thioinosine and 9-(2-hydroxy-3-nonyl)adenine

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

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

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

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

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

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

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

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

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

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 (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

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 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

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 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 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

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

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

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

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

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