell Differentiation; Dexamethasone; Embryonic and Fetal Development; Female; Fetus; Gene Expression Regulation, Developmental; Hepatocytes; In Vitro Techniques; Liver; Membrane Transport Modulators; Membrane Transport Proteins; Pregnancy; Rats; Rats, Wistar; RNA, Messenger; Thioinosine; Triiodothyronine; Uridine

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.

Topics: Adenosine; Algorithms; Amino Acids; Animals; Antineoplastic Agents; Biological Transport; Cell Membrane; Cytoplasm; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Equilibrative Nucleoside Transporter 1; Glycosylation; Humans; Immunoblotting; Immunohistochemistry; Membrane Transport Proteins; Protein Structure, Tertiary; Recombinant Proteins; Software; Thioinosine; Xenopus

1. Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5' iodotubercidin (ITU) and 5-amino-5'-deoxyadenosine (NH(2)dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells. 3. Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters. 3. We tested the effects of ITU and NH(2)dAdo on [(3)H]-adenosine uptake and conversion to [(3)H]-adenine nucleotides in the three cell types. NH(2)dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [(3)H]-adenosine uptake and [(3)H]-adenine nucleotide formation at concentrations < or =100 nM in rENT1-C6 cells, while concentrations > or =3 microM were required for C6 or rCNT2-C6 cells. 4. Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells. 5. This study demonstrates that the effects of ITU, but not NH(2)dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.

Topics: Adenine Nucleotides; Adenosine; Adenosine Kinase; Animals; Carrier Proteins; Deoxyadenosines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Membrane Proteins; Membrane Transport Proteins; Nucleoside Transport Proteins; Thioinosine; Tritium; Tubercidin; Tumor Cells, Cultured

The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K(+) channels, most likely Ca(2+)-dependent intermediate-conductance K(+) (I(K)) channels. A(1) but not A(2) receptor antagonists blocked the effects of adenosine. RT-PCR studies showed that A549 cells expressed mRNA for I(K)-1 channels as well as A(1), A(2A), and A(2B) but not A(3) receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of K(+) channels in A549 cells and that hENTs play a crucial role in this process.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine; Affinity Labels; Amiloride; Autocrine Communication; Cell Line; Cell Polarity; Clotrimazole; Diuretics; Epithelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Growth Inhibitors; Humans; Membrane Transport Proteins; Patch-Clamp Techniques; Potassium; Potassium Channels; Quinazolines; Receptors, Purinergic P1; Respiratory Mucosa; Theobromine; Thioinosine; Triazoles; Uridine; Xanthines

1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Carrier Proteins; Extracellular Space; Membrane Transport Proteins; Muscle Contraction; Muscle, Skeletal; Nucleoside Transport Proteins; Rats; Rats, Wistar; Thioinosine

Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome. The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder. Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs). The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1. However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1. NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs. The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

Topics: Adenosine Triphosphate; Apoptosis; Cell Survival; Dose-Response Relationship, Drug; Drug Antagonism; Endothelium, Vascular; Humans; Nucleoside Transport Proteins; Protective Agents; Shiga Toxin; Thioinosine; Umbilical Veins

While a great deal is known about stimuli that can induce the release of adenosine from brain tissue, relatively little is known about the regulation of the basal extracellular concentration of adenosine that is present in the absence of stimulation. Under normal conditions, enough adenosine is present to tonically activate a significant portion of the high-affinity adenosine A1 receptors. The present experiments demonstrated that the estimated basal concentration of extracellular adenosine in rat hippocampal slices maintained at 21 degrees C (430 nM) is approximately twice that at 32 degrees C (220 nM). The sensitivity of presynaptic modulatory adenosine A1 receptors was not significantly different at 21 degrees C or at 32 degrees C. Slices maintained at 21 degrees C also showed a reduced ability to inactivate extracellular adenosine, which reflects a reduction in adenosine transport across cell membranes. This effect appears to be primarily due to a reduction in the function of the equilibrative, dipyridamole-sensitive (ei) adenosine transporter; the nitrobenzylthioinosine-sensitive equilibrative transporter (es transporter) appears to be relatively less affected by temperature than is the ei transporter. These experiments demonstrate that extracellular concentrations of adenosine in the brain are sensitive to temperature, and suggest that some of the neurological effects of hypothermia might be mediated via increased concentrations of adenosine in the extracellular space.

Topics: Adenosine; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Animals; Carrier Proteins; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Excitatory Postsynaptic Potentials; Extracellular Space; Hippocampus; In Vitro Techniques; Male; Membrane Proteins; Rats; Rats, Sprague-Dawley; Temperature; Thioinosine

In leukemic cells exposed to 2-chlorodeoxyadenosine (2-CdA), levels of the nucleoside drug and its phosphate metabolites decay with time in the absence of external 2-CdA; an intrinsic part of this process is the efflux of 2-CdA. The effects of nitrobenzylthioinosine (NBMPR) and of dipyridamole (DPM), both potent inhibitors of es (e, equilibrative; s, sensitive to NBMPR) nucleoside transport processes, were studied in four lines of cultured leukemic lymphoblasts. Suspensions of 2-CdA-loaded cells were diluted 10-fold with 2-CdA-free medium to initiate the cellular 2-CdA decay processes, which followed a biexponential time course. When diluting media contained NBMPR or DPM, intracellular levels of 2-CdA and its metabolites were substantially increased (P < 0.001) compared with cells in media lacking the transport inhibitors, and 2-CdA loss followed a monoexponential time course. As a consequence, the AUCs (area under time-course plots of intracellular 2-CdA and its metabolites) were significantly (P < 0.001) lower in untreated control cells compared to inhibitor-treated cells. These results suggest that nucleoside transport processes contribute to the efflux of 2-CdA from the cultured lymphoblasts. The cytotoxicity of 1-h exposure to 2-CdA of Reh-A2 and CCRF-CEM cells was enhanced three-fold by subsequent exposure to 0.5 microM NBMPR relative to that of control cells subjected to the same manipulations without NBMPR exposure. However, before such a strategy may be considered to have a therapeutic application, careful examination of effects in normal lymphocytes and ex vivo leukemic lymphoblasts must first be undertaken. Leukemia (2000) 14, 52-60.

Topics: Antineoplastic Agents; Biological Transport; Chromatography, High Pressure Liquid; Cladribine; Drug Synergism; Humans; Nucleosides; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioinosine; Tumor Cells, Cultured

WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.

Topics: Aged; Antineoplastic Agents; Biological Transport, Active; Bryostatins; Carrier Proteins; Cladribine; Deoxycytidine Kinase; Dipyridamole; Down-Regulation; Drug Resistance, Neoplasm; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Male; Membrane Proteins; Nucleoside Transport Proteins; Phosphorylation; Thioinosine; Tumor Cells, Cultured; Vidarabine

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.

Topics: Biological Transport; Carrier Proteins; Cell Line; Cytidine; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Genetic Complementation Test; Guanosine; Humans; Inosine; Kinetics; Membrane Proteins; Nucleosides; Thioinosine

Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of the es nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive ei transporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG-transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR-P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells.

Topics: 3T3 Cells; Animals; Bone Marrow Cells; Carrier Proteins; Cell Death; DNA, Complementary; Drug Resistance; Drug Synergism; Equilibrative-Nucleoside Transporter 2; Female; Folic Acid Antagonists; Gene Transfer Techniques; Genetic Vectors; Glutamates; Guanine; Hematopoietic Stem Cells; Membrane Proteins; Mice; Mice, Inbred C57BL; Pemetrexed; Quinazolines; Retroviridae; Thioinosine; Thiophenes

Ageing is a multifactorial, inevitable event of life span, which affects neurotransmission in the CNS. Since adenosine is a major neuromodulator of the synaptic activity, it was of interest to investigate the possible modification of the adenosinergic system in the brain during ageing. Using "in vitro" quantitative autoradiography and the radioactive ligands [(3)H]Cyclohexyladenosine and [(3)H]Nitrobenzylthioinosine, we have studied the distribution of A1 adenosine receptors and adenosine uptake sites in the aged mice (26 months) compared to the young ones (3 months). Our results showed a widespread reduction in A1 receptor binding in the aged animals, which was brain area-specific, occurring in areas where adenosine plays a significant neuromodulatory role such as the hippocampus, cortex, basal ganglia, and thalamus. Interestingly, the significant reduction in NBI-sensitive adenosine uptake sites was restricted to few areas of the aged brain, mainly in thalamic nuclei. Since the alterations in the density of A1 receptors and adenosine uptake sites showed no regional correlation and since no significant changes in either neuronal or glial cell number are observed, at least in hippocampus and cortex in this mouse strain during ageing, our findings could be explained by a selective age-dependent reduction of these adenosinergic components rather than by a general neuronal cell degeneration. As adenosine depresses electrical activity in hippocampus, a downregulation of adenosinergic function could probably be related to enhanced excitability seen in hippocampal neurons of the CA1 subregion and dentate gyrus of aged animals.

Topics: Adenosine; Aging; Animals; Autoradiography; Binding Sites; Brain; Mice; Mice, Inbred C57BL; Radioligand Assay; Receptors, Purinergic P1; Thioinosine

To clone and sequence the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (es) from the human small intestine and to examine the capacities of nucleosides and nucleoside analogs to inhibit the uptake of uridine by this transporter.. Using PCR, es was cloned from a cDNA library of the human small intestine. The uptake of 3H-uridine (10 microM) by the recombinant es, expressed in Xenopus oocytes, was measured in the presence (2 mM) and absence of nucleosides and nucleoside analogs.. The amino acid sequence of this es transporter was identical to that of the human placental es transporter. Uptake of 3H-uridine by this es transporter was inhibitable by 1 microM NBMPR. Removal of the oxygen from the 3' position or from both the 2' and 3' positions, but not from 2' or 5' position, resulted in a partial or total loss of the capacity of the nucleosides to inhibit 3H-uridine uptake. No modifications of the adenosine base or of the uridine base (except for 3 and 6 positions on uracil) affected nucleoside inhibitory capacity.. The es transporters of the human intestine and placenta are identical in their amino acid sequences. Moreover, the inhibitory profiles of various nucleoside analogs in inhibiting the uptake of uridine by the intestinal es transporter are similar to those obtained with the as-yet-uncloned human erythrocyte es transporter. Collectively, these findings suggest that the es transporter does not appear to be functionally variant in the human placenta, small intestine or erythrocytes.

Topics: Animals; Carrier Proteins; Gene Library; Humans; Intestine, Small; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; Thioinosine; Uridine; Xenopus laevis

The equilibrative nucleoside transporters of mammalian cells play an important role in the regulation of extracellular adenosine concentrations, and inhibition of these transporters potentiates the biological effects of adenosine. Two subtypes of equilibrative transporters have been defined by their differential sensitivities to inhibition by nitrobenzylthioinosine (NBMPR; es/ENT1, sensitive; ei/ENT2, insensitive). In addition, significant species differences have been noted in es/ENT1 transporter affinity for a subset of inhibitors including draflazine and dipyridamole. Draflazine and a series of 15 chemically related compounds were compared for their abilities to: (a) inhibit the binding of [3H]NBMPR to the es/ENT1 transporter in mouse Ehrlich cell and human erythrocyte membranes, and (b) inhibit the es/ENT1 and ei/ENT2 transporter-mediated uptake of [3H]uridine in Ehrlich cells. Compounds within this series represented over a 1000-fold range of affinities for the es/ENT1 and ei/ENT2 transporters with subtype selectivities (ENT1/ENT2) ranging from 370 for R70527 to 0.17 for soluflazine. Five other analogues were identified, in addition to soluflazine, that had significantly higher affinity for the ei/ENT2 transporter compared with es/ENT1. Structure activity analyses of these data identified the requirement of a hydrophobic group connected to a 2-aminocarbonyl piperazine by a 5-carbon chain for high-affinity interactions with es/ENT1. This hydrophobic moiety was not as important for ei/ENT2 affinity and, in contrast to es/ENT1, a shorter alkyl chain enhanced binding to ei/ENT2. These draflazine analogues also varied in their differential affinities for mouse vs. human es/ENT1 transporters, and the degree of species discrimination was strongly dependent on the position of the aminocarbonyl group on the piperazine ring. This information, combined with structural data derived from molecular studies with ENT1 and ENT2 recombinant proteins, should guide further development of subtype-selective inhibitors of the equilibrative nucleoside transporters.

Topics: Animals; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Erythrocytes; Humans; In Vitro Techniques; Membrane Proteins; Mice; Piperazines; Radioligand Assay; Species Specificity; Thioinosine; Tumor Cells, Cultured; Uridine

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Benzodiazepinones; Biological Transport; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Guanosine; Hexobendine; Hydrogen-Ion Concentration; Inosine; Isoquinolines; Kinetics; Male; Mitochondria; Osmolar Concentration; Phenylisopropyladenosine; Rats; Rats, Sprague-Dawley; Sodium; Subcellular Fractions; Temperature; Testis; Thioinosine; Thionucleosides; Time Factors; Tritium; Uridine

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

Topics: Adenine; Amino Acid Sequence; Animals; Antimalarials; Biological Transport; Blotting, Southern; Carrier Proteins; Cations; Cell Membrane; Cloning, Molecular; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins; Nucleosides; Phylogeny; Piperazines; Plasmodium falciparum; Platelet Aggregation Inhibitors; Protein Structure, Secondary; Protozoan Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Thioinosine; Time Factors; Uridine; Vasodilator Agents; Xenopus

The effect of repeated oral administration of ATP on purine transport and metabolism was investigated in rats. An increased ability of the gut to capture intraluminal purine nucleosides and to export ATP and nucleosides toward portal bloodstream was observed in rats after 30 days of treatment with 5 mg/kg/day ATP. This was accompanied in erythrocytes by an increased transport of adenosine rapidly transformed into ATP, which in turn was exported toward extracellular fluid. However, these metabolic changes were associated with a paradoxical and progressive diminution of plasma ATP level below that found in control rats and that was not strictly dependent on the ATP dose administered, whereas plasma adenosine concentration remained unchanged. This diminution likely resulted from an increased ectonucleotidase activity, suggesting that the chronic administration of ATP seems to induce a progressive adaptation of purine metabolism. This adaptive response to free purine supplementation affects both intracellular metabolism and purine exchange between intracellular and extracellular compartments. This modification of free purine turnover and delivery may affect physiological parameters under the control of P(1) and P(2) purinoceptors described in different experimental models.

Topics: Adenosine; Adenosine Triphosphate; Administration, Oral; Animals; Biological Transport; Dose-Response Relationship, Drug; Male; Purines; Rats; Rats, Sprague-Dawley; Thioinosine

We recently suggested that, in muscular dystrophies, the excessive accumulation of adenosine as a result of an altered purine metabolism may contribute to progressive functional deterioration and muscle cell death. To verify this hypothesis, we have taken advantage of C2C12 myoblastic cells, which can be differentiated in vitro into multinucleated cells (myotubes). Exposure of both proliferating myoblasts and differentiated myotubes to adenosine or its metabolically-stable analog, 2-chloro-adenosine, resulted in apoptotic cell death and myotube disruption. Cytotoxicity by either nucleoside did not depend upon extracellular adenosine receptors, but, at least in part, by entry into cells via the membrane nitro-benzyl-thio-inosine-sensitive transporter. The adenosine kinase inhibitor, 5-iodotubercidin, prevented 2-chloro-adenosine-induced (but not adenosine-induced) effects, suggesting that an intracellular phosphorylation/activation reaction plays a key role in 2-chloro-adenosine-mediated cytotoxicity. Conversely, adenosine cytotoxicity was aggravated by the addition of homocysteine, suggesting that adenosine effects may be due to the accumulation of S-adenosyl-homocysteine, which blocks intracellular methylation-dependent reactions. Both nucleosides markedly disrupted the myotube structure via an effect on the actin cytoskeleton; however, also for myotubes, there were marked differences in the morphological alterations induced by these two nucleosides. These results show that adenosine and 2-chloro-adenosine induce apoptosis of myogenic cells via completely different metabolic pathways, and are consistent with the hypothesis that adenosine accumulation in dystrophic muscles may represent a novel pathogenetic pathway in muscle diseases.

Topics: 2-Chloroadenosine; Acetylcysteine; Actin Cytoskeleton; Adenosine; Adenosine Kinase; Animals; Apoptosis; Cell Adhesion; Cell Line; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Homocysteine; Intracellular Fluid; Mice; Microscopy, Electron, Scanning; Muscle, Skeletal; Purinergic P1 Receptor Antagonists; Reactive Oxygen Species; Thioinosine; Tubercidin

Adenosine levels increase in brain during cerebral ischemia, and adenosine has receptor-mediated neuroprotective effects. This study was performed to test the hypothesis that nitrobenzylthioinosine (NBMPR), a selective and potent inhibitor of one adenosine transporter subtype termed ENT1, or es, can protect against ischemic neuronal injury by enhancing adenosine levels and potentiating adenosine receptor-mediated effects, including attenuation of the cellular production and release of tumor necrosis factor-alpha (TNF-alpha). In rats, the phosphorylated prodrug form of NBMPR, NBMPR-phosphate, or saline was administered by intracerebroventricular injection 30 min before forebrain ischemia. Seven days following the ischemic episode, rats were killed, and neuronal damage in the CA1 region of the hippocampus was assessed. The number of pyramidal neurons was significantly (p < 0.001) greater in the NBMPR-P treatment group. A trend toward protection was still evident at 28 days postreperfusion. Adenosine increased significantly during ischemia to levels eight- to 85-fold above basal. NBMPR-P treatment did not cause statistically significant increases in ischemic adenosine levels; however, this treatment tended to increase adenosine levels in all brain regions at 7 min postreperfusion. Ischemia-induced expression of TNF-alpha was not altered by NBMPR-P treatment, and the nonselective adenosine receptor antagonist 8-(p-sulfophenyl) theophylline did not abolish the neuroprotective effects of NBMPR-P treatment. These data indicate that NBMPR can protect CA1 pyramidal neurons from ischemic death without statistically significant effects on adenosine levels or adenosine receptor-mediated inhibition of the proinflammatory cytokine TNF-alpha.

Topics: Adenosine; Affinity Labels; Animals; Cerebral Ventricles; Gene Expression Regulation; Injections, Intraventricular; Ischemic Attack, Transient; Male; Neurons; Prodrugs; Prosencephalon; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion; Thioinosine; Thionucleotides; Transcription, Genetic; Tumor Necrosis Factor-alpha

Adenosine transport was characterized in human umbilical artery smooth muscle cells isolated from non-diabetic and diabetic pregnant subjects. Transport of adenosine was mediated by a Na+-independent transport system inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) in both cell types. Diabetes increased adenosine transport, an effect that was associated with a higher maximal velocity (Vmax) for NBMPR-sensitive (es) saturable nucleoside transport (18 +/- 2 vs. 61 +/- 3 pmol (microgram protein)-1 min-1, P < 0.05) and the maximal number of binding sites (Bmax) for specific [3H]NBMPR binding (74 +/- 4 vs. 156 +/- 10 pmol (microgram protein)-1, P < 0.05), with no significant changes in the Michaelis-Menten (Km) and dissociation (Kd) constants, respectively. Adenosine transport was unaltered by inhibition of nitric oxide (NO) synthase (with 100 microM NG-nitro-L-arginine methyl ester, L-NAME) or protein synthesis (with 1 microM cycloheximide), but was increased by inhibition of adenylyl cyclase activity (with 100 microM, SQ-22536) in non-diabetic cells. Diabetes-induced adenosine transport was blocked by L-NAME and associated with an increase in L-[3H]citrulline formation from L-[3H]arginine and intracellular cGMP, but with a decrease in intracellular cAMP compared with non-diabetic cells. Expression of inducible NO synthase (iNOS) was unaltered by diabetes. Dibutyryl cGMP (dbcGMP) increased, but dibutyryl cAMP (dbcAMP) decreased, adenosine transport in non-diabetic cells. dbcGMP or the NO donor S-nitrosoacetylpenicillamine (SNAP, 100 microM) did not alter the diabetes-elevated adenosine transport. However, activation of adenylyl cyclase with forskolin (1 microM), directly or after incubation of cells with dbcAMP, inhibited adenosine transport in both cell types. Our findings provide the first evidence that adenosine transport in human umbilical artery smooth muscle cells is mediated by the NBMPR-sensitive transport system es, and that its activity is upregulated by gestational diabetes.

Topics: Adenosine; Biological Transport; Cells, Cultured; Cyclic AMP; Cyclic GMP; Diabetes, Gestational; Female; Humans; Intracellular Membranes; Kinetics; Muscle, Smooth, Vascular; Nitric Oxide; Nucleotides, Cyclic; Pregnancy; Reference Values; Thioinosine; Umbilical Arteries

Previous studies indicated that the peptide neurotensin (NT) stimulates Cl(-) secretion in animal small intestinal mucosa in vitro. In this study, we investigated whether NT causes Cl(-) secretion in human colonic mucosa and examined the mechanism of this response.. Human mucosal preparations mounted in Ussing chambers were exposed to NT. Drugs for pharmacologic characterization of NT-induced responses were applied 30 minutes before NT.. Serosal, but not luminal, administration of NT (10(-8) to 10(-6) mol/L) induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive short-circuit current (Isc) increase that was inhibited by the specific nonpeptide NT receptor antagonists SR 48692 and SR 142948A, the neuronal blocker tetrodotoxin, and the prostaglandin synthesis inhibitor indomethacin. The mast cell stabilizer lodoxamide and the histamine 1 and 2 receptor antagonists pyrilamine and ranitidine, respectively, did not significantly alter NT-induced Isc increase. In contrast, the adenosine receptor 1 and 2 antagonists inhibited this secretory response, whereas the adenosine uptake inhibitors S-(4-nitrobenzyl)-6-thioguanosine and S-(4-nitrobenzyl)-6-thioinosine and the adenosine deaminase inhibitor deoxycoformycin potentiated NT-induced Isc increase. Serosal adenosine induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive Isc increase.. NT stimulates chloride secretion in human colon by a pathway(s) involving mucosal nerves, adenosine, and prostaglandins.

Topics: Adamantane; Adenosine; Adenosine Deaminase Inhibitors; Affinity Labels; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Chlorides; Colon; Electrophysiology; Enteric Nervous System; Enzyme Inhibitors; Guanosine; Histamine; Humans; Imidazoles; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Mast Cells; Membrane Potentials; Neurotensin; Pentostatin; Pyrazoles; Quinolines; Receptors, Neurotensin; Tetrodotoxin; Thioinosine; Thionucleosides

Cilostazol (Pletal), a quinolinone derivative with a cyclic nucleotide phosphodiesterase type 3 (PDE3) inhibitory activity, was recently approved by the Food and Drug Administration for treatment of symptoms of intermittent claudication (IC). However, the underlying mechanisms of action are not entirely clear. In this study, we showed that cilostazol inhibited adenosine uptake into cardiac ventricular myocytes, coronary artery smooth muscle, and endothelial cells with a median effective concentration (EC50) approximately 10 microM. In vivo, cilostazol increased cardiac interstitial adenosine levels after a 2-min ischemia in rabbit hearts (329 +/- 92% increase vs. 102 +/- 29% ischemia alone). The combination of cilostazol and 2-min ischemia reduced infarction from subsequent 30-min regional ischemia and 3 h of reperfusion (infarct size was 18 +/- 4% vs. 53 +/- 3% in the hearts with 2-min ischemia alone or 48 +/- 2% in the hearts treated with cilostazol alone). In contrast, milrinone had no effect on either adenosine uptake or interstitial adenosine levels. These data show that cilostazol, unlike milrinone, inhibits adenosine uptake, and thus potentiates adenosine accumulation from a 2-min ischemia. Future studies are needed to investigate the role of adenosine in the treatment of IC by cilostazol.

Topics: Adenosine; Animals; Blood Pressure; Cilostazol; Dose-Response Relationship, Drug; Heart Rate; Humans; Intermittent Claudication; Male; Milrinone; Myocardial Ischemia; Pentoxifylline; Phosphodiesterase Inhibitors; Rabbits; Tetrazoles; Thioinosine

The present study was designed to assess whether adenosine A(2a) receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A(1) receptors were characterised in brain membranes and on slide-mounted sections. The B(MAX) for [(3)H]nitrobenzylthioinosine ([(3)H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560+/-52 fmol/mg protein, n=5, P<0.05, Kruskal-Wallis ANOVA) compared to wildtype (1239+/-213 fmol/mg protein) and heterozygous mice (1300+/-558 fmol/mg protein). Quantitative autoradiography data indicated that [(3)H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly (P<0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [(3)H]1, 3-dipropyl-8-cyclopentylxanthine ([(3)H]DPCPX) (adenosine A(1) receptor density) binding increased significantly (P<0.05, Kruskal-Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [(3)H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [(3)H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [(3)H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A(2a) receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.

Topics: Affinity Labels; Animals; Binding Sites; Brain; Brain Chemistry; Carrier Proteins; Cell Membrane; Genotype; Male; Mice; Mice, Knockout; Neurons; Radioligand Assay; Receptor, Adenosine A2A; Receptors, Purinergic P1; Thioinosine; Tritium; Xanthines

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.

Topics: Adenine; Adenosine; Adenosine Triphosphate; AMP Deaminase; Animals; Carrier Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Equilibrative Nucleoside Transport Proteins; Equilibrative-Nucleoside Transporter 2; Formycins; Glioma; Hypoxanthine; Inosine; Iodoacetates; Nucleosides; Phosphodiesterase Inhibitors; Purines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Sodium; Sodium Cyanide; Thioinosine; Tumor Cells, Cultured

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Animals; Biological Transport; Carrier Proteins; Coronary Vessels; Dinoprost; Dipyridamole; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Female; In Vitro Techniques; Muscle, Smooth, Vascular; Purinergic P1 Receptor Agonists; Substrate Specificity; Swine; Thioinosine; Vasoconstriction; Vasodilation; Vasodilator Agents

The existence of adenosine A1 receptors and adenosine transporters in the central nervous system has been well demonstrated, although their possible modulation by hormones and/or exogenous drugs is poorly understood. To further analyze these modulatory mechanisms, the effects of prolactin and cyclosporine (CyA) on adenosine A1 receptors and transporters were analyzed in the central nervous system. For this purpose the number and affinity of adenosine A1 receptors were measured using the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and the transporters with the high affinity ligand nitrobenzylthioinosine (NBTI). This procedure was carried out in hyperprolactinemic and control male rats treated with CyA or its vehicle for 8 days. As expected, pituitary grafting increased plasma prolactin levels (p<0.01). CyA treatment reduced but did not normalize (p<0.05) this parameter in hyperprolactinemic rats and did not modify circulating prolactin in control animals. Both hyperprolactinemia and CyA treatment reduced the number of adenosine transporters by 70% and by 40% the number of A1 receptors. The Kd for transporters was also reduced in all experimental groups. Hyperprolactinemia increased the affinity of A1 receptors (p<0.01) and CyA treatment did not further modify this parameter. These data demonstrated that prolactin and CyA influence adenosine transporters and A1 receptors at the central nervous system and suggest the existence of an interaction between prolactin and CyA may be operating to modulate these processes.

Topics: Adenosine; Animals; Brain; Carrier Proteins; Cyclosporine; Female; Hyperprolactinemia; Male; Membrane Proteins; Nucleoside Transport Proteins; Pituitary Gland, Anterior; Prolactin; Purinergic P1 Receptor Antagonists; Radioligand Assay; Rats; Rats, Wistar; Receptors, Purinergic P1; Thioinosine; Xanthines

The aim of this study was to characterise the receptor(s) mediating relaxations to adenosine and its analogues in the hamster isolated aorta. Adenosine relaxed the aorta but there was no significant difference between pIC20 values in the absence and presence of 8-sulphophenyltheophylline (8-SPT, 50 microM), although there was a small right-shift (approximately threefold) of the lower portion of the curve in the presence of 8-SPT. However, in the presence of the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1 microM), curves to adenosine were left-shifted by approximately 100-fold and an apparent pK(B) for 8-SPT of 5.79+/-0.05 was obtained. Likewise, 5'-N-ethylcarboxamidoadenosine (NECA) relaxed the aorta but curves were biphasic. The first phase of the curve was blocked by 8-SPT (10-100 microM, pA2 = 5.75+/-0.14) and the A2A-selective antagonist 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylaminolethyl) phenol (ZM 241385, 3 nM-1 microM, pK(B)=9.17+/-0.10). Similarly, the A2A-selective agonist 2-[p)-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxam idoadenosine (CGS 21680) relaxed the tissues but curves were biphasic and the first phase was again blocked by ZM 241385 (10 nM, apparent pK(B)=9.06+/-0.34). In contrast, relaxations to N6-R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), 2-chloroadenosine (2-CADO) and N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were not blocked by 8-SPT (50 microM). Responses to IB-MECA were also not blocked by the A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihyd ropyridine-3,5-dicarboxylate (MRS 1191, 30 microM). The asymptote of the first phase of curves to NECA was markedly reduced (and in some preparations the first phase was completely abolished) both in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), and in the absence of endothelium. Likewise, the first phase of curves to CGS 21680 was abolished both in the presence of L-NAME (0.1 mM) and in the absence of endothelium. In contrast, there were only relatively small shifts to the right of curves to adenosine and the other analogues in the presence of L-NAME or the absence of endothelium (between three- and fivefold). The data suggest the presence of A2A receptors which are located on the endothelium and mediate release of nitric oxide. These receptors are activated by NECA, CGS 21680 and adenosine (in the presence of uptake blockade). The resi

Topics: 2-Chloroadenosine; Adenosine; Animals; Aorta; Cricetinae; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Mesocricetus; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phenethylamines; Receptors, Purinergic P1; Theophylline; Thioinosine; Vasodilation

The effects of elevated D-glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies. Elevated D-glucose resulted in a time- (8-12 h) and concentration-dependent (half-maximal at 10+/-2 mM) inhibition of adenosine transport, which was associated with a reduction in the Vmax for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside with no significant change in Km. d-Fructose (25 mM), 2-deoxy-D-glucose (25 mM) or D-mannitol (20 mM) had no effect on adenosine transport. Adenosine transport was inhibited following incubation of cells with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min to 24 h). D-Glucose-induced inhibition of transport was abolished by calphostin C (100 nM, an inhibitor of PKC), and was not further reduced by PMA. Increased PKC activity in the membrane (particulate) fraction of endothelial cells exposed to D-glucose or PMA was blocked by calphostin C but was unaffected by NG-nitro-L-arginine methyl ester (L-NAME; 100 microM, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 microM, an inhibitor of mitogen-activated protein kinase kinase 1). D-Glucose and PMA increased endothelial NOS (eNOS) activity, which was prevented by calphostin C or omission of extracellular Ca2+ and unaffected by PD-98059. Adenosine transport was inhibited by S-nitroso-N-acetyl-l, d-penicillamine (SNAP; 100 microM, an NO donor) but was increased in cells incubated with L-NAME. The effect of SNAP on adenosine transport was abolished by PD-98059. Phosphorylation of mitogen-activated protein kinases p44mapk (ERK1) and p42mapk (ERK2) was increased in endothelial cells exposed to elevated D-glucose (25 mM for 30 min to 24 h) and the NO donor SNAP (100 microM, 30 min). The effect of D-glucose was blocked by PD-98059 or L-NAME, which also prevented the inhibition of adenosine transport mediated by elevated D-glucose. Our findings provide evidence that D-glucose inhibits adenosine transport in human fetal endothelial cells by a mechanism that involves activation of PKC, leading to increased NO levels and p42-p44mapk phosphorylation. Thus, the biological actions of adenosine appear to be altered under conditions of sustained hyperglycaemia.

Topics: Adenosine; Biological Transport; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Fetus; Flavonoids; Glucose; Humans; Mitogen-Activated Protein Kinases; Naphthalenes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Penicillamine; Protein Kinase C; S-Nitroso-N-Acetylpenicillamine; Tetradecanoylphorbol Acetate; Thioinosine

Adenosine is a biologically active metabolite that evokes numerous potent actions in the heart and other tissues. A better understanding of the regulation of the local adenosine concentration seems mandatory to permit specific manipulation of the adenosine tissue concentration. To achieve this a combined experimental and model analysis approach was developed. Experiments carried out in isolated perfused guinea pig hearts, coronary endothelial, and smooth muscle cells and data were analyzed with an axially distributed, 4-region mathematical model of adenosine metabolism and transport. This approach permitted us to obtain a comprehensive parameter set that adequately described cardiac adenosine metabolism. The parameter values that gave the optimal fits to experimental results indicated that adenosine production was largest in the cytosol, while extracellular adenosine production accounted for approximately 8% of total cardiac adenosine production. However, despite the much higher intracellular rate of adenosine production, the concentration gradient of adenosine across cell membranes was directed toward the cytosol under physiological conditions, i.e., when the cytosolic adenosine concentration was low. This was due to the high rate of intracellular adenosine removal which exceeded intracellular production. The endothelial region contributed approximately 5% to total cardiac adenosine production. Despite this small contribution endothelial cells may effectively control the vascular adenosine concentration over a wide concentration range (5-500 nM). In conclusion, a combination of experimental and modeling approaches may provide unique insights into capillary-tissue exchange and metabolism of adenosine. In the future this may reveal realistic concentration-effect relationships for adenosine in the heart. These achievements seem critical in order to design strategies which permit a specific manipulation of substrates with high turnover rates in biological tissues.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Binding, Competitive; Biological Transport; Blood Flow Velocity; Cell Membrane; Cells, Cultured; Coronary Circulation; Coronary Vessels; Endothelium, Vascular; Enzyme Inhibitors; Guinea Pigs; Homocysteine; In Vitro Techniques; Models, Cardiovascular; Muscle, Smooth, Vascular; Myocardium; S-Adenosylhomocysteine; Thioinosine

3-Deazaadenosine (DZA), one of the potent inhibitors of S-adenosylhomocysteine hydrolase, is known to possess several biological properties including an induction of apoptosis. To evaluate a possibility that DZA may be utilized for the treatment of human leukemia, we studied molecular events of cell death induced by DZA in human leukemia HL-60 and U-937 cells. DZA induced a specific cleavage of poly ADP-ribose polymerase (PARP) and an activation of the cysteine protease caspase-3/CPP32 which is known to cleave PARP. DZA-mediated nuclear DNA-fragmentation was completely blocked in the presence of a universal inhibitor of caspases (z-VAD-fmk) or the specific inhibitor of caspase-3 (z-DEVD-fmk) unlike of cycloheximide (CHX). DNA fragmentation was preceded by the lowering of c-myc mRNA in the DZA treated cells. In addition, DZA-induced apoptosis was blocked by pretreatment with adenosine transporter inhibitors such as nitrobenzylthioinosine (NBTI) and dipyridamole (DPD). Taken together, these results demonstrate that DZA-induced apoptosis initiated through an active transport of DZA into human leukemia cells, is dependent on the caspase-3-like activity without de novo synthesis of proteins and possibly involves c-myc down-regulation.

Topics: Adenosine; Apoptosis; Biological Transport, Active; Carrier Proteins; Caspase 3; Caspases; Down-Regulation; Enzyme Activation; Genes, myc; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Thioinosine; Transcription Factors; Tubercidin; U937 Cells

This study assessed the cardioprotective effects of inhibitors of adenosine metabolism in an isolated perfused rat heart model. Specifically, we studied the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine and the selective nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine, in terms of their potential to enhance protection when added to Bretschneider's cardioplegic solution.. Rat hearts were infused for 5 min with Krebs-Henseleit buffer solution (group 1), Bretschneider's cardioplegic solution (group 2), Bretschneider's cardioplegic solution with the addition of 25 microM erythro-9-(2-hydroxy-3-nonyl)-adenine and 5 microM S-(p-nitrobenzyl)-6-thioinosine (group 3), and Bretschneider's cardioplegic solution with the addtion of 25 microM erythro-9-(2-hydroxy-3-nonyl)-adenine only (group 4). After cardioplegic arrest and 45 min of ischemic storage at 25 degrees C, the functional recovery of the hearts was tested during 15 min of Langendorff reperfusion and then 45 min of working heart reperfusion.. In relation to the cardioprotective effects of Bretschneider's cardioplegic solution alone, we observed an improved recovery of hemodynamic function of the hearts with the addition of both erythro-9-(2-hydroxy-3-nonyl)-adenine and S-(p-nitrobenzyl)-6-thioinosine. However, the myocardial adenosine triphosphate (ATP) concentration remained unchanged. Bradycardia observed under the addition of erythro-9-(2-hydroxy-3-nonyl)-adenine alone was prevented by the addition of S-(p-nitrobenzyl)-6-thioinosine.. A combination of both substances may be tested further for cardiac preservation, as it might improve the recovery from ischemia at moderate temperatures.

Topics: Adenine; Adenosine Deaminase; Affinity Labels; Animals; Cardioplegic Solutions; Disease Models, Animal; Enzyme Inhibitors; Glucose; In Vitro Techniques; Male; Mannitol; Myocardial Reperfusion Injury; Myocardium; Potassium Chloride; Procaine; Rats; Rats, Sprague-Dawley; Thioinosine

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.

Topics: Adenosine; Animals; Carrier Proteins; Diazepam; Dinucleoside Phosphates; Dipyridamole; Dose-Response Relationship, Drug; Female; Heart; Heart Atria; Muscle Relaxation; Phenethylamines; Pulmonary Artery; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptor, Adenosine A2A; Receptors, Purinergic P1; Thioinosine; Triazines; Triazoles; Verapamil

The aim of this study was to determine whether endogenous adenosine has antiarrhythmic effects on ischemia-induced ventricular tachyarrhythmias. We therefore modulated the effect of endogenous adenosine in isolated rat hearts using four different approaches. First, interstitial adenosine was elevated by metabolic inhibition with either EHNA (erythro-9-(2-hydroxy-3-nonly)adenine) or acadesine [5-amino-1-beta-D-imidazole-4-carboxamide). Second, cardiac effects of A1 adenosine receptors were allosterically enhanced with PD81,723 (2-amino-4,5-dimethyl-3-thienyl)[3-(trifluoromethyl)phenyl]-methanone . Third, endogenous adenosine release was suppressed with NBMPR (S-(4-nitrobenzyl)-6-thioinosine), and fourth, adenosine receptor subtypes were blocked with antagonists of different selectivity. Regional ischemia, induced by coronary artery ligation, caused ventricular fibrillation of a reproducible kind in about 20% of untreated hearts with a low calcium concentration in the perfusion medium (0.80 mmol/l CaCl2) and in about 75% with high calcium (1.85 mmol/l) within an observation period of 30 min. At high calcium, EHNA (1 and 10 micromol/l) and acadesine (500 micromol/l) suppressed the occurrence of ventricular fibrillation from 68% (controls) to 47%, 33% and 38%, respectively. Conversely, PD81,723 (10 micromol/l) did not influence the occurrence of ventricular fibrillation. At low calcium, NBMPR (0.1 and 1 micromol/l) resulted in a concentration-dependent rise of ventricular fibrillation from 13% (controls) to 40% and 57%, respectively. The adenosine receptor antagonists theophylline (100 micromol/l), XAC (Xanthine Amine Congener; 1 micromol/l) and 8-PT (8-phenyltheophylline; 1 micromol/l) caused a rise in the occurrence of ventricular fibrillation from 25%, 15% and 18% (controls) to 57%, 39% and 44%, respectively, and the selective A2a receptors antagonist CSC (8-(3-chlorostyryl)caffeine; 5 micromol/l) from 20% to 56%. Conversely, the selective A1 receptor blocker DPCPX (8-cyclopentyl-1,3-dipropyl-xanthine; 1 micromol/l) was ineffective. NBMPR or EHNA concentration-dependent suppressed or increased ischemia-induced adenosine overflow, respectively, in a concentration-dependent manner, whereas the adenosine receptor antagonists did not influence adenosine overflow. We conclude that endogenous adenosine is an antiarrhythmic mediator accumulating in acute ischemic myocardium to a level which effectively decreases the occurrence of ventricular fibrillation by an A2

Topics: Adenine; Adenosine; Aminoimidazole Carboxamide; Animals; Caffeine; Creatine Kinase; Electrocardiography; Enzyme Inhibitors; Hypoxanthine; Inosine; Male; Myocardial Ischemia; Myocardium; Perfusion; Rats; Rats, Wistar; Reperfusion Injury; Ribonucleosides; Theophylline; Thioinosine; Thiophenes; Time Factors; Ventricular Fibrillation; Xanthines

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of [3H]NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10. 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0. 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0. 04)x10(8) M-1.min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

Topics: Base Sequence; Biological Transport; Carrier Proteins; Dilazep; Dipyridamole; DNA Primers; Equilibrative Nucleoside Transporter 1; Genetic Complementation Test; Glycosylation; Humans; Kinetics; Membrane Proteins; Proteolipids; Recombinant Proteins; RNA, Messenger; Saccharomyces cerevisiae; Thioinosine; Thymidine

Adenosine levels present in the interstitial fluid and coronary effluent of the aged heart exceed those of the young adult heart. The present study investigated mechanisms in the Fischer 344 rat heart which may be responsible for the observed differences. (1) Total production of adenosine was determined in isolated perfused hearts by measuring coronary effluent adenosine content while inhibiting adenosine deamination and rephosphorylation with erythrohydroxy-nonyladenosine (EHNA) and iodotubercidin (ITC), respectively. Total adenosine production was similar in both young (3-4 month) and aged (20-21 month) hearts at 31.8 +/- 6.6 and 38.4 +/- 3.3 nmol/min/g dry wt, respectively. However, stimulation with the beta-adrenergic agent, isoproterenol, elicited a significantly greater increase in adenosine production in the young vs. aged heart. (2) Adenosine transport was evaluated in isolated perfused hearts by determining 14C uptake by the myocardium after 20 min of 14C-adenosine perfusion. Adenosine uptake in the agent-free heart was found to be decreased 17 to 25% in aged compared to young adult hearts. (3) Adenosine transport characteristics were determined with nitrobenzylthioinosine saturation-binding studies in ventricular membrane preparations. The Bmax values were significantly lower in aged than young adult hearts (140.2 +/- 1.5 fmol/mg and 191.9 +/- 2.3 fmol/mg in aged and young hearts, respectively) indicating a decreased number of transporter sites in the aged heart. However, the values for Kd were decreased with aging, suggesting an increase in the affinity of the transporter for adenosine in the aged vs. young adult heart. (4) The activities and kinetics of adenosine kinase were determined in homogenates of aged and young adult ventricular myocardium. No statistical difference was found between the two activities. Taken together these results suggest that increased interstitial adenosine levels in the aged heart result from decreased uptake of adenosine by the ventricular myocardium.

Topics: Adenosine; Adenosine Kinase; Aging; Animals; Carbon Radioisotopes; Heart; Inosine; Male; Myocardial Contraction; Myocardium; Rats; Rats, Inbred F344; Receptors, Adrenergic, beta; Thioinosine

Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions.. In isolated, isovolumically working guinea pig hearts (n=50), the release rate of adenosine and accumulation of S-adenosylhomocysteine (after 20 minutes of 200 micromol/L homocysteine), a measure of the free cytosolic adenosine concentration, were determined in the absence and presence of specific and powerful blockers of adenosine membrane transport (nitrobenzylthioinosine 1 micromol/L), adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5 micromol/L), and adenosine kinase (iodotubericidine 10 micromol/L). Data analysis with a distributed multicompartment model revealed a total cardiac adenosine production rate of 2294 pmol. min-1. g-1, of which 8% was produced in the extracellular region. Because of a high rate of intracellular metabolism, however, 70.3% of extracellularly produced adenosine was taken up into cellular regions, an effect that was effectively eliminated by membrane transport block. The resulting approximately 2.8-fold increase of the interstitial adenosine concentration evoked near-maximal coronary dilation.. We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Biological Transport; Bradykinin; Coronary Circulation; Cytosol; Depression, Chemical; Dipyridamole; Drug Synergism; Enzyme Inhibitors; Extracellular Space; Guinea Pigs; Heart; Models, Biological; Myocardium; Osmolar Concentration; Oxygen; Oxygen Consumption; Piperazines; Thioinosine; Tubercidin

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.

Topics: Caco-2 Cells; Carrier Proteins; Colon; Epithelial Cells; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine

1. Intracellularly recorded excitatory junction potentials (ej.ps) were used to study the effects of adenosine receptor antagonists on neurotransmitter release from postganglionic sympathetic nerve terminals in the guinea-pig vas deferens in vitro. 2. The A1 adenosine receptor antagonists, 8-phenyltheophylline (10 microM) and 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), increased the amplitude of e.j.ps evoked during trains of 20 stimuli at 1 Hz in the presence, but not in the absence, of the alpha2-adrenoceptor antagonist, yohimbine (1 microM) or the non-selective alpha-adrenoceptor antagonist, phentolamine (1 microM). 3. Adenosine (100 microM) reduced the amplitude of e.j.ps, both in the presence and in the absence of phentolamine (1 microM). This inhibitory effect of adenosine is most likely caused by a reduction in transmitter release as there was no detectable change in spontaneous ej.p. amplitudes. 4. In the presence of phentolamine, application of the adenosine uptake inhibitor, S-(p-nitrobenzyl)-6-thioinosine (0.1 microM), had no effect on ej.p. amplitudes. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 microM), significantly increased the amplitudes of all e.j.ps evoked during trains of 20 stimuli at 1 Hz, both in the presence and in the absence of phentolamine (1 microM). 6. These results suggest that endogenous adenosine modulates neurotransmitter release by an action at prejunctional A1 adenosine receptors only when alpha2-adrenoceptors are blocked.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine Triphosphate; Adrenergic alpha-Antagonists; Animals; Guinea Pigs; In Vitro Techniques; Male; Membrane Potentials; Neuroeffector Junction; Phentolamine; Phosphodiesterase Inhibitors; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Synaptic Transmission; Theophylline; Thioinosine; Vas Deferens; Xanthines; Yohimbine

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [3H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35S-labeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra- and extracellular levels of adenosine in brain.

Topics: Adenosine; Affinity Labels; Animals; Autoradiography; Blotting, Northern; Brain Chemistry; Carrier Proteins; DNA Probes; DNA, Complementary; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Humans; In Situ Hybridization; Male; Membrane Proteins; Radioligand Assay; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfur Radioisotopes; Thioinosine; Tritium

Nucleoside transport regulation in undifferentiated Neuro-2A cells has been studied and found to include Na+-dependent adenosine transport and facilitated diffusion adenosine transport. The latter corresponded to nitrobenzylthioinosine-sensitive nucleoside transport. Short-term treatment of Neuro-2A cells with physiologically relevant signals only modulated the facilitated diffusion component. The stimulation of undifferentiated cells with forskolin or other activators of the protein kinase A pathway, decreased NBTI-sensitive adenosine transport. Treatment of cells with an inactive analogue of forskolin, 1,9-dideoxi-forskolin, had no effect on NBTI-sensitive nucleoside transport. Therefore, the inhibition of protein kinase A activity by pre-incubation with H-89 or the cAMP antagonist, Rp-8-Br-cAMPS, completely prevented the inhibitory effect of forskolin. Similarly, the activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore A-23187 decreased NBTI-sensitive adenosine transport. The effect of PDBu was reversed by pre-incubation of cells with staurosporine. Maximal transport inhibition was obtained by the simultaneous stimulation of cells with a phorbol ester and A-23187 or a phorbol ester and forskolin. The modulation of NBTI-sensitive nucleoside transport corresponded to changes in specific [3H]NBTI binding to Neuro-2A cells. Maximal inhibition correlated well with a maximal enhancement of cAMP production. However, the Na+-dependent adenosine transport in Neuro-2A cells was not modulated by any of these signals.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Mice; Neuroblastoma; Protein Kinase C; Sodium; Thioinosine; Tritium; Tumor Cells, Cultured

The purine nucleoside analogue NBMPR (nitrobenzylthioinosine or 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine) was selectively phosphorylated to its nucleoside 5'-monophosphate by Toxoplasma gondii but not mammalian adenosine kinase (EC 2.7.1.20). NBMPR was also cleaved in toxoplasma to its nucleobase, nitrobenzylmercaptopurine. However, nitrobenzylmercaptopurine was not a substrate for either adenosine kinase or hypoxanthine-guanine-xanthine phosphoribosyltransferase (EC 2.4.2.8). Because of this unique and previously unknown metabolism of NBMPR by the parasite, the effect of NBMPR as an antitoxoplasmic agent was tested. NBMPR killed T. gondii grown in human fibroblasts in a dose-dependent manner, with a 50% inhibitory concentration of approximately 10 microM and without apparent toxicity to host cells. Doses of up to 100 microM had no significant toxic effect on uninfected host cells. The promising antitoxoplasmic effect of NBMPR led to the testing of other 6-substituted 9-beta-D-ribofuranosylpurines, which were shown to be good ligands of the parasite adenosine kinase (M. H. Iltzsch, S. S. Uber, K. O. Tankersley, and M. H. el Kouni, Biochem. Pharmacol. 49:1501-1512, 1995), as antitoxoplasmic agents. Among the analogues tested, 6-benzylthioinosine, p-nitrobenzyl-6-selenopurine riboside, N(6)-(p-azidobenzyl)adenosine, and N(6)-(p-nitrobenzyl)adenosine, like NBMPR, were selectively toxic to parasite-infected cells. Thus, it appears that the unique characteristics of purine metabolism in T. gondii render certain 6-substituted 9-beta-D-ribofuranosylpurines promising antitoxoplasmic drugs.

Topics: Adenosine Kinase; Animals; Antiprotozoal Agents; Cell Survival; Drug Evaluation, Preclinical; Humans; Nucleotides; Phosphorylation; Purines; Thioinosine; Toxoplasma

The role of adenosine receptor-mediated signaling was examined in the alcohol withdrawal syndrome. CD-1 mice received a liquid diet containing ethanol (6.7%, v/v) or a control liquid diet that were abruptly discontinued after 14 days of treatment. Mice consuming ethanol showed a progressive increase in signs of intoxication throughout the drinking period. Following abrupt discontinuation of ethanol diet, mice demonstrated reversible signs of handling-induced hyperexcitability that were maximal between 5-8 h. Withdrawing mice received treatment with adenosine receptor agonists at the onset of peak withdrawal (5.5 h) and withdrawal signs were blindly rated (during withdrawal hours 6 and 7). Adenosine A1-receptor agonist R-N6(phenylisopropyl)adenosine (0.15 and 0.3 mg/ kg) reduced withdrawal signs 0.5 and 1.5 h after drug administration in a dose-dependent fashion. Adenosine A2A-selective agonist 2-p-(2-carboxyethyl)phenylethyl-amino-5'-N-ethylcarboxamidoadenosine (0.3 mg/kg) reduced withdrawal signs at both time points. In ethanol-withdrawing mice, there were significant decreases in adenosine transporter sites in striatum without changes in cortex or cerebellum. In ethanol-withdrawing mice, there were no changes in adenosine A1 and A2A receptor concentrations in cortex, striatum, or cerebellum. There appears to be a role for adenosine A1 and A2A receptors in the treatment of the ethanol withdrawal syndrome. Published by Elsevier Science Inc.

Topics: Adenosine; Animals; Brain; Central Nervous System Depressants; Ethanol; Male; Mice; Phenethylamines; Purinergic P1 Receptor Agonists; Receptor, Adenosine A2A; Receptors, Purinergic P1; Substance Withdrawal Syndrome; Thioinosine

The effects on ATP breakdown of some modulators of adenosine transport or metabolism were studied in the rat colon muscularis mucosae, a tissue which contracts to ATP and is thought to contain P2Y1 receptors. The compounds tested were the xanthine oxidase inhibitor allopurinol, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine uptake blocker S-(4-nitrobenzyl)-6-thioinosine (NBTI). The degradation of adenosine 5'-triphosphate (ATP) (100 microM) and the appearance of metabolites was followed by high pressure liquid chromatography during incubation of isolated tissue preparations alone or in the presence of the drugs, following preincubation with the drugs for 1 h. In the absence of drugs ATP was rapidly degraded by the rat colon muscularis mucosae with a half-life of 6.1 +/- 0.7 min, the major breakdown product being inosine rather than adenosine. Allopurinol (1 microM) and NBTI (10 microM) had no effect on the rate of breakdown of ATP or on the pattern of metabolites produced. EHNA (1 or 10 microM) also had no effect on the half-life of ATP, but in the presence of EHNA (1 microM) the rate of production of inosine was significantly reduced and some adenosine was detected, while in the presence of 10 microm EHNA the production of inosine was abolished and adenosine became the final breakdown product. These results indicate that allopurinol (1 microM) and NBTI (10 microM) have no detectable effect on extracellular purine metabolism in this tissue, and that the build-up of adenosine produced by treatment with EHNA does not have a feedback effect on ATP breakdown.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Allopurinol; Animals; Colon; Enzyme Inhibitors; Half-Life; In Vitro Techniques; Intestinal Mucosa; Muscle, Smooth; Rats; Rats, Wistar; Receptors, Purinergic P1; Receptors, Purinergic P2; Thioinosine

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

Topics: Affinity Labels; Animals; Cell Communication; Cell Death; Cell Division; Culture Media; Escherichia coli; Humans; Mice; Prodrugs; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines; Thioinosine; Tumor Cells, Cultured

Adenosine is a potent bronchoconstricting agent that is released by activated mast cells and hypoxic lung tissue. However, both inhibition and stimulation of mediator release from human lung mast cells by adenosine have been described, and this discrepancy seems to be due to contaminating cells or the effects of enzymatic treatment. We, therefore, investigated the effects of adenosine and its receptor-specific analogues on human cultured mast cells (HCMC). Adenosine inhibited Fc epsilon RI-mediated tryptase release from HCMC in a dose-dependent manner, and this inhibitory effect was completely blocked by the A2a receptor antagonist ZM241385. The specific agonist of A2a adenosine receptors CGS21680 inhibited the release of tryptase more potently than A1 and A3 agonists, and A2a receptor mRNA was detected by RT-PCR, suggesting the involvement of A2a receptors in the inhibitory effects of adenosine. In addition, adenosine increased intracellular cAMP level in a dose-dependent manner and inhibited protein tyrosine phosphorylation including that of ERK-2. These results suggest that adenosine acts via A2 receptors to inhibit Fc epsilon RI-mediated mediator release from human mast cells.

Topics: Adenosine; Antibodies; Chymases; Cross-Linking Reagents; Cyclic AMP; Guanosine; Humans; Immunoglobulin E; Mast Cells; Mitogen-Activated Protein Kinase 1; Phenethylamines; Phosphorylation; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, IgE; RNA, Messenger; Serine Endopeptidases; Thioinosine; Thionucleosides; Triazines; Triazoles; Tryptases

Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBankTM data base (human HNP36, 326 residues) that has been identified as a growth factor-induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5'-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter.

Topics: Affinity Labels; Amino Acid Sequence; Biological Transport; Carrier Proteins; Cloning, Molecular; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; Membrane Proteins; Molecular Sequence Data; Nuclear Proteins; Nucleosides; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Thioinosine; Tumor Cells, Cultured; Uridine

Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl- secretion. Previous data suggest that A2b receptor-mediated Cl- secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na(+)-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5'-(N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl- secretory responses and hence diarrheal disorders.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Biological Transport; Carrier Proteins; Cells, Cultured; Colforsin; Down-Regulation; Humans; Intestinal Mucosa; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Sodium; Tetradecanoylphorbol Acetate; Thioinosine; Vasodilator Agents

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K[D] = 6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.

Topics: Adenosine; Animals; Carrier Proteins; Cattle; Cells, Cultured; Chromaffin Cells; Flow Cytometry; Fluoresceins; Membrane Proteins; Nucleoside Transport Proteins; Purine Nucleosides; Thioinosine

The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals. Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation. Equilibrium binding assays were performed using [3H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (Bmax) and dissociation constant (Kd) of [3H]-NBMPR binding of human myocardial preparations. Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a Bmax of 283+/-32 fmol/mg protein and Kd of 0.56+/-0.12 nM. These values are lower than those obtained from guinea pigs (Bmax = 1440+/-187 fmol/mg protein and Kd = 0.21+/-0.03 nM) and canine atrium (Bmax 594+/-73 fmol/mg protein, and Kd = 1.12+/-0.22 nM). Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes.

Topics: Affinity Labels; Animals; Binding Sites; Carrier Proteins; Dogs; Guinea Pigs; Humans; In Vitro Techniques; Kinetics; Membrane Proteins; Membranes; Myocardium; Nucleoside Transport Proteins; Thioinosine

Previously, we have demonstrated the role of nucleoside transport and purine release in post-ischemic reperfusion injury (myocardial stunning) in several canine models of ischemia. Since rabbits are deficient of xanthine oxidase, it is not known whether selective blockade of purine release is beneficial in a rabbit model of coronary artery occlusion and reperfusion (stunning). Therefore, we determined the hemodynamic and metabolic correlates in response to myocardial stunning in the presence or absence of selective nucleoside transport blocker (p-nitrobenzylthioinosine, NBMPR) and adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA). Sixty adult anaesthetized rabbits were surgically prepared for hemodynamic measurements. After stabilization period, the left anterior descending coronary artery was occluded for 15 min and reperfused for 30 min. Transmural myocardial biopsies were obtained from the ischemic LAD area and from the non-ischemic posterior (circumflex, CFX) segment of the myocardium. Rabbits (n = 60) were randomly assigned to either the control or the EHNA/NBMPR-treated group (n = 30 each). Each group was further divided to either functional or metabolic groups (n = 15 each subgroup). Each animal received intravenously 30 ml of either a vehicle solution or 100 M EHNA and 25 M NBMPR 10 min before ischemia. Although administration of EHNA/NBMPR did not affect the heart rate, it did cause mild hypotension (about 20-30%). Fifteen minutes of LAD occlusion resulted in significant ATP depletion and concomitant accumulation of nucleosides in both groups (p < 0.05 vs. baseline and non-ischemic CFX segment). AMP was higher in the LAD compared to the CFX segment. Significant accumulation of adenosine was observed in the treated group compared to the control group. It is concluded that EHNA/NBMPR induced site specific entrapment of adenosine of nucleoside transport in the rabbit heart, in vivo.

Topics: Adenine; Adenine Nucleotides; Adenosine Deaminase Inhibitors; Animals; Carrier Proteins; Dogs; Enzyme Inhibitors; Female; Heart; Hemodynamics; Male; Membrane Proteins; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Stunning; Myocardium; Nucleoside Transport Proteins; Purines; Rabbits; Thioinosine

In a previous report, we have demonstrated that simultaneous inhibition of nucleoside transport and adenosine deaminase accumulates endogenous adenosine and protects the myocardium against stunning. The differential cardioprotective effects of erythro-9(2-hydroxy-3-nonyl)-adenine (EHNA), a potent inhibitor of adenosine deamination but not transport, and p-nitrobenzylthioinosine (NBMPR), a selective blocker of adenosine and inosine transport, are not known. Thirty-seven anaesthetized adult dogs were instrumented to monitor left ventricular performance using sonomicrometery. Dogs were randomly assigned into four groups. The control group (n = 8) received only the vehicle solution. Treated groups received saline containing 100 microM EHNA (EHNA-group, n = 7), 25 microM NBMPR (NBMPR-group, n = 7), or a combination of 100 microM EHNA and 25 microM NBMPR (EHNA/NBMPR-group, n = 10). Hearts were subjected to 30 min of normothermic global ischaemia and 60 min of reperfusion while on bypass. Adenine nucleotides, nucleosides, oxypurines and NAD+ were determined in extracts of transmural myocardial biopsies using HPLC. TTC staining revealed the absence of necrosis in this model. Drug administration did not affect myocardial ATP metabolism and cardiac function in the normal myocardium. Ischemia caused about 50% ATP depletion and accumulation of nucleosides. The ratio between adenosine/inosine at the end of ischemia was 1:10, 1:1, 1:1 and 10:1 in the control, EHNA-, NBMPR- and EHNA/NBMPR-group, respectively. Upon reperfusion, both nucleosides washed out from the myocardium in the control and EHNA-group while retained in the myocardium in the NBMPR and EHNA/NBMPR groups. Ventricular dysfunction 'stunning' persisted in the control group (52%) and in the EHNA-treated group (32%) after 30 min of reperfusion. Significant improvement of function was observed in the EHNA group only after 60 min of reperfusion. LV function recovered in the NBMPR- and EHNA/NBMPR-treated groups during reperfusion. ATP recovery occurred only when animals were pretreated with the combination of EHNA/NBMPR and remained depressed in the control group and EHNA and NBMPR-treated groups. At post mortem, TTC staining revealed the absence of myocardial necrosis. Superior myocardial protection was observed with inhibition of nucleoside transport by NBMPR alone or in combination with inhibition of adenosine deaminase by EHNA. Selective blockade of nucleoside transport by NBMPR is more cardioprotective than

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Animals; Biological Transport; Carrier Proteins; Dogs; Enzyme Inhibitors; Membrane Proteins; Models, Cardiovascular; Myocardial Ischemia; Myocardial Reperfusion Injury; Nucleoside Transport Proteins; Purines; Thioinosine

Adenosine plays an important role in protection of the heart before, during and after ischemia. Nucleoside transport inhibitors (NTI) increase adenosine concentration without inducing ischemia by preventing its uptake and metabolism in cardiac cells. However, prolonged effects of nucleoside transport inhibitors on adenosine and nucleotide metabolism and its combined effect with nucleotide precursors has not been established in cardiomyocytes. The aim of this study was to investigate the effect of two nucleoside transport inhibitors, dipyridamole (DIPY) and nitrobenzylthioinosine (NBTI) alone or combined with adenine and ribose on adenosine production and ATP content in cardiomyocytes. Rat cardiomyocytes were isolated using collagenase perfusion technique. Isolated cell suspensions were incubated for up to 480 min with different substrates and inhibitors as follows: (1) control; (2) 100 microM adenine and 2.5 mM ribose; (3) 10 microM DIPY; (4) 1 microM NBTI; (5) DIPY, adenine and ribose and (6) NBTI, adenine and ribose. Five microM EHNA (erythro-9(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase) was added to all incubations. After incubation, extracts of myocyte suspension were analysed by HPLC for adenine nucleotides and metabolite concentrations. ATP content decreased in cardiomyocytes after 8 h of incubation with DIPY, while no change was observed with NBTI or without inhibitors. Adenosine concentration increased with both DIPY and NBTI. In the presence of adenine and ribose an elevation in ATP concentration was observed, but no significant change in adenosine content. In the presence of DIPY or NBTI together with adenine and ribose, an enhancement in cardiomyocyte ATP concentration was observed together with an increase in adenosine content. This increase in adenosine production was especially prominent with DIPY. In conclusion, dipyridamole causes a decrease in ATP concentration in isolated cardiomyocytes by mechanisms other than nucleoside transport inhibition. Addition of adenine/ribose with dipyridamole prevents the depletion of ATP. Combination of adenine/ribose with nucleoside transport inhibitors may also further enhance adenosine concentration and thus, could be more effective as pharmacological agents for treatment.

Topics: Adenine; Adenosine; Adenosine Triphosphate; Animals; Biological Transport; Carrier Proteins; Dipyridamole; Enzyme Inhibitors; Membrane Proteins; Myocardium; NAD; Nucleoside Transport Proteins; Rats; Rats, Sprague-Dawley; Ribose; Thioinosine

The affinity of the human red cell nucleoside transporter for the transport inhibitor nitrobenzylthioinosine decreases upon protein purification. The affinity was highest for the whole cells (Kd, 0.04 nM), lowered upon cytoskeleton depletion (Kd, 0.2 nM) and lowest after partial purification and reconstitution (Kd, 0.3 nM), as determined by frontal affinity chromatography.

Topics: Carrier Proteins; Chromatography, Affinity; Cytochalasin B; Erythrocyte Membrane; Erythrocytes; Glucose Transporter Type 1; Humans; Kinetics; Membrane Proteins; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Proteolipids; Thioinosine

The inward transport of two purines, adenosine and hypoxanthine, at 37 degrees C by horse erythrocytes was compared. No mediated transport of adenosine was detected in horse erythrocytes, nor was saturable, high-affinity binding of the potent facilitated-diffusion inhibitor nitrobenzylthioinosine demonstrable in horse erythrocyte membranes. In contrast, erythrocytes from most horses possessed a saturable sodium-dependent hypoxanthine transporter (apparent K(m), 100 +/- 28 microM; Vmax, 0.20 +/- 0.08 mmol (l cells)-1 h-1; means +/- S.E.M., n = 5). Guanine inhibited hypoxanthine influx (apparent Ki, 24 +/- 6 microM), but adenine and xanthine had no effect. Unlike human erythrocytes, no sodium-independent hypoxanthine transporter was detected in horse erythrocytes. There are, however, a small number of animals (approximately 15%) whose erythrocytes fail to transport hypoxanthine. This variation appears to be under genetic control, but the precise nature of the control is unknown.

Topics: Adenosine; Animals; Carrier Proteins; Erythrocyte Membrane; Erythrocytes; Horses; Hypoxanthine; Kinetics; Membrane Transport Proteins; Protein Binding; Sodium; Thioinosine

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Biological Transport; Cells, Cultured; Dipyridamole; Heart; Myocardium; Rats; Rats, Sprague-Dawley; Thioinosine

1. The major toxicity associated with oral therapy with ribavirin is anaemia, which has been postulated to occur as a result of accumulation of ribavirin triphosphate interfering with erythrocyte respiration. The objective of this study was to determine the mechanism by which ribavirin enters into erythrocytes. 2. Entry into human erythrocytes was examined by measuring influx rates of [3H]-ribavirin alone and with the inhibitor nitrobenzylthioinosine (NBMPR), and by investigating the inhibitory effects of nucleoside and nucleobase permeants on ribavirin transport, by use of inhibitor oil-stop methods. Transport mechanisms were further characterized by assessment of substrates to cause countertransport of ribavirin in preloaded erythrocytes, and by measuring the effects of ribavirin on [3H]-NBMPR binding to erythrocyte membranes. 3. Human erythrocytes had a saturable influx mechanism for ribavirin (Km at 22 degrees C of 440+/-100 microM) which was inhibited by nanomolar concentrations of NBMPR (IC50 0.99+/-0.15 nM). Nucleosides also inhibited the influx of ribavirin (adenosine more effective than uridine) but the nucleobases hypoxanthine and adenine had no effect. In addition, uridine caused the countertransport of ribavirin in human erythrocytes. Entry of ribavirin into horse erythrocytes, a cell type that lacks the NBMPR-sensitive (es) nucleoside transporter, proceeded slowly and via a pathway that was resistant to NBMPR inhibition. Ribavirin was a competitive inhibitor of adenosine influx (mean Ki 0.48+/-0.14 mM) and also inhibited NBMPR binding to erythrocyte membranes (mean Ki 2.2+/-0.39 mM). 4. These data indicate that ribavirin is a transported permeant for the es nucleoside transporter of human erythrocytes. There was no evidence for ribavirin entering cells via a nucleobase transporter.

Topics: Adenosine; Animals; Antiviral Agents; Carrier Proteins; Equilibrative-Nucleoside Transporter 2; Erythrocyte Membrane; Erythrocytes; Horses; Humans; In Vitro Techniques; Membrane Proteins; Ribavirin; Thioinosine; Uridine

Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP by a series of nucleotidase reactions. Adenosine formed intracellularly can be released by bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT1 MF-2 smooth muscle cells, which possess nitrobenzylthioinosine-sensitive (ES) transporters as well as both adenosine A1 and A2 receptors, were loaded with the metabolically stable nucleoside analogue [3H]formycin B. N6-cyclohexyladenosine (CHA), a selective adenosine A1 receptor agonist, produced a concentration-dependent inhibition of [3H]formycin B release with an IC50 value of 2.7 microM. Further investigation revealed CHA interacts directly with nucleoside transporters with a Ki value of 3.3 microM. Neither 5'-N-ethylcarboxamidoadenosine (NECA), a mixed adenosine A1 and A2 receptor agonist, nor CGS 21680, a selective adenosine A2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside formycin B from DDT1 MF-2 cells is not regulated by adenosine A1 or A2 receptor activation.

Topics: Affinity Labels; Animals; Cell Line; Cricetinae; Cyclic AMP; Formycins; Male; Mesocricetus; Muscle, Smooth; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Thioinosine; Vas Deferens

The possible involvement of the adenosinergic modulatory system in the pathogenesis of absence seizures was investigated in genetic absence epilepsy rats from Strasbourg (GAERS). Using in vitro quantitative autoradiography, the distribution of A1 adenosine receptors and adenosine uptake sites in the brain of GAERS was studied and compared to that of control animals. An area-specific lower density of A1 receptors (15% decrease) was detected in reticular (nRT) and anterior ventral (AV) thalamic nuclei as well as basal ganglia in the brains of GAERS animals compared with control animals. Since adenosine exerts an anti-oscillatory effect on the thalamic nuclei by suppressing (via A1 receptors) excitatory as well as inhibitory neurotransmitter release, the impairment in A1 receptor density seen here, especially in nRT, could be implicated in the thalamic rhythmicity underlying spike and wave discharges present in this absence epilepsy model.

Topics: Adenosine; Affinity Labels; Animals; Autoradiography; Brain Chemistry; Epilepsy, Absence; Male; Purinergic P1 Receptor Antagonists; Rats; Receptors, Purinergic P1; Thalamic Nuclei; Thioinosine

We have recently isolated cDNAs from human placenta and rat jejunum encoding the prototypic human and rat equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporters hENT1 and rENT1. The two proteins (456 and 457 residues, Mr 50,000) are 78% identical in amino acid sequence and contain 11 potential transmembrane segments (TMs) with a large putative extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. When expressed in Xenopus oocytes, recombinant hENT1 and rENT1 transport both purine and pyrimidine nucleosides, including adenosine, and are inhibited by nanomolar concentrations of NBMPR. hENT1 is also potently inhibited by coronary vasodilator drugs (dipyridamole, dilazep, and draflazine), whereas rENT1 is insensitive to inhibition by these compounds (dipyridamole IC50 values 190 nM (hENT1) and >/=10 microM (rENT1) at 10 microM uridine). In the present study, we have generated reciprocal chimeras between hENT1 and rENT1, using splice sites at residues 99 (end of TM 2) and 231 (end of TM 6), to identify structural domains of hENT1 responsible for transport inhibition by vasoactive compounds. Transplanting the amino-terminal half of hENT1 into rENT1 converted rENT1 into a dipyridamole/dilazep-sensitive transporter, whereas the amino-terminal half of rENT1 rendered hENT1 dipyridamole/dilazep-insensitive. Domain swaps within the amino-terminal halves of hENT1 and rENT1 identified residues 100-231 (incorporating TMs 3-6) of hENT1 as the major site of vasodilator interaction. Since these drugs function as competitive inhibitors of nucleoside transport and NBMPR binding, TMs 3-6 are likely to form part of the substrate-binding site.

Topics: Affinity Labels; Amino Acid Sequence; Animals; Binding Sites; Cardiovascular Agents; Carrier Proteins; Dipyridamole; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Humans; Membrane Proteins; Molecular Sequence Data; Oocytes; Protein Conformation; Protein Structure, Secondary; Rats; Recombinant Fusion Proteins; Thioinosine; Vasodilator Agents; Xenopus

Adenosine has been shown to play a significant role as a modulator of neuronal activity in convulsion disorders, acting as an endogenous anticonvulsant agent. In the present study, we have investigated in mice the effect of acute tonic-clonic seizures induced by a single Pentylenetetrazol (PTZ)-injection (a) on the time development of adenosine uptake site binding after seizures in membranes of hippocampus, cortex, cerebellum, and striatum, and (b) on the regional distribution of adenosine uptake sites in the mouse brain by using "in vitro" quantitative autoradiography. As radioligand, the specific adenosine uptake blocker [3H]N-9-nitrobenzylthioinosine ([3H]NBI) was used. Acute seizures induced a rapid significant increase in [3H]NBI uptake site binding in hippocampus and cerebellum within 5 min, in cortex within 10 min after seizures, which reached a maximum level at 1 hr and reversed to control levels in about 150 min after seizures. On the contrary, in striatum a significant decrease of [3H]NBI uptake site binding was observed within 10 min after seizures, which reached its maximum at 1 hr and reversed to control levels at 150 min after seizures. With this single exception of striatum the "in vitro" quantitative autoradiography revealed a rather widespread upregulation of [3H]NBI uptake site density in the mouse brain, which was specifically enhanced in certain areas known to mediate seizure activity, such as hippocampus, specific thalamic nuclei, temporal cortex, and substantia nigra. The pattern of increases in [3H]NBI uptake site binding as they develop after acute seizures correlates well in time with the rapid enhancement of endogenous adenosine concentration released during epileptic activity. Since extracellular adenosine levels seem to be regulated by a rapid reuptake system, it seems likely that in our study, the [3H]NBI adenosine uptake system is acutely activated by seizures in order to compensate for the excess of endogenous adenosine. Furthermore, the upregulation of [3H]NBI uptake sites as revealed by the "in vitro" quantitative autoradiography seems to be organized in selective brain areas related to seizure propagation.

Topics: Adenosine; Affinity Labels; Animals; Autoradiography; Binding Sites; Brain; Cell Membrane; Cerebellum; Cerebral Cortex; Corpus Striatum; Epilepsy, Tonic-Clonic; Female; Hippocampus; In Vitro Techniques; Mice; Mice, Inbred BALB C; Organ Specificity; Pentylenetetrazole; Receptors, Purinergic P1; Reference Values; Seizures; Thioinosine; Time Factors; Tissue Distribution; Tritium

Little is known regarding the status and implications of altered retinal blood flow (RBF) following a period of temporary retinal ischemia. We undertook studies to measure acute changes in RBF after ischemia, and the mechanisms responsible for mediating these changes.. Retinal ischemia was induced in anesthetized, mechanically ventilated newborn pigs by severe hypoxia, hypotension, and bradycardia secondary to 9 min of asphyxia by discontinued ventilation. Using fluorescein videoangiography, we calculated stimulus-induced changes in RBF by measuring changes in arteriovenous transit times and arteriolar and venular diameters from the angiogram videorecordings.. Asphyxia led to a progressive reduction in RBF during early reperfusion, with RBF decreasing 24 +/- 6% and 34 +/- 5% below baseline 1 h and 2 h, respectively, after asphyxia (n = 6). Intravitreal administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (25 nmol) at 15 min of postischemic reperfusion did not increase the magnitude of hypoperfusion (n = 6), and intravitreal acetylcholine (20 nmol) was no longer able to increase RBF at 1.5-2.0 h of postasphyxic reperfusion. The endothelin A receptor antagonist TBC 11251z attenuated the response by 53% at 2 h (n = 5). The adenosine transport inhibitor 4-nitrobenzyl-6-thioinosine reversed the hypoperfusion response (n = 5), whereas ventilating animals with 100% oxygen during reperfusion exacerbated the flow deficit, with RBF reduced to 49 +/- 5% below baseline at 2 h post-asphyxia (n = 6).. These findings indicate that (1) constriction by endothelin, together with a loss of nitric oxide's tonic dilatative effect, contributes importantly to mediating postischemic hypoperfusion in retina; (2) improvements in retinal perfusion can be realized with endothelin receptor blockade or potentiation of extracellular adenosine; and (3) additional reductions in postischemic RBF can occur in response to resuscitation with 100% oxygen because retinal microcirculatory reactivity to hyperoxia remains intact during the hypoperfusion period.

Topics: Acetylcholine; Adenosine; Animals; Animals, Newborn; Blood Flow Velocity; Endothelin Receptor Antagonists; Endothelium, Vascular; Enzyme Inhibitors; Fluorescein Angiography; Hyperoxia; Hypoxia; Ischemia; Isoxazoles; Nitric Oxide Synthase; omega-N-Methylarginine; Reperfusion; Retinal Vessels; Swine; Thioinosine; Thiophenes

The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]formycin B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2',2'-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 approximately 400 microM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.

Topics: Animals; Antimetabolites, Antineoplastic; Carcinoma, Ehrlich Tumor; Carrier Proteins; Deoxycytidine; Formycins; Gemcitabine; Male; Membrane Proteins; Mice; Nucleoside Transport Proteins; Sodium; Thioinosine; Uridine

The nucleus accumbens (NAc) is a site mediating the rewarding properties of drugs of abuse, such as cocaine, amphetamine, opiates, nicotine, and alcohol (Wise and Bozarth, 1987; Koob, 1992; Samson andHarris, 1992; Woolverton and Johnson, 1992; Self and Nestler, 1995; Pontieri et al., 1996). Acute cocaine has been shown to decrease excitatory synaptic transmission mediated by the cortical afferents to the NAc (Nicola et al., 1996), but the effects of long-term cocaine treatment and withdrawal have not been explored. Here, we report that long-term (1 week) withdrawal from chronic cocaine reduced the potency of adenosine to presynaptically inhibit glutamate (Glu) release by activating adenosine A1 receptors. Adenosine A1 receptors were not desensitized, because the potency of the metabolically stable adenosine analog N6-cyclopentyl-adenosine was unchanged after chronic cocaine withdrawal. When adenosine transporters were blocked, the potency of adenosine to inhibit Glu release from naive and cocaine-withdrawn NAc slices was similar. These results suggest that one of the long-term consequences of cocaine withdrawal is an augmented uptake of adenosine. This long-lasting change expressed at the presynaptic excitatory inputs to the medium spiny output neurons in the NAc may help identify new therapeutic targets for the treatment of drug abuse.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine; Affinity Labels; Animals; Cocaine; Dipyridamole; Dopamine Uptake Inhibitors; Excitatory Postsynaptic Potentials; Glutamic Acid; Hippocampus; Male; Nucleus Accumbens; Phosphodiesterase Inhibitors; Presynaptic Terminals; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Substance Withdrawal Syndrome; Substance-Related Disorders; Theophylline; Thioinosine; Vasodilator Agents; Xanthines

We have used rats with streptozotocin-induced diabetes to investigate the effects of hyperglycaemia-mediated impaired nucleoside uptake on the actions of endogenous adenosine in hippocampal slices. In control tissue under conditions of anoxia and aglycaemia the rise in the extracellular adenosine concentration resulted in complete inhibition of synaptic activity in about 2 min. In slices from previously hyperglycaemic rats the inhibition of synaptically mediated responses occurred significantly faster, although this change could be prevented by insulin treatment. Application of the selective adenosine A1 receptor antagonist [8-cyclopentyl-1,3-dipropylxanthine (DPCPX)] prevented the anoxia/aglycaemia-mediated inhibition and, furthermore, abolished the differences in the electrophysiological responses between control and diabetic tissue. The effects of impaired nucleoside uptake could be mimicked in control slices by applying the nucleoside uptake blocker hydroxynitrobenzylthioinosine (HNBTI). This had the effect of speeding up the rate of anoxia/aglycaemia-induced synaptic inhibition in control tissue to that seen in diabetic tissue. However, such treatment had no effect on the responses in diabetic tissue as expected if the HNBTI-sensitive uptake process was already inhibited by the chronic hyperglycaemia. The impairment of nucleoside uptake by chronic hyperglycaemia results in the potentiation of the modulatory actions of endogenous adenosine in the central nervous system. Such an alteration in adenosine function may be important in explaining behavioural and pathological changes associated with diabetes mellitus.

Topics: Action Potentials; Adenosine; Affinity Labels; Animals; Brain Ischemia; Diabetes Mellitus, Experimental; Hippocampus; Hypoglycemia; Hypoxia, Brain; Male; Rats; Rats, Wistar; Synaptic Transmission; Thioinosine; Xanthines

We have examined the cytotoxic effects of cyclic adenosine-3', 5'-monophosphate (cAMP) derivatives on multiple myeloma cells lines and determined that the 8-Chloro substituted derivative (8Cl-cAMP) is one of the most potent. We report here that 8Cl-cAMP is cytotoxic to both steroid sensitive and insensitive myeloma cells with a half maximal concentration of approximately 3 micromol/L. 8Cl-cAMP toxicity in myeloma cells is dependent on phosphodiesterase activity in the serum of cell culture medium. A metabolite of 8Cl-cAMP, 8-Chloro-adenosine (8Cl-AD), kills myeloma cells as effectively as 8Cl-cAMP. Adenosine deaminase (ADA) converts 8Cl-AD into 8Cl-inosine and abrogates the cytotoxic effects of 8Cl-cAMP, 8Cl-AMP, and 8Cl-AD, as does 5-(p-Nitrobenzyl)-6-Thio-Inosine (NBTI), an inhibitor of nucleoside uptake. These data suggest that 8Cl-cAMP must be converted to 8Cl-AD and that 8Cl-AD is the compound that enters the cell. Contrary to glucocorticoid-mediated cell death in myeloma cells, the pathway of 8Cl-AD-mediated cell death appears to be independent of interleukin-6 (IL-6) actions. Although the exact mode of action for this agent is currently unknown, its ability to kill steroid sensitive and insensitive multiple myeloma cells in an IL-6 independent fashion may offer exciting new therapeutic options.

Topics: 1-Methyl-3-isobutylxanthine; 2-Chloroadenosine; 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Deaminase; Animals; Antimetabolites, Antineoplastic; Apoptosis; Biological Transport; Biotransformation; Cattle; Culture Media; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Interleukin-6; Multiple Myeloma; Phosphoric Diester Hydrolases; Prodrugs; Thioinosine; Tumor Cells, Cultured

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and (b) treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es approximately = cit > ei > cib >>> cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.

Topics: Animals; Antimetabolites, Antineoplastic; Biological Transport; Carrier Proteins; Cell Division; Cell Survival; Deoxycytidine; Dipyridamole; DNA, Complementary; Gemcitabine; HeLa Cells; Humans; K562 Cells; Kinetics; Membrane Proteins; Membrane Transport Proteins; Nucleoside Transport Proteins; Recombinant Proteins; Thioinosine; Transfection; Tumor Cells, Cultured

We investigated whether xanthine oxidase-derived superoxide radical generation could be modified by interfering with adenosine transport and metabolism in reducing myocardial injury during post-ischemic reperfusion. Isolated rat hearts perfused at constant pressure were subjected to 20 min of pretreatment with test agents, followed by 40 min global ischemia and 30 min reperfusion with or without test agents. In hearts treated with adenosine deaminase inhibitor, erythro 9-(2-hydroxy-3-nonyl) adenine (EHNA), alone or together with a selective nucleoside transport blocker, p-nitrobenzylthioinosine (NBMPR), the accumulated amount of O-2. was significantly reduced [10.2+/-0.97, 11.6+/-2.4, 8.1+/-0.51, respectively, v 31.6+/-2.1 (s. e.) nmol/wet g/30 min in ischemic control, P<0.01]. A positive correlation between O-2. and inosine release was observed in the initial 5 min of reperfusion in hearts treated with either EHNA or NBMPR ( r=0.475, P<0.05). Furthermore, the accumulated amount of LDH release showed positive correlation with that of O-2. among the same groups (r=0.474, P<0.05). Both EHNA and NBMPR had the cardioprotective effect on the recovery of left ventricular end-diastolic pressure (LVEDP), ATP repletion, and build up of endogenous adenosine. This study suggests that : (1) adenosine metabolism can be manipulated towards the formation of O-2. during reperfusion, and it has an important bearing on the cardiac recovery of ischemic myocardium, (2) the generation of O-2. is related to only inosine release during initial reperfusion.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Animals; Enzyme Inhibitors; Heart; Heart Rate; In Vitro Techniques; Isoenzymes; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Organophosphates; Purines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stroke Volume; Superoxides; Thioinosine; Xanthine Oxidase

Adenosine is an antiarrhythmic substance particularly effective in catecholamine-dependent tachycardias. Although endogenous adenosine substantially accumulates in catecholamine-stimulated hearts, little is known about the antiarrhythmic potency of endogenous adenosine in this condition. Therefore, we sought to demonstrate a potential antifibrillatory effect of endogenous adenosine either by blockade of adenosine receptors with 8-phenyltheophylline (8-PT) or by suppression of endogenous adenosine release with nitrobenzyl-6-thioinosine (NBTI). The study was performed in spontaneously beating Langendorff-perfused rat hearts. Adenosine release into the effluent was determined by HPLC methods. Catecholamine stimulation was induced by perfusing the hearts with norepinephrine (1 mumol/l) for 30 min, which caused ventricular tachycardia (VT) in 31% and ventricular fibrillation (VF) in 25% of control hearts (n = 35). When 8-PT (10 mumol/l) was added to the perfusion buffer prior to norepinephrine, the incidence of VT and VF increased to 79 and 68%, respectively. The addition of 8-PT did not affect the catecholamine-dependent formation of adenosine. Perfusion of the hearts with NBTI (10 mumol/l) prior to norepinephrine reduced adenosine release and increased the occurrence of both VT (65%) and VF (40%). In summary, the results indicate that adenosine is an endogenous antiarrhythmic substance, which accumulates in catecholamine-stimulated myocardium to a level, which effectively suppresses the occurrence of ventricular arrhythmias.

Topics: Adenosine; Animals; Electrocardiography; Heart; In Vitro Techniques; Male; Myocardium; Norepinephrine; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Theophylline; Thioinosine; Vasoconstrictor Agents; Ventricular Fibrillation

The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters.

Topics: Adenosine; Amino Acid Sequence; Base Sequence; Candida albicans; Carrier Proteins; Cloning, Molecular; Dipyridamole; Genetic Complementation Test; Guanosine; Membrane Proteins; Molecular Sequence Data; Nucleoside Transport Proteins; Purine Nucleosides; Saccharomyces cerevisiae; Substrate Specificity; Thioinosine; Uridine

The equilibrative nucleoside transporters (ENTs) are a newly recognized family of membrane proteins of which hENT1 is the nitrobenzylmercaptopurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-insensitive (ei) transporter of human cells. BeWo cells exhibit large numbers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we have demonstrated that proliferating BeWo cells possess (i) mRNA encoding hENT1 and hENT2 and (ii) hENT1-specific immunoepitopes. We examined NBMPR binding and its inhibition of uridine transport in various BeWo membrane fractions and proteoliposomes derived therefrom to determine if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fractions enriched 5-fold in plasma membranes relative to postnuclear supernatants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites with affinities similar to those of enriched plasma membranes (Kd = 0.4-0.9 nM). When proteoliposomes were made from octyl glucoside-solubilized membranes, high affinity NBMPR-binding sites were not only observed in crude membrane preparations and plasma membrane-enriched fractions but also in nuclear envelope fractions. Proteoliposomes prepared from either unfractionated membranes or nuclear envelopes exhibited both hENT1-mediated (82-85%) and hENT2-mediated (15-18%) transport of [3H]uridine. These results provided evidence for the presence of functional es and ei transporters in nuclear membranes and endoplasmic reticulum, suggesting that hENT1 and hENT2 may function in the translocation of nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.

Topics: Affinity Labels; Binding Sites; Carrier Proteins; Cell Membrane; Cell Nucleus; Choriocarcinoma; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Membrane Proteins; Nuclear Envelope; Proteolipids; RNA, Messenger; Thioinosine; Tumor Cells, Cultured; Uridine

A stability study of adenosine receptor agonists in rat and human whole blood was performed. The compounds were incubated at 37 degrees in fresh blood, and aliquots of the incubation mixture were hemolyzed at regular time intervals and analyzed with HPLC. N6-cyclopentyladenosine (CPA) and N6-cyclobutyladenosine (CBA) were degraded, whereas N6-cyclohexyladenosine, N6-cycloheptyladenosine and N6-sulfophenyladenosine were not. 2-Chloroadenosine had a half-life very similar to that of CPA. However, the 2'-, 3'-, and 5'-deoxyribose derivatives of CPA remained intact. The nucleoside transport inhibitor nitrobenzylthioinosine attenuated CBA and CPA metabolism in rat blood as did the inhibitor of adenosine deaminase erythro-9-(2-hydroxy-3-nonyl)adenine, albeit at relatively high concentrations. Complete blockade of CBA and CPA degradation was achieved by a preincubation of rat and human blood with the adenosine kinase (AK) inhibitor 5'-amino-5'-deoxyadenosine. We conclude that the two adenosine analogues are metabolized by AK both in rat and in human whole blood.

Topics: Adenine; Adenosine; Adenosine Kinase; Aminohydrolases; Animals; Blood; Deoxyadenosines; Enzyme Inhibitors; Humans; Purinergic P1 Receptor Agonists; Rats; Thioinosine

Solute interactions with membrane proteins can be analyzed by biomembrane affinity chromatography (BAC), previously applied to the human red cell glucose transporter. As a novel example, frontal BAC analysis of interactions between the nucleoside transport inhibitor nitrobenzylthioinosine (NBTI) and immobilized reconstituted nucleoside and glucose transporters from human red cells revealed two binding sites, presumably corresponding to the two transporters. The affinities and amounts of sites were determined by use of a double rectangular hyperbolic equation. The Kd value for NBTI binding to the nucleoside transporter in egg phospholipid proteoliposomes was 0.38 +/- 0.08 nM (22 degrees C, I = 0.16, pH 7.4), lower than previously reported for reconstituted systems. The molar ratio between the amounts of nucleoside transporter sites for NBTI and glucose transporter sites for cytochalasin B was 4.5 +/- 0.6%.

Topics: Affinity Labels; Binding Sites; Blood Proteins; Carrier Proteins; Chromatography, Affinity; Cytochalasin B; Erythrocytes; Humans; In Vitro Techniques; Kinetics; Membrane Proteins; Membranes, Artificial; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Proteolipids; Thioinosine

Studies using choroid plexuses incubated in vitro have led to the conclusion that pyrimidine deoxyribonucleosides, such as thymidine, enter the brain predominantly through the blood-cerebrospinal fluid (CSF) barrier across the choroid plexuses. In order to examine this hypothesis, ventriculocisternal perfusions were carried out to determine the magnitude of the passage of [3H]thymidine from the CSF into the brain and blood. These experiments demonstrated that approximately 50% of the [3H]thymidine was eliminated from the CSF perfusate, some 41.6 +/- 5.6% passing into the blood and only 7.6 +/- 0.6% to the brain. Efflux into both the blood and brain was saturable, with a Km of 17.8 microM and a Vmax of 0.46 nM min-1, and partially nitrobenzylthioinosine (NBMPR) sensitive. However, a non-saturable component did exist (Kd, 13.8 microliters min-1). Overall, the rapid removal of [3H]thymidine from the CSF and its low uptake from the CSF into the brain suggests that the choroid plexuses would be an inefficient pathway for the entry of this pyrimidine deoxyribonucleoside into the brain.

Topics: Affinity Labels; Anesthesia; Animals; Brain; Choroid Plexus; Female; Injections, Intraventricular; Kinetics; Male; Polyethylene Glycols; Rabbits; Thioinosine; Thymidine

Isolated myenteric ganglion networks were prepared from guinea pig ileum and were used in a perifusion protocol to examine the effects of interstitial adenosine on evoked release of substance P-like immunoreactivity (SPLI). The release of SPLI evoked by elevated extracellular K+ concentration was increased in the presence of tetrodotoxin (TTX), indicating tonic inhibition of SPLI release and revealing net inhibitory interganglionic transmission. Perifusion in the presence of the adenosine A1 receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine enhanced evoked SPLI release, which was further enhanced in the additional presence of TTX, indicating that adenosine contributes some, but not all, of the overall inhibitory tone within the networks. In addition to neural release of adenosine per se, an additional source was investigated. Perifusion in the presence of alpha, beta-methylene-ADP plus guanosine 5'-monophosphate, which inhibits ecto-adenosinetriphosphatase (ATPase) activity, enhanced SPLI release, indicating that hydrolysis of released ATP contributes to the total interstitial nucleoside concentration and thereby to the overall inhibitory tone. It is concluded that endogenous adenosine, some of which arises from ATP metabolism, is an important contributor to the overall inhibitory tone present in myenteric ganglion networks.

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphatases; Animals; Biological Transport; Dipyridamole; Ganglia; Guinea Pigs; Ileum; In Vitro Techniques; Myenteric Plexus; Nerve Net; Radioimmunoassay; Receptors, Purinergic P1; Substance P; Tetrodotoxin; Thioinosine; Xanthines

We investigated adenosine stimulation of DNA synthesis in human endothelial cells by measuring [3H]thymidine incorporation in cultures derived from human umbilical veins. After 18 h of exposure to adenosine in serum-free medium, endothelial cell [3H]thymidine incorporation was increased by 30-64%. Adenosine-induced DNA synthesis was not mimicked by adenosine receptor agonists and was not inhibited by adenosine receptor antagonists. Adenosine-induced DNA synthesis was inhibited 81% by 100 microM 5'-(N,N-dimethyl)amiloride, an inhibitor of Na+/H+ exchange, and was totally inhibited by 10 microM 2',4'-dibromoacetophenone, an inhibitor of phospholipase A2 (PLA2). Adenosine increased adenosine 3',5'-cyclic monophosphate levels in endothelial cells, but adenosine-induced DNA synthesis was not inhibited by the protein kinase A (PKA) inhibitor Rp-cAMPS. Both ATP and the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PMA) increased DNA synthesis in human endothelial cells. Stimulation by ATP was inhibited by the P2-receptor antagonist suramin, and PMA stimulation was inhibited by the protein kinase C (PKC) inhibitor H-7. Neither suramin nor H-7 inhibited adenosine-stimulated DNA synthesis. The results suggest that Na+/H+ exchange and PLA2 are involved in adenosine-induced DNA synthesis in cultures of human endothelial cells independently of adenosine receptor, PKA, or PKC activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine Triphosphate; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA; Endothelium, Vascular; Humans; Kinetics; Poly A; Purinergic P1 Receptor Antagonists; Quinazolines; Tetradecanoylphorbol Acetate; Thioinosine; Thionucleotides; Thymidine; Triazoles; Umbilical Veins

Purification of functional nucleoside transporters has been hampered by the instability of detergent-solubilized proteins. The present study was undertaken to determine if the presence of specific transporter ligands in the solubilization medium could enhance the functional stability of the isolated proteins. Ehrlich cell plasma membranes were solubilized with 1% (w/v) octylglucoside (+/- transporter ligands) and reconstituted into liposomal membranes either immediately after solubilization or after storage for 48 hr at 6 degrees. Storage resulted in a parallel loss (approximately 60%) of [3H]nitrobenzylthioinosine (NBMPR) binding and reconstituted [3H]uridine uptake activities. furthermore, upon storage, the relative amount of NBMPR-resistant [3H]uridine uptake by the reconstituted system dropped from 19 +/- 2 to 8 +/- 1% of the total mediated influx. The inclusion of high concentrations (> 10 mM) of adenosine in the solubilization medium completely prevented the storage-induced loss of both [3H]NBMPR binding and [3H]uridine influx activity, and prevented the shift in NBMPR sensitivity. In addition, inclusion of adenosine in the solubilization procedure increased the relative amount of NBMPR-resistant [3H]uridine uptake to 33 +/- 2% of the total influx in proteoliposomes prepared immediately after the proteins were extracted from the plasma membrane (i.e. no storage). A partial protection of [3H]NBMPR binding activity was also obtained using 2'-deoxyadenosine, 2-chloroadenosine, uridine, and non-radiolabelled NBMPR, but not with cytidine, inosine, diazepam, dipyridamole, or dilazep. These results suggest that both NBMPR sensitivity and transporter stability are dependent upon the conformational state of the protein. The protective effects of adenosine analogues and other nucleosides are likely due to their binding to the substrate translocation site, thereby effectively "locking" the transporter in a stable conformation.

Topics: Adenosine; Animals; Binding Sites; Carrier Proteins; Membrane Proteins; Mice; Nucleoside Transport Proteins; Thioinosine; Uridine

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 microM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%), thymidine (2%) and formycin B (1%). The Km for adenosine was 160 +/- 42 microM. Unlabeled nucleosides (100 microM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5'-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50% or more. However, inosine related compounds guanosine, 3'-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0-30% at 10 microM, but dilazep, a known inhibitor, was inactive upto 10 microM. Increase in temperature from 22 degrees C to 33 degrees C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.

Topics: Animals; Biological Transport; Dilazep; Entamoeba histolytica; Nucleosides; Purine Nucleosides; Pyrimidine Nucleosides; Thioinosine

beta-L-(-)-2',3'-Dideoxy-3'-thiacytidine (3TC) is a cytosine nucleoside analog that potently inhibits the replication of human and duck hepatitis B viruses and human immunodeficiency virus through the activity of its 5'-triphosphate ester metabolite. The present study examined the intracellular decay of 3TC 5'-phosphates and tested strategies for modulating the cellular content of those nucleotides in primary cultures of duck hepatocytes and in human hepatoma 2.2.15 cells and CCRF-CEM T lymphoblasts. Inhibition by deoxycytidine of the 5'-phosphorylation of 3TC in duck hepatocytes confirmed that, as in mammalian cells, deoxycytidine kinase catalyzed 3TC activation. The 5'-mono, 5'-di-, and 5'-triphosphates of 3TC underwent monoexponential elimination from duck hepatocytes and 2.2.15 cells (half-lives, 3.6 to 8.0 h). Thymidine and fludarabine, which are agents that enhance the activity of deoxycytidine kinase, were tested in strategies for increasing the cellular content of 3TC 5'-phosphates. Coordinate treatment of cells with 3TC and thymidine (50 microM) increased the content of 3TC 5'-monophosphate in duck hepatocytes and the content of 3TC 5'-di- and 5'-triphosphates in 2.2.15 cells, but enhancement of 3TC 5'-phosphate levels in CCRF-CEM cells required a higher thymidine concentration (100 microM). Fludarabine (5 microM) did not affect the contents of 3TC 5'-di- and 5'-triphosphates in duck hepatocytes, but modestly increased the contents of those nucleotides in 2.2.15 cells and CCRF-CEM cells. Nitrobenzylthioinosine (NBMPR), an inhibitor of the es facilitated diffusion nucleoside transporter, reduced the level of entry of 3TC into 2.2.15 cells and abolished inward fluxes of thymidine, adenosine, and deoxycytidine. In 2.2.15 cells and CCRF-CEM cells, NBMPR reduced the formation of 3TC 5'-di- and 5'-triphosphates and reversed the thymidine- and fludarabine-induced increases in the formation of those nucleotides. NBMPR protected against the cytotoxicity of 3TC in CCRF-CEM cells, whereas thymidine potentiated that toxicity, apparently by enhancing the formation of 3TC 5'-triphosphate. Taken together, these results indicate that deoxycytidine kinase and the es nucleoside transporter are targets for manipulation of the metabolism and activity of 3TC.

Topics: Affinity Labels; Animals; Anti-HIV Agents; Antineoplastic Agents; Carcinoma, Hepatocellular; Cells, Cultured; Drug Interactions; Ducks; Hepadnaviridae Infections; Hepatitis B Virus, Duck; Humans; Thioinosine; Thymidine; Tumor Cells, Cultured; Vidarabine; Zalcitabine

Both subjective and electroencephalographic arousal diminish as a function of the duration of prior wakefulness. Data reported here suggest that the major criteria for a neural sleep factor mediating the somnogenic effects of prolonged wakefulness are satisfied by adenosine, a neuromodulator whose extracellular concentration increases with brain metabolism and which, in vitro, inhibits basal forebrain cholinergic neurons. In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness as contrasted with slow wave sleep; exhibited progressive increases during sustained, prolonged wakefulness; and declined slowly during recovery sleep. Furthermore, the sleep-wakefulness profile occurring after prolonged wakefulness was mimicked by increased extracellular adenosine induced by microdialysis perfusion of an adenosine transport inhibitor in the cholinergic basal forebrain but not by perfusion in a control noncholinergic region.

Topics: Adenosine; Animals; Cats; Electrophysiology; Microdialysis; Prosencephalon; Sleep; Sleep Deprivation; Thioinosine; Time Factors; Wakefulness

Entry of adenosine, and thymidine, into erythrocytes from adult dolphins was rapid, showed saturation at higher substrate concentrations, and was strongly inhibited by low concentrations of nitrobenzylthioinosine (NBMPR). Kinetic parameters were estimated from the concentration dependence of initial rates of tracer entry at 21 degrees C, as K(m) 0.14 +/- 0.05 mM and Vmax 24.4 +/- 1.9 mumol/litre cell water/sec for zero trans entry of adenosine, and K(m) 0.96 +/- 0.21 mM and Vmax 25.4 +/- 1.7 mumol/litre cell water/sec for thymidine. Adenosine, and thymidine, entry were inhibited by both purine and pyrimidine nucleosides. Mass law analysis of a saturable component of nitrobenzylthioinosine binding to dolphin red cell membranes gave values of Bmax 65.4 +/- 1.2 pmol/mg protein, and K(d) of 1.53 +/- 0.08 nM for a single class of sites. Photo-irradiation of dolphin red cell membranes in the presence of tritiated nitrobenzylthioinosine led to radioactive labeling of polypeptides M(r) 52, 500-58,000, on SDS-PAGE.

Topics: Affinity Labels; Animals; Binding, Competitive; Biological Transport; Dolphins; Erythrocytes; Humans; Nucleosides; Photochemistry; Purine-Nucleoside Phosphorylase; Rabbits; Rats; Thioinosine

NB4 cells are the only bona fide in vitro model of human acute promyelocytic leukemia. We have examined cytidine and guanosine transport in this cell line and characterized a novel guanosine-specific transporter. Cytidine transport occurred predominately by equilibrative nitrobenzylthioinosine (NBMPR)-sensitive (es) transport. In the presence of Na+, guanosine at various concentrations accumulated at least 6-fold above equilibrium. The initial rate of guanosine transport in Na+ buffer decreased by 75% with the addition of 1 microM NBMPR and the IC50 for NBMPR inhibition was 0.7 +/- 0.1 nM. Replacement of Na+ with choline also resulted in a 75% decrease in total guanosine transport. The potent inhibition of guanosine transport by NBMPR and the loss of transport in choline suggested that a Na+-dependent NBMPR-sensitive transporter was responsible for the majority of guanosine uptake. This concentrative, sensitive transporter is Na+ dependent with a stoichiometric coupling ratio of 1:1. This novel transporter, referred to as csg, is guanosine-specific with total guanosine transport inhibited by only 50% in the presence of 1 mM competing nucleosides. HL-60, acute myelocytic leukemia cells, do not exhibit csg activity while L1210, murine acute lymphocytic leukemia cells, exhibit csg transport. The presence of the csg transporter suggests an important role for guanosine in particular forms of leukemia and may provide a new target for cytotoxic therapy.

Topics: Biological Transport; Carrier Proteins; Choline; Cytidine; Guanosine; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Nucleosides; Sodium; Substrate Specificity; Thioinosine; Tumor Cells, Cultured

Nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in many cell types, modulates the in vivo disposition of several cytotoxic nucleoside analogs. In this study, a radioligand binding assay was developed for measurement of the NBMPR content of plasma and erythrocytes.. The assay was based on the competition between NBMPR and [3H]NBMPR for high-affinity sites on human erythrocytes membranes. With this assay, we followed in mice changes in the NBMPR content of blood plasma and erythrocytes, following the intraperitoneal injection of the disodium salt of NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR.. The radioligand binding assay was able to measure precisely as little as 2.5 pmol of NBMPR, allowing the direct determination of NBMPR concentrations in plasma as low as 16 nM. As few as 8 x 10(3) molecules of NBMPR per cell could be determined in erythrocytes. The NBMPR content of plasma from mice injected with NBMPR-P was maximal at about 20 min after injection and declined to < 0.2% of the peak value by 10 h. Erythrocyte-associated NBMPR was also maximal at 20 min, and declined to 11% of the peak value by 10 h after injection. Time courses for the disappearance of NBMPR from plasma and erythrocytes were monoexponential and yielded half-life values of 0.39 h and 0.68 h, respectively, an apparent volume of distribution of 0.61 l/kg, and a clearance of 1.1 l/h per kg.. The radioligand binding assay is a sensitive and facile method for monitoring NBMPR concentrations in mammalian plasma and tissue extracts.

Topics: Affinity Labels; Animals; Blood Chemical Analysis; Erythrocytes; Female; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Radioligand Assay; Reproducibility of Results; Sensitivity and Specificity; Thioinosine

It was not until the development of a technique that could measure the brain uptake of slowly moving substrates, that the saturable transport system at the blood-brain barrier (BBB) for the pyrimidine deoxyribonucleoside, thymidine, was demonstrated. The aim of this present study was to further characterize this saturable uptake system at the blood-brain and blood-CSF barriers in terms of specificity, 6-(4-nitrobenzyl)thio-9-beta-D-ribofuranosylpurine (NBMPR) sensitivity and saturation kinetics by means of the in situ brain perfusion technique in anaesthetized guinea pigs. The results indicated that the transport system identified for [3H]thymidine can also transport other pyrimidine deoxyribonucleosides (deoxycytidine) and pyrimidine ribonucleosides (uridine) and is partially NBMPR-sensitive. In addition, guanosine, monocarboxylic acids, hexoses or amino acids were not substrates for the transport system. Further studies revealed that the transport system for [3H]thymidine at the BBB has a low affinity (Km 0.20 +/- 0.06 mM), but a relatively high capacity (Vmax 1.06 +/- 0.08 nmol min(-1) g(-1)). Overall, this study is indicative of a NBMPR-sensitive (es) facilitative transport system for [3H]thymidine and the likely presence of a NBMPR-insensitive and/or sodium-dependent transport system of the N2 (cit) type at the blood-brain and blood-CSF barriers of the guinea pig.

Topics: Affinity Labels; Animals; Biological Transport; Central Nervous System; Dose-Response Relationship, Drug; Guinea Pigs; Kinetics; Sensitivity and Specificity; Thioinosine; Thymidine

We have shown previously that the human jejunal brush border membrane expresses both the N1 (cif) and the N2 (cit) Na+-dependent (concentrative) nucleoside transporters but not the Na+-independent (facilitative) nitrobenzylmercaptopurineriboside (NBMPR)-sensitive (es) transporter (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). In the present study, we have demonstrated that when Xenopus laevis oocytes are microinjected with human jejunal mRNA, four nucleoside transporters are expressed simultaneously, namely the N1 and N2 Na+-dependent nucleoside transporters and the es and the NBMPR-insensitive (ei) Na+-independent transporters. The expressed Na+-dependent nucleoside transporters showed substrate specificity identical to that previously described by us using jejunal brush border membrane vesicles (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). The expressed es and ei Na+-independent transporters demonstrated broad substrate selectivity with both purines and pyrimidines capable of inhibiting the uptake of guanosine and thymidine mediated by this transporter. The expressed Na+-dependent nucleoside transporters mediated the transport of their respective nucleoside substrates with a high affinity and a low capacity, whereas the es and the ei transporters mediated the transport of nucleosides with a low affinity and a high capacity. Collectively, these observations suggest that the Na+-independent nucleoside transporters are expressed in the basolateral membrane of the human jejunal epithelium. Based on these data, we hypothesize that the concentrative transporters in the brush border membrane and equilibrative transporters in the basolateral membrane are arranged in series in the human jejunal epithelium to allow efficient vectorial transport of nucleosides from the lumen to the blood. The simultaneous expression of four nucleoside transporters in X. laevis oocytes establishes a basis for molecular cloning of these four human nucleoside transporters.

Topics: Animals; Carrier Proteins; DNA, Complementary; Humans; Intestinal Mucosa; Membrane Proteins; Membrane Transport Proteins; Nucleoside Transport Proteins; Nucleosides; Oocytes; Substrate Specificity; Thioinosine; Transfection; Xenopus laevis

Dipyridamole (37.5 mg/kg, s.c., b.i.d.), a potent inhibitor of nucleoside transport, was administered to guinea pigs for 14 days in order to investigate the effects of: 1) chronic dipyridamole treatment on [3H]nitrobenzylthioinosine ([3H]NBMPR) binding: 2) chronically released endogenous adenosine on adenosine A1 and A2 receptors. Comparisons of the binding capacities (Bmax) and equilibrium dissociation constants (Kd) in vehicle-treated (VTA) and dipyridamole-treated animals (DTA), revealed a 100 percent increase in Kd of [3H]NBMPR binding in the kidney of DTA but not in heart or brain. There were no changes in adenosine A1 or A2 receptor activities in kidney and brain as measured by [3H]R-phenylisopropyladenosine and [3H]5'-N-ethyl-carboxamidoadenosine binding, respectively. The data suggest that cardiac and central nucleoside transporters may be either less susceptible to chronic dipyridamole administration or have a different adaptive mechanism. Also, endogenous adenosine, which may be chronically released upon dipyridamole treatment, has no effect on adenosine receptors.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Biological Transport; Brain; Cardiovascular System; Cell Membrane; Dipyridamole; Guinea Pigs; Kidney; Male; Myocardium; Nucleosides; Rats; Receptors, Purinergic P1; Thioinosine

The alteration in energy metabolic products was analyzed in cultured retinal cells submitted to oxidative stress, hypoxia, glucopenia, or ischemia-like conditions. Ischemia highly reduced cellular ATP and increased AMP formation, without significant changes in ADP. Ischemia induced a significant increase in extracellular adenosine (ADO) and hypoxanthine (HYP), and to a lesser extent inosine (INO). Glucopenia reduced cellular ATP by about two- to threefold, which was not compensated for by AMP formation. Under glucopenia, extracellular ADO and HYP were significantly increased, although a major increase in extracellular INO was observed. 5-(4-Nitrobenzyl)-6-thioinosine (10 microM) reduced extracellular ADO during glucopenia or ischemia by approximately 80%, indicating that ADO accumulation occurs mainly via the transporter. Intracellular ATP, ADP, or AMP and extracellular ADO, INO, or HYP were not apparently changed after oxidative stress or hypoxia. Nevertheless, in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenosine, oxidative stress was shown to increase significantly the accumulation of ADO, which was reduced in the presence of 200 microM alpha,beta-methyleneadenosine 5'-diphosphate, suggesting that ADO accumulation after oxidative stress may result from extracellular degradation of adenine nucleotides. The increase in ADO accumulation resulting from the depletion of cellular ATP was directly related to the release of endogenous glutamate occurring through a Ca2+-independent pathway after ischemia. Increased metabolic products derived from ATP are suggested to exert a modulating effect against excitotoxic neuronal death.

Topics: Adenine; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Cell Hypoxia; Cells, Cultured; Chick Embryo; Enzyme Inhibitors; Glucose; Hypoxanthine; Inosine; Iodoacetates; Iodoacetic Acid; Ischemia; Kinetics; Oligomycins; Oxidative Stress; Retina; Thioinosine

Influx of the purine nucleoside, adenosine, was assessed in erythrocytes from both normal subjects and from subjects with a range of genetically determined erythrocyte disorders from Myanmar. The latter included alpha-thalassemia major (Myanmar variant), beta-thalassemia major (Myanmar variant), beta-thalassemia trait, HbEE and HbAE erythrocytes and two variants of glucose-6-phosphate dehydrogenase (G6PDH) deficiency. Significant reductions (p < 0.01) of adenosine influx were observed in erythrocytes from individuals with alpha- and beta-thalassemia major and severe G6PDH deficiency. Abnormal erythrocytes infected with the malarial parasites, Plasmodium falciparum or Plasmodium vivax, demonstrated a reduction in adenosine transport which correlated with the proportion of abnormal erythrocytes present in the samples obtained. The effect of nitrobenzylthioinosine (NBMPR) on adenosine influx was explored in normal and abnormal erythrocytes. In all these cases, NBMPR completely inhibited the transport of adenosine. However, transport of adenosine into P. falciparum and P. vivax-infected normal erythrocytes and abnormal cells was only inhibited 50-60% by NBMPR. The combination of tubercidin and NBMPR completely blocked adenosine transport into both normal and abnormal erythrocytes infected with either P. falciparum or P. vivax.

Topics: Adenosine; Adult; Affinity Labels; Child; Erythrocytes; Erythrocytes, Abnormal; Female; Glucosephosphate Dehydrogenase Deficiency; Hemoglobinopathies; Humans; Malaria, Falciparum; Malaria, Vivax; Male; Myanmar; Thioinosine; Tubercidin

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

Topics: Affinity Labels; Amino Acid Sequence; Animals; Carrier Proteins; Cloning, Molecular; Dilazep; Dipyridamole; Electrophoresis, Polyacrylamide Gel; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Membrane Proteins; Molecular Sequence Data; Nuclear Proteins; Nucleoside Transport Proteins; Peptide Mapping; Protein Structure, Secondary; Rats; Sequence Alignment; Thioinosine; Vasodilator Agents; Xenopus

MCF-7 cells display both nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative, but not the Na+-dependent, nucleoside transport. Transport of uridine by es is more sensitive to inhibition by purine nucleosides, whereas the ei component is more sensitive to nucleosides without an amino side group, such as inosine and thymidine. When exposed to 10 microM tamoxifen for 5 days, MCF-7 cells displayed a 44% decrease in the total number of NBMPR-binding sites [Bmax from 245000+/-18000 to 136000+/-25000 sites per cell (mean+/-S.E.M.; n=5; P<0.05)], and a 57% decrease in cell growth with no significant change in binding affinities [Kd from 0.37+/-0.05 to 0.45+/-0.08 nM (n=5; P>0.05)]. Kinetic studies of [3H]uridine transport showed a decrease in the Vmax of the es component from 21.7+/-0.3 (n=8) to 8.4 +/- 2.2 microM/s (n=4; P < 0.05), whereas the Vmax of the ei component [from 4.7 +/- 0.5 (n=8) to 5.8 +/- 1.6 microM/s (n=4; P > 0.05)] and Km values for both components [es from 460 +/- 80 to 630 +/- 280 microM (n>/=4; P > 0.05) and ei from 355 +/- 115 to 440 +/- 220 microM (n>/=4; P > 0.05)] did not change significantly. Oestradiol at 100 nM reversed almost completely the NBMPR-binding site decrease and growth retardation in tamoxifen-treated cells. Thus tamoxifen is shown to cause an oestrogen-reversible decrease of es nucleoside transporters in MCF-7 cells.

Topics: Binding Sites; Biological Transport; Breast Neoplasms; Carrier Proteins; Cell Division; Estradiol; Humans; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Protein Binding; Substrate Specificity; Tamoxifen; Thioinosine; Tumor Cells, Cultured; Uridine

Equilibrative nucleoside transport processes in mammalian cells are categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Inhibition of the es process arises from binding of NBMPR to a high-affinity site(s) on the es transporter that can be identified by photoaffinity labeling with [3H]NBMPR. This study examined the equilibrative nucleoside transport processes of cultured human erythroleukemia (K562) cells. The presence of NBMPR binding sites (4.8 +/- 0.9 x 10(5)/cell, Kd = 0.3 nM), together with the identification of polypeptides by specific photolabeling of membranes with [3H]NBMPR, indicated that K562 cells possess es nucleoside transporters (ca 500,000 copies/cell). The photolabeled polypeptides of K562 cells migrated with lower relative mobility (peak M(r) value, 63,000) than did those of human erythrocytes (peak M(r) value, 53,000). This difference in apparent M(r) was abolished by prolonged treatment of membrane proteins with N-glycosidase F, suggesting that equilibrative nucleoside transport in K562 cells and erythrocytes is mediated by the same, or a closely related, es isoform. A cDNA encoding the es nucleoside transporter of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human erythrocytes. hENT-like mRNA species were detected in K562 cells, as well as in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji, SW480), by high-stringency northern analysis with a placental hENT1 probe. A cDNA that encoded a protein identical to hENT1 was isolated by reverse transcriptase polymerase chain reaction with primers specific for hENT1. NBMPR inhibited zero-trans influx of 3H-labeled adenosine, uridine and thymidine by 50% (IC50 values) at 0.4-1.0 nM, confirming the presence of an NBMPR-sensitive (es) transport process, which accounted for 80-90% of total transport activity. The remaining component was identified as the equilibrative NBMPR-insensitive (ei) transport process since it: (i) exhibited low (IC50 > 1.0 microM) sensitivity to NBMPR; (ii) was not concentrative; and (iii) was unchanged by elimination of the sodium gradient. The kinetic parameters (determined at 37 degrees C) for the es- and ei-mediated processes differed markedly. Values for transport of uridine by the es- and ei-mediated processes were, respectively: K(m) = 229 +/- 39 and 1077 +/- 220 microM; Vmax, 186 +/- 31 and 40 +/- 5 pmol/

Topics: Adenosine; Affinity Labels; Binding Sites; Blotting, Northern; Carrier Proteins; Cell Division; Cell Line; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Gene Amplification; Humans; In Vitro Techniques; Leukemia, Erythroblastic, Acute; Membrane Proteins; Nucleoside Transport Proteins; Polymerase Chain Reaction; Protein Binding; Thioinosine; Thymidine; Tubercidin; Uridine

Mammalian equilibrative nucleoside transporters are typically divided into two classes, es and ei, based on their sensitivity or resistance respectively to inhibition by nitrobenzylthioinosine (NBMPR). Previously, we have reported the isolation of a cDNA clone encoding a prototypic es-type transporter, hENT1 (human equilibrative nucleoside transporter 1), from human placenta. We now report the molecular cloning and functional expression in Xenopus oocytes of a cDNA from the same tissue encoding a homologous ei-type transporter, which we designate hENT2. This 456-residue protein is 46% identical in amino acid sequence with hENT1 and corresponds to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in a form bearing a sequence deletion. In addition to placenta, hENT2 is found in brain, heart and ovarian tissue. Like hENT1, hENT2 mediates saturable transport of the pyrimidine nucleoside uridine (Km 0.2+/-0.03 mM) and also transports the purine nucleoside adenosine. However, in contrast with hENT1, which is potently inhibited by NBMPR (Ki 2 nM), hENT2 is NBMPR-insensitive (IC50<1 microM). It is also much less sensitive to inhibition by the coronary vasoactive drugs dipyridamole and dilazep and to the lidoflazine analogue draflazine, properties that closely resemble those reported for classical ei-type transport in studies with intact cells.

Topics: Adenosine; Amino Acid Sequence; Animals; Biological Transport; Cardiotonic Agents; Carrier Proteins; Cloning, Molecular; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; Kinetics; Membrane Proteins; Molecular Sequence Data; Nuclear Proteins; Nucleoside Transport Proteins; Oocytes; Placenta; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Thioinosine; Uridine; Vasodilator Agents; Xenopus

Nucleoside transport processes can be classified by the transport mechanism, e=equilibrative and c=concentrative, by the sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), s=sensitive and i=insensitive, and also by permeant selectivity. To characterize nucleoside transport in acute promyelocytic NB4 cells, nucleoside transport was resolved into different components by selective elimination of transport processes with NBMPR and with Na+-deficient media. Initial transport rates were estimated from time course experiments. For adenosine, uridine, and formycin B, equilibrative transport accounted for approximately 60% of their uptake, with ei and es transport contributing almost equally, and Na+-dependent transport accounting for the remaining 40% of the total uptake. Thymidine uptake was mediated exclusively by equilibrative systems with ei and es systems each contributing 50% to total uptake. Adenosine accumulated above equilibrative concentrations, suggesting that a concentrative transport process was active and/or that metabolism led to adenosine's accumulation. Formycin B, a nonmetabolizable analog, also accumulated in the cells, supporting the concentrative potential of the Na+-dependent transporter. Kinetic analyses also provided evidence for three distinct high affinity transport mechanisms. NBMPR binding assays indicated the presence of two high affinity (Km 0.10 and 0.35 nM) binding sites. In conclusion, NB4 cells express ei and es transport, as well as a large ci transport component, which appears to correspond to cif (f=formycin B or purine selective) nucleoside transport, not previously described in human cells.

Topics: Affinity Labels; Binding Sites; Biological Transport; Carrier Proteins; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Sodium; Thioinosine; Tumor Cells, Cultured

The presence of [3H] NBMPR binding sites in the mitochondrial fraction of rat testis is described. The dissociation constant (KD) from saturation studies was 0.16 +/- 0.04 nM. The association and dissociation rate constants (k1 and k-1) were 3.95 +/- 0.57 x 10(8) M(-1) min(-1) and 0.025 +/- 0.002 min(-1), respectively. The number of binding sites was 2,100 +/- 163 fmols/mg protein. [3H] NBMPR binding was inhibited, in a nanomolar range, by NBMPR (KI= 0.23 +/- 0.02 nM), OH-NBMPR (KI= 2.30 +/- 0.55 nM) and HNBTG (KI= 2.58 +/- 0.33 nM). In the micromolar range, adenosine receptor ligands such as PIA (3.46 +/- 1.36 microM), 2-chloroadenosine (18.81 +/- 3.36 microM) and NECA (8.26 +/- 3.90 microM), and mitochondrial benzodiazepine receptor ligands such as Ro 5-4864 (5.15 +/- 1.82 micrmoM and PK 11195 inhibited the specific binding of [3H] NBMPR. These results suggest the existence of a nucleoside transport system in the mitochondrial fraction of rat testis.

Topics: Affinity Labels; Animals; Binding, Competitive; Carrier Proteins; Male; Mitochondria; Molecular Structure; Radioligand Assay; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Testis; Thioinosine; Tritium

In contrast with other mammalian granulocytes, human granulocytes rapidly cleave the transferrin receptor (TFR) from sheep exosomes. Proteolysis of TFR from exosomes is more rapid and more extensive than that from the sheep reticulocyte cell surface itself, although little difference in cleavage is seen when immunoprecipitates or when immobilized, solubilized receptors from the two sources are compared. Circulating exosomes but not the plasma membrane fraction from seven species of immature red cells or erythropoietic cells show the presence of a peptide of approximately 18 kD recognized by an antibody to the cytoplasmic domain of the TFR. This 18 kD peptide is virtually absent from the corresponding cellular plasma membranes including human reticulocyte membranes. Taken together, the data are consistent with the conclusion that the exosomes released to the circulation from maturing red cells are the principal source of the soluble, circulating, truncated TFR. The granulocyte protease appears to be present on the cell surface and not released into the medium after short (30 min) periods of incubation at 37 degrees C. The protease is inhibited by PMSF but only at high (1 mM) concentrations. Using sheep exosome bound-TFR as substrate, human granulocytes exceed other granulocytes in their capacity to cleave TFR, suggesting that this may be a key factor for the prominent amount of circulating, soluble receptor found in human sera during periods of elevated reticulocyte levels. Human exosomes, unlike those from other species, contain little native size TFR. Truncated receptor containing the cytoplasmic domain being predominant in human exosomes.

Topics: Acetylcholinesterase; Affinity Labels; Animals; Blotting, Western; Cell Membrane; Cytoplasm; Endopeptidases; Enzyme Stability; Granulocytes; Humans; Membrane Proteins; Organelles; Protease Inhibitors; Receptors, Transferrin; Reticulocytes; Sheep; Temperature; Thioinosine; Transferrin; Trypsin

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the 'equilibrative sensitive' or 'es' transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximately Km values of 21 microM, 12.5 microM and 15 microM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Topics: Adenosine; Amino Acid Sequence; Animals; Base Sequence; Biological Transport; Carrier Proteins; Cells, Cultured; Dilazep; Dipyridamole; Genes, myc; Guanosine; Haplorhini; Intestines; Kidney; Membrane Transport Proteins; Molecular Sequence Data; Nucleosides; Pyrimidine Nucleosides; Rats; Recombinant Proteins; Thioinosine; Transfection; Uridine

1. The isolated uterus of the non-pregnant guinea-pig has been suggested to contain P1-, and possibly P2-purinoceptors mediating spasm. The presence of P1-purinoceptors has been confirmed and these receptors have been further characterized. 2. In the presence of the adenosine uptake inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1-purinoceptor antagonist 8-sulphophenyltheophylline (140 microM), the potency order of agonists as spasmogens was: 2 methylthio ATP >> alpha,beta methylene ATP = UTP = ATP >> beta,gamma methylene ATP. This order is not consistent with any single recognised P2-purinoceptor subtype. 3. Indomethacin (1 microM) treatment abolished responses to 2 methylthio ATP, alpha,beta methylene ATP and UTP, while spasm to ATP was significantly inhibited. When the endometrial and circular smooth muscle cell layers were removed, spasmogenic responses to ATP, 2 methylthio ATP, alpha,beta methylene ATP and UTP were significantly reduced. 4. 2-methylthio ATP was able to cause desensitization to itself, but not to UTP, indicating that these agonists act at different receptor sites. 5. The P2-purinoceptor antagonist, suramin antagonized 2 methylthio ATP with a PA2 of 5.9 +/- 0.3. Suramin was also an antagonist of ATP and UTP. In the case of ATP, the antagonism was not dependent on suramin concentration, while for UTP the interaction appeared to be non-equilibrium. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) had no effect on spasm to ATP, UTP or 2 methythio ATP. 6. In the presence of indomethacin, responses to ATP were unaffected by 8-sulphophenyltheophylline (140 microM) or by suramin (100 microM), but PPADS (10 microM) antagonized ATP. 7. These results suggest that the isolated uterus of the non-pregnant guinea-pig contains a mixture of P2-purinoceptors. P2U- (or UTP-selective pyrimidinoceptors) and P2Y-purinoceptors appear to be present, probably mainly located on the endometrial or circular smooth muscle layer. Activation of these receptors leads to spasm via increases in prostanoid generation. There appears also to be a third class of non-P2X-, non p2Y-purinoceptor present, at which ATP is an agonist and PPADS is an antagonist, located on the longitudinal smooth muscle, activation of which causes spasm independent of changes in prostanoids.

Topics: Adenosine Triphosphate; Animals; Female; Guinea Pigs; Indomethacin; Muscle Contraction; Muscle, Smooth; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Receptors, Purinergic P1; Receptors, Purinergic P2; Spasm; Suramin; Theophylline; Thioinosine; Thionucleotides; Uridine Triphosphate; Uterus

The kinetic characteristics of [3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of KT and Vmax, and the sensitivities with which nucleoside transport inhibitors blocked [3H] adenosine accumulations were determined in cultured human fetal astrocytes. KT and Vmax values for accumulations of [3H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and the adenosine kinase inhibitor 5'-iodotubercidin (ITU) were 6.2 microM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components, respectively. In the presence of EHNA and ITU, where < 4% of accumulated [3H] adenosine was metabolized, transport per se was measured, and kinetic values for KT and Vmax were 179 microM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [3H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of "concentrative" uptake in that the intracellular levels of [3H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [3H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H]-adenosine transport; for the inhibitor-sensitive components the IC50 values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC50 values were 2.5 microM for NBI, 5.1 microM for dilazep, and 39.0 microM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.

Topics: Adenine; Adenosine; Affinity Labels; Astrocytes; Biological Transport; Carrier Proteins; Cells, Cultured; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fetus; Humans; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine; Tritium; Tubercidin; Vasodilator Agents

Rat liver parenchymal cells express Na(+)-dependent and Na(+)- independent nucleoside transport activity. The Na(+)-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227-233]. This transport activity shows apparent K(m) values for uridine in the range 8-13 microM and a Vmax of 246 pmol of uridine per 3 min per 10(5) cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na(+)-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na(+)-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na(+)-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na(+)-dependent transport systems. Na(+)-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na(+)-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na(+)-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na(+)-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na(+)-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

Topics: Affinity Labels; Animals; Biological Transport; Ethylmaleimide; Humans; Liver; Male; Nucleosides; Rats; Rats, Wistar; Sodium; Thioinosine; Tumor Cells, Cultured

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.

Topics: Adenosine; Biological Transport; Carrier Proteins; Cell Line; Endothelium, Vascular; Humans; Hypoxanthine; Hypoxanthines; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine

The present investigation characterizes the nucleoside transporters in bovine adrenomedullary endothelial cells and their possible regulation by the action of protein kinases A and C to establish comparisons with the nucleoside transport system in chromaffin cells. The nucleoside transport proved to be a nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with high affinity for adenosine. These endothelial cells had a high density of nucleoside transporters (660,000 +/- 130,000 transporters/ cell), measured by NBTI binding, and the efficiency was close to 2 adenosine molecules internalized transporter-1.s-1. The stimulation of the cells with bradykinin and P1,P4-di(adenosine-5')tetraphosphate, which raise the intracellular Ca2+ concentration, did not modulate the adenosine transport. When the cells were stimulated with signals coupled to adenosine 3',5'-cyclic monophosphate intracellular production, such as norepinephrine and isoproterenol, the adenosine transport was not modified. Furthermore, the treatment of the cells with direct activators of both protein kinases A and C had no effect on adenosine transport, in contrast to that reported in chromaffin cells.

Topics: Adenosine; Adrenal Glands; Animals; Biological Transport; Carrier Proteins; Cattle; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Endothelium; Protein Kinase C; Thioinosine

The actions of adenosine, adenosine deaminase, the adenosine uptake blocker, S-(p-nitrobenzyl)-6-thioinosine, and of the adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, on electrically evoked [3H]acetylcholine release were investigated in rat phrenic nerve-hemidiaphragm preparations. Adenosine deaminase (0.25-2.5 U/ml) increased [3H]acetylcholine release. S-(p-Nitrobenzyl)-6-thioinosine (3-30 microM) and erythro-9(2-hydroxy-3-nonyl)adenine (25 nM-50 microM) caused biphasic effects on [3H]acetylcholine release: at low concentrations S-(p-nitrobenzyl)-6-thiomosine (5 microM) and erythro-9(2-hydroxy-3-nonyl)adeNine (50 nM) decreased [3H]acetylcholine release, and at concentrations higher than 10 microM S-(p-nitrobenzyl)-6-thioinosine and 0.5 microM for erythro-9(2-hydroxy-3-nonyl)adenine facilitated [3H]acetylcholine release. Both S-(p-nitrobenzyl)-6-thioinosine-induced inhibition and facilitation of [3H]acetylcholine release resulted from extracellular endogenous adenosine accumulation, because they were blocked after inactivation of endogenous adenosine with adenosine deaminase (0.5 U/ml). The inhibitory actions of both S-(p-nitrobenzyl)-6-thioinosine (5 microM) and erythro-9(2-hydroxy-3-nonyl)adenine (50 nM) were antagonized by the A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), whereas the blockade of A2a adenosine receptors with PD 115,199 (25 nM) prevented the facilitatory effects of S-(p-nitrobenzyl)-6-thioinosine (30 microM) and erythro-9(2-hydroxy-3-nonyl)adenine (50 microM). The adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine (25 nM), potentiated the effect of S-(p-nitrobenzyl)-6-thioinosine (3-30 microM), and this adenosine uptake blocker, when applied at a concentration (3 microM) that by itself was devoid of effect, potentiated both the inhibitory (25 nM) and excitatory (0.5 microM) effects of erythro-9(2-hydroxy-3-nonyl)adenine, on evoked [3H]acetylcholine release. Exogenously applied adenosine (10-500 microM) had biphasic effects similar to those of S-(p-nitrobenzyl)-6-thioinosine and erythro-9(2-hydroxy-3-nonyl)adenine. Adenosine (30 microM) reduction of evoked [3H]acetylcholine release was prevented after pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM); when applied at high concentrations (100-500 microM), adenosine consistently increased evoked [3H]acetylcholine release in a PD 115,199 (25 nM)-sensitive manner. It is concluded that both uptake and deaminatio

Topics: Acetylcholine; Adenine; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Affinity Labels; Animals; Deamination; Diaphragm; Electric Stimulation; Enzyme Inhibitors; Female; In Vitro Techniques; Male; Motor Neurons; Phrenic Nerve; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, Purinergic P1; Thioinosine

Purification of equilibrative nucleoside transporters has been hampered by the functional instability of the detergent-solubilized proteins. A variety of nonionic detergents were compared with octylglucoside (the most commonly used detergent in this regard) for their abilities to solubilize functionally stable nucleoside transporter proteins from Ehrlich cell plasma membranes. Transporter stability was assessed through the binding of the specific probe [3H]nitrobenzylthioinosine to freshly solubilized and stored (48 h/6 degrees C) preparations. The most promising detergents were decylmaltoside and cyclohexylbutylmaltoside, both of which, like octylglucoside, solubilized over 70% of the transporters from the membrane. Decylmaltoside- and cyclohexylbutylmaltoside-solubilized transport proteins retained 61 and 83%, respectively, of their [3H]-nitrobenzylthioinosine binding activity upon storage, compared to about 30% using octylglucoside. Decylmaltoside was also superior to octylglucoside in its capacity to solubilize the transporter in a state that retained its high affinity for the transport inhibitors dilazep (Ki = 11 nM, vs 75 nM in octylglucoside) and dipyridamole (Ki = 260 nM, vs 12 microM in octylglucoside). Reconstitution studies indicated that both the decylmaltoside- and cyclohexylbutylmaltoside-solubilized transporters were capable of mediating the uptake of [3H]uridine. Decylmaltoside was superior to cyclohexylbutylmaltoside, however, in both the enhanced transport activity of the resulting proteoliposomes (Vi = 21 pmol/mg/s vs 13 pmol/mg/s, respectively) and the lower nonmediated uptake observed in the decylmaltoside-derived vesicles (27% of total uptake at 4 min incubation). Nevertheless, cyclohexylbutylmaltoside may be useful in initial solubilization procedures due to its ability to selectively solubilize the nucleoside transporter from the plasma membrane. The rational use of these detergents will enable a more extensive purification of functional nucleoside transporters.

Topics: Animals; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane; Detergents; Glucosides; In Vitro Techniques; Kinetics; Ligands; Male; Membrane Proteins; Mice; Molecular Probes; Nucleoside Transport Proteins; Nucleosides; Solubility; Thioinosine

We examined the effects of Ca(2+)-free medium containing 20 mM Mg2+, a non-selective adenosine receptor antagonist, theophylline, and adenosine transport inhibitors, dipyridamole and nitrobenzylthioinosine, on high K(+)-evoked spreading depression, glutamate, and taurine release from the rat hippocampus using brain microdialysis. High K+ alone perfusion evoked spreading depression and increased glutamate release followed by taurine efflux. Perfusion of Ca(2+)-free medium with high K+ never evoked spreading depression and decreased the high K(+)-evoked taurine release. Perfusion of theophylline (1 mM) increased the occurrence of high K(+)-evoked spreading depression and glutamate release, but did not modify taurine release. In contrast, simultaneous perfusion of dipyridamole (100 microM) and nitrobenzylthioinosine (50 microM) reduced the occurrence of spreading depression and the high K(+)-evoked glutamate release, but enhanced significantly the taurine efflux. These findings suggest that endogenous taurine with adenosine may have neuroprotective actions against high K(+)-evoked glutamate release and spreading depression in the rat hippocampus, in addition to its osmoregulatory action.

Topics: Adenosine; Animals; Biological Transport; Calcium; Cortical Spreading Depression; Dipyridamole; Glutamic Acid; Hippocampus; Male; Microdialysis; Potassium; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Taurine; Theophylline; Thioinosine

We investigated the effects of dietary MaxEPA (a major source of eicosapentaenoic acid in fish oil) supplementation on blood pressure (BP) responses and heart rate (HR) of Dahl salt-sensitive (SS) rats fed low (0.4% NaCl) and high (8.0% NaCl) sodium diets. During a four week treatment period, BP remained normotensive in rats on low salt diet but was significantly elevated in those on high salt diet, causing 50% mortality. MaxEPA diminished the BP elevation and prevented the high salt-induced mortality. HR was not affected by either salt diet alone but was reduced in the presence of MaxEPA. At the end of the treatment period, the distribution of [3H]nitrobenzylthioinosine ([3H]NBMPR) binding, a putative marker of adenosine transport and metabolism, was estimated in selected rat tissues in order to evaluate the role of the purinergic system in the BP lowering effect of MaxEPA. Maximal [3H]NBMPR binding capacity (Bmax) in the kidney and platelets were 39% and 82% lower, respectively, in rats on high salt diet than in those on low salt diet. MaxEPA significantly blunted the decrease in Bmax in the kidney but not in platelets and increased Bmax in heart (48%) of low salt group. There were no changes in dissociation constants (Kd). The results suggest that MaxEPA can attenuate salt-induced hypertension, reduce salt-induced mortality and protect the integrity of kidney NBMPR binding sites in salt-induced hypertension.

Topics: Affinity Labels; Animals; Binding Sites; Blood Platelets; Blood Pressure; Body Weight; Dietary Fats, Unsaturated; Docosahexaenoic Acids; Drug Combinations; Eicosapentaenoic Acid; Fatty Acids, Omega-3; Heart Rate; Hypertension; Kidney; Male; Rats; Rats, Sprague-Dawley; Sodium Chloride, Dietary; Thioinosine

1. The effects of analogues of adenosine and ATP on noradrenaline release elicited by electrical stimulation (5 Hz, 2700 pulses) were studied in superfused preparations of rat tail artery. The effects of purinoceptor antagonists, of adenosine deaminase and of adenosine uptake blockade were also examined. Noradrenaline was measured by h.p.l.c. electrochemical detection. 2. The A1-adenosine receptor agonist, N6-cyclopentyladenosine (CPA; 0.1-100 nM) reduced, whereas the A2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3-30 nM) increased evoked noradrenaline overflow. These effects were antagonized by the A1-adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 nM) and the A2-adenosine receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), respectively. The P2Y-purinoceptor agonist, 2-methylthio-ATP (1-100 microM) reduced noradrenaline overflow, an effect prevented by the P2-purinoceptor antagonist, cibacron blue 3GA (100 microM) and suramin (100 microM). 3. Adenosine deaminase (2 u ml-1), DMPX (100 nM) and inhibition of adenosine uptake with S-(p-nitrobenzyl)-6-thioinosine (NBTI; 50 nM) decreased evoked noradrenaline overflow. DPCPX alone did not change noradrenaline overflow but prevented the inhibition caused by NBTI. The P2Y-purinoceptor antagonist, cibacron blue 3GA (100 microM) increased evoked noradrenaline overflow as did suramin, a non-selective P2-antagonist. 4. It is concluded that, in rat tail artery, inhibitory (A1 and P2Y) and facilitatory (A2A) purinoceptors are present and modulate noradrenaline release evoked by electrical stimulation. Endogenous purines tonically modulate noradrenaline release through activation of inhibitory P2Y and facilitatory A2A purinoceptors, whereas a tonic activation of inhibitory A1 purinoceptors seems to be prevented by adenosine uptake.

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Arteries; Male; Norepinephrine; Phenethylamines; Rats; Rats, Wistar; Receptors, Purinergic; Tail; Thioinosine; Thionucleotides; Xanthines

Adenosine 5'-triphosphate (ATP), beta, gamma-methylene ATP and alpha, beta-methylene ATP produced relaxation of carbachol-precontracted isolated trachealis muscle from the guinea-pig in the presence of indomethacin (2.8 microM) and the adenosine uptake inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI; 300 nM). The potency order for ATP and analogues was: beta, gamma-methylene ATP = ATP > alpha, beta-methylene ATP = uridine 5'-triphosphate (UTP) = 2-methylthio ATP. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) also caused relaxation. Relaxations to ATP, beta, gamma-methylene ATP, adenosine and NECA were not inhibited by the P2 purinoceptor antagonist suramin (100 microM), but were inhibited by the P1 purinoceptor antagonist 8-sulphophenyltheophylline (140 microM). NBTI significantly potentiated adenosine and ATP but not beta, gamma-methylene ATP or NECA. The data are compatible with the idea that beta, gamma-methylene ATP could interact directly with P1 purinoceptors while ATP acts indirectly at P1 purinoceptors via conversion to adenosine.

Topics: Adenosine Triphosphate; Animals; Carbachol; Female; Guinea Pigs; In Vitro Techniques; Indomethacin; Male; Muscle Contraction; Muscle Relaxation; Parasympatholytics; Receptors, Purinergic P1; Receptors, Purinergic P2; Suramin; Theophylline; Thioinosine; Trachea

Adenosine receptor signal transduction mechanisms have previously been characterized in Syrian hamster smooth muscle DDT1 MF-2 cells but adenosine transport in these cells has not. DDT1 MF-2 cells possess a high density (370,000 sites/cell) of high affinity (Kd value of 0.26 nM) binding sites for [3H]nitrobenzylthioinosine, a marker for the equilibrative and inhibitor-sensitive subtype of nucleoside transporters. Transport of [3H]adenosine was insensitive to Na+ and was inhibited by the nucleoside transport inhibitors nitrobenzylthioinosine, dilazep and dipyridamole with IC50 values of 1, 13 and 270 nM, respectively. Propentofylline, a neuroprotective compound that can inhibit nucleoside transporters, is rapidly metabolized in vivo to the racemate (+/-)-A720287. Based on recent findings that some transport inhibitors exhibit marked stereoselectivity, we tested the degree to which individual stereoisomers of (+/-)-A720287 affect adenosine transport. Propentofylline inhibited [3H]adenosine transport in DDT1 MF-2 cells with an IC50 value of 24 microM. (+/-)-A720287 and the individual stereoisomers (+)-833791 and (-)-844261 had similar potency to propentofylline for inhibition of [3H]adenosine transport in DDT1 MF-2 cells as well as in clonal mouse leukemia L1210/B23.1 cells, cells which possess only the equilibrative and inhibitor-sensitive subtype of nucleoside transporters. Thus, the neuroprotective effects of propentofylline may be due, in part, to the primary metabolites of propentofylline.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Cricetinae; Dilazep; Dipyridamole; Genital Neoplasms, Male; Kinetics; Leiomyosarcoma; Leukemia L1210; Male; Mesocricetus; Muscle, Smooth; Rats; Thioinosine; Tumor Cells, Cultured; Vas Deferens; Xanthines

Cerebral ischemia is often followed by a period of delayed hypoperfusion that may contribute to tissue injury. We tested the hypothesis that augmentation of interstitial adenosine can improve tissue perfusion under this condition 10 min global ischemia was produced in two groups of isoflurane-anesthetized newborn pigs by occlusion of subclavian and brachiocephalic arteries, and changes in local cortical blood flow and cortical interstitial purine metabolites were measured using the combined hydrogen clearance-microdialysis technique. In one group, the dialysis probe was perfused with artificial cerebrospinal fluid buffer containing nitrobenzyl-thioinosine (NBT1, 100 mumol/l), a competitive inhibitor of adenosine transport. In the untreated group (n = 9), baseline cortical blood flow (39 +/- 3 ml/min/100 g) was depressed by 51 +/- 5% and 42 +/- 6% at 40 and 60 min, respectively, of postischemic reperfusion. NBTI increased baseline interstitial adenosine levels 2.4-fold which increased baseline cortical blood flow 1.5-fold to 60 +/- 4 ml/min/100 g, and increased both absolute adenosine levels as well as adenosine as a percentage of total purine metabolites throughout ischemia and reperfusion. As a result, the extent of postischemic hypoperfusion was significantly lessened in NBTI-treated animals (n = 9), with reductions in cortical blood flow of only 28 +/- 3% and 24 +/- 5% at 40 and 60 min of reperfusion, respectively. These results indicate that inhibition of adenosine transport by NBTI elevates interstitial adenosine concentration during and following cerebral ischemia, and concomitantly improves cortical perfusion in the post-ischemic period. The latter effect may contribute to the documented neuroprotective efficacy of adenosinergic therapy in cerebral ischemia.

Topics: Adenosine; Animals; Biological Transport; Brain; Brain Ischemia; Cerebrovascular Circulation; Purines; Reperfusion; Swine; Thioinosine

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Apoptosis; Chromaffin Cells; Deoxyadenosines; Dilazep; Enzyme Inhibitors; Epinephrine; Mutagens; Norepinephrine; Phosphorylation; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Thioinosine; Tubercidin; Vasodilator Agents

The synthesis of S4-substituted nucleosides possessing the imidazo- and v-triazolo[4,5-d]pyridazine ring systems was undertaken and the compounds prepared were evaluated as inhibitors of nucleoside transport into human erythrocytes. 1-(2,3,5-Tri-O-acetyl-beta-D-ribofuranosyl)-v-triazolo[4,5-d]pyridazine- 4 (5H)-thione and 1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4 (5H)-thione were each synthesized by two different routes and served as precursors for the title analogues. The nitrobenzylmercaptopurine riboside (NBMPR) analogues, 4-(p-nitrobenzylthio)-1-(beta-D-ribofuranosyl)imidazo[4,5-d]pyrida zine and 4-(p-nitrobenzylthio)-1-(beta-D-ribofuranosyl)-v-triazolo[4,5-d]py ridazine, inhibited the transport of adenosine, but were approximately 4- and 28-fold less active, respectively, than NBMPR and nitrobenzylthioformycin, known potent and specific inhibitors of carrier-mediated transport.

Topics: Affinity Labels; Biological Transport; Erythrocytes; Humans; Nucleosides; Thioinosine; Thionucleosides

Specific binding of the nucleoside transporter probe, [3H]nitrobenzylthioinosine, ([3H]NBMPR) was measured in washed guinea pig cardiac, renal and forebrain membranes after 14 days of treatment with dipyridamole (37.5 mg/kg, s.c., b.i.d.) or vehicle. When compared to values in vehicle-treated animals, a 100 percent increase in equilibrium dissociation constant (Kd) was observed in the kidney of dipyridamole-treated animals (0.51 +/- 0.04 to 1.0 +/- 0.06, p < 0.01). The maximal binding capacity (Bmax) was unaltered. No changes were observed in the heart or forebrain. The increase in Kd suggests that chronic dipyridamole treatment decreases the apparent binding affinity of NBMPR for kidney nucleoside transporters. Cardiac and brain nucleoside transporters may be either less susceptible to chronic dipyridamole administration or have a different adaptive mechanism.

Topics: Animals; Binding Sites; Brain; Dipyridamole; Guinea Pigs; Kidney; Male; Myocardium; Radioligand Assay; Rats; Rats, Sprague-Dawley; Thioinosine; Tritium

The iris-ciliary body (ICB) is a site of action for topically applied antiglaucoma drugs. Moreover, adenosine has been implicated as a modulator of aqueous humor dynamics. The present study compares the binding of the nucleoside transporter probe, [3H]nitrobenzylthioinosine ([3H]NBMPR), to homogenates prepared from rabbit ICB and a cultured rabbit nonpigmented ciliary epithelial cell line (NPE) to determine whether NPE can be used as an experimental model to study the nucleoside transporter. Linear transformation of the saturation binding data revealed that [3H]NBMPR binds to a homogeneous population of binding sites with similar binding affinities (Kd = 0.3 +/- 0.1 and 0.6 +/- 0.1 nM in NPE and ICB, respectively). However, the maximal binding capacity in NPE (Bmax = 288 +/- 54 fmol/mg protein) was significantly higher than that in ICB (Bmax = 154 +/- 17 fmol/mg protein). Selected inhibitors of the nucleoside transport system and structural analogs of adenosine inhibited the binding in both homogenate preparations with a similar rank order of potency: NBMPR > DPY > CV-1808 > CHA > R-PIA > S-PIA > 2-CADO > NECA. The results suggest that NPE is a useful model which could be used for characterizing the nucleoside transporter in ICB and for the screening of nucleoside transport inhibitors as potential antiglaucoma drugs.

Topics: Adenosine; Affinity Labels; Animals; Binding Sites; Binding, Competitive; Blood Proteins; Carrier Proteins; Cells, Cultured; Ciliary Body; Dipyridamole; Epithelium; Female; Iris; Male; Membrane Proteins; Nucleoside Transport Proteins; Pigment Epithelium of Eye; Rabbits; Thioinosine

Adenosine mechanisms are altered in brain stem nuclei associated with cardiovascular control in spontaneously hypertensive rats (SHR). Therefore, in the present study we used a number of techniques to compare the binding of the adenosine transport inhibitor [3H]nitrobenzylthioinosine ([3H]NBMPR) as well as adenosine deaminase immunoreactivity (ADA-IR) in brain stems and nodose ganglia of SHR and age-matched normotensive Donryu rats (DRY). Saturation binding revealed a single class of [3H]NBMPR binding sites in the dorsal brain stem of both strains, with Kd and Bmax values of 65 +/- 9 pmol/L and 282 +/- 31 fmol/mg protein, respectively, in SHR and 129 +/- 2 pmol/L and 217 +/- 23 fmol/mg protein in DRY. The Kd for [3H]NBMPR was significantly lower in SHR than in DRY. In competition assays, NBMPR, dilazep, dipyridamole, and adenosine displaced [3H]NBMPR binding, with Kd values of 0.21 +/- 0.04, 57.16 +/- 16.20, 1340 +/- 100, and 87000 +/- 12500 nmol/L, respectively, in DRY and 0.17 +/- 0.04, 28.24 +/- 3.60, 621 +/- 100, and 32000 +/- 6820 in SHR. Kd values for all displacers were lower in SHR; however, only values for dipyridamole and adenosine reached statistical significance. Autoradiography of adenosine transport sites with [3H]NBMPR revealed that unilateral nodose ganglionectomy reduced [3H]NBMPR binding on the denervated side of the nucleus tractus solitarius by 20.6 +/- 1.1% in DRY and 18.7 +/- 2.3% in SHR. The density of [3H]NBMPR binding in nodose ganglia was significantly lower in SHR (0.99 +/- 0.06 Bq/mm2) than in DRY (1.25 +/- 0.08). Immunohistochemical studies demonstrated ADA-IR in the dorsal vagal complex, associated with both nerve cells and fibers. Measurement of ADA-IR in the dorsal vagal complex with an 125I-labeled secondary antibody revealed a significantly higher level of ADA-IR in SHR (122%) than in DRY. In the nodose ganglia, ADA-IR was associated with a population of vagal perikarya. The present study helps provide a molecular explanation for the previously reported impaired cardiovascular responses to intra-nucleus tractus solitarius microinjection of adenosine in hypertensive rats.

Topics: Adenosine; Adenosine Deaminase; Animals; Autoradiography; Binding Sites; Brain Stem; Male; Rats; Rats, Inbred SHR; Species Specificity; Thioinosine

Using quantitative receptor autoradiographic methods we have examined A1 adenosine receptors, adenosine uptake sites, benzodiazepine receptors, NMDA, AMPA, and kainic acid receptors in temporal lobes removed from patients suffering from complex partial seizures and in normal control post-mortem temporal cortex. Binding to A1 adenosine receptors and NMDA receptors was reduced in epileptic temporal cortex, while the other neurochemical parameters were unchanged. The reason for this A1 receptor loss is unclear as it occurred in both idiopathic and symptomatic cases and thus may be a consequence rather than an initial cause of seizures. However, because adenosine is a powerful anticonvulsant substance, loss of anticonvulsant A1 receptors may contribute to the human epileptic condition. It is also possible that the observed differences in A1 binding are due to autopsy vs. biopsy changes in the levels of A1 adenosine receptors.

Topics: Adolescent; Adult; Aged; Autoradiography; Cell Count; Epilepsy, Temporal Lobe; Female; Hippocampus; Humans; Male; Middle Aged; Receptors, Amino Acid; Receptors, GABA; Receptors, Purinergic P1; Reference Values; Temporal Lobe; Thioinosine

Nucleoside transport inhibitors that cross the blood-brain barrier may be able to potentiate the neuroprotective effects of adenosine. We tested whether nitrobenzylthioinosine (NBMPR) crosses the blood-brain barrier in three types of experiments. First, intravenous injection of [3H]NBMPR and [14C]sucrose was performed. Brain volume of distribution and brain delivery were greater for [3H]NBMPR than for [14C]sucrose. Second, rats were injected intraperitoneally with NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR, or vehicle. Perchloric acid extracts of brains from rats treated with NBMPR-P inhibited [3H]NBMPR binding in competition binding assays nearly 3-fold more than extracts from brains of vehicle-treated animals. Third, cerebrospinal fluid (CSF) extracted from rats treated with NBMPR-P (10 mg/kg i.p.) contained 24.1 +/- 4.4 nM NBMPR while levels were undetectable in CSF from vehicle-treated rats. From these data, we conclude that NBMPR crosses the blood-brain barrier.

Topics: Animals; Binding, Competitive; Blood-Brain Barrier; Brain; Injections, Intraperitoneal; Injections, Intravenous; Male; Rats; Rats, Sprague-Dawley; Sucrose; Thioinosine; Thionucleotides

Tamoxifen inhibits the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) to human MCF-7 breast cancer cells with an IC50 of 8 microM. Tamoxifen at 30 microM changed the apparent Kd for [3H]NBMPR binding from 0.63 +/- 0.12 to 4.75 +/- 0.58 nM, with little effect on the Bmax (311000 +/- 76000 and 263000 +/- 46000 sites per cell for untreated and tamoxifen-treated cells respectively). Corresponding to this decrease in binding of [3H]NBMPR in the presence of tamoxifen was an inhibition of NBMPR-sensitive equilibrative transport of 50 microM [3H]uridine (IC50 7-10 microM). In the presence of 15 microM tamoxifen, the apparent K(m) for [3H]uridine transport was increased from 390 +/- 30 to 1500 +/- 250 microM, with no change in Vmax (12.0 +/- 0.1 and 11.3 +/- 4.3 microM/s for untreated and tamoxifen-treated cells respectively). The inhibitory effect of tamoxifen on NBMPR-sensitive equilibrative uridine transport was specific, as similar results were also observed in HL-60 leukaemia and EL4 lymphoma cells. Furthermore a similar concentration of tamoxifen had no effect on the NBMPR-insensitive equilibrative transport of uridine in MCF-7, HL-60 and Morris 7777 hepatoma cells, and on the Na(+)-dependent transport of uridine in murine splenocytes. In this paper we demonstrate that tamoxifen by itself might have some antiproliferative effects through inhibition of DNA synthesis by blocking the nucleoside salvage pathway.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Cells, Cultured; Humans; Kinetics; Membrane Proteins; Mice; Neoplasms; Nucleoside Transport Proteins; Nucleosides; Protein Binding; Sodium; Spleen; Tamoxifen; Thioinosine; Uridine

[3H]L-Adenosine, an enantiomer of the physiological D-adenosine, was shown previously to be taken up and released, at least in part, through nucleoside transporters in rat brain preparations. In the present study, we used clonal smooth muscle DDT1 MF-2 cells that contain almost exclusively equilibrative inhibitor-sensitive (es) nucleoside transporters to test the hypothesis that L-adenosine is a permeant for these bidirectional nucleoside transporters. DDT1 MF-2 cells accumulated approximately 3 times more [3H]D- than [3H]L-adenosine; 10 microM nitrobenzylthioinosine significantly (P < 0.01) inhibited the accumulation of [3H]D-adenosine by 86% and of [3H]L-adenosine by 63%. The IC50 values for the nitrobenzylthioinosine-sensitive portions of [3H]L- and [3H]D-adenosine accumulation were 1.6 and 2.0 nM, respectively. [3H]D-Adenosine accumulation was significantly (P < 0.05) inhibited by up to 72% with L-adenosine and [3H]L-adenosine accumulation was significantly (P < 0.01) inhibited by up to 52% with D-adenosine. Release of accumulated [3H]L-adenosine was temperature- and time-dependent, and was significantly (P < 0.05) reduced by 47% with dipyridamole, 39% with dilazep, and 45% with nitrobenzylthioinosine; the apparent IC50 value for nitrobenzylthioinosine was < 1 nM. These data indicate that a significant proportion of [3H]L-adenosine uptake and release in DDT1 MF-2 cells is mediated by es nucleoside transporters.

Topics: Adenosine; Animals; Carrier Proteins; Cells, Cultured; Cricetinae; Dose-Response Relationship, Drug; Male; Membrane Proteins; Mesocricetus; Muscle, Smooth; Nucleoside Transport Proteins; Stereoisomerism; Thioinosine; Tritium

Endogenous purinergic systems are important in spinal mechanisms of antinociception. Antinociception induced by spinal mu opioid receptor-selective agonists, in particular, appears to be mediated in part by opioid-stimulated adenosine release. Nucleoside transport system(s) have been implicated both in adenosine release and in its reuptake at spinal sites. The present investigations were designed to determine the significance of nucleoside transport system(s) inhibition in vivo in antinociception induced by opioids administered intrathecally in mice. Dilazep, but not dipyridamole or s (4-nitrobenzyl)-6-thioinosine, nucleoside transport system(s) inhibitors, induced time- and dose-dependent antinociception in the tail-flick test, putatively via spinal adenosine reuptake inhibition. Each nucleoside transport system(s) inhibitor, at doses that have no significant effects alone, enhanced adenosine-mediated antinociception when coadministered intrathecally. Concurrent treatment of mice with opioid receptor-selective agonists and nucleoside transport system(s) inhibitors had varying effects on antinociception, depending on the timing of the nucleoside transport inhibitor. In general, antinociception induced by mu opioid receptor-selective agonists was inhibited by pretreatment, was not affected after coadministration and was enhanced by post-treatment, with nucleoside transport system(s) inhibitors. In contrast, antinociception induced by delta opioid receptor-selective agonists was enhanced by nucleoside transport system(s) inhibitors in all treatment protocols. These results provide in vivo evidence that alterations in adenosine movements into or out of spinal neurons via the nucleoside transport systems can induce antinociception and enhance or inhibit opioid-mediated antinociception. These data also support the hypothesis that adenosine plays significant but independent roles in antinociception induced by mu and delta opioid receptor-selective agonists.

Topics: Adenosine; Analgesics; Animals; Biological Transport; Dilazep; Dipyridamole; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Narcotics; Nucleosides; Receptors, Opioid; Thioinosine; Time Factors

2-Chlorodeoxyadenosine (2-CdA) is an important agent in the treatment of hairy cell leukemia and chronic lymphocytic leukemia (CLL). Others have reported that levels of 2-CdA phosphates present in human leukemia cells decline rapidly when the cells are in 2-CdA-free medium (Santana et al. J Clin Oncol 1991; 9: 416-422). In the present study, time-courses of 2-CdA loss from CLL cells were biexponential: the mean half-life of the initial phase was 0.30 +/- 0.18 h; the presence of 0.5 microM nitrobenzylthioinosine (NBMPR, a classical inhibitor of nucleoside transport) in the suspending medium, significantly decreased the initial rate of 2-CdA efflux (mean half-life, 0.43 +/- 0.22 h). As a consequence, AUCs (areas under time-course plots) were significantly higher in the NBMPR-treated cells (4.56 +/- 2.01 pmol.h/10(6) cells, n = 19) than in untreated control cells (3.83 +/- 1.74 pmol.h/10(6) cells; n = 19). 2-CdA was the principal efflux product released into the medium from 2-CdA-loaded CLL cells. We conclude that nucleoside transport processes contribute to the efflux of 2-CdA from CLL cells and that NBMPR may be useful as a retentive agent.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Biological Transport; Cladribine; Female; Half-Life; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Thioinosine

Prolonged high-frequency orthodromic stimulation of superior cervical ganglia is known to result in increased acetylcholine (ACh) synthesis and ACh content after the period of stimulation. In a previous study, we provided evidence to suggest that adenosine acts as an extracellular signal to activate this increased ACh synthesis and we proposed that the source of that adenosine might be postsynaptic. Thus, the purpose of the present study was to test whether direct stimulation of the post-ganglionic nerves could affect ganglionic ACh content. Antidromic conditioning of ganglia (15 Hz, 45 min) did not affect significantly their ACh content. However, if ganglia were allowed a 15-min rest period after this antidromic conditioning, their ACh stores were increased by 20%; a similar increase was induced by 4-Hz stimulation before the rest period. During the 15-Hz antidromic stimulation, ACh release was not clearly increased above the basal level, suggesting that preganglionic nerve endings were not stimulated to an extent that could explain the increased ACh content. Orthodromic stimulation (5 Hz) of ganglia 15 min after they had been subjected to antidromic conditioning (15 Hz, 45 min) showed increased ACh release in comparison with that from control unconditioned ganglia. Moreover, the extra ACh released by the conditioned ganglia was quantitatively similar to the increase in the ACh stores, as if most, or all, of the additional ACh was released by preganglionic stimulation. If the antidromic conditioning and the rest period were done during perfusion with Ca(2+)-free medium, the ganglia did not accumulate extra ACh.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Adenosine; Animals; Calcium; Carrier Proteins; Cats; Culture Media; Dipyridamole; Electric Stimulation; Female; Ganglia, Sympathetic; Male; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine

We have developed and utilized a photoaffinity probe to identify the adenosine transporter in cardiac sarcolemmal (SL) vesicles. The probe is an azidosalicylate derivative of adenosine made by reacting adenosine with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA). Following synthesis and radiolabeling of the probe (ASA-adenosine), 125I-ASA-adenosine was purified by high pressure liquid chromatography. Iodine-125-ASA-adenosine, upon irradiation with uv light, covalently labeled a 65 kDa protein in bovine cardiac SL vesicles. Labeling of this protein was greatly diminished in the presence of nonradiolabeled adenosine, 5'-amino adenosine, or guanosine (inhibitors of purine nucleoside transport) but not by glucose (which does not inhibit transport). We conclude that the cardiac adenosine transporter is a protein with an apparent Mr of 65 kDa.

Topics: Adenosine; Affinity Labels; Animals; Azides; Carrier Proteins; Cattle; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Myocardium; Salicylates; Thioinosine

1. Adenosine-5'-triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2x- or G-protein coupled P2Y-purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol-prestimulated tissues. 2. ATP exerted a clear concentration-dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contraction, followed by a slower sustained relaxation. The receptor mediating contraction was susceptible to desensitization by ATP and by the ATP analogue, alpha,beta-methyleneATP (alpha,beta-meATP) showing the typical features of the P2x-purinoceptor; conversely, ATP-evoked relaxation did not undergo tachyphylaxis following either ATP or alpha,beta-meATP. 3. The slower and sustained relaxant phase seemed to be due to activation of P2Y-purinoceptors, based on responses obtained with the P2Y agonist, 2-methyl-thioATP (2-meSATP) and, more importantly, based on the clear involvement of the G-proteins. In fact, the G-protein activator, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) significantly potentiated and the G-protein blocking agent, guanosine 5'-O-(2-thio-diphosphate) (GDP beta S) completely abolished the ATP-induced relaxation. No effects were exerted by these two G-protein modulators on the ATP-induced contraction. 4. The relaxant component of the ATP response of bladder tissue was not significantly influenced by nitro-benzyl-thioinosine (NBTI) or by 8-phenyltheophylline (8-PT), suggesting that the contribution of the ATP metabolite adenosine to this response was negligible. Moreover, relaxation evoked by ATP and by the adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA) was additive.5. Suramin was unable to modify either the relaxant or the contractile responses of bladder strips to ATP. However, when tested on the concentration-response curve to the slowly hydrolysable P2x-agonist alpha,beta-meATP, a rightward shift was detected, suggesting that ATP contractile responses are mediated by suramine-sensitive P2x-purinoceptors.6. Uridine-5'-triphosphate (UTP) only induced a rapid and concentration-dependent contraction of the rat bladder preparation, which was not desensitized by pre-exposure to alpha,beta-meATP, suggesting that UTP responses were not mediated by the 'classical' P2X-purinoceptor.7. It is therefore concluded that both P2x- and P2y-purinocepto

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Dose-Response Relationship, Drug; Guanosine Triphosphate; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Thioinosine; Uridine Triphosphate; Urinary Bladder; Vasodilator Agents

1. Adenosine is known to stimulate capillary outgrowth and endothelial cell proliferation, but the underlying mechanism has not been identified. In order to identify the receptor subtype involved, the effects of adenosine receptor agonists and antagonists on human umbilical vein endothelial cell (HUVEC) proliferation were investigated. 2. Raising intracellular adenosine levels by use of the adenosine transport inhibitor, 4-nitrobenzylthioinosine (NBMPR) did not affect cell growth. This observation suggests that stimulation of an extracellular adenosine receptor generates the mitogenic signal. 3. In the presence of adenosine deaminase (ADA), which was used to remove adenosine present in the culture medium, the adenosine receptor agonists N-ethylcarboxamidoadenosine (NECA, non-selective) and CGS21680 (A2A-receptor-selective) stimulated [3H]-thymidine incorporation with a half-maximum effect at about 10 nM, while N6-cyclopentyladenosine (CPA, A1-selective) was about 100 fold less potent. The adenosine receptor antagonist, xanthine amine congener (XAC) produced a concentration-dependent decrease in endothelial cell proliferation with a half-maximum effect at about 10 nM. Hence, stimulation of an endothelial A2A-adenosine receptor seems responsible for the mitogenic signal. 4. In the presence of ADA, isoprenaline is also able to stimulate [3H]-thymidine incorporation with a half maximal effect of about 3 nM, an effect, which is reversed by the highly beta 2-selective antagonist, ICI 118,551. In the absence of ADA, isoprenaline exerts only a minor stimulatory effect. Combination of A2A adenosine and beta 2-adrenoceptor agonists did not further enhance [3H]-thymidine incorporation when compared to the sole addition of each agonist. We therefore conclude that both receptors stimulate endothelial cell proliferation via a common signal transduction pathway. 5. Both receptors are coupled to stimulation of adenylyl cyclase via the stimulatory G protein G8.However, direct activation of downstream effectors in the cyclic AMP-signalling cascade (G8 with cholera toxin, adenylyl cyclase with forskolin, protein kinase A with 8Br-cyclic AMP) not only failed to mimic the action of receptor-activation, but even reduced cell proliferation.6. Similarly, pertussis toxin-treatment which inactivated the Gi 2 protein present in HUVEC and thus inhibited cell proliferation per se, did not impair the ability of A2A-receptor agonists to stimulate cell proliferation. This suggests that

Topics: Adenosine; Adenosine Deaminase; Cell Division; Cyclic AMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Receptors, Adrenergic, beta-2; Receptors, Purinergic P1; Thioinosine; Thymidine; Umbilical Veins; Virulence Factors, Bordetella

This study was designed to determine whether intermittent warm aortic crossclamping induces cumulative myocardial stunning or if the myocardium becomes preconditioned after the first episode of ischemia in canine models in vivo. The role of adenosine triphosphate catabolism and subsequent release of purines on reperfusion-mediated postischemic ventricular dysfunction and arrhythmias was assessed with the use of selective inhibitors of nucleoside transport, p-nitrobenzylthioinosine (NBMPR), and a specific adenosine deaminase inhibitor, erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA). Thirty-two anesthetized dogs were instrumented to monitor left ventricular contractility, off bypass, by sonomicrometry. During cardiopulmonary bypass dogs were treated before ischemia with either saline solution (control group, n = 8) or EHNA (100 mumol/L) and NBMPR (25 mumol/L) (EHNA/NBMPR group, n = 8). Hearts were subjected to either 60 minutes of global ischemia and 120 minutes of reperfusion (n = 16) or 6 episodes of 10 minutes of global ischemia and 10 minutes of reperfusion, followed by 60 minutes of reperfusion (n = 16). Sixty minutes of sustained ischemia resulted in 80% loss of adenosine triphosphate and induced reperfusion-mediated ventricular fibrillation and severe left ventricular dysfunction in the control group. EHNA/NBMPR treatment augmented myocardial adenosine trapping during ischemia, attenuated ventricular fibrillation, and enhanced left ventricular functional recovery, despite similar depletion of adenosine triphosphate (80% loss). In the intermittent ischemia experiment, the first episode of 10 minutes of ischemia and reperfusion caused significant adenosine triphosphate depletion, ventricular fibrillation, and left ventricular stunning in both control and drug-treated groups. The prevalence of ventricular fibrillation was greater in the control group than in the drug-treated group after the first episode of ischemia (p < 0.05). Adenosine was the major nucleoside accumulated in the myocardium at the end of 10 minutes of ischemia in the EHNA/NBMPR-treated group (p < 0.05 versus control). Subsequent episodes of ischemia prevented ventricular fibrillation and did not cause cumulative left ventricular stunning in either group. Left ventricular function fully recovered in the EHNA/NBMPR-treated group after intermittent ischemia, but remained stunned in the control group. Unlike sustained ischemia, intermittent ischemia and reperfusion preserved myocardial adeno

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Aorta; Constriction; Dogs; Inosine; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardial Stunning; Myocardium; Thioinosine; Ventricular Dysfunction, Left; Ventricular Fibrillation; Ventricular Function, Left

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [3H]adenosine 1.8-fold within 30 s, approx. half of the [3H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [14C]Inosine, [14C]hypoxanthine and [3H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC50 approx. 0.7 microM), but not nitrobenzylthioinosine (15 microM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 microM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [3H]adenosine and [14C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purines may also be present.

Topics: Adenosine; Adenosine Kinase; Animals; Binding, Competitive; Biological Transport; Carrier Proteins; Dipyridamole; Membrane Proteins; Nucleoside Transport Proteins; Protozoan Proteins; Purines; Thioinosine; Toxoplasma

Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na(+)-dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC50 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent Km values of 500 and 1600 microM and Vmax values of 13 and 12 microM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the Km of NBMPR-insensitive transporter, with apparent Km values of 100 and 110 microM and Vmax values of 3.0 and 2.5 microM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent Kd values of 0.32 and 2.3 nM with Bmax values of 48,000 and 44,000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 microM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.

Topics: Animals; Biological Transport; Carrier Proteins; Cell Line; Ethylmaleimide; Kinetics; Membrane Proteins; Mice; Nucleoside Transport Proteins; Thioinosine; Tumor Cells, Cultured; Uridine

Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with L-[3H]adenosine, our objectives here were to determine (1) if L-[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological D-adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. L-[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. L-[3H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 microM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with L-[3H]adenosine alone or I-[3H]adenosine plus unlabeled L-adenosine, D-adenosine, or uridine, L-[3H]adenosine release was inhibited 42% by L-adenosine, 69% by uridine, and 81% by D-adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Biological Transport; Brain; Carrier Proteins; Dilazep; Dipyridamole; Kinetics; Male; Membrane Proteins; Nucleoside Transport Proteins; Potassium Chloride; Rats; Rats, Sprague-Dawley; Synaptosomes; Thioinosine; Tritium

Findings from previous investigations conducted at this institution and others have suggested that University of Wisconsin solution (UWS) is preferable for the prolonged hypothermic storage of hearts before transplantation. The benefit seen with UWS may in part be related to the inclusion of adenosine (5 mmol/L) in the UWS. To investigate whether further manipulations of adenosine metabolism might enhance myocardial protection, studies were initially conducted using cultured myocytes, followed by confirmatory experiments using isolated rat hearts. Cultured human ventricular myocytes (7 to 8 dishes/group) were stored for 12 hours at 0 degrees C in unmodified UWS or UWS supplemented with increasing concentrations (1 to 100 mumol/L) of the nucleoside-transport blocker p-nitrobenzylthioinosine. The adenosine triphosphate concentrations were found to be enhanced with nucleoside-transport inhibition, with the best results achieved with the 1- and 3-mumol/L groups (control, 3.37 +/- 0.41 nmol/micrograms DNA; UWS, 2.89 +/- 1.31 nmol/micrograms DNA; 1 mumol/L, 5.91 +/- 3.23 nmol/micrograms DNA; 3 mumol/L, 7.86 +/- 3.45 nmol/micrograms DNA; p < 0.05 versus control or UWS group). Isolated rodent hearts from Sprague-Dawley rats were prepared on a Langendorff apparatus with an intraventricular balloon and subsequently stored for 8 hours at 0 degrees C in unmodified UWS (13 hearts/group) or UWS supplemented with 1 or 3 mumol/L of p-nitrobenzylthioinosine (9 to 10 hearts/group).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Carrier Proteins; Cells, Cultured; Coronary Circulation; Creatine Kinase; DNA; Glutathione; Heart Transplantation; Humans; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Membrane Proteins; Myocardium; Nucleoside Transport Proteins; Organ Preservation; Organ Preservation Solutions; Purine Nucleosides; Raffinose; Rats; Rats, Sprague-Dawley; Thioinosine

Polyclonal antibodies raised against the human erythrocyte nucleoside transporter were used to investigate the distribution of the nucleoside transporters in the placenta. Immunoblots of brush-border membranes isolated from the human syncytiotrophoblast revealed a cross-reactive species that co-migrated with the erythrocyte nucleoside transporter as a broad band of apparent M(r) 55,000. In contrast, no labelling was detected in basal membranes containing a similar number of equilibrative nucleoside transporters as assessed by nitrobenzylthioinosine (NBMPR)-binding. The absence of cross-reactive epitopes in basal membranes and their presence in brush-border membranes was confirmed by confocal immunofluorescence microscopy. These results suggest that at least two isoforms of the NBMPR-sensitive nucleoside transporter are present in the human placenta. The lumenal surfaces of fetal capillaries, small placental vessels and umbilical vein were also strongly labelled by the antibody, a finding that suggests that the high fetal-placental adenosine uptake previously reported is due to endothelial transporters.

Topics: Affinity Labels; Carrier Proteins; Cell Membrane; Endothelium; Female; Humans; Immunoblotting; Immunohistochemistry; Isomerism; Membrane Proteins; Microscopy, Confocal; Microvilli; Nucleoside Transport Proteins; Placenta; Pregnancy; Thioinosine; Trophoblasts; Umbilical Cord

The objective of this study was to determine whether chronic infusion (7 days) of an A1-adenosine receptor (A1-AdoR) agonist, R-N6-phenylisopropyladenosine (R-PIA), causes homologous and/or heterologous desensitization of the atrioventricular (AV) nodal A1-AdoR system in the guinea pig. The negative dromotropic effects of adenosine, the A1-AdoR agonist N6-cyclopentyladenosine and the muscarinic cholinergic agonist carbachol on hearts from control and treated animals were compared. The potencies of adenosine and N6-cyclopentyladenosine to prolong AV nodal conduction time of hearts from treated animals were significantly reduced compared with control. In contrast, no difference in the potency of carbachol to prolong AV nodal conduction time in the hearts from control and R-PIA-treated guinea pigs was found. The densities of A1-AdoRs in atria and ventricles of treated hearts were 54% (P < .05) and 36% (P < .05) lower than in control hearts, respectively. The number of A1-AdoRs with high affinity for agonists was 37% lower (P < .05) in ventricular membranes prepared from hearts of R-PIA-treated guinea pigs than in membranes from control hearts. In contrast, atrial or ventricular muscarinic acetylcholine receptor densities were not different in hearts of control compared with hearts of treated animals. An up-regulation of the density of nucleoside transporter sites was observed in hearts of treated animals. The quantities of Gi and G(o) in atrial membranes prepared from hearts of treated guinea pigs were lower by 46% and 80%, respectively, than the quantities of Gi and G(o) in atrial membranes prepared from hearts of control animals. It was concluded that chronic administration of R-PIA to guinea pigs desensitized the AV node to the negative dromotropic effect of adenosine in a homologous but not a heterologous manner and desensitization of the AV node response to adenosine was associated with down-regulation of A1-Ado receptors, a decrease in the fraction of A1-Ado receptors in the high-affinity state and a decrease in the contents of Gi and G(o) proteins.

Topics: Adenosine; Adenosine Diphosphate Ribose; Animals; Atrioventricular Node; Binding Sites; Biological Transport; Carbachol; Cell Membrane; Dipyridamole; Down-Regulation; Female; Gallopamil; GTP-Binding Proteins; Guinea Pigs; Magnesium Chloride; Male; Myocardium; Phenylisopropyladenosine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Signal Transduction; Thioinosine; Time Factors; Virulence Factors, Bordetella

Uptake and metabolism of adenosine by human placenta were studied using the single-circulation paired-tracer technique. When isolated cotyledons were perfused through the fetal (basal) circulation at mean pressures of 36 +/- 3.3 mmHg and mean flow rates of 6.6 +/- 0.3 ml/min the maximal [3H]adenosine uptake was 51.3 +/- 3.9 per cent. The uptake was not changed when the vascular resistance was pharmacologically increased. Adenosine uptake was significantly inhibited by adenosine, inosine and nitrobenzylthioinosine (NBMPR), but was unaffected by hypoxanthine. The kinetic analysis of adenosine transport showed it to be a saturable and, Na(+)-independent process, with a Km of 60.8 microM and a Jmax of 0.148 mumol/min. Thin layer chromatographic analysis showed that about 65 per cent of [3H]adenosine was metabolized (10-30 sec) in a single passage through the fetoplacental circulation. [3H]hypoxanthine and [3H]adenine were the major products recovered in the venous perfusate. In the presence of NBMPR the fractional recovery of [3H]adenine and [3H]phosphorylated derivatives was reduced while that of [3H]hypoxanthine was increased. These overall results show that the uptake of adenosine is a Na(+)-independent, NBMPR-sensitive, carrier-mediated process, which appears to be specific for nucleosides, and suggests that metabolization of adenosine proceeds both intra- and extracellularly.

Topics: Adenosine; Biological Transport; Female; Humans; Hypoxanthine; Hypoxanthines; In Vitro Techniques; Perfusion; Placenta; Pregnancy; Purine Nucleosides; Sodium; Thioinosine; Vasoconstriction

Pregnancy complicated by diabetes is a relatively frequent event and may result in fetal embriopathy. However, little is known regarding whether placental transport functions are altered. In this study, we have compared the activity of the nitrobenzylthioinosine (NBMPR)-sensitive adenosine transporter and adenosine metabolism in human placental brush-border- and basal-membrane vesicles from placentas of normal and diabetic pregnancies. Neither [3H]NBMPR binding, a marker of the facilitative-diffusion nucleoside transporter in the human placenta, nor adenosine metabolism exhibited a significant difference in either the brush-border- or the basal-membrane vesicles between the normal and diabetic group, except for an increased affinity in [3H]NBMPR binding at the basal side in diabetic placenta. This result contrasts with an earlier finding using the same group of patients that adenosine transport is downregulated in umbilical vein endothelial cells from diabetic pregnancies. It is concluded that adenosine transport is modulated selectively in different tissues in diabetic pregnancies.

Topics: Adenosine; Affinity Labels; Biological Transport; Female; Humans; Microvilli; Placenta; Pregnancy; Pregnancy in Diabetics; Thioinosine; Tritium

Topics: Adenosine; Affinity Labels; Alkaloids; Animals; Antimalarials; Benzodioxoles; Benzoquinones; Biological Transport; Ciona intestinalis; Crithidia; Dilazep; Erythrocytes; Furosemide; HeLa Cells; Humans; Malaria, Falciparum; Phlorhizin; Piperidines; Plasmodium falciparum; Polyunsaturated Alkamides; Stereoisomerism; Thioinosine; Trichomonas vaginalis

The salvage of nucleosides dominates over de novo biosynthesis in lymphocytes, polymorphonuclear cells (PMN) and in central neutral nervous system (CNS) in higher organisms. Earlier works in our laboratory have shown that the salvage of deoxycytidine (dCyd) did not correlate with DNA synthesis. The uptake and metabolism of dCyd was higher in undifferentiated germinal center lymphocytes and in follicles comparing to more differentiated cells. Recently we have compared the transport of thymidine (dThd), dCyd, uridine (Urd) and adenosine (Ado) in the three cell systems in which the salvage of nucleosides is dominating. It was found that dCyd was transported 30 times more effectively into lymphocytes than into PMN and synaptosomes, while Urd was transported about the same rate into the two cells and into synaptosomes. All transport processes could be inhibited by dipyridamole, NBRPR, papaverine and dilazep. The dCyd and dThd was phosphorylated even at 0 degrees C up to TTP and dCTP without incorporation into DNA and into liponucleotides. Our results show that the processes of transport-phosphorylation, as well as the processes of DNA-CDP-phospholipid synthesis are tightly coupled to each other in intact cells and organelles.

Topics: Adenosine; Animals; Biological Transport; Cerebral Cortex; Child; Deoxycytidine; Dilazep; Dipyridamole; DNA; Humans; Kinetics; Lymphocytes; Neutrophils; Nucleosides; Palatine Tonsil; Papaverine; Rats; Synaptosomes; Thioinosine; Thymidine; Uridine

Topics: Adenosine; Animals; Biological Transport; Cattle; Cell Line; Dipyridamole; Endothelium, Vascular; Heart; Hydrogen Peroxide; Hypochlorous Acid; Inosine; Kinetics; Nucleosides; Reactive Oxygen Species; Thioinosine; Uridine; Vitamin K

Adenosine transport and adenosine A1 receptors in rat brain are subjected to regulation by thyroid hormone levels. The studies were carried out with brain stem synaptosomal preparations from rat brain in euthyroid and various hypothyroid situations. The maximum velocity of the nitrobenzylthioinosine (NBTI)-sensitive adenosine transport was 3.3 +/- 0.3 pmol.mg protein-1.s-1 in euthyroid rats. The transport in 1-wk thyroidectomized rats was decreased by 45.8% with respect to controls. No changes were found in the affinity of euthyroid and hypothyroid rats, with the Michaelis-Menten constant values equal to 1.9 +/- 0.9 and 2.0 +/- 0.5 microM, respectively. The transporter number measured by NBTI binding also decreased; the maximum binding capacity (Bmax) was 112.9 +/- 21.9 and 31.3 +/- 4.1 fmol/mg protein for euthyroid and hypothyroid rats, respectively. The adenosine A1 receptors were measured in synaptosomal membrane preparations in the presence of 100 microM guanosine-5'-O-3-thiotriphosphate for cylopenthyl-1,3-dipropylxanthine 8-[dipropyl 2,3-3H(N)] ([3H]DPCPX) binding. In euthyroid rats, the Bmax value was 227.6 +/- 27.6 fmol/mg protein, a significant decrease of 23% was obtained in 1-wk hypothyroid rats. In all other thyroid situations studied, adenosine transport capacity, adenosine transporter number, and adenosine A1 receptor number were restored to control levels.

Topics: Adenosine; Animals; Biological Transport; Brain; Male; Rats; Rats, Wistar; Receptors, Purinergic P1; Thioinosine; Thyroid Hormones; Xanthines

The nucleoside transport characteristics of undifferentiated and differentiated LA-N-2 human neuroblastoma cells were compared through measurement of the cellular accumulation of [3H]formycin B in the absence and presence of specific nucleoside transport blockers such as dipyridamole and nitrobenzylthioinosine (NBMPR). [3H]NBMPR was also used as a high affinity probe to obtain an estimate of the number of NBMPR-sensitive nucleoside transport proteins. Undifferentiated LA-N-2 cells accumulated [3H]formycin B (25 microM) via a NBMPR/dipyridamole sensitive, Na(+)-independent, nucleoside transport system (Vi = 1.52 pmol/microliters/s; maximum intracellular concentration = 45 pmol/microliters cell water). The undifferentiated cells also had a high density of site-specific [3H]NBMPR binding sites (135,000 sites/cell; KD = 0.4 nM). When cell differentiation was induced by exposure to a serum-free defined medium, the initial rate of transporter-mediated [3H]formycin B uptake increased to 1.92 pmol/microliters/s, and the steady-state intracellular concentration of [3H]formycin B also increased significantly to 73 pmol/microliters. However, there was no concomitant change in the number of [3H]NBMPR binding sites, and the additional uptake was not Na(+)-dependent. This enhanced uptake in the differentiated cells appeared to be due, in part, to an increased functional expression of a NBMPR-resistant form of facilitated nucleoside transporter. Approximately 18% of the transporter-mediated uptake in the differentiated cells was resistant to inhibition by NBMPR at concentrations that blocked transport completely in the undifferentiated cells. This cell model may prove useful for basic studies on regulation of nucleoside transporter subtype expression in neural tissues, and for evaluation of the efficacy and potential host toxicity of cytotoxic nucleoside analogues (+/- specific transport blockers) in the treatment of neuroblastoma.

Topics: Biological Transport; Cell Differentiation; Formycins; Humans; Neuroblastoma; Nucleosides; Thioinosine; Tumor Cells, Cultured

In the present work we studied the effect of adenosine and various adenosine analogues on cAMP level in guinea pig coronary endothelial cells of microvascular origin. The tested adenosine agonist mediate a concentration-dependent increase in cAMP level. The rank order of potency was 5'-N-ethylcarboxamidoadenosine (NECA) > CGS 21680 > N6-phenylisopropyladenosine (R-PIA) > 2-chloro-N6-cyclopentyladenosine (CCPA) which is typical for an adenosine A2 receptor. Their respective concentrations for half maximal stimulation of cAMP formation were 0.36 microM, 0.82 microM, 4.7 microM and 9.8 microM. The tested agonists showed differences in efficacy, NECA being the most efficacious. R-PIA, CCPA and adenosine were less efficacious, suggesting partial agonism. The efficacy of adenosine was unchanged by the addition of the nucleoside transport inhibitor S(4-nitrobenzyl)-6-thioinosine (NBTI, 10 microM) suggesting that inhibition of adenylyl cyclase through P-site activation is not responsible for the observed low efficacy of adenosine. We could demonstrate CGS 21680 activation of adenylyl cyclase in a peripheral receptor. We therefore suggest that the endothelial adenosine receptor resembles the striatal adenosine A2a receptor.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Antihypertensive Agents; Cells, Cultured; Colforsin; Coronary Vessels; Cyclic AMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Guinea Pigs; Isoproterenol; Myocardium; Phenethylamines; Phenylisopropyladenosine; Receptors, Purinergic P1; Thioinosine; Vasodilator Agents

The effect of endogenous adenosine on frequency-induced long-term potentiation of the responses evoked by stimulation of the Schaffer fibres and recorded in the CA1 area was studied in hippocampal slices of the rat. Long-term potentiation was facilitated in the presence of the selective A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (10-20 nM), and was reduced in the presence of the adenosine uptake blocker, nitrobenzylthioinosine (5 microM), suggesting that endogenous adenosine exerted a tonic inhibitory role on long-term potentiation, which was mediated through adenosine A1 receptors. We also found that long-term potentiation was increased in the presence of the selective A2 receptor agonist, CGS 21680 (30 nM), suggesting that the activation of adenosine A2 receptors may have excitatory effects on long-term potentiation. We suggest that, endogenous adenosine is able to modulate mechanisms of synaptic plasticity, such as long-term potentiation, in the hippocampus.

Topics: Adenosine; Animals; Antihypertensive Agents; Electric Stimulation; In Vitro Techniques; Long-Term Potentiation; Nerve Fibers; Neuronal Plasticity; Phenethylamines; Purinergic P1 Receptor Antagonists; Purinergic P2 Receptor Antagonists; Pyramidal Cells; Rats; Rats, Wistar; Synapses; Thioinosine; Xanthines

Spreading depression (SD) is known to be involved in the N-methyl-D-aspartate receptor-mediated neuronal damage. In urethane-anesthetized rats, we examined the release of adenosine and glutamate during SD induced by microdialysis of high K+ perfusate through the hippocampal CA1 area. The effects of endogenous adenosine upon SD were studied by applying an adenosine antagonist, theophylline (1 mM) and by a simultaneous application of adenosine uptake blockers, dipyridamole (DPR) (100 microM) and nitrobenzylthioinosine (NBI) (50 microM). The dialysates were sampled every 5 or 10 min and analyzed by HPLC. SD was identified by flattening of background EEg and disappearance of population spikes recorded from the pyramidal cell layer of CA1 area by a glass microelectrode. Adenosine and glutamate release was enhanced significantly in association with the occurrence of SD. Theophylline increased the release of glutamate and the incidence of SD and decreased the latency of the SD occurrence. DPR+NBI decreased the release of glutamate and the occurrence of SD, but increased extracellular adenosine concentration. The effects of DPR+NBI were blocked by application of a selective antagonist of adenosine A1 receptor, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM). These findings suggest that endogenous adenosine exerts inhibitory influences upon the development of SD and the glutamate release through the A1 receptor in rat hippocampus.

Topics: Adenosine; Animals; Cortical Spreading Depression; Dipyridamole; Glutamic Acid; Hippocampus; Male; Microdialysis; Potassium; Purinergic P1 Receptor Antagonists; Purines; Rats; Rats, Wistar; Theophylline; Thioinosine; Xanthines

Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were Km 150 +/- 45 microM, and Vmax 414 +/- 17 pmol . 10(6) cells-1 . min-1. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (Ki 3 nM) and dipyridamole (Ki 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC50 value for the adenosine transport inhibition by uridine was very high (240 microM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a Km value of 185 +/- 11 microM and a Vmax value of 4.24 +/- 0.10 pmol . mg protein-1 . s-1. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the Vmax value but the Km value was not significantly modified. Adenosine 5'-[beta,gamma-imino]triphosphate, 1,N6-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine(Ap4A) at 100 microM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.

Topics: Adenine Nucleotides; Adrenal Medulla; Affinity Labels; Animals; Biological Transport; Carrier Proteins; Cattle; Cell Membrane; Cells, Cultured; Dipyridamole; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine; Uridine

Although dipyridamole has been extensively studied as an anti-aggregating agent, its mechanism of action has not been elucidated. Cultured mesangial cells were treated with dipyridamole 1-100 microM from 6-72 h. Ecto-5'-nucleotidase activity approximately doubled (from 115 +/- 11 to 226 +/- 14 nmol/min/mg) after treatment with 100 microM dipyridamole for 72 h. This effect was concentration- and time-dependent. Cycloheximide, an inhibitor of protein synthesis, did not alter basal 5'-nucleotidase activity. However, it prevented stimulation by 5 microM dipyridamole. Adenosine availability at the receptor sites was increased by dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), which inhibit adenosine uptake into the cell. Addition of dipyridamole or NBTI to the adenosine-treated mesangial cells produced an additive increase in ecto-5'-nucleotidase activity. Dipyridamole, through its effect on extracellular adenosine and ecto-5'-nucleotidase, may have an influence upon regulation of the glomerular microcirculation.

Topics: 5'-Nucleotidase; Adenosine; Affinity Labels; Animals; Cycloheximide; Dactinomycin; Dipyridamole; Glomerular Mesangium; Rats; Rats, Sprague-Dawley; Thioinosine

We examined the functional and pharmacological characteristics of liposome-reconstituted nucleoside transporter proteins obtained by detergent (octylglucoside) extraction from Ehrlich ascites tumor cell plasma membranes. Optimal reconstitution was achieved using a lipid composition of phosphatidylcholine, cholesterol, phosphatidylethanolamine and phosphatidylserine in a molar ratio of 33:33:26:8 and with a lipid-to-protein ratio of 30. This preparation had Km and Vmax values of 280 microM and 570 pmol/mg/sec, respectively, for the transporter-mediated uptake of [3H]uridine and bound 8.3 pmol of [3H]nitrobenzyl-thioinosine per milligram of protein. In general, the reconstituted system had kinetic and pharmacological characteristics comparable to those of the native membrane-located system, including an 80:20 ratio of nitrobenzylthioinosine-sensitive to -resistant [3H]uridine influx. The uridine translocation capacity of the optimally reconstituted system was 56 molecules per transporter per second compared with 104 molecules per transporter per second in intact cells, indicating that more than half of the inserted proteins were capable of mediating the influx of [3H]uridine (assuming that each functioning transporter was operating at optimal efficiency). Differences between the native and reconstituted transporters included the appearance of a [3H]uridine influx component (inhibited by adenosine) that was resistant to inhibition by R75231 (18%) and dipyridamole (10%). Dilazep was also significantly less effective in inhibiting the nitrobenzylthioinosine-sensitive transporter in the reconstituted preparations (IC50 = 41 nM) relative to that seen in intact Ehrlich cells (IC50 = 1.4 nM). These results suggest that inhibitor sensitivity may be a factor of the lipid microenvironment of the transporter or may involve other cellular components that could dissociate from the complex on detergent solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carrier Proteins; Glucosides; Kinetics; Lipids; Liposomes; Male; Membrane Proteins; Mice; Nucleoside Transport Proteins; Thioinosine; Uridine

We compared the binding sites of the adenosine transport inhibitors (3H)dipyridamole (DPR) and (3H)nitrobenzylthioinosine (NBI) in human parietal cortex and erythrocytes. In comparison with guinea pig (3H)DPR marked only slightly more binding sites than (3H)NBI with a Bmax of 1080 +/- 29 and 780 +/- 7 fmol/mg protein respectively in parietal cortex and 24288 +/- 2725 and 20875 +/- 1905 fmol/mg protein respectively in erythrocytes. NBI displaced (3H)DPR binding completely from its binding sites at about KD/2 concentrations in parietal cortex as well as erythrocytes with inhibition constants comparable to its dissociation constants. Lineweaver-Burke analysis in erythrocytes indicated a competitive inhibition of (3H)DPR binding by NBI. Pharmacological characterization of (3H)DPR binding sites in human erythrocytes is consistent with their localization on adenosine transporters. These findings provide evidence that as opposed to guinea pig (3H)DPR and (3H)NBI largely label binding sites to the same adenosine transporter in human erythrocytes and parietal cortex.

Topics: Adenosine; Binding Sites; Binding, Competitive; Biological Transport; Dipyridamole; Erythrocytes; Humans; Parietal Lobe; Thioinosine

The effect of nitrobenzylthioinosine (NBTI) on [3H]adenosine uptake and the characterization of the [3H]NBTI binding in cell (primary cultures and LLC-PK1 cell line) plasma membrane and brush-border membrane (BBM) vesicles from pig renal cortices and LLC-PK1 cells was analyzed. [3H]adenosine uptake was strongly inhibited by NBTI in nonconfluent cells, whereas it was totally insensitive to the reagent in BBM. The concentration dependence of [3H]adenosine uptake in BBM was linear, suggesting simple diffusion. In both cell membranes and BBM high-affinity [3H]NBTI binding was observed. [3H]NBTI binding as well as NBTI-sensitive [3H]adenosine uptake was strongly reduced when cells grew to confluence. Both reduction effects were reproduced by treatment of nonconfluent cells with chlorophenyl adenosine 3',5'-cyclic monophosphate (cAMP), which indicates that the transporter is regulated by a cAMP-dependent protein kinase. To confirm this hypothesis, the binding of [3H]NBTI was analyzed in pig kidney BBM obtained in the presence of orthovanadate and alkaline phosphatase. With respect to control membranes, BBM obtained in the presence of orthovanadate showed a lower maximum number of binding sites (Bmax), whereas those obtained in the presence of alkaline phosphatase showed a slight increase in Bmax for [3H]NBTI binding. Taken together, these results suggest that the reduction in both [3H]NBTI-binding capacity and NBTI-sensitive [3H]adenosine uptake takes place by a mechanism that involves phosphorylation of the transporter molecule or of a protein that interacts with it.

Topics: Adenosine; Affinity Labels; Animals; Biological Transport; Cell Line; Cell Membrane; Cells, Cultured; Cytochalasin B; Epithelium; Glucose; Kidney; Kidney Cortex; Kidney Tubules, Proximal; Kinetics; Swine; Thioinosine; Time Factors; Tritium

The dose-response curves for the inhibition of equilibrative uridine transport by dilazep, dipyridamole and nitrobenzylthioinosine (NBMPR) in undifferentiated HL-60 cells were biphasic. Some 70% of the transport activity was inhibited with IC50 values of 0.7, 1 and 7 nM respectively. No inhibition of the remaining 30% of transport activity was observed until the dilazep, dipyridamole and NBMPR concentrations exceeded 1, 0.1 and 3 microM respectively. Exposure to phorbol 12-myristate 13-acetate (PMA) for 48 h, to induce monocytic differentiation, caused a 20-fold decrease in Vmax. of both NBMPR-sensitive and NBMPR-insensitive equilibrative uridine transport. The decrease in NBMPR-sensitive uridine transport induced by PMA corresponded to a decrease in NBMPR binding sites. A 30% decrease in specific NBMPR binding sites occurred within 6 h of PMA exposure, and could be prevented by uridine and thymidine at concentrations as low as 100 microM, and by staurosporine at 40 nM. However, the protective effects of these compounds diminished with prolonged PMA exposure. No protection was observed with uracil. Exogenous protein kinase C (PKC) in the presence of ATP and PMA decreased the number of specific NBMPR-binding sites in purified HL-60 cell plasma membranes. These results suggest that a PKC-induced conformational change in substrate-binding/transporting site may be responsible for the decrease in NBMPR-sensitive nucleoside transport during PMA-induced monocytic differentiation of HL-60 cells.

Topics: Affinity Labels; Alkaloids; Binding Sites; Biological Transport; Cell Differentiation; Dilazep; Dipyridamole; Humans; Kinetics; Leukemia; Monocytes; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Thioinosine; Thymidine; Tumor Cells, Cultured; Uracil; Uridine

We have investigated the hypothesis that adenosine, a purine nucleoside produced within hypoxic regions of solid tumors, may interfere with the recognition of tumor cells by cytolytic effector cells of the immune system. We measured the adhesion of murine spleen-derived anti-CD3-activated killer (AK) lymphocytes to syngeneic MCA-38 colon adenocarcinoma cells in a model system. Adenosine, in the presence of the adenosine deaminase inhibitor coformycin to prevent the breakdown of adenosine, inhibited adhesion by up to 60%. The inhibitory effect of adenosine was exerted on the AK cells and not on the MCA-38 targets. The response to adenosine was generated at the cell surface, since the inhibition of adhesion was not abrogated by S-(4-nitrobenzyl)-6-thioinosine or dipyridamole, which block adenosine uptake. The inhibition of adhesion due to adenosine was not blocked by either the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine. This suggested that a non-A1, A2 receptor might be involved. The relative order of potencies of adenosine and common analogues was: 5'-N-ethylcarboxamidoadenosine = adenosine = (R)-phenylisopropyladenosine > N6-cyclopentyladenosine > 2-chloro-N6-cyclopentyladenosine = 2-p-(2- carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine. This agonist potency profile was again inconsistent with either the A1 or the A2 receptor subtype but indicated that the recently described A3 receptor subtype might be responsible for the inhibition of adhesion. Consistent with this suggestion, aminophenylethyladenosine, an adenosine analogue that binds with high affinity to A3 receptors, inhibited the adhesion of AK cells to MCA-38 tumor cells with high potency (50% inhibitory concentration approximately 1 nM). Adenosine, therefore, interferes with the AK cell recognition of colorectal tumor targets by acting through an A3 receptor on the effector cells. We suggest that this mechanism of immunosuppression, secondary to tissue hypoxia, may be important in the resistance of colorectal and other solid cancers to immunotherapy.

Topics: Adenocarcinoma; Adenosine; Animals; Antibodies; CD3 Complex; Cell Adhesion; Colonic Neoplasms; Killer Cells, Natural; Mice; Theobromine; Thioinosine; Tumor Cells, Cultured; Xanthines

1. The aim of the present study was to determine the effect of the nucleoside transporter inhibitor, draflazine, on the force of contraction in human myocardium and the affinity of the compound for the nucleoside transporter. Nucleoside transport inhibitors, like draflazine, are of potential importance for cardiopreservation of donor hearts for heart transplantation. 2. Functional experiments were performed in isolated electrically driven (1 Hz, 1.8 mmol l-1 Ca2+) human atrial trabeculae and ventricular papillary muscle strips. The affinity of draflazine for the myocardial nucleoside transporter was studied in isolated membranes from human ventricular myocardium and human erythrocytes in radioligand binding experiments using [3H]-nitrobenzylthioinosine ([3H]-NBTI). Dipyridamole was studied for comparison. 3. In membranes from human myocardium and erythrocytes, [3H]-NTBI labelled 1.18 pmol mg-1 protein and 23.0 pmol mg-1 protein, respectively, nucleoside transporter molecules with a KD value of 0.8 nmol l-1. Draflazine concentration-dependently inhibited binding of [3H]-NBTI to myocardial and erythrocyte membranes with a K(i)-value of 4.5 nmol l-1. The potency as judged from the K(i) values was ten times greater than that of dipyridamole in both myocardial and erythrocyte membranes. 4. Draflazine, at concentrations up to 100 mumol l-1, did not produce negative inotropic effects in atrial and ventricular myocardium. (-)-N6-phenylisopropyladenosine (R-PIA) and carbachol did not reduce force of contraction in ventricular myocardium, but exerted concentration-dependent direct negative inotropic effects in atrial myocardium. 5. The data provide evidence that draflazine specifically binds to the nucleoside transporter of the human heart and erythrocytes with high affinity. The compound does not produce negative inotropic effects at concentrations as high as 100 micromol 1-1.6. Draflazine could be a useful agent for cardio preservation because it does not produce cardio depressant effects. Thus, it may be possible to perfuse explanted hearts directly with this agent without the hazard of cardiodepression.

Topics: Adult; Affinity Labels; Aged; Carbachol; Cardiac Pacing, Artificial; Carrier Proteins; Dipyridamole; Erythrocyte Membrane; Female; Heart; Humans; In Vitro Techniques; Male; Membrane Proteins; Middle Aged; Myocardial Contraction; Myocardium; Nucleoside Transport Proteins; Phenylisopropyladenosine; Piperazines; Radioligand Assay; Thioinosine

This study investigates the hypothesis that inhibition of nucleoside transport during hypothermic storage elevates tissue adenosine (ADO) content and improves the function of the isolated rat heart. The hearts, flushed with a cardioplegic solution containing varying concentrations (0-100 nM) of a nucleoside transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI), were immersion-stored at 0 degrees C for 9 hr. Function was assessed after 30 min of working reperfusion. Function of unstored fresh hearts served as controls and poststorage recovery is reported as percentage of control function. Poststorage heart rate in all groups returned to control level after reperfusion. Recovery of other functional parameters in the no-NBTI group was as follows: aortic flow (AF), 56.2 +/- 4.6%; coronary flow (CF), 53.9 +/- 3.2%; cardiac output (CO), 55.5 +/- 4.0%; systolic pressure, 81.6 +/- 2.5%; work, 47.0 +/- 4.2%; and coronary vascular resistance (CVR), 157.1 +/- 7.8% of control. NBTI improved functional recovery in a dose-dependent fashion; the maximal improvement was seen at a dose of 5 nM, in which the recovery was: AF, 78.1 +/- 3.4%; CF, 73.5 +/- 4.4%; CO, 76.7 +/- 3.6%; work, 70.7 +/- 5.0%; and CVR, 127.5 +/- 4.5% of control (P < 0.05 vs. no-NBTI). The ADO A1-receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (0.1 microM) blocked the effects of 5 nM NBTI; the recovery of AF, CF, CO, work, and CVR decreased to 62.8 +/- 8.0%, 58.3 +/- 5.0%, 61.5 +/- 3.9%, 54.4 +/- 4.5%, and 163.8 +/- 12.7% of control, respectively (P < 0.05 vs. 5 nM NBTI). Tissue ADO content in 5 nM NBTI hearts at the end of storage was 0.075 +/- 0.025 mumol/g dry wt, which was significantly elevated from 0.016 +/- 0.004 mumol/g dry wt in no-NBTI hearts. Purine release during initial reperfusion was delayed in 5 nM NBTI hearts, indicating the inhibition of nucleoside transport by NBTI. But NBTI treatment did not improve end-storage or end-reperfusion myocardial ATP. In conclusion, the addition of NBTI to cardioplegic solution enhanced tissue ADO and improved poststorage function of the hypothermically stored rat heart. The effect is ADO A1-receptor mediated without invoking energy conservation.

Topics: Adenosine; Adenosine Triphosphate; Animals; Biological Transport; Cardioplegic Solutions; Cryopreservation; Dose-Response Relationship, Drug; Heart; Male; Myocardium; Organ Preservation; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion; Thioinosine; Xanthines

A previous study has shown that endogenous adenosine trapping during ischemia (by blocking adenine nucleoside transport and inhibiting adenosine breakdown) prevents myocardial stunning. In this study, we tested the hypothesis that delay of administration of inhibitors until reperfusion would similarly prevent myocardial stunning in the absence of entrapped adenosine. In both studies, a selective nucleoside transport blocker, p-nitrobenzyl-thioinosine, was used in combination with a potent adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, to entrap adenosine (preischemic treatment) or inosine (postischemic treatment) in an in vivo canine model of reversible global ischemia. Twenty-five anesthetized adult dogs were instrumented (by sonomicrometry) to monitor left ventricular performance from the relationship between stroke work and end-diastolic length as a sensitive and load-independent index of contractility. Hearts of animals supported by cardiopulmonary bypass were subjected to 30 minutes of normothermic global ischemia and 60 minutes of reperfusion. Saline solution containing the pharmacologic agents were infused into the bypass circuit before ischemia (group 1) or during reperfusion (group 2). Control group (group 3) received saline before and after ischemia. Myocardial biopsy specimens were obtained before, during, and after ischemia, and levels of adenine nucleotides, nucleosides, oxypurines, and the oxidized form of nicotinamide-adenine dinucleotide were determined. Left ventricular contractility fully recovered within 30 minutes of reperfusion in the groups treated with erythro-9-(2-hydroxy-3-nonyl)adenine and p-nitrobenzyl-thioinosine (p < 0.05 versus control group). Myocardial adenosine triphosphate was depleted by 50% in all groups at the end of ischemia. Adenosine triphosphate recovered during reperfusion only in the group that was treated with inhibitors before ischemia (group 1). At the end of ischemia, adenosine levels were low (< 10% of total nucleosides) in the control group (group 3) and in the group treated only after ischemia (group 2). A high level of adenosine (> 90% of total nucleosides) was present in group 1. We infer that selective pharmacologic blockade of nucleoside transport, only after ischemic injury, accelerated functional recovery during reperfusion, even without trapping of endogenous adenosine during ischemia and without adenosine triphosphate recovery during reperfusion. Recovery of myocardial adenosi

Topics: Adenine; Adenine Nucleotides; Adenosine; Adenosine Deaminase Inhibitors; Affinity Labels; Animals; Biological Transport, Active; Dogs; Female; Inosine; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Stunning; Purinones; Random Allocation; Thioinosine; Ventricular Function, Left

Adenosine transport in cultured human umbilical vein endothelial cells (HUVEC) was characterized and shown to be mediated by a single facilitated diffusion mechanism. Initial rates of adenosine influx at 22 degrees C were saturable [apparent Michaelis constant, 69 +/- 10 microM; maximum velocity (Vmax), 600 +/- 70 pmol.10(6) cells-1.s-1] and inhibited by nitrobenzylthioinosine (NBMPR). Formycin B had an unusually high affinity [inhibitory constant (Ki), 18 +/- 4.3 microM], whereas inosine had a low affinity (Ki, 440 +/- 68 microM) and nucleobases were without effect on adenosine influx. The number of transporters (1.2 x 10(6) sites/cell) was estimated by NBMPR equilibrium binding (apparent dissociation constant, 0.11 +/- 0.01 nM; maximum binding, 2.0 +/- 0.15 pmol/10(6) cells). In addition, we compared these endothelial cells with those obtained from cords from pregnancies complicated by diabetes (HUVEC-D), since embriopathy may occur in these conditions. HUVEC-D exhibited a 2.3-fold reduction in both the Vmax for adenosine influx and the maximum number of NBMPR binding sites (260 +/- 40 pmol.10(6) cells-1.s-1 and 0.86 +/- 0.08 pmol/10(6) cells, respectively). However, the turnover number for each nucleoside transporter in normal and diabetic HUVEC was similar (approximately 300 adenosine molecules/s). Adenosine metabolism at 10 microM in HUVEC-D was modified compared with normal cells. Intracellular phosphorylation (> 90%) was the predominant pathway in normal HUVEC, whereas in HUVEC-D, substantial levels of adenine and adenosine were detected. The present results demonstrate therefore the downregulation of the NBMPR-sensitive nucleoside transporter and changes in adenosine metabolism in HUVEC from diabetic pregnancies.

Topics: Adenosine; Biological Transport; Cell Division; Cells, Cultured; Endothelium, Vascular; Female; Humans; Pregnancy; Pregnancy in Diabetics; Reference Values; Thioinosine; Time Factors; Umbilical Veins

Although myocardial ATP is essential for myocardial viability and ventricular function, it is a major source of free radical substrates for endothelial xanthine oxidase. Correlation between myocardial ATP and ventricular function has been hindered by the impact of ATP catabolites on ventricular function during reperfusion.. This work results from four separate experiments assessing the role of nucleoside efflux in reperfusion mediated injury to determine the dual role of myocardial ATP in postischemic ventricular dysfunction. An adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and an adenine nucleoside transport blocker, p-nitrobenzylthioinosine (NBMPR), were used to specifically inhibit adenosine deamination and block nucleoside release, respectively. This pharmacological intervention results in site-specific entrapment of intramyocardial adenosine and inosine, generated during ischemia, and blocks degradation to free radical substrates during reperfusion, thereby limiting the impact of reperfusion mediated injury.. Forty-three anesthetized dogs were instrumented to monitor left ventricular performance from the slope of the relationship between stroke work and end-diastolic length (SW/EDL). Hearts were subjected to varying periods (30, 60, or 90 min) of global ischemia and 60 or 120 minutes of reperfusion. Two control groups for 30 and 60 minutes of ischemia (16 dogs) received only saline solution. Four treated groups (27 dogs) received saline containing 100 microM EHNA and 25 mM NBMPR prior to ischemia or only during reperfusion (n = 7). Myocardial biopsies were analyzed for ATP catabolites and NAD+.. Myocardial ATP and left ventricular function were severely depressed by 50% and 80% in the untreated controls, following 30 and 60 minutes of ischemia (37 degrees C), respectively. Ventricular dysfunction was inversely related to inosine levels in the myocardium at the end of the ischemic period. Administration of EHNA/NBMPR before ischemia or only during reperfusion resulted in significant accumulation of mainly adenosine or inosine, respectively. Entrapment of nucleosides was associated with complete recovery of ventricular function after 30 or 60 minutes of ischemia. Hearts subjected to 90 minutes of ischemia developed contracture.. Despite severely reduced ATP levels, ventricular function significantly recovered to preischemic values only in the EHNA/NBMPR-treated groups. Selective blockade of purine release during reperfusion is cardioprotective against post-ischemic reperfusion mediated injury. It is concluded that nucleoside transport plays an important role in regulation of endogenous adenosine and inosine affecting the degree of myocardial injury or protection from reperfusion mediated injury.

Topics: Adenine; Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Dogs; Female; Heart; Inosine; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Thioinosine; Ventricular Function, Left

In cultured cells, dipyridamole, in a dose-dependent manner, prevented the enhancement by 1-beta-D-arabinofuranosylcytosine (AraC) of either N-phosphonoacetyl-L-aspartate (PALA)- or methotrexate- resistance frequency. Maximal blockade of enhancement of PALA-resistance frequency occurred if the dipyridamole was added with or within about 20 hr of PALA addition. Thereafter, the effect of dipyridamole decreased. Nitrobenzylthioinosine similarly reduced AraC enhancement of PALA- and methotrexate-resistance frequency. Both dipyridamole and nitrobenzylthioinosine inhibited uridine and thymidine uptake into cells to a similar extent in cells pretreated or not with AraC. Thus, although inhibition of nucleoside uptake would seem a reasonable explanation for the effect on PALA-resistance frequency by dipyridamole, there is no obvious explanation at present of how dipyridamole selectively affects resistance frequency in AraC-pretreated cells.

Topics: Animals; Aspartic Acid; Cell Line, Transformed; Cricetinae; Cytarabine; Dipyridamole; Drug Resistance; Drug Synergism; Humans; Methotrexate; Nucleosides; Phosphonoacetic Acid; Thioinosine

The influence of inosine on DNA synthesis by Chinook salmon embryo cells (CHSE-214) was investigated because previously cell number was shown to increase from six- to thirtyfold if inosine was added to the basal medium (L-15) supplemented with either dialyzed fetal bovine serum (dFBS), calf serum (CS), or dCS. Relative to L-15, 3H-thymidine incorporation was inhibited by these sera alone but elevated in nondialyzed (intact) FBS. Inosine at 10 microM stimulated 3H-thymidine incorporation from ten- to seventyfold in dFBS, CS, and dCS but was only slightly stimulatory in FBS and in L-15 alone. As well as inosine, hypoxanthine, cIMP, IMP, IDP, and ITP were just as stimulatory, but the nonsalvageable purines (xanthine, xanthosine, and XMP) were not. The stimulatory action of inosine was highest in low density cultures. Dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibitors of facilitated nonconcentrative nucleoside transport, did not completely block the enhancement of cell number by inosine and by themselves increased proliferation in CS and dCS. Overall, these results suggest that exogenous inosine promoted CHSE-214 proliferation by overcoming factors in the nondialyzable fraction of sera that led to purine loss and by raising intracellular purine nucleotides to levels necessary for cells to respond to growth factors in the nondialyzable fraction of sera.

Topics: Animals; Biological Transport; Cattle; Cell Division; Cell Line; Dipyridamole; Fetal Blood; Inosine; Nucleosides; Purines; Pyrimidines; Salmon; Thioinosine; Thymidine; Time Factors

The presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue is reported. Displacement studies using 15 nM [3H]dipyridamole revealed 50-60% of the sites were sensitive to nitrobenzylthioinosine. Non-linear analysis of binding isotherms to estimate the density of nitrobenzylthioinosine-sensitive and -insensitive sites revealed a maximum number of nitrobenzylthioinosine-sensitive binding sites (Bmax) of 395 +/- 19 fmol/mg protein and a nitrobenzylthioinosine-insensitive Bmax of 3910 +/- 700 fmol/mg protein (corresponding Kd values of 0.1 nM and 114 nM respectively). Thus there are approximately 10 times as many nitrobenzylthioinosine-insensitive sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes.

Topics: Adult; Affinity Labels; Aged; Aged, 80 and over; Binding Sites; Dipyridamole; Ependyma; Female; Humans; Male; Middle Aged; Regression Analysis; Thioinosine

Adenosine influx and formycin B influx and efflux were characterized in guinea-pig ventricular myocytes at 22 degrees. Transport by both modes was saturable and inhibited by nitrobenzylthioinosine (NBMPR), indicating the presence of an equilibrative NBMPR-sensitive nucleoside transporter in the cardiomyocytes. The kinetic constants for influx and efflux of formycin B, a non-metabolized nucleoside, were similar, suggesting that the nucleoside transporter exhibits symmetrical kinetics (apparent Km 490 +/- 160 and 700 +/- 140 microM; Vmax 6.5 +/- 1.7 and 3.5 +/- 0.3 nmol/10(6) cells per min for influx and efflux, respectively). No evidence was found of either NBMPR-insensitive equilibrative nucleoside transport or sodium-dependent concentrative nucleoside transport. Inhibition of adenosine influx (apparent Km100 +/- 33 microM), by lidoflazine and the analogues mioflazine, soluflazine and R73-335, gave average Ki values of 730, 100, 64 and 2.9 nM, respectively. These compounds also inhibited formycin B efflux with a similar potency to that of adenosine influx. NBMPR-sensitive nucleoside transport was associated with high affinity binding of NBMPR (apparent Kd approximately 1 nM; 9.6 x 10(5) sites/cell). Specific binding of NBMPR was also inhibited by lidoflazine and its analogues. Mioflazine and soluflazine were 20-30-fold more potent at inhibiting NBMPR-sensitive nucleoside influx in guinea-pig erythrocytes than ventricular myocytes, indicating that the potency of some of the compounds studied is tissue dependent.

Topics: Adenosine; Affinity Labels; Animals; Biological Transport; Cells, Cultured; Formycins; Guinea Pigs; Heart Ventricles; In Vitro Techniques; Kinetics; Lidoflazine; Myocardium; Thioinosine

In this study, we determined whether R75231, (+/-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis( 4-fluoro- phenyl)pentyl]-1-piperazineacetamide, and its two enantiomers, all nucleoside transport inhibitors, could play a role as anti-aggregatory agents. First, we determined the binding characteristics of [3H]nitrobenzylthioinosine, also a nucleoside transport inhibitor, on intact human erythrocytes. The Kd value was 0.27 +/- 0.04 nM and the Bmax was 23.5 +/- 5.1 pmol/10(9) erythrocytes. Second, we studied the ability of R75231 and its enantiomers R88021 ((-)-R75231, or draflazine) and R88016 ((+)-R75231), to displace [3H]nitrobenzylthioinosine. R75231 had an IC50 value of 2.2 +/- 0.3 nM. R88021 was twice as potent as R75231 and R88016 was approximately 20-fold less potent than R75231. Finally, the ability of these nucleoside transport inhibitors to enhance anti-aggregatory effects of adenosine was examined in whole human blood. Adenosine alone, 10 microM, had no effect on ADP-induced platelet aggregation. However, in the presence of 1 microM R75231, 10 microM of adenosine inhibited the aggregatory response completely. Dose-response curves indicated that the IC50 values of draflazine and R88016 were approximately 0.5 microM and 10 microM, respectively. R75231 and its enantiomers are valuable research tools to assess the role of the nucleoside transporter. Moreover, R75231 and draflazine (R88021) may prove to be useful as anti-aggregatory agents.

Topics: Adenosine; Adenosine Diphosphate; Binding Sites; Biological Transport; Blood Platelets; Erythrocytes; Humans; Piperazines; Platelet Aggregation; Platelet Aggregation Inhibitors; Radioligand Assay; Stereoisomerism; Thioinosine

The potential cytoprotective effect of the purine transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI) in a model of myocardial ischaemia and reperfusion was investigated in the anaesthetised ferret. The left anterior descending coronary artery (LAD) was occluded for 90 min, producing ischaemia in 53 +/- 3% of the left ventricular free wall, followed by 240 min reperfusion. NBTI (0.5 mg kg-1, i.v.) was given prior to ischaemia or prior to reperfusion. In addition the effect of purine transport inhibition was investigated in animals subjected to ischaemia without reperfusion. NBTI reduced infarct size from 84.0 +/- 1.7 to 71.4 +/- 3.7% of the area at risk (P < 0.05) when given prior to occlusion of the LAD. NBTI was ineffective however when given 15 min prior to reperfusion. NBTI had no effect upon infarct size produced by ischaemia without reperfusion. The effect of NBTI was independent of significant changes in myocardial blood flow during ischaemia and reperfusion or upon neutrophil infiltration.

Topics: Adenosine; Animals; Atenolol; Blood Gas Analysis; Blood Pressure; Coronary Circulation; Ferrets; Heart Rate; Hemodynamics; Injections, Intravenous; Leukocyte Count; Male; Microspheres; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neutrophils; Peroxidase; Thioinosine

The nitrobenzylthioinosine binding sites from luminal membranes of proximal tubule of pig kidney were solubilized by treatment of the brush-border membrane vesicles with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) in 2% solution. The high yield solubilization of a stable form of the transporter took place in the presence of adenosine in the medium of incubation with the detergent and the additional presence of glycerol as stabilizer. The solubilization of the NBTI-sensitive nucleoside transporter from pig kidney brush-border membranes did not change the nitrobenzylthioinosine (NBTI) binding characteristics; the only major change was a 3-fold decrease in the affinity. The carrier molecule was cross-linked to [3H]NBTI and by electrophoretic characterization under reducing conditions it displayed a molecular mass of 65 kDa. Treatment of the samples at low temperature prior to electrophoresis gave rise to the appearance of further bands corresponding to dimeric and tetrameric forms which interacted non-covalently. The removal of the N-linked oligosaccharides by treatment with endoglycosidase F shifted the molecular mass to 57 kDa. The chromatographic behaviour of the solubilized transporter was similar to that of human erythrocytes and differed from that found in pig erythrocytes. Since the molecular mass of the monomer before and after treatment with endoglycosidase F is the same for pig erythrocytes and pig kidney luminal membranes, the different chromatographic behaviour might result from tissue differences due to transcriptional variations or to posttranscriptional modifications of the transporter molecule.

Topics: Affinity Labels; Animals; Binding Sites; Carrier Proteins; Humans; Kidney Tubules, Proximal; Membrane Proteins; Microvilli; Nucleoside Transport Proteins; Photochemistry; Solubility; Swine; Thioinosine; Tritium

Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated nitrobenzylthioinosine (NBMPR)-sensitive transport activity and large numbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hogue and Cass (1992) Biochem. J. 288, 987-996]. The present study investigates whether NBMPR-sensitive nucleoside transport activity could be induced in Xenopus laevis oocytes by microinjection of poly(A)+ RNA isolated from proliferating cultures of BeWo cells. Expression of uridine transport activity was assayed by comparing rates of uptake (22 degrees C) of 100 microM [3H]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridine uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocytes were injected with 50 ng of BeWo poly(A)+ RNA, and this stimulation was abolished when the RNA-injected oocytes were assayed in the presence of 10 microM NBMPR. The expressed uridine transport activity in oocytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 nM NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 microM [3H]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [3H]uridine in the RNA-injected oocytes were greatly reduced in the presence of high concentrations (2 mM) of non-radioactive nucleosides (adenosine, thymidine, inosine) that are known permeants of NBMPR-sensitive nucleoside transport processes. These results establish that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native transporters.

Topics: Animals; Binding Sites; Biological Transport; Carrier Proteins; Choriocarcinoma; Humans; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Oocytes; Thioinosine; Tumor Cells, Cultured; Xenopus laevis

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.

Topics: Adenosine; Affinity Labels; Antigens, Polyomavirus Transforming; Blood Proteins; Carrier Proteins; Cell Transformation, Neoplastic; Fetus; Humans; Membrane Proteins; Nerve Tissue Proteins; Nucleoside Transport Proteins; Retina; Thioinosine; Transfection; Tumor Cells, Cultured

The HD3 cell, a chicken erythroblast cell line infected with a temperature-sensitive avian erythroblastosis virus, becomes committed to differentiate to an erythrocyte upon temperature shift in presence of inducers. Before induction, the HD3 cell transports glucose and 2-deoxyglucose (2-DG). 3-O-methylglucose is poorly taken up. Upon induction of differentiation, glucose and 2-DG transport activity fall. Twenty-four hours postinduction, up to 75% of the glucose transport activity may disappear. By use of cDNA probes for chicken glucose transporters, two species of mRNA of 3.1 and 1.7 kb (equivalent to mammalian GLUT1 and GLUT3 mRNA, respectively) are detected. Both messages virtually disappear within 48 h after induction. Run-on assays show the cessation of synthesis of the corresponding RNAs parallel to the loss of glucose transport. In contrast to the glucose transporters, the nucleoside transporter level increases after induction of hematopoiesis. This developmental pattern is consistent with earlier studies showing that mature chicken erythrocytes have little glucose transport activity but retain appreciable levels of the nucleoside transporter and that nucleosides and glutamine provide major sources of oxidizable carbon compounds to sustain metabolism in circulating chicken erythrocytes.

Topics: 3-O-Methylglucose; Affinity Labels; Animals; Biological Transport, Active; Blood Proteins; Carrier Proteins; Cell Differentiation; Cell Division; Cell Line; Chickens; Deoxyglucose; Erythroblasts; Gene Expression; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 3; Glutamine; Kinetics; Membrane Proteins; Methylglucosides; Monosaccharide Transport Proteins; Nerve Tissue Proteins; Nucleoside Transport Proteins; Reticulocytes; RNA, Messenger; Sheep; Thioinosine; Time Factors

Adenosine (Ado) is a potent vasodilator that has occasionally been shown to cause vasoconstriction. Constrictor responses are generally attributed to A1-receptor stimulation or interactions with the renin-angiotensin system. We describe a previously unreported vasoconstrictor action of Ado and inosine (Ino) in hamster cheek pouch arterioles and examine the mechanism by which these nucleosides induce constriction. Arterioles were dissected from male Golden hamster cheek pouches, transferred to a 37 degrees C tissue chamber, and cannulated at both ends. Changes of luminal diameter in response to Ado were measured to generate cumulative concentration-response curves. The concentration-response curves were biphasic: 10(-6) M Ado elicited an intense, transient constriction, and higher concentrations induced dilator responses. Pretreatment with 8(p-sulfophenyl)theophylline, an Ado receptor antagonist, inhibited the dilator responses but did not alter the constriction. Inhibition of Ado uptake with S-(4-nitrobenzyl)-6-thio-inosine eliminated the constrictor response without altering dilator responses. Similar effects were found after pretreatment with an Ado deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Finally, Ino, a metabolite of Ado, induced constrictions of similar magnitude to those seen with Ado, but at higher concentrations. The constrictor response was focal in nature, suggesting discrete sites of action of Ado. Methylene blue staining after Ado application revealed degranulated mast cells closely associated with the vessel wall, indicating a possible role for mast cell degranulation in the constrictor response. Supporting this idea were the observations that inhibition of degranulation by 10 microM cromolyn blocked the constrictor response, and compound 48/80 (a mast cell secretagogue) caused constriction similar to that elicited by Ado.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Affinity Labels; Animals; Arterioles; Cricetinae; Dose-Response Relationship, Drug; In Vitro Techniques; Inosine; Male; Mast Cells; Mesocricetus; Methylene Blue; Mouth; Muscle, Smooth, Vascular; p-Methoxy-N-methylphenethylamine; Purinergic P1 Receptor Antagonists; Thioinosine; Vasoconstriction

Adenosine transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, is mediated by a saturable transport system that has not been characterized extensively. Moreover, the inability of adenosine to augment cerebral blood flow in most species after intracarotid adenosine administration suggests the presence of an enzymatic BBB to circulating adenosine. Therefore, the present studies investigate the Michaelis-Menten kinetics of BBB adenosine transport and the rate of cerebral metabolism of circulating adenosine after internal carotid artery perfusion in anesthetized rats. The studies also assess the ability of various adenosine analogues to inhibit [3H]adenosine transport at the BBB in vivo. Initial rates of BBB transport of adenosine in vivo were observed for at least 15 sec of internal carotid artery perfusion. BBB adenosine transport was partially sodium-dependent and was saturable with the following kinetic parameters: Km = 1.1 +/- 0.2 microM; Vmax = 202 +/- 44 pmol/min/g; and KD = 34 +/- 6 microliters/min/g. BBB transport of [3H]adenosine was not inhibited by cyclohexyladenosine or S-(4-nitrobenzyl)-6-thioinosine, but was inhibited by dipyridamole (Ki = 2.2 +/- 0.9 microM). Capillary depletion studies were performed, which demonstrated sequestration of [3H] radioactivity by the microvascular pellet after carotid arterial infusion of [3H]adenosine. Only 10 +/- 3% of cerebral [3H] radioactivity resided in the free adenosine pool after 15 sec of internal carotid artery perfusion of [3H]adenosine and rapid termination of brain metabolism with microwave irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Biological Transport; Blood-Brain Barrier; Brain; Carrier Proteins; Perfusion; Rats; Thioinosine; Tritium

Adenosine uptake via nucleoside transporters is inhibited when S49 and NG108-15 cell lines cells are exposed to ethanol. This inhibition leads to an accumulation of extracellular adenosine that binds to adenosine A2 receptors and increases cAMP production. Subsequently, there is a heterologous desensitization of receptors coupled to adenylyl cyclase for which adenosine also is required. There are multiple classes of facilitative and concentrative nucleoside transporters that could be inhibited by ethanol to initiate this cascade of events. In this paper, we establish that adenosine uptake by only one type of nucleoside transporter, an NBMPR-sensitive facilitative transporter, is inhibited by ethanol. There is no effect on other classes of nucleoside transporters even when present in the same cell. Thus, ethanol-induced extracellular accumulation of adenosine results specifically from inhibition of NBMPR-sensitive facilitative nucleoside transporters. We also find that human lymphocytes express only facilitative nucleoside transporters and that the NBMPR-sensitive type is predominant. Thus, inhibition of this type of transporter by ethanol may be related to the desensitization of cAMP signal transduction that we have reported in lymphocytes from alcoholics.

Topics: Adenosine; Adult; Biological Transport; Blood Proteins; Carrier Proteins; Ethanol; Formycins; Glutamates; Glutamic Acid; Humans; In Vitro Techniques; Isoleucine; Lymphocytes; Male; Membrane Proteins; Middle Aged; Nucleoside Transport Proteins; Sodium; Thioinosine

1. Adenosine transport is subjected to regulation by hormones. Glucocorticoids, sexual steroids, and retinoic acid inhibit adenosine transport in chromaffin cells after a long-term incubation period (24 hr). No effects were observed after a short-term incubation period (10 min). 2. The kinetic parameters of transporters were studied. No significant changes were observed for the affinity constant (Km), whose value remains at 1 +/- 0.2 microM after 24-hr incubation in the presence of these compounds. The maximal velocity (Vmax) was significantly modified, with a decrease of about 20% in all cases. 3. NBTI binding was not modified in its affinity constant or maximal bound capacity (Bmax) by the presence of these compounds for a 24-hr incubation period. Thus the efficiency of transporters (quotient Vmax/Bmax) changed from 10.9 +/- 0.08 adenosine molecules transported per transporter per sec in the control cells to 9.1 +/- 0.07 in hormone-treated cultured cells. 4. The thyroid hormone (T3) significantly increased adenosine transport in a long-term incubation period in chromaffin cells (24 hr). This activatory effect is antagonized by steroid hormones and retinoic acid.

Topics: Adenosine; Adrenal Medulla; Affinity Labels; Animals; Biological Transport; Carrier Proteins; Cattle; Cells, Cultured; Dexamethasone; Hormones; Kinetics; Steroids; Thioinosine; Tretinoin; Triiodothyronine

Nitrobenzylthioinosine (NBTI) is a high affinity probe for facilitated diffusion nucleoside transporters. Kinetic analysis of the binding of [3H]NBTI to plasma membranes of chromaffin cells was conducted in the presence or absence of adenosine 5'-triphosphate (ATP). Similar curvilinear plots with a Hill number of 1.32 were obtained in both conditions. ATP significantly increased the number of NBTI binding sites in these preparations showing Bmax values of 1.62 +/- 0.20 pmol/mg protein for controls and 3.22 +/- 0.31 pmol/mg protein in the presence of ATP. However, the affinity constant (KD) was not significantly modified. The non-metabolizable ATP analogue, 5'-adenylyl imidodiphosphate (AMP-PNP) and diadenosine tetraphosphate (Ap4A) can mimic the stimulatory ATP effect, but adenosine monophosphate (AMP) has no effect on the NBTI binding to plasma membranes. These results indicate a modulatory role for ATP, non-hydrolysis dependent, on nucleoside transport in chromaffin cells. Therefore, a nucleotide binding site on the nucleoside transporter similar to that described for glucose transporter could be suggested.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Cattle; Cell Membrane; Centrifugation, Density Gradient; Enterochromaffin Cells; Kinetics; Thioinosine

Mounting evidence suggests a protective effect of exogenous adenosine in myocardial ischemia and reperfusion. We tested the hypothesis that augmentation of endogenous adenosine levels, achieved by inhibiting adenosine catabolism and washout, is beneficial in postischemic myocardial dysfunction ("stunning").. In phase I of the study, open-chest dogs undergoing a 15-minute coronary artery occlusion and 4 hours of reperfusion received an intracoronary infusion of either saline (controls, n = 23) or 6-(4-nitrobenzyl)-mercapto: purine ribonucleoside (NBMPR, a selective nucleoside transport inhibitor) combined with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, a potent adenosine deaminase inhibitor) (EHNA + NBMPR, n = 15) starting 15 minutes before coronary occlusion and ending 15 minutes after the initiation of reflow. Regional myocardial function (assessed as systolic wall thickening) was similar in control and treated groups at baseline and during ischemia. After reperfusion, however, the dogs treated with EHNA + NBMPR exhibited a significant improvement in the recovery of function, which was evident as early as 30 minutes after restoration of flow and was sustained throughout the rest of the reperfusion phase. The enhanced recovery effected by EHNA + NBMPR could not be attributed to nonspecific factors such as differences in collateral flow during occlusion, coronary flow after reperfusion, arterial pressure, heart rate, or other hemodynamic variables. In phase II of the study, the myocardial content of adenine nucleotides and nucleosides was measured by high performance liquid chromatography in myocardial biopsies obtained serially from open-chest dogs undergoing the same protocol used in phase I. There were no significant differences between control (n = 8) and treated (n = 9) dogs with respect to myocardial levels of adenosine triphosphate (ATP) at 30 and 60 minutes after reperfusion, indicating that the beneficial effects of EHNA + NBMPR cannot be ascribed to repletion of ATP stores. Compared with controls, dogs treated with EHNA + NBMPR exhibited a much larger increase in myocardial adenosine (6.07 +/- 1.47 vs 1.03 +/- 0.16 nmol/mg protein, P < .05) and a much smaller increase in inosine (0.52 +/- 0.27 vs 3.04 +/- 0.54 nmol/mg protein, P < .05) at the end of ischemia, such that the inosine-to-adenosine ratio noted in controls was completely reversed (approximately 6:1 vs approximately 1:6, respectively). In the treated group, adenosine levels remained markedly increased compared with controls up to 1 hour after reperfusion.. This study demonstrates that (1) administration of an adenosine deaminase inhibitor plus a nucleoside transport blocker is remarkably effective in augmenting myocardial adenosine levels during regional ischemia and subsequent reperfusion in vivo, (2) this augmentation of adenosine results in a significant and sustained attenuation of myocardial stunning, and (3) the attenuation of stunning is not due to ATP repletion or to nonspecific actions on hemodynamic variables or coronary flow. These findings suggest that endogenous adenosine production during ischemia serves as an important pathophysiological mechanism that protects against myocardial stunning. The results also suggest that augmentation of endogenous adenosine (without exogenous adenosine administration) represents an effective therapeutic approach to the alleviation of reversible postischemic dysfunction.

Topics: Adenine; Adenine Nucleotides; Adenosine; Animals; Coronary Circulation; Dogs; Heart; Hemodynamics; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Stunning; Nucleosides; Thioinosine

Metabolic interventions capable of preventing ventricular dysfunction "stunning" or accelerating its functional recovery have potential clinical importance. Myocardial protection of the stunned myocardium has not been documented when drugs were administered only during postischemic reperfusion. The role of ATP depletion and release of purines in myocardial injury was assessed using the selective nucleoside transport blocker p-nitrobenzylthioinosine (NBMPR) in a combination with specific adenosine deaminase inhibitor erythro-9-[hydroxy-3-nonyl]adenine (EHNA) administered during reperfusion after reversible ischemic injury.. Sixteen anesthetized dogs were instrumented with minor axis sonocrystals and intraventricular Millar. Ventricular performance was determined, off bypass, from the slope of the relationship between stroke-work and end-diastolic length as a sensitive and load-independent index of contractility within physiological range. Hearts were subjected to 20 minutes' warm global ischemia and reperfused with warm blood treated with either saline (control group, n = 8) or saline containing 100 mumol/L EHNA and 25 mumol/L NBMPR (EHNA/NBMPR-treated group, n = 8). Myocardial biopsies were collected and analyzed for ATP and metabolites using high-performance liquid chromatography. Warm ischemia induced significant depletion of ATP (P < .05 versus preischemia) and accumulation of inosine at the end of ischemia (> 90% of total nucleosides) in both groups. Complete functional recovery was observed in the EHNA/NBMPR-treated group (P < .05 versus control group).. Selective entrapment of adenine nucleosides during postischemic reperfusion attenuated ventricular dysfunction (stunning) after brief global ischemia. It is concluded that nucleoside transport plays an important role in myocardial stunning, and its blockade augmented myocardial protection against reperfusion injury. Selective entrapment of endogenous inosine, generated during ischemia, represents an attractive therapeutic approach to the alleviation of postischemic dysfunction mediated by reperfusion in a wide spectrum of ischemic syndromes, including percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery.

Topics: Adenine; Adenosine Triphosphate; Affinity Labels; Animals; Chromatography, High Pressure Liquid; Dogs; Drug Therapy, Combination; Female; Inosine; Male; Myocardial Reperfusion; Myocardial Stunning; Myocardium; Thioinosine; Time Factors

Binding expressions are derived and analytical procedures developed for the quantitative characterization of inhibitor binding that is only partially competitive with the interaction between an acceptor and the ligand that is being monitored. Two such situations are considered: (i) that in which the partial competition reflects binding of inhibitor to fewer acceptor sites than available to ligand; and (ii) that in which the partial competition reflects binding of inhibitor to acceptor sites in addition to those occupiable by ligand. The potential efficacy of the suggested analyses is then explored by their application to simulated data that span the likely range of experimental behavior. Quantitative analysis of the displacement of [3H]nitrobenzylthioinosine from cultured leukemic cells by an adduct of 5'-S-(2-amino-ethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine with fluorescein-5-isothiocyanate is used to establish that the cells possess 6% fewer sites (150,000 cf. 159,000 sites/cell) for the fluorescent adduct than for the tritiated ligand, and that the binding is 10-fold weaker (binding constant of 0.28 cf. 2.8 nM-1). Corresponding analysis of results obtained with bovine aorta endothelial cells indicates that a 3-fold weaker interaction (binding constant of 1.1 cf. 3.3 nM-1) occurs between the fluorescent adduct and 79% of the cell sites accessible to the tritiated ligand. The present analytical procedures extend the utility of competitive binding assays for the quantitative screening of potential inhibitors by removing the inherent limitation of existing analyses that all acceptor sites be accessible to both the competing solute and the indicator ligand.

Topics: Adenosine; Affinity Labels; Binding Sites; Binding, Competitive; Humans; Kinetics; Leukemia, Myelomonocytic, Acute; Mathematical Computing; Models, Biological; Receptors, Cell Surface; Thioinosine; Thionucleosides; Tritium; Tumor Cells, Cultured

The present study aimed at determining the modulation by adenosine of the release of noradrenaline in the epididymal portion of the rat vas deferens. The tissues were treated with pargyline and perifused in the presence of desipramine and yohimbine. Up to four periods of electrical stimulation were applied (5 Hz, 9 min). The A1-adenosine receptor selective agonist R-N6-phenylisopropyladenosine (R-PIA; 100-900 nmol.l-1) reduced, whereas the A2A-receptor selective agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3-30 nmol.l-1) increased the electrically-evoked noradrenaline overflow in a concentration-dependent manner. The nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA; 30-300 nmol.l-1) reduced noradrenaline overflow, but the effect did not depend on the concentration. Adenosine deaminase at the concentration of 0.5 mu.ml-1 decreased but at that of 2.0 mu.ml-1 increased noradrenaline overflow. The inhibitors of adenosine uptake, S-(4-nitrobenzyl)-6-thioinosine (NBTI; 50 nmol.l-1) and dipyridamole (3 mumol.l-1), increased the electrically-evoked noradrenaline overflow. The A1-adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 20 nmol.l-1) caused an increase whereas the A2-adenosine receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX; 0.1 mumol.l-1) caused a decrease. NBTI (50 nmol.l-1), partially antagonized the effect of both DPCPX (20 nmol.l-1) and DMPX (0.1 mumol.l-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine Deaminase; Animals; Dipyridamole; Electric Stimulation; Epididymis; In Vitro Techniques; Lactates; Male; Muscle, Smooth; Norepinephrine; Purinergic P1 Receptor Antagonists; Purinergic P2 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Receptors, Purinergic P2; Thioinosine; Vas Deferens

The purpose of this study was to compare the responses of isolated hearts of the diving muskrat with the nondividing guinea pig (GP) to determine the contribution of adenosine (ADO) to the profound bradycardia that was seen in isolated muskrat hearts during exposure to hypoxia. Muskrat hearts were more sensitive than GP hearts to the heart rate-lowering effects of exogenously applied ADO or a stable ADO analogue, (R)-N6-(phenylisopropyl)adenosine. The hearts of both species were unpaced, and the bradycardia appeared to be due to high degree of atrioventricular block. Radioligand binding with 8-cyclopentyl-1,3-[3H]dipropylxanthine to A1-ADO receptors was greater in cardiac membranes prepared from GP hearts than from muskrat hearts. Nucleoside transporter antagonist binding was also greater in GP hearts compared with muskrats. This was determined by membrane binding of [3H]-nitrobenzylthioinosine, an antagonist of nucleoside transport. Both muskrat and GP hearts responded to 30 min of hypoxic perfusion by releasing ADO into the coronary effluent; however, the muskrat hearts released approximately five times more than the GP hearts. When hearts were subjected to hypoxia in the presence of ADO deaminase, theophylline, or 8-(p-sulfophenyl)theophylline, the hypoxia-induced bradycardia was blocked in the GP hearts and either slightly reduced or not affected in muskrat hearts. In contrast to GP hearts, muskrat hearts release larger amounts of ADO during hypoxia and are more sensitive to the negative chronotropic effects of exogenously administered ADO; yet the hypoxia-induced bradycardia does not appear to be exclusively mediated by ADO in the muskrat as it is in the isolated GP heart.

Topics: Adenosine; Adenosine Deaminase; Animals; Arvicolinae; Guinea Pigs; Heart Rate; In Vitro Techniques; Reference Values; Theophylline; Thioinosine; Xanthines

To determine if the purine vasodilator adenosine participates in mediating autoregulatory dilations of the retinal microcirculation in vivo.. The retinal microcirculation of isoflurane-anesthetized newborn pigs was observed by videomicroscopy (x310). Systemic hypoxia (PaO2 = 24 +/- 1 mm Hg; n = 8) or hemorrhagic hypotension (MABP = 41 +/- 1 mm Hg; n = 5) was induced, and the effect of intravitreal microsuffusion of 0.4 nmol of the adenosine receptor antagonist 8-sulfophenyltheophylline (8SPT) on retinal arteriolar dilations resulting from these stimuli were measured. The effect of potentiation of endogenous interstitial adenosine concentrations with 0.2 nmol 4-nitrobenzyl-6-thioinosine (NBTI) on the response to hypotension (MABP = 43 +/- 2 mm Hg; n = 4) was also determined.. The significant vasodilatative response of the retinal arterioles to systemic hypoxia (36 +/- 8% increase in diameter above baseline; P = 0.0012) was attenuated 55% (P < 0.0001) by the adenosine antagonist 8SPT. Similarly, the significant arteriolar vasodilation induced by systemic hypotension (29 +/- 3% increase in diameter; P < 0.0001) was inhibited 76% by 8SPT (P = 0.0002). When adenosine reuptake was inhibited with NBTI, the arteriolar dilation induced by hypotension (32 +/- 5% increase in diameter; P = 0.0234) was potentiated 100% (P = 0.0117).. Our finding that inhibition or potentiation of endogenous adenosine action uniquely affected retinal arteriolar dilatative responses to hypoxia and hypotension suggests that adenosine is a key participant in mediating autoregulatory adjustments in retinal blood flow in the eye of the newborn.

Topics: Adenosine; Animals; Arterioles; Homeostasis; Microcirculation; Muscle, Smooth, Vascular; Ocular Hypotension; Oxygen Consumption; Receptors, Purinergic; Retinal Artery; Swine; Theophylline; Thioinosine; Vasodilation; Video Recording

We have investigated the potential role of a bi-directional nucleoside carrier in the release of endogenous adenosine from spinal cord synaptosomes by examining the effects of dipyridamole and nitrobenzylthioinosine (NBI) on evoked release of adenosine. When 40 pmol adenosine were added to synaptosomes, only 70 +/- 2% was recovered, suggesting 30% uptake of adenosine. Dipyridamole (0.1-10 microM) reduced this uptake and also increased basal adenosine release, probably due to inhibition of the re-uptake of adenosine derived from released nucleotide. In contrast, NBI (0.1-10 microM) had no effect on either uptake of added adenosine or on basal release of adenosine. Addition of K+ (24 mM) and morphine (10 microM) produced a 50-60% increase in the release of adenosine, and this was reduced 35-98% by both dipyridamole and NBI (0.01-10 microM). Dipyridamole (0.01-1 microM) had no effect on the release of nucleotides (detected as adenosine) induced by noradrenaline, 5-hydroxytryptamine (5-HT) and capsaicin (50 microM each), although 10 microM dipyridamole significantly reduced release evoked by noradrenaline and 5-HT. This latter effect of dipyridamole was determined not to be due to inhibition of ATP release when measured directly. Within the spinal cord, there is a removal system for adenosine which is dipyridamole-sensitive but NBI-insensitive. Release of adenosine, but not nucleotides, appears to occur via this carrier system. The inhibition of release by NBI, but its lack of effect on uptake, suggests the involvement of heterogeneous carrier molecules in adenosine uptake and release from the spinal cord.

Topics: Adenosine; Animals; Capsaicin; Dipyridamole; In Vitro Techniques; Male; Morphine; Norepinephrine; Nucleosides; Potassium; Rats; Rats, Sprague-Dawley; Serotonin; Spinal Cord; Synaptosomes; Thioinosine

The transport of adenosine into blood from beta-thalassaemia subjects was measured to provide a background to the relationship between resistance of malaria infection and beta-thalassaemia. Adenosine transport was significantly reduced in the abnormal cells in the blood samples. As adenosine is one of the major purines salvaged by P. falciparum malaria, we suggest that the resistance to malaria in beta-thalassaemia subjects may be due to a nutrient deficiency in the abnormal red cells.

Topics: Adenosine; Adult; beta-Thalassemia; Biological Transport; Child; Erythrocytes; Humans; Malaria; Thioinosine

The neuroprotective effects of adenosine are well-recognized. Recently, propentofylline, a xanthine derivative, has been shown to increase extracellular concentrations of adenosine in ischemic brain and to limit the extent of neuronal damage in experimental models of cerebral ischemia. Since the concentration of adenosine in brain is controlled, in part, by nucleoside transporter proteins, the action of propentofylline was proposed to be due to inhibition of mediated transfer of adenosine across cell membranes. To determine the likelihood of this mechanism, we examined the inhibitory effects of propentofylline on [3H]adenosine transport by the three best-characterized nucleoside transport processes, es, ei, and cif in cultured cell lines under conditions where only a single transporter type was operative. Propentofylline inhibited [3H]adenosine uptake by each of the three transport processes in a concentration-dependent manner. The greatest inhibitory potency was for es transporters (L1210/B23.1 cells), with an IC50 value of 9 microM, followed by ei transporters, with IC50 values of 170 microM (L1210/C2 cells) and 166 microM (Walker 256 cells). Propentofylline was a weak inhibitor of cif transporter, with an IC50 value of 6 mM. These results demonstrate that propentofylline is an inhibitor of adenosine transport processes and suggest that its neuroprotective effects may be due to an increase in extracellular concentrations of adenosine by virtue of inhibition of es transporter function.

Topics: Adenosine; Animals; Biological Transport; Brain Ischemia; Cell Line; Mice; Rats; Thioinosine; Tritium; Tumor Cells, Cultured; Xanthines

This study investigated the interaction of the mioflazine derivative R75231 with the nucleoside transport system of rabbit cortical synaptosomes, and assessed the binding of [3H]R75231 to human erythrocyte ghost membranes. R75231 was a potent inhibitor of [3H]nitrobenzylthioinosine binding and [3H]uridine uptake in synaptosomes (Ki < 10 nM). This inhibition was evident even after extensive washing of the synaptosomes, subsequent to exposure to R75231. In addition to its tight binding characteristics, R75231 was shown to be a 'mixed' type inhibitor of [3H]nitrobenzylthioinosine binding (increased KD, decreased Bmax). [3H]R75231 bound with high affinity (KD = 0.4 nM) to erythrocyte membranes with a Bmax of 44 pmol/mg protein, which is comparable to the number of [3H]nitrobenzylthioinosine binding sites in this preparation. Binding of [3H]R75231 to these membranes was reversible, but the rate of dissociation was dependent upon the displacer used. Nitrobenzylthioinosine and dipyridamole each induced a complete dissociation of site-bound [3H]R75231 at rates not significantly different from those observed using a protocol involving a 100-fold dilution with buffer (no displacer). However, R75231 and mioflazine slowed the rate of dissociation of [3H]R75231 and actually caused an initial increase in the amount of site-bound [3H]R75231. Adenosine, on the other hand, enhanced the rate of [3H]R75231 dissociation. These results indicate that R75231 binding to the nucleoside transporter is a complex reaction, which may involve multiple interacting sites demonstrating positive cooperativity.

Topics: Adenosine; Animals; Binding, Competitive; Carrier Proteins; Cerebral Cortex; Erythrocyte Membrane; Half-Life; Humans; In Vitro Techniques; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Piperazines; Rabbits; Synaptosomes; Thioinosine; Uridine

Two distinct transporters for nucleobases have been characterized in LLC-PK1 cells. The first system accumulates hypoxanthine against a concentration gradient in the presence of sodium. The sodium-dependent uptake of hypoxanthine was saturable at 22 degrees C with a Km value of 0.79 +/- 0.43 microM, a Vmax of 15 +/- 4 pmol/mg protein/60 s, and a Na+:hypoxanthine coupling stoichiometry of 1.27 +/- 0.20. Uptake of hypoxanthine was inhibited by 5-fluorouracil, uracil, thymine, and guanine (Ki values 3-6 microM). Adenine and nucleosides were without effect. Using cell monolayers grown on a permeable filter support, Na(+)-dependent hypoxanthine uptake occurred preferentially from the apical surface. The second system exhibited no cation specificity and was saturable with a low affinity for hypoxanthine (Km of 124 +/- 22 microM) and a high Vmax of 275 +/- 38 pmol/mg protein/60 s. Adenine and guanine inhibited Na(+)-independent hypoxanthine uptake (Ki values 30 +/- 15 and 18 +/- 7 microM, respectively). Other nucleobases and nucleosides exhibited little or no inhibition of equilibrative hypoxanthine influx. Dipyridamole, dilazep, and phloridzin were effective inhibitors of Na(+)-dependent hypoxanthine uptake but had little effect on the Na(+)-independent flux. This study represents the first direct demonstration of a unique high affinity Na+ nucleobase co-transporter system in cultured animal cells.

Topics: Animals; Biological Transport; Cations, Monovalent; Cell Line; Dilazep; Dipyridamole; Epithelium; Hypoxanthine; Hypoxanthines; Kidney; Kinetics; Nucleosides; Purines; Pyrimidines; Sodium; Thioinosine; Tritium

Adenosine (ADO) has been shown previously to inhibit p-nitrophenol glucuronidation in a concentration-dependent manner when concurrently incubated in isolated rat hepatocytes for 30-60 min. In the current study, preincubation of ADO (500 microM) in isolated hepatocytes for 30 min prior to addition of 100 microM p-nitrophenol resulted in a greater inhibition of glucuronidation when compared to that without preincubation (80 vs 50% inhibition). The inhibitory effect of 250 microM ADO on glucuronidation was decreased from 60 to 10% in the presence of the ADO transport inhibitor nitrobenzyl thioinosine during the 30-min preincubation period. Without prior incubation, 100 microM dibutyryl cyclic AMP (DBcAMP) produced an inhibition of glucuronidation similar to that of 500 microM ADO. In contrast to ADO, there was no significant difference in the inhibitory effect of DBcAMP on p-nitrophenol glucuronidation with or without a 30-min preincubation. Thus, DBcAMP and ADO appear to inhibit glucuronidation through different mechanisms. Furthermore, these results indicate that the inhibitory effect of ADO on p-nitrophenol glucuronidation is dependent to a large degree on the cellular uptake of ADO into hepatocytes, while a portion of the inhibitory effect may arise from the generation of intracellular cyclic AMP.

Topics: Adenosine; Animals; Bucladesine; Cells, Cultured; Liver; Male; Nitrophenols; Rats; Rats, Sprague-Dawley; Thioinosine; Uridine Diphosphate Glucuronic Acid

The inhibition of [3H]nitrobenzylthioinosine ([3H]NBI) binding to human parietal cortex membranes by adenosine transport inhibitors, adenosine receptor agonists and antagonists and dihydropyridines was investigated. While the adenosine transport inhibitors inhibited [3H]NBI binding with Ki values in the low nanomolar range and the adenosine A1 receptor agonist, cyclopentyladenosine, with a Ki in the low micromolar range, no IC50 values could be obtained for the adenosine receptor antagonists at concentrations up to 100,000 nM. Among the dihydropyridines (+)-nimodipine was the most potent with a Ki of 201 +/- 55 nM. Inhibition of adenosine transport thus may contribute to the clinical effects of nimodipine in the central nervous system.

Topics: Dihydropyridines; Humans; In Vitro Techniques; Nimodipine; Parietal Lobe; Receptors, Purinergic P1; Thioinosine

We have used enzymic cleavage by trypsin in conjunction with glycosidase digestion to probe the transmembrane topologies and molecular structures of mammalian equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive, nucleoside transport systems. Transporters from four species (human, pig, guinea pig, and rat) and three tissues (erythrocyte, liver, and lung), which differ from each other in size and in their sensitivity to inhibition by the vasodilator dipyridamole, were investigated. Broadly equivalent sites of [3H]NBMPR photolabeling, carbohydrate attachment, and trypsin cleavage were observed for all systems. Results from these experiments demonstrate that molecular weight differences between rat transporters and those from two other species (human and guinea pig) are due largely to oligosaccharide heterogeneity and that the low dipyridamole sensitivity of rat nucleoside transporters is probably a consequence of relatively minor differences in molecular structure. In marked contrast, carbohydrate removal increases the molecular weight difference between the pig erythrocyte transporter and, for example, that in human erythrocytes. This polypeptide difference is limited largely, if not completely, to one tryptic fragment of the protein. In the case of the human erythrocyte transporter, the site of N-linked glycosylation has been located very close to one end of the protein, and the site of NBMPR photolabeling to within 16 kDa of that site. Trypsin cleavage occurs endofacially. Our results provide evidence of substantial structural conservation among mammalian NBMPR-sensitive nucleoside transporters.

Topics: Affinity Labels; Animals; beta-Galactosidase; Blood Proteins; Blotting, Western; Carrier Proteins; Cell Membrane; Erythrocyte Membrane; Glycoside Hydrolases; Glycosylation; Guinea Pigs; Humans; Liver; Lung; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Membrane Proteins; Nucleoside Transport Proteins; Rats; Rats, Sprague-Dawley; Swine; Thioinosine; Trypsin

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

Topics: Adenosine; Binding Sites; Carrier Proteins; Cell Line; Cytarabine; Fluoresceins; Humans; Leukemia, Myeloid; Ligands; Membrane Proteins; Nucleoside Transport Proteins; Purine Nucleosides; Thioinosine; Thionucleosides

A1 adenosine receptors in the rat prepiriform cortex play an important role in the inhibition of bicuculline methiodide-induced convulsions. In the present study we evaluated manipulation of endogenous adenosine in this brain area as a strategy to effect seizure suppression. All compounds evaluated were unilaterally microinjected into the rat prepiriform cortex. Administration of exogenous adenosine afforded a dose-dependent protection (ED50 = 48.1 +/- 8.4 nmol) against bicuculline methiodide-induced seizures, and these anticonvulsant effects were significantly potentiated by treatment with an adenosine kinase inhibitor, 5'-amino-5'-deoxyadenosine; by the adenosine transport blockers, dilazep or nitrobenzylthioinosine 5'-monophosphate; and by an adenosine deaminase inhibitor, 2'-deoxycoformycin. When administered alone, 5'-amino-5'-deoxyadenosine, 5'-iodotubercidin and dilazep were found to be highly efficacious as anticonvulsants with respective ED50 values of 2.6 +/- 0.8, 4.0 +/- 2.7 and 5.6 +/- 1.5 nmol. In contrast, 2'-deoxycoformycin was both less potent and less efficacious. These results suggest that accumulation of endogenous adenosine may contribute to seizure suppression, and that adenosine kinase and adenosine transport may play a pivotal role in the regulation of extracellular levels of adenosine in the central nervous system. The adenosine antagonist, 8-(p-sulfophenyl)theophylline, increased markedly the severity of bicuculline methiodide-induced seizures. Moreover, reduction of extracellular adenosine formation by a focal injection of an ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine diphosphate, produced generalized seizures (ED50 = 37.3 +/- 22.7 nmol). Together the proconvulsant effect of an adenosine receptor antagonist and the convulsant action of an ecto-5'-nucleotidase inhibitor further support the role of endogenous adenosine as a tonically active antiepileptogenic substance in the rat prepiriform cortex.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Kinase; Animals; Bicuculline; Cerebral Cortex; Male; Pentostatin; Rats; Rats, Sprague-Dawley; Receptors, Purinergic; Seizures; Theophylline; Thioinosine

The characteristics of Na(+)-dependent and Na(+)-independent uridine uptake at 22 degrees C were determined for monolayers of OK renal epithelial cells. The majority of uridine influx in subconfluent to early confluent (day 1 postconfluency) OK monolayers was mediated via a facilitated-diffusion pathway (apparent Km 160 +/- 41 microM, Vmax 610 +/- 100 pmol/mg protein per min). This system was inhibited with high affinity by nitrobenzylthioinosine (NBMPR) (IC50 value 1.5 nM) and by purine and pyrimidine nucleosides. Specific [3H]NBMPR binding sites were detected in OK monolayers (apparent Kd 0.67 +/- 0.25 nM, Bmax 90 +/- 19 fmol/mg protein) yielding a turnover number for the carrier of 112 uridine molecules/site per s at 22 degrees C. Na(+)-dependent uridine uptake was minor in subconfluent OK monolayers, but increased 8-fold with time after confluency reaching a stable plateau at 8 days postconfluency. Inhibition of Na(+)-dependent 1 microM uridine uptake by inosine, guanosine, adenosine and uridine was biphasic with approx. 40% of the total uptake inhibited with high affinity (IC50 value 2 to 14 microM). Concentrations of thymidine and cytidine up to 1 mM had no effect on Na(+)-dependent uridine uptake and no Na(+)-dependent thymidine influx by confluent OK monolayers was detected. Using cell monolayers grown on a permeable filter support, Na(+)-dependent uridine uptake occurred preferentially from the apical surface. This high affinity component of Na(+)-dependent uridine uptake is suggested to represent the Na(+)-dependent purine preferring N1 nucleoside transporter. The Na+/uridine stoichiometry for this system was consistent with 1:1. The remaining component of Na(+)-dependent uridine uptake was inhibited by some nucleosides, such as guanosine and inosine, with low affinity (IC50 values of 0.6 to 5 mM). Other nucleosides showed little specific inhibition. We propose that this component of uridine uptake represents a mutated carrier that binds nucleosides but is defective in the translocation of permeant.

Topics: Animals; Carrier Proteins; Cell Line; Cellular Senescence; Epithelium; Kidney Tubules, Proximal; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Opossums; Purine Nucleosides; Sodium; Thioinosine; Uridine

The goal of this study was to determine whether specific transport systems are involved in nucleoside elimination from the cerebrospinal fluid (CSF). First, in vitro studies were carried out in isolated choroid plexus tissue slices from rat to ascertain the mechanisms of transport of formycin B, a model nucleoside analogue. 3H-Formycin B accumulated against a concentration gradient in the presence of an Na+ gradient in the isolated ATP-depleted choroid plexus tissue slices. This accumulation was reduced by high concentrations of unlabeled formycin B. Nitrobenzylthioinosine (NBMPR), an equilibrative nucleoside transport inhibitor, inhibited the uptake of formycin B in the absence of an Na+ gradient. These data suggest that both equilibrative and secondary active Na(+)-nucleoside transport systems are present in rat choroid plexus. In vivo, formycin B, together with inulin as a bulk flow marker, was injected into the lateral ventricle of the anesthetized rat with the aid of a stereotaxic device, and CSF was sampled from the cisterna magna at various times after injection. Twelve rats were randomized and divided into a low- and a high-dose group. The CSF clearance (CLCSF) of formycin B was significantly higher than the CLCSF of inulin in both animal groups (P < 0.01), indicating that formycin B is cleared from CSF by a pathway(s) in addition to bulk flow. Formycin B CLCSF was significantly lower in the high-dose group than in the low-dose group (P < 0.05), suggesting a saturable CSF elimination. The CLCSF of formycin B was also significantly reduced in animals treated with NBMPR (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Biological Transport; Choroid Plexus; Formycins; In Vitro Techniques; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Thioinosine

We have examined nucleoside transport (NT) in a cell line derived from primary day 7 murine bone marrow macrophages (S1 macrophages) in response to the macrophage growth factor, colony-stimulating factor 1 (CSF-1). Adenosine and uridine transport in quiescent S1 macrophages occurred primarily by two facilitated diffusional routes, one that was sensitive and one that was relatively resistant to the inhibitor nitrobenzylthioinosine (NBMPR). Addition of CSF-1 to quiescent cultures resulted in increased adenosine and uridine transport with biphasic kinetics with respect to the cell cycle. Basal NT activity was elevated (about twofold) within 15 min of CSF-1 addition, returned to near basal levels by 1 h, and then increased again (three- to fourfold) 8-12 h later, returning again to basal levels by 48 h post CSF-1 stimulation. We propose that the large increase in NT activity at 8-12 h corresponded with the time when cultures synchronously began to enter the S phase of the cell cycle. In addition to these changes in the absolute rates, the proportions of NBMPR-sensitive and NBMPR-insensitive transport also change after CSF-1 addition. Quiescent cultures exhibited primarily NBMPR-insensitive transport while logrithmically growing cultures exhibited primarily NBMPR-sensitive nucleoside transport activity. The increase in the NBMPR-sensitive component of the transport process paralleled a similar increase in the number of high-affinity NBMPR binding sites, suggesting that the mechanism for upregulating NBMPR-sensitive NT activity involves increases in the number of NBMPR-sensitive transporter sites. Interestingly, we were unable to detect Na(+)-dependent concentrative uptake of adenosine, uridine, or formycin-B either in the S1 macrophage cell line or in primary (day 7) murine macrophages. Thus these bone marrow derived macrophages did not display the characteristically large Na(+)-dependent transport systems observed by others in peritoneal macrophages, implying that these two populations of macrophages are, indeed, functionally distinct.

Topics: 3-O-Methylglucose; Adenosine; Animals; Binding Sites; Biological Transport; Bone Marrow; Cell Division; Cell Line, Transformed; Diffusion; Kinetics; Macrophage Colony-Stimulating Factor; Macrophages; Methylglucosides; Mice; Nucleosides; S Phase; Thioinosine; Uridine

Human cardiac valves are increasingly used in the reconstruction of ventricular outflow tracts and offer performance advantages over porcine and mechanical prostheses; the durability of these replacements has been associated with leaflet interstitial cell viability and a presumed sustained function after implantation. Preimplantation tissue preparation entails sequential steps that are potentially cytotoxic and may therefore affect functional cell survival at thaw. We defined the metabolic consequences of each interval using semilunar cusps from 118 porcine valves to model a homograft preparation with 40 minutes of fixed cadaveric (harvest) ischemia. Fifty-eight valves served as controls and were first processed according to standard cryopreservation protocol; nucleosides were extracted at the end of each step to differentiate independent contributions to high-energy phosphate depletion. Sixty simultaneously harvested leaflets were administered the nucleoside transport inhibitor p-nitrobenzy-thionosine (NBMPR) and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) at procurement, to attempt adenosine salvage and restitution of processing-incurred adenine nucleotide losses. High-performance liquid chromatography was used to compare adenosine triphosphate, diphosphate, and monophosphate and diffusible nucleopurines of the control and EHNA/NBMPR-treated groups. Control results indicate that disruption of the adenosine triphosphate-diphosphate cycle occurs independently with antibiotic disinfection and cryopreservation. However, throughout all preparation steps, adenine nucleotides were maintained at harvest (baseline) concentrations in the EHNA/NBMPR valves. This suggests that salvage therapy may protect a significant number of cells from net high-energy phosphate catabolism. If, with further study, the durability of transplanted valves is concluded to benefit from retained leaflet interstitial cell viability, such enhancement of metabolic tolerance to the obligatory processing may facilitate functional recovery.

Topics: Adenine; Adenine Nucleotides; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Biological Transport, Active; Female; Heart Valves; Organ Preservation; Purine Nucleosides; Swine; Thioinosine; Transplantation, Homologous

The effects of several ions on [3H]NBMPR specific binding were characterized in guinea pig cardiac membranes. Binding of [3H]NBMPR was enhanced by Mg2+ Ca2+, Na+ and K+ but inhibited by Mn2+, Cu2+ and Zn2+. The effects of Cu2+ and Zn2+ were observed in micromolar concentrations while the effects of the other cations were observed between 1 and 300 mM. The anions tested had no significant effect on the binding. Scatchard analyses of the [3H]NBMPR saturation equilibrium data showed that the enhancement by Na+ and Mg2+ was due to an increase in the number of binding sites number with no change in apparent affinity. The inhibition of the binding by Cu2+ and Zn2+ was due to a decrease in both the apparent affinity and the number of binding sites. The regulation by cations imply that [3H]NBMPR binding sites are physiological receptors that may be involved in postreceptor coupling.

Topics: Animals; Anions; Binding Sites; Cations; Cell Membrane; Guinea Pigs; Heart Ventricles; Ion Channels; Ligands; Male; Thioinosine

A single dose of the adenosine analogue Formycin A (FoA) (20 mg/kg), combined with nitrobenzyl mercaptopurine ribonucleoside 5'-monophosphate (NBMPR-P) (10 mg/kg), a prodrug of nitrobenzylthioinosine (NBMPR), was effective in reducing the size of the foot pad lesions from 7.4 +/- 0.2 to 3.9 +/- 0.2 of Syrian golden hamsters infected with Leishmania major. There was a statistical difference (p < 0.01) in the size of the foot pad by the fifth day between the infected groups that received treatment and the controls, as well as between the groups that were treated with combined drugs and FoA only. The initial reduction in size of the foot pad noted in the group that received only FoA was transient. The effect of FoA or FoA combined with NBMPR on the in vitro cultured promastigotes was similar, indicating that the transport inhibitor might be manipulating the availability of FoA in the host's macrophages where the leishmania amastigotes are resident. The results further indicate the need to explore the usefulness of combining cytotoxic nucleoside analogues with host protecting nucleoside transport inhibitors in the treatment of protozoan parasitic infections.

Topics: Animals; Cricetinae; Drug Combinations; Female; Foot; Formycins; Leishmania; Leishmaniasis; Mesocricetus; Microbial Sensitivity Tests; Thioinosine

Comparisons of the site specific binding of nitrobenzylthioinosine (NBMPR) to intact and lysed red cells from various mammalian and avian species suggest the presence of a cytoplasmic pool of nucleoside transporters. In some species the cytoplasmic pool is about 50% of the total (mouse). On the average, the cytoplasmic pool is approx. 20% of the surface pool of NBMPR-binding sites. In sheep reticulocytes, both pools disappear in an energy-dependent manner during the maturation of the reticulocyte in vitro.

Topics: 4-Chloromercuribenzenesulfonate; Affinity Labels; Animals; Binding Sites; Blood Proteins; Carrier Proteins; Cells, Cultured; Chick Embryo; Erythrocyte Membrane; Membrane Proteins; NADH Dehydrogenase; Nucleoside Transport Proteins; Reticulocytes; Sheep; Thioinosine; Trypsin

The aims were, first, to detect and quantify the release of ATP from human erythrocytes in response to a brief exposure to a hypoxic/hypercapnic environment, similar to that found in vigorously exercising skeletal or cardiac muscle; and second, to explore the mechanism of ATP release in response to hypoxia.. Washed human erythrocytes suspended in Krebs-Henseleit solution were exposed for 50 s to an atmosphere of approximately 8.0 kPa PCO2 and 2.7 kPa PO2; ATP released into the suspension was assayed using the firefly luminescence technique. Samples of human blood were obtained by venepuncture of the median cubital vein from male and female volunteers ranging in age from 21 to 74 years. Anticoagulation was with EDTA.. A background of 0.49 x 10(6) (SEM 0.037 x 10(6)) ATP molecules.cell-1 was attributed to spontaneous haemolysis of 1% of the cell population, as estimated by levels of haemoglobin measured in the suspension fluid. When the erythrocytes were exposed to the hypoxic/hypercapnic gas mixture at 37 degrees C the ATP concentration in the suspension fluid rose to 2.67 x 10(6) (0.27 x 10(6)) molecules.cell-1. An efflux rate of 276(37) molecules.mu-2.s-1 was calculated. The hypoxia induced ATP release was blocked in three different ways: first, by application of 50 microM of the specific band 3 anion channel blocking agents niflumic acid (a translocation inhibitor), DIDS (a transport site inhibitor), or dipyridamole (a channel blocker); secondly, by replacement of extracellular chloride and bicarbonate with the impermeant anion methanesulphonate; and thirdly, by application of 5 nM of the nucleoside transport blocker nitrobenzylthioinosine. None of these blocking techniques affected the background levels of ATP attributed to haemolysis.. A situation of hypoxia/hypercapnia, such as would be found in exercising muscle, induces release of ATP from the erythrocyte via the plasma membrane protein moiety known as band 4.5 (a nucleoside transporter) and electrical balance across the erythrocyte membrane is maintained by the simultaneous influx of extracellular chloride and/or bicarbonate via the plasma membrane protein known as band 3 (anion channel). The circulation of erythrocytes into a region of hypoxia in vivo could promote an increase in local blood flow through release of endothelium dependent relaxing factor in response to released ATP.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Adult; Aged; Carbon Dioxide; Cell Hypoxia; Cells, Cultured; Dipyridamole; Erythrocytes; Female; Humans; Male; Mesylates; Middle Aged; Models, Biological; Muscles; Niflumic Acid; Physical Exertion; Thioinosine

The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A23187 or with concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyloxy]-phenyl]-1, 3-dipropylxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence of XAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a site antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Calcimycin; Carrier Proteins; Concanavalin A; Histamine Release; Humans; In Vitro Techniques; Lung; Mast Cells; Membrane Proteins; Nucleoside Transport Proteins; Receptors, Purinergic; Thioinosine

Nucleoside transport was determined in the cloned porcine kidney cell line PK-15 which exhibits properties of tubular cells. The cells did not express any Na(+)-dependent, concentrative nucleoside transport. They exhibited only nitrobenzylthioinosine-sensitive equilibrative nucleoside transport. Their transport activity, however, was only about 10% of that observed in many other mammalian cell lines. This low transport activity correlated with a relatively low number of high-affinity nitrobenzylthioinosine binding sites (5 x 10(3)/cell). Furthermore, although the equilibrative transporter of PK-15 cells exhibited a similar broad substrate specificity as the equilibrative nucleoside transporters of other mammalian cells, it exhibited a much higher affinity for certain nucleosides, especially cytidine and uridine, than the latter. The Michaelis-Menten constants for zero-trans transport and equilibrium exchange of uridine in ATP-depleted cells were about the same (about 40 microM), indicating equal mobility of the nucleoside-loaded and empty carrier. Concentrative nucleoside transport was detected in one set of PK-15 cultures, but was found to be due to mycoplasma contamination.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Carrier Proteins; Cell Line; Dipyridamole; Formycins; Gramicidin; Kidney; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Swine; Thioinosine

Nitrobenzylthioinosine, dilazep, and dipyridamole are potent inhibitors of equilibrative transport of nucleosides that may have pharmacological applications in modulating the therapeutic index of nucleoside antimetabolites used in cancer chemotherapy. We have compared the relative abilities of these inhibitors to reduce the toxicity of in vitro exposures to tubercidin against clonogenic progenitor cells of normal human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two leukemic human cell lines (HL-60/C1, CCRF-CEM) that differ in their expression of transporter subtypes. Short (1-h) exposures to 1 microM tubercidin alone inhibited colony formation (a) of normal human hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and (b) of HL-60/C1 and CCRF-CEM cells by > 90%. Pretreatment (30 min) with nitrobenzylthioinosine, dilazep, or dipyridamole followed by simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoietic progenitors and cell lines. Greater reductions of toxicity were consistently seen with bone marrow progenitors and CCRF-CEM cells than with HL-60/C1 cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in tubercidin toxicity of 50-100% were achieved at these concentrations: > or = 0.1 microM (nitrobenzylthioinosine); > or = 0.1 microM (dilazep); and > or = 3.0 microM (dipyridamole). Pretreatment (30 min) followed by simultaneous treatment (1 h) with any of the transport inhibitors (> or = 0.1 microM) and 0.1 microM [3H]-tubercidin blocked the uptake of radioactivity completely in CCRF-CEM cells and only partially in HL-60/C1 cells. These effects, which were consistent with the nucleoside transport phenotypes of CCRF-CEM cells (inhibitor-sensitive) and HL-60/C1 cells (inhibitor-sensitive and inhibitor-resistant), suggested that protection was due to the inhibition of tubercidin uptake via equilibrative nucleoside transport system(s). Light-density mononuclear cells from human bone marrow, of which the clonogenic progenitors represented only a minor (< 0.01%) subpopulation, possessed far fewer nitrobenzylthioinosine-binding sites (2 x 10(4) sites/cell, Kd = 0.7 nM) than either HL-60/C1 cells (1.7 x 10(5) sites/cell, Kd = 0.9 nM) or CCRF-CEM cells (3.3 x 10(5) sites/cell, Kd = 0.5 nM). Initial rates of uptake of 1 microM [3H]adenosine (0-6 s, 20 degrees C) by human bone marrow mononuclear cells were reduced partially by 0.1 microM inhibitor (nitrobenzylthioinosine > dipyrid

Topics: Adenosine; Colony-Forming Units Assay; Dilazep; Dipyridamole; Hematopoietic Stem Cells; Humans; Leukemia, Promyelocytic, Acute; Thioinosine; Tubercidin; Tumor Cells, Cultured

Polyclonal antibodies were raised against the nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter of human erythrocyte membranes. On Western blots of these membranes they labeled the broad "band 4.5" region (average apparent M(r) 55,000), which contains both the nucleoside and glucose transport proteins. However, they did not recognize the glucose transporter when this was prepared free of nucleoside transporter by expression from a cDNA clone. Their specificity for the nucleoside transporter was confirmed by the ability to immunoadsorb NBMPR- but not cytochalasin B-binding sites from a detergent-solubilized mixture of band 4.5 proteins. Although a large proportion of the antibodies recognized extracellular epitopes, these appeared to be located primarily on the polypeptide moiety of the glycoprotein, as demonstrated by the ability of the antibodies strongly to label the deglycosylated transporter (apparent M(r) 45,000) on Western blots. The antibodies were species-cross-reactive, recognizing nucleoside transporters from pig and rabbit erythrocytes and from rat liver. The pig protein is similar to the human transporter in its inhibitor sensitivity but is considerably larger (apparent M(r) 57,000 after deglycosylation). In contrast, the rat protein is similar in size to the human transporter (apparent M(r) 45,000 after deglycosylation) but much less sensitive to the inhibitors dilazep and dipyridamole. These findings indicate that despite their differences in size and inhibitor specificity, the NBMPR-sensitive nucleoside transporters of these mammalian species are related in amino acid sequence.

Topics: Affinity Labels; Animals; Antibodies; Blood Proteins; Blotting, Western; Carrier Proteins; Cells, Cultured; Cross Reactions; DNA; Enzyme-Linked Immunosorbent Assay; Erythrocyte Membrane; Humans; Membrane Proteins; Nucleoside Transport Proteins; Rabbits; Sequence Homology, Nucleic Acid; Substrate Specificity; Swine; Thioinosine

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biological Transport; Cerebral Cortex; Dipyridamole; Female; Filtration; Glass; Guinea Pigs; Male; Rabbits; Synaptosomes; Thioinosine; Tritium; Uridine

The nucleoside transporter present in chromaffin tissue membranes has been studied by [3H]nitrobenzylthioinosine (NBTI) binding. This ligand presents a high affinity, with a Kd value of 2.1 +/- 0.2 nM and a Bmax of 1.7 +/- 0.2 pmol/mg protein. From the Scatchard and the semilogarithmic graphical representations a positive cooperativity was deduced, with a Hill coefficient of 1.7 +/- 0.4. In displacement studies of NBTI by the non labelled compound, the Hill coefficient was also higher than 1 (1.44 +/- 0.11) in the presence of ATP. This nucleotide seems necessary to maintain the number of high affinity binding sites.

Topics: Adenosine Triphosphate; Adrenal Medulla; Animals; Carrier Proteins; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Protein Binding; Thioinosine

Nitrobenzylthioinosine (NBTI) was systematically modified by attachment of substituents at the 2-, 5'-, 3'- and 2'-positions in order to assess the importance of these positions in the binding of NBTI to high-affinity membrane binding sites (Kd < or = 1 nM) and the inhibition of NBTI-sensitive, equilibrative nucleoside transport by mammalian cells. We determined the effect of the derivatives on the equilibrium binding of 1 nM [3H]NBTI to human erythrocytes and mouse P388 leukemia cells and on the inhibition of zero-trans influx of formycin B in P388 cells and equilibrium exchange of uridine in human erythrocytes. Placement of substituent groups at the 5'-position of NBTI had relatively little effect on its binding to high-affinity binding sites or its inhibition of nucleoside transport, regardless of the size of the substituent group (up to about 1000 kDa). All substituents at the 2-position considerably reduced the affinity of NBTI to membrane binding sites and its potency as an inhibitor of nucleoside transport, but some substituent groups reduced the affinity of binding more than the inhibition of nucleoside transport. The effect of the 2-substituents was not directly related to their size. Attachment of a succinate at the 3'- or 5'-position also reduced to a greater extent the binding of NBTI than its inhibition of nucleoside transport, which was relatively little affected. Attachment of succinates at both the 3' and 5'-positions almost completely abolished both binding to high-affinity sites and inhibition of nucleoside transport. Both functions of NBTI were abolished completely by the simultaneous blockage of the 2'- and 3'-positions. None of the NBTI derivatives significantly inhibited NBTI-resistant equilibrative formycin B transport in P388 and Novikoff rat hepatoma cells at concentrations of < or = 1 microM.

Topics: Animals; Binding Sites; Biological Transport; Cell Membrane; Erythrocyte Membrane; Formycins; Humans; Mice; Nucleosides; Rats; Structure-Activity Relationship; Thioinosine; Tumor Cells, Cultured; Uridine

The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg-1 min-1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p less than 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces post-ischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

Topics: Adenosine; Animals; Cell Adhesion; Cricetinae; Endothelium, Vascular; Ischemia; Leukocytes; Mesocricetus; Muscles; Phenethylamines; Receptors, Purinergic; Reperfusion Injury; Thioinosine; Xanthines

Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5'-phosphate of nitrobenzylthioinosine, a potent inhibitor of the es equilibrative nucleoside transport (NT) system. NBTGR-P, the 5'-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of "cured" mice than were the same treatment regimens without NBMPR-P.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Drug Synergism; Female; Hindlimb; Leukemia L1210; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Transplantation; Nervous System Diseases; Paralysis; Prodrugs; Thioinosine; Thionucleotides; Vidarabine

The adenosine transport in cultured chromaffin cells was increased by the presence of triiodo-L-thyronine (T3) throughout the prolonged period studied. The Vmax values of this transport obtained in absence and presence of 1 microM T3 were 36.21 +/- 2.1 and 44.17 +/- 3.5 (means +/- SD) pmol/10(6) cells/min respectively for 26 hours incubation-time with the hormone. The Km values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by [3H]nitrobenzylthioinosine (NBTI) binding, was increased by 1 microM T3 for 26 hours incubation-time. The values of binding sites per cell were 33,500 +/- 3,000 and 40,153 +/- 3,700 in absence and presence of T3 respectively, without changing the Kd constant. When the transport studies were carried out in presence of cycloheximide, an inhibitor of protein synthesis, the adenosine transport capacity decreased with a half-life values of 23.9 +/- 2.8 and 24.3 +/- 2.1 hours both in the presence or absence of T3 respectively. When cells were incubated in the presence of both T3 and cycloheximide, not only the activatory effect of T3 was completely abolished but also adenosine transport was decreased to the same extent as with cycloheximide alone. These results indicated that T3 activation of adenosine transport in chromaffin cells required the protein-synthesizing mechanism.

Topics: Adenosine; Adrenal Medulla; Affinity Labels; Animals; Biological Transport; Catecholamines; Cattle; Cells, Cultured; Cycloheximide; Radioligand Assay; Thioinosine; Time Factors; Triiodothyronine

A kinetic study of the uptake of nicotinamide by reconstituted liposomes containing the human erythrocyte glucose transporter, compared with that of D-glucose, demonstrated that the Km and Vmax. values were almost the same for each compound, and that the uptake of D-glucose was competitively inhibited by nicotinamide. At 20 mM concentration, 2-deoxy-D-glucose, 3-O-methyl-D-glucose and 4,6-O-ethylidene-D-glucose all caused 50% inhibition of nicotinamide uptake, but L-glucose and nicotinic acid were not inhibitory. Similar results were obtained for the uptake of D-glucose. Cytochalasin B binding to the liposomes was inhibited in a dose-dependent manner by either nicotinamide or D-glucose. Antibody for glucose transporter detected in band 4.5 by SDS/PAGE inhibited the uptake of D-glucose and nicotinamide. A possible uptake of nicotinamide by nucleoside transporter was excluded. In human erythrocytes, cytochalasin B binding was inhibited dose-dependently by either nicotinamide or D-glucose, and cytochalasin B depressed the uptake of both nicotinamide and 2-deoxy-D-glucose. These findings were well reproduced in the reconstituted liposomes. The very close similarities between uptake of nicotinamide and D-glucose suggest that the glucose transporter plays a direct role in transport of nicotinamide, which is structurally quite different from monosaccharides, and thus that the transporter is probably multifunctional.

Topics: Biological Transport; Cytochalasin B; Erythrocytes; Glucose; Humans; In Vitro Techniques; Kinetics; Liposomes; Monosaccharide Transport Proteins; Niacinamide; Thioinosine

We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with 'high' affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with 'low' affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the 'NBMPR-sensitive' component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus 'NBMPR-insensitive'. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of 'erythrocyte-like' transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transpor

Topics: Binding Sites; Biological Transport; Carrier Proteins; Cell Membrane; Choriocarcinoma; Electrophoresis; HeLa Cells; Humans; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Sensitivity and Specificity; Sodium; Thioinosine; Thymidine; Time Factors; Tritium; Tumor Cells, Cultured

Although adenosine A1 receptors mediate the inhibition of dopamine-dependent stimulation of adenylate cyclase activity in the developing chick retina, their localization and function are unknown. We have examined the localization of these receptors, and of endogenous adenosine and adenosine uptake sites at several stages of chick retinal development. A1 receptors were already localized predominantly to plexiform regions by embryonic day 12 (E12) with no gross changes at subsequent stages. Adenosine immunoreactivity was absent from retina at E8 but was detected at E12 in the ganglion cell layer, as well as cells in the inner nuclear cell layer and photoreceptors. At more advanced developmental stages the immunoreactivity was greater, but displayed similar localizations. Uptake sites labeled with [3H]nitrobenzylthioinosine (NBI) were detected even earlier using binding and autoradiographic methods. [3H]NBI binding was saturable, and Scatchard analysis demonstrated a single class of sites with a Kd of 0.91 nM and Bmax of 298 fmol/mg protein in E15 retinal membranes. The binding was displaced by unlabeled NBI and dipyridamole. NBI binding sites differentiated earlier than adenosine A1 receptors or endogenous adenosine immunoreactivity, showing a diffuse distribution at E8, but predominating in the plexiform layers of more developed retinas. The results indicate that elements of a putative purinergic system differentiate at specific localizations early in retinal development.

Topics: Adenosine; Affinity Labels; Animals; Autoradiography; Binding, Competitive; Biological Transport; Cell Membrane; Chick Embryo; Immunohistochemistry; Kinetics; Phenylisopropyladenosine; Receptors, Purinergic; Retina; Thioinosine; Tritium

Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L1210/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydorxy-3-nonyl)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleotide transporters). The selection medium did not allow es activity and selected against cells that expressed the Na(+)-linked cif process. Cells of the L1210/B23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of [3H]formycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L1210/B23.1 cells indicates that (i) the mutation/selection procedure impaired or deleted the Na(+)-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.

Topics: Adenine; Adenosine; Animals; Antiviral Agents; Biological Transport; Carrier Proteins; Cell Membrane; Clone Cells; Formycins; Kinetics; Leukemia L1210; Membrane Proteins; Mice; Mutagenesis; Nucleoside Transport Proteins; Nucleosides; Thioinosine; Thymidine; Tumor Cells, Cultured; Vidarabine

The tritiated analogue of R75231 ((+-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis (4-fluorophenyl)pentyl]-1-piperazineacetamide) has been examined as a new radioligand for (nitrobenzylthioinosine sensitive) nucleoside transport proteins. [3H]R75231 was prepared in two steps from R69064 ((+-)-4-[5,5-bis[4-fluorophenyl)-4-pentenyl]-2-piperazinecarboxamide+ ++ dihydrochloride) with a specific activity of 0.23 TBq/mmol (6.3 Ci/mmol). [3H]R75231 bound in a pseudo-irreversible and saturable manner to a membrane preparation of calf lung tissue. The new radioligand displayed high affinity (KD = 0.32 +/- 0.06 nmol/l at 25 degrees C) and capacity (Bmax = 6.1 +/- 0.3 pmol/mg protein). Specific [3H]R75231 binding could be fully displaced by both structural analogues and reference inhibitors such as dipyridamole, NBI, dilazep and hexobendine, as well as by various nucleosides. The two stereoisomers of R75231, R88016 ((+)-R75231) and R88021 ((-)-R-75231), potently displaced specific [3H]R75231 and [3H]NBI binding, R88021 being 30-fold more active than R88016. Pseudo-Hill coefficients derived from the shape of all the [3H]R75231 displacement curves were approximately unity. In contrast, R75231 and most of its analogues displaced specific [3H]NBI binding with pseudo-Hill coefficients consistently larger than unity under identical experimental conditions. This latter finding is suggestive for the existence of two distinct binding sites for the two radioligands, which may or may not overlap to some extent.

Topics: Affinity Labels; Animals; Cattle; In Vitro Techniques; Lung; Membranes; Piperazines; Radioligand Assay; Stereoisomerism; Thioinosine; Tritium

Important cellular nutrients, including nucleosides and hexose sugars, are rapidly taken up by cells, largely through mediated carrier systems. The present study examined nucleoside and hexose transport activity in normal Rat-2 fibroblasts and clonal derivatives that expressed either the wild-type (C10) or a temperature-sensitive mutant (NA9) form of v-fps, a transforming protein-tyrosine kinase. Initial uptake rates (transport) of adenosine, thymidine, 3-O-methylglucose and 2-deoxyglucose were greater in v-fps-transformed cells than in normal cells. Elevated transport rates were seen in cells that expressed the temperature-sensitive mutant v-fps only after growth at a temperature that was permissive for protein-tyrosine kinase activity. Nucleoside transport rates declined with increasing cell density in both normal and v-fps transformed cells. Analysis of the sensitivity of adenosine transport to inhibition by nitrobenzylthioinosine (NBMPR) indicated that Rat-2 fibroblasts, like many other rat cell types, possess at least two nucleoside transport systems, which can be distinguished by differences in sensitivity to NBMPR. Although both transport activities were elevated in v-fps-transformed cells, a greater increase was seen in the NBMPR-sensitive component than in the NBMPR-insensitive component. Mass law analysis of the binding of [3H]NBMPR indicated that transformed cells had either the same number (NA9) or a smaller number (C10) of NBMPR-binding sites than normal cells, and photolabelling of membrane proteins with [3H]NBMPR identified polypeptides with similar electrophoretic mobilities (55-75 kDa) in both normal and transformed cells. Thus transformation by v-fps resulted in an increase in NBMPR-sensitive transport activity which was not related to either the number of NBMPR-binding sites or the apparent molecular mass of NBMPR-binding polypeptides.

Topics: 3-O-Methylglucose; Adenosine; Affinity Labels; Animals; Biological Transport; Cell Line; Cell Line, Transformed; Cell Membrane; Cell Transformation, Neoplastic; Deoxyglucose; Fibroblasts; Fusion Proteins, gag-onc; Genes, gag; Kinetics; Methylglucosides; Nucleosides; Protein-Tyrosine Kinases; Rats; Thioinosine; Thymidine

The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells. Photolabelling of transporters with [3H]NBTI induced a down-regulation of this protein from the plasma membrane with a half-life value of 2.31 +/- 0.61 h, measured by specific isolation of plasma membrane on polycationic beads. In this internalization step 50-60% of transporters were destroyed. The remaining labelled protein reappeared in plasma membranes and underwent a new disappearance cycle with a longer half-life period (34.65 +/- 3.9 h). A similar pattern of internalization and reappearance of nucleoside transporters was observed in cells cross-linked with non-labelled NBTI, with a half value of reappearance of 33 h. Chromaffin cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, had a component of disappearance for NBTI binding sites with a half-life value of 24.6 +/- 1.4 h.

Topics: Affinity Labels; Biological Transport; Carrier Proteins; Cell Membrane; Cells, Cultured; Chromaffin System; Cycloheximide; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Half-Life; Membrane Proteins; Models, Biological; Neurons; Nucleoside Transport Proteins; Thioinosine

Recent evidence suggests that adenosine mediates many of the acute and chronic effects of ethanol in both cultured cells and whole animals. These adenosine-mediated effects of ethanol result from ethanol-induced increases in extracellular adenosine. Acute exposure of primary cultures of rat hepatocytes to 12.5-200 mM ethanol increased extracellular adenosine concentrations by 20-35%. Pretreatment of hepatocytes with 100 microM 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, completely blocked ethanol-induced increases in extracellular adenosine at 12.5 and 25 mM ethanol. However, even in the presence of 4-methylpyrazole, ethanol at concentrations greater than 50 mM still increased extracellular adenosine concentrations. This increase appears to be due to ethanol inhibition of adenosine uptake via the nucleoside transporter (50% inhibitory concentration, 28 mM). After chronic treatment with 100 mM ethanol for 48 h, acute challenge with ethanol no longer inhibited adenosine uptake, i.e., the nucleoside transporter had become tolerant to ethanol. Moreover, in these chronically treated cells, ethanol-induced increases in extracellular adenosine were completely blocked by treatment with 4-methylpyrazole at all concentrations of ethanol. Taken together, these results suggest that increased extracellular adenosine in hepatocytes is dependent on both ethanol oxidation and inhibition of adenosine uptake via the nucleoside transporter.

Topics: Adenosine; Animals; Carrier Proteins; Ethanol; Extracellular Space; Fomepizole; Liver; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Oxidation-Reduction; Pyrazoles; Thioinosine; Time Factors

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.

Topics: Affinity Labels; Animals; Biological Transport; Cell Line; Dilazep; Epithelium; Kidney Cortex; Kidney Medulla; Kinetics; Microvilli; Nucleosides; Swine; Thioinosine; Uridine

The role of nucleoside uptake in the enhanced metabolic recovery seen with postischemic ATP.MgCl2 was assessed by determining the effect of S-(p-nitrobenzyl)-6-thioinosine (NBTI) on postischemic ATP recovery in rats given normal saline (NS), ATP.MgCl2, or adenosine after 45 min of bilateral renal ischemia. In NS-infused animals, postischemic administration of NBTI (250 nmol) had no significant effect on the pattern of ATP recovery. In animals given 50 mumol ATP.MgCl2, coinfusion of NBTI significantly reduced the renal ATP content 2 h after reperfusion but blocked only one-half of the enhancement in renal ATP content compared with animals given ATP.MgCl2 alone. In animals postischemically infused with [2,5,8-3H]ATP.MgCl2 (50 mumol) there was significant labeling of nucleotides, nucleosides, and bases after 2 h of reperfusion. The specific activity of the adenosine pool was consistent with significant label uptake in the form of adenosine. Coinfusion of NBTI led to a significant reduction in label incorporation into renal ATP and total adenine nucleotide pools. These data are consistent with an important role for an NBTI-sensitive nucleoside uptake mechanism in the enhanced metabolic and functional recovery observed in ischemically injured kidney treated by postischemic infusion of ATP.MgCl2.

Topics: Adenosine; Adenosine Triphosphate; Animals; Blood; Ischemia; Kidney; Magnetic Resonance Spectroscopy; Nucleosides; Rats; Renal Circulation; Thioinosine

In an attempt to elucidate the types of nucleoside transporters present in bone marrow stem cells, this study examined the effect of nucleoside transport inhibitors on the toxicity of nucleoside analogs and on the salvage of thymidine by mouse bone marrow granulocyte and macrophage progenitor cells using the CFU-GM assay. Concentrations of NBMPR (nitrobenzylmercaptopurine riboside) as low as 10 nM protected these cells from the toxicity of the adenosine analog tubercidin and provided a partial block of thymidine-rescue of the granulocyte-macrophage progenitor cells from methotrexate toxicity. Dipyridamole had similar effects but generally required higher concentrations. These results suggested that the major nucleoside transporter in these cells is the NBMPR-sensitive equilibrative carrier, es. In contrast to the results with tubercidin, the toxicity of 2-chlorodeoxyadenosine was increased 8- to 10-fold by 1 microM NBMPR. These results suggested that the bone marrow granulocyte-macrophage progenitor cells also have a concentrative nucleoside transporter that is capable of pumping 2-chlorodeoxyadenosine into the cells while efflux of the nucleoside via es is blocked by NBMPR.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Biological Transport; Bone Marrow Cells; Cells, Cultured; Cladribine; Female; Granulocytes; Hematopoietic Stem Cells; Macrophages; Methotrexate; Mice; Mice, Inbred CBA; Nucleoside Transport Proteins; Thioinosine; Thymidine; Tubercidin

The effects of coformycin, alpha,beta-methylene ADP, dipyridamole in the absence and presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), nitrobenzylthioinosine (NBTI), mioflazine and ouabain on the metabolic pathways of exogenously applied ATP and its metabolites in the frog innervated sartorius muscle were investigated. ATP catabolism yielded ADP, AMP, IMP, adenosine and inosine; the ecto-ATPase in situ was shown to be Ca(2+)- or Mg(2+)-activated with a Kmapp for ATP of 767 +/- 48 microM. AMP catabolism yielded IMP, adenosine and inosine; inosine was formed from either exogenous IMP or exogenous adenosine. Catabolism of AMP into IMP was blocked by coformycin, which enhanced adenosine and inosine formation from AMP. alpha,beta-Methylene ADP blocked adenosine formation from AMP and inosine formation from IMP; formation of IMP from AMP was enhanced by alpha,beta-methylene ADP. Complete blockade of AMP degradation was achieved with the simultaneous use of coformycin and alpha,beta-methylene ADP. Dipyridamole attenuated but did not completely block extracellular adenosine removal and inosine appearance in the bath. EHNA, applied in the presence of dipyridamole, did not cause any further attenuation of extracellular adenosine removal. Mioflazine, NBTI and ouabain did not affect adenosine disappearance from the bath. The results suggest that, in the frog innervated sartorius muscle, ATP can be sequentially catabolized into AMP which is then catabolized either into IMP or into adenosine. This extracellular degradation of AMP into IMP might then constitute a shunt-like mechanism to control the levels of adenosine formed from adenine nucleotides.

Topics: Adenine; Adenine Nucleotides; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cations; Coformycin; Dipyridamole; In Vitro Techniques; Inosine Monophosphate; Kinetics; Muscles; Piperazines; Rana ridibunda; Sodium-Potassium-Exchanging ATPase; Thioinosine

Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.

Topics: Adsorption; Antibodies, Monoclonal; Blood Proteins; Carrier Proteins; Cross Reactions; Cytochalasin B; Detergents; Epitopes; Erythrocyte Aggregation; Erythrocytes; Humans; Membrane Proteins; Micelles; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Thioinosine

Previous studies have demonstrated that the xanthine compound, propentofylline, has beneficial effects in models of cerebral ischemia and can enhance some and exhibit other effects of adenosine. We investigated the in vitro effects of propentofylline and its hydroxy metabolite, A72,0287, on the binding of [3H]cyclohexyladenosine ([3H]CHA), [3H]2-[p-(2-carbonyl-ethyl)-phenylethyl-amino]-5'-N- ethylcarboxamido adenosine ([3H]CGS 21680) and [3H]nitrobenzylthioinosine ([3H]NBMPR) to adenosine A1 and A2 receptors and NBMPR-sensitive nucleoside transporters, respectively, in 10-microns coronal rat brain sections. Both xanthines had micromolar affinity for each of these sites with approximately 10-fold lower affinity for A2 receptors than for A1 receptors and [3H]NBMPR binding sites. Saturation analysis of [3H]CHA or [3H]CGS 21680 binding in the presence of increasing concentrations of propentofylline produced significant increases in KD values without affecting Bmax values; thus propentofylline is a competitive inhibitor at A1 and A2 receptors. The effects on A2 receptors apparently require higher concentrations (Ki approximately 200 microM) than the effects on A1 receptors (Ki approximately 20 microM). Propentofylline was also found to be a competitive inhibitor of [3H]NBMPR binding. Therefore we conclude that propentofylline interacts with adenosine-responsive systems to increase interstitial adenosine concentrations and to selectively inhibit A1 receptors.

Topics: Adenosine; Animals; Autoradiography; Binding Sites; Brain; Carrier Proteins; Male; Membrane Proteins; Nucleoside Transport Proteins; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thioinosine; Xanthines

The in-vitro effects of hydroxyurea 5-FU and 5-FUdR have been extensively studied in experimental systems employing cell-line techniques. In this study we investigated the effects of these drugs on the levels of incorporation of labeled nucleosides into DNA in explants of intact rat colonic mucosa maintained in organ culture. The effects of the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR) and dipyridamole--which are modulators of antimetabolite cytotoxicity--on the incorporation of tritiated thymidine ([3H]TdR) into DNA were also studied. The incorporation of tritiated TdR into DNA was reduced by hydroxyurea but was not altered by either 5-FU or 5-FUdR. The levels of tritiated deoxyuridine were reduced by 5-FU and 5-FUdR in separate experiments; this is in keeping with thymidylate synthase inhibition. NBMPR and dipyridamole also reduced 3H-TdR incorporation into DNA. These results can be explained in terms of the known mechanisms of action of these drugs. This experimental model is therefore useful in assessing the effects of antimetabolites and nucleoside transport inhibitors in intact colonic mucosa.

Topics: Animals; Biological Transport; Colon; Dipyridamole; DNA; Hydroxyurea; Intestinal Mucosa; Male; Models, Biological; Nucleosides; Organ Culture Techniques; Pyrimidines; Rats; Rats, Inbred Strains; Thioinosine; Thymidine; Time Factors; Tritium; Uridine

The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport.

Topics: Adenosine; Adenosine Deaminase; Animals; Biological Transport; Chlorides; Colforsin; Dogfish; Feedback; In Vitro Techniques; Inosine; Male; Salt Gland; Theophylline; Thioinosine

Bone marrow leukemia cells from eight adults with untreated acute myeloid leukemia (AML) were evaluated before and after three daily leukaphereses to determine if mechanical cytoreduction can modulate the cell cycle distribution. The percentage of cells in S-phase and the proliferative fraction (PF = %S + %G2M) were determined by flow cytometry after dual labeling with bromodeoxyuridine and propidium iodide. Prior to pheresis the median %S and PF were 5.4 and 15.4%, respectively. The median change in %S was +2.5% (range -5.5 to +18.8) with increases greater than or equal to 3.7% in 4/8 patients. The median change in PF was +6.1% (range -13.8 to +25.3) with an increase of greater than or equal to 3.6% in 6/8 patients. The median absolute changes of 2.5 and 6.1% represent increases of 47% for %S and 40% for PF compared to the day 1 (pre-pheresis) median values. As the number of nucleoside transporters in the cell membrane [nitrobenzylmercaptopurine riboside (NBMPR) binding sites] has been related to the percentage of cells in S-phase and to cytosine arabinoside (ara-C) cellular pharmacology, these were also measured before and after leukapheresis. Changes in the number of NBMPR binding sites varied widely with a median increase of 365 sites per cell (range -26,061 to +10,396). The change in NBMPR sites was significantly and positively correlated with changes in %S (r = 0.829, p = 0.042). These data suggest that mechanical cytoreduction by leukapheresis can increase the fraction of leukemia cells in S-phase and the PF in some patients with AML. The increase in %S is accompanied by an increase in NBMPR binding sites per cell. These changes in leukemia cell characteristics would be expected to result in an increase in efficacy of ara-C or other S-phase specific agents.

Topics: Binding Sites; Carrier Proteins; Cell Cycle; Humans; Leukapheresis; Leukemia, Myeloid, Acute; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine

Topics: Affinity Labels; Animals; Binding, Competitive; Carrier Proteins; Cattle; Cell Membrane; Kinetics; Lung; Membrane Proteins; Nucleoside Transport Proteins; Radioligand Assay; Thioinosine; Tritium

Topics: Animals; Biological Transport; Dilazep; Dipyridamole; Epinephrine; Erythrocytes; Humans; In Vitro Techniques; Nucleosides; Phosphates; Piperazines; Rabbits; Thioinosine

Topics: Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane; Kinetics; Membrane Lipids; Membrane Proteins; Mice; Nucleoside Transport Proteins; Phospholipids; Protein Binding; Proteolipids; Thioinosine; Uridine

The results above show that mammalian cells, as exemplified by MELC, can be selected to be resistant to Adox, and that the resistant cells have greatly decreased nucleoside transport capacity. Since no mutagen was used prior to the selection process and Adox resistance was genetically stable, it appears that within a population of normal cells there is a genetically controlled range of expression of the nucleoside transporter. On the basis of the present data we cannot determine if the low level of nucleoside transporter in AR MELC is due to an altered form of the protein or a decreased amount of the normal protein. However the similarity of the Kd for NBTI in normal and AR MELC suggests that the latter is the case. Considerable indirect evidence is presented that Adox, with its ribose converted to an acyclic dialdehyde, is a substrate for the nucleoside transporter, adding a new type of compound to this list.

Topics: Adenosine; Animals; Carrier Proteins; Cell Division; Drug Resistance; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Membrane Proteins; Mice; Nucleoside Transport Proteins; S-Adenosylhomocysteine; Thioinosine; Tumor Cells, Cultured

Cultured chick heart muscle cells degrade ATP during metabolic inhibition via ADP to AMP. Whether AMP is primarily deaminated to IMP or dephosphorylated to adenosine depends on the 'metabolic block' (glycolysis vs. oxidative phosphorylation). Inhibition of glycolysis (deoxyglucose) results in an inosine/adenosine ratio greater than 1 in the supernatant, whereas the nucleoside ratio is less than or equal to 1 during inhibition of oxidative phosphorylation (hypoxia, rotenone). EHNA, a blocker of adenosine deaminase, has little effect on inosine release during metabolic inhibition, consistent with the reported low activity of adenosine deaminase in cardiac muscle cells. The amount of adenosine and inosine released can be largely attenuated by two nucleoside carrier inhibitors, nitrobenzyl-thioinosine and dipyridamole, which suggests that nucleosides are produced intracellularly and subsequently released. These results indicate that the amount of inosine or adenosine released from the cardiomyocyte during impaired energy metabolism (e.g. ischemia) can be controlled by the metabolic state of the cell.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Cells, Cultured; Chick Embryo; Chromatography, High Pressure Liquid; Dipyridamole; Glycolysis; Inosine; Myocardium; Oxidative Phosphorylation; Phosphorylation; Thioinosine

Hippocampal cells were cultured in 24-well culture plates with enriched populations of neuron or glial cells. The [3H]adenosine uptake by 7-10-day cultures of these cells was dependent on temperature, but independent of extracellular Na+. The uptake of adenosine (10 microM) for 15 s was greatly blocked by addition of 100 microM dipyridamole, 50-200 microM propentofylline or 50 microM of 2-chloroadenosine or nitrobenzylthioinosine in both cells and by 100 microM pentoxifylline in neuron. Either caffeine or theophylline (50 microM each) had no effect on the uptake by these cells. Inhibition of the adenosine uptake by propentofylline was demonstrated to be competitive in both cells.

Topics: 2-Chloroadenosine; Adenosine; Affinity Labels; Animals; Biological Transport; Caffeine; Cells, Cultured; Cytarabine; Dipyridamole; Embryo, Mammalian; Hippocampus; Kinetics; Neuroglia; Neurons; Pentoxifylline; Rats; Sodium; Theophylline; Thioinosine; Tritium; Xanthines

3'-Deoxythymidin-2'-ene (d4T) is a potent and selective inhibitor of human immunodeficiency virus replication in a variety of human cell types and is currently undergoing phase I clinical trials for the treatment of acquired immunodeficiency syndrome. As part of our ongoing studies of the cellular pharmacology of d4T, and in light of recent reports in which such nucleoside analogs as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyadenosine were shown to permeate cells by the unusual mechanism of nonfacilitated diffusion, we have investigated the uptake of d4T in the human lymphocyte cell line H9. Several lines of evidence suggest that d4T permeation of H9 cells occurs by nonfacilitated diffusion; 1) [3H]d4T influx was linear for the first 10 sec and was nonconcentrative, reaching equilibrium with the extracellular drug concentration in 2-3 min, 2) the initial rates of influx were a linear function of concentration over the range from 1 microM to 5 mM, with no sign of uptake by a saturable mechanism, and 3) the uptake of [3H]d4T was insensitive to the nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole, as well as a large molar excess of AZT, thymidine, or adenosine. The octanol/water partition coefficient of d4T was 0.179, intermediate between those of thymidine and AZT. Thus, d4T does not appear to be a substrate for the nucleoside transport system responsible for the uptake of physiological nucleosides as well as most nucleoside analogs, and it enters the cell by nonfacilitated diffusion.

Topics: Antiviral Agents; Cell Membrane Permeability; Cells, Cultured; Dideoxynucleosides; Diffusion; Dipyridamole; Humans; Kinetics; Lymphocytes; Octanols; Solubility; Stavudine; Thioinosine; Thymidine; Time Factors; Water

The in vivo nucleoside transport inhibitory effects of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), used as its 5'-monophosphate derivative (NBMPR-P), dilazep, mioflazine and its derivatives soluflazine, R57974 and R75231, were investigated in BALB/c mice. The extent and duration of action were followed by assaying adenosine transport in blood cells sampled at time intervals following i.p. administration (ca. 20 mg/kg). Dilazep and R57974 were found to be short-acting inhibitors, while NBMPR-P and R75231 were similar in their action and caused essentially full inhibition of adenosine transport over a 4- to 5-h period. Mioflazine and soluflazine were rather ineffective, causing only partial inhibition. R75231 was also active after oral administration which, when repeated three times in 4-h intervals, resulted in essentially full transport inhibition up to 20 h following the initial dose. Effects of NBMPR-P, R57974 and dilazep on adenosine transport in blood cells were also measured in blood cells of hamsters after i.p. administration of the same doses. All three drugs caused full transport inhibition, but the action of dilazep and R75231 showed reversal within about 30 min and 2 h, respectively, while NBMPR-P caused full inhibition for at least 3-4 h. These results demonstrate the potential of the mioflazine derivative R75231 to be useful in vivo, possibly even after p.o. administration, for host protection against the actions of cytotoxic nucleosides used in experimental antiparasitic therapy or other studies requiring suppression of nucleoside transport.

Topics: Adenosine; Administration, Oral; Animals; Biological Transport; Cardiovascular Agents; Cricetinae; Dilazep; Injections, Intraperitoneal; Mesocricetus; Mice; Mice, Inbred BALB C; Piperazines; Species Specificity; Thioinosine

Topics: Biological Transport; Carrier Proteins; Humans; Leukemia; Lymphocytes; Lymphoma; Membrane Proteins; Nucleoside Transport Proteins; Pentostatin; Thioinosine; Tumor Cells, Cultured

In mammalian cells, nucleoside transport usually is mediated by facilitated diffusion. In addition, a Na(+)-dependent, concentrative nucleoside transport system has been detected in several tissues but not the liver. To further clarify hepatic nucleoside transport mechanisms, we measured the uptake of [2-14C]uridine (2 to 100 mumol/L) and of [8-14C]adenosine (10 to 75 mumol/L) by the isolated perfused rat liver in the presence or absence of extracellular sodium or specific inhibitors of facilitated nucleoside diffusion. Uridine transport and metabolism were monitored by the release of labeled catabolites including 14CO2, which indicated complete degradation of the pyrimidine. Adenosine, uridine and uridine catabolites were measured in the effluent perfusate by reversed-phase high-performance liquid chromatography and a radioactivity flow monitor. The existence of a Na(+)-dependent nucleoside transport system could be inferred from the following observations: (a) Sodium depletion caused a strong inhibition of nucleoside transport reflected by an up to threefold and 15-fold increase in extracellular uridine and adenosine, respectively. The sodium-dependent transport of uridine was saturated when the influent uridine concentration was raised beyond 20 mumol/L. No such saturation was observed for much higher concentrations of adenosine used (10 to 75 mumol/L). (b) Na(+)-free perfusion resulted in a strong suppression of the release of uridine catabolites by the liver. Complete uridine breakdown was depressed to 7% of the amount of 14CO2 released in the presence of sodium and at influent uridine concentrations below 20 mumol/L. (c) Inhibition of uridine (10 mumol/L) transport and degradation was observed after coperfusion with adenosine, deoxyadenosine, guanosine and deoxyguanosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biological Transport; Diffusion; Dipyridamole; In Vitro Techniques; Liver; Male; Nucleosides; Perfusion; Purine Nucleosides; Pyrimidine Nucleosides; Rats; Rats, Inbred Strains; Sodium; Thioinosine; Uridine

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.

Topics: Aminoisobutyric Acids; Animals; Biological Transport, Active; Carrier Proteins; Dipyridamole; Formycins; Kinetics; Lymphoma; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; Sodium; Thioinosine; Tumor Cells, Cultured

The present study compares the characteristics of radioligand binding to adenosine receptors and adenosine uptake sites in 100- and 50-day-old normal and narcoleptic dogs. Binding to A1 receptors was quantified using a selective A1 agonist ([3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine, [3H]R-PIA) and an antagonist ([3H]dipropyl-8-cyclopentyl-xanthine, [3H]CPX). Differences in the binding of [3H]R-PIA and that of [3H]5'-ethylcarboxamide adenosine ([3H]NECA), which binds to both A1 and A2 receptors with similar affinities, were used to quantify A2 receptors. Nucleoside transport sites were labeled with [3H]nitrobenzylthioinosine ([3H]NBTI), a potent inhibitor of nucleoside transport systems. The present study offered no evidence that either adenosine A1 receptors and adenosine uptake sites in the frontal cortex or adenosine A2 receptors in the putamen were altered in narcoleptic dogs. However, we found that adenosine A1 receptors in the dog exist in different affinity states and that the affinity state in which the receptor is found depends on the brain region examined. A characterization of these low- and high-affinity sites was performed and results indicated that these sites cannot be explained by a single interaction of the A1 receptor with a single G-protein population.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Aging; Animals; Brain; Cataplexy; Dogs; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Kinetics; Membranes; Narcolepsy; Phenylisopropyladenosine; Radioligand Assay; Receptors, Purinergic; Thioinosine; Xanthines

The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2'-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2'-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparent Km approx. 150 microM) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparent Kd 0.98 +/- 0.21 nM). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparent Ki values were 0.23 +/- 0.012, 0.36 +/- 0.035, 0.78 +/- 0.1, 0.70 +/- 0.12 (mM), and 0.12 and 4.2 +/- 1.4 (nM). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brush-border membrane vesicles (apparent Kd 1.05 +/- 0.13 nM and apparent Ki values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14 +/- 0.045, 0.54 +/- 0.046, 1.26 +/- 0.20, 1.09 +/- 0.18 mM and 0.14 and 3.7 +/- 0.5 nM, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparent Mr on SDS gel electropherograms of 77,000-45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surfaces of the human placental syncytiotrophoblast possess broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.

Topics: Adenosine; Affinity Labels; Basement Membrane; Biological Transport, Active; Cell Membrane; Glycoside Hydrolases; Humans; In Vitro Techniques; Kinetics; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Microvilli; Nucleosides; Osmosis; Placenta; Thioinosine; Trophoblasts

The effects of adenosine receptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea-pig papillary muscles preincubated with [3H] noradrenaline were studied. N6-cyclopentyladenosine (CPA), N6-(R-phenylisopropyl)-adenosine, and 5'-N-ethylcarboxamidoadenosine caused concentration-dependent inhibition of evoked overflow with a rank order of potency typical for interaction of the compounds with the A1-subtype of adenosine receptors. Maximum inhibition was 80%. The A1-selective antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) induced a rightward shift of the concentration-response curve for CPA with a pA2 of 8.35. However, DPCPX per se had no effect on stimulation-evoked tritium overflow. On the other hand, in the presence of 4-nitrobenzylthioinosine (2 mumol/l) and deoxycoformycin (1 mumol/l), inhibitors of adenosine uptake and deamination, respectively, DPCPX produced a concentration-dependent increase in overflow with a pD2 of 8.1. Pretreatment of the animals with pertussis toxin caused a substantial reduction in the activity of toxin-sensitive G proteins, as indicated by a lack of [32P]ADP ribosylation in a ventricular membrane preparation. Nevertheless, the inhibitory effect of the adenosine receptor agonists on stimulus-evoked overflow remained unaffected. These results are compatible with the existence of inhibitory prejunctional adenosine receptors in guinea-pig papillary muscle, which appear to be coupled to a pertussis toxin-insensitive G protein. The role of endogenous adenosine in occupying these receptors seems minimal under basal conditions.

Topics: Animals; Electric Stimulation; Female; GTP-Binding Proteins; Guinea Pigs; Male; Norepinephrine; Nucleosides; Papillary Muscles; Pertussis Toxin; Purinergic Antagonists; Receptors, Purinergic; Thioinosine; Virulence Factors, Bordetella

The nucleoside transport characteristics of two strains of Leishmania donovani promastigotes were studied. Strain S1, growing in fully defined medium, and strain S2 (MHOM/ET/67/HA3) both transported adenosine and inosine, but only strain S1 transported uridine and thymidine. Competition studies in the presence of 100 microM of unlabeled adenosine, inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, 3'-deoxyinosine as well as uridine, thymidine and cytidine, with either 1 microM [3H]adenosine or [3H]inosine as permeant, were carried out. The inhibition profile with [3H]inosine as permeant was essentially identical in S1 and S2 promastigotes, indicating that the same inosine transporter was present in both strains. However, with [3H] adenosine as permeant, significant differences were noted between the two strains. Thus, only adenosine, 2'-deoxyadenosine, tubercidin, uridine, and thymidine were strongly inhibitory in S1 promastigotes, while essentially all nucleosides tested were effective in S2 promastigotes. This indicates that adenosine transport in S2 promastigotes seems to involve a transporter differing from that described for S1 promastigotes.

Topics: Adenosine; Animals; Biological Transport; Diffusion; Dilazep; Inosine; Kinetics; Leishmania donovani; Nucleosides; Thioinosine; Thymidine; Uridine

We have examined the effects of some analogues of adenosine upon the circadian rhythm in rate of thymidine incorporation by cultured chick pineal glands. Incorporation in the early period of the photoperiod on day 2 of culture was slightly inhibited by the adenosine analogue N-ethylcarboxamido-adenosine, but this effect was not countered by the antagonist 8-phenyl-theophylline. Thymidine incorporation was inhibited when glands were continuously exposed to the adenosine transport inhibitor nitrobenzyl-thioinosine, but ongoing incorporation was not inhibited by addition of this agent. Removal of adenosine and deoxyadenosine supplements from the medium, with or without further addition of adenosine deaminase, had no appreciable effects upon thymidine incorporation. We conclude that adenosine and analogues probably play no role in regulation of the rhythm in rate of thymidine incorporation.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Animals; Cells, Cultured; Chickens; Circadian Rhythm; Pineal Gland; Receptors, Purinergic; Theophylline; Thioinosine; Thymidine; Vasodilator Agents

Topics: Adenine; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Biological Transport; Free Radicals; Myocardial Reperfusion Injury; Thioinosine

Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.

Topics: Adenosine; Animals; Binding Sites; Carrier Proteins; Cattle; Cells, Cultured; Chromaffin System; Enzyme Activation; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Protein Kinase C; Tetradecanoylphorbol Acetate; Thioinosine

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.

Topics: Adenosine; Animals; Biological Transport; Brain; Kinetics; Male; Molecular Structure; Rats; Rats, Inbred Strains; Stereoisomerism; Synaptosomes; Thioinosine

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Topics: Adenosine; Affinity Labels; Carrier Proteins; Cell Line; Cytarabine; Flow Cytometry; Fluorescent Dyes; Humans; Kinetics; Membrane Proteins; Molecular Structure; Nucleoside Transport Proteins; Spectrometry, Fluorescence; Thioinosine; Thionucleosides

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.

Topics: Adenosine; Animals; Biological Transport; Formycins; Furosemide; Leukemia L1210; Mice; Nucleosides; Nystatin; Sodium; Thioinosine; Tumor Cells, Cultured

Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Transport Systems; Amino Acids; Animals; Biological Transport; Blotting, Western; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane Permeability; Electrophoresis, Polyacrylamide Gel; Molecular Weight; Radiation Dosage; Sodium; Thioinosine; Tumor Cells, Cultured

Uptake of [3H]adenosine and [3H]R-phenylisopropyladenosine (R-PIA) into retinal cells was assessed autoradiographically, in the presence and absence of the purine nucleoside transport inhibitor, nitrobenzylthioinosine (NBI). Under control conditions, both purine nucleosides were accumulated in cell bodies localized to the ganglion cell layer, and the inner nuclear layer. In the presence of NBI, significantly less accumulation of nucleosides within cell bodies was observed, particularly within the inner nuclear layer, suggesting that most of the uptake occurred via the transport of both substrates. The stereoisomer of adenosine, L-[3H]adenosine, was not accumulated into retinal cells consistent with the view that the accumulation of both adenosine and R-PIA occurs via the purine nucleoside transporter.

Topics: Adenosine; Animals; Autoradiography; Cornea; Lens, Crystalline; Neurons; Phenylisopropyladenosine; Rabbits; Retina; Retinal Ganglion Cells; Stereoisomerism; Thioinosine; Vitreous Body

The membrane transport of ara-C and intracellular ara-CTP accumulation were investigated in P388 murine leukemic cells resistant to vincristine (P388/VCR) and its parent cell line. The transport of ara-C in P388/VCR cell line was a 1.4-fold increase at 30 sec compared to that in P388 parent cell line (P less than 0.01). The increase of the transport of ara-C in P388/VCR cell line, however, was not completely abolished by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBTI) to the level in parent cell line. Scatchard analyses revealed that the resistant cells had significantly less NBTI binding sites than the parent cells had. These results suggested that the changes in ara-C transport in P388/VCR cells were due, in part, to increase of NBTI-insensitive transport sites in the membrane. The measurement of the intracellular ara-CTP concentration by high-performance liquid chromatography revealed that the intracellular ara-CTP level in P388/VCR cells was also significantly higher than that in parent cells (1.4-fold, P less than 0.01). As the transport of ara-C is rate limiting at a concentration of 1 microM in the both cell lines, we concluded that the accumulation of ara-CTP in P388/VCR cells might have partially resulted from the enhancement of the ara-C transport.

Topics: Animals; Arabinofuranosylcytosine Triphosphate; Binding Sites; Biological Transport; Cell Membrane; Cytarabine; Drug Resistance; Leukemia P388; Mice; Mice, Inbred Strains; Thioinosine; Tumor Cells, Cultured; Vincristine

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.

Topics: Animals; Carrier Proteins; Cell Differentiation; Cell Membrane; Chickens; Exocytosis; Hot Temperature; Membrane Proteins; Nucleoside Transport Proteins; Receptors, Transferrin; Reticulocytes; Sheep; Thioinosine; Transferrin

Kinetic analysis of the binding of [3H]nitrobenzylthioinosine ([3H] NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of [3H] NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the [3H]NBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of [3H]NBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of [3H]NBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of [3H]NBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of [3H]NBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both [3H]NBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of [3H]NBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of [3H]NBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of [3H]NBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of [3H]NBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam. Comparable effects of adenosine and dipyridamole on [3H]NBMPR dissociation rate were obtained upon solubilization of the membranes with octylglucoside, suggesting that this phenomenon was not due to changes in membrane fluidity. These results are compatible with the existence of specific ligand recognition sites on the nucleoside transport complex of Ehrlich cells that are pharmacologically distinct from, but allosterically linked to, the high affinity binding sites for [3H]NBMPR. The marked effects on [3H]NBMPR binding kinetics that result from ligand interactions with these sites must be considered in the design and analysis of all studies involving the use of [3H]NBMPR as a high affinity probe for the nucleosid

Topics: Affinity Labels; Allosteric Regulation; Animals; Binding Sites; Blood Proteins; Carcinoma, Ehrlich Tumor; Carrier Proteins; Kinetics; Membrane Proteins; Mice; Nucleoside Transport Proteins; Thioinosine; Tritium; Tumor Cells, Cultured

The expression of nucleoside carrier [nitrobenzylmercaptopurine riboside (NBMPR) binding] sites has been related to proliferative fraction in cell lines and in patient myeloid and lymphoid blasts. This correlation was examined in patients with untreated acute myeloid leukemia (AML). Bone marrow blasts were incubated with 8 microM bromodeoxyuridine (BrdUrd) and dual-labeled with propidium iodide and anti-BrdUrd monoclonal antibody. Flow cytometry was used to determine the percentage of cells with detectable BrdUrd incorporation into DNA (%S) and the proliferative fraction (PF = %S+%G2M) in 63 patients; NBMPR binding sites were quantitated in samples from 29 patients. The median %S was 6.1% (range 0.6-25.9%) and the median PF was 13.0% (range 2.4-36.1%), with a median of 7243 NBMPR binding sites per cell (range 1716-27247). In contrast to a previous report which included bone marrow and peripheral blood blasts, %S in marrow blasts did not correlate with NBMPR binding sites per cell (r = 0.005, p = 0.979). Similarly, PF did not correlate with NBMPR sites per cell (r = 0.190, p = 0.325). This lack of correlation between leukemia cell proliferation and NBMPR binding sites per cell suggests that DNA synthesis in AML blasts depends primarily on de novo nucleoside synthesis rather than the usage of salvage pathways.

Topics: Adult; Aged; Aged, 80 and over; Binding Sites; Bone Marrow; Cell Line; Humans; Leukemia, Myeloid, Acute; Middle Aged; Nucleosides; S Phase; Thioinosine

The presence of 10 microM dipyridamole in incubation media of L1210/C2 cells decreased initial rates of zero-trans influx of formycin B (FB, 50 microM), a poorly metabolized inosine analogue, from 4.84 pmol/microliters cell water/s to 0.87 pmol/microliter cell water/s. However, after a 5-min interval of uptake, free FB levels in dipyridamole-treated cells were 165 pmol/microliters cell water, 2.3-fold greater than in dipyridamole-free cultures. This indicated the presence of a concentrative, dipyridamole-insensitive nucleoside transport (NT) system in L1210 cells, in addition to the equilibrative NT systems known to be expressed in these cells. The concentrative system was demonstrable only in the presence of NT inhibitors and required extracellular Na+. The presence of 8 microM 6-[(4-nitrobenzyl)thio]-9-beta-D- ribofuranosylpurine or 15 microM dilazep also induced an accumulation of free FB above steady-state levels, although of a lesser magnitude than that observed with dipyridamole. It appears that NT inhibitors induced nucleoside accumulation by inhibiting bidirectional nucleoside movements mediated by the equilibrative component of nucleoside transport in L1210/C2 cells without interfering with inward FB fluxes mediated by the Na(+)-dependent transporter. The presence of NT inhibitors also enhanced the cellular accumulation and retention of arabinosyladenine and its 5'-triphosphate in these cells. The increased cellular accumulation of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosyladenine triphosphate by dipyridamole was associated with enhanced antiproliferative activity of 9-beta-D-arabinofuranosyladenine towards the leukemia cells.

Topics: Animals; Biological Transport; Dilazep; Dipyridamole; Formycins; Leukemia L1210; Mice; Nucleosides; Sodium; Thioinosine; Vidarabine; Vidarabine Phosphate

Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.

Topics: 2-Chloroadenosine; Affinity Labels; Animals; Carrier Proteins; Cell Division; Cell Membrane; Cell Survival; Cladribine; Deoxyadenosines; Kinetics; Leukemia L1210; Membrane Transport Proteins; Methotrexate; Mice; Mutation; Nucleosides; Sodium; Thioinosine; Thymidine; Tumor Cells, Cultured

The potency of mioflazine and related drugs (Janssen Pharmaceutica, Belgium) as inhibitors of adenosine transport in isolated erythrocytes from several species were measured and compared with those of dilazep and 6-(4-nitrobenzylmercapto)purine ribonucleoside (NBMPR). [8-3H]Adenosine was used as the permeant at 1 microM and incubation times were 10 s, and assays were conducted in the presence and absence of varying doses of potential transport inhibitors. The species investigated included mouse, hamster, rabbit, baboon and man. Dilazep was the most potent compound throughout with an IC50 of about 2 nM. In the mouse and hamster mioflazine and its derivatives were considerably less potent (IC50 values greater than 200 nM) with the exception of R57974 with IC50 values of about 150 and 60 nM in mouse and hamster, respectively. In the man and baboon the derivatives had IC50 values in the same order of magnitude as NBMPR (less than 100 nM), and in the rabbit they had potencies close to that of NBMPR, ranging between 10-60 nM. Nucleoside transport inhibitors are of potential importance as host protectors during treatment of parasitic infections with cytotoxic nucleosides. Present data indicate that mioflazine and its derivatives are not very potent in some of the preferred animal models for parasitic infections (mouse, hamster) but are more effective in primates such as man and baboon.

Topics: Adenosine; Animals; Cricetinae; Dilazep; Dimethyl Sulfoxide; Erythrocytes; Humans; In Vitro Techniques; Male; Mesocricetus; Mice; Mice, Inbred BALB C; Papio; Piperazines; Rabbits; Species Specificity; Thioinosine

The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.

Topics: Affinity Labels; Biological Transport; Blood Proteins; Carrier Proteins; Erythrocyte Membrane; Glycoside Hydrolases; Humans; Kinetics; Leukemia, T-Cell; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Membrane Proteins; Molecular Weight; Nucleoside Transport Proteins; Phospholipids; Thioinosine; Tumor Cells, Cultured; Uridine

This study investigated the mechanisms by which adenosine stimulates proliferation of microvascular endothelial cells. The metabolic byproducts of adenosine, inosine and hypoxanthine were unable to stimulate proliferation. When adenosine uptake was prevented, the stimulation of proliferation was unchanged, suggesting that uptake of adenosine with subsequent incorporation into the nucleotide pool is not the mechanism for increasing proliferation. Treatment of endothelial cells with adenosine analogues, presumably selective for either the A1 or A2 receptor, stimulated proliferation equally. This suggested that adenosine 3', 5'-cyclic monophosphate (cAMP) might not mediate the proliferative response to adenosine. However, radioimmunoassay of cell extracts after treatment with either analogue showed an increase in cAMP. In addition, adenylate cyclase blockade with 2', 5'-dideoxyadenosine prevented the proliferative response brought about by these analogues. These data suggest that the proliferative response to adenosine depends on an increase in cAMP. A 2-h pulse of cholera toxin stimulated endothelial cell proliferation, further supporting a role for cAMP. Pretreatment of endothelial cells with pertussis toxin blocked the stimulation of proliferation, indicating that a Gi or similar G protein is also involved in proliferation. We conclude that the proliferative response to adenosine involves a pertussis toxin-sensitive substrate as well as an increase in cAMP.

Topics: Adenosine; Animals; Biomechanical Phenomena; Cell Division; Cyclic AMP; Dideoxyadenosine; Dipyridamole; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypoxanthine; Hypoxanthines; Inosine; Isomerism; Microcirculation; Molecular Weight; Thioinosine

The initial velocity of thymidine uptake was measured in HeLa S3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h post-infection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified proteins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection.

Topics: Affinity Labels; Biological Transport; Cell Transformation, Viral; Dipyridamole; HeLa Cells; Humans; Kinetics; Methionine; Simplexvirus; Sucrose; Thioinosine; Thymidine

The glycogenolytic potency of adenosine and ATP was studied in adult rat hepatocytes and compared with the action of glucagon and noradrenaline. In cells cultured for 48 h, adenosine and ATP as well as their analogues 2-chloroadenosine, phenylisopropyladenosine, N-ethylcarboxamidoadenosine and beta-gamma-methylene-substituted ATP (p[CH2]ppA) increased glycogen phosphorylase alpha to levels indistinguishable from those obtained by the addition of glucagon or noradrenaline. The P1 receptor antagonist 8-phenyltheophylline abolished the activation of phosphorylase by adenosine and by p[CH2]ppA, but not that by ATP. Protein kinase A was activated by p[CH2]ppA and ATP via their breakdown to adenosine. [14C]Glucose production from glycogen was stimulated only 3-fold by ATP and adenosine, compared with a 7-fold increase produced by the hormones. Stimulation of glucose production by glucagon or noradrenaline was almost completely abolished by ATP or adenosine, with half-maximal effects at around 10 microM. The non-degradable adenosine analogues were equipotent with glucagon with respect to stimulation of glucose production, and their action was also inhibited by adenosine. ATP and p[CH2]ppA, which were both degraded to adenosine, showed comparable metabolic effects, whereas the alpha, beta-methylene analogue was without biological action and also was not degraded to adenosine. In the presence of the adenosine transport inhibitor nitrobenzyl thioinosine (NBTI), adenosine exerted an increased glycogenolytic potency, reaching 80% of the maximal stimulation obtained by glucagon. The glucagon-antagonistic effect of adenosine could be completely abolished by NBTI, but was not affected by phenyltheophylline. It is concluded that, in the hepatocyte culture system, adenosine and ATP decrease the catalytic efficiency of phosphorylase alpha through signals arising from their uptake into the cell.

Topics: Adenosine; Adenosine Triphosphate; Animals; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon; Glucose; Glycogen; Inositol Phosphates; Liver; Male; Norepinephrine; Rats; Rats, Inbred Strains; Thioinosine

The depressant effect of adenosine (Ad) was studied electrophysiologically in hippocampal slices from 5-, 10-, 15-, 20-, 30- and 120-day-old rats. Ad (10 microM) depressed the field EPSP in CA1 to the same extent in all age groups. Caffeine (Caf), an Ad receptor antagonist, enhanced and nitrobenzylthioinosine (NBI), an Ad uptake blocker, depressed the field EPSP. Both these effects were, however, less prominent in slices from younger animals, a finding consistent with lower extracellular levels of endogenous Ad in neonatal rats.

Topics: Action Potentials; Adenosine; Animals; Caffeine; Female; Hippocampus; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thioinosine

DEAE-column-purified band 4.5 polypeptides of human erythrocyte membranes are mostly glucose transporters with nucleoside transporters as a minor component. The purpose of the present work was to differentially identify and isolate the nucleoside transporters in band 4.5 free from glucose transporters. Equilibrium binding studies demonstrated that the band 4.5 preparation binds nibrobenzylthioinosine (NBTI), a potent nucleoside transport inhibitor, at two distinct sites, one with a high affinity (dissociation constant, KD of 1 nM) with a small capacity, BT (0.4 nmol/mg protein), and the other with a low affinity (KD of 15 microM) with a large BT (14-16 nmol/mg protein). The BT of the low-affinity site was equal to that of the cytochalasin B binding site in the preparation. A gel-filtration chromatography of band 4.5 photolabeled with [3H]NBTI and [3H]cytochalasin B identified three polypeptides of apparent Mr 55,000, 50,000 and 40,000. Of these, the 55 kDa polypeptide was specifically labeled by cytochalasin B (p55GT), indicating that it is a glucose transporter. Both the 50 and 40 kDa polypeptides were labeled with NBTI at low ligand concentrations (less than 0.1 microM), which was abolished by an excess (20 microM) of nitrobenzylthioguanosine, indicating that they are two forms (p50NT and p40NT, respectively) of the high affinity NBTI binding protein or nucleoside transporter. At higher (not less than 10 microM) NBTI concentrations, however, p55GT was also labeled with NBTI, indicating that the low-affinity NBTI binding is due to a glucose transporter. Treatment of band 4.5 with trypsin reduced the p50NT labeling with a concomitant and stoichiometric increase in the p40NT NBTI labeling without affecting the high-affinity NBTI binding of the preparation. These findings indicate that the nucleoside transporter is slightly smaller in mass than the glucose transporter and that trypsin digestion produces a truncated nucleoside transporter of apparent Mr 40,000 which retains the high-affinity NBTI binding activity of intact nucleoside transporter. Both p55GT and p50 NT were coeluted in a major protein fraction, P1 in the chromatography, while p40NT was eluted separately as a minor protein fraction, P1a. All three polypeptides formed mixed dimers, which were eluted in a fraction PO. We have purified and partially characterized the truncated nucleoside transporter, p40NT. The purified p40NT may be useful for biochemical characterization of the nucleoside transp

Topics: Blood Proteins; Carrier Proteins; Chromatography, Gel; Erythrocyte Membrane; Humans; Membrane Proteins; Molecular Weight; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Thioinosine

The aim of this study was to determine the dual role of ATP as an energy substrate and as a major source of oxygen-derived free-radical-mediated reperfusion injury by using adenine nucleoside blocker, p-nitrobenzylthioinosine (NBMPR), and adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). In a randomized study, 16 dogs were instrumented with minor-axis LTZ-piezoelectric crystals and intraventricular pressure transducers to monitor, off bypass, left ventricular performance by using a sensitive and load-independent index of contractility (slope of the stroke work-end-diastolic length relation). Hearts were subjected to 60 minutes of normothermic global ischemia and 120 minutes of reperfusion. Normal saline without (Group 1, n = 8) or with (Group 2, n = 8) NBMPR and EHNA was infused in three boluses into the cardiopulmonary bypass reservoir before ischemia and reperfusion. Transmural serial biopsies were obtained before and during ischemia and reperfusion and analyzed for myocardial adenine nucleotide pool intermediates by using high-performance liquid chromatography. In the control group, three hearts developed ischemic contracture and another three hearts exhibited cardiogenic shock during reperfusion. In the EHNA/NBMPR-treated group, left ventricular performance recovered within 30 minutes of reperfusion (p less than 0.05 vs. control). Myocardial ATP was depleted to 20% of normal in both groups by the end of ischemia (p less than 0.05). Intramyocardial adenosine in the EHNA/NBMPR-treated group was 12-fold greater (15.09 +/- 1.6 nmol/mg protein) than the control group at the end of the ischemic period (p less than 0.05). Inosine was about fourfold higher in the control group (19.07 +/- 1.50 nmol/mg protein) compared with the drug-treated group (p less than 0.05). During reperfusion, myocardial ATP levels increased to approximately 50% of normal in the EHNA/NBMPR group while remaining depressed (20% of normal) in the control group. Thus, despite the dramatic loss of myocardial ATP during ischemia, complete recovery of ventricular performance and significant repletion of ATP during reperfusion were observed when adenosine transport and deamination were modulated during ischemia and reperfusion. These results suggest that 1) the myocardium may have more ATP than is needed for basic cardiac functions and 2) washout of ATP diffusible catabolites is detrimental to ventricular performance during reperfusion. Specific blockade of nucleosid

Topics: Adenine; Adenine Nucleotides; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Affinity Labels; Animals; Dogs; Female; Free Radicals; Male; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Thioinosine; Ventricular Function, Left

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.

Topics: Adenosine; Affinity Labels; Animals; Biological Transport; Carrier Proteins; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Glucosides; Ligands; Membrane Glycoproteins; Membrane Proteins; Molecular Weight; Nucleoside Transport Proteins; Nucleosides; Solubility; Swine; Thioinosine; Thionucleosides

The sensitivity of facilitated-diffusion and Na(+)-dependent nucleoside transporters to inhibition by a series of novel compounds related to lidoflazine and mioflazine was investigated. Uridine transport by rabbit erythrocytes, which proceeds solely by the nitrobenzylthioinosine (NBMPR)-sensitive facilitated-diffusion system, was inhibited with apparent Ki values of less than 10 nM by lidoflazine, mioflazine, soluflazine and R73-335. These compounds also blocked site-specific [3H]NBMPR binding to rabbit erthrocyte membranes in a competitive fashion. The NBMPR-sensitive system in rat erythrocytes was also inhibited by lidoflazine, mioflazine, soluflazine and R73-335 but was two to three orders of magnitude less sensitive to inhibition than the system in rabbit erythrocytes (apparent Ki 7.3, 2.4, 5.7 and 0.1 microM, respectively). Lidoflazine, mioflazine and R73-335 exhibited a similar potency for the NBMPR-sensitive and -insensitive nucleoside transporters in rat erythrocytes. In contrast, soluflazine was 20- to 100-fold more potent as an inhibitor of the NBMPR-insensitive nucleoside transport component in rat erythrocytes (IC50 of 0.08-0.2 microM) compared to the NBMPR-sensitive nucleoside carrier in these cells (IC50 approximately 10 microM). None of the test compounds were potent inhibits of Na(+)-dependent uridine transport in bovine renal brush-border membrane vesicles. These results indicate that lidoflazine, mioflazine, soluflazine and R73-335 are selective inhibitors of nucleoside transport in animal cells and that the potency of these compounds as nucleoside transport inhibitors is species dependent.

Topics: Affinity Labels; Animals; Biological Transport; Cardiovascular Agents; Cattle; Cells, Cultured; Erythrocytes; Humans; Kidney; Lidoflazine; Nucleosides; Piperazines; Placenta; Rabbits; Rats; Sodium; Thioinosine; Uridine

Previous studies have demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) both increases and decreases levels of 3'-azido-3'-deoxythymidine (AZT) nucleotides in certain human myeloid cells. The present studies have examined the effects of GM-CSF on AZT metabolism in U-937 cells. The results demonstrate that GM-CSF stimulated AZT nucleotide formation in these cells. This stimulation was detectable during concurrent exposure to GM-CSF and AZT or as a result of pretreatment with GM-CSF. The GM-CSF-induced enhancement in AZT nucleotide formation was associated with a 4-fold increase in AZT uptake. The finding that uptake of AZT into U-937 cells was only partially sensitive to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) suggested a process primarily involving nonfacilitated diffusion. The results also demonstrate that treatment of U-937 cells with GM-CSF was associated with nearly a 2-fold increase in thymidine kinase activity. Moreover, the findings indicate that retention of AZT-MP and AZP-TP was prolonged significantly (P less than 0.05 and P less than 0.01 respectively) in association with GM-CSF treatment. Taken together, these results suggest that GM-CSF enhances the formation of AZT nucleotides by increasing AZT uptake and phosphorylation, as well as increasing retention of phosphorylated derivatives.

Topics: Biological Transport; Cell Division; Dideoxynucleotides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; In Vitro Techniques; Monocytes; Phosphorylation; Thioinosine; Thymidine Kinase; Thymine Nucleotides; Tumor Cells, Cultured; Zidovudine

In isolated perfused rat kidneys the medullary thick ascending limb (mTAL) is uniquely vulnerable to cellular injury produced by its hypoxic milieu and exacerbated by active transport. Endogenous inhibitors of transport might therefore be expected to reduce cell injury. We studied the possible role of adenosine in altering mTAL damage in isolated rat kidneys perfused for 60 min. When adenosine deaminase was added to the recirculating perfusate in 8 experiments, severe damage to mTAL cells adjacent to the inner medulla was significantly exacerbated to involve 89.4 +/- 3% of them as compared to 74.9 +/- 4.7% in 9 controls (p less than 0.025). Similar results were obtained when 6-nitrobenzylthioinosine, which inhibits adenosine efflux from hypoxic cells, was added to the perfusion (n = 5, p less than 0.025). The addition of the adenosine analogue R(-)-phenylisopropyladenosine [R(-)-PIA] conferred protection, so that now only 12.5 +/- 2.5% of deep mTAL cells exhibited severe morphological damage (n = 7, p less than 0.005). The protective effect of R(-)-PIA was minimized by 8-phenyltheophylline, which blocks adenosine receptors. The S(+)-isomer of PIA was significantly less potent than R(-)-PIA in protecting against hypoxic injury. These results suggest that endogenous adenosine may play a role in modifying the injurious effects of anoxia on medullary cells, by inhibiting active transport.

Topics: Adenosine; Adenosine Deaminase; Animals; Biological Transport; Kidney Diseases; Kidney Medulla; Male; Perfusion; Rats; Rats, Inbred Strains; Theophylline; Thioinosine

The displacement of [3H]nitrobenzylthioinosine from nucleoside transporter-associated binding sites on calf lung tissue membranes by several transport inhibitors appeared to be pH dependent. These blockers, viz. dilazep, hexobendine, soluflazine and ST7092, provided to be more potent in this respect at pH 9.5 than at pH 6.6, although to differing extents. A further analysis showed that this behaviour could be related to the ionization characteristics of the compounds. For a quantitative evaluation of the findings presented in this study, we paid attention to the degradation of these labile compounds by esterases present in the membrane preparation. Degradation was almost fully prevented by the use of 10 mumol/l physostigmine, a potent ester hydrolase inhibitor, in the binding experiments. This concentration of physostigmine did not displace any [3H]NBI binding. From the aqueous ionization constants of the compounds the relative distribution of the diprotonated, monoprotonated and uncharged molecules at pH 6.6 and pH 9.5 was calculated. Combination of these data with the Ki values of the compounds, obtained from [3H]NBI displacement studies at both pH values and corrected for degradation, yielded the 'true' affinities of the monoprotonated and uncharged species. The relative paucity of the diprotonated species at pH 6.6 and its virtual absence at pH 9.5 made us assume that this species has only negligible affinity for the nucleoside transport protein. For all compounds it was found that the uncharged species has higher affinity than the monoprotonated molecule. This phenomenon was most pronounced for hexobendine, the difference in 'true' Ki values being more than 80-fold.

Topics: Animals; Binding, Competitive; Cattle; Dilazep; Hexobendine; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Membranes; Nucleosides; Piperazines; Spectrophotometry, Ultraviolet; Thermodynamics; Thioinosine

Chronic exposure to ethanol results in heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory guanine nucleotide regulatory protein. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization (Nagy, L. E., Diamond, I., Collier, K., Lopez, L., Ullman, B., and Gordon, A. S., Mol. Pharmacol., in press). To understand the mechanism underlying ethanol-induced increases in extracellular adenosine, we examined the interaction of ethanol with the adenosine transport system in S49 lymphoma cells. We found that ethanol inhibited nucleoside uptake without affecting deoxyglucose or isoleucine transport. Inhibition of adenosine uptake was due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake. Consequently, ethanol no longer increased extracellular adenosine concentrations. Taken together with our previous studies, these results suggest that ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which causes an initial increase in intracellular cAMP levels and subsequent development of heterologous desensitization of cAMP signal transduction.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Adenosylhomocysteinase; Affinity Labels; Animals; Biological Transport; Cell Line; Deoxyadenosines; Ethanol; Humans; Hydrolases; Kinetics; Receptors, Purinergic; Thioinosine

Nitrobenzylthioinosine (NBMPR), dipyridamole, and dilazep, potent inhibitors of nucleoside transport, were found to be ineffective in preventing 9-beta-D-arabinofuranosylguanine (ara-G)-induced inhibition of MOLT 4 and CCRF CEM cell growth, ara-G (2.0 microM) was metabolized to 9-beta-D-arabinofuranosylguanine 5'-triphosphate in MOLT 4 cells, and the levels of this metabolite were not affected by the presence of 5.0 microM NBMPR in the incubation medium. Permeation of the MOLT 4 cell membrane by ara-G occurred primarily by means of the NBMPR-sensitive nucleoside transport system. However, a residual transport component accounting for 10-20% of the total transport activity was demonstrated in the presence of NBMPR. This component was inhibited by adenine and hypoxanthine but not by dilazep, dipyridamole, or other nucleosides. In contrast, inhibitors of nucleoside transport readily reversed the cytotoxic effect of 7-deazaadenosine (tubercidin) in both MOLT 4 and CCRF CEM cells. The levels of tubercidin 5'-triphosphate formed from 2.0 microM tubercidin in MOLT 4 cells were reduced by 80% in the presence of 5.0 microM NBMPR. The influx of tubercidin into MOLT 4 cells was found to occur primarily by means of the NBMPR-sensitive nucleoside transport system. This same system mediated the transport of ara-G into human erythrocytes.

Topics: Arabinonucleosides; Biological Transport; Cell Division; Cell Line; Dilazep; Dipyridamole; Erythrocytes; Humans; Inosine; Kinetics; Leukemia-Lymphoma, Adult T-Cell; Purines; Ribonucleosides; Thioinosine; Tubercidin; Tumor Cells, Cultured

The potency of nucleoside transport inhibitors, including 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), dilazep, mioflazine and its derivatives soluflazine and R57974 as inhibitors of the binding of [3H(G)]NBMPR to intact erythrocytes and respective ghost membranes from human, mouse and hamster was determined. There was no close agreement between the IC50 profiles for the different inhibitors when comparing values obtained for intact cells and membranes from each species, and there was no consistent profile of differences when considering individual drugs and comparing their actions in the three species. Present data also were compared with potency values obtained previously with the same drugs directly in nucleoside transport inhibition studies with erythrocytes from the same species as well as with [3H(G)]NBMPR binding studies in isolated liver and lung membranes from hamster. The overall conclusion from this and previous studies is that the evaluation of relative potencies in screening of potential nucleoside transport inhibitors is best carried out at the level actual nucleoside transport studies in intact cells, since [3H(G)]NBMPR binding studies yield discrepant data.

Topics: Adenosine; Animals; Binding, Competitive; Carrier Proteins; Cricetinae; Dilazep; Erythrocyte Membrane; Erythrocytes; Hot Temperature; Humans; Hydrogen-Ion Concentration; Inosine; Membrane Proteins; Mice; Nucleoside Transport Proteins; Piperazines; Rats; Thioinosine

In an attempt to investigate the role of nucleoside transporter function in the hypertensive state, we have compared the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR), a nucleoside transporter probe, in membranes prepared from platelet, renal, pulmonary, cardiac and brain tissues of spontaneously hypertensive rats (SHR) to those of age-matched Wistar-Kyoto (WKY) controls. At 4 weeks of age, [( 3H]NBMPR) binding sites (Bmax) increased in the kidney of SHR but decreased in platelets, whereas no changes were found in the heart, lung or brain. At 18 weeks of age, [3H]NBMPR binding sites (Bmax) remained increased in the kidney and decreased in platelets with no changes in the other tissues. The only change in apparent binding affinity (KD) was an increase in the heart of SHR at 4 weeks. Age-dependent decreases were also observed in the heart and platelets of both SHR and WKY at 18 weeks. The results indicate that the changes in binding characteristics may be due to a combination of the pharmacodynamic differences between the strains, age, as well as to the pathogenesis of hypertension. Consequently, it cannot be concluded that the altered binding characteristics are the result of the elevated blood pressure.

Topics: Affinity Labels; Animals; Binding Sites; Blood Platelets; Blood Pressure; Brain; Hypertension; Inosine; Kidney; Least-Squares Analysis; Male; Myocardium; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Regression Analysis; Thioinosine

Extracellular adenosine is transported into chromaffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5'-(N-Ethylcarboxamido)adenosine (NECA), an agonist of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls and 100 nM NECA, respectively. The A1 agonist N6-cyclohexyladenosine, the A1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine, and the A1-A2 antagonist 1,3-dipropyl-8-(4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl)- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]-nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 +/- 800 and 23,200 +/- 700 transporters/cell for controls and 100 nM NECA, respectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Animals; Biological Transport; Cells, Cultured; Chromaffin System; Colforsin; Dipyridamole; Kinetics; Nucleosides; Receptors, Purinergic; Thioinosine

Accumulation of cytosine arabinoside triphosphate (araCTP) from a range of cytosine arabinoside (araC) concentrations (1-50 microM) was measured during incubations of leukemic cells freshly isolated from patients with acute leukemia. In all but one patient, increments in extracellular araC above 10 microM did not increase intracellular araCTP levels. This maximal level of araCTP accumulation ranged from 254 to 1607 pmol/10(7) cells attained after 1 h incubation and did not correlate with either the number of nucleoside transporters on the cell membrane or the Vmax of araC phosphorylation in cell free extracts. Extremely low araCTP accumulation (103 pmol/10(7) cells/h at 50 microM araC) was observed in an AML patient with the unusual finding of micromyeloblasts. These cells also had very low numbers of nucleoside transport sites (less than 500 sites/cell) and were mitotically inactive. The unique feature of the myeloblasts from this patient was that intracellular araCTP accumulation showed a linear dependence on extracellular araC up to 50 microM with no evidence of saturation.

Topics: Acute Disease; Arabinofuranosylcytosine Triphosphate; Arabinonucleotides; Binding Sites; Biological Transport; Cell Membrane; Cytarabine; Deoxycytidine Kinase; Extracellular Space; Humans; Leukemia; Lymphocytes; Nucleosides; Phosphorylation; Thioinosine; Tumor Cells, Cultured

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.

Topics: Animals; Biological Transport; Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Glycosylation; Leukemia L1210; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Protein Binding; Thioinosine; Tumor Cells, Cultured; Tunicamycin; Uridine

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.

Topics: Affinity Labels; Animals; Antibiotics, Antineoplastic; Biological Transport; Formycins; Inosine; Kinetics; Leukemia L1210; Mice; Nucleosides; Thioinosine

Nitrobenzylthioinosine is an effective inhibitor of adenosine transport in chromaffin cells. When adenosine transport was measured at a 0.15 microM adenosine concentration, in the presence of variable concentrations of nitrobenzylthioinosine, ranging from 10(-14) to 10(-6) M, a half-maximal inhibitory concentration value (IC50) of 1 +/- 0.3 nM was deduced. This compound has the capacity to inhibit the total adenosine transport at 10(-7) M concentration. Nitrobenzylthioinosine acts in a non-competitive manner in blocking adenosine transport, as deduced from a Dixon plot, with a constant inhibition value (K1) of 0.01 +/- 0.003 nM. The results suggest that all nucleoside transporters present in bovine chromaffin cells are sensitive to the nitrobenzylthioinosine inhibition.

Topics: Adenosine; Adrenal Medulla; Animals; Carrier Proteins; Cattle; Cells, Cultured; Dipyridamole; Inosine; Thioinosine

Adenosine transport by rat and guinea pig synaptosomes was studied to establish the basis for the marked differences in the potency of some transport inhibitors in these species. An analysis of transport kinetics in the presence and absence of nitrobenzylthioinosine (NBTI) using synaptosomes derived from several areas of rat and guinea pig brain indicated that at least three systems contributed to adenosine uptake, the Km values of which were approximately 0.4, 3, and 15 microM in both species. In both species, the system with the Km of 3 microM was potently (IC50 of approximately 0.3 nM) and selectively inhibited by NBTI. This NBTI-sensitive system accounted for a greater proportion of the total uptake in the guinea pig than in the rat and was inhibited by dipyridamole, mioflazine, and related compounds more potently in the guinea pig. Preliminary experiments with other species indicate that adenosine transport in the mouse is similar to that in the rat, whereas in the dog and rabbit, it is more like that in the guinea pig. In the rat, none of the systems appeared to require Na+, but the two systems possessing the higher affinities for adenosine were inhibited by veratridine- and K(+)-induced depolarization. The transport systems were active over a broad pH range, with maximal activity between pH 6.5 and 7.0. Our results are consistent with the possibility that adenosine transport systems may be differentiated into uptake and release systems.

Topics: Adenosine; Animals; Biological Transport; Brain; Dipyridamole; Dogs; Female; Guinea Pigs; Hydrogen-Ion Concentration; Kinetics; Male; Membrane Potentials; Mice; Piperazines; Potassium; Rabbits; Rats; Rats, Inbred Strains; Sodium Chloride; Species Specificity; Synaptosomes; Thioinosine; Tissue Distribution

[8-3H]Adenosine uptake in mouse peritoneal exudate cells, harvested following i.p. challenge with Complete Freund's Adjuvant from BALB/c mice, was found to be insensitive to common nucleoside transport inhibitors such as dilazep or 6-[(4-nitrobenzyl)mercapto]purine ribonucleoside and to require sodium ion, being inactive when sodium was replaced by lithium or potassium. These findings also applied to the adherent (macrophages) and nonadherent (polymorphonuclear cells) cell fractions prepared from the peritoneal cell mixture. Uptake was inhibited by several nucleosides including deoxyadenosine, inosine, uridine, thymidine and, to a lesser extent, by the adenosine analog tubercidin, while adenine, fructose, glucose and ribose were without effect. Uptake [8-3H]adenosine was fully matched by rapid intracellular phosphorylation to AMP, ADP and ATP. Inosine was a substrate for the transporter, but tubercidin was not. The system clearly is distinct from carrier-mediated, nonconcentrative transport and has similarities to concentrative, sodium-dependent nucleoside transporters described in other cell types.

Topics: Adenosine; Animals; Biological Transport, Active; Cell Adhesion; Dilazep; Erythrocytes; In Vitro Techniques; Macrophages; Mice; Mice, Inbred BALB C; Peritoneal Cavity; Sodium; Thioinosine; Time Factors

The formycin analogs of nitrobenzylthioinosine and nitrobenzylthioguanosine were synthesized and evaluated as nucleoside transport inhibitors. These analogs have a potential therapeutic advantage over their parent compounds in that their C-nucleosidic linkages prevent them from being degraded to the immunosuppressive agents, 6-mercaptopurine and 6-thioguanine. 7-[(4-Nitrobenzyl)-thio]-3-(beta-D-ribofuranosyl)pyrazolo[4,3- d]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(beta-D- ribofuranosyl)pyrazolo[4,3-d]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC50 value for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [3H]NBTF yielded a Kd of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with Kd values of 8.1 and 0.81 nM respectively. The octanol/water partition coefficients of nitrobenzylthioinosine, NBTF and NBTGF were 3.5, 3.2, and 2.8 respectively. NBTF could be expected to be equipotent with nitrobenzylthioinosine in whole blood where inhibitor concentrations of 10(-7) to 10(-6) M are required in order to saturate erythrocytic binding sites; hence, it may exhibit the advantages inherent in a C-nucleoside.

Topics: Adenosine; Biological Transport; Erythrocytes; Humans; Leukemia, Promyelocytic, Acute; Nucleosides; Thioinosine; Thionucleosides; Tumor Cells, Cultured

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.

Topics: 1-Methyl-3-isobutylxanthine; 2',3'-Cyclic-Nucleotide Phosphodiesterases; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cerebral Cortex; In Vitro Techniques; Kinetics; Male; Myocardium; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Purinergic; Theophylline; Thioinosine; Xanthines

1. Adenosine and its metabolically stable analogue N-ethyl-carboxamidoadenosine (NECA) enhance histamine release from rat peritoneal mast cells when these are stimulated by calcium-mobilizing agents. NECA and adenosine shift the concentration-response curve of the calcium ionophore A23187 to lower concentrations. 2. The potencies of NECA or adenosine in enhancing A23187-induced histamine release are dependent on the level of stimulated release in the absence of adenosine analogues. At high levels of release their potencies are up to 20 times higher than at low levels. Consequently, averaged concentration-response curves of adenosine and NECA for enhancing histamine release are shallow. 3. The adenosine transport blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) has no effect by itself at low levels of stimulated histamine release, but abolishes the enhancing effect of adenosine. At high levels of release, however, NBTI alone enhances the release of histamine. 4. It is concluded that adenosine and calcium reciprocally enhance the sensitivity of the secretory processes to the effects of the other agent. The levels of intracellular adenosine obtained by trapping adenosine inside stimulated mast cells are sufficient to enhance histamine release substantially, suggesting that this effect may play a physiological and pathophysiological role.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Calcimycin; Calcium Channel Agonists; Histamine Release; In Vitro Techniques; Male; Mast Cells; Peritoneal Cavity; Rats; Thioinosine

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Topics: Antigens, Differentiation, T-Lymphocyte; Biological Transport; Carrier Proteins; CD3 Complex; Cell Division; Cell Separation; Humans; In Vitro Techniques; Lymphocytes; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Receptors, Antigen, T-Cell; Thioinosine; Thymus Gland

Dipyridamole (DPM) enhanced the sensitivity of human ovarian carcinoma 2008 cells to etoposide (VP-16) producing a 5.5-fold reduction in 50% inhibitory concentration at a DPM concentration of 20 microM. This interaction was shown to be truly synergistic by isobologram and median effect analysis. DPM increased the steady-state VP-16 content of 2008 cells; a DPM concentration of 4 microM increased VP-16 content by 2-fold. DPM was 25 times less potent when cells were incubated in human plasma. In tissue culture medium 96% of the DPM was free, whereas in plasma only 15% was non-protein bound. DPM did not displace VP-16 from proteins under either condition. DPM did not increase the initial influx of VP-16 but did inhibit the initial efflux, reducing the efflux rate constant by 27%. DPM had no effect on the later stages of drug efflux, nor did it irreversibly bind VP-16 in the cell. The effect of DPM was evident within 1 min; once removed, the effect disappeared within 2 min. DPM is a potent nucleoside membrane transport inhibitor and can also inhibit cyclic AMP (cAMP) phosphodiesterase in platelets. Nitrobenzylthioinosine, another nucleoside transport inhibitor which competes for binding with DPM, did not enhance sensitivity to VP-16 or increase VP-16 cellular accumulation and did not block the effect of DPM. In 2008 cells, DPM did not increase cAMP; when cAMP was increased by incubation with dibutyryl cyclic 3':5'-AMP, there was no synergy with VP-16. The results indicate that enhanced sensitivity to VP-16 was not due to an effect of DPM on the protein binding of VP-16 or on cellular cAMP and suggest that it is not directly related to inhibition of nucleoside transport. This effect appears to be a newly identified mechanism of action for this agent.

Topics: Blood Proteins; Cell Survival; Cyclic AMP; Dipyridamole; Drug Synergism; Etoposide; Female; Humans; Protein Binding; Thioinosine; Tumor Cells, Cultured

The mechanism by which 2,3'-dideoxycytidine, an inhibitor of HIV-I infectivity, permeates the cell membrane was investigated. The influx of ddCyd into human erythrocytes was nonconcentrative. The initial velocity of both ddCyd influx and efflux was, in contrast to compounds that permeate the cell membrane via the nucleoside transporter, a linear function of nucleoside concentration in the 1 microM to 10 mM range and relatively insensitive to temperature. Furthermore, potent inhibitors of nucleoside transporter and other nucleosides were found to inhibit ddCyd influx only partially or not at all suggesting that ddCyd permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic seems to be due to the lack of 3'-hydroxyl moiety of ddCyd which appears to be an important determinant for the nucleoside carrier specificity rather than to lipid solubility itself. As far as permeation of the cell membrane is concerned ddCyd shares these properties with 2',3'-dideoxythymidine and 3'-azido-3'-deoxythymidine.

Topics: Acquired Immunodeficiency Syndrome; Biological Transport; Diffusion; Dipyridamole; Erythrocyte Membrane; Hot Temperature; Humans; In Vitro Techniques; Thioinosine; Zalcitabine

Transmembrane equilibration of dideoxycytidine (ddCyd) in P388 mouse leukemia cells and human erythrocytes was only 1% as rapid as that of uridine and 2'-deoxycytidine which is mediated by the facilitated nucleoside transporter of these cells. ddCyd entry was nonsaturable up to a concentration of 1 mM but was partially inhibited by dipyridamole, nitrobenzylthioinosine and nucleosides, but not by nucleobases. Thus, entry was partly (70-80%) mediated, though very inefficiently, by the nucleoside carrier. Intracellular phosphorylation of ddCyd in P388 cells was also very inefficient compared to that of 2'-deoxycytidine and uridine and not rate limited by its slow entry into the cells.

Topics: Animals; Blood Proteins; Carrier Proteins; Dipyridamole; Erythrocytes; Humans; Hydroxyurea; Leukemia, Experimental; Membrane Proteins; Mice; Nucleoside Transport Proteins; Permeability; Solubility; Structure-Activity Relationship; Thioinosine; Uridine; Zalcitabine

The effects induced by adenosine and some related compounds upon Mg2+-free epileptogenesis were studied in slices of human epileptogenic neocortex maintained in vitro. Extracellular recordings revealed stimulus-induced and spontaneous epileptiform activity within 1-2 h of perfusion with Mg2+-free medium. A 30-90% decrease of the frequency of occurrence of spontaneous epileptiform discharges was induced by 40-50 microM adenosine while the analog 2-Cl-adenosine exerted a depressant effect (greater than 75% reduction in frequency of occurrence) at 0.3-3 microM. 2-Cl-adenosine also depressed stimulus-induced epileptiform responses and often blocked spontaneous epileptiform activity. Similar effects were seen during bath application of the adenosine uptake inhibitor nitrobenzylthioinosine (10-50 microM) indicating that endogenous adenosine can by itself influence epileptogenicity. Our data demonstrate that in the human epileptogenic neocortex a purinergic mechanism can control Mg2+-free epileptiform activity.

Topics: Adenosine; Cerebral Cortex; Epilepsy; Humans; In Vitro Techniques; Thioinosine

The uptake of 2',3'-dideoxyadenosine was examined in a human immunodeficiency virus (HIV) infected and uninfected T cell line (H9 cells), a B cell line (Namalwa), and in normal peripheral blood mononuclear cells. After a 10-minute incubation at ambient temperature, the intracellular 2',3'-dideoxyadenosine-derived radioactivity was 8- to 16-fold higher than the extracellular radioactivity. In metabolically inactive cells (0 degrees C), the intracellular and extracellular 2',3'-dideoxyadenosine-derived radioactivities were nearly equal. In infected and noninfected H9 cells, a large excess of p-nitrobenzylmercaptopurine riboside or pyrimidine nucleosides weakly inhibited the uptake of 2',3'-dideoxyadenosine (7-30%), whereas deoxycoformycin was a stronger inhibitor (50-80%). Purine nucleosides minimally enhanced the uptake (10-20%). The cellular uptake was not associated with the accumulation of dideoxyadenosine triphosphate. In normal peripheral blood mononuclear cells, the uptake of 2',3'-dideoxyadenosine was inhibited by all agents except 2'-deoxyadenosine (15% enhancement). In contrast to H9 cells, the formation and accumulation of dideoxyadenosine triphosphate paralleled the uptake of dideoxyadenosine. The results of these studies suggest that the major route of transport of 2',3'-dideoxyadenosine into cells is by simple diffusion and that different metabolic patterns exist among cell lines and normal peripheral blood mononuclear cells. An understanding of these cellular differences could aid in the development of therapeutic strategies directed against HIV.

Topics: Acquired Immunodeficiency Syndrome; Antiviral Agents; Cell Line; Deoxyadenosines; Dideoxyadenosine; Diffusion; HIV; Humans; Nucleosides; Osmolar Concentration; Pentostatin; Temperature; Thioinosine

In mediating the entry of adenosine into mouse erythrocytes and mouse leukaemia L1210 cells, nucleoside transport systems were stereoselective, showing a marked preference for the D-enantiomer of adenosine (D-Ado). Inward zero-trans fluxes of the mirror-image isomer, L-adenosine (L-Ado), in those cells were slow relative to those of D-Ado. Contributing to L-Ado fluxes in both cell types were (i) a transporter-mediated process of high nitrobenzylthioinosine-sensitivity and (ii) simple diffusion.

Topics: Adenosine; Animals; Biological Transport; Erythrocyte Membrane; In Vitro Techniques; Kinetics; Leukemia L1210; Mice; Sodium; Stereoisomerism; Thioinosine

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Topics: Animals; Biological Transport; Cations, Monovalent; Cell Compartmentation; Cell Membrane; Glucose; In Vitro Techniques; Kidney Cortex; Kidney Tubules, Proximal; Microvilli; Nucleosides; Rabbits; Sodium; Thioinosine; Uridine; Valinomycin

Topics: Biological Transport; Cell Differentiation; Cytarabine; Deoxycytidine Kinase; Dipyridamole; Humans; Inosine; Leukemia, Promyelocytic, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thioinosine; Tretinoin; Tumor Cells, Cultured

A novel type of somatic mutation that causes the expression of a high affinity purine base permease has been inserted into murine S49 lymphoma cells that are deficient in nucleoside transport. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possesses an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possesses an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells can transport nucleosides suggesting that the expression of the high affinity base transporter did not revert the mutation in the nucleoside transport system. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild type cells indicated that the expression of the high affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high affinity purine base transporter of mutant cells and the nucleoside transport function of wild type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild type cells.

Topics: Adenine; Animals; Biological Transport; Clone Cells; Dipyridamole; Dose-Response Relationship, Drug; Gene Expression; Guanine; Hybridization, Genetic; Hypoxanthine; Hypoxanthines; Inosine; Lymphoma; Mice; Mice, Inbred BALB C; Mutation; Phenotype; T-Lymphocytes; Thioinosine; Tumor Cells, Cultured

Cytosine arabinoside (ARA C), a competitive inhibitor of the incorporation of 2'-deoxycytidine into DNA in other cell types, caused a concentration-dependent inhibition of KCl- and insulin-stimulated survival of postmitotic ciliary parasympathetic ganglion neurons, and the nerve growth factor (NGF)-stimulated survival of postmitotic dorsal root ganglion (DRG) sensory neurons in vitro. The IC50 for survival was 2 x 10(-8) M for both types of neurons after 4 d under the culture conditions used. The inhibition of DRG survival by ARA C in the presence of varying concentrations of NGF indicated that ARA C acted as an apparent noncompetitive antagonist of NGF. This cytotoxic effect of ARA C was blocked by 2'-deoxycytidine, but not by cytosine, 2'-deoxyadenosine, 2'-deoxyguanosine, or 2'-deoxythymine, indicating that ARA C was interfering with a deoxycytidine-specific survival process. Cytidine could block ARA C toxicity, but it was 40 times less potent than 2'-deoxycytidine. The blockade of the cytotoxic effect of ARA C by 2'-deoxycytidine indicated that 2'-deoxycytidine was an apparent competitive antagonist of ARA C toxicity. 2'-Deoxycytidine, by itself, was not survival-promoting. Other antimitotic agents, such as adenine arabinoside, thymine arabinoside, 5-fluorodeoxyuridine, 5-bromodeoxycytidine, 5-azadeoxycytidine, and aphidicolin had no effect on neuronal survival at a concentration 5000 times the EC50 of ARA C, indicating that inhibition of DNA synthesis or repair was probably not the mechanism by which ARA C inhibited neuronal survival and that other 2'-deoxynucleosides were not involved in the survival-promoting process. Nitrobenzylthioinosine, an inhibitor of 2'-deoxycytidine and ARA C membrane transport in other cell types, inhibited the cytotoxic effect of ARA C in neurons, suggesting that ARA C entered the neurons through a similar transport mechanism and that ARA C needed to gain access to the inside of the neuron to be effective. These results indicate that ARA C, in addition to being an antimitotic agent for dividing cells, is also cytotoxic for postmitotic neurons. This inhibition of neuronal survival by ARA C is hypothesized to be due to inhibition of a 2'-deoxycytidine-dependent process that is independent of DNA synthesis or repair. Thus, 2'-deoxycytidine may have an important and previously unrecognized role in cellular function that in the case of neurons is critical for survival.

Topics: Animals; Antineoplastic Agents; Cell Survival; Chick Embryo; Ciliary Body; Cytarabine; Deoxycytidine; DNA; Dose-Response Relationship, Drug; Ganglia, Spinal; Insulin; Mitosis; Nerve Growth Factors; Neurons; Parasympathetic Nervous System; Thioinosine

The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.

Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Biological Transport; Cell Membrane Permeability; Cells, Cultured; Chromatography, High Pressure Liquid; Dilazep; Dipyridamole; Erythrocytes; Guanosine; Humans; Inosine; Molecular Structure; Nucleosides; Plasmodium falciparum; Thioinosine; Thionucleosides; Tubercidin

Rapid kinetic techniques were used to study the transport and salvage of uridine and other nucleosides in mouse spleen cells. Spleen cells express two nucleoside transport systems: (1) the non-concentrative, symmetrical, Na+-independent transporter with broad substrate specificity, which has been found in all mammalian cells and is sensitive to inhibition by dipyridamole and nitrobenzylthioinosine; and (2) a Na+-dependent nucleoside transport, which is specific for uridine and purine nucleosides and resistant to inhibition by dipyridamole and nitrobenzylthioinosine. The kinetic properties of the two transporters were determined by measuring uridine influx in ATP-depleted cells and dipyridamole-treated cells, respectively. The Michaelis-Menten constants for Na+-independent and -dependent transport were about 40 and 200 microM, respectively, but the first-order rate constants were about the same for both transport systems. Nitrobenzylthioinosine-sensitivity of the facilitated nucleoside transporter correlated with the presence of about 10,000 high-affinity (Kd = 0.6 nM) nitrobenzylthioinosine-binding sites per cell. The turnover number of the nitrobenzylthioinosine-sensitive nucleoside transporter was comparable to that of mouse P388 leukemia cells. The activation energy of this transporter was 20 kcal/mol. Entry of uridine via either of the transport routes was rapidly followed by its phosphorylation and conversion to UTP. The Michaelis-Menten constant for the in situ phosphorylation of uridine was about 50 microM and the first-order rate constants for phosphorylation and transport were about the same. The spleen cells also efficiently salvaged adenosine, adenine, and hypoxanthine, but not thymidine.

Topics: Animals; Biological Transport; Carrier Proteins; Dipyridamole; In Vitro Techniques; Kinetics; Membrane Proteins; Mice; Nucleoside Transport Proteins; Nucleosides; Phosphorylation; Purines; Pyrimidines; Sodium; Spleen; Thioinosine; Uridine

We evaluated the interaction of several nucleoside transport inhibitors and substrates with the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) to nucleoside transport sites in guinea pig cardiac sections. Using quantitative autoradiography, we determined inhibition constants for inhibition of [3H]NBMPR binding to both coronary endothelial cells and cardiac myocytes. We studied the interactions of NBMPR, nitrobenzylthioguanosine, dipyridamole, dilazep, hexobendine, lidoflazine, mioflazine, soluflazine, adenosine, inosine and uridine for these two cell types. Of the compounds tested in this study, lidoflazine (8.2X) and hexobendine (6.3X) have the greatest selectivity for coronary endothelial cell nucleoside transporters. All other compounds had 3-fold or less selectivity. Therefore, there is evidence of nucleoside transporter subtypes between endothelial cells and myocytes. This heterogeneity of transport inhibitory sites on nucleoside transporters may allow the development of agents to modulate selectively some of the cardiovascular effects of adenosine.

Topics: Animals; Autoradiography; Binding, Competitive; Endothelium, Vascular; Guinea Pigs; In Vitro Techniques; Male; Myocardium; Nucleosides; Thioinosine

Dilazep, a tertiary amine that is greater than 96% protonated at pH 7.4, is a potent inhibitor of facilitated diffusion (equilibrative) nucleoside transport (NT) in animal cells. In this study, saturable reversible binding of [3H]dilazep was demonstrated at sites on S49 mouse lymphoma cells but not in AE1 cells, an NT-deficient mutant of S49 cells. Mass law analysis of dilazep binding under equilibrium conditions revealed two saturable components, representing binding sites that differed about 50-fold in affinity for dilazep (Kd values of 0.21 and 10 nM). At pH 7.4, the low affinity sites were more abundant (Bmax, 3.5 X 10(5) sites/cell) than the high affinity site (Bmax, 3.0 X 10(4) sites/cell). Binding of dilazep was pH dependent; at pH 9.0, binding at the high affinity sites predominated, whereas, at pH 5.0, the low affinity component predominated, suggesting that these components represented binding of nonprotonated and protonated dilazep molecules, respectively. Nitrobenzylthioinosine (NBMPR) and physostigmine selectively blocked binding of nonprotonated and protonated species of dilazep, respectively, at pH 7.4, yielding Scatchard plots that were similar to control plots obtained at pH 5.0 and 9.0. First-order plots of the dissociation of [3H]dilazep-binding site complexes in the presence of excess nonradioactive dilazep at pH 7.4 were nonlinear and were resolved into rapid (rate constant, 3.4-4.7 min-1) and slow (rate constant, 0.13-0.15 min-1) components. In the presence of site-saturating concentrations of NBMPR or high concentrations of nucleoside permeants, dissociation of site-bound [3H]dilazep was incomplete and only the slow component of dissociation was apparent (rate constant, 0.11-0.19 min-1). The combined presence of nonradioactive dilazep and NBMPR yielded time courses of [3H]dilazep-site dissociation equivalent to those obtained in the presence of nonradioactive dilazep alone. These results are consistent with a model in which protonated and nonprotonated species of dilazep bind at separate sites on S49 cells. The absence of both high and low affinity sites on AE1 cells suggests that, in S49 cells, both populations of sites are associated with NT polypeptides. The high affinity sites that bind nonprotonated species of dilazep appear to overlap with NBMPR binding sites on these cells.

Topics: Animals; Azepines; Binding Sites; Carrier Proteins; Dilazep; Hydrogen-Ion Concentration; Inosine; Kinetics; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Physostigmine; Thioinosine; Tumor Cells, Cultured

Normal bovine erythrocytes have negligible ability to transport adenosine and related nucleosides across their cell membrane. However, infection with the intraerythrocytic parasite Babesia bovis was found to induce a nucleoside permeation site into the host cell membrane. Transport experiments over periods of up to 30 s determined that the transport rate of 1 microM adenosine into the infected cell was 1.72 +/- 1.2 pmol incorporated (microliter cell water)-1s-1, a rate three times higher than for normal human erythrocytes. Incorporation studies over 6 h with labelled adenosine indicated that the purine moiety was incorporated into parasite nucleic acids. The mammalian nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole inhibited the induced nucleoside transport mechanism in the Babesia-infected erythrocytes, though at higher concentrations than those required to inhibit normal human erythrocyte transport. An ID50 value for NBMPR of 0.36 microM was determined. Phloretin and 5'-p-fluorosulphonyl benzoyl adenosine-HCl (5FSBA) were also shown to be inhibitory, with ID50 values of 0.11 and 0.18 microM, respectively, whilst phlorizin and verapamil at 1 microM had no effect. Binding studies with [3H]NBMPR indicated that high-affinity NBMPR binding sites could not be detected in either normal or B. bovis infected bovine erythrocytes. The results indicate that the induced nucleoside permeation site(s) in B. bovis infected erythrocytes has characteristics different from either human erythrocytes or erythrocytes infected with the malarial parasites Plasmodium falciparum or Plasmodium yoelii.

Topics: Adenosine; Animals; Babesia; Binding Sites; Biological Transport; Cattle; Dilazep; Dipyridamole; Erythrocyte Membrane; Erythrocytes; Guanosine; Humans; Kinetics; Phloretin; Phlorhizin; Thioinosine; Thionucleosides; Verapamil

A new series of compounds related to the nucleoside transport inhibitors, lidoflazine and mioflazine, is introduced. The influence of these derivatives on nucleoside-specific transport proteins was studied in two ways. First, a rapid, non-radioactive assay was developed for the screening of this type of material for actual transport inhibition in human erythrocytes. The method is based on the dose-dependent reversal of the inhibition of inorganic phosphate release induced by inosine when human erythrocytes are suspended in a phosphate-free medium. It enables the estimation of the potency and specificity of this new series of nucleoside transport inhibitors, most of which are highly active (EC50 values as low as 13 nM). Second, the displacement of a radiolabeled transport inhibitor, [3H]nitrobenzylthioinosine, was examined. All compounds were capable of displacing specific [3H]nitrobenzylthioinosine binding to crude and solubilized plasma membranes of calf lung tissue, displaying affinities in the nanomolar range. Pseudo-Hill coefficients derived from the shape of the displacement curves were significantly greater than unity for most derivatives, in contrast to values of approximately unity obtained for dipyridamole and analogs. These findings were incorporated in a mathematical model describing the interaction of mioflazine analogs with the transport protein, suggesting that one molecule of mioflazine is capable of displacing two or more molecules of [3H]nitrobenzylthioinosine at a time. The consequences of this model regarding the nature of the transport protein are discussed.

Topics: Animals; Binding, Competitive; Cattle; Dipyridamole; Erythrocytes; In Vitro Techniques; Lidoflazine; Lung; Membranes; Nucleosides; Phosphates; Piperazines; Thioinosine

The binding of [G-3H]-6-(4-nitrobenzylmercapto)purine ribonucleoside [( G-3H]NBMPR) was investigated using a centrifugation assay with membrane preparations from hamster tissues including liver, lung, kidney and heart. Only liver and lung membranes showed high specific binding, with dissociation constants (Kd) values of 2.4 +/- 0.4 and 0.44 +/- 0.05 nM, and maximal binding (Bmax) of 3.7 +/- 0.4 and 1.04 +/- 0.01 pmol/mg, respectively. The binding of [G-3H]NBMPR was inhibited in a concentration dependent manner by unlabelled NBMPR, dilazep and a new group of chemically related nucleoside transport inhibitors, mioflazine, soluflazine and R57974, the latter being the most potent derivative. R57974 displaced bound [G-3H]NBMPR as effectively as unlabelled NBMPR suggesting a common binding site. The assay procedure used appears useful for the rapid screening of the effectiveness of nucleoside transport inhibitors which will be of value for the selection of inhibitors suitable for combination with cytotoxic nucleosides in the treatment of selected cancers or parasitic diseases.

Topics: Animals; Binding Sites; Cardiovascular Agents; Cell Membrane; Cricetinae; Dilazep; In Vitro Techniques; Inosine; Liver; Lung; Male; Mesocricetus; Piperazines; Thioinosine

1. The distribution of nucleoside transport inhibitory sites in rat and guinea-pig cardiac sections was investigated by use of [3H]-nitrobenzylthioinosine ([3H]-NBMPR) autoradiography. The distribution of these sites was heterogeneous in guinea-pig sections and homogeneous in rat sections. 2. The areas of high density of nucleoside transport inhibitory sites found in guinea-pig cardiac sections were compared to the distribution of an endothelial cell marker, von Willebrand Factor, determined by radioimmunocytochemistry. These two markers were co-localized suggesting that coronary endothelial cells from guinea-pig have a high density of NBMPR binding sites and thus may have a high nucleoside transport capacity. 3. Nucleoside transporter subtypes with differing affinity for NBMPR or dipyridamole were investigated by quantitative autoradiography. Sites in rat tissues had high affinity for NBMPR (KD = 0.6 nM) but were of low sensitivity to dipyridamole (Ki = 3.1 microM). In guinea-pig sections, areas of high and low [3H]-NBMPR binding site density were analyzed separately. In both areas, sites had high affinity for NBMPR (KD = 1.4 nM, 4.5 nM, respectively) and dipyridamole (Ki = 108 nM, 245 nM, respectively). 4. While differences in density of nucleoside transport inhibitory sites are detectable between distinct regions of the heart, subtypes differing in affinity for NBMPR or dipyridamole were not evident. Therefore, more detailed structure activity studies are required to determine if subtypes of these sites exist within a single heart.

Topics: Animals; Autoradiography; Dipyridamole; Guinea Pigs; Immunohistochemistry; In Vitro Techniques; Inosine; Male; Myocardium; Nucleosides; Rats; Thioinosine; von Willebrand Factor

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Topics: Adenosine; Animals; Binding Sites; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Cell Membrane; Dipyridamole; Erythrocyte Membrane; Glucosides; Humans; Mice; Thioinosine; Tumor Cells, Cultured; Uridine

Sodium-dependent 3H-labeled nucleoside transport was studied using a mixed population of dissociated brain cells from adult rats. The accumulation of [3H]adenosine during brief (15-s) incubation periods was significantly greater in the presence of 110 mM Na+ than in its absence. This occurred at substrate concentrations that ranged from 0.25 to 100 microM. Similar findings were observed for the rapid accumulation of [3H]uridine. Kinetically, the rapid accumulation of [3H]adenosine in both the absence and the presence of Na+ was best described by a two-component system. In the presence of Na+, the KT and Vmax values for the high-affinity affinity component were 0.9 microM and 8.9 pmol/mg of protein/15 s, and those for the low-affinity component were 313 microM and 3,428 pmol/mg of protein/15 s, respectively. In the absence of Na+, the KT value for the high-affinity component was significantly higher (1.8 microM). [3H]Uridine accumulation was best described kinetically by a one-component system that in the presence of Na+ had KT and Vmax values of 1.0 mM and 2.6 nmol/mg of protein/15 s, respectively. As was found for [3H]adenosine, in the absence of Na+, the KT value was significantly higher (1.8 mM). The sodium-dependent transport of [3H]adenosine was inhibitable by ouabain and 2,4-dinitrophenol. Of the three nucleoside transport inhibitors tested, only nitrobenzylthioninosine demonstrated high affinity and selectivity in blocking the sodium component. Thus, high-affinity sodium-dependent nucleoside transport systems, in addition to facilitated diffusion systems, exist on brain cells from adult rats.

Topics: Adenosine; Animals; Brain; Dilazep; Dipyridamole; Kinetics; Male; Nucleosides; Ouabain; Rats; Rats, Inbred Strains; Sodium; Thioinosine; Uridine

1-beta-D-Arabinofuranosylcytosine (araC) is an effective drug in the i.p. therapy of ovarian carcinoma but little is known of its transport and metabolism in this tumor. Influx of araC at 1 microM into cultured human ovarian carcinoma cells (CI 80-13S) was largely inhibited by nanomolar concentrations of the nucleoside transport inhibitor, nitrobenzylthioinosine, while the residual influx (approximately 10%) was inhibited only by micromolar concentrations of nitrobenzylthioinosine. There was a two fold greater density of specific [3H]nitrobenzylthioinosine binding to the nucleoside transporters on the ovarian than on cultured human leukemic cells (RC2a). Calculated turnover rates of the nucleoside transporter for 1 microM araC were 5-fold less in ovarian than in leukemic cells. The major metabolic product of araC was 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) which accumulated in the ovarian cells to levels half those achieved in the leukemic cells. AraC was the major product of araCTP degradation in ovarian cells consistent with a pathway (araCTP--------araCMP----araC) which is different from that previously found in leukemic cells (araCTP--------araCMP----araUMP----araU). Despite these differences, ovarian carcinoma cells show substantial accumulation of araCTP from extracellular araC.

Topics: Adenocarcinoma; Aged; Arabinofuranosylcytosine Triphosphate; Binding Sites; Cell Line; Cytarabine; Female; Humans; Leukemia, Myelomonocytic, Acute; Male; Ovarian Neoplasms; Thioinosine

The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Animals; Brain; Cerebellum; Cerebral Cortex; Colforsin; Corpus Striatum; Electroshock; Hippocampus; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thioinosine

A refined method for the photoaffinity labelling of the NBI-sensitive nucleoside transport protein is described. It involves the use of low concentrations of the photolabile probe [3H]nitrobenzylthioinosine ([3H]NBI), whereas the usual inclusion of dithiothreitol in the protocol is omitted. The method was successfully applied to cell membranes of calf lung tissue, which was shown to be a rich source of this physiologically important protein with all the characteristics (both in membrane bound and solubilized form) known from similar proteins on other cell types. Specific covalent incorporation of radioactivity appeared to be pH independent. SDS-polyacrylamide gel electrophoresis revealed a specifically labelled protein with an apparent molecular weight of 55 kDa.

Topics: Adenosine; Affinity Labels; Animals; Carrier Proteins; Cattle; Cell Membrane; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Inosine; Lung; Membrane Proteins; Nucleoside Transport Proteins; Photochemistry; Thioinosine

Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.

Topics: Adenine; Adenosine; Amino Acid Transport Systems; Biological Transport; Carrier Proteins; Cell Line; Dipyridamole; Fibroblasts; Humans; Kinetics; Lysosomes; Nucleosides; Substrate Specificity; Thioinosine

The effect of a chronic hypoxic stimulus and of altered adenosine metabolism on vascular density was studied in the chick chorioallantoic membrane (CAM). Eggs were incubated in 15% oxygen/85% nitrogen for 3 or 7 days beginning at 7 days of age. Vessel density of the CAM was estimated by counting the number of vessels intersecting 4 concentric circles (72 mm total circumference) placed over the formalin-fixed membrane. The 15% oxygen stimulated 34-41% increases (P less than 0.001) in CAM vascularity after 3 or 7 days. Nitrobenzylthioinosine (NBTI), an adenosine re-uptake inhibitor, augmented the hypoxia-induced angiogenesis an additional 17% and 14% (P less than 0.001) at Days 10 and 14, respectively. Methyl-isobutylxanthine (MIX), an adenosine receptor blocker, reduced the vasoproliferation by 66% (P less than 0.001) at both times. Topically suffused adenosine elicited a local concentration-related increase in vascularity. This response was completely blocked by MIX. Exposure to 15% oxygen for 7 days stimulated a 13.7% increase in the hematocrit (P less than 0.001). Embryo weights were reduced 20.7% (P less than 0.001). These findings point to a modulatory role for adenosine in hypoxia-induced angiogenesis, and support the broader hypothesis that vasoactive metabolites produced in response to hypoxic conditions partially mediate a structurally based long-term autoregulatory response.

Topics: Adenosine; Allantois; Animals; Chick Embryo; Chorion; Extraembryonic Membranes; Neovascularization, Pathologic; Oxygen Consumption; Receptors, Purinergic; Thioinosine

A rapid sampling technique was used to follow nucleoside uptake by Trichomonas vaginalis. The results indicated that nucleoside uptake is biphasic with time. Adenosine, guanosine, and uridine uptake is carrier mediated, transported substrate is rapidly metabolised to nucleotides. Two separate carriers appear to exist, one which transports all nucleosides and a second which transports adenosine, guanosine and uridine. Both carriers have more than one binding site for nucleosides. The first carrier has sites for adenosine and pyrimidine nucleosides, and a separate site for purine nucleosides. The second carrier has a site for adenosine and uridine and a separate site for guanosine. Adenosine uptake could not be completely inhibited by nitrobenzylthionucleosides. The rate of nucleoside uptake by T. vaginalis is sufficient to sustain growth.

Topics: Adenosine; Animals; Binding, Competitive; Biological Transport; Carrier Proteins; Cytidine; Dilazep; DNA; Guanosine; Inosine; Kinetics; Nucleosides; RNA; Thioinosine; Thionucleosides; Thymidine; Trichomonas vaginalis; Uridine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Apyrase; Biological Transport; Blood; Cell Division; Cell Line, Transformed; Dipyridamole; Fibroblasts; Mice; Nucleotidases; Thioinosine; Uridine Triphosphate

In order to analyze the cellular determinants that mediate the action of 2',3'-dideoxycytidine, the growth inhibitory and cytotoxic effects and the metabolism of the dideoxynucleoside were examined in wild type human CEM T lymphoblasts and in mutant populations of CEM cells that were genetically deficient in either nucleoside transport or deoxycytidine kinase activity. Whereas 2',3'-dideoxycytidine at a concentration of 5 microM inhibited growth of the wild type CEM parental strain by 50%, two nucleoside transport-deficient clones were 4-fold resistant to the pyrimidine analog. The deoxycytidine kinase-deficient cell line was virtually completely resistant to growth inhibition by the dideoxynucleoside at a concentration of 1024 microM. An 80% diminished rate of 2',3'-[5,6-3H]dideoxycytidine influx into the two nucleoside transport-deficient lines could account for their resistance to the dideoxynucleoside, while the resistance of the deoxycytidine kinase-deficient cells to 2',3'-dideoxycytidine toxicity could be explained by a virtually complete failure to incorporate 2',3'-[5,6-3H]dideoxycytidine in situ. Two potent inhibitors of mammalian nucleoside transport, 4-nitrobenzylthioinosine and dipyridamole, mimicked the effects of a genetic deficiency in nucleoside transport with respect to 2',3'-dideoxycytidine toxicity and incorporation. These data indicate that the intracellular metabolism of 2',3'-dideoxycytidine in CEM cells is initiated by the nucleoside transport system and the cellular deoxycytidine kinase activity.

Topics: Acquired Immunodeficiency Syndrome; Biological Transport; Cell Division; Cell Survival; Deoxycytidine; Deoxycytidine Kinase; Dipyridamole; Humans; Kinetics; Leukemia, Lymphoid; Mutation; Nucleosides; Phosphorylation; Phosphotransferases; T-Lymphocytes; Thioinosine; Tumor Cells, Cultured; Zalcitabine

L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.

Topics: Animals; Biological Transport; Carrier Proteins; Cytarabine; Floxuridine; Inosine; Leukemia L1210; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; Thioinosine; Thymidine; Tubercidin; Tumor Cells, Cultured; Uridine

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.

Topics: Affinity Labels; Animals; Animals, Newborn; Antibodies, Monoclonal; Blood Proteins; Blotting, Western; Carrier Proteins; Cytochalasin B; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Glycoside Hydrolases; Humans; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Membrane Proteins; Molecular Weight; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Peptide Fragments; Photochemistry; Swine; Thioinosine; Trypsin

The aim of this study was to differentiate myocardial reperfusion injury from that of ischemia. We assessed the role of the myocardial adenosine 5'-triphosphate (ATP) catabolites, hypoxanthine and xanthine, generated during ischemia and the early phase of reperfusion, in reperfusion injury by modulating adenosine transport and metabolism with specific metabolic inhibitors. This was followed by intracoronary infusion of exogenous hypoxanthine and xanthine. Twenty-four dogs instrumented with minor-axis piezoelectric crystals and intraventricular pressure transducers were subjected to 30 minutes of normothermic global myocardial ischemia and 60 minutes of reperfusion. In Group 1 (n = 7), normal saline was infused into the cardiopulmonary bypass reservior before ischemia and before reperfusion. Saline solution containing 25 microM p-nitrobenzylthioinosine (NBMPR) and 100 microM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was infused in Group 2 (n = 10) dogs. Group 3 (n = 7) dogs were treated exactly like those in Group 2 except, at the end of the ischemic period and immediately before releasing the cross-clamp, a solution of EHNA-NBMPR containing 100 microM hypoxanthine and 100 microM xanthine was infused into the aortic root. Left ventricular performance and myocardial adenine nucleotide pool intermediates were determined before and after ischemia. ATP was depleted by about 50% (p less than 0.05 vs. preischemia) in all groups after 30 minutes of ischemia. Inosine was the major ATP catabolite (9.29 +/- 1.2 nmol/mg protein) in Group 1, while adenosine (9.91 +/- 0.7 nmol/mg protein) was the major metabolite in EHNA-NBMPR-treated dogs (Groups 2 and 3). Hypoxanthine levels were fivefold more in Group 1 compared with Groups 2 and 3 (p less than 0.05). Left ventricular performance in Group 1 decreased from 76.8 +/- 7.6 to 42.9 +/- 9.8 and 52.3 +/- 8.4 dynes/cm2 x 10(3) (p less than 0.05), while myocardial ATP decreased from 30.9 +/- 2.2 to 17.2 +/- 1.0 and 16.5 +/- 1.0 nmol/mg protein during 30 and 60 minutes of reperfusion, respectively (p less than 0.05 vs. preischemia). Ventricular function in Group 2 dogs completely recovered within 30 minutes of reperfusion, and myocardial ATP recovered to the preischemic level at 60 minutes of reperfusion. In Group 3, left ventricular performance was depressed by 39% and 30% during 30 and 60 minutes of reperfusion (p less than 0.05), respectively, and myocardial ATP did not recover during reperfusion despite a significant int

Topics: Adenine; Adenine Nucleotides; Animals; Coronary Disease; Dogs; Female; Free Radicals; Heart; Heart Ventricles; Hypoxanthine; Hypoxanthines; Male; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Thioinosine; Xanthine; Xanthines

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.

Topics: Antibodies, Monoclonal; Binding Sites; Blood Proteins; Carrier Proteins; Cytochalasin B; Erythrocyte Membrane; Filtration; Humans; Membrane Proteins; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Peptide Fragments; Thioinosine; Trypsin

The nucleoside transporter has been purified by passage of a preparation of human erythrocyte-membrane band-4.5 proteins through a column of immobilized antibodies specific for the glucose transporter. This procedure removed greater than 99.8% of the glucose transporters and achieved an approx. 18-fold purification of the nucleoside transporter, constituting a 478-fold purification from erythrocyte membranes. The isolated protein migrated as a single broad band of average apparent Mr 55,000 on SDS/polyacrylamide gels and bound approx. 0.6 mol of nitrobenzylthioinosine/mol of polypeptide, with a Kd of 1.1 +/- 0.14 (S.E.M.) nM. Upon reconstitution into large unilamellar phospholipid vesicles it catalysed the uptake of uridine with an apparent specific activity 6-fold greater than that of the unfractionated band-4.5 proteins. Furthermore, the purified nucleoside transporter was not labelled on Western blots by monoclonal antibody raised against the glucose transporter. It is concluded that the nucleoside transporter has been purified to near homogeneity.

Topics: Antibodies, Monoclonal; Blood Proteins; Blotting, Western; Carrier Proteins; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Glucose; Humans; Membrane Proteins; Nucleoside Transport Proteins; Peptide Fragments; Protein Binding; Thioinosine; Uridine

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.

Topics: Animals; Biological Transport; Epithelium; Female; Formycins; In Vitro Techniques; Intestine, Small; Kinetics; Mice; Mice, Inbred CBA; Nucleosides; Sodium; Substrate Specificity; Thioinosine

The ontogenetic profile of adenosine uptake sites was investigated in guinea pig cerebral cortex and cerebellum using as ligand probes the uptake inhibitors, [3H]nitrobenzylthioinosine ([3H]NBI) and [3H]dipyridamole ([3H]DPR). In cerebral cortex [3H]NBI binding was highest at E50 and decreased subsequently until P28 while in cerebellum after a first peak at E50 and a subsequent decline it increased again until P28. [3H]DPR binding increased by 25% from E40 to P28 in cerebral cortex while in cerebellum hardly any binding could be detected before E50 and it afterwards increased by more than 250% until P28. Scatchard analysis demonstrated that [3H]NBI labeled approximately as many sites as [3H]DPR in cerebral cortex at E44 while at P28 [3H]DPR labeled more than double as many sites. Accordingly, NBI was more potent in displacing [3H]DPR binding at E44 than at P28. These findings suggest that part of the [3H]DPR binding sites, i.e. the NBI-insensitive one develops later than [3H]NBI binding sites during ontogeny in guinea pig cerebral cortex and cerebellum.

Topics: Aging; Animals; Cerebellum; Cerebral Cortex; Dipyridamole; Gestational Age; Guinea Pigs; Inosine; Kinetics; Membranes; Receptors, Purinergic; Thioinosine

Adenosine uptake sites have been characterized and localized in guinea pig and pointer dog brain by in vitro autoradiography, using as probes 3H-nitrobenzylthioinosine (3H-NBI) and the recently available 3H-dipyridamole (3H-DPR). In guinea pig brain and, to a lesser extent, in pointer dog brain, 3H-DPR was found to label more high-affinity binding sites than 3H-NBI and NBI inhibited 3H-DPR binding having pseudo-Hill coefficients smaller than 0.5. 3H-DPR and 3H-NBI labeled brain structures with different intensities in guinea pig brain, as was revealed by quantitative analysis. While the intensity of 3H-DPR binding varied about 4-fold in neuron-containing structures, 8-fold differences were observed for 3H-NBI binding with phylo- and ontogenetically older brain areas such as hypothalamus and various brain stem structures showing relatively higher densities. These findings raise the interesting possibility of adenosine uptake site heterogeneity (NBI-sensitive and insensitive) in guinea pig brain, complementing the well-established adenosine receptor heterogeneity (A1 and A2). As adenosine's neurodepressant effects are believed to be mainly mediated by adenosine A1-receptors, these were localized using 3H-cyclohexyl-adenosine (3H-CHA) as a ligand probe. In guinea pig brain, the highest receptor densities were seen in hippocampus and claustrum, while only relatively low levels were found in hypothalamus and various brain stem structures. As was previously described for rat brain, major discrepancies in the regional distribution of adenosine A1-receptors and adenosine uptake sites, as labeled by 3H-NBI, were seen in guinea pig brain. These discrepancies were only partly abolished (e.g., in cerebellum) by the use of 3H-DPR as an additional ligand probe for adenosine uptake sites. Adenosine uptake site heterogeneity, therefore, probably does not explain the previously described discrepancies in rodent brain between the distribution of adenosine A1-receptors and uptake sites. Because of the low affinity of 3H-DPR for adenosine uptake sites in rat and mouse brain, these species could not be investigated with this new radioligand probe. In pointer dog brain, as compared to guinea pig brain, a more similar distribution pattern of adenosine A1-receptors and adenosine uptake sites in the brain structures investigated (e.g., hippocampus) could be observed. The situation in guinea pig brain can, therefore, not be universalized to other species.(ABSTRACT TRUNCATED AT 400

Topics: Adenosine; Animals; Binding Sites; Brain; Dipyridamole; Dogs; Guinea Pigs; In Vitro Techniques; Male; Receptors, Purinergic; Thioinosine; Tissue Distribution

1. Uptake and metabolism of adenosine were investigated from both maternal (M) and fetal (F) circulations of the isolated, dually perfused guinea-pig placenta by using a single-circulation paired-tracer [( 14C]sucrose as extracellular reference, and [3H]adenosine) dilution technique. 2. Maximal [3H]adenosine uptakes (percentage of dose) from adenosine-free perfusates were 75 +/- 1 and 87 +/- 2% (mean +/- S.E. of mean) at maternal and fetal blood-tissue interfaces respectively. Rapid backflux (percentage of influx) of tritium (labelled adenosine and/or adenosine derivatives) from the placental tissue into the ipsilateral circulation was higher at the fetal (24 +/- 2%) than at the maternal side (11 +/- 2%). 3. Tritium uptakes were reduced to 50 +/- 4 (M) and 60 +/- 6% (F) when the perfusion medium contained 100 microM-unlabelled adenosine; backflux was highly stimulated (44% M and 84% F). Neither uptake nor backflux were affected by inosine, uridine, adenine or hypoxanthine present in the perfusion medium (1 mM). 4. Tissue sequestration of tritium (5-6 min) was approximately 60% of the injected dose when perfusates were adenosine-free and 20% or less in the presence of 100 microM-adenosine. 5. Cellular uptake of [3H]adenosine at both sides of the placenta was markedly reduced by the nucleoside transport inhibitors dipyridamole (DIP, 10 microM) and nitrobenzylthioinosine (NBMPR, 5 microM). 6. Thin-layer chromatographic separation of [3H]inosine, [3H]hypoxanthine and [3H]phosphorylated derivatives in venous effluents following a bolus arterial injection of [3H]adenosine showed a greater fraction of metabolites at the fetal side (about 0.75) than at the maternal side (about 0.50). The percentage of [3H]inosine increased when perfusates contained 100 microM-adenosine and the effect was more marked in the fetal circulation. In the presence of DIP and NBMPR the fractional recovery of 3H-labelled metabolites was greatly reduced. 7. During steady-state perfusion of [3H]adenosine (100 microM) a maintained (5-60 min) tritium uptake of about 55% was observed and all the effluent activity was 3H-labelled metabolites [( 3H]adenosine was only 2.8 +/- 0.2%). Under these conditions high-performance liquid chromatography (HPLC) showed that effluents contained xanthine and urate at 16 +/- 1 and 23 +/- 2 microM respectively. 8. Transplacental transfer (6 min) of tritiated compounds (of which only 10-20% was [3H]adenosine) was often less than that of the extracellular marker [

Topics: Adenosine; Animals; Biological Transport; Dipyridamole; Female; Guinea Pigs; Hypoxanthine; Hypoxanthines; Inosine; Maternal-Fetal Exchange; Placenta; Pregnancy; Thioinosine; Tritium; Uric Acid

The effects of adenosine, of its non-metabolizable analogs, and of compounds related to its metabolism, were investigated in the photosensitive chicken pineal, maintained in static culture or in superfusion. Stimulation or inhibition of melatonin production was obtained, depending on the experimental conditions tested. Endogenous adenosine is involved in the regulation of the melatonin output. The effects of the nucleoside might depend on the balance between its intra- and extracellular pools; (re)uptake mechanisms are most probably involved. It is suggested that cell surface receptors mediate adenosine effects, but intracellular actions (P-site, transmethylation pathways) are not excluded. This investigation is a breakthrough in the field of pineal physiology which offers new perspectives in the study of the control of melatonin production.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Deaminase; Animals; Chickens; Colforsin; Deoxyadenosines; Dipyridamole; Melatonin; Organ Culture Techniques; Phenylisopropyladenosine; Pineal Gland; Thioinosine

In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.

Topics: 3-O-Methylglucose; Adipose Tissue; Animals; Biological Transport; Cytochalasin B; Dipyridamole; Guanosine; In Vitro Techniques; Inosine; Insulin; Kinetics; Male; Methylglucosides; Methylglycosides; Nucleosides; Papaverine; Rats; Rats, Inbred Strains; Thioinosine; Thionucleosides

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.

Topics: Adenosine; Aminoglycosides; Animals; Anti-Bacterial Agents; Cell Membrane Permeability; Erythrocyte Membrane; Erythrocytes; Humans; Plasmodium falciparum; Thioinosine; Tubercidin

Evidence suggests that adenosine modulates neuronal and cerebral vascular functions by interacting with specific receptors on brain cells and blood vessels. Adenosine and other nucleosides are also transported across the blood-brain barrier via a saturable, carrier-mediated mechanism. Using direct ligand binding methods, we studied the two adenosine receptor subtypes, A1 and A2 and the nucleoside transporter moiety in human brain microvessels, pial vessels, choroid plexus, and cerebral cortex membranes. The following specific tritiated ligands were used: cyclohexyladenosine (CHA) for A1 receptors; 5'-N-ethylcarboxamide adenosine (NECA) for A2 receptors; nitrobenzylthioinosine (NBMPR) and dipyridamole (DPY) for nucleoside transporters. We find that cerebral microvessels, pial vessels, and choroid plexus have few, if any, A1 receptors, in contradistinction to cerebral membranes, which have a 10-20-fold higher density of A1 receptor sites. Specific high-affinity NECA binding to A2 receptors in cerebral microvessels, pial vessels, and choroid plexus was saturable and was equivalent to that of cerebral cortical membranes. The Bmax and Kd of the high-affinity NECA binding to vessel preparations were approximately 1.3 pmol/mg protein and approximately 250 nM, respectively, which is similar to our previous findings in the rat and pig. NBMPR and DPY binding were also saturable and were consistent with a single class of high-affinity binding sites. The density of nucleoside transporters was approximately four-fold higher in cerebral microvessels than in cerebral cortex, pial vessels, and choroid plexus. These results suggest that human cerebral microvessels have A2, but not A1, receptors and are particularly enriched with the adenosine transporter moiety.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adult; Blood Proteins; Brain; Carrier Proteins; Cerebral Cortex; Choroid Plexus; Dipyridamole; Female; Humans; Membrane Proteins; Microcirculation; Middle Aged; Nucleoside Transport Proteins; Pia Mater; Receptors, Purinergic; Thioinosine

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.

Topics: Biological Transport; Cytochalasin B; Erythrocyte Membrane; Erythrocytes; Galactosemias; Glucose; Humans; Monosaccharide Transport Proteins; Nucleosides; Thioinosine; Thymidine; Uridine

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.

Topics: Animals; Biological Transport; Cerebral Cortex; Dilazep; Dipyridamole; Male; Nucleosides; Rats; Rats, Inbred Strains; Synaptosomes; Thioinosine; Uridine

Adenosine (Ado, 10 microM) was metabolized in whole blood within 1 min, primarily to hypoxanthine and ATP. The concentration of Ado, the activities of adenosine deaminase (ADA) and Ado kinase, the Km values for Ado with ADA and Ado kinase, and the substrate inhibition of Ado kinase are factors that govern the Ado metabolism between deamination and phosphorylation. If ADA activity was blocked by 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of ADA, most of the Ado (96%) was incorporated into adenine nucleotides, whereas if Ado kinase activity was blocked with 5-iodotubercidin (10 microM), Ado was mainly (95%) metabolized into hypoxanthine. A high phosphate concentration (25 mM) caused marked increases in the formation of IMP. The nucleoside transport inhibitors dilazep (1 microM), dipyridamole (10 microM) and nitrobenzylthioinosine (NBMPR, 1 microM) strongly blocked cellular Ado metabolism. In the presence of nucleoside transport inhibitors, Ado which slowly enters the cell was metabolized principally by Ado kinase rather than ADA. Dilazep, NBMPR and dipyridamole were more effective in blocking Ado uptake and metabolism by erythrocytes suspended in a protein-free medium than by cells suspended in plasma.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Biological Transport; Dilazep; Dipyridamole; Humans; Phosphates; Thioinosine

We used video-microscopic techniques to study responses of rat cremaster muscle arterioles to adenosine (ADO) placed in a bathing solution in an effort to determine 1) the sensitivity of these vessels to local interstitial ADO concentration and 2) the parameters of interstitial adenosine handling. Two vessels, located at different depths (approximately 40 and 115 microns) below the surface of the tissue, were studied simultaneously. Invariably, a higher bath ADO concentration was required to induce vasodilation in the deeper vessel; the concentration required for 50% dilation response (EC50) for ADO dilation increased at an average of 1.8 +/- 0.2 log10 U/100 microns of depth into the tissue. This result was shown to be due to a standing gradient in interstitial ADO concentration. By extrapolating results to the tissue surface, we estimate that the EC50 for arteriolar dilation to local interstitial ADO is approximately 0.1 microM. The steepness of the tissue ADO gradient indicates that the rate constant for interstitial ADO loss is near 0.24/s. The gradients for nonmetabolizable adenosine analogues were less than 1/10th as steep as that for ADO itself. Qualitatively similar results were obtained from experiments on hamster cremaster muscle preparations.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Arterioles; Biological Assay; Cricetinae; Diffusion; Dipyridamole; Extracellular Space; Kinetics; Methods; Muscles; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Thioinosine; Vasodilation

Differences in sensitivity of uridine transport in erythrocytes and cultured cells to inhibition by dipyridamole, dilazep and lidoflazine were largely species-specific; uridine transport in human cells, and probably in pig and rabbit cells, was 2-3- and 10-times more sensitive to inhibition by dipyridamole (IC50 approx. 50 nM) and about 10- and 20-times more sensitive to dilazep inhibition (IC50 approx. 5 nM) than transport in mouse and rat cells, respectively. Uridine transport in human erythrocytes and HeLa cells was strongly inhibited by lidoflazine (IC50 10-140 nM), whereas that in both mouse and rat cells was highly resistant (IC50 greater than 10 microM). Superimposed on species-specific differences were some cell type specific differences in sensitivity of nucleoside transport to these inhibitors. Uridine transport in Walker 256 rat carcinoma cells was more resistant to dipyridamole and dilazep than that of other rat cells. Transport in human Hep-2 cells was more resistant to lidoflazine (IC50 2000 nM) than that of human erythrocytes and HeLa cells, whereas it showed similar sensitivity to dilazep and dipyridamole. Uridine transport in Chinese hamster cells was also more resistant to dilazep than that of baby hamster kidney cells. In addition HeLa cells and clones thereof expressed uridine transporters (about 50% each) with difference of about 1000-fold in sensitivity to inhibition by dilazep (IC50 approx. 5 nM and 5 microM, respectively).

Topics: Animals; Azepines; Biological Transport; Carrier Proteins; Cells, Cultured; Dilazep; Dipyridamole; Erythrocytes; Humans; Inosine; Lidoflazine; Membrane Proteins; Nucleoside Transport Proteins; Piperazines; Species Specificity; Thioinosine; Uridine

Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of [3H]NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content of the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane.

Topics: Animals; Binding Sites; Biological Transport; Erythrocyte Membrane; In Vitro Techniques; Inosine; Nucleosides; Saponins; Swine; Thioinosine; Tritium

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.

Topics: Adrenal Medulla; Affinity Labels; Animals; Cattle; Cell Fractionation; Cell Membrane; Cells, Cultured; Chromaffin Granules; Cytosol; Dipyridamole; Kinetics; Receptors, Purinergic; Subcellular Fractions; Thioinosine

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Cerebellum; Deoxyadenosines; In Vitro Techniques; Jejunum; Mice; Muscle Contraction; Neuroblastoma; Rabbits; Rats; Receptors, Purinergic; Thioinosine; Thionucleosides

Mechanically dissociated brain cells from adult rats were used to study biochemically and pharmacologically their capacity to accumulate rapidly [3H]adenosine. The assay, which used an inhibitor-stop method to prevent further uptake into cells, was characterized with respect to protein and optimal substrate concentrations, and incubation times that ranged from 5 to 180 s. The accumulation of [3H]adenosine using 15-s incubation periods, conditions under which less than 10% of accumulated [3H]adenosine was metabolized, was best described kinetically by a two-component system with Km and Vmax values for the high-affinity component of 0.8 microM and 6.2 pmol/mg protein/15 s and for the low-affinity component 259 microM and 2,217 pmol/mg protein/15 s, respectively. The potencies with which nucleosides, adenosine deaminase resistant adenosine receptor agonists, and nucleoside uptake inhibitors competed for these uptake components were determined. Of the nucleosides examined, adenosine was the "preferred" substrate for the uptake site. The Ki value of adenosine for the high-affinity component was 10.7 microM. Inosine and uridine competed for a single lower affinity uptake system: Ki values were 142 and 696 microM, respectively. Nucleoside uptake inhibitors--nitrobenzylthioinosine, dipyridamole, and dilazep--were the most potent inhibitors of [3H]adenosine accumulation tested: the Ki values for the high-affinity system were 0.11, 1.3, and 570 nM, respectively. The adenosine analogs S-phenylisopropyladenosine, R-phenylisopropyladenosine, and cyclohexyladenosine inhibited the high-affinity component with Ki values of 2.3, 9.3, and 14.5 microM, respectively. N-Ethylcarboxamidoadenosine competed for a single lower affinity uptake system: Ki, 292 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine Monophosphate; Animals; Binding, Competitive; Brain; Dilazep; Dipyridamole; Inosine; Kinetics; Male; Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Thioinosine; Uridine

Purine nucleotides, nucleosides, nucleobases, dinucleotides and nucleosides derivatives from acid-extracted rat liver and diaphragm were separated and quantitated by reversed-phase ion-pair high-performance liquid chromatography with a mobile phase composed of 90 mM potassium phosphate, 15 mM tetrabutylammonium hydroxide and a 1-30% methanol gradient. During 5 min of ischemia, adenine and guanine nucleotides decreased along with significant declines in NAD and increases in adenosine, inosine, hypoxanthine, xanthine, NADP and adenylosuccinate. Nitrobenzylthioinosine by gavage (5 mg/kg per day for five days) increased adenosine levels but without any alteration in nucleobase levels. Adenosine was shuttled to every available intracellular reservoir which included in declining order of magnitude GDP greater than adenosylhomocysteine greater than adenosine greater than ADP greater than AMP greater than IMP = XMP = GMP.

Topics: Adenine Nucleotides; Adenosine; Animals; Chromatography, High Pressure Liquid; Diaphragm; Inosine; Ischemia; Liver; Male; Rats; Rats, Inbred F344; Respiratory Muscles; Solvents; Spectrophotometry, Ultraviolet; Thioinosine

Extracellular and intracellular recordings from CA1 pyramidal neurones of rats in vitro were used to study the effects of endogenous and exogenously applied adenosine. The adenosine receptor antagonist, caffeine, enhanced the intracellular recorded e.p.s.p.-i.p.s.p. sequence evoked by stimulation of the stratum radiatum which is antagonized by exogenous adenosine. The late, potassium dependent i.p.s.p. was not antagonized. The adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI), mimicked the effects of exogenously applied adenosine. The effects of NBTI and of exogenously applied adenosine were antagonized by caffeine in the same manner. Exposure to adenosine deaminase enhanced the evoked field e.p.s.p. During this enhancement caffeines effects were significantly reduced. In low calcium high magnesium medium which abolishes synaptic activity, adenosine deaminase increased, NBTI decreased cell firing. We conclude that endogenous adenosine, release by a calcium independent mechanism, can exert an inhibitory tone on CA1 neurones in vitro. This is consistent with a role for adenosine as a mediator of negative feedback between the metabolic state and electrophysiological activity of nervous tissue.

Topics: Adenosine; Adenosine Deaminase; Animals; Caffeine; Electrophysiology; Evoked Potentials; Hippocampus; In Vitro Techniques; Male; Neurons; Rats; Rats, Inbred Strains; Thioinosine

The purine ribonucleoside inosine is known to be metabolized in islet cells (its ribose moiety feeds into the pentose-phosphate cycle) and stimulate insulin release, but the mechanisms of this stimulation have not been established. These were investigated with mouse islets. In the absence of glucose, 5 mM inosine decreased 86Rb+ efflux from islet cells, depolarized the B-cell membrane, induced electrical activity (slow waves of membrane potential with bursts of spikes on the plateau), accelerated 45Ca2+ efflux and stimulated insulin release with the same efficiency as 10 mM glucose. Raising the concentration of inosine to 20 mM only had a slight further effect and, in particular, failed to cause persistent depolarization of the B-cell membrane. The electrical activity triggered by inosine was blocked by cobalt, and the stimulation of 45Ca2+ efflux and insulin release was abolished in a Ca2+-free medium. The effects of 10 mM glucose on electrical activity, 45Ca2+ efflux and insulin release were augmented by as little as 0.5 mM inosine. All effects of inosine were abolished by an inhibitor of nucleoside transport (nitrobenzylthioguanosine) and markedly impaired by inhibitors of nucleoside phosphorylase (formycin B) or of glycolysis (iodoacetate). In conclusion, inosine metabolism in B-cells induces insulin release by triggering the same sequence of events as glucose metabolism: a decrease of K+ permeability of the B-cell membrane, leading to depolarization and activation of voltage-dependent Ca channels.

Topics: Animals; Electrophysiology; Female; Glucose; Hypoxanthines; Inosine; Insulin; Iodoacetates; Ion Channels; Islets of Langerhans; Mice; Ribose; Thioinosine

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+-glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+-glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.

Topics: Animals; Biological Transport, Active; Carrier Proteins; Erythrocyte Membrane; Glycine; Nucleosides; Ouabain; Protease Inhibitors; Sheep; Sodium; Sodium-Potassium-Exchanging ATPase; Thioinosine

The ontogenesis of adenosine transport sites as labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined using radioligand binding and membrane preparations from whole brain and 4 brain regions of rats between the postnatal ages of one day through to adulthood. In whole brain, cerebral cortex and cerebellum, [3H]NBI binding was two-fold higher in 6-day-old than in 50-day-old rats. In contrast, [3H]NBI binding was higher in adults than in one-day-old rats by 4-fold in hypothalamus and 8-fold in superior colliculus. In cortex and hypothalamus, the levels of [3H]NBI binding in newborn and adult rats were reflected by changes in Bmax and not Kd values. As a measure of the utility of [3H]NBI as a probe for identifying functional adenosine transport sites, we examined [3H]NBI binding to and [3H]adenosine accumulation by intact brain cells prepared from adult and newborn rats. For [3H]NBI binding to brain cells from adult rats, the values of Kd were 0.092 nM and of Bmax were 274 fmol/mg protein. For newborns, slightly higher Kd and Bmax values were observed; 0.2 nM and 395 fmol/mg protein, respectively. [3H]Adenosine accumulation was higher in brain cells from one-day-old than from adult rat brains. Kinetically this uptake was best described by a two-component model: the Vmax values for the high- and low-affinity uptake, and the Km value for the high-affinity component in one-day-old rats were greater than in adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Aging; Animals; Binding Sites; Brain; Cerebellum; Cerebral Cortex; Hypothalamus; Inosine; Male; Rats; Rats, Inbred Strains; Superior Colliculi; Thioinosine

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.

Topics: Animals; Antibodies, Monoclonal; Biological Transport; Chemical Precipitation; Cytochalasin B; Erythrocyte Membrane; Erythrocytes; Immunoelectrophoresis; Nucleosides; Peptide Fragments; Peptides; Thioinosine

The intracellular half-life for retention of the active triphosphate metabolite 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) of 1-beta-D-arabinofuranosylcytosine was measured in vitro in blast cells from patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, and T-cell lymphoblastic lymphoma. araCTP accumulation from 1 microM 1-beta-D-arabinofuranosylcytosine in leukemic blast cells was closely correlated with the nucleoside transport capacity as measured by equilibrium binding of [3H]nitrobenzylthioinosine. The half-life of araCTP retention was related to araCTP accumulation only when the level of araCTP was expressed as a percentage of total intracellular 1-beta-D-arabinofuranosylcytosine metabolites. Accumulation of 1-beta-D-arabinofuranosyluracil 5'-monophosphate was inversely related to the half-life of araCTP retention and directly related to dCMP deaminase activity in cell free extracts. No conversion of 1-beta-D-arabinofuranosyluracil to 1-beta-D-arabinofuranosyluracil 5'-monophosphate was detectable in intact cells. The end product of araCTP degradation was 1-beta-D-arabinofuranosyluracil and it is proposed that conversion of 1-beta-D-arabinofuranosylcytosine 5'-monophosphate to 1-beta-D-arabinofuranosyluracil 5'-monophosphate is a step in the degradative pathway of araCTP. However, it is the cells' nucleoside transport capacity which primarily determines the level of intracellular araCTP accumulation.

Topics: Arabinofuranosylcytosine Triphosphate; Arabinonucleotides; Cytarabine; DCMP Deaminase; Guanosine; Half-Life; Humans; Kinetics; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Lymphocytes; Phosphorylation; Thioinosine; Thionucleosides

The uptake of various nucleosides by S49 mouse T-lymphoma cells and that by a single-step nucleoside transport-defective mutant thereof (AE1) were compared. Residual nucleoside entry into AE1 cells occurred via two routes, nonmediated permeation and saturable, non-concentrative transport with broad substrate specificity and a Michaelis-Menten constant approximating that for thymidine transport in wild-type cells. However, in contrast to nucleoside transport in wild-type cells, residual nucleoside transport in AE1 cells was resistant to inhibition by nitrobenzylthioinosine. In its properties the latter resembled nitrobenzylthioinosine-resistant nucleoside transport observed in other types of mammalian cells. It amounted to less than 1% of the total nucleoside transport activity of wild-type S49 cells. The results indicate that nitrobenzylthioinosine-resistant and -sensitive nucleoside transports are genetically distinguishable. In wild-type cells, the salvage of thymidine, when present at concentrations higher than 1 to 10 microM, was limited by phosphorylation, because of the saturation of thymidine kinase. In AE1 cells, entry into the cells mainly limited thymidine salvage, but at high thymidine concentrations the combined entry via residual transport and nonmediated permeation was sufficiently rapid to support intracellular thymidine phosphorylation at rates comparable to those observed in wild-type cells.

Topics: Animals; Carrier Proteins; Cell Line; Drug Resistance; Inosine; Kinetics; Lymphoma; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; T-Lymphocytes; Thioinosine; Thymidine; Thymine Nucleotides

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.

Topics: Animals; Biological Transport; Calcium Channel Blockers; Cell Line; Erythrocytes; HeLa Cells; Humans; Leukemia P388; Lidoflazine; Liver Neoplasms, Experimental; Mice; Nucleosides; Rats; Species Specificity; Thioinosine; Uridine

A previous report from this laboratory indicated that the concentration of free uridine (Urd) in many normal murine tissues greatly exceeds that in plasma. We now report that Urd uptake by isolated murine splenocytes is concentrative, and that the rate of uptake from medium containing 10 to 500 microM [3H]Urd conforms to a process that is saturable with a Km of 38.0 +/- 4.1 (SE) microM and Vmax of 2.70 +/- 0.27 pmol/s/microliter cell water. Other ribosyl and deoxyribosyl pyrimidine nucleosides or their analogues were not concentrated by splenocytes; however, ribosyl and deoxyribosyl purine nucleosides and, to a lesser extent, deoxyuridine did inhibit Urd uptake. In this system Urd uptake was not inhibited by 1 microM nitrobenzylthioinosine or 10 microM dipyridamole but was significantly inhibited by 5 mM NaN3 or 250 microM KCN. Transport of Urd involves Na+ cotransport as evidenced by complete inhibition when Na+ is replaced by Li+ in the incubation medium, and it is also inhibited by 3 mM ouabain. Active Urd transport coexists with the nonspecific, carrier mediated, facilitated diffusion of nucleosides as demonstrated by the inhibition of Urd efflux and thymidine influx in splenocytes by nitrobenzylthioinosine and dipyridamole. Under identical conditions, Urd entry into L1210 leukemia cells was nonconcentrative and non-Na+ dependent but inhibited by nitrobenzylthionosine. That nucleosides enter most cultured neoplastic cell lines by facilitated diffusion and not the active transport mechanism for Urd confirms earlier findings and may represent an exploitable target for chemotherapy.

Topics: Animals; Biological Transport, Active; Kinetics; Leukemia L1210; Mice; Mice, Inbred C57BL; Sodium; Spleen; Substrate Specificity; Thioinosine; Thymidine; Uridine

The affinities of adenosine and 2-chloroadenosine for the nucleoside transport system of guinea pig myocytes were evaluated indirectly by studying the inhibition of the binding of [3H]nitrobenzylthioinosine and directly by measuring the influx of [3H]radiolabeled substrates. Maximal transport velocities of the two nucleosides were also obtained. [3H]Nitrobenzylthioinosine bound to a single class of high-affinity sites (KD of 0.8 nM) which possessed a maximal binding capacity (Bmax) of 870,000 sites/cell. Adenosine, 2-chloroadenosine or the nucleoside transport inhibitor, dipyridamole, competitively inhibited the site-specific binding of [3H]nitrobenzylthioinosine with Ki values of 318 microM, 22 microM and 75 nM respectively. Both [3H]adenosine and [3H]2-chloroadenosine entered myocytes in a saturable and inhibitible manner. Observed transport kinetic constants (Km and Vmax) were 146 microM and 24.2 pmoles/10(6) cells/sec, respectively, for adenosine and 36 microM and 11.7 pmoles/10(6) cells/sec, respectively for 2-chloroadenosine. Affinities of adenosine, 2-chloroadenosine, nitrobenzylthioinosine and dipyridamole for the nucleoside transport system derived from binding and influx methodologies were equivalent which confirms that [3H]nitrobenzylthioinosine binding sites are closely associated with the nucleoside transporter.

Topics: 2-Chloroadenosine; Adenosine; Animals; Binding Sites; Biological Transport; Guinea Pigs; In Vitro Techniques; Kinetics; Male; Myocardium; Thioinosine; Tritium

Vesicles are released during the in vitro culture of sheep reticulocytes which can be harvested by centrifugation at 100,000 X g for 90 min. These vesicles contain a number of activities, characteristic of the reticulocyte plasma membrane, which are known to diminish or disappear upon reticulocyte maturation. The activities include acetylcholinesterase, cytochalasin B binding (glucose transporter) nucleoside binding (i.e. nucleoside transporter), Na+-independent amino acid transport, and the transferrin receptor. Enzymes of cytosolic origin are not detectable or are present at low activity in the vesicles. Cultures of whole blood, mature red cells, or white cells do not yield comparable levels of these activities, supporting the conclusion that the activities arise from the reticulocytes. In addition, the lipid composition of the vesicles shows the high sphingomyelin content characteristic of sheep red cell plasma membranes, but not white cell or platelet membranes, also consistent with the conclusion that the vesicles are of reticulocyte origin. It is suggested that vesicle externalization may be a mechanism for shedding of specific membrane functions which are known to diminish during maturation of reticulocytes to erythrocytes.

Topics: Acetylcholinesterase; Amino Acids; Animals; Carrier Proteins; Cell Membrane; Cells, Cultured; Cytochalasin B; Glyceraldehyde-3-Phosphate Dehydrogenases; Leucine; Membrane Proteins; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Receptors, Transferrin; Reticulocytes; Sheep; Sodium; Thioinosine

In normal mouse erythrocytes, nucleoside permeation was almost completely blocked in the presence of binding site-saturating concentrations of nitrobenzylthioinosine, whereas permeation in erythrocytes infected with the malarial parasite, Plasmodium yoelii, was substantial under these conditions, suggesting the presence of a permeation mechanism of low sensitivity to nitrobenzylthioinosine in the infected cells. Binding sites for nitrobenzylthioinosine were more numerous on infected erythrocytes than on uninfected cells. When mice infected with P. yoelii were treated with combinations of tubercidin and nitrobenzylthioinosine 5'-monophosphate, progression of parasitemia was delayed and survival times were increased.

Topics: Adenosine; Affinity Labels; Animals; Biological Transport; Erythrocytes; Female; Inosine; Kinetics; Malaria; Mice; Mice, Inbred Strains; Plasmodium; Ribonucleosides; Thioinosine; Tritium; Tubercidin

The ontogenesis of rat forebrain adenosine uptake sites labelled by [3H]nitrobenzylthioinosine ([3H]NBI) was determined and compared to that of rat forebrain adenosine receptors labelled by N6-cyclohexyl[3H]adenosine ([3H]-CHA). [3H]NBI binding is highly invariant with similar levels of [3H]NBI binding sites from embryonic day 19 to day 30 postpartum. Scatchard and Hill analyses reveal the binding of [3H]NBI in 6-day-old tissue to be indistinguishable from such binding in 30-day-old tissue. In contrast, [3H]-CHA binding is highly variant. [3H]CHA binding develops slowly but steadily from about embryonic day 19, with adult binding levels being achieved at around 25 days postpartum. The ontogenetic profile of [3H]CHA appears to coincide with synaptogenesis whereas that of [3H]NBI does not.

Topics: Adenosine; Affinity Labels; Animals; Binding Sites; Brain; Female; Kinetics; Pregnancy; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thioinosine

Topics: Animals; Autoradiography; Binding Sites; Guinea Pigs; In Vitro Techniques; Inosine; Kidney; Rats; Thioinosine

Rapid kinetic techniques were used to measure the transport of uridine in pig erythrocytes in zero-trans entry and exit and equilibrium exchange protocols. The kinetic parameters were computed by fitting appropriate integrated rate equations to the time-courses of transmembrane equilibration of radiolabeled uridine. Transport of uridine conformed to the simple carrier model with directional symmetry, but differential mobility of substrate-loaded and empty carrier. At 5 degrees C, the carrier moved about 30-times faster when loaded than when empty. Uridine transport was inhibited in a concentration-dependent manner by nitrobenzylthioinosine and dipyridamole and the inhibition correlated with the binding of the inhibitors to high-affinity binding sites on the cells (Kd about 1 and 10 nM, respectively). Thus, in its kinetic properties, differential mobility when empty and loaded, and sensitivity to inhibition by nitrobenzylthioinosine and dipyridamole, the transporter of pig erythrocytes is very similar to that of human erythrocytes. Also, the total number of high-affinity binding sites for nitrobenzylthioinosine and dipyridamole/cell were similar for the two cell types and the [3H]nitrobenzylthioinosine-labeled carrier of pig erythrocytes, just as that of human red cells, was mainly recovered in the band 4.5 protein fraction of Triton X-100-solubilized membranes. However, sodium dodecylsulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled band 4.5 membrane proteins indicated a slightly higher molecular weight for the transporter from pig than human erythrocytes. We have also confirmed the lack of functional sugar transport in erythrocytes from adult pigs by measuring the uptake of various radiolabeled sugars. But in spite of the lack of functional sugar transport we recovered as much band 4.5 protein from pig as from human erythrocyte membranes.

Topics: Affinity Labels; Animals; Biological Transport; Carbohydrates; Carrier Proteins; Dipyridamole; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Inosine; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Photochemistry; Swine; Thioinosine; Uridine

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.

Topics: Affinity Labels; Animals; Carrier Proteins; Chromatography, DEAE-Cellulose; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Glucosides; Guanosine; Membrane Proteins; Molecular Weight; Nucleoside Transport Proteins; Photochemistry; Solubility; Swine; Thioinosine; Thionucleosides; Uridine

Guanosine transport and metabolism were examined in adult rat cardiac myocytes. Myocytes transported guanosine via saturable [Km = 18 microM, maximum velocity (Vmax) = 3.61 pmol.mg-1.s-1] and nonsaturable (rate constant = 1.47 X 10(-2] processes. The saturable process was inhibited by nitrobenzyl-thioinosine, inosine [inhibition constant (Ki) = 180 microM], and adenosine (Ki = 112 microM). Extracellular guanosine taken up by myocytes was slowly phosphorylated to guanine nucleotides. The majority of guanosine (98%) existed as free intracellular guanosine after 60 s. Countertransport of nucleosides could not be demonstrated in these cells at physiological concentrations in the presence of up to a 10-fold gradient of nucleoside. These studies indicate that adult rat cardiac myocytes can be used to assess myocardial guanosine transport separate from its metabolism. Comparable inhibition of guanosine and adenosine transport by each other and by inosine support the hypothesis that guanosine and adenosine are transported by a common carrier.

Topics: Adenosine; Animals; Binding, Competitive; Biological Transport; Guanine Nucleotides; Guanosine; Inosine; Kinetics; Male; Myocardium; Phosphorylation; Rats; Rats, Inbred Strains; Thioinosine

Based on the importance of myocardial adenosine and adenine nucleotide metabolism, the adenosine salvage pathway in ventricular myocytes was studied. Accurate estimates of transport rates, separate from metabolic flux, were determined. Adenosine influx was constant between 3 and 60 s. Adenosine metabolism maintained intracellular adenosine concentrations less than 10% of the extracellular adenosine concentrations and thus unidirectional influx could be measured. Myocytes transported adenosine via saturable [Michaelis constant = 6.2 +/- 2.1 microM and maximal velocity (Vmax) = 9.58 +/- 0.98 X 10(-1) pmol X mg protein-1 X s-1] and nonsaturable (rate constant = 1.8 X 10(-3)/s) processes. A minimum estimate of the Vmax of myocytic adenosine kinase (2 pmol X mg protein-1 X s-1) indicated the saturable component of adenosine influx was independent of adenosine kinase activity. Saturable transport was inhibited by nitrobenzylthioinosine and verapamil (inhibitor constant = 17 +/- 5 microM). Extracellular adenosine taken up by myocytes was rapidly phosphorylated to adenine nucleotides. Not all extracellular adenosine, though, was phosphorylated on entering myocytes, since free, as opposed to protein-bound, intracellular adenosine was detected after digitonin extraction of cells in the presence of 1 mM ethylene-diaminetetraacetic acid.

Topics: Adenosine; Affinity Labels; Animals; Heart; In Vitro Techniques; Kinetics; Male; Myocardium; Rats; Rats, Inbred Strains; Receptors, Purinergic; Temperature; Thermodynamics; Thioinosine; Verapamil

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28 degrees C is about 180 microM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.

Topics: Adenine; Adenosine Triphosphate; Aedes; Animals; Biological Transport; Carrier Proteins; Hypoxanthine; Hypoxanthines; Kinetics; Membrane Proteins; Mutation; Nucleoside Transport Proteins; Nucleosides; Thioinosine; Thymidine; Uracil; Uridine

The binding of [3H]dipyridamole ([3H]DPR) to guinea pig brain membranes is described and compared to that of [3H]nitrobenzylthioinosine ([3H]NBI). The binding of [3H]DPR is saturable, reversible, and specific with pharmacologic evidence indicating that this ligand is binding to the adenosine uptake site. Compared to [3H]NBI the binding of [3H]DPR is of higher capacity (Bmax = 208 +/- 16 fmol/mg protein for [3H]NBI and 530 +/- 40 fmol/mg protein for [3H]DPR) and lower affinity (KD = 0.35 +/- 0.02 nM for [3H]NBI and 7.6 +/- 0.7 nM for [3H]DPR). The adenosine uptake inhibitors are the most potent inhibitors of binding (Ki of 10(-8)-10(-7) M) whereas adenosine receptor ligands such as cyclohexyladenosine, 2-chloroadenosine, and various methylxanthines are several orders of magnitude less potent (Ki 10(-5)-10(-2). The inhibition of [3H]DPR binding by NBI is biphasic, with only 40% of binding being susceptible to inhibition of NBI concentrations less than 10(-5) M. The tissue distribution of [3H]DPR binding parallels that of [3H]NBI although in most cases significantly more sites are observed with [3H]DPR. Calcium channel blocking agents such as nifedipine, nimodipine, and verapamil are also inhibitors of [3H]DPR binding with potencies in the micromolar range. The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI-sensitive.

Topics: Adenosine; Animals; Brain; Calcium Channel Blockers; Cell Membrane; Cerebral Cortex; Dipyridamole; Guinea Pigs; Kinetics; Male; Thioinosine

The localization of adenosine receptors labelled with [3H]cyclohexyladenosine ([3H]CHA) and adenosine transport sites labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined in striatum and superior colliculus (SC) using radioligand binding and lesioning methods. Striatal kainic acid lesions significantly reduced the number (Bmax) of a single class of high affinity binding sites for [3H]CHA by 50% and that for [3H]NBI by 15% without altering Kd values for either ligand. In SC, enucleations significantly reduced both high and low affinity [3H]CHA binding sites by about 60% while levels of [3H]NBI binding were unaffected. Thus, adenosine receptors are present on striatal interneurons and retinal projections to the SC and some [3H]NBI binding sites are located on striatal interneurons.

Topics: Adenosine; Animals; Binding Sites; Corpus Striatum; Inosine; Kainic Acid; Male; Ophthalmologic Surgical Procedures; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Superior Colliculi; Thioinosine

Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Cytopathogenic Effect, Viral; Deltaretrovirus; Deoxycytidine; Deoxycytidine Kinase; Female; Goats; Humans; Leukemia P388; Mice; Structure-Activity Relationship; Thioinosine; Zalcitabine

8-Azidoadenosine was employed as a possible covalent probe of the erythrocyte nucleoside transporter. 8-Azidoadenosine was shown to enter human erythrocytes by a saturable mechanism (apparent Km for influx 80 microM) that was inhibited by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, and competitively inhibit uridine influx and NBMPR binding. Irradiation with UV light of human erythrocyte membranes or a partially purified preparation of the nucleoside transporter in the presence of [3H]8-azidoadenosine and dithiothreitol (as a free radical scavenger) resulted in selective covalent incorporation into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr 66,000-45,000). Covalent labeling of band 4.5 was inhibited by adenosine, uridine, and inosine, but NBMPR had no effect. Surprisingly, D-glucose and cytochalasin B, but not L-glucose and cytochalasin E, blocked covalent attachment of the ligand. No incorporation of radioactivity into membranes from rabbit and pig erythrocytes was observed, cells which transport nucleosides rapidly, but have little or no functional glucose carrier. Limited treatment with trypsin of unsealed human erythrocyte membranes photolabeled with [3H]8-azidoadenosine yielded a single radioactive fragment of Mr 19,000, a pattern identical to that obtained with [3H]cytochalasin B-labeled membranes. These results suggest that, despite 8-azidoadenosine being a permeant for the nucleoside transporter, under photoactivation 8-azidoadenosine preferentially labeled the glucose carrier.

Topics: Adenosine; Affinity Labels; Azides; Blood Proteins; Coformycin; Cytochalasin B; Dose-Response Relationship, Drug; Erythrocytes; Humans; Kinetics; Membrane Proteins; Molecular Weight; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Pentostatin; Phenylisopropyladenosine; Phloretin; Photochemistry; Receptors, Cell Surface; Receptors, Purinergic; Thioinosine; Uridine

We have examined adenosine (ADO) production and transport in a preparation of isolated adult cardiocytes which attach to and form a monolayer on culture dishes. This preparation contains 85% viable cells which are greater than 50% rod shaped and maintain an ATP/ADP ratio of nine. Incubation under control conditions for 15 mins results in a net release of 240 +/- 47 pmol ADO/mg protein (final adenosine concentration in the medium = 47 +/- 9 nM). Both 0.1 mM dinitrophenol (DNP) and 10 mM iodoacetate (IAA) cause a significant increase in ADO (DNP = 1763 +/- 147 and IAA = 612 +/- 90 pmol/mg). Both 20 microM nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside carrier, and 0.1 mM alpha,beta-methylene adenosine diphosphate (AOPCP), an inhibitor of 5'-nucleotidase activity, attenuate DNP-stimulated ADO release (NBMPR by 62% and ADOCP by 76%). The results are consistent with the hypothesis that under the conditions of our experiments, adenosine is formed by a 5'-nucleotidase in association with transport across the cell membrane, perhaps by an enzyme-carrier complex. In addition, we have examined the effect of 0.1 mM dipyridamole on the extracellular appearance of adenosine in this preparation and found that it causes a significant increase in the amount of adenosine released. These results are consistent with the hypothesis that dipyridamole inhibits adenosine's uptake more than its release in cardiac myocytes.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Biological Transport, Active; Dinitrophenols; Dipyridamole; Heart; In Vitro Techniques; Iodoacetates; Iodoacetic Acid; Myocardium; Nucleotidases; Rats; Rats, Inbred Strains; Thioinosine

The loss of the catabolic products of adenosine triphosphate in the form of purine nucleosides and oxypurines during ischemia and subsequent reperfusion may limit adenine nucleotide regeneration. This study compared the effects of infusion of inhibitors of the major reactions involved in the degradation of adenosine triphosphate to inosine on the postischemic recovery of high energy phosphate and myocardial function. Inhibitors of adenylate kinase, 5'nucleotidase, adenosine translocase and adenosine deaminase were studied. Following 30 minutes of ischemia, only hearts infused with alpha, beta, methylene adenosine diphosphate (5' nucleotidase inhibitor) recovered significantly better ventricular function than control (p less than 0.05), but all hearts had increased adenosine triphosphate regeneration (p less than 0.05). The formation and washout of greater than 30% of the total adenine pool metabolites was not prevented by any drug. Nevertheless all manipulations of adenine metabolism resulted in recruitment of high energy phosphate during preischemic infusion.

Topics: 5'-Nucleotidase; Acute Disease; Adenine; Adenine Nucleotides; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Triphosphate; Adenylate Kinase; Animals; Coronary Circulation; Coronary Disease; Dinucleoside Phosphates; Male; Nucleotidases; Rats; Rats, Inbred Strains; Thioinosine

The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial, p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 microM) by NBMPR was biphasic--35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 microM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparent Km for uridine 50 +/- 18 and 163 +/- 28 microM, respectively). The two transport systems also differed in their sensitivity to pCMBS. NBMPR-insensitive uridine transport was inhibited by pCMBS with an IC50 of approximately 25 microM, while 1 mM pCMBS had little effect on NBMPR-sensitive transport by intact cells. pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by beta-mercaptoethanol, suggesting that the pCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparent Kd 46 +/- 25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25 +/- 6 molecules/site per sec at 22 degrees C. pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude

Topics: 4-Chloromercuribenzenesulfonate; Animals; Biological Transport; Dipyridamole; Erythrocyte Membrane; Erythrocytes; Inosine; Kinetics; Male; Phenylmercury Compounds; Rats; Rats, Inbred Strains; Thioinosine; Uridine

Genetic deficiencies in the nucleoside transport function markedly altered the abilities of cultured mutant S49 T lymphoblasts to transport, incorporate, and salvage exogenous hypoxanthine. The concentrations of exogenous hypoxanthine required to reverse azaserine toxicity and replenish azaserine-depleted nucleoside triphosphate pools in AE1 cells, a nucleoside transport-deficient clone, were about 10-fold higher than those required for wild type cells. In a similar fashion, guanine could reverse mycophenolic acid toxicity in wild type but not in AE1 cells. Surprisingly, a second nucleoside transport-deficient clone, 80-5D2, which had lost 80-90% of its ability to transport nucleosides, required lower hypoxanthine concentrations than the wild type parent to reverse these azaserine-mediated effects. The addition of submicromolar concentrations of either p-nitrobenzylthioinosine or dipyridamole, two potent inhibitors of nucleoside transport, to wild type cells mimicked the phenotype of the AE1 cells with respect to hypoxanthine. AE1 cells or p-nitrobenzylthioinosine-treated wild type cells could only transport hypoxanthine at 10-25% the rate of untreated wild type cells, whereas 80-5D2 cells could transport hypoxanthine more efficiently. Adenine transport was also diminished in AE1 and FURD-80-3-6 cells, but not to sufficiently low levels to interfere with their ability to salvage adenine to overcome azaserine toxicity. These studies on S49 cells altered in their nucleoside transport capacity provide powerful genetic evidence that purine nucleobases share a common transport function with nucleosides in these mammalian T lymphoblasts.

Topics: Adenine; Animals; Antimetabolites, Antineoplastic; Azaserine; Biological Transport; Cell Line; Dipyridamole; Hypoxanthine; Hypoxanthines; Mice; Nucleosides; Purines; T-Lymphocytes; Thioinosine

The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Cell Differentiation; Cell Line; Coformycin; Deoxyadenosines; Dimethylformamide; Humans; Leukemia, Myeloid, Acute; Nucleosides; Pentostatin; Purines; Thioinosine

Rapid kinetic techniques were applied to determine the effect of transport inhibitors on the transport and metabolism of adenosine in human red cells. Dipyridamole inhibited the equilibrium exchange of 500 microM adenosine by deoxycoformycin-treated cells in a similar concentration dependent manner as the equilibrium exchange and zero-trans influx of uridine with 50% inhibition being observed at about 20 nM. Intracellular phosphorylation of adenosine at an extracellular concentration of 5 microM was inhibited only by dipyridamole concentrations greater than or equal to 100 nM, which inhibited transport about 95%. Lower concentrations of dipyridamole actually stimulated adenosine phosphorylation, because the reduced influx of adenosine lessened substrate inhibition of adenosine kinase. When the cells were not treated with deoxycoformycin, greater than 95% of the adenosine entering the cells at a concentration of 100 microM became deaminated. A 95-98% inhibition of adenosine transport by treatment with dipyridamole, dilazep, or nitrobenzylthioinosine inhibited its deamination practically completely, whereas adenosine phosphorylation was inhibited only 50-85%. Whether adenosine entering the cells is phosphorylated or deaminated is strictly based on the kinetic properties of the responsible enzymes, substrate inhibition of adenosine kinase, and the absolute intracellular steady state concentration of adenosine attained. The latter approaches the extracellular concentration of adenosine, since transport is not rate limiting, except when modulated by transport inhibitors. In spite of the extensive adenosine deamination in cells incubated with 100 microM adenosine, little IMP accumulated intracellularly when the medium phosphate concentration was 1 mM, but IMP formation increased progressively with increase in phosphate concentration to 80 mM. The intracellular phosphoribosylation of adenine and hypoxanthine were similarly dependent on phosphate concentration. The results indicate that adenosine is the main purine source for erythrocytes and is very efficiently taken up and converted to nucleotides under physiological conditions, whereas hypoxanthine and adenine are not significantly salvaged. Hypoxanthine resulting from nucleotide turnover in these cells is expected to be primarily released from the cells. Adenosine was also dephosphorylated in human red cells presumably by 5'-methylthioadenosine phosphorylase, but this reaction seems without physiological sign

Topics: Adenine Phosphoribosyltransferase; Adenosine; Adenosine Kinase; Biological Transport; Coformycin; Dilazep; Dipyridamole; Erythrocytes; Humans; Pentostatin; Phosphates; Phosphorylation; Thioinosine

The single nucleoside transport function of mouse S49 lymphoblasts also transports purine bases (B. Aronow and B. Ullman, J. Biol. Chem. 261:2014-2019, 1986). This transport of purine bases by S49 cells is sensitive to inhibition by dipyridamole (DPA) and 4-nitrobenzylthioinosine, two potent inhibitors of nucleoside transport. Therefore, wild-type S49 cells cannot salvage low hypoxanthine concentrations in the presence of 10 microM DPA and 11 microM azaserine; the latter is a potent inhibitor of purine biosynthesis. Among a mutagenized wild-type population, a cell line, JPA2, was isolated which could proliferate in 50 microM hypoxanthine-11 microM azaserine-10 microM DPA. The basis for the survival of JPA2 cells under these selective conditions was expression of a unique, high-affinity purine nucleobase transport function not present in wild-type cells. JPA2 cells could transport 5 microM concentrations of hypoxanthine, guanine, and adenine 15- to 30-fold more efficiently than parental cells did. Kinetic analyses revealed that the affinity of the JPA2 transporter for all three purine bases was much greater than that of the wild-type nucleobase transport system. Moreover, nucleobase transport in JPA2 cells, unlike that in parental cells, was insensitive to inhibition by DPA, 4-nitrobenzylthioinosine, sulfhydryl reagents, and nucleosides. No alterations in nucleoside transport capability, phosphoribosylpyrophosphate levels, or purine phosphoribosyltransferase enzymes were detected in JPA2 cells. Thus, JPA2 cells express a novel nucleobase transport capability which can be distinguished from the nucleoside transport function by multiple biochemical parameters.

Topics: Adenine; Animals; Azaserine; Biological Transport, Active; Cell Line; Dipyridamole; Guanine; Hypoxanthine; Hypoxanthines; Kinetics; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutation; Phenotype; Purine Nucleosides; T-Lymphocytes; Thioinosine

Erythrocytes from mice infected with Trypanosoma brucei brucei showed a higher rate of efflux of labelled thymidine than did control erythrocytes from uninfected mice (0.56 +/- 0.10 and 0.38 +/- 0.06 mumole min-1 ml-1 packed cells respectively). Efflux of the nucleoside from erythrocytes of normal and infected mice were inhibited to the same extent by a specific nucleoside transport inhibitor, nitrobenzylthioinosine. Enumeration of nitrobenzylthioinosine binding sites on the erythrocytes showed that both have similar numbers of sites (6.2-6.6 X 10(3) sites/erythrocyte). It is concluded that the membrane permeability of the erythrocytes from infected mice was affected by the trypanosome in such a way as to enhance the purine nucleoside transport capacity. This may result in an increased supply of vital purine bases and nucleosides to trypanosomes which depend on their hosts for these nutrients.

Topics: Animals; Binding Sites; Erythrocytes; Mice; Purines; Thioinosine; Thymidine; Tritium; Trypanosoma brucei brucei; Trypanosomiasis, African

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.

Topics: Affinity Labels; Animals; Biological Transport; Carbon Radioisotopes; Erythrocytes; Guinea Pigs; Kinetics; Temperature; Thermodynamics; Thioinosine; Uridine

The potential role of plasma nucleosides as metabolic energy substrates for pig erythrocytes, which are impermeable to glucose, was investigated in vivo by infusion of anesthetized pigs with nitrobenzylthioinosine phosphate (NBMPR-P), a soluble prodrug form of the specific nucleoside transport inhibitor, nitrobenzylthioinosine. NBMPR-P administration (1 or 10 mg X kg-1 X h-1) led to complete in vivo blockade of erythrocyte nucleoside transport activity and was associated with a dramatic decrease in the erythrocyte [ATP]-to-[ADP] ratio from 11.4 at time 0 to 2.9 after 4 h (mean results from 3 animals). Plasma inosine concentrations increased progressively from 2-4 microM at time 0 to 20-70 microM after 4 h of drug administration. In contrast, plasma adenosine concentrations remained less than 0.4 microM in all samples. These data suggest that pig erythrocytes utilize plasma inosine as their physiological energy substrate.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; Animals; Blood Glucose; Energy Metabolism; Erythrocytes; Inosine; Swine; Thioinosine; Thionucleotides

The transport of 500 microM uridine by human erythrocytes and S49, P388 and L1210 mouse leukemia cells, Chinese hamster ovary (CHO) cells and Novikoff rat hepatoma cells was inhibited strongly by dilazep and hexobendine with similar concentration dependence, but the sensitivity of transport in the various cell types varied greatly; IC50 values ranged from 5-30 nM for human erythrocytes and S49 and P388 cells to greater than 1 microM for CHO and Novikoff cells. The binding of nitrobenzylthioinosine (NBTI) to high-affinity sites on these cells (Kd approximately equal to 0.5 nM) was inhibited by hexobendine and dilazep in a similar pattern. Furthermore, these drugs, just as dipyridamole and papaverine, inhibited the dissociation of NBTI from high-affinity binding sites but only at concentrations 10-100 times higher than those inhibiting uridine transport. In contrast, high uridine concentrations (greater than 2 mM) accelerated the dissociation of NBTI. Dilazep also inhibited the transport of hypoxanthine, but only in those cell lines whose transporter is sensitive to inhibition by uridine and dipyridamole. Adenine transport was not inhibited significantly by dilazep in any of the cell lines tested, except for a slight inhibition in Novikoff cells. [14C]Hexobendine equilibrated across the plasma membrane in human erythrocytes within 2 sec of incubation at 25 degrees, but accumulated to 6-10 times the extracellular concentration in cells of the various cultured lines. Uptake was not affected by high concentrations of uridine, NBTI or dipyridamole. Hexobendine inhibited the growth of various cell lines to a lesser extent (IC50 = greater than or equal to 100 microM) than dipyridamole (IC50 = 15-40 microM). At 40 microM, however, it completely inhibited the growth of S49 cells that had been made nucleoside dependent by treatment with methotrexate or pyrazofurin.

Topics: Animals; Azepines; Benzoates; Biological Transport; Carbon Radioisotopes; Cell Line; Cricetinae; Cricetulus; Dilazep; Dipyridamole; Erythrocytes; Hexobendine; Humans; Hypoxanthine; Hypoxanthines; Inosine; Mice; Nucleosides; Rats; Thioinosine; Uridine

Topics: Cell Line; Cell Survival; Colonic Neoplasms; Fluorouracil; Humans; Inosine; Thioinosine; Thymidine

Tubercidin, an adenosine analogue, is toxic to human neuroblastoma cell lines, to peripheral blood mononuclear cells (PBMCs), and to myeloid colony-forming cells (CFU-C) as tested by a short-term labeled precursor uptake and by a clonogenic assay. When it was co-administered with a potent purine transport inhibitor, nitrobenzyl thioinosine (NBTI), the cytotoxic effect of tubercidin was abolished in PBMCs but not in neuroblastoma cells. Studies of nucleoside transport in neuroblastoma cells demonstrate that although [3H]NBTI binds to the plasma membrane of these cells, the transport of thymidine into the cells is only partially inhibited in the presence of excess NBTI. These data imply that neuroblastoma cells contain a nucleoside transport mechanism which is insensitive to NBTI. "Host protection" with a nucleoside transport inhibitor such as NBTI, may allow effective therapy with otherwise toxic dosages of tubercidin and other cytotoxic nucleosides in patients with neuroblastoma.

Topics: Biological Transport; Cell Line; Colony-Forming Units Assay; Humans; Inosine; Lymphocytes; Neuroblastoma; Ribonucleosides; Thioinosine; Thymidine; Tubercidin; Tumor Stem Cell Assay

From a mutagenized population of wildtype S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). NBMPR protected wildtype cells from the cytotoxic effects of a spectrum of nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. In addition, NBMPR prevented wildtype cells from surviving in hypoxanthine-amethopterin-thymidine containing medium, whereas KAB1 and KAB5 cells grew normally. Rapid sampling transport studies indicated that mutant cells, unlike wildtype parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wildtype complement of NBMPR binding sites. The characterization of the KAB1 and KAB5 cell lines suggested that the NBMPR binding site in wildtype S49 cells is genetically distinguishable from the nucleoside carrier site.

Topics: Animals; Binding Sites; Blood Proteins; Cell Line; Inosine; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Ribonucleosides; Thioinosine

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Biological Transport, Active; Carrier Proteins; Cytochalasin B; Deoxyguanosine; Deoxyribonucleosides; Guanosine; Inosine; Intracellular Membranes; Kinetics; Membrane Proteins; Mitochondria, Liver; Rats; Thioinosine; Thionucleosides

The nucleoside transporter of cerebral microvessels and choroid plexus was identified and characterized using [3H]nitrobenzylthioinosine (NBMPR) as a specific probe. [3H]NBMPR bound reversibly and with high affinity to a single specific site in particulate fractions of cerebral microvessels, choroid plexus, and cerebral cortex of the rat and the pig. The dissociation constants (KD 0.1-0.7 nM) were similar in the various tissue preparations from each species, but the maximal binding capacities (Bmax) were about fivefold higher in cerebral microvessels and choroid plexus than in the cerebral cortex. Nitrobenzylthioguanosine and dipyridamole were the most potent competitors for [3H]NBMPR binding. Several naturally occurring nucleosides displaced specific [3H]NBMPR binding to cerebral microvessels in vitro, in a rank order that correlated well with their ability to cross the blood-brain barrier in vivo. Adenosine analogues and theophylline were less effective in displacing [3H]NBMPR binding than in displacing adenosine receptor ligands. Photoactivation of cerebral microvessels and choroid plexus bound with [3H]NBMPR followed by solubilization and polyacrylamide gel electrophoresis labeled a protein(s) with a molecular weight of approximately 60,000. These results indicate that cerebral microvessels and choroid plexus have a much higher density of the nucleoside transporter moiety than the cerebral cortex and that this nucleoside transporter has pharmacological properties and a molecular weight similar to those of erythrocytes and other mammalian tissues.

Topics: Adenosine; Affinity Labels; Animals; Blood Proteins; Blood-Brain Barrier; Brain; Capillaries; Choroid Plexus; Dipyridamole; Endothelium; Female; Guanosine; Male; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Rats; Rats, Inbred Strains; Swine; Theophylline; Thioinosine; Thionucleosides

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.

Topics: Animals; Biological Transport; Cells, Cultured; Epithelium; Formycins; Intestinal Mucosa; Nucleosides; Rats; Sodium; Thioinosine

Dipyridamole-mediated inhibition of nucleoside transport by the nitrobenzylthioinosine (NBMPR)-sensitive facilitated diffusion system in mammalian erythrocytes was investigated. [3H]Dipyridamole was a competitive inhibitor of uridine equilibrium exchange influx into guinea pig erythrocytes (apparent Ki 1 nM). Analysis of the results using total inhibitor levels instead of cell-free inhibitor concentrations increased the apparent Ki value to 7 nM. Similarly, [3H]dipyridamole inhibition of zero-trans-[14C] uridine influx was consistent with simple competitive inhibition (apparent Ki 1.4 +/- 0.7 nM). In contrast, [3H]dipyridamole behaved as a noncompetitive inhibitor of zero-trans-[14C]uridine efflux (apparent Ki 0.7 +/- 0.2 nM). In a second series of experiments, [3H]dipyridamole was found to bind to a single class of high affinity sites on plasma membranes from human erythrocytes (apparent Kd 0.65 +/- 0.07 nM) with a maximum number of binding sites similar to that determined with the nucleoside transport inhibitor NBMPR. Binding of dipyridamole to these sites was blocked by the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine, and dilazep and in a competitive manner by adenosine and uridine (apparent inhibition constants 0.1 and 0.9 mM, respectively). These inhibition constants are similar to the apparent Km for adenosine and uridine equilibrium exchange in human erythrocytes. These results are consistent with the notion that, in mammalian erythrocytes, dipyridamole interacts with the NBMPR-sensitive transporter at the same site as NBMPR, which is preferentially located on the outer surface of the cell membrane totally or partially within the permeation site.

Topics: Animals; Binding, Competitive; Dipyridamole; Erythrocytes; Female; Guinea Pigs; Humans; Inosine; Kinetics; Thioinosine; Uridine

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Topics: Adenosine; Affinity Labels; Animals; Binding Sites; Biological Transport; Cell Membrane; Cells, Cultured; Dipyridamole; Electrophoresis, Polyacrylamide Gel; Inosine; Liver Neoplasms, Experimental; Neoplasm Proteins; Rats; Thioinosine; Uridine

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.

Topics: Animals; Carrier Proteins; Cell Membrane; Dilazep; Dipyridamole; Guinea Pigs; Lidoflazine; Lung; Male; Membrane Proteins; Nucleoside Transport Proteins; Nucleosides; Rats; Rats, Inbred Strains; Thioinosine

The olfactory cortex slice preparation from guinea-pig has been used to test compounds which inhibit the cellular uptake of adenosine. The uptake inhibitors dipyridamole (0.1-10 mumol/l), dilazep (1-10 mumol/l) nitrobenzylthioguanosine (1-10 mumol/l), nitrobenzylthioinosine (0.1-5 mumol/l), and hexobendine (1-100 mumol/l) increased the potency of adenosine (0.1-30 mumol/l) by up to 5-fold but did not potentiate cyclohexyladenosine (0.01-10 mumol/l). The benzodiazepine, diazepam (1 mumol/l) slightly increased the potency of adenosine (by 1.7-fold) whereas flurazepam (3 mumol/l) had no effect, suggesting that inhibition of adenosine uptake is probably not the major therapeutic action of these compounds. The uptake inhibitors depressed the amplitude of the monosynaptic epsp when added alone, an effect reversed by adenosine deaminase (1 unit/ml) whereas the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (10 mumol/l) had no effect on adenosine action. These results show that in this preparation (a) adenosine action is attenuated by an uptake mechanism and (b) endogenous adenosine release normally has no apparent effects on synaptic transmission at low stimulus rates. Nitrobenzylthioinosine and nitrobenzylthioguanosine are probably the best uptake blockers.

Topics: Adenosine; Adenosine Deaminase; Animals; Diazepam; Dilazep; Dipyridamole; Evoked Potentials; Flurazepam; Guanosine; Guinea Pigs; Hexobendine; In Vitro Techniques; Limbic System; Olfactory Pathways; Thioinosine; Thionucleosides

Previous studies have demonstrated that high concentrations of adenosine interact with both a cell surface receptor and with an intracellular site to evoke relaxation of the guinea-pig aorta. The intracellular action of adenosine was investigated in the present study. The purine sensitive 'P-site' did not appear to be involved since other P-site agonists did not consistently evoke relaxation. A major interaction with intracellular S-adenosylhomocysteine hydrolase also appeared unlikely since 1-homocysteine had only minor effects on adenosine-evoked responses. Inhibition of adenosine deaminase attenuated responses evoked by high concentrations of adenosine. The deaminated metabolite of adenosine, inosine, also evoked aortic relaxation. These responses were mediated solely via an intracellular site since they were blocked by an inhibitor of nucleoside-facilitated diffusion but were unaffected by an adenosine receptor antagonist. These results indicate that a major part of the intracellular effect of adenosine is mediated by its deaminated metabolite inosine.

Topics: Adenosine; Animals; Aorta, Thoracic; Female; Guinea Pigs; In Vitro Techniques; Inosine; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Norepinephrine; Theophylline; Thioinosine

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than (-)-N6-(R-phenyl-isopropyl)-adenosine greater than (+)-N6-(S-phenylisopropyl)-adenosine, in that order of potency, inhibited in vitro antigen-induced histamine release from human basophils in a dose-dependent fashion. Inhibition occurred only during the first stage of antigen-induced histamine release and the nucleosides failed to inhibit the release caused by the Ca2+ ionophore, A23187. 6-nitrobenzylthioinosine and dipyridamole, which inhibit adenosine uptake, and erythro-9-(2-hydroxy-3-nonyl)adenine, which blocks adenosine metabolism, did not impair the inhibition caused by NECA and adenosine. 8-phenyltheophylline and theophylline, two competitive antagonists of adenosine receptors, blocked the inhibition caused by NECA and adenosine. These data suggest that NECA and other adenosine analogs activate a specific cell surface adenosine receptor which possesses properties similar to those of an adenosine A2/Ra receptor.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Basophils; Biological Transport; Calcimycin; Histamine Release; Humans; Receptors, Cell Surface; Receptors, Purinergic; Structure-Activity Relationship; Theophylline; Thioinosine

There is evidence to suggest that several different groups of drugs including the so-called coronary vasodilators, benzodiazepines, and calcium channel inhibitors may owe their vasoactivity, in part, to the potentiation of the vasorelaxant effects of endogenous adenosine. To measure the affinity of some of these agents for the membrane-located nucleoside transport system, competition binding assays have been performed using the high-affinity radioligand [3H]nitrobenzylthioinosine (NBMPR). Experiments were performed on human erythrocytes and cardiac membranes from guinea pigs and rats. Recognized nucleoside transport inhibitors had high affinity (less than 50 nM) for NBMPR recognition sites associated with the nucleoside transporter complex in human erythrocytes, whereas calcium channel inhibitors and benzodiazepines had predominantly low affinity (greater than 1 microM). Although some recognized transport inhibitors, such as dipyridamole, show marked differences in affinity for NBMPR sites in guinea pig and rat tissues, benzodiazepines and calcium channel blockers displayed no such species selectivity and had low affinity (greater than 1 microM) for NBMPR sites in both guinea pig and rat cardiac membranes. Consequently, it is unlikely that agents such as benzodiazepines and calcium channel inhibitors cause significant inhibition of adenosine transport, and hence potentiate adenosine actions, at the concentrations required to induce effects through occupation of their respective, specific high-affinity sites.

Topics: Animals; Anti-Anxiety Agents; Benzodiazepines; Biological Transport; Calcium Channel Blockers; Erythrocytes; Guinea Pigs; Humans; In Vitro Techniques; Kinetics; Membranes; Myocardium; Nucleosides; Rats; Thioinosine; Vasodilator Agents

Prostaglandin A1 (PGA1) inhibits energy-dependent cyclic AMP export by pigeon red cells [Brunton and Mayer, J. Biol. Chem. 254:9714 (1979)]. To assess the specificity of this action, we observed the effect of 10 microM PGA1 (a concentration that inhibits cyclic AMP efflux greater than 95%) on a variety of membrane-protein-mediated processes that we could readily characterize and quantify in the pigeon red cell. Included in this study were isoproterenol-sensitive cyclic AMP production, ouabain-inhibitable 86Rb+ influx, furosemide-sensitive NaCl-KCl symport, 4-acetamido-4'-isothiocyano-2, 2'-disulfonic stilbene (SITS)-sensitive sulfate exchange, Na+-dependent alpha-aminoisobutyrate influx, and glucose and adenosine uptake. Remarkably, none of these membrane activities is significantly affected by PGA1. Furthermore, SITS, nitrobenzylthioinosine, cytochalasin B, and Na+-free extracellular medium (inhibitors of band 3, the nucleoside transporter, the hexose transporter, and amino acid uptake, respectively), failed to inhibit cyclic AMP export by pigeon red cells. On the basis of this data, we conclude that PGA1 does not act via a generalized alteration of a basic property at the plasma membrane, such as its fluidity; rather, PGA1 acts specifically to inhibit cyclic AMP extrusion. The data also imply that a transporter not relying on the Na+ gradient and distinct from transporters of cations, anions, amino acids, sugars, and nucleosides mediates cyclic AMP export.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Blood Glucose; Carrier Proteins; Columbidae; Cyclic AMP; Cyclic AMP Receptor Protein; Cytochalasin B; Erythrocyte Membrane; Erythrocytes; Kinetics; Male; Ouabain; Potassium Cyanide; Prostaglandins A; Rubidium; Thioinosine

Nucleoside transport sites in rat brain membrane preparations were labeled with [3H]nitrobenzylthioinosine ([3H] NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with KD and Bmax values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of [3H]NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The Ki values (micromolar concentration) of [3H]NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parallel displacement curves indicating their interaction with a common site labeled by [3H]NBI. The nucleobases, hypoxanthine and adenine, exhibited Ki values of 220 and 3640 microM, respectively. Adenosine receptor agonists exhibited moderate affinities for the [3H]NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The Ki values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 microM, respectively. These affinities and the rank order of potencies indicate that [3H]NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The Ki values of other nucleoside transport inhibitors were: nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 microM; and 2'-deoxyadenosine, 22 microM. These results indicate that [3H]NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control "adenosinergic" function.

Topics: Adenosine; Animals; Binding Sites; Binding, Competitive; Brain; Inosine; Male; Nucleosides; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Thioinosine; Time Factors; Tritium

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Biological Transport, Active; Female; In Vitro Techniques; Myocardium; Nucleotidases; Rats; Rats, Inbred Strains; Thioinosine

Topics: Binding Sites; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Erythrocyte Membrane; Humans; In Vitro Techniques; Nucleosides; Pyridines; Receptors, Nicotinic; Thioinosine

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.

Topics: Adenosine; Animals; Benzodiazepines; Binding Sites; Brain; Cell Membrane; Dogs; Erythrocytes; Glycerol; Guinea Pigs; HeLa Cells; Humans; Kinetics; Rats; Receptors, Cell Surface; Receptors, Purinergic; Thioinosine

This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-beta-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (approximately equal to 16-fold) and dipyridamole (approximately equal to sevenfold) for the [3H]Ro 15-1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.

Topics: Affinity Labels; Animals; Benzodiazepinones; Biological Transport, Active; Carbolines; Cerebral Cortex; Dipyridamole; Flumazenil; Flunitrazepam; Guinea Pigs; Nucleosides; Photochemistry; Receptors, GABA-A; Thioinosine

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.

Topics: 4-Chloromercuribenzenesulfonate; Biological Transport, Active; Blood Proteins; Dithiothreitol; Erythrocyte Membrane; Guanosine; Humans; Membrane Proteins; Nucleoside Transport Proteins; Phenylmercury Compounds; Structure-Activity Relationship; Thioinosine; Thionucleosides; Uridine

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Biological Transport; Carcinoma 256, Walker; Cell Line; Dipyridamole; Inosine; Kinetics; Lymphoma; Mice; Nucleosides; Rats; Sulfhydryl Compounds; Thioinosine; Uridine

In vitro resistance of HCT-8 cells to 5-fluoro-2'-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 microM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd phosphorylase, 5'-fluorouridine kinase, 5-fluorouridine phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 microM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2'-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25 degrees C and at 4 degrees C over a range of extracellular FdUrd concentrations (0.1 to 10 microM). Rapid entrance of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.

Topics: Adenocarcinoma; Biological Transport; Cell Line; Colonic Neoplasms; Diffusion; Drug Resistance; Floxuridine; Humans; Methotrexate; Nucleosides; Sucrose; Thioinosine

From a mutagenized population of wild-type S49 T lymphoblasts, cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, and 30 microM p-nitrobenzyl-6-thioinosine (NBMPR), a potent inhibitor of nucleoside transport. Unlike wild-type parental cells, two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild-type cells, KAB1, and KAB5 cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that nucleoside incorporation was much less sensitive to inhibition by NBMPR in the mutant cells. Rapid transport studies indicated that the mutant cell lines, unlike the wild-type parent, had acquired an NBMPR-insensitive nucleoside transport component which was similar to the NBMPR-sensitive wild-type transporter with respect to affinities for nucleosides and sensitivities toward N-ethylmaleimide and dipyridamole. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild-type complement of NBMPR binding sites with wild-type binding characteristics. These data suggest that the NBMPR binding site in wild-type S49 cells is genetically distinguishable from the nucleoside carrier site and that the former may be a regulatory site.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Blood Proteins; Cytidine; Drug Resistance; Inosine; Kinetics; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleotides; T-Lymphocytes; Thioinosine

Incubation of deoxycoformycin-treated L1210 leukemia cells with dipyridamole or nitrobenzylthioinosine, inhibitors of nucleoside transport, enhanced the long-term incorporation of 2'-deoxyadenosine and adenosine into the nucleotide pool and the toxicity of 2'-deoxyadenosine for the cells. In contrast, 2'-deoxyadenosine uptake in deoxycoformycin-treated P388 leukemia cells, which was about 10 times greater than that in L1210 cells, was inhibited by dipyridamole and nitrobenzylthioinosine, and 2'-deoxyadenosine toxicity was not significantly affected by the transport inhibitors. P388 cells also were about 6 times more resistant to 2'-deoxyadenosine than were L1210 cells, in spite of the greater uptake of the nucleoside. We found that purine nucleoside transport in L1210 and P388 cells exhibited similar kinetic properties and sensitivity to dipyridamole and nitrobenzylthioinosine (both influx and efflux) and that the stimulation of 2'-deoxyadenosine uptake by the inhibitors in L1210 cells is not mediated at the level of its transport into the cells but rather reflects an enhanced intracellular net accumulation of deoxyadenosine nucleotides.

Topics: Adenosine; Animals; Biological Transport; Coformycin; Deoxyadenosines; Dipyridamole; Inosine; Leukemia L1210; Leukemia P388; Leukemia, Experimental; Mice; Pentostatin; Thioinosine

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.

Topics: Adenosine; Affinity Labels; Animals; Brain; Dipyridamole; Female; Guinea Pigs; Membranes; Molecular Weight; Photochemistry; Thioinosine

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ( [3H]NBI) to cerebral cortical membrane preparations from human, dog, guinea pig, rat, and mouse was investigated. Reversible, specific, saturable, high affinity binding was found in all five species with similar kinetic parameters. (Kd = 0.16-0.44 nM; Bmax = 128-196 fmol/mg prot.). Dilazep, hexobendine, and dipyridamole were all high potency inhibitors of [3H]NBI binding in human, dog, guinea pig and mouse preparations but not in rat. These compounds showed a competitive inhibition of [3H]NBI binding indicating that they are acting at the same site. Discrepancies seen in the rat appear to be a unique, species related anomaly. The dihydropyridine calcium antagonists also inhibited binding with lower potency than the adenosine uptake blockers. This inhibition was most potent in dog and human and suggests a relationship between the calcium channel and the adenosine uptake site.

Topics: Animals; Brain; Calcium Channel Blockers; Cerebral Cortex; Dihydropyridines; Dogs; Guinea Pigs; Humans; Inosine; Kinetics; Mice; Pyridines; Rats; Rats, Inbred Strains; Species Specificity; Thioinosine

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.

Topics: Biological Transport, Active; Blood Proteins; Erythrocytes; Glucose; Guanosine; Humans; Membrane Proteins; Molecular Weight; Nucleoside Transport Proteins; Octoxynol; Phospholipids; Photolysis; Polyethylene Glycols; Sonication; Stereoisomerism; Thioinosine; Thionucleosides; Time Factors; Uridine

The adenosine uptake site has been localized in rat brain by an in vitro light microscopic autoradiographic method, using [3H]nitrobenzylthioinosine ( [3H]NBI) as the probe. The binding characteristics of [3H]NBI on slide-mounted sections are comparable to those seen in studies performed on brain homogenates. A very high density of uptake sites occurs in the nucleus tractus solitarius, in the superficial layer of the superior colliculus, in several thalamic nuclei, and also in geniculate body nuclei. A high density of sites are also observed in the nucleus accumbens, the caudate putamen, the dorsal tegmentum area, the substantia nigra, and the central gray. The localization of the adenosine uptake site in brain may provide information on the functional activity of the site and suggests the involvement of the adenosine system in the central regulation of cardiovascular function.

Topics: Adenosine; Animals; Autoradiography; Binding Sites; Inosine; Male; Rats; Rats, Inbred Strains; Thioinosine; Tissue Distribution; Tritium

A mutant clone (NT-1) of a T-cell lymphoma was selected for its ability to grow in HAT medium (hypoxanthine, aminopterin and thymidine) in the presence of the nucleoside transport inhibitor P-nitrobenzyl-6-mercaptoinosine (NBMI). NT-1 cells contain half the number of NBMI binding sites present on the parental S49 cells and are partially able to transport nucleosides in the presence of the transport inhibitor (NBMI). These observations suggest that the mutant cells are heterozygous for nucleoside transport proteins and contain two types of transport proteins: the first protein can both bind and is inhibited by NBMI similar to the wild type phenotype, and the second is an altered protein. The altered transport protein apparently lost its NBMI binding sites without a parallel loss of nucleoside transport ability suggesting that the nucleoside transported sites are separate from the binding sites of the transport inhibitor.

Topics: Adenosine; Animals; Biological Transport; Cell Division; Cells, Cultured; Deoxycytidine; Kinetics; Lymphoma; Mice; Nucleosides; Thioinosine; Thymidine

The binding characteristics of [3H]nitrobenzylthioinosine ([3H]NBI) to rat brain membrane preparations was examined, and the autoradiographic distribution of this ligand in brain sections was compared with the immunohistochemical localization of adenosine deaminase (ADA). It was found that [3H]NBI labels sites for which adenosine has far higher affinity than do other nucleosides, that these sites are heterogeneously distributed and that there is an exact correspondence between areas containing [3H]NBI sites and ADA-immunoreactive neurons. Our results indicate that [3H]NBI and ADA are potential markers for revealing anatomical sites at which actions of adenosine may be expressed.

Topics: Adenosine; Adenosine Deaminase; Animals; Autoradiography; Brain; Inosine; Male; Neurons; Nucleoside Deaminases; Rats; Rats, Inbred Strains; Thioinosine

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.

Topics: Animals; Biological Transport, Active; Blood Proteins; Clone Cells; Hybrid Cells; Inosine; Lymphoma; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; Thioinosine

Site-specific binding to human erythrocyte membranes of nitrobenzylthioinosine (NBMPR), un-ionized at physiological pH, was compared with that of hydroxynitrobenzylthioinosine (HNBMPR), pKa 6.4, at graded pH values. Binding of [3H]NBMPR was measured directly, and that of HNBMPR was assayed by competitive inhibition by HNBMPR of [3H]NBMPR binding. Kd and Bmax values for binding of [3H]NBMPR to erythrocyte membranes were independent of pH. Kd values for the competing ligand were determined by mass law analysis of equilibrium binding data using either (a) apparent ligand concentration (dissociated plus undissociated forms of HNBMPR) or (b) the concentration of undissociated HNBMPR. Kd values for HNBMPR calculated with the apparent ligand concentration increased 10-fold as the fraction of HNBMPR molecules present in the dissociated form was increased (by pH changes) from 14 to 88%, whereas Kd values for the undissociated form of HNBMPR were independent of pH. The results presented here demonstrate that the undissociated form of HNBMPR binds more tightly to the transport-inhibitory sites of erythrocytes than NBMPR and suggest that ionization of S6-substituted thiopurine ribonucleosides eliminates or greatly decreases their ability to interact with the binding sites.

Topics: Affinity Labels; Binding, Competitive; Blood Proteins; Carrier Proteins; Erythrocyte Membrane; Guanosine; Humans; Hydrogen-Ion Concentration; Inosine; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine; Thionucleosides

Cultured Novikoff rat hepatoma and Walker 256 carcinoma cells have previously been reported to express only nitrobenzylthioinosine (NBTI)-resistant uridine transport and to lack high affinity NBTI-binding sites, whereas the latter are common on all other types of cultured mammalian cells from different species [1-7) X 10(5) sites/cell) which have been investigated with the exception of a transport-deficient cell variant which lacks high-affinity NBTI-binding sites. The present study shows that lack of NBTI sensitivity of transport and of NBTI-binding sites in Novikoff and Walker 256 cells are not related to the species or tissue origin of these cells. Uridine transport in a variant (NRM) of Novikoff hepatoma cells, in HTC rat hepatoma cells, normal rat kidney (NRK) cells, rat erythrocytes and rat hepatocytes was inhibited 15-60% by 10-500 nM NBTI and the cells expressed high-affinity NBTI-binding sites (Kd = 0.1-0.6 nM). The apparent turnover numbers for the NBTI-sensitive nucleoside carriers fell into two classes, with those for transformed cells about 10-times higher than those for the normal rat cells.

Topics: Animals; Binding Sites; Biological Transport; Carcinoma 256, Walker; Dipyridamole; Inosine; Kidney; Liver; Liver Neoplasms, Experimental; Male; Mathematics; Nucleosides; Rats; Thioinosine; Uridine

The interactions of the epipodophyllotoxins, teniposide (VM-26) and etoposide (VP-16), with the nucleoside carrier were examined with emphasis on their effects on 1-beta-D-arabinofuranosylcytosine (ara-C) transport and net accumulation. VM-26 inhibited ara-C transport by Ehrlich ascites cells within 1 min of exposure, and inhibition was only partially reversed after 45 min in VM-26-free medium. ara-C transport was slowed by 50% by 7 microM VM-26 or by 35 microM VP-16. Since epipodophyllotoxins were noncompetitive inhibitors, fractional inhibition was independent of the ara-C concentration. Analysis of ara-C transport kinetics revealed only a single saturable transport route, and there was no indication of VM-26-insensitive transport. VM-26, VP-16, and ara-C were competitive inhibitors of the specific binding of nitrobenzylthioinosine to the nucleoside carrier with Ki values of 7.4 microM, 23 microM, and 2.2 microM, respectively. The rate of dissociation of nitrobenzylthioinosine (t 1/2 = 20.6 min) was accelerated by 5 microM ara-C (t 1/2 = 18.5 min) but slowed by 100 microM VM-26 (t 1/2 = 34.6). By these criteria, the interaction of VM-26 with the nucleoside carrier was qualitatively similar to that of dipyridamole. Although VM-26 inhibited ara-C transport, it did not significantly slow the rate of net intracellular accumulation of ara-C by Ehrlich cells, presumably because transport capacity far exceeds the capacity for phosphorylation in these cells. In freshly isolated human leukemic blasts, which have far less nucleoside transport activity, inhibition of ara-C accumulation by VM-26 was dependent on the ara-C concentration. At 1 microM ara-C, a concentration where transport was rate limiting for net uptake, VM-26 inhibited accumulation of ara-C over a 60-min time course. At 50 microM ara-C, transport was in excess, and VM-26 did not slow ara-C metabolism.

Topics: Animals; Binding Sites; Biological Transport; Carcinoma, Ehrlich Tumor; Cells, Cultured; Cytarabine; Etoposide; Humans; Leukemia; Male; Mice; Mice, Inbred Strains; Podophyllotoxin; Teniposide; Thioinosine; Tritium

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site-specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high-affinity (KD 0.11-4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 less than 20 nM and IC50 greater than 6 microM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitor-induced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.

Topics: Adenosine; Animals; Binding Sites; Biological Transport, Active; Brain; Cell Membrane; Diazepam; Dipyridamole; Dogs; Erythrocytes; Female; Guinea Pigs; Humans; Inosine; Male; Mice; Nucleosides; Rabbits; Rats; Rats, Inbred Strains; Species Specificity; Thioinosine

Uptake of [14C]-adenosine into freshly dispersed rat hepatocytes was inhibited 44% by dipyridamole (50 microM) and 60% by nitrobenzylthioinosine (NBTI, 20 microM). The results are consistent with the known ability of these drugs to inhibit adenosine transport in other cell types. The nucleotide analogue, alpha, beta-methylene adenosine diphosphate (AOPCP, 50 microM), inhibited by 84% the degradation of exogenous 5' AMP that occurred rapidly when this substrate alone was presented to isolated hepatocytes. This confirms the ecto-5'-nucleotidase inhibitory properties of this analogue in isolated hepatocytes. During hypoxic incubation, isolated hepatocytes released adenosine, which accumulated in the extracellular volume. Dipyridamole and NBTI each markedly attenuated this extracellular adenosine accumulation. In contrast, AOPCP had no inhibitory effect on net hypoxic adenosine release. It is concluded that hypoxic rat hepatocytes produce adenosine intracellularly and that this adenosine is released via facilitated diffusion to the extracellular space, based on the inhibition observed with the transport inhibitors. The plasma membrane enzyme ecto-5'-nucleotidase does not appear to participate in hypoxic adenosine release from these cells as indicated by the lack of effect of the nucleotidase inhibitor, AOPCP.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Animals; Dipyridamole; Liver; Oxygen; Rats; Thioinosine

Human erythrocytes were shown to possess a saturable uptake mechanism for 2-chloroadenosine (apparent Km 23 microM, 22 degrees). Uptake by this route was inhibited by nitrobenzylthioinosine, uridine and adenosine, but adenine had no effect. In addition, uridine caused the countertransport of 2-chloroadenosine and vice versa. 2-Chloroadenosine was also shown to be an apparent competitive inhibitor of uridine influx (apparent Ki value of 33 microM) and high-affinity nitrobenzylthioinosine binding (apparent Ki 0.18 mM). The apparent Ki value for inhibition of uridine influx was close to the apparent Km value for 2-chloroadenosine uptake. Previous studies [Jarvis et al., Biochem. J. 208, 83 (1982)] have demonstrated that dog erythrocytes do not possess a saturable transport system for uridine and adenosine. Similarly, in the present study, the entry of 2-chloroadenosine into dog erythrocytes was slow and linear with concentration. Nitrobenzylthioinosine (NBMPR) had no effect on the uptake of 2-chloroadenosine into dog erythrocytes. These results demonstrate that 2-chloroadenosine enters human erythrocytes by the same nucleoside carrier as other nucleosides. It is suggested from these data that the previous explanation that the inability of nucleoside transport inhibitors to potentiate the pharmacological effects of 2-chloroadenosine was due to the failure of the nucleoside carrier to accept 2-chloroadenosine as a permeant may have to be reassessed.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Biological Transport; Blood Proteins; Carrier Proteins; Erythrocyte Membrane; Erythrocytes; Humans; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine; Uridine

The transmembrane topology of the nucleoside transporter of human erythrocytes, which had been covalently photolabelled with [3H]nitrobenzylthioinosine, was investigated by monitoring the effect of proteinases applied to intact erythrocytes and unsealed membrane preparations. Treatment of unsealed membranes with low concentrations of trypsin and chymotrypsin at 1 degree C cleaved the nucleoside transporter, a band 4.5 polypeptide, apparent Mr 66 000-45 000, to yield two radioactive fragments with apparent Mr 38 000 and 23 000. The fragment of Mr 38 000, in contrast to the Mr 23 000 fragment, migrated as a broad peak (apparent Mr 45 000-31 000) suggesting that carbohydrate was probably attached to this fragment. Similar treatment of intact cells under iso-osmotic saline conditions at 1 degree C had no effect on the apparent Mr of the [3H]nitrobenzylthioinosine-labelled band 4.5, suggesting that at least one of the trypsin cleavage sites resulting in the apparent Mr fragments of 38 000 and 23 000 is located at the cytoplasmic surface. However, at low ionic strengths the extracellular region of the nucleoside transporter is susceptible to trypsin proteolysis, indicating that the transporter is a transmembrane protein. In contrast, the extracellular region of the [3H]cytochalasin B-labelled glucose carrier, another band 4.5 polypeptide, was resistant to trypsin digestion. Proteolysis of the glucose transporter at the cytoplasmic surface generated a radiolabelled fragment of Mr 19 000 which was distinct from the Mr 23 000 fragment radiolabelled with [3H]nitrobenzylthioinosine. The affinity for the reversible binding of [3H]cytochalasin B and [3H]nitrobenzylthioinosine to the glucose and nucleoside transporters, respectively, was lowered 2-3-fold following trypsin treatment of unsealed membranes, but the maximum number of inhibitor binding sites was unaffected despite the cleavage of band 4.5 to lower-Mr fragments.

Topics: Affinity Labels; Blood Proteins; Carrier Proteins; Chymotrypsin; Cytochalasin B; Erythrocyte Membrane; Humans; Membrane Proteins; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Thioinosine; Trypsin; Uridine

Adenosine (Ado, 10 microM) did not inhibit ADP-induced human platelet aggregation in whole blood. However, if the blood was preincubated with dipyridamole (10 microM), a potent inhibitor of the erythrocytic nucleoside transport system (NTS), Ado acted as a strong inhibitor of platelet aggregation. Similarly, Ado inhibited platelet aggregation in whole blood in the presence of other potent NTS inhibitors, dilazep (1 microM) and p-nitrobenzylthioinosine (NBMPR, 1 microM). RA 233 (10 microM), an analog of dipyridamole which is a potent inhibitor of platelet cAMP phosphodiesterase (PDE), did not evoke the Ado effect in whole blood. However, in platelet-rich plasma (PRP), RA 233 potentiated strongly Ado-mediated inhibition, whereas dipyridamole, dilazep and NBMPR were without activity. 5'-Methylthioadenosine (MTA), an Ado receptor antagonist, reversed the inhibition produced by a nucleoside transport system inhibitor plus Ado in whole blood. Dipyridamole (10 microM), dilazep (1 microM) or NBMPR (1 microM) blocked [14C]Ado (10 microM) uptake by blood cells in whole blood, whereas RA 233 (10 microM) was not effective. The combination of 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of adenosine deaminase (ADA), plus 5-iodotubercidin (ITu, 10 microM), a potent inhibitor of adenosine kinase (Ado kinase), gave comparable Ado-mediated inhibition of platelet aggregation in whole blood as was obtained when the blood was pretreated with dilazep. These studies suggest that the in vivo antiplatelet actions of drugs such as dipyridamole and dilazep result from their abilities to block erythrocytic Ado uptake and subsequent metabolism, thus elevating the extracellular steady-state concentration of the physiologically occurring, antiplatelet agent, Ado.

Topics: Adenosine; Azepines; Blood Platelets; Dilazep; Dipyridamole; Erythrocytes; Humans; In Vitro Techniques; Inosine; Mopidamol; Platelet Aggregation; Thioinosine

The distribution of [3H]nitrobenzylthioinosine ([3H]NBI) binding to nucleoside transport sites in rat spinal cord and spinal roots was examined using membrane binding and autoradiographic techniques. A single class of high affinity binding sites having dissociation constants (KD) between 0.42 +/- 0.05 and 0.088 +/- 0.012 nM was observed in dorsal and ventral spinal cord and their associated roots. The maximal number of binding sites (Bmax) in dorsal and ventral spinal cord was 110.1 +/- 7.1 and 73.6 +/- 7.5 fmol/mg protein, respectively. The highest levels of [3H]NBI binding were found in the dorsal grey matter of the cervical and lumbar enlargements. Autoradiographic studies showed that [3H]NBI sites were especially concentrated in the substantia gelatinosa of the dorsal spinal cord and the nucleus caudalis of the spinal trigeminal nucleus. The level of these binding sites in dorsal roots was nearly 4 times that observed in ventral roots; 98.5 and 23.0 fmol/mg protein, respectively. Adult animals depleted of unmyelinated sensory fibers by neonatal capsaicin treatment showed significantly reduced numbers of [3H]NBI sites (35%) in dorsal roots but not ventral roots, while KD values were unaffected. These results indicate that [3H]NBI sites are enriched in areas of the spinal cord and brainstem which subserve sensory functions and that these sites are located, in part, on unmyelinated primary afferent fibers.

Topics: Adenosine Deaminase; Animals; Autoradiography; Binding Sites; Capsaicin; Ganglia, Spinal; Inosine; Male; Neurons, Afferent; Rats; Rats, Inbred Strains; Spinal Cord; Thioinosine

Isolated guinea pig hearts were used to determine whether an extracellular (interstitial) or intracellular pool of myocardial adenosine is most important in attenuating the catecholamine-induced enhancement of cardiac contractile state and glycogenolysis. Isoproterenol (2 X 10(-8) M) stimulation of hypoxic (30% O2) perfused hearts produced a marked elevation in tissue and effluent perfusate adenosine levels that were greater than the increases observed with the isoproterenol stimulation of oxygenated hearts (95% O2). In the isoproterenol stimulated hypoxic hearts nitrobenzylthioinosine (NBMPR), a potent inhibitor of adenosine cellular transport, further increased tissue adenosine content and markedly decreased the perfusate level of the nucleoside. Assuming that perfusate levels of adenosine correlate directly with extracellular levels, NBMPR was used as a tool to increase the intracellular and decrease the extracellular content of the nucleoside. When compared to responses in oxygenated hearts, hypoxia reduced the isoproterenol-produced increase in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractility but enhanced the increase in glycogen phosphorylase alpha formation. NBMPR completely prevented the reduction of the isoproterenol-induced cyclic AMP and cyclic AMP-dependent protein kinase responses but only partially prevented the attenuation of the contractile response. The increase in phosphorylase alpha formation in the hypoxic isoproterenol stimulated hearts was not influenced by NBMPR. The results suggest that an increase in extracellular adenosine is more influential than an elevation of intracellular adenosine in attenuating beta-adrenoceptor-elicited increases in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractile state.

Topics: Adenosine; Animals; Catecholamines; Cyclic AMP; Extracellular Space; Female; Glycogen; Guinea Pigs; Heart; Hypoxia; Intracellular Fluid; Isoproterenol; Myocardial Contraction; Myocardium; Thioinosine

HeLa cells as well as human and mouse erythrocytes possess membrane sites which bind the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), reversibly but tightly (KD, 10(-9)-10(-10) M). Site-specific binding of the ligand correlates with inhibition of nucleoside transport. The present study showed that the 5'-phosphate of NBMPR, NBMPR-P, was not transport inhibitory. Upon exposure to [35S]NBMPR-P or [G-3H]NBMPR-P, HeLa cells retained the isotopic labels virtually exclusively in the form of NBMPR. The dephosphorylation of [G-3H]NBMPR-P by HeLa cells, assayed by the production of extracellular [G-3H]NBMPR, was competitively inhibited by AMP, but was not affected by the presence of 5 microM NBMPR, a concentration sufficient to completely occupy the transport inhibitory sites. Thus, the sites at which dephosphorylation of NBMPR occurs in HeLa cells are separate from and function independently of the high affinity sites which bind NBMPR.

Topics: 5'-Nucleotidase; Animals; Binding Sites; Cell Membrane; Erythrocytes; HeLa Cells; Humans; Inosine; Kinetics; Male; Mice; Nucleotidases; Protein Binding; Thioinosine; Thionucleotides; Uridine

A highly heterogeneous distribution of [3H]nitrobenzylthioinosine [( 3H]NBI) binding sites was observed using both autoradiographic and membrane binding methodology. Of the 24 brain regions examined in the radio-ligand binding studies, the highest levels of [3H]NBI sites were found in the thalamus, followed by midbrain, superior colliculus, olfactory cortex and hypothalamus. The thalamus contained over 5 times more sites than cerebellum which exhibited the lowest [3H]NBI binding levels. The results obtained from autoradiographic analysis agreed well with quantitative measurements and revealed that subnuclei of thalamus and hypothalamus as well as specific layers of the superior colliculus contained particularly high concentrations of [3H]NBI sites. When the [3H]NBI autoradiograms were compared with the distribution of adenosine deaminase in brain it was found that brain regions richest in neural elements immunoreactive for adenosine deaminase contained the greatest numbers of [3H]NBI sites. In contrast, a poor correlation was found between the distribution of [3H]NBI binding and adenosine receptors labelled with [3H]cyclohexyladenosine. The co-localization of [3H]NBI binding and adenosine deaminase in brain indicates the existence of neural systems having a high capacity to take up and metabolize adenosine.

Topics: Adenosine; Animals; Autoradiography; Brain; Brain Chemistry; Cell Membrane Permeability; Inosine; Male; Membranes; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Thioinosine

The effect of chronic caffeine treatment on three different binding sites in five brain areas of mice is characterized. The sites studied were the adenosine receptor, using [3H] diethylphenylxanthine, the benzodiazepine receptor, using [3H] diazepam and the adenosine uptake site, using [3H] nitrobenzylthioinosine. Significant increases were only observed in adenosine receptors with the greatest degree of change seen in the cerebellum and brain stem at both 16 and 23 days of caffeine treatment. The lack of significant effects of chronic caffeine on benzodiazepine receptors and adenosine uptake sites indicates that the caffeine effect is specific. The effect of chronic caffeine treatment on the ontogeny of adenosine receptors was also studied with the result showing a significantly accelerated development of the receptor in the caffeine treated animals. The adult adenosine receptor levels were 20-30% higher than those observed in control animals. The observed alterations in adenosine receptor number which occur as a consequence of caffeine consumption may underlie some of the behavioral effects of this cortical stimulant as well as provide insights concerning the mechanisms of tolerance to and dependence on caffeine.

Topics: Adenosine; Animals; Brain; Brain Stem; Caffeine; Cerebellum; Female; Male; Mice; Pregnancy; Prenatal Exposure Delayed Effects; Receptors, Cell Surface; Receptors, GABA-A; Receptors, Purinergic; Thioinosine; Time Factors

5'-nucleotidase activity, arachidonate metabolism and adenosine uptake were measured in P388 murine leukaemia cells and in a subline resistant to doxorubicin. These membranes related activities were found to be increased in the doxorubicin resistant cell line, compared to the drug sensitive cells. It is suggested that these differences do not play a role in the mechanism of resistance to doxorubicin. Rather they reflect alterations in plasma membrane composition and structure between these cell lines. This study also suggests that the use of decreased 5'-nucleotidase activity as a marker of certain leukaemias should be reviewed with caution. An increase in cell enzyme activity in treated patients may not necessarily indicate a shift toward normal behaviour of these cells, but rather a selection of certain cell subpopulations.

Topics: 5'-Nucleotidase; Adenosine; Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Cells, Cultured; Doxorubicin; Drug Resistance; Indomethacin; Leukemia P388; Leukemia, Experimental; Mice; Nucleotidases; Thioinosine

The assay of residual adenosine deaminase (ADA) activity was used as a sensitive measure of the transport of deoxycoformycin (dCF) into human erythrocytes. Contrary to prior reports from this laboratory, the inactivation of intraerythrocytic ADA by dCF was linear rather than log-linear, with time. Linear inactivation rates were also seen when erythrocytes were preloaded with a 5-fold excess of calf intestinal ADA. The uptake of tritium-labeled dCF molecules and the rate of inactivation of ADA molecules showed an approximate 1:1 stoichiometry. The nucleoside transport inhibitors, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and dipyridamole, and the permeant, uridine, inhibited dCF transport with Ki values of 35 nM, 45 nM, and 340 microM respectively. The affinity of dCF for the nucleoside transporter was low with a Ki of approximately 10 mM for the inhibition of adenosine influx.

Topics: Adenosine Deaminase Inhibitors; Biological Transport; Coformycin; Dipyridamole; Erythrocytes; Humans; Kinetics; Nucleoside Deaminases; Nucleosides; Pentostatin; Ribonucleosides; Thioinosine; Tritium; Uridine

The availability of a human lymphoma cell line deficient in adenosine deaminase, adenosine kinase and methylthioadenosine phosphorylase enabled us to compare the effects of nucleoside transport inhibitors on the excretion of endogenously generated adenosine, deoxyadenosine and 5'-methylthioadenosine. The nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole blocked the efflux of adenosine, but not deoxyadenosine or 5'-methylthioadenosine. The inhibitors also prevented the uptake of exogenous adenosine, but not deoxyadenosine or 5'-methylthioadenosine, by human lymphoblasts. The results show (i) that the transport inhibitors modify adenine nucleoside efflux and influx similarly, and (ii) that the effects of the compounds on the excretion and uptake of these three physiologically important adenine nucleosides are distinctly different.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Biological Transport; Cell Division; Cell Line; Deoxyadenosines; Dipyridamole; Humans; Lymphocytes; Lymphoma; Nucleoside Deaminases; Purine-Nucleoside Phosphorylase; Thioinosine; Thionucleosides

Topics: Animals; Biological Transport; Inosine; Liver; Mice; Mice, Inbred ICR; Nucleosides; Thioinosine; Tissue Distribution; Uridine

[3H] Nitrobenzylthioinosine (NBI) binding is characterized in dog heart and brain. Evidence is presented suggesting that [3H]NBI is binding to the adenosine uptake site in both tissues. Physiologic studies in open-chested dogs clearly demonstrate that NBI acts as a coronary vasodilator, consistent with an action at the adenosine uptake site. The binding is reversible, saturable and of high affinity (KD = 0.78 +/- .06 nM for heart and 0.52 +/- .05 nM for brain). Both dipyridamole and hexobendine are high potency inhibitors of [3H]NBI binding in heart and brain while other antihypertensives and vasodilators such as propranolol and nitroglycerin have no effect. The inhibition of [3H]NBI binding observed with dipyridamole was competitive indicating that both agents are acting at the same site. The dihydropyridine calcium antagonists also inhibited binding with a lower potency than the adenosine uptake blockers. Non-dihydropyridine calcium antagonists were much less potent in this regard. The inhibition of [3H]NBI binding observed with the dihydropyridine calcium antagonists was non-competitive suggesting that the calcium channel and adenosine uptake site may be coupled to each other.

Topics: Adenosine; Animals; Blood Pressure; Brain; Calcium Channel Blockers; Coronary Circulation; Dipyridamole; Dogs; Heart Rate; Inosine; Kinetics; Myocardium; Thioinosine; Vascular Resistance

N-Phosphonacetyl-L-aspartic acid (PALA) resistance may be due to the ability of tumor cells to utilize preformed circulating pyrimidine nucleosides, thereby overcoming the block of de novo pyrimidine biosynthesis which PALA causes. To test this hypothesis we examined the effects of PALA and nitrobenzylthioinosine (NBMPR) alone and in combination on B16 melanoma cells in vitro using a clonogenic assay and in vivo using growth delay. In medium containing purine and pyrimidine nucleosides at a final concentration of 28 microM, exposure to PALA (100 microM) alone or to NBMPR (10 microM) alone for periods up to 72 hr did not result in any cytotoxicity. However, exposures to PALA (100 microM) plus NBMPR (10 microM) resulted in a decrease in clonogenic survival to 0.011 at 72 hr. In medium without nucleosides, PALA (100 microM) exposure for 72 hr caused a similar decrease in survival to 0.015, whereas NBMPR (10 microM) had no effect on survival. The addition of uridine resulted in a concentration-dependent reversal of the cytotoxic effects of PALA. C57 Bl female mice bearing B16 melanoma were treated intraperitoneally daily for 4 days with PALA, the phosphate of NBMPR (NBMPR-P), or PALA plus NBMPR-P. PALA, 300 mg/kg daily X 4, resulted in a 6-day tumor growth delay but NBMPR-P, 100 mg/kg daily X 4, had no effect. PALA, 150 mg/kg daily X 4, plus NBMPR, 50 or 100 mg/kg daily X 4, resulted in a 6-day tumor growth delay also. These studies demonstrate that: (1) circulating pyrimidine nucleosides are determinants of the cytotoxic effects of PALA; (2) in vitro PALA and NBMPR combine to cause significant cytotoxicity whereas either agent alone has no effect; (3) in vivo the combination of PALA and NBMPR-P results in the same antitumor affect as PALA alone at twice the dose; and (4) due to an increase in animal toxicity, no therapeutic advantage could be demonstrated for the combination over PALA alone in vivo. We conclude that the cytotoxic effect of PALA is modulated by the levels of the preformed circulating nucleosides and that combining PALA with an inhibitor of salvage pyrimidine uptake would not increase the therapeutic efficacy of PALA because of an increase in toxicity.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Cell Line; Dose-Response Relationship, Drug; Female; Inosine; Melanoma; Mice; Mice, Inbred C57BL; Nucleosides; Organophosphorus Compounds; Phosphonoacetic Acid; Thioinosine; Thionucleotides

Specific binding of [3H]nitrobenzylthioinosine to cortical membranes from several mammalian species was investigated. Rat, mouse, guinea pig, and dog membranes contained an apparent single class of binding sites; there was, however, a marked species-dependent variation in their affinity for [3H]nitrobenzylthioinosine. Rabbit cortical membranes contained two classes of binding sites and the high and low affinity components were similar to those found in guinea pig and dog cortical membranes, respectively. The [3H]nitrobenzylthioinosine binding sites in rat and the low affinity sites in rabbit were atypical in that they exhibited a low affinity for dipyridamole. It is proposed that these latter sites may represent a form of the central nervous system nucleoside transport system which is less susceptible to inhibition by dipyridamole.

Topics: Animals; Binding Sites; Binding, Competitive; Biological Transport, Active; Cerebral Cortex; Dipyridamole; Dogs; Guinea Pigs; In Vitro Techniques; Inosine; Kinetics; Membranes; Mice; Nucleosides; Rabbits; Species Specificity; Thioinosine

Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [3H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that 3H was present in polypeptides which migrated as a major band with an apparent Mr of 45000-65000.

Topics: Affinity Labels; Animals; Blood Proteins; Cell Line; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Inosine; Leukemia L5178; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Photochemistry; Thioinosine; Ultraviolet Rays

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.

Topics: Adenosine; Animals; Benzodiazepines; Binding, Competitive; Biological Transport; Brain; Cell Membrane; Female; Guinea Pigs; Inosine; Kinetics; Nucleosides; Phenothiazines; Subcellular Fractions; Synaptosomes; Thioinosine

The potent inhibitor of nucleoside transport, 6-[(4-nitrobenzyl) mercapto]-9-beta-D-ribofuranosylpurine (NBMPR), binds reversibly, but with high affinity (KD 0.3-1.4 nM) to plasma membrane sites on human erythrocytes, and occupancy of those sites by NBMPR correlates with inhibition of transport. An earlier study from this laboratory showed that, upon photoactivation of site-bound [3H]NBMPR on erythrocyte membranes by exposure to UV light, isotopic ligand molecules became covalently linked to membrane polypeptides, which migrated as a discrete band on gel electrophoretograms, thereby identifying nucleoside transport polypeptides. The present study showed that erythrocytes on which the high affinity sites were occupied by reversibly bound [3H]NBMPR were freed of the latter by slow passage at 37 degrees C through 9 X 300 mm columns of Sephadex G-200 gel. Photoactivation of site-bound [3H]NBMPR on the intact cells caused apparent covalent attachment of the ligand because cells so treated retained the isotopic label during passage through the gel columns. Apparent covalent binding to erythrocytes also resulted from photoactivation of site-bound [3H]nitrobenzylmercaptopurine arabinoside.

Topics: Chromatography, Gel; Erythrocytes; Humans; Inosine; Thioinosine

Attempts were made to measure adenosine transport in isolated smooth muscle preparations including guinea-pig taenia caeci, beef coronary arteries and longitudinal muscle of rabbit small intestine. Because adenosine-mediated relaxation is potentiated by nucleoside transport inhibitors such as dipyridamole and 6- thiobenzylpurine ribosides in the first two systems but not in rabbit intestinal muscle, possible differences in transport capacities and in the effects of these inhibitors in the three tissues were examined. Transport was to be measured by assessing metabolic products of adenosine including adenine nucleotides and inosine plus hypoxanthine in both tissues and incubation media. Despite extensive rinsing of tissues, adenosine deaminase leaked into the incubation media, requiring its inhibition by 5 nM deoxycoformycin. When measuring apparent transport rates by quantitating metabolic products in the presence of 5 nM deoxycoformycin, no saturation of uptake at 100 to 400 microM adenosine was observed in taenia caeci and rabbit muscle. Comparing these results with literature reports on transport rates in single cell preparations, it appears that the obtained values (20-40 pmol/mg/min) may be at least 100-fold lower, suggesting that rates of diffusion through tissue and intracellular deamination of adenosine were the limiting functions measured by the methodology used in this study, requiring a careful definition for the terms transport and uptake and suggesting that it is practically not possible to measure true transport of adenosine in intact tissues. The uptake of adenosine was inhibited in all three tissues by dipyridamole and 6- thiobenzylpurine ribosides (10 microM) to a similar extent, leaving open the question of why potentiation of the relaxant effects of adenosine is seen in taenia caeci and coronary arteries but not in rabbit intestinal muscle.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Animals; Biological Transport; Cattle; Coformycin; Coronary Vessels; Dipyridamole; Dose-Response Relationship, Drug; Female; Guanosine; Guinea Pigs; In Vitro Techniques; Inosine; Intestinal Mucosa; Male; Muscle, Smooth; Pentostatin; Rabbits; Thioinosine; Thionucleosides

Binding of the potent nucleoside transport inhibitor [3H]nitrobenzylthioinosine to rat and guinea pig lung membranes was investigated. Reversible high-affinity binding was found in both species (apparent KD approximately 0.3nM). Binding was inhibited by nitrobenzylthioguanosine, adenosine and uridine. Dipyridamole was also an effective inhibitor of [3H]nitrobenzylthioinosine binding to guinea pig membranes. In contrast, rat membranes were relatively insensitive to dipyridamole. Exposure of site-bound [3H]nitrobenzylthioinosine to high intensity U.V. light resulted in the photoaffinity labelling of lung proteins with apparent molecular weights similar to that of the human erythrocyte nucleoside transporter (45,000-65,000).

Topics: Adenosine; Affinity Labels; Animals; Binding Sites; Biological Transport; Cell Membrane; Dipyridamole; Guinea Pigs; Inosine; Kinetics; Lung; Male; Nucleosides; Rats; Rats, Inbred Strains; Thioinosine; Uridine

The site-specific binding of the potent and selective nucleoside transport inhibitor, [3H]nitrobenzylthioinosine (NBMPR), to the nucleoside transport system of cardiac membranes of several species was investigated. The affinity of [3H]NBMPR for these sites ranged from 0.03 nM in rat to 0.78 nM in dog. The maximal binding capacity of cardiac membranes for [3H]NBMPR was also species dependent and was greatest in bovine and guinea pig heart (2551 and 1700 fmol/mg protein, respectively) and least in rat (195 fmol/mg protein). The affinities of recognized nucleoside transport inhibitors and benzodiazepines for these transport inhibitory sites in guinea pig and rat heart were estimated by studying the inhibition of the site-specific binding of [3H]NBMPR in competition experiments. These values were compared with their inhibitory effects on the transporter-dependent accumulation of [3H]adenosine in guinea pig and rat cardiac muscle segments and with their ability to potentiate the negative inotropic action of adenosine in electrically driven guinea pig and rat left atria. In guinea pig heart, the recognized nucleoside transport inhibitors and benzodiazepines had an order of affinity (dilazep greater than hydroxynitrobenzylthioguanosine greater than dipyridamole greater than hexobendine much greater than lidoflazine much greater than flunitrazepam greater than diazepam greater than lorazepam greater than flurazepam) for the NBMPR site which was similar to those for the inhibition of [3H]adenosine accumulation and for potentiation of adenosine action. In contrast, in rat heart, where the maximal binding capacity of [3H]NBMPR was lower (eightfold), the nucleoside transporter dependent accumulation of [3H]adenosine was also lower (sixfold) and the negative inotropic action of adenosine was not significantly potentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Benzodiazepines; Biological Transport; Cattle; Drug Synergism; Guinea Pigs; In Vitro Techniques; Inosine; Male; Mice; Myocardial Contraction; Myocardium; Rabbits; Rats; Species Specificity; Thioinosine; Tritium

The kinetic and molecular properties of the adenosine transporter in guinea pig cardiac membranes were studied using nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport. [3H]-NBMPR bound tightly but reversibly to guinea pig cardiac membranes (apparent dissociation constant 0.24 +/- 0.07 nM; maximum binding capacity 1.24 +/- 0.45 pmol of NBMPR bound/mg protein). Reversible high-affinity [3H]NBMPR binding was inhibited in an apparent competitive manner by adenosine (apparent inhibition constant 0.14 mM). L-N-phenylisopropyladenosine (L-PIA) had no effect on NBMPR binding. Exposure of cardiac membranes in the presence of [3H]-NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent molecular weight 66,000-50,000. Covalent attachment of [3H]NBMPR under equilibrium binding conditions was inhibited by adenosine, nitrobenzylthioguanosine , and dipyridamole but was unaffected by the adenosine receptor agonist L-PIA. These data suggest that the photolabeled molecular weight protein (apparent mol wt 66,000-50,000) is involved in adenosine permeation by guinea pig cardiac membranes.

Topics: Adenosine; Affinity Labels; Animals; Biological Transport; Female; Guinea Pigs; Membranes; Myocardium; Photolysis; Thioinosine; Tritium

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Topics: Biological Transport; Dose-Response Relationship, Radiation; Erythrocyte Membrane; Freezing; Humans; Inosine; Kinetics; Thioinosine; Uridine

The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.

Topics: Animals; Biological Transport; Cell Line; Cricetinae; Cricetulus; Female; Hypoxanthine; Hypoxanthines; Inosine; Kinetics; L Cells; Leukemia L1210; Leukemia P388; Liver Neoplasms, Experimental; Lymphoma; Mice; Ovary; Rats; Thioinosine; Tritium; Uridine

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.

Topics: Adenosine; Affinity Labels; Animals; Binding, Competitive; Blood Proteins; Cell Line; Cell Membrane; Dipyridamole; Guanosine; Inosine; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Thioinosine; Thionucleosides; Tritium

Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.

Topics: Affinity Labels; Animals; Carrier Proteins; Cell Fractionation; Cell Membrane; Erythrocyte Membrane; Guinea Pigs; In Vitro Techniques; Liver; Male; Membrane Proteins; Nucleosides; Rats; Rats, Inbred Strains; Thioinosine; Uridine

Topics: Animals; Binding Sites; Biological Transport, Active; Brain; Female; Guinea Pigs; Kinetics; Nucleosides; Thioinosine

The site-specific binding of the potent nucleoside transport inhibitor, [3H]nitrobenzylthioinosine ([3H]NBMPR), to guinea pig cardiac membranes was rapid, reversible and saturable. [3H]NBMPR bound with high affinity to a single class of sites at which the KD was 0.23 +/- 0.07 nM and which had a Bmax of 1700 +/- 290 fmol/mg protein. Several recognized nucleoside transport inhibitors and benzodiazepines inhibited the binding of [3H]NBMPR with an order of potency similar to that observed for the inhibition of the binding of [3H]NBMPR to human erythrocytes and guinea pig synaptosomes.

Topics: Animals; Anti-Anxiety Agents; Benzodiazepines; Binding Sites; Biological Transport; Dilazep; Dipyridamole; Female; Guinea Pigs; In Vitro Techniques; Inosine; Membranes; Myocardium; Thioinosine

Topics: Adenosine; Animals; Brain; Guinea Pigs; In Vitro Techniques; Inosine; Synaptosomes; Temperature; Thioinosine

Both human lymphoblastoid (RPMI 6410) and murine leukemia (L1210) cells were found to have a component of uridine transport which is insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In both cell lines NBMPR-insensitive uridine transport is inhibited by other nucleosides and by the sulfhydryl reagent p-chloromercuribenzenesulfonate. In RPMI 6410 cells NBMPR-insensitive transport accounts for only 2% of the initial rate of uridine transport. In contrast, 20% of the initial rate of transport of L1210 cells is insensitive to NBMPR, and uridine uptake over longer periods (10 min) is completely insensitive to NBMPR.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Biological Transport, Active; Humans; In Vitro Techniques; Inosine; Leukemia L1210; Lymphocytes; Mice; Nucleosides; Thioinosine; Uridine

A major action of adenosine is the induction of profound behavioral inactivity. 6-(4-Nitrobenzyl)-thioinosine (NBI) and 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBG) have been shown to inhibit adenosine uptake. To investigate the possible synergism between exogenous ligand and reuptake inhibition, mice were treated with NBI or NBG + adenosine. NBI and NBG potentiated the effects of adenosine at doses which did not in themselves induce behavioral inactivity. These behavioral results support the proposed role of NBI and NBG as adenosine uptake site blockers which increase synaptic concentrations of adenosine and postsynaptic responses to adenosine in vivo.

Topics: Adenosine; Animals; Arousal; Brain; Guanosine; Inosine; Male; Mice; Mice, Inbred C57BL; Motor Activity; Receptors, Cell Surface; Receptors, Purinergic; Synapses; Thioinosine; Thionucleosides

The characteristics of nucleoside transport were examined in L1210 murine leukemia cells and five other cultured neoplastic cells. The initial rates of uridine, adenosine, and thymidine transport in L1210 cells were only partially inhibited by 1 microM nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in other cells. The IC50 for NBMPR inhibition of uridine transport was 5 nM, but 20% of the activity remained insensitive to concentrations as high as 3 microM. Uridine uptake in the presence of 1 microM NBMPR was saturable and was inhibited by other nucleosides, suggesting the participation of an NBMPR-insensitive transport mechanism. There appeared to be little difference in the specificity of NBMPR-sensitive and -insensitive transport for the physiological nucleosides. Uridine, adenosine, and thymidine were all substrates for both mechanisms, and the Km values for total and NBMPR-insensitive uridine transport were the same (250 microM). Furthermore, little difference was found in the ability of several other nucleosides to inhibit total or NBMPR-insensitive uridine transport. In both cases, adenosine was the most effective inhibitor while cytidine and deoxycytidine were the least effective. The two transport processes did, however, differ from each other in their sensitivity to p-mercuribenzenesulfonate (pMBS). NBMPR-insensitive uridine transport was inhibited by pMBS with an IC50 less than 25 microM, while the IC50 for NBMPR-sensitive transport was greater than 400 microM. Cloning of the parent L1210 cell line indicated that both NBMPR-sensitive and -insensitive transport occurred in the same cell. Both types of uridine transport activity were also observed in three other cell lines (RPMI 6410, L5178Y, and P388), while two lines, S49 and Walker 256, exhibited only NBMPR-sensitive and -insensitive transport, respectively. The level of NBMPR-insensitive transport was an important determinant in the ability of NBMPR to inhibit uridine uptake over prolonged periods (10 min), with as little as 20% NBMPR-insensitive transport sufficient to render uptake over 10 min virtually insensitive to NBMPR. The existence of these two types of nucleoside transport activity in mammalian cells may have important implications in the chemotherapeutic use of transport inhibitors in combination with cytotoxic nucleosides or with inhibitors of pyrimidine and purine biosynthesis.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Biological Transport; Cell Line; Diffusion; Dipyridamole; Leukemia L1210; Mice; Neoplasms, Experimental; Nucleosides; Rats; Thioinosine; Uridine

Nitrobenzylthioinosine (NBMPR) was employed as a covalent probe of the erythrocyte nucleoside transporter. This nucleoside analogue, a potent inhibitor of nucleoside transport, binds tightly (KD = 10(-10) - 10(-9) M) but reversibly to specific sites on the carrier mechanism. High intensity UV irradiation of intact human erythrocytes, isolated "ghosts," and "protein-depleted" membranes in the presence of [3H]NBMPR and dithiothreitol (as a free radical scavenger) under nonequilibrium and equilibrium binding conditions resulted in selective covalent incorporation of 3H into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr = 45,000-65,000). Covalent labeling of band 4.5 protein(s) under equilibrium binding conditions was inhibited by nitrobenzylthioguanosine, dipyridamole, uridine, and adenosine. A similar photolabeling pattern was observed using membranes from pig erythrocytes. In contrast, no incorporation of radioactivity into band 4.5 was observed under equilibrium binding conditions with membranes from nucleoside-impermeable sheep erythrocytes. These experiments suggest that the human and pig erythrocyte nucleoside transporters are band 4.5 polypeptides, a conclusion supported by previous isolation studies based on the assay of reversible [3H]NBMPR binding activity.

Topics: Affinity Labels; Blood Proteins; Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Inosine; Kinetics; Membrane Proteins; Molecular Weight; Nucleoside Transport Proteins; Photolysis; Thioinosine; Uridine

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site--NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.

Topics: Animals; Binding Sites; Biological Transport; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Viral; Clone Cells; Cricetinae; Dilazep; Fibroblasts; Inosine; Kinetics; Mesocricetus; Nucleosides; Thioinosine; Uridine

The kinetics of [3H]nitrobenzylthioinosine binding to human erythrocyte membranes was studied. The pseudo-first-order association was linear and consistent with a simple bimolecular reaction mechanism between nitrobenzylthioinosine and the nucleoside-transport mechanism. Dissociation of the [3H]nitrobenzylthioinosine complex at 22 degrees C was also linear (apparent k-1 congruent to 0.20 min-1). Adenosine was a competitive inhibitor of equilibrium high-affinity [3H]nitrobenzylthioinosine-binding activity (apparent Ki 0.1 mM). Dissociation of the [3H]nitrobenzylthioinosine-membrane complex was faster in the presence of adenosine and uridine, and this effect was proportional to the nucleoside concentration. Nucleoside concentrations less than 1 mM had no significant effect on the dissociation rate constant. In contrast, dissociation was slower in the presence of high concentrations (micromolar) of dipyridamole. Low concentrations of dipyridamole (2-200 nM) and nitrobenzylthioinosine concentrations as high as 2.5 microM had no effect on the rate of [3H]nitrobenzylthioinosine dissociation. These results are discussed in terms of possible distinct inhibitor and permeation sites, and are suggested to be consistent with both a single-site model for the binding of nitrobenzylthioinosine and permeant to the same site, or an allosteric-site model in which permeant and inhibitor bind to different sites.

Topics: Adenosine; Binding Sites; Carrier Proteins; Dipyridamole; Erythrocyte Membrane; Humans; In Vitro Techniques; Inosine; Kinetics; Models, Biological; Nucleosides; Protein Binding; Thioinosine; Uridine

The membrane transport of cytosine arabinoside (araC) has been studied in blasts freshly isolated from a variety of acute leukaemias. The major fraction of araC influx was facilitated and this fraction was 80-87% at l microM araC and 68-80% at 200 microM araC. Competitive kinetics were observed between araC and deoxycytidine for entry into leukaemic blasts and, moreover, araC influx was blocked by phloretin, a broad-spectrum inhibitor of facilitated transport systems. Kinetic analysis of facilitated araC influx gave KmS which varied over a 10-fold range between patients and which were positively correlated to the Vmax. Nucleoside influx Vmax also varied over an 80-fold range between individuals, although the mean araC transport was 4-fold greater in myeloblasts than in lymphoblasts. Larger transport of araC may explain the greater sensitivity of acute myeloid leukaemia to this drug.

Topics: Acute Disease; Adolescent; Adult; Aged; Biological Transport; Cell Membrane; Child; Child, Preschool; Cytarabine; Deoxycytidine; Humans; Infant; Kinetics; Leukemia; Leukocytes; Middle Aged; Phloretin; Thioinosine

Human erythrocyte membranes and partially purified nucleoside transporter (band 4.5 and 7) were photoaffinity labelled with 3H-labelled 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine under equilibrium binding conditions. Band 4.5 was the major site of radiolabelling in both preparations. These experiments provide additional evidence to implicate band 4.5 polypeptides in nucleoside permeation, proteins previously shown to be involved in hexose transport.

Topics: Affinity Labels; Biological Transport; Blood Glucose; Carrier Proteins; Erythrocyte Membrane; Humans; In Vitro Techniques; Membrane Proteins; Monosaccharide Transport Proteins; Nucleosides; Thioinosine

N6-(p-Azidobenzyl)adenosine (ABA) and nitrobenzylthioinosine (NBMPR) were employed as covalent probes of the nucleoside transport mechanism in human erythrocytes. NBMPR, a potent inhibitor of nucleoside transport, binds tightly (KD 0.3-1 nM) to specific sites on nucleoside transporter elements. ABA, a less potent inhibitor of uridine influx, competitively inhibited NBMPR binding (Ki 15 nM). [3H]ABA was bound tightly (KD 13.4 nM) but reversibly to sites on erythrocytes which appeared to be those which bind NBMPR. ABA binding was inhibited by uridine and adenosine. Irradiation with UV light caused site-bound [3H]ABA on erythrocyte membranes to become covalently bound and, similarly, photoactivation resulted in covalent attachment of membrane-bound [3H]NBMPR. In the presence of dithiothreitol, a free radical scavenger, photoactivation of the site-bound 3H-ligand on membranes depleted of extrinsic membrane proteins resulted in selective incorporation of 3H into band 4.5 of the membrane polypeptides which were resolved on sodium dodecyl sulfate-polyacrylamide gel electropherograms. This result, when considered with previous findings, indicates that the NBMPR-binding component of the nucleoside transport mechanism (or the entire mechanism, if the NBMPR site is an integral part) is a band 4.5 polypeptide.

Topics: Affinity Labels; Azides; Dithiothreitol; Erythrocyte Membrane; Erythrocytes; Humans; Inosine; Nucleosides; Photochemistry; Thioinosine; Time Factors; Ultraviolet Rays; Uridine

The interaction of several benzodiazepines (BDZs) with the nucleoside transport system of fresh erythrocytes from humans was investigated. The affinities of BDZs for the nucleoside transport system were estimated by measuring BDZ inhibition of (a) the site-specific binding of nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport, and (b) the uridine transport processes, zero-trans influx, zero-trans efflux, and equilibrium exchange influx. The BDZs inhibited both the inward and outward transport processes, and, for individual agents, inhibition constants (Ki) were similar for the inhibition of each transport process and for the inhibition of the site-specific binding of nitrobenzylthioinosine. The order of potencies of the BDZs in their interactions with the nucleoside transport mechanism (Ro 5-4864 greater than diazepam greater than clonazepam greater than lorazepam greater than flurazepam) is distinct from the potencies of these compounds at BDZ recognition sites. The affinities of the BDZs for the nucleoside transport system, which are about 1000-fold lower than for BDZ recognition sites, suggest that significant inhibition is unlikely to occur with the plasma concentrations (less than 1 microM) that result from usual anxiolytic doses of these agents.

Topics: Benzodiazepines; Binding, Competitive; Biological Transport; Erythrocytes; Humans; Kinetics; Thioinosine; Uridine

The binding of [G-3H]nitrobenzylthioinosine to intact Chinese hamster ovary cells has been studied kinetically and thermodynamically. The association of nitrobenzylthioinosine with cells is a second-order process which proceeds at 24 degrees C with a rate constant of 2 X 10(7) M-1 X S-1. Dissociation of the complex was characterized as a simple first-order process with rate constant on the order of 7 X 10(-3)S-1. The quotient of these is comparable to the dissociation constant as measured in equilibrium binding studies, 2.2 X 10(-10) M. The temperature dependence of the rate of association indicated an Arrhenius activation energy of 8.4 kcal X mol-1, while that of the equilibrium constant for dissociation indicated a standard enthalpy change of 8.8 kcal X mol-1. The large increase in affinity of nitrobenzylthioinosine as compared to natural nucleosides is attributable to an entropy-driven interaction with the binding site. Thymidine, dipyridamole and papaverine each decrease the apparent dissociation constant for the nitrobenzylthioinosine-cell complex; the latter, inhibitors of nucleoside transport, decrease the rate of dissociation of the complex.

Topics: Animals; Carrier Proteins; Cell Line; Cricetinae; Cricetulus; Dipyridamole; Female; Inosine; Kinetics; Nucleosides; Ovary; Papaverine; Temperature; Thermodynamics; Thioinosine; Thymidine

Kinetic characteristics of the transport of uridine, a non-metabolized permeant in human erythrocytes, have been compared in erythrocytes from fresh and outdated stored blood. Uridine transport kinetics in fresh cells conformed to the predictions of a simple carrier model operating with directional symmetry, but in erythrocytes from outdated blood the kinetic characteristics of uridine transport were those of an asymmetric system. The latter result agrees with earlier reports by others. The mobility of the loaded and empty carriers differed by about 6- and 12-fold in fresh and outdated blood, respectively.

Topics: Binding Sites; Biological Transport, Active; Blood Preservation; Erythrocytes; Humans; In Vitro Techniques; Kinetics; Thioinosine; Time Factors; Uridine

Topics: Biological Transport; Dipyridamole; Erythrocytes; Humans; In Vitro Techniques; Inosine; Kinetics; Nucleosides; Temperature; Thermodynamics; Thioinosine

This study demonstrates the existence of adenosine receptor(s) in a homogeneous population of cultured smooth muscle cells and intact muscle strips from rabbit aorta. Binding assays with 3H-adenosine were performed using the crude microsomal fraction of the cultured cells. Adenosine binding was reversible and displayed a single species of saturable binding sites. The nuclear and soluble fractions showed negligible binding. Adenosine uptake blockers (e.g., nitrobenzylthioinosine, dipyridamole) had no effect on its binding. Theophylline inhibited the binding and the relaxation of aortic strips to adenosine. The data suggest the existence of specific binding sites for adenosine on the surface of aortic smooth muscle cells.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Aorta, Thoracic; Dipyridamole; Kinetics; Microsomes; Muscle Relaxation; Muscle, Smooth, Vascular; Rabbits; Receptors, Cell Surface; Receptors, Purinergic; Subcellular Fractions; Thioinosine

1. Nucleoside transport by fetal erythrocytes from nucleoside-permeable and nucleoside-impermeable type new-born lambs and by reticulocytes from adult sheep was compared with that of mature erythrocytes from adult sheep of the two phenotypes.2. Fetal cells and reticulocytes transported [U-(14)C]uridine rapidly, with little difference between cells from the two types of sheep. Transport occurred by a saturable uptake mechanism with similar properties to that present in mature cells from adult nucleoside-permeable type animals, except for an approximately 100-fold higher V(max).3. This increased translocation capacity was associated with increased numbers of high-affinity [(3)H]nitrobenzylthioinosine binding sites ( approximately 2000-3000 sites/cell compared with approximately 20 sites/cell for mature nucleoside-permeable sheep erythrocytes).4. The calculated transport capacity for each nucleoside translocation site is therefore similar in all cell types (140-180 molecules/site. s at 25 degrees C, assuming that each transport site binds a single molecule of inhibitor). These values compare favourably with turnover estimates for the nucleoside transporter from human and pig erythrocytes.5. Loss of nucleoside transport activity after birth closely paralleled loss of [(3)H]nitrobenzylthioinosine binding sites and the progressive loss of fetal cells from the circulation. Similarly, reticulocyte maturation in vitro was also associated with rapid loss of both nucleoside transport capacity and inhibitor binding activity.6. p-Chloromercuriphenylsulphonate and trypsin had no effect on [(3)H]nitrobenzylthioinosine binding to intact fetal cells. In contrast, both agents markedly inhibited binding to isolated ;ghosts' where both sides of the cell membrane were accessible to reagent. p-Chloromercuriphenylsulphonate inhibition was markedly reduced in the presence of uridine, and reversed by addition of dithiothreitol.7. We conclude that nucleoside transport changes during ontogeny and reticulocyte maturation in the sheep as well as species differences in nucleoside transport capacity are regulated by variations in the numbers of functional transport sites per cell rather than by changes in the activity of a constant number of sites. It is also likely that the nucleoside carrier exhibits chemical asymmetry.8. A simple molecular model of the erythrocyte nucleoside transporter consistent with these and other known properties of the carrier is proposed.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Binding Sites; Biological Transport; Cell Membrane Permeability; Erythrocytes; Fetal Blood; Kinetics; Models, Biological; Nucleosides; Reticulocytes; Sheep; Thioinosine; Trypsin; Uridine

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [(3)H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/10(7) cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.

Topics: Acute Disease; Adolescent; Adult; Aged; Binding Sites; Bone Marrow Cells; Carrier Proteins; Cell Transformation, Neoplastic; Child; Child, Preschool; Cytarabine; Humans; Infant; Leukemia; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Middle Aged; Nucleosides; Thioinosine; Time Factors

A procedure is described for determining early time courses of nucleoside uptake by cultured cells in suspension. Replicate samples of cell suspensions were exposed to medium containing 3H-nucleosides for brief intervals (sec) ended by addition of nitrobenzylthioinosine, a potent inhibitor of nucleoside transport that terminated nucleoside uptake virtually instantaneously. Time courses of nucleoside uptake were constructed from the cellular content of nucleoside acquired by the replicate samples during graded intervals of exposure to the labeled permeant. Such time courses were definitive of cellular uptake of nucleosides during the first few sec of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(beta-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin). Defining initial rates of nucleoside uptake as rates of inward transport, relationships between transport rates and extracellular concentrations of these permeants were evaluated in HeLa cells and in two cultured lines of mouse lymphoma L5178Y cells that differ in their abilities to phosphorylate adenosine and tubercidin. Transport rates for these permeants were similar in the two L5178Y cell types and were saturable in the 3 cell lines with Km values between 14 and 38 microM. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.

Topics: Adenosine; Animals; Biological Transport; Cells, Cultured; Humans; Kinetics; Mice; Ribonucleosides; Thioinosine; Tubercidin

Saturable, specific, high affinity binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ([3H]NBI) to brain membranes has been demonstrated. In an effort to test the hypothesis that benzodiazepine action may be due to the inhibition of adenosine uptake, the inhibition of [3H]NBI binding by diazepam, clonazepam and chlordiazepoxide was tested. The benzodiazepines are very weak inhibitors of [#H]NBI binding, having IC50 values several orders of magnitude above their therapeutic levels.

Topics: Adenosine; Animals; Benzodiazepines; Binding Sites; Brain; Cell Membrane; Inosine; Male; Rats; Rats, Inbred Strains; Synaptosomes; Thioinosine; Tritium

Mouse livers were perfused at 22 degrees C with an oxygenated salts medium containing [5-3H]cytidine and [carboxyl-14C]inulin. The cellular uptake of cytidine was determined from the 3H content of liver samples less that present in the extracellular (inulin) space of the samples. Time courses of cytidine uptake were biphasic with initial phases which were approximately linear for 15 s and had time zero values that approximated the extracellular space. Rates of cytidine uptake derived from the initial phase of uptake evidently represented rates of membrane transport because (i) initial rates were saturable, and (ii) cytidine uptake was blocked by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). Treatment of mice with the 5'-monophosphate of NBMPR (greater than 0.2 mg/kg, injected i.p.) 30-40 min prior to the perfusion also blocked cytidine entry into livers. An apparent half-saturation constant of about 10(-3) M and a maximum rate of about 1 mumol . g-1 . min-1 were estimated for the NBMPR-sensitive transport of cytidine into mouse liver cells.

Topics: Adenine Nucleotides; Animals; Cytidine; Erythrocytes; HeLa Cells; In Vitro Techniques; Liver; Male; Mice; Thioinosine

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ([3H]NBI) to brain membrane fractions was investigated. Reversible, saturable, specific, high-affinity binding was demonstrated in both rat and human brain. The KD in both was 0.15 nM with Bmax values of 140-200 fmol/mg protein. Linear Scatchard plots were routinely obtained, indicating a homogeneous population of binding sites in brain. The highest density of binding sites was found in the caudate and hypothalamus in both species. The binding site was heat labile and trypsin sensitive. Binding was also decreased by incubation of the membranes in 0.05% Triton X-100 and by treatment with dithiothreitol and iodoacetamide. Of the numerous salt and metal ions tested, only copper and zinc had significant effects on [3H]NBI binding. The inhibitory potencies of copper and zinc were IC50 = 160 microM and 6 mM, respectively. Subcellular distribution studies revealed a high percentage of the [3H]NBI binding sites on synaptosomes, indicating that these sites were present in the synaptic region. A study of the tissue distribution of the [3H]NBI sites revealed very high densities of binding in erythrocyte, lung, and testis, with much lower binding densities in brain, kidney, liver, muscle, and heart. The binding affinity in the former group was approximately 1.5 nM, whereas that in the latter group was 0.15 nM, suggesting two types of binding sites. The pharmacologic profile of [3H]NBI binding was consistent with its function as the adenosine transport site, distinct from the adenosine receptor, since thiopurines were very potent inhibitors of binding whereas adenosine receptor ligands, such as cyclohexyladenosine and 2-chloroadenosine, were three to four orders of magnitude less potent. [3H]NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain.

Topics: Adenosine; Animals; Biological Transport; Brain; Caudate Nucleus; Cell Membrane; Heart; Humans; Hypothalamus; Inosine; Kinetics; Male; Rats; Rats, Inbred Strains; Synaptosomes; Thioinosine; Tissue Distribution; Tritium

1. Nitrobenzylthioinosine is a potent and specific inhibitor of nucleoside translocation in animal cells. Kinetic and inhibitor binding studies were undertaken to clarify how this inhibitor interacts with the nucleoside transporter from human and nucleoside-permeable type sheep erythrocytes.2. [(3)H]nitrobenzylthioinosine inhibition of zero-trans [U-(14)C]uridine influx into nucleoside-permeable type sheep cells was consistent with simple competitive inhibition (apparent K(i) 1 nmol/l). Analysis of results using total inhibitor levels instead of cell-free inhibitor concentrations did not affect the inhibition pattern, but increased the apparent K(i) value by 5-fold.3. In contrast, [(3)H]nitrobenzylthioinosine was a non-competitive inhibitor of zero-trans [U-(14)C]uridine efflux (apparent Ki 1.5 nmol/l). Dipyridamole, another potent inhibitor of nucleoside translocation, also inhibited zero-trans [U-(14)C]uridine influx in a competitive manner (apparent K(i) 20-40 nmol/l).4. [(3)H]nitrobenzylthioinosine bound to high-affinity sites on cell membranes from human and nucleoside-permeable type sheep cells (apparent K(D) values approximately 1 nmol/l). Binding of inhibitor to these sites was competitively blocked by uridine, a well characterized substrate for the nucleoside transporter (apparent K(i) 1.25 and 0.9 mmol/l, respectively). These apparent K(i) values are close to the apparent K(m) for uridine equilibrium exchange in human erythrocytes.5. Similarly, deoxycytidine was found to be a competitive inhibitor of high-affinity [(3)H]nitrobenzylthioinosine binding activity (apparent K(i) 1.0 and 1.2 mmol/l for human and nucleoside-permeable type sheep cell membranes, respectively). This contrasts with a previous report that this nucleoside had no effect on inhibitor binding activity. Transport studies confirmed that deoxycytidine is a substrate for the erythrocyte nucleoside transporter. Apparent K(m) and V(max) values for [U-(14)C]-deoxycytidine zero-trans influx into human and nucleoside-permeable type sheep cells were comparable to those obtained for [U-(14)C]uridine.6. It is suggested from these results that nitrobenzylthioinosine competes directly with nucleosides for the permeation site of the nucleoside transporter, but that inhibitor binds preferentially to the external membrane surface.

Topics: Animals; Binding Sites; Binding, Competitive; Biological Transport; Cell Membrane Permeability; Deoxycytidine; Dipyridamole; Erythrocytes; Humans; Inosine; Kinetics; Nucleosides; Sheep; Thioinosine; Uridine

Topics: Animals; Biological Transport; Choroid Plexus; Deoxyuridine; Kinetics; Nucleosides; Rabbits; Structure-Activity Relationship; Thioinosine; Thymidine; Tritium

Topics: Adenosine; Affinity Labels; Animals; Brain; In Vitro Techniques; Inosine; Rats; Thioinosine; Tritium

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.

Topics: Animals; Binding Sites; Biological Transport; Dogs; Erythrocytes; Guinea Pigs; Humans; In Vitro Techniques; Inosine; Kinetics; Mice; Rabbits; Rats; Species Specificity; Swine; Thioinosine; Uridine

1. The nucleoside transport system in rat cerebral cortical synaptosomes was investigated using [H3]p-nitrobenzylthioinosine (NBMPR) as a high affinity probe. 2. There are high affinity and low affinity binding sites for NBMPR on rat synaptosomal membranes. The high affinity sites showed a KD value of 0.05 nM and a Bmax value of 113 fmol/mg protein. 3. Biochemical characterization of the high affinity [H3]NBMPR binding sites indicated that they probably correspond to nucleoside transport sites. 4. Several known adenosine uptake inhibitors including clonazepam were tested for their interaction with this high affinity binding site. 5. The results suggest that hexobendine and papaverine inhibit adenosine uptake by occupying the [H3]NBMPR high affinity binding sites. 6. Clonazepam and dipyridamole appear to inhibit adenosine uptake in rat cerebral cortical synaptosomes via an interaction at a different site.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Cerebral Cortex; Inosine; Male; Rats; Rats, Inbred Strains; Synaptic Membranes; Synaptosomes; Thioinosine

Topics: Adenosine; Anti-Anxiety Agents; Benzodiazepines; Binding, Competitive; Biological Transport; Brain; Erythrocytes; Humans; In Vitro Techniques; Inosine; Membranes; Thioinosine

Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.

Topics: 4-Chloromercuribenzenesulfonate; Binding Sites; Biological Transport; Cell Membrane; Drug Synergism; Ethylmaleimide; HeLa Cells; Humans; Inosine; Kinetics; Nucleosides; Thioinosine; Uridine

Previous studies from this laboratory demonstrated that a potent inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), protected cultured cells against cytotoxic nucleosides (nebularine, tubercidin, and toyocamycin). NBMPR and its 5'-monophosphate (NBMPR-P) also protected mice against potentially lethal dosage of these agents. This report describes protection of mice from potentially lethal dosages of tubercidin by administration of NBMPR-P and the use of combinations of these agents in treatments of mice bearing transplanted neoplasms. Treatment of mice bearing i.p. implants of the Ehrlich ascites carcinoma, leukemia L1210/TG8, and colon carcinoma 26 with potentially lethal dosages of tubercidin administered together with host-protecting dosages of NBMPR-P resulted in substantial kill of neoplastic cells and long-term survivors. In these experiments, therapeutic effects were achieved at optimal dosages of NBMPR-P, which protected host vital tissues but did not protect neoplastic cells in ascitic fluids (Ehrlich ascites carcinoma cells and leukemia L1210/TG8 cells). However, at supraoptimal dosages of NBMPR-P, the occurrence of therapeutic failures which were neoplastic deaths indicated that NBMPR-P also protected the neoplastic ascites cells against tubercidin cytotoxicity. Thus, the selectivity of tubercidin toxicity toward cells of the Ehrlich ascites carcinoma and leukemia L1210/TG8 was modified by NBMPR-P dosage.

Topics: Animals; Dose-Response Relationship, Drug; Drug Therapy, Combination; Mice; Neoplasms, Experimental; Ribonucleosides; Thioinosine; Tubercidin

Nitrobenzylthioinosine, a potent nucleoside-transport inhibitor, binds to high-affinity sites on the human erythrocyte membrane. This binding is a specific interaction with functional nucleoside-transport sites. The protein(s) responsible for high-affinity nitrobenzylthioinosine binding was purified 13-fold by treatment of haemoglobin-free 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with Triton X-100 and passage of the soluble extract through a DEAE-cellulose column equilibrated with Triton X-100. Void-volume fractions were collected and treated with Bio-Beads SM-2 to remove detergent. These fractions contained 31% of the starting nitrobenzylthioinosine-binding activity. They also contained D-glucose-sensitive cytochalasin B-binding activity. Nitrobenzylthioinosine binding to the partially purified preparation was saturable (apparent Kd 1.6 nM) and inhibited by nitrobenzylthioguanosine, dipyridamole and uridine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pooled void-volume fractions revealed the presence of only two detectable protein bands, the broad zone 4.5 (containing glucose-transport protein) and a small amount of band 7.

Topics: Biological Transport; Carrier Proteins; Chromatography, DEAE-Cellulose; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Erythrocytes; Humans; In Vitro Techniques; Inosine; Nucleosides; Polyethylene Glycols; Thioinosine

Topics: Adenosine; Animals; Antibiotics, Antineoplastic; Biological Transport, Active; Cell Survival; Cells, Cultured; Leukemia L1210; Membranes; Mice; Nucleosides; Showdomycin; Thioinosine; Time Factors

Cells of an adenosine-resistant clone (AE1) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2'-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE1 cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 X 10(4) sites/cell, Kd = 0.2 nM), whereas site-specific binding of NBMPR to AE1 cells was not demonstrable, indicating that loss of nucleoside transport activity in AE1 cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE1 cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine.

Topics: Adenosine; Animals; Anti-Bacterial Agents; Biological Transport; Cell Division; Cell Line; Clone Cells; Inosine; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Ribonucleosides; Thioinosine

Pig erythrocytes are unable to metabolize glucose and their physiological energy source is unknown. These cells have a high-capacity nucleoside transport system with similar properties to that responsible for nucleoside transport in other species. Nucleoside transport is sufficiently rapid to allow the possibility that inosine and/or adenosine may represent major energy substrates for pig erythrocytes in vivo. Normal and adenosine deaminase-deficient pig erythrocytes have similar ATP levels, suggesting that adenosine is not important in this respect. However, it was calculated that an extracellular inosine concentration of only 40 nM could support the cells' entire energy requirement, a value 40-fold lower than plasma levels of this nucleoside.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Animals; Biological Transport; Dipyridamole; Energy Metabolism; Erythrocytes; Inosine; Kinetics; Swine; Thioinosine

Topics: Biological Transport; Cholic Acids; Erythrocyte Membrane; Erythrocytes; Humans; Inosine; Nucleosides; Thioinosine

Topics: Animals; Cell Membrane Permeability; Erythrocyte Membrane; Erythrocytes; Guanosine; Humans; Inosine; Kinetics; Nucleosides; Sheep; Thioinosine; Thionucleosides

Topics: Binding, Competitive; Dipyridamole; HeLa Cells; Humans; Inosine; Lidoflazine; Nucleosides; Thioinosine; Uridine

Nitrobenzylthioninosine, a potent nucleoside-transport inhibitor, binds specifically to functional nucleoside transport sites. Irradiation of freeze-dried human erythrocyte membranes with high-energy electrons was used to estimate the apparent molecular weight of the nitrobenzylthioninosine-binding complex in situ. The nitrobenzylthioinosine-binding complex had an apparent mol.wt. of 122000.

Topics: Binding Sites; Biological Transport; Electrons; Erythrocyte Membrane; Erythrocytes; Freeze Drying; Humans; In Vitro Techniques; Inosine; Molecular Weight; Nucleosides; Particle Accelerators; Thioinosine

Binding of the potent nucleoside transport inhibitor 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and rates of uptake of several nucleosides were examined at 4-hr intervals during the replication cycle of HeLa S3 cells. Monolayer cultures of synchronous cells, obtained by mitotic detachment, were assayed for high-affinity binding of NBMPR and for rates of uptake of thymidine, uridine, cytidine, adenosine, inosine, and guanosine. The number of NBMPR binding sites per cell doubled between 4 and 16 hr after detachment (late G1 and S phase); during this interval, V max 'S for uptake of cytidine and adenosine doubled, and for uridine and thymidine uptake increased about 4- and 8-fold, respectively. Rates of inosine and guanosine uptake at extracellular concentrations below saturation increased 2-fold between G1 and S phase of the cell cycle. Km 'S for cellular uptake of thymidine, uridine, cytidine, and adenosine did not change with progress through the cycle. The results presented suggest that changes in nucleoside uptake during the HeLa cell cycle were due, in part, to changes in the activity of NBMPR-sensitive transport elements in the membrane.

Topics: Binding Sites; Biological Transport, Active; Cell Cycle; Cell Membrane; HeLa Cells; Humans; Inosine; Nucleosides; Thioinosine

The vascular effects of several purine compounds were evaluated using isolated arteries from bovine heart and tongue. At almost all concentrations tested, adenosine, AMP, ADP, ATP, guanosine, GMP, GDP and inosine produced significant relaxation of the lingual artery. In general, these compounds were much less effective in the coronary artery. Dipyridamole and nitrobenzylthioinosine (NBMPR), compounds which block the cellular uptake of nucleosides, partially prevented the actions of these compounds in the lingual artery but not in the coronary artery. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a potent inhibitor of adenosine deaminase also altered the relaxant effect of adenosine. These results suggest that at least part of the action of purine compounds on the vascular smooth muscle of the lingual artery is a result of an intracellular effect.

Topics: Adenine; Animals; Arteries; Blood Vessels; Cattle; Coronary Vessels; Dipyridamole; Drug Interactions; In Vitro Techniques; Purine Nucleosides; Purine Nucleotides; Thioinosine; Tongue

In the presence of nitrobenzylthioinosine (NBMPR) a potent inhibitor of nucleoside transport, Roswell Park Memorial Institute 6410 cells proliferating in culture were protected from otherwise inhibitory concentrations of 9-beta-D-ribofuranosylpurine (nebularine); cellular uptake of nebularine was greatly reduced under these circumstances. Initial rates of nebularine uptake by Roswell Park Memorial Institute 6410 cells were inhibited by NBMPR, indicating that the latter interfered with nebularine transport. NBMPR protected mice against potentially lethal treatment regimens with nebularine, 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin) or 4-amino-5-cyano-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (toyocamycin); protection resulted when NBMPR was administered i.p. in advance of or simultaneously with nebularine, but not when NBMPR followed nebularine by 1 hr. Both NBMPR and its 5'-monophosphate protected mice against nebularine lethality when administered s.c.

Topics: Animals; Biological Transport; Cells, Cultured; Female; Inosine; Mice; Mice, Inbred Strains; Nucleosides; Purine Nucleosides; Ribonucleosides; Thioinosine

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport, was tested in combination with 1-beta-D-arabinofuranosylcytosine (ara-C) for therapeutic activity against mouse leukemia L1210. NBMPR alone had no activity, whereas therapy with NBMPR and ara-C in combination was significantly better than with ara-C alone. The therapeutic potentiation resulting from the combination of NBMPR and ara-C appeared to be host mediated since NBMPR alone was not toxic to cultured L1210 cells. NBMPR treatment of normal mice increased the plasma half-time of ara-C and decreased rates of urinary excretion of ara-C and 2'-deoxycytidine; however, these effects were not large enough to explain the therapeutic potentiation. Because the drug combination appeared to be no more effective than ara-C alone in therapy of mouse leukemia L1210/TG (a thiopurine-resistant L1210 subline lacking hyposanthine-guanine phosphoribosyltransferase), the host-mediated therapeutic potentiation was attributed in in vivo breakdown of NBMPR to 6-mercaptopurine.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Cytarabine; Drug Synergism; Drug Therapy, Combination; Female; Half-Life; Inosine; Leukemia L1210; Mice; Nitrobenzenes; Sulfides; Thioinosine

Topics: Biological Transport; Carbon Isotopes; Erythrocytes; Humans; Inosine; Kinetics; Methods; Nitrobenzenes; Nucleosides; Sulfides; Thioinosine; Uridine