adenosine-kinase and 4-nitrobenzylthioinosine

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

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

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

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

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 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

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

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

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

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

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

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

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

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 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