n(6)-cyclopentyladenosine 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

The current understanding of the effects of hypoxia on early embryogenesis is limited. Potential mediators of hypoxic effects include adenosine, which increases dramatically during hypoxic conditions and activates A(1) adenosine receptors (A(1)ARs).. To examine the influences of hypoxia and adenosine signaling on cardiac development, chicken embryos were studied. Real time RT-PCR assay was used to examine the A(1)AR gene expression during embryogenesis and after siRNA- mediated knock down. Cell proliferation was determined by counting cell nuclei and PhosphoHistone H3 positive cells. Apoptosis was determined by TUNEL assay.. A(1)ARs were found to be expressed in chicken embryos during early embryogenesis. Treatment of Hamburger and Hamilton stage 4 embryos with the A(1)AR agonist N(6)-cyclopentyladenosine caused cardiac bifida and looping defects in 55% of embryos. Hamburger and Hamilton stage 4 embryos exposed to 10% oxygen for 6, 12, 18, and 24 h followed by recovery in room air until stage 11, exhibited cardia bifida and looping defects in 34, 45, 60, and 86% of embryos respectively. Hypoxia-induced abnormalities were reduced when A(1)AR signaling was inhibited by the A(1)AR antagonist 1,3 dipropyl-8-cyclopentylxanthine or by siRNA-targeting A(1)ARs. Hypoxia treatment did not increase apoptosis, but decreased embryonic cell proliferation.. These data indicate that hypoxia adversely influences cardiac malformations during development, in part by A(1)AR signaling.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Base Sequence; Chick Embryo; Heart Defects, Congenital; Hypoxia; In Situ Nick-End Labeling; Receptor, Adenosine A1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering

Failure to autoresuscitate from apnea has been suggested to play a role in sudden infant death. Little is known, however, about factors that influence the gasping and heart rate response to severe hypoxia that are fundamental to successful autoresuscitation in the newborn. The present experiments were carried out on 184 rat pups to investigate the influence of the parasympathetic nervous system, as well as adenosine, in mediating the profound bradycardia that occurs with the onset of hypoxic-induced primary apnea and in modulating hypoxic gasping. On days 1 to 2, days 5 to 6, and days 10 to 11 postpartum and following bilateral cervical vagotomy (VAG) or administration of a selective adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine; DPCPX), each pup was exposed to a single period of severe hypoxia produced by breathing an anoxic gas mixture (97% N(2)-3% CO(2)). Exposure to severe hypoxia resulted in an age-dependent decrease in heart rate (P < 0.001), accentuated with increasing postnatal age, that was attenuated in all age groups by DPCPX but not by VAG. Furthermore, DPCPX but not VAG decreased the time to last gasp but increased the total number of gasps in the 1- to 2-day-old and 5- to 6-day-old pups but not in the 10- to 11-day-old pups during exposure to severe hypoxia. Thus our data provide evidence that adenosine acting via adenosine A(1) receptors plays a role in modulating hypoxic gasping and in mediating the profound bradycardia that occurs coincident with hypoxic-induced primary apnea in rats during early postnatal life.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Animals, Newborn; Dose-Response Relationship, Drug; Forelimb; Heart Rate; Hypoxia; Parasympathetic Nervous System; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Reflex; Remission, Spontaneous; Vagotomy; Xanthines

Adenosine and adenosine A1 receptor agonists are often, but not always, protective against metabolic insults. The effects of an A1 agonist and antagonist on neuronal death were determined in cortical cell cultures. The A1 agonist cyclohexyladenosine did not attenuate neuronal death induced by oxygen-glucose deprivation, but did attenuate death caused by glucose deprivation or NMDA. Extracellular adenosine levels during oxygen-glucose deprivation were significantly higher than those during glucose deprivation or NMDA exposure. The A1 antagonist 8-cyclopentyltheophylline increased death induced by oxygen-glucose deprivation, but not that caused by glucose deprivation or NMDA exposure. Thus, while activation of A1 receptors can provide neuroprotection, the protective effect appears to become saturated by high levels of endogenous extracellular adenosine during oxygen-glucose deprivation.

Topics: Adenosine; Animals; Cell Culture Techniques; Cell Death; Cerebral Cortex; Excitatory Amino Acid Agonists; Extracellular Space; Glucose; Hypoxia; Mice; N-Methylaspartate; Neurons; Neuroprotective Agents; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline

We compared the modulation of synaptic transmission by adenosine A(1) receptors in the hippocampus of aged (24 months) and young adult rats (6 weeks). The adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, was less potent (P:<0.05) to inhibit synaptic transmission in aged (EC(50)=53 nM) than young adult (EC(50)=14 nM) hippocampal slices, these effects being prevented by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). In contrast with the lower effect of the A(1) receptor agonist, it was observed that blockade of A(1) receptors with DPCPX (50 nM), or removal of endogenous extracellular adenosine with adenosine deaminase (2 u ml(-1)), caused a more pronounced disinhibition of synaptic transmission in aged rats. Also consistent with a more intense A(1) receptor-mediated inhibitory tonus by endogenous adenosine in aged rats was the finding that to fully prevent the depression of synaptic transmission induced by 3 min hypoxia, a higher concentration of DPCPX was required in slices from aged (100 nM) than from young (50 nM) rats. It is concluded that in hippocampal slices of aged rats the efficiency of A(1) receptors to modulate synaptic transmission is reduced, but this may be compensated by an enhanced inhibitory tonus by endogenous adenosine.

Topics: Adenosine; Adenosine Deaminase; Aging; Animals; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; Hippocampus; Hypoxia; In Vitro Techniques; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Synaptic Transmission; Xanthines

1. The effects of adenosine receptor agonists were examined on isolated rings of guinea-pig pulmonary artery under normoxic and hypoxic conditions. The rings were denuded of endothelium and tissues were precontracted with phenylephrine (3 x 10(-6) M) before constructing cumulative concentration-response curves to the agonists. 2. 5'-(N-ethylcarboxamido)adenosine (NECA) caused concentration-dependent contractions of the pulmonary artery which were not different between hypoxia and normoxia. The contractions were converted to a relaxation in the presence of the cyclooxygenase inhibitor, indomethacin, and again these were unaffected by hypoxia. 3. Examination of a range of agonists under normoxic conditions in the presence of indomethacin revealed relaxations, except for the A2a receptor-selective agonist, CGS 21680. The vasorelaxation was therefore A2b receptor-mediated. 4. In hypoxia, however, in the presence of indomethacin, vasoconstriction occurred to R(-)-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and, to a greater extent, to Nb-cyclopentyladenosine (CPA). In the absence of indomethacin, the constriction by CPA during hypoxia was significantly greater. 5. The indomethacin-resistant contraction by CPA was abolished by the A1 receptor antagonist, 8-cyclopentyltheophylline (CPT, 3 x 10(-6) M). 6. This study has demonstrated cyclooxygenase-dependent and-independent vasoconstrictions to adenosine agonists in guinea-pig pulmonary artery under hypoxic conditions. The cyclooxygenase-independent contraction is mediated via A1 receptors. 7. These results suggest that endogenous adenosine released in the pulmonary circulation under hypoxic conditions will cause vasoconstriction and may contribute to the pulmonary hypertension associated with acute respiratory failure.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Guinea Pigs; Hypoxia; In Vitro Techniques; Indomethacin; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Phenethylamines; Phenylisopropyladenosine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Vasoconstriction; Vasodilation

The effects of adenosinergic antagonists caffeine and DPCPX, and of the adenosinergic agonists L-PIA, CPA and CGS 21680 were investigated on fully and partially reversible hypoxia-induced electrophysiological changes in rat hippocampal slices. The influence of a high potassium solution and of the N-methyl-D-aspartate antagonist dizocilpine (MK 801) was also tested. The latency to obtain a 50% decrease in the amplitude of the CA1 population spike (CA1 PS) during a short- (5-10 min) lasting hypoxic period was significantly increased (P less than 0.01) by slice perfusion with caffeine (50 microM), DPCPX (0.2 microM), and by increasing (from 3 to 4 mM) the potassium concentration in the medium bathing the hippocampal slices. The latency was significantly decreased (P less than 0.01) by slice perfusion with L-PIA (0.2 microM) and CPA (0.05 microM). It was not significantly modified by CGS 21680 (5 microM). The incidence of reappearance of the CA1 PS during reoxygenation after long- (45 min) lasting hypoxia was significantly increased (P less than 0.05) by slice perfusion with MK 801 (50 microM), while it was not significantly affected by slice perfusion with caffeine (50 microM) or DPCPX (0.2 microM) or L-PIA (0.2 microM) or CPA (0.05 microM) or CGS 21680 (5 microM). The results indicate a prevalent involvement of the A1 adenosine receptors in the early mechanisms underlying hypoxia-induced reversible changes. Adenosine seems to have a limited role in the late mechanisms occurring after a long-lasting hypoxic period.

Topics: Adenosine; Animals; Caffeine; Dizocilpine Maleate; Electrophysiology; Hippocampus; Hypoxia; In Vitro Techniques; Male; Membrane Potentials; Phenethylamines; Phenylisopropyladenosine; Potassium; Purinergic Antagonists; Rats; Rats, Inbred Strains; Synapses; Xanthines