Adenosine depresses spontaneous transmitter release from frog motor nerve terminals by acting at an A1-like receptor

Adenosine (1 microM to 1 mM) depressed spontaneous transmitter release from frog motor nerve terminals without producing any observable postsynaptic effects. Since this action of adenosine was blocked by 20 microM theophylline and 1 microM 8-phenyltheophylline, adenosine probably acts at a specific receptor on motor nerve terminals to reduce spontaneous transmitter output. The effects of the adenosine analogs, L-N6-phenylisopropyladenosine (L-PIA, 100 pM to 1 microM), D-PIA (100 nM to 100 microM), and 5'-N-ethylcarboxamidoadenosine (NECA, 10nM to 100 microM), were tested on spontaneous transmitter release at the frog neuromuscular junction. L-PIA depressed mepp frequency at a threshold concentration of about 1 nM, was thirteen times more potent than NECA, and was 294 times more effective than D-PIA. The rank-order potency of these analogs indicates that adenosine acts at an A1-like receptor to depress spontaneous transmitter release. Inhibitory actions of maximally effective concentrations of adenosine and L-PIA were also blocked by the A1-specific antagonist, 1-3-dipropyl-8-cyclopentylxanthine (DPCPX) at a concentration of 100 nM. Micromolar concentrations of NECA, an agonist with approximately equal affinity for the A1 and A2 receptors, produced biphasic effects on mepp frequency. Thus, a second adenosine receptor, perhaps of the A2 subtype, may be present on motor nerve terminals and may mediate an increase in spontaneous transmitter release.     

Comparison of the inhibition of insulin release by activation of adenosine and alpha 2-adrenergic receptors in rat beta-cells.

Rat islets were used to compare the mechanisms whereby adenosine and adrenaline inhibit insulin release. Adenosine (1 microM-2.5 mM) and its analogue N6(-)-phenylisopropyladenosine (L-PIA) (1 nM-10 microM) caused a concentration-dependent but incomplete (45-60%) inhibition of glucose-stimulated release. L-PIA was more potent than D-PIA [the N6(+) analogue], but much less than adrenaline, which caused nearly complete inhibition (85% at 0.1 microM). 8-Phenyltheophylline prevented the inhibitory effect of L-PIA and 50 microM-adenosine, but not that of 500 microM-adenosine or of adrenaline. In contrast, yohimbine selectively prevented the inhibition by adrenaline. Adenosine and L-PIA thus appear to exert their effects by activating membrane A1 receptors, whereas adrenaline acts on alpha 2-adrenergic receptors. Adenosine, L-PIA and adrenaline slightly inhibited 45Ca2+ efflux, 86Rb+ efflux and 45Ca2+ influx in glucose-stimulated islets. The inhibition of insulin release by adenosine or L-PIA was totally prevented by dibutyryl cyclic AMP, but was only attenuated when adenylate cyclase was activated by forskolin or when protein kinase C was stimulated by a phorbol ester. Adrenaline, on the other hand, inhibited release under these conditions. It is concluded that inhibition of adenylate cyclase, rather than direct changes in membrane K+ and Ca2+ permeabilities, underlies the inhibition of insulin release induced by activation of A1-receptors. The more complete inhibition mediated by alpha 2-adrenergic receptors appears to result from a second mechanism not triggered by adenosine.     

Adenosine analogs: potentiation of bradykinin-induced plasma exudation in rat skin and prevention by caffeine and theophylline

Adenosine and various analogs potentiated plasma exudation elicited by bradykinin in rat skin using 125I-labelled bovine serum albumin (125I-BSA) as a tracer. L-N6-Phenylisopropyladenosine (L-PIA) was much more effective than D-PIA, adenosine, N6-cyclohexyladenosine (CHA) and 2-chloroadenosine, all of which were comparable in activity. Adenosine 5'-cyclopropylcarboxamide was the least effective analog. Caffeine and theophylline had no effect on basal or bradykinin-elicited plasma exudation, while inhibiting plasma exudation elicited by L-PIA, CHA or a combination of bradykinin and L-PIA. 8-Phenyltheophylline was more potent than caffeine or theophylline versus the bradykinin and L-PIA combination. 2',5'-Dideoxyadenosine, a P-site inhibitor of adenylate cyclase, had no effect on plasma exudation elicited by bradykinin, L-PIA or a combination of bradykinin and L-PIA, but did inhibit plasma exudation elicited by prostaglandin E1 (PGE1) or a bradykinin-PGE1-combination. The antihistamine cyproheptadine slightly reduced plasma exudation elicited by a bradykinin-PGE1 combination. The results suggest that adenosine potentiates bradykinin-induced plasma exudation via an adenosine receptor and that histamine may be involved to some extent in the phenomenon.     

Influence of adenosine analogs on morphine tolerance and dependence in mice

A number of adenosine agonists were investigated for possible actions on tolerance to morphine withdrawal in mice. The induction of tolerance to a sustained release preparation of morphine was assessed by measuring the analgesic effect induced by a test dose of the drug. The concomitant treatment with L- and D-phenylisopropyl adenosine, (L- and D-PIA), cyclopentyladenosine (CPA) or chloroadenosine (CADO) during the period of morphine absorption did not alter the induction of the process. In contrast cyclohexyladenosine (CHA) significantly decreased the intensity of tolerance. The administration of naloxone 30 hrs, after the priming dose of morphine induced an intense withdrawal reaction. The intensity of the abstinence syndrome was decreased by the administration of L-PIA, CHA or CADO; CPA and D-PIA were ineffective. These results suggest that adenosine analogs may interfere with the known morphine effects on calcium disposition in nerve terminals.     

N6-substituted 9-methyladenines: a new class of adenosine receptor antagonists

A series of 15 N6-substituted 9-methyladenines have been assessed as antagonists of A2-adenosine receptor-mediated stimulation of adenylate cyclase in membranes of human platelets and rat PC12 cells and of A1-adenosine receptor-mediated inhibition of adenylate cyclases in membranes of rat fat cells and as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine to A1-adenosine receptors in rat brain membranes. N6 substitution can markedly increase the potency of 9-methyladenine at A1 receptors, while having lesser effects or even decreasing potency at A2 receptors. Effects of N6 substituents on adenosine receptor activity of the 9-methyladenines are reminiscent of effects of N6 substituents on activity of adenosine, suggesting that N6 substituted 9-methyladenines bind to adenosine receptors in the same orientation as do N6-substituted adenosines. N6-Cyclopentyl-9-methyladenine with Ki values at the A1 receptors of 1.3 microM (fat cells) and 0.5 microM (brain) is at least 100-fold more potent than 9-methyladenine (Ki 100 microM, both receptors), while at the A2 receptors KB values of 5 microM (platelets) and 25 microM (PC12 cells) make it 5-fold more potent and equipotent, respectively, compared to 9-methyladenine (KB 24 microM, both receptors). N6-Cyclopentyl and several other N6-alkyl and N6-cycloalkyl analogs are selective for A1 receptors while 9-methyladenine is the most A2 receptor selective antagonist. The N6-R- and N6-S-(1-phenyl-2-propyl)-9-methyladenines, analogous to N6-R- and N6-S-phenylisopropyladenosines, exhibit stereoselectivity at both A1 and A2 receptors. Marked differences in potency of certain N6-substituted 9-methyladenines at the A2 receptors of human platelets and rat PC12 cells provide evidence that these are not identical receptors.     

Discriminative stimulus properties of L-phenylisopropyl adenosine: blockade by caffeine and generalization to 2-chloroadenosine

Recent neurochemical data on the effects of activation and blockade of adenosine A1 receptors has suggested a direct role of adenosine in neurotransmission. The present research used a drug discrimination procedure to test the hypotheses that A1 adenosine receptor activation could serve as a discriminative stimulus and that caffeine, a drug believed to be an A1 receptor antagonist, could block the adenosine discrimination. Food-deprived rats were trained to press one of two levers on an FR 10 schedule of food-pellet delivery. Responses on one lever were reinforced following i.p. injection of N6 - (L-phenylisopropyl) adenosine (L-PIA); responses on the other lever were reinforced following i.p. injection of saline. L-PIA training dose was increased from 0.064 to 0.08 mg/kg L-PIA in the course of the study. Subjects required an average of 91 sessions to acquire this discrimination. Stimulus control by L-PIA was dose-dependent, with the ED-50 being approximately 0.03 mg/kg. 2-Chloroadenosine (2CA) generalized to L-PIA with a tenth the potency. Caffeine blocked L-PIA-induced lever selection. These results indicate that 1) rats can be trained to discriminate L-PIA from saline in a two-lever food-reinforced task and 2) the discriminative stimuli produced by L-PIA are based on its agonistic action at the adenosine A1 receptor.     

Pertussis toxin pretreatment reveals differential effects of adenosine analogs on IgE-dependent histamine and peptidoleukotriene release from RBL-2H3 cells

The effects of adenosine (A) and the nonmetabolizable adenosine analogs, N-ethylcarboxamidoadenosine (NECA), L-phenylisopropyladenosine (L-PIA), D-PIA and 2-chloroadenosine (2CHA) were examined on the IgE-dependent mediator release from RBL-2H3 cells, a model for mast-cell function. Adenosine and the adenosine analogs failed to influence mediator release from cells, previously sensitized with monoclonal anti-TNP mouse immunoglobulin E (anti-TNP IgE), when added alone. When added prior to conjugated trinitrophenol-ovalbumin (TNP-OVA), adenosine and the adenosine analogs (10(-8)-10(-4) M) significantly potentiated the release of both histamine (marker for degranulation) and peptidoleukotrienes (LT) (marker for de novo synthesized mediators). The effects were concentration-dependent with the potency order being L-PIA greater than NECA greater than A greater than D-PIA, 2CHA. The stimulatory effect on both histamine and LT release were reversed by prior treatment of the cells with pertussis toxin but not by the purinoceptor antagonists, theophylline and 8-phenyltheophylline, nor adenosine uptake blockers. At higher concentrations (above 10(-5) M), adenosine and adenosine analogs were also inhibitory on LT but not on histamine release. This inhibition was more evident on pertussis-toxin-treated cells in which there was no effect of adenosine or adenosine analogs on histamine release, but a concentration-dependent inhibition of IgE-dependent LT release. These findings demonstrate that adenosine analogs have two distinct mechanisms on mediator release from RBL-2H3 cells; a stimulatory effect on both histamine and LT release, mediated via a pertussis-toxin-sensitive G protein and an inhibitory effect on LT release via a pertussis-toxin-insensitive pathway. An abstract of this work has been published.     

Effects of adenosine on the secretions of glucagon and insulin of isolated ad perfused pancreas of the rat

The effects of adenosine on insulin and glucagon secretions were studied using the isolated perfused rat pancreas. The secretion of glucagon was stimulated by adenosine at concentrations ranging from 1.65 to 165 mumol/l, in the presence of glucose 0.5 g/l; the stimulation was immediate, but transient and was dose-dependent. Insulin secretion was not changed by adenosine in the presence of glucose 0.5 g/l; in the presence of glucose 1.5 g/l, adenosine at 1.65 and 16.5 mumol/l did not significantly modify insulin secretion. But at 165 mumol/l adenosine induced a progressive increase in time after the 5th minute. The A cell appears then to be much more sensitive to adenosine than the B cell.     

Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.     

L-phenylisopropyladenosine (L-PIA) diminishes halothane anesthetic requirements and decreases noradrenergic neurotransmission in rats

The effect of L-phenylisopropyladenosine (L-PIA), the A1 adenosine agonist, on the depth of anesthesia was investigated in halothane-anesthetized rats. L-PIA treatment reduced the minimum anesthetic concentration (MAC) of halothane that prevented 50% of animals from moving in response to a painful stimulus by 49%. MAC experiments performed with L-PIA given in conjunction with A1 adenosine receptor antagonists which either permeate the blood-brain barrier (8-phenyltheophylline [8-PT] or do not (8-sulphophenyltheophylline [8-So-PT]) indicate that central mechanisms are involved. Noradrenergic neurotransmission was diminished following L-PIA administration in halothane-anesthetized rats in all brain regions. These data suggest that acute L-PIA treatment decreases central noradrenergic neurotransmission and may represent the mechanism for the decrease in halothane dose to achieve an anesthetic endpoint anesthetic response to halothane.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Adenosine: a novel ulcer modulator in stomachs.

Adenosine has been demonstrated for its actions on gastric secretion and stress-induced gastric ulceration in animals. We examined the pharmacological actions of adenosine on ethanol-evoked gastric lesions and gastric mucosal blood flow (GMBF) in rats, because both of them are closely related. Adenosine pretreatment, in dose of 7.5 mg/kg increased GMBF and protected against ethanol-evoked gastric lesion formation. However, this antiulcer action was followed by an aggravation of gastric lesions and reduction in GMBF. We further investigated whether these actions could act through the adenosine A1 or A2 receptors, therefore L-phenylisopropyladenosine (L-PIA) or N-ethylcarboxamidoadenosine (NECA), the adenosine A1 or A2 receptor agonists, respectively, were used. The drugs given in doses of 10 or 50 micrograms/kg for L-PIA and 1 or 5 micrograms/kg for NECA, dose-dependently inhibited GMBF and potentiated ethanol-induced gastric damage. When the two drugs were given together to animals, they did not further aggravate the severity of ulceration and reduction of GMBF. These findings indicate that the antiulcer action of adenosine is not mediated via the adenosine A1 and A2 receptors but if acts through different adenosine receptor subtypes. It was because the lesion worsening effects of adenosine at the second stage of the biphasic responses were similar to the actions of L-PIA and NECA, the ulcer potentiating effect is probably acting through adenosine A1 and A2 receptors in anaesthetised rats.     

Adenosine inhibits the renal plasma-membrane (Ca2+ + Mg2+)-ATPase through a pathway sensitive to cholera toxin and sphingosine.

Adenosine, a potent autacoid produced and released in kidneys, affects nearly all aspects of renal function, and an increase in cytosolic calcium has been implicated in adenosine effects. The aim of this work was to investigate whether adenosine modifies the calcium pump present in basolateral membranes of kidney proximal tubule cells. Adenosine exerts a biphasic influence on (Ca2+ + Mg2+)-ATPase activity. Inhibition occurs up to 0.1 microM and then gradually disappears as the adenosine concentration increases to 100 microM, an effect mimicked by the adenosine analog N6-cyclohexyladenosine, which preferentially binds to A1-type receptors. In contrast, the A2 receptor agonist 5', N-ethylcarboxamideadenosine is ineffective. The A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine blocks the inhibitory effect of 0.1 microM adenosine and stimulates (Ca2+ + Mg2+)-ATPase activity in the presence of 1 mM adenosine, a concentration high enough to occupy the low-affinity A2 receptors. Inhibition by adenosine increases as medium ATP is lowered to micromolar concentrations, is maintained in the presence of pertussis toxin, and is completely abolished with 0.1 microM cholera toxin or 1 microM sphingosine. The inhibitory effect of adenosine can be reproduced by guanosine 5'-[gamma-thio]triphosphate, inositol 1,4, 5-trisphosphate or the diacylglycerol analog 12-O-tetradecanoylphorbol 13-acetate. In conjunction with the selectivity for its analogs and for its receptor agonist, the concentration profile of adenosine effects indicates that both inhibitory (A1) and stimulatory (A2) receptors are involved. The results obtained with the toxins indicate that a pathway that is modulated by G-proteins, involves a phospholipase C and a protein kinase C, and is affected by local variations in adenosine concentrations participates in the regulation of the (Ca2+ + Mg2+)-ATPase resident in basolateral membranes of kidney proximal tubules.     

Regulation of Cyclic AMP Formation in Cultures of Human Foetal Astrocytes by β2-Adrenergic and Adenosine Receptors

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.     

Specific photoaffinity labelling of inhibitory adenosine receptors

N6(L-phenylisopropyl)adenosine (L-PIA) and N6(3-iodo-4-azido benzyl)-adenosine (IAzBA) inhibit the adenylate cyclase activity in synaptic membranes of chick cerebellum via Ri adenosine receptors. [3H]L-PIA and [125I]AzBA bind to these membranes with Kd values of approximately 1 nM and Bmax values of approximately 1000 fmol/mg protein. Photolysis of [125I]AzBA bound to synaptic membranes results in the specific incorporation of radioactivity into a protein with Mr = 36,000. This photoincorporation is blocked by simultaneous exposure to L-PIA, theophylline, an adenosine receptor antagonist, or Gpp(NH)p, but not by cytosine, suggesting that the 36,000 dalton protein is the Ri adenosine receptor or a subunit of the receptor that contains the adenosine binding site.     

Deaza-analogues of Adenosine and 2-Chloroadenosine as Agonists of Adenosine Receptors

Abstract

A variety of adenosine analogues have been recently evaluated in order Lo find more potent and selective agonists on adenosine receptors. The most potent adenosine analogues acting on A₁ receptor, a high affinity receptor inhibitory to adenylate cyclase, are N⁶-substituted compounds. So ⁶-cyclohexyladenosine (CHA) and ⁶-L-phenylisopropyladenosine (L-PIA) are extremely potent agonists on A₂ receptor, whereas they are relatively weak agonists on A receptor, a lower affinity receptor which is stirnulatory to cyclase, and they have no effect on the adenosine P site.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Adenosine receptor mediated inhibition of noradrenaline release from slices of the rat hippocampus

Adenosine and adenosine analogues inhibited electrically evoked 3H-noradrenaline (3H-NA) release from slices of the rat hippocampus in vitro in a dose -dependent manner in the concentration range 0.01–100 M. L-phenylisopropyladenosine (L-PIA) was more potent than 5′-N-carboxamidoadenosine (NECA), which was more potent than adenosine. The adenosine uptake blocker dipyridamole (3 M) enhanced the effect of exogenous adenosine, and had a slight inhibitory effect per se. The effect of L-PIA on NA release was competitively antagonized by 8-phenyltheopylline; pA2=7.1. Enprophylline (300 M), theophylline (300 M) and 8-phenyltheophylline (1–10 M) enhanced the evoked 3H-NA release per se, while no such enhancement was seen with the non-xanthine phosphodiesterase inhibitor ZK 62.711 (Rolipram) (30 M).It is concluded that adenosine, at physiologically relevant concentrations, inhibits electrically evoked NA release from terminals in the central nervous system. Alkylxanthines increase evoked NA release from hippocampal terminals, wich probably not related to cyclic AMP but may partly involve inhibition of endogenous adenosine acting as a modulator of transmitter release in the hippocampal slice preparation.     

Inhibition of Ca2+ signaling and glucagon secretion in mouse pancreatic alpha-cells by extracellular ATP and purinergic receptors

Glucagon secreted from pancreatic alpha-cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since alpha-cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and beta-cell secretory granules. Using immunocytochemistry, we identified in alpha-cells the P2 receptor subtype P2Y1, as well as the P1 receptors A1 and A2A. In contrast, only P2Y1 and A1 receptors were localized in beta-cells. To analyze the role of purinergic receptors in alpha-cell function, we studied their participation in Ca2+ signaling. At low glucose concentrations, mouse alpha-cells exhibited the characteristic oscillatory Ca2+ signals that lead to secretion. Application of ATP (1-10 microM) abolished these oscillations or reduced their frequency in alpha-cells within intact islets and isolated in culture. ATPgammaS, a nonhydrolyzable ATP derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1-10 microM) was also found to reduce Ca2+ signals. ATP-mediated inhibition of Ca2+ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y1 and A2A were likely involved in the inhibitory effect on Ca2+ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the alpha-cell function.