thioinosine and 6-(4-nitrobenzylthio)guanosine

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

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

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 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

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

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 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

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

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

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 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

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

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

Topics: Animals; Cell Membrane Permeability; Erythrocyte Membrane; Erythrocytes; Guanosine; Humans; Inosine; Kinetics; Nucleosides; Sheep; Thioinosine; Thionucleosides