Adenosine Al Receptors Research Articles

Differential effects of repeated imipramine on hippocampal responsiveness to adenosine and serotonin

Imipramine-induced enhancement of the inhibitory action of 5-HT lA receptor activation in hippocampal pyramidal neurons has been attributed to alterations in the transduction mechanism that involves G protein-dependent opening of K + channels. Postsynaptic 5-HT lA and adenosine Al receptors may share that transduction pathway. We investigated the influence of repeated imipramine administration on 5-HT lA and adenosine A1 receptor-mediated effects in rat hippocampal slices. Repeated imipramine selectively enhanced the postsynaptic effects of 5-HT 1A receptor activation, including hyperpolarization and reduction of input resistance of neurons and reduction of the population spike amplitude. In contrast, after imipramine treatment only the presynaptic effect of adenosine receptor agonists, a decrease of the field excitatory postsynaptic potential, was enhanced. The data demonstrate that alterations in the presumed common transduction mechanism that was postulated for the 5-HT lA and adenosine A1 receptor-mediated activation of K + channels are not involved in the effect of repeated imipramine administration.

SEVOFLURANE PROTECTS HUMAN MYOCARDIUM FROM ISCHEMIA VIA ACTIVATION OF ATP-SENSITIVE POTASSIUM CHANNELS

Introduction: Myocardial protection afforded by the volatile anesthetic isoflurane has been demonstrated to be mediated through activation of adenosine Al receptors and ATP-sensitive potassium (KATP) channels [1,2]. We evaluated the protective role of sevoflurane (Sevo) in ischemic human myocardium. Methods: Trabecular muscles were dissected from human atrial appendages acquired with the permission of the University Human Investigational Committee. Muscles were superfused in a 32 [degree sign]C organ bath with oxygenated Krebs-Henseleit buffer with pH maintained at 7.4 by bubbling with 95% O2/5%CO2. Isometric contractions were electrically stimulated at 1 Hz. Fifteen min. prior to a 60 min. anoxia, muscles were either non-preconditioned or preconditioned for 5 min. with anoxia by replacing 95% O2/5%CO2 with 95% N2/5%CO2. Anesthetic-treated muscles received 2% Sevo during the 5 min. pretreatment interval in the presence or absence of 1 [micro sign]M glibenclamide (Glib), an antagonist of KATP channels or 100 nM 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), an antagonist at adenosine A1 receptors. Post-ischemic recovery of force was measured 90 min. after conclusion of the ischemic insult and is reported as percent of pretreatment force of contraction. Results: In non-preconditioned muscles, post-anoxic force of contraction recovered to only 41.6 +/- 4.4% of pretreatment control. Pretreatment of muscles with anoxia initially decreased contractile force to 28.6 +/- 7.5% and post-anoxic recovery was enhanced to 58.2 +/- 5.7%. Sevo pretreatment resulted in a contractile force reduction to 82.8 +/- 6.6%. Post-anoxic recovery of force in these muscles was 72.0 +/- 6.5% of pretreatment control. Concurrent administration of Glib or DPCPX during the Sevo pretreatment did not alter the initial decrease in force caused by Sevo. However, post-anoxic recovery of Sevo-pretreated muscles was significantly decreased to 41.9 +/- 7.8% pretreatment control by the inclusion of Glib. In contrast, the A1 antagonist DPCPX did not completely inhibit the protection afforded by Sevo. Conclusions: The volatile anesthetic Sevo possesses direct cardioprotective properties in the human myocardium. The protection afforded by Sevo appears to be due to an effect mediated via KATP channels which may not be induced entirely through adenosine A1 receptors. Figure 1Figure 1: Post-Anoxic Recovery of ForceSupported by a grant from Abbott Laboratories and NIH Grant HL09825-02.

No Participation of Adenosine A1 Receptor in Acute Nephrotoxicity by 4-Pentenoic Acid Administration in Dogs

Intrarenal infusion of 4-pentenoic acid is known to lower renal cortical ATP content and cause a reduction in glomerular filtration rate (GFR). The alteration in nucleotide metabolism might augment the production of adenosine, thereby eliciting the fall in GFR. This study was conducted to examine whether 4-pentenoic acid stimulates renal production of adenosine, and if so, to examine the role of adenosine Al receptor in the reduction of GFR by 4-pentenoic acid. With infusion of 4-pentenoic acid (1 μmol-kg^-min-1) into the renal artery of anesthetized dogs, GFR gradually decreased and reached minimum at 60 min with values ranging from 33.9±2.2 to 20.2±2.8 ml/min. Neither renal blood flow nor mean arterial pressure was affected, but tubular reabsorption of water and sodium was significantly attenuated. Renal venous plasma concentration and urinary excretion of adenosine rose markedly (20-fold) without any change in arterial concentration, suggesting that renal adenosine production was augmented by 4-pentenoic acid. However, KW-3902 (8-(noradamantan-3-yl)-l,3-dipropylxanthine), a selective antagonist of the adenosine Al receptor, did not affect the action of 4-pentenoic acid on GFR or renal handling of water and sodium. It is concluded that 4-pentenoic acid markedly increases renal adenosine production, but adenosine Al receptor is not involved in the 4-pentenoic acid-induced nephrotoxicity.

Evidence for physiologically active axonal adenosine receptors in the rat corpus callosum

Several neurotransmitter receptors have been identified on axons, and emerging evidence suggests that central axonal conduction may be modulated by neurotransmitters. We have recently demonstrated the presence of extra-synaptic adenosine Al receptors along rat hippocampal axons. We now present immunocytochemical evidence for Al receptors on rat corpus callosum axons and show that these receptors actively modulate axon physiology. Using rat brain coronal slices, we stimulated the corpus callosum and recorded the evoked extracellular compound action potential. The lipid-soluble, Al-specific adenosine receptor agonist cyclopentyladenosine, dose-dependently decreased the compound action potential amplitude, an effect reversed by the specific Al antagonist 8-cyclopentyl-1,3-dipropylxanthine. These data provide the first direct evidence that axonal Al adenosine receptors modulate axon physiology in the adult mammalian brain. Influencing axonal transmission is a potentially powerful mechanism of altering information processing in the nervous system.

Stress-dependent antinociceptive effects of carbamazepine: A study in stressed and nonstressed rats

1. 1. The present study examined the antinociceptive effects of carbamazepine on the tail flick test in stressed and nonstressed rats. 2. 2. Carbamazepine produced a bimodal antinociceptive effect in stressed rats, the first peak appearing 30 min and the second 4 h after injection. Antinociceptive effect was not observed in nonstressed rats. 3. 3. The secondary, but not the initial, carbamazepine antinociception in stressed rats was blocked by naloxone (0.2 mg/Kg I.P.), an opioid receptor antagonist. 4. 4. Caffeine (5 mg/Kg I.P.), an adenosine A1 A2 receptor antagonist, inhibited the both initial and secondary antinociceptive effects of carbamazepine in stressed rats. 5. 5. Carbamazepine increased the antinociceptive effect induced by either i.p. or i.c.v. administration of N 6-cyclohexyl adenosine (CHA), an adenosine Al receptor agonist, in stressed rats, but decreased it in nonstressed rats. 6. 6. These results suggest that the initial antinociceptive effect of carbamazepine in stressed rats may be produced via an activation of the adensosine A1 receptors, such as was produced by CHA. The secondary long-lasting antinociceptive effects of carbamazepine may be mediated by an activation of opioid systems. 7. 7. Furthermore, the initial activation of the adenosine A1 receptors by carbamazepine may be a triggering factor for the subsequent long-lasting activation of the opioid system, which results in the antinociception effects.

Adenosine A1 receptor agonist GR79236 suppresses apnea during all sleep stages in the rat.

We tested the hypothesis that N-[(1S, trans)-2-hydroxycyclopentyl]adenosine (GR79236), a novel adenosine A1 receptor agonist, would suppress sleep-related apnea in the rat at doses not associated with hypotension or hypothermia. Nine adult Sprague-Dawley rats were instrumented for chronic recording of sleep by electroencephalographic and electromyographic monitoring. Respirations were measured by single chamber plethysmograph, and blood pressure and heart period were transduced by a telemetric implant. Each rat was polygraphically recorded for 6 hours on four occasions in random order, with recordings for an individual animal separated by at least 3 days. Fifteen minutes prior to each recording (0945 hours) each animal received a 1 ml/kg intraperitoneal bolus injection of one of four injectates: saline (control) or 0.03 mg/kg, 0.3 mg/kg, or 3 mg/kg of GR79236. The study was a repeated-measures balanced design such that each animal was recorded exactly once for each injectate. The rate of spontaneous apneas (pauses > 2.5 seconds) was significantly reduced during all sleep stages by all doses of GR79236. At the highest dose, apnea index was reduced by over 70% in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. In contrast, GR79236 had no effect on sleep stage volumes or blood pressure at any dose tested. Heart rate and core temperature were reduced only at the highest dose (3 mg/kg). We conclude that the adenosine A1 receptor agonist GR79236 significantly suppresses apnea expression in all sleep stages at doses not associated with significant changes in sleep architecture, blood pressure, heart rate, or core temperature.

Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum.

The effects of adenosine and magnesium ion (Mg2+) on striatal dopamine release were studied in awake rats by in vivo microdialysis. The mean striatal basal levels of dopamine release at Mg2+ free perfusate were 56.95 +/- 5.30 fmol/sample (for 20 min). By varying the Mg2+ levels in perfusate from 0 mmol/L to 1, 10 or 40 mmol/L, the dopamine release was inhibited by Mg2+ in a level-dependent manner. Perfusion with modified Ringer's solution containing zero Mg2+ and from 5 to 50 mumol/L adenosine, non-selective adenosine agonist, as well as 0.1 mumol/L 2-chloro-N6-cyclopentyladenosine (CCPA), selective adenosine A1 agonist, showed no effect on dopamine release. However, from 5 to 50 mumol/L adenosine and from 0.1 to 1 mumol/L CCPA plus Mg2+ (1 and 40 mumol/L) perfusion decreased the dopamine release. This inhibitory effect of adenosine and CCPA on striatal dopamine release was enhanced by an increase in extracellular Mg2+ levels. Levels of 50 mumol/L of 8-cyclopentyl-1,3-dimethylxanthine (CPT), a selective adenosine A1 receptor antagonist, in perfusate increased the dopamine release under conditions both with and without Mg2+. This stimulatory effect of CPT on striatal dopamine release was reduced by an increase in extracellular Mg2+ levels. As a result, CPT antagonized the inhibitory effects of adenosine and CCPA on dopamine release under conditions of the presence and absence of Mg2+. These results suggest that the inhibition of striatal dopamine release by adenosine was mediated by adenosine A1 receptor. This inhibition was intensified by Mg2+. This study also revealed that the concentrations of Mg2+, which ranged from physiological to supraphysiological, reduced the striatal dopamine release; furthermore it was found that the physiological concentration of Mg2+ potentiated the effects of adenosine agonists, but inhibited adenosine antagonist. Thus, the present study, using in vivo microdialysis preparations, suggests Mg2+ inhibits the calcium ion channels and enhances the adenosinergic function in the central nervous system.

Localisation of the adenosine uptake site in the human brain: a comparison with the distribution of adenosine Al receptors

Using quantitative receptor autoradiography we investigated the distribution of the adenosine uptake site labelled with [ 3H]NBTI in post-mortem human brain and compared its distribution with that of the A1 adenosine receptor labelled with [ 3H]CHA. The highest levels of [3H]NBTI binding were found in the cortex and striatum, with moderate levels in the hippocampus, globus pallidus, cerebellum and some midbrain and spinal cord nuclei. The distribution of A1 receptors and this adenosine uptake site differed in the hippocampus where A1 receptors were highest in CAI but the uptake site was low in CA1 and higher in the molecular layer of the dentate gyrus. These results define the anatomical distribution of the high affinity NBTI sensitive adenosine uptake site in the normal human brain.

P-922 - Protection against cisplatin-induced acute renal failure by KW-3902, an adenosine Al-receptor antagonist, and its combined effect with hydration regimen in rats.

P-922 - Protection against cisplatin-induced acute renal failure by KW-3902, an adenosine Al-receptor antagonist, and its combined effect with hydration regimen in rats.

The roles of spinal adenosine receptors in the control of acute and more persistent nociceptive responses of dorsal horn neurones in the anaesthetized rat.

1. We describe here the effects of intrathecal selective adenosine receptor agonists on acute and more persistent evoked responses of dorsal horn nociceptive neurones recorded in intact rats anaesthetized with halothane. 2. The effects of the A1 receptor agonist, N6-cyclopentyladenosine and the non-selective agonist 2-chloroadenosine as well as the A2a receptor agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride were gauged on the C-, A delta-, A beta-fibre, post-discharge and wind-up responses produced by peripheral tanscutaneous stimulation. The antagonists, theophylline and 8(p-sulphophenyl) theophylline were also tested alone and to reverse the agonist effects. 3. Subcutaneous formalin (5%) was used to produce a more prolonged nociceptive response initiated by peripheral inflammation. 4. Both N6-cyclopentyladenosine and 2-chloroadenosine produced inhibitions of the C-fibre evoked responses, wind-up and post-discharge of the neurones with no significant effects on the A beta responses. By contrast, the A delta evoked responses were facilitated over the same time course and dose-range as the inhibitions. N6-cyclopentyladenosine was more potent and effective than 2-chloroadenosine. In marked contrast to these agonists, the A2a agonist produced only weak non-specific inhibitions. Theophylline and 8(p-sulphophenyl) theophylline alone had no effect on the acute responses but prevented or reversed inhibitory effects of N6-cyclopentyladenosine. 5. The formalin response was markedly inhibited by spinal N6-cyclopentyladenosine with both the acute first phase and more prolonged second phase being dose-dependently inhibited. N6-cyclopentyladenosine was considerably more potent on the formalin response than on the other neuronal measures. 6 The results suggest a role of adenosine Al receptors in the modulation of both acute and inflammatory nociception in the spinal cord.

8-(Noradamantan-3-yl)-1,3-dipropylxanthine

The title compound, KW-3902 {1,3-dipropyl-8-(3-tricyclo[3.3.1.0 3,7 ]nonyl)-3,7-dihydro-1H-purine-2,6-dione, C 20 H 28 N 4 O 2 }, is a selective adenosine Al-receptor antagonist. In the crystal of KW-3902, the mirror plane of the 3-noradamantyl group is nearly in the plane of the xanthine moiety. The two propyl side chains have fully extended conformations and are on the same side of the xanthine plane.

Adenosine A1 receptor-mediated changes in basal and histamine-stimulated levels of intracellular calcium in primary rat astrocytes.

1. The effects of adenosine A1 receptor stimulation on basal and histamine-stimulated levels of intracellular free calcium ion concentration ([Ca2+]i) have been investigated in primary astrocyte cultures derived from neonatal rat forebrains. 2. Histamine (0.1 microM-1 mM) caused rapid, concentration-dependent increases in [Ca2+]i over basal levels in single type-2 astrocytes in the presence of extracellular calcium. A maximum mean increase of 1,468 +/- 94 nM over basal levels was recorded in 90% of type-2 cells treated with 1 mM histamine (n = 49). The percentage of type-2 cells exhibiting calcium increases in response to histamine appeared to vary in a concentration-dependent manner. However, the application of 1 mM histamine to type-1 astrocytes had less effect, eliciting a mean increase in [Ca2+]i of 805 +/- 197 nM over basal levels in only 30% of the cells observed (n = 24). 3. In the presence of extracellular calcium, the A1 receptor-selective agonist, N6-cyclopentyladenosine (CPA, 10 microM), caused a maximum mean increase in [Ca2+]i of 1,110 +/- 181 nM over basal levels in 30% of type-2 astrocytes observed (n = 53). The size of this response was concentration-dependent; however, the percentage of type-2 cells exhibiting calcium increases in response to CPA did not appear to vary in a concentration-dependent manner. A mean calcium increase of 605 +/- 89 nM over basal levels was also recorded in 23% of type-1 astrocytes treated with 10 microM CPA (n = 30). 4. In the absence of extracellular calcium, in medium containing 0.1 mM EGTA, a mean increase in [Ca2+]i of 504 +/- 67 nM over basal levels was recorded in 41% of type-2 astrocytes observed (n = 41) after stimulation with 1 microM CPA. However, in the presence of extracellular calcium, pretreatment with the A1 receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, for 5-10 min before stimulation with 1 microM CPA, completely antagonized the response in 100% of the cells observed. 5. In type-2 astrocytes, prestimulation with 10 nM CPA significantly increased the size of the calcium response produced by 0.1 microM histamine and the percentage of responding cells. Treatment with 0.1 microM histamine alone caused a mean calcium increase of 268 +/- 34 nM in 41% of the cells observed (n = 34). After treatment with 10 nM CPA, mean calcium increase of 543 +/- 97 nM was recorded in 100% of the cells observed (n = 33). 6. These data indicate that adenosine Al receptors couple to intracellular calcium mobilization and extracellular calcium influx in type-1 and type-2 astrocytes in primary culture. In addition, the simultaneous activation of adenosine Al receptors on type-2 astrocytes results in an augmentation of the calcium response to histamine H1 receptor stimulation.

P2-purinoceptor-mediated inhibition of noradrenaline release in rat atria.

1. We looked for P2-purinoceptors modulating noradrenaline release in rat heart atria. Segments of the atria were preincubated with [3H]-noradrenaline and then superfused with medium containing desipramine (1 microM) and yohimbine (1 microM) and stimulated electrically, by 30 pulses/1 Hz unless stated otherwise. 2. The adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; EC50 9.7 nM) and the nucleotides, ATP (EC50 6.6 microM) and adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; EC50 4.8 microM), decreased the evoked overflow of tritium. The adenosine A2a-agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.03-0.3 microM) and the P2x-purinoceptor agonist beta, gamma-methylene-L-ATP (30 microM) caused no change. 3. The concentration-response curve of CPA was shifted to the right by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX; 3 nM; apparent pKB value 9.7) but hardly affected by the P2-purinoceptor antagonist, cibacron blue 3GA (30 microM). In contrast, the concentration-response curves of ATP and ATP gamma S were shifted to the right by DPCPX (3 nM; apparent pKB values 9.3 and 9.4, respectively) as well as by cibacron blue 3GA (30 microM; apparent pKB values 5.0 and 5.1, respectively). Combined administration of DPCPX and cibacron blue 3GA caused a much greater shift of the concentration-response curve of ATP than either antagonist alone. The concentration-response curve of ATP was not changed by indomethacin, atropine or the 5'-nucleotidase blocker alpha, beta-methylene-ADP. 4. Cibacron blue 3GA (30 microM) increased the evoked overflow of tritium by about 70%. The increase was smaller when the slices were stimulated by 9 pulses/O00 Hz instead of 30 pulses/I Hz.5. The results indicate that the postganglionic sympathetic axons in rat atria possess P2-purinoceptors in addition to the known adenosine Al-receptor. Both mediate inhibition of noradrenaline release. Some adenine nucleotides such as ATP and ATP gamma S act at both receptors. The presynaptic P2-purinoceptor seems to be activated by an endogenous ligand, presumably ATP, under the condition of these experiments. This is the first evidence for presynaptic P2-purinoceptors at cardiac postganglionic sympathetic axons.

Biochemical and pharmacological evidence for the presence of A1 but not A2 a adenosine receptors in the brain of the low vertebrate teleost Carassius auratus (Goldfish)

In whole brain membranes of goldfish, 3H-chlorocyclopentyladenosine bound to adenosine A1 receptors. The A1 receptors were ubiquitously distributed in the brain with a maximum in the hypothalamus and a minimum in the spinal cord. In superfused goldfish cerebellar slices, cyclohexyladenosine inhibited the cyclic AMP accumulation stimulated by forskolin and the selective adenosine Al receptor antagonist, 8-cyclopentyltheophylline, reversed this effect. In the same brain preparation, 30 mM K + stimulated the release of glutamate, glutamine, glycine and GABA in a Ca 2+-dependent manner, whereas the aspartate and taurine release was Ca 2+-independent. Cyclohexyladenosine, in a dose-dependent manner, inhibited the 30 mM K +-evoked release of glutamate whereas that of aspartate was unaffected. The CHA inhibition of glutamate-evoked release was reversed by 8-cyclopentyltheophylline. The adenosine A2 a receptors were not detectable in whole brain membranes of goldfish either using the specific agonist 3H-CGS 21680 or 3H-5′- N-ethylcarboxamidoadenosine. The presence of A2 b seems to be suggested by the NECA stimulation of cyclic AMP accumulation, which was reversed by 8-cyclopentyltheophylline. The results, taken together, indicate that adenosine has a neuromodulatory function in the nervous system of lower vertebrates which is comparable to that described in mammalian brain.

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia. Two site analysis revealed that the agonists bind to both a high and low affinity state of the receptor.4. The existence of Al binding sites in the rat vasa deferentia, colon muscularis mucosae and duodenum, and their absence in the urinary bladder, is consistent with previous functional studies.However, in contrast to the findings of the [3H]-DPCPX binding assay, no functional response mediated by adenosine Al receptors could be detected by measuring contractile or relaxant responses to CPA in the colon longitudinal muscle. The functional significance of the binding sites in this tissue has therefore yet to be determined.

Endogenous adenosine exerts inhibitory effects upon the development of spreading depression and glutamate release induced by microdialysis with high K + in rat hippocampus

Spreading depression (SD) is known to be involved in the N-methyl- D-aspartate receptor-mediated neuronal damage. In urethane-anesthetized rats, we examined the release of adenosine and glutamate during SD induced by microdialysis of high K + perfusate through the hippocampal CA1 area. The effects of endogenous adenosine upon SD were studied by applying an adenosine antagonist, theophylline (1 mM) and by a simultaneous application of adenosine uptake blockers, dipyridamole (DPR) (100 μM) and nitrobenzylthioinosine (NBI) (50 μM). The dialysates were sampled every 5 or 10 min and analyzed by HPLC. SD was identified by flattening of background EEG and disappearance of population spikes recorded from the pyramidal cell layer of CA1 area by a glass microelectrode. Adenosine and glutamate release was enhanced significantly in association with the occurrence of SD. Theophylline increased the release of glutamate and the incidence of SD and decreased the latency of the SD occurrence. DPR + NBI decreased the release of glutamate and the occurrence of SD, but increased extracellular adenosine concentration. The effects of DPR + NBI were blocked by application of a selective antagonist of adenosine Al receptor, 8-cyclopentyl-l,3-dipropylxanthine (DPCPX, 0.1 μM). These findings suggest that endogenous adenosine exerts inhibitory influences upon the development of SD and the glutamate release through the Al receptor in rat hippocampus.

Functional adenosine al receptors in goldfish brain: Regional distribution and inhibition of K +-evoked glutamate release from cerebellar slices

In goldfish brain, [ 3H]cyclohexyladenosine binding sites are ubiquitously distributed with a maximum in the hypothalamus and a minimum in the spinal cord. The binding parameters measured in cerebellar membranes ( K d = 0.88 ± 0.08nM; B max = 59.65 ± 2.62fmol/mg protein) are not significantly different from those of the whole brain. In perfused goldfish cerebellar slices, stimulation of cyclic AMP accumulation by 10 −5 M forskolin was markedly reduced (58.7%) by treatment with 10 −4 M cyclohexyl-adenosine, an adenosine Al receptor agonist, and the reduction was reversed in the presence of 10 −4 M 8-cyclopentyltheophylline, a selective Al receptor antagonist. In the same brain preparation, 30 mM K + stimulated the release of glutamate, glutamine, glycine and GABA in a Ca 2+-dependent manner, whereas the aspartate and taurine release was Ca 2+-independent. Cyclohexyladenosine inhibited the 30 mM K +-evoked release of glutamate in a dose-related manner. This effect was reversed by 8-cyclopentyltheophylline. These results support the hypothesis that adenosine A1 receptors present in goldfish cerebellum are involved in the modulation of glutamate transmitter release.

Characterization of the adenosine receptor mediating contraction in rat colonic muscularis mucosae

1. The objective of this study was to characterize the adenosine receptor mediating contraction in rat isolated colonic muscularis mucosae (RCMM). 2. Sequential additions of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.01-10 microM) elicited reproducible, concentration-related contractions in RCMM. The effects of NECA were mimicked by the adenosine A1 receptor-selective agonists cyclopentyladenosine (CPA), R-phenylisopropyladenosine (R-PIA) and N-[1S, trans)2-hydroxycyclopentyl] adenosine (GR79236) and by S-PIA (the stereoisomer of R-PIA). The adenosine A2 agonists N-[(2-methylphenyl)methyl] adenosine (metrifudil) and 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS21680) also produced contractions in RCMM but were 54 and 165 times less potent respectively than NECA. The rank order of agonist potency for contraction of RCMM was CPA > or = GR79236 = R-PIA > or = NECA > > S-PIA = metrifudil > CGS21680, which is identical to that reported for the inhibition of spontaneous rate in rat isolated right atria and inhibition of lipolysis in rat isolated adipocytes by these same agonists. 3. R-PIA, S-PIA and metrifudil behaved as partial agonists in RCMM. 4. The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3- dipropylxanthine (DPCPX) inhibited the contractions produced by all the adenosine agonists tested, with pKB values between 9.2 and 9.5. The non-selective adenosine antagonist 8-phenyltheophylline (8-PT) antagonized the effects of NECA but also markedly potentiated (by 93.0 +/- 10.2% at 3 microM) the maximum contractile response to NECA in RCMM. Neither 8-PT (3 microM) nor DPCPX (0.1 microM) had any effect on the contractions produced by carbachol. 5. The contractile responses to NECA in RCMM were not affected by atropine (1 microM), tetrodotoxin(0.3 microM) or the P2 antagonist, suramin (100 microM).6. The present study confirms that contractions to adenosine agonists in the RCMM are mediated via adenosine Al receptors.

Effect of an intracellular calcium chelator on the regulation of electrically evoked [3H]‐noradrenaline release from rat hippocampal slices

1. The electrically (3 Hz, 5 min) evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal slices was reduced by prior treatment of the slices with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetomethylester (BAPTA/AM) in a concentration-(10 to 500 microM) dependent manner (40% at 30 microM). Reduction of medium calcium from 1.3 to 0.5 mM caused a larger (70%) decrease. BAPTA free acid (100 mM), a non-permeable Ca(2+)-chelator had no significant effect. 2. Basal [3H]-noradrenaline release was reduced by BAPTA/AM in a concentration-dependent manner (50% at 30 microM), but reduction of external Ca2+ from 1.3 to 0.5 mM did not alter basal release. 3. About 10% of total [3H]-NA in the slices was released at 3 Hz stimulation in 1.3 mM Ca2+ buffer. Addition of the alpha 2-adrenoceptor antagonist, idazoxan (1 microM), increased electrically evoked [3H]-NA release to 26% but stimulated release was not altered by the adenosine A1-receptor antagonist, 8-cyclopentyl theophylline (8-CPT) (1 microM). 4. Evoked release was reduced by the alpha 2-receptor agonist, UK 14,304, in a concentration-dependent manner in the presence of 8-CPT (1 microM). The magnitude of this effect was not altered by the treatment of slices with 30 microM BAPTA/AM. 5. The adenosine A1 receptor agonist, N6-cyclohexyl adenosine (CHA) (1 microM) inhibited electrically evoked [3H]-NA release by about 40% in the presence of idazoxan (1 microM). The effect of CHA was not significantly altered by treatment of slices with BAPTA/AM. 7. The present results show that spontaneous [3H]-NA release is affected by reduction of intracellular Ca2+, but not by reduction of extracellular Ca2+ or by the presynaptic agonists or w-conotoxin. By contrast, electrically evoked release was affected more strongly by alterations of extracellular Ca2+ than by buffering intracellular Ca2+. The reduction of electrically evoked [3H]-NA release by agonists at the adenosine Al-receptor and a2-adrenoceptor is probably mediated through the control of Ca2+ entry via membrane ion channels or at a low affinity Ca2'-site governing evoked release.

Role of adenosine receptor activation in myocardial infarct size limitation by ischaemic preconditioning.

The aims were to examine the role of adenosine receptors in the mechanism of preconditioning in a chronic rabbit model of myocardial infarction; to assess whether the preconditioning effect is blocked by an adenosine receptor antagonist, 8-phenyltheophylline; and to determine whether an adenosine A1 receptor agonist, R(-)N6-2-phenylisopropyl adenosine (R-PIA), mimics infarct size limitation by preconditioning. Myocardial infarction was induced in male rabbits by occlusion of the left coronary artery for 30 min, which was followed by 72 h reperfusion. Before the 30 min ischaemia, rabbits were subjected to one of the following six protocols: (1) untreated control; (2) intravenous injection of 8-phenyltheophylline; (3) preconditioning with 5 min ischaemia; (4) pretreatment with 8-phenyltheophylline plus preconditioning; (5) intravenous injection of R-PIA; or (6) R-PIA plus atrial pacing (240.min-1). Infarct size and area at risk were determined by histology and fluorescent particles, respectively. Preconditioning significantly limited infarct size, normalised as a percent of area at risk (%IS/AR), to 19.2 (SEM 2.3)% v control value of 46.5(2.8)%. 8-Phenyltheophylline alone did not modify the %IS/AR, but its injection before preconditioning attenuated the preconditioning effect such that IS/AR = 34.4(2.5)%. While R-PIA did not achieve statistically significant myocardial salvage, R-PIA plus atrial pacing limited infarct size to 33.7(3.0)% (p<0.05 v control). The R-PIA group had severe hypotension and their infarct sizes were inversely correlated with diastolic blood pressure at reperfusion. There was no such correlation in the R-PIA plus pacing group in which bradycardia and hypotension induced by R-PIA were attenuated by atrial pacing. The infarct size limiting effect of preconditioning was attenuated by 8-phenyltheophylline, and pretreatment with R-PIA was able to limit myocardial infarct size when severe hypotension was avoided by atrial pacing. These findings suggest that adenosine receptor activation plays a crucial role in the mechanism of preconditioning.