thioinosine and Hypoxia

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

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

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

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

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

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

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

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

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

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

Isolated guinea pig hearts were used to determine whether an extracellular (interstitial) or intracellular pool of myocardial adenosine is most important in attenuating the catecholamine-induced enhancement of cardiac contractile state and glycogenolysis. Isoproterenol (2 X 10(-8) M) stimulation of hypoxic (30% O2) perfused hearts produced a marked elevation in tissue and effluent perfusate adenosine levels that were greater than the increases observed with the isoproterenol stimulation of oxygenated hearts (95% O2). In the isoproterenol stimulated hypoxic hearts nitrobenzylthioinosine (NBMPR), a potent inhibitor of adenosine cellular transport, further increased tissue adenosine content and markedly decreased the perfusate level of the nucleoside. Assuming that perfusate levels of adenosine correlate directly with extracellular levels, NBMPR was used as a tool to increase the intracellular and decrease the extracellular content of the nucleoside. When compared to responses in oxygenated hearts, hypoxia reduced the isoproterenol-produced increase in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractility but enhanced the increase in glycogen phosphorylase alpha formation. NBMPR completely prevented the reduction of the isoproterenol-induced cyclic AMP and cyclic AMP-dependent protein kinase responses but only partially prevented the attenuation of the contractile response. The increase in phosphorylase alpha formation in the hypoxic isoproterenol stimulated hearts was not influenced by NBMPR. The results suggest that an increase in extracellular adenosine is more influential than an elevation of intracellular adenosine in attenuating beta-adrenoceptor-elicited increases in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractile state.

Topics: Adenosine; Animals; Catecholamines; Cyclic AMP; Extracellular Space; Female; Glycogen; Guinea Pigs; Heart; Hypoxia; Intracellular Fluid; Isoproterenol; Myocardial Contraction; Myocardium; Thioinosine