R-phenylisopropyladenosine Research Articles

Adenosine enhances antigen-induced bronchoconstriction and histamine release in rat isolated lungs

The effects of adenosine and some of its analogues on bronchoconstriction and mediator release were studied in isolated lungs of actively sensitized rats. Adenosine (ADO) and its analogues R-phenyl-iso-propyl-adenosine (R-PIA) and N-ethyl-carboxamide-adenosine (NECA), enhanced antigen-induced bronchoconstriction in a dose-dependent manner. The enhancement of anaphylactic bronchoconstriction by adenosine and its analogues was accompanied by a rise in histamine release. The observed rank order of potency for adenosine and analogues (NECA greater than or equal to R-PIA greater than ADO) did not permit an unequivocal classification of the adenosine receptor involved. Dipyridamole and S-(p-nitrobenzyl-6-thioinosine) (NBTI), both inhibitors of adenosine uptake, had no inhibitory influence on the adenosine-induced enhancement of anaphylactic bronchoconstriction. Therefore, this enhancement was likely to be mediated through an extra-cellular receptor. Theophylline inhibited the enhancement of anaphylactic bronchoconstriction by adenosine in a concentration-dependent manner, without affecting preformed mediator release.

Activation of adenosine A1 receptor by N 6-(R)-phenylisopropyladenosine (R-PIA) inhibits forskolin-stimulated tyrosine hydroxylase activity in rat striatal synaptosomes

We investigated the effect of the relatively selective A1 adenosine receptor agonist N 6-(R)-phenylisopropyladenosine (R-PIA) on tyrosine hydroxylase activity (TH) of synaptosomes obtained from rat striatum. TH activity was assayed in supernatant obtained following sonication and centrifugation of the tissue preincubated with the test compounds. R-PIA produced a modest decrease of basal enzyme activity, but significantly reduced the activation of the enzyme by submaximal (0.1–0.5 μM) concentrations of forskolin (FSK) a stimulator of adenylate cyclase. The IC 50 value of R-PIA was 17 nM and the maximal inhibition corresponded to 30–40% decrease of the enzyme activity stimulated by FSK. The S-isomer of PIA failed to affect TH activity under control and stimulated conditions. Moreover, the inhibitory effect of R-PIA was completely antagonized by 8-cyclopentyl- 1,3 -dimethylxanthine, an adenosine receptor blocker. R-PIA inhibited both basal and FSK-stimulated adenylate cyclase activity. These results indicate that in striatal dopaminergic terminals TH activity can be modulated in an inhibitory manner by activation of presynaptic A1 adenosine receptors.

Subsensitivity of presynaptic adenosine A 1-receptors in caudal arteries of spontaneously hypertensive rats

(-)-N 6-(R-phenylisopropyl)-adenosine (R-PIA) depressed tritium overflow and vasoconstriction evoked by electrical stimulation to a similar extent in isolated tail arteries of Wistar rats (WR) preincubated with [ 3H]noradrenaline. The inhibitory effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) and R-PIA were determined on the constrictor responses of tail arteries obtained from WR, as well as spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). In WR and WKY, the rank order of agonist potency (R-PIA > NECA > adenosine) was compatible with the presence of adenosine A 1-receptors. Whereas adenosine, NECA and R-PIA were equiactive in WR and WKY, they produced no or only slight changes in SHR. The left renal arteris of some WR were partially occluded to induce hypertension. R-PIA had the same effect in the tail arteries of these animals as in preparations obtained from sham-operated WR. The above results suggest that the subsensitivity of presynaptic A 1-receptors in the blood vessels of SHR is genetically determined. This could contribute in vivo to enhanced transmitter release from terminals of perivascular nerves and subsequent increases in vascular resistance.

Adenosine as substrate and receptor agonist in the ovary

In the present study the possible dual effects of adenosine as substrate and adenosine receptor agonist in rat granulosa cells, cumulus-oocyte complexes, luteal cells and ovarian membranes are discussed. Adenosine is an indispensable compound in cell energy metabolism, as precursor to cofactors, second messenger and nucleic acids. Adenosine is also an agonist to adenosine receptors. The adenosine receptor can either inhibit (A 1) or stimulate (A 2) adenylate cyclase. Alternatively, in some cells adenosine receptor activation is linked to other cellular events like inhibition of Ca 2+ fluxes. Adenosine is taken up by isolated preovulatory granulosa and luteal cells from pregnant mare serum gonadotropin-treated immature rats, but follicle stimulating hormone (FSH) decreases the uptake by granulosa cells. Adenosine, but not the non-metabolizable adenosine analogs 5'-(N-ethyl) carboxamido-adenosine (NECA), 2-chloro-adenosine (2-Clado), N 6-(R-phenyl-isopropyl) -adenosine (R-PLA) and N 6-(S-phenyl-isopropyl)-adenosine (S-PLA), increase granulosa cell ATP levels. FSH and luteinizing hormone (LH) decrease granulosa cell ATP levels in the presence or absence of adenosine. It has previously been shown that FSH and LH decrease oxygen consumption by cumulus-oocyte complexes and increase their lactate production. These effects have been suggested to be due to a competition of cofactors (e.g. ADP) common to glycolysis and the respiratory chain. The fact that adenosine reverse the gonadotropin-induced effects on oxygen consumption and lactate production support this theory. Adenosine and its analogs increase cAMP accumulation in luteal and granulosa cells only in the presence of gonadotropins, and this effect is antagonized by the adenosine receptor antagonist 8-phenyl-theophylline (8-PHT). Furthermore, adenylate cyclase is stimulated by adenosine analogs in membranes from non-luteinized and luteinized ovarian membranes and in luteal cell homogenates. The effect of NECA is antagonized by 8-PHT. In the membranes, the rank order of potency was NECA>2-Clado>R-PLA>S-PLA, suggesting adenosine A 2 receptors. In summary, it is suggested that adenosine can act both as a substrate to intracellular metabolism and as an adenosine A 2 receptor agonist in granulosa and luteal cells. A paracrine short loop positive feedback model is proposed where extracellular adenosine, derived from a gonadotropin-induced extracellular increase in cAMP and a decrease in cellular ATP, enhances gonadotropin stimulation in granulosa and luteal cells.

Effects of adenosine analogues on basal, prostaglandin E 1- and forskolin-stimulated cyclic AMP formation in intact neuroblastoma cells

We have examined the effects of R-phenylisopropyladenosine ( R-PIA) and other adenosine analogues on basal, prostaglandin E 1 (PGE 1)- and forskolin-stimulated cyclic AMP (cAMP) formation in intact N1E-115 neuroblastoma cells, to determine whether the cells contain A 1 adenosine receptors that are negatively coupled with adenylate cyclase. Basal levels of cAMP (68 ± 7 pmol/mg protein; mean ± SE, N = 15) were not altered by low concentrations of R-PIA. The apparent lack of inhibition was not due to increases in cAMP due to activation of a stimulatory A 2 receptor by endogenously-synthesized adenosine. By comparison, low levels of R-PIA did reduce significantly (P < 0.05) PGE 1-dependent increases in cAMP formation (maximum response to PGE 1, 972 ± 77 pmol cAMP/mg protein; ec 50 for PGE 1, 0.2 μM). Inhibition was dose dependent, and resulted in a 30–50% maximum reduction in production stimulated by PGE 1. Nanomolar concentrations of R-PIA elicited half-maximal inhibition; the inhibitory response was blocked by 8-phenyltheophylline (8-PT). The order of potencies of several adenosine analogues in eliciting this response suggested that inhibition was mediated by an A 1 adenosine receptor. Examination of the effects of R-PIA on forskolin-stimulated cAMP formation yielded several interesting findings. First, stimulation by the diterpene by itself was blocked by both adenosine deaminase (ADA) and 8-PT (40 and 25% inhibition respectively). Low concentrations of R-PIA (< 10 −6M) had no effect on forskolin-stimulated cAMP production. At higher levels (⩾ 10 −6M) the analogues acted synergistically with the diterpene, to yield cAMP levels that were up to 3-fold higher than the additive effect of the two agents. Potentiation was stereospecific, Ca 2+ dependent, and was blocked by 8-PT. The results of this study suggest that, in N1E-115 neuroblastoma cells, inhibitory A 1 receptors are not stimulated in response to non-specific elevations in cAMP, but are associated with specific stimulatory receptors such as those activated by PGE 1.

Functional role of cholinoceptors and purinoceptors in human isolated atrial and ventricular heart muscle.

1. The effects of cholinergic and purinergic stimulation on action potential, force of contraction and 86Rb efflux were investigated in human atrial and ventricular heart muscle. 2. In atrial heart muscle, carbachol and (-)-N6-(R-phenyl-isopropyl)-adenosine (R-PIA) and 5'-(N-ethyl)-carboxamido-adenosine (NECA) evoked transient decreases of action potential duration and force of contraction; the steady-state effects on force of contraction were virtually identical to control values. In the presence of propranolol, steady-state values after carbachol, R-PIA or NECA amounted to about 50% of control values. 3. In ventricular heart muscle, carbachol, NECA and R-PIA did not significantly affect the action potential configuration or force of contraction. 4. Carbachol, NECA and R-PIA induced a maintained depression of the positive inotropic response to isoprenaline in both atrial and ventricular heart muscle. 5. The rate constant of 86Rb efflux was slightly increased by carbachol, NECA and R-PIA in atrial (10-20%) but not in ventricular heart muscle. 6. In the presence of isoprenaline, carbachol, NECA and R-PIA did not significantly affect the rate constant of 86Rb efflux in both atrial and ventricular heart muscle. Isoprenaline alone increased the rate constant of 86Rb by about 25% in both tissues.

Pronounced cholinergic but only moderate purinergic effects in isolated atrial and ventricular heart muscle from cats

1. The effects of cholinergic and purinergic stimulation on action potential, force of contraction and 86Rb efflux were investigated in cat atrial and/or ventricular heart muscle. 2. Acetylcholine and carbachol exerted a concentration-dependent negative inotropic effect in cat atrial heart muscle. Carbachol 10 mumol l-1 completely abolished the force of contraction and increased the rate constant of 86Rb efflux 2-3 fold, whereas the action potential duration was shortened to about 1/10 of its length under control conditions. 3. The effects of acetylcholine and carbachol in cat atrial heart muscle were mimicked, qualitatively, by adenosine and its analogues 5'-(N-ethyl)-carboxamido-adenosine (NECA) and (-)-N6-(R-phenyl-isopropyl)-adenosine (R-PIA). Maximal purinergic effects, however, amounted to about 15-50% in comparison to those of cholinergic stimulation. 4. In cat ventricular heart muscle, cholinergic or purinergic stimulation had no significant effects on the force of contraction in the absence of a cyclic AMP-dependent positive inotropic effect. Carbachol antagonized the positive inotropic effect elicited by either 3-isobutyl-1-methylxanthine, isoprenaline or cyclic 8-(4-chlorphenylthio)adenosine-3':5'-monophosphate; NECA and R-PIA were less effective. The inhibition by carbachol of the effects of isoprenaline was not related to a change in the rate constant of 86Rb efflux. 5. It is concluded that the effects of cholinoceptor and purinoceptor agonists in the cat heart involve a change in the potassium conductance in the atrium, whereas the effects in the ventricle may be related to changes of intracellular cyclic AMP levels. It seems reasonable to assume that, in comparison to cholinergic stimulation, a low density of purinoceptors in the cat heart is responsible for the relatively weak effects of adenosine agonists in this species.

Inhibition by adenosine of histamine and leukotriene release from human basophils

Adenosine inhibited the release of histamine and leukotriene C 4 (LTC 4) from immuno-logically-activated basophils in a dose-dependent manner. Structural congeners of adenosine also attenuated the elaboration of these two mediators from stimulated basophils and a rank order of potency for the inhibition was observed following the sequence 2-chloroadenosine ⩾ N-methylcarbox-amidoadenosine (NECA) > adenosine ⩾ R-phenylisopropyladenosine ( R-PIA) ⩾ S-PIA. These same nucleosides modulated the generation of LTC 4 more potently than the release of histamine. A number of methylxanthines, which are antagonists of cell surface adenosine receptors, reversed the inhibition by adenosine and its congeners of the release of both histamine and LTC 4 to varying extents. Dipy-ridamole and nitrobenzylthioinosine (NBTI), agents that block the intracellular uptake of adenosine, antagonized the inhibition of histamine release by adenosine (and 2-chloroadenosine) but failed to reverse the attenuation of LTC 4 generation by the nucleoside. These same uptake blockers were unable to antagonize the inhibitory effects of NECA on either histamine or LTC 4 release. In purified basophils, NECA and R-PIA, and in that order of decreasing reactivity, increased total cell cyclic adenosine monophosphate (cAMP) levels and inhibited the stimulated release of mediators. In total, these results suggest that the basophil possesses a cell surface adenosine receptor which, on the basis of both pharmacological and biochemical criteria, most closely conforms to an A 2/R a-like receptor. However, in addition to an interaction at the cell surface, studies with agents that block the intracellular uptake of adenosine suggest that the nucleoside may also exert intracellular effects when countering the release of histamine (but not LTC 4).

Role of Adenosine 3',5'-Monophosphate and the Ri-Receptor Gi-Coupled Adenylate Cyclase Inhibitory Pathway in the Mechanism Whereby Adrenalectomy Increases the Adenosine Antilipolytic Effect in Rat Fat Cells*

The aim of this study was to establish the mechanism by which adrenalectomy promotes the antilipolytic effect of the adenosine analog (-)-N6-(R-phenyl-isopropyl)adenosine (R-PIA) in rat fat cells. This action of adrenalectomy was not specific for R-PIA, since it was also observed with nicotinic acid and was prevented by phosphodiesterase inhibitors. In contrast, the inhibitory effect of R-PIA and nicotinic acid toward isoproterenol-stimulated cAMP accumulation was unaltered by adrenalectomy regardless of whether phosphodiesterase inhibitors were present. Whatever the conditions used, however, the cAMP levels in adrenalectomized rat adipocytes were one quarter to one third of those in sham-operated rats and remained below the limit over which variations in cAMP had no more influence in lipolysis. Both total and particulate low Km cAMP phosphodiesterase activities per adipocyte were decreased in adrenalectomized rats, but the stimulatory responses of the particulate enzyme to R-PIA remained unchanged. Pertussis toxin-catalyzed ADP ribosylation studies revealed a marked decrease in the total amount of the alpha-subunits of Go and the adenylate cyclase inhibitory regulatory protein Gi after adrenalectomy. However, the inhibitory dose-response curves of adenylate cyclase to R-PIA, nicotinic acid, GTP, guanylylimidodiphosphate, and guanosine 5'-O-(3-thiotriphosphate) were unaltered by adrenalectomy, indicating that the inhibitory function of Gi is unimpaired by adrenalectomy. Lastly, adrenalectomy resulted in a 60% reduction of the Mn2+-stimulated adenylate cyclase activity/adipocyte, which indicates that adrenalectomy causes a defect in adenylate cyclase catalytic activity. Thus, enhanced antilipolytic effects of R-PIA induced by adrenalectomy do not involve increased function of the adenosine receptor Gi-coupled adenylate cyclase inhibitory pathway, but are related to abnormally low intracellular cAMP levels due to defective adenylate cyclase catalytic activity.

Adenosine analogs inhibit gastric acid secretion

The potencies with which four adenosine deaminase-resistant analogs of adenosine affected the volume, pH and acid output of basal gastric acid secretions were examined in unanesthetized rats with chronic indwelling gastric cannulas. All four adenosine receptor agonists, R-phenylisopropyladenosine (R-PIA), S-phenylisopropyladenosine (S-PIA), N-ethylcarboxamide adenosine (NECA), and 2-chloroadenosine (CADO) significantly decreased gastric acid output in a dose-dependent manner. The rank order of potency was NECA, R-PIA > CADO > S-PIA. NECA and R-PIA were approximately equipotent in reducing gastric acid output. The levels of gastric acid output tended to increase at the lowest doses of the agonists. NECA decreased the volume of gastric secretion, whereas R-PIA had no effect on volume, but significantly increased the pH of the secretions. Valid measurements of pH in NECA-treated rats were not always obtainable because of near total inhibition of gastric secretions. S-PIA did not significantly affect volume, but increased pH at the higher doses tested. CADO decreased volume, but did not affect pH. These results indicate that adenosine analogs regulate not only the hydrogen ion concentration, but also the volume of gastric secretions.

Human basophil/mast cell releasability. VII. Heterogeneity of the effect of adenosine on mediator secretion

5'-N-ethylcarboxamideadenosine (NECA) > 2-chloroadenosine > adenosine > N 6-(R-phenyl-isopropyl)-adenosine (R-PIA) inhibited in vitro anti-IgE-induced histamine and peptide leukotriene C 4 (LTC 4) release from human basophils in a concentration- dependent fashion. Micromolar concentrations of adenosine, NECA and R-PIA potentiated the anti-IgE-stimulated release of histamine and LTC 4 from human lung parenchymal mast cells. Submillimolar concentrations of adenosine, NECA and R-PIA inhibited in a concentration dependent manner the release of histamine and prostaglandin D 2 (PGD 2) from skin mast cells challenged with anti-IgE. These results demonstrate marked heterogeneity of the modulatory effect exerted by adenosine on mediator release from human basophils and mast cells.

Inhibition of nigrostriatal release of dopamine in the rat by adenosine receptor agonists: A 1 receptor mediation

The stable analogues of adenosine, N-ethylcarboxamidoadenosine (NECA), R-phenylisopropyladenosine (R-PIA) and cyclohexyladenosine (CHA), dose-dependently decreased levels of 3-methoxytyramine (3-MT) in the striatum and antagonized pargyline-dependent accumulation of 3-methoxytyramine. These agents were equipotent with ED 25 values of approximately 1 mg/kg, (p.o.) in inhibiting pargyline-dependent accumulation of 3-methoxytyramine. Since CHA and R-PIA are relatively selective for A 1 receptors and NECA is almost equipotent at A 1 and A 2 sites, the data of undifferentiated potency for these 3 agents on release of dopamine (levels of 3-MT) would argue in favor of mediation of A 1 receptors in this phenomenon. This conclusion was further supported by experiments with the A 1-selective antagonist, 8-cyclopentyl-l,3-dipropylxanthine (CPDX), which antagonized the actions of CHA. Similar antagonism of CHA-dependent decreases in levels of cyclic GMP in the cerebellum, an action known to be mediated by A 1 receptors, was also observed. These data support previous studies which indicated an adenosine receptor-mediated modulation of nigrostriatal release of dopamine. In addition, the present data indicate that this is an action on A 1 receptors.

Adenosine Potentiates Mediator Release from Human Lung Mast Cells

Micromolar concentrations of adenosine were found to potentiate the release of histamine and leukotriene C4 (LTC4) from immunologically activated human lung mast cells (HLMC). Structurally modified congeners of adenosine including 5'-N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyladenosine (R-PIA) also potentiated mediator release. A rank order of potency was established where NECA greater than R-PIA for the potentiation of both LTC4 production and histamine secretion. Mast cells isolated by either enzymatic or mechanical means from human lung parenchyma were both similarly responsive to the modulatory effects of adenosine and analogues, and the potency series of NECA greater than R-PIA also applied. Moreover, histamine release induced by the calcium ionophore A23187 was augmented by NECA, R-PIA, and adenosine and in that potency order. Dipyridamole, an agent thought to impede the intracellular uptake of adenosine, failed to reverse the nucleoside's enhancement of IgE-mediated secretion. The irreversible inhibitor of adenosine deaminase, deoxycoformycin, did not modify the adenosine enhancement of stimulated secretion. Low concentrations of methylxanthines, which antagonize responses mediated at cell surface adenosine receptors, were inconsistent in their effects. Theophylline modestly reversed the adenosine-induced potentiation of IgE-mediated LTC4 generation but not histamine release. Studies employing 8-phenyltheophylline were complicated by the methylxanthine possessing inhibitory properties of its own at concentrations expected to antagonize a nucleoside-mediated effect. In total, these results suggest that the response of HLMC to adenosine describes properties most consistent with an A2/Ra-like process, although an interaction via an, as yet, uncharacterized cell surface receptor cannot be excluded.

A1 adenosine receptor-G protein coupling in bovine brain membranes: effects of guanine nucleotides, salt, and solubilization.

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N‐ethylmaleimide‐sensitive G‐protein, but not to adenylate cyclase or dihydropyridine‐sensitive Ca2+‐channeIs

Electrically evoked [3H]acetylcholine ([3H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A1-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1-10 microM. The maximal effect was observed with 1 microM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 microM, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 +/- 0.01% v. 0.10 +/- 0.003% in the control slices), but did not affect stimulated release (0.73 +/- 0.05% vs. 0.74 +/- 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [3H]ACh release in a non-competitive manner. The S2/S1 ratio was 0.92 +/- 0.03 in controls and was reduced to 0.32 +/- 0.02 by 1 microM R-PIA in the control slices and to 0.57 +/- 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (1 microM) and rolipram (30 microM) before the second stimulation (S2) enhanced the S2/S1 ratio by about 30% to 1.26 +/- 0.12, but did not reduce the inhibitory effect of R-PIA (1 microM). The Ca2+-channel agonist Bay K 86(44) (1 microM), a concentration that increases K+-evoked noradrenaline release, did not affect the basal or electrically evoked [3H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and 1 microM) on [3H]ACh release. Our results suggest that the presynaptic inhibitory effects of A1-receptor agonists on [3H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Mediation of acute ethanol-induced motor disturbances by cerebellar adenosine in rats.

The possible involvement of brain adenosine in acute ethanol-induced motor incoordination (MI) and inhibition of spontaneous motor activity (SMA) was investigated in male Sprague-Dawley rats. Pretreatment with theophylline or 7-(2-chloroethyl)-theophylline, adenosine antagonists, markedly reduced ethanol-induced MI and inhibition of SMA during a 60 min test period compared with saline + ethanol group. On the contrary, pretreatment with (-)-N6(R-phenylisopropyl)adenosine (R-PIA), an adenosine agonist, or dilazep, an adenosine uptake blocker, markedly potentiated the ethanol-induced MI as well as inhibition of SMA in a 60 min test period compared with saline + ethanol group. No effect on motor coordination was seen when the drug pretreatment was not followed by ethanol. However, the adenosine agonists and antagonists did alter SMA when the pretreatment with these drugs was not followed by ethanol. Ethanol clearance was not altered by the drug pretreatment as blood ethanol levels were similar in all groups except for lower ethanol levels in the R-PIA-treated group. Adenosine A1 binding studies, using 3H-R-PIA as the radioligand and crude membrane preparation from cerebellar cortex, revealed an increase in Bmax with no significant change in Kd in ethanol-treated animals vs. saline control. Theophylline pretreatment prevented the increase in Bmax elicited by ethanol. Collectively, the data suggest that endogenous cerebellar adenosine may be a participating factor in ethanol-induced motor dysfunctions.

Presynaptic P1-purinoceptors in jejunal branches of the rabbit mesenteric artery and their possible function.

1. Excitatory junction potentials (EJPs) evoked by nerve stimulation with fifteen pulses at 1 Hz were recorded from smooth muscle cells of the rabbit isolated mesenteric artery. The effects of P1-purinoceptor agonists and antagonists, as well as of substances which interfere with the inactivation of endogenous adenosine, were tested. 2. Adenosine and its analogues depressed the EJPs in the train in a concentration-dependent manner. The percentage inhibition of the first EJP and that of the later ones was similar; some early EJPs, however, were inhibited more markedly. The rank order of potency of the agonists was (-)-N6-(R-phenylisopropyl)-adenosine (R-PIA) congruent to 5'-N-ethylcarboxamidoadenosine (NECA) greater than (+)-N6-(S-phenylisopropyl)-adenosine (S-PIA) greater than adenosine. The respective IC40 values (the concentrations producing 40% inhibition of the first EJP in the train) were 0.018, 0.028, 0.83 and 4.7 mumol/l. 3. Three methylxanthines, namely 8-phenyltheophylline (1, 10 mumol/l), 8-cyclopentyltheophylline (0.1, 1 mumol/l) and 8-(p-sulphophenyl)-theophylline (100 mumol/l), antagonized the effect of R-PIA (0.1 mumol/l). When given alone they also enhanced the amplitudes of all EJPs in the train. The percentage facilitation of the first EJP and that of the later ones was similar. Some early EJPs, however, were potentiated more markedly. 8-Phenyltheophylline was less potent than 8-cyclopentyltheophylline both in preventing the action of R-PIA and in enhancing the EJPs. A concentration (100 mumol/l) of 8-(p-sulphophenyl)-theophylline, which strongly antagonized the R-PIA effect, produced only a moderate facilitation of EJPs. 4. S-(p-nitrobenzyl)-6-thioguanosine (10 mumol/l) both depressed the EJPs, and enhanced the inhibitory effect of adenosine. Adenosine deaminase (10 micrograms/ml) caused some potentiation of EJPs; this action was prevented by a concentration (10 mumol/l) of deoxycoformycin, which had no effect of its own. AH21-132 (10 mumol/l) enhanced all EJPs in the train. 5. None of the above substances influenced the resting membrane potential of the smooth muscle cells. In addition, R-PIA (0.1 mumol/l) did not change the depolarization induced by noradrenaline (3 mumol/l). 6. We suggest that the axon terminals of postganglionic sympathetic neurones in the rabbit mesenteric artery possess P1-purinoceptors of the A1-type. The activation of these presynaptic receptors by endogenous adenosine may inhibit the release of the main neuroeffector transmitter, which is probably ATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine receptor-mediated effects by nonmetabolizable adenosine analogs in preovulatory rat granulosa cells: a putative local regulatory role of adenosine in the ovary.

The influence of nonmetabolizable adenosine analogs on cAMP production was investigated in preovulatory rat granulosa cells. 5'-(N-ethyl)Carboxamido-adenosine (NECA), a stimulatory A2-adenosine receptor agonist, stimulated cAMP accumulation, and NECA and 2-chloro-adenosine also potentiated the response to FSH. The adenosine receptor antagonist 8-phenyltheophylline antagonized the effect of NECA, shown by a shift in the dose-response curve to the right. The stimulatory effect of NECA was also seen in an ovarian membrane preparation, where NECA stimulated adenylate cyclase in both the presence and absence of FSH. The stimulatory effect of NECA was also decreased by 8-phenyltheophylline in this preparation. The A1-receptor agonists N6-(R-phenyl-isopropyl)-adenosine (R-PIA) and N6-(S-phenyl-isopropyl)-adenosine (S-PIA) both inhibited FSH-stimulated cAMP accumulation. The inhibitory effects of R-PIA and S-PIA, but not the stimulatory effects of NECA, could be counteracted by dipyridamole, a nucleoside transport inhibitor. Furthermore, R-PIA and S-PIA inhibited adenosine uptake into granulosa cells. Thus, the inhibitory effects of R-PIA and S-PIA are not likely to be mediated via membrane-bound inhibitory A1-adenosine receptors. Neither the stimulatory effects of NECA nor the inhibitory effects of R- and S-PIA could be attributed to changes in ATP levels, since the ATP levels were unaffected by these analogs. The results of this study indicate the existence of stimulatory A2-adenosine receptors in preovulatory rat granulosa cells and suggest a membrane-associated modulatory role of adenosine in preovulatory granulosa cells.

Species differences in high‐affinity adenosine A2 binding sites in striatal membranes from mammalian brain

Abstract[3H]5′‐N‐Ethylcarboxamidoadenosine (NECA) labels both A1 and A2 adenosine receptors in mammalian brain tissue. In the presence of 50 nM N6‐cyclopentyladenosine (CPA), however, to block selectively the A1 component of binding, [3H]NECA labels two binding components in striatal tissue. Such binding is dependent on the presence of Mg2+ ions and the removal of endogenous adenosine, using the catabolic enzyme, adenosine deaminase. The two binding components were observed in six mammalian species–‐rabbit, man, rat, mouse, guinea pig, and calf–‐with dissociation constants varying between 3.2 and 9.6 nM for the higher‐affinity site and 54 and 232 nM for the lower‐affinity site. In rat brain tissue, specific binding was reduced by the presence of 5′ guanylyimidodiphosphate Gpp(NH)p, indicating the involvement of a G‐protein. Gpp(NH)p effects were attributable to a conversion of a proportion of high‐affinity to low‐affinity components, as determined by saturation experiments. These results, together with the observation that Gpp(NH)p shifted the inhibition curve for the agonist, 2‐chloro‐adenosine(2‐CADO), to the right indicate that the sites labeled by [3H]NECA in the presence of 50 nM CPA represent multiple affinity states rather than multiple receptor subtypes.Pharmacological examination of the higher‐affinity binding component using 2 nM [3H]NECA showed that binding was consistent with the labeling of an A2 adenosine receptor in all species. The order of activity for adenosine agonists was NECA &gt; 2‐CADO &gt; R‐phenylisopropyladenosine (R‐PIA) &gt; N6cyclohexyladenosine ≥ CPA » S‐PIA. The activity ratios of the diastereomers of PIA (SIR) varied between 7.9 (mouse) and 14.5 (rat). The interaction of a series of 8‐phenyl‐substituted xanthines showed marked species differences with 1,3‐dipropyl‐8‐(2‐amino‐4‐chloro) phenylxanthine (PACPX) being the most active at 14‐15 nM in rabbit and man, 3‐fold less active in calf, and 24‐40‐fold less active in guinea pig, mouse, and rat. A similar trend was seen for 8‐phenyltheophylline (8‐PT), 8‐para‐sulfo‐phenyltheophylline (8‐PST), and 1,3‐diethyl‐8‐phenylxanthine (DPX), the relative order of activity of these adenosine antagonists being PACPX &gt; 8‐PT 2 DPX 2 8‐PST. Caffeine and theophylline were much less active (1C50 &gt; 10,000 nM), while the phosphodiesterase inhibitor 3‐isobutyl‐1 ‐methyl xanthine (IBMX IC50, 2,257‐4,410 nM) was 2‐3 times more potent in man and calf than in the other species studied.

Evidence for adenylate cyclase-coupled A 1-adenosine receptors on ventricular cardiomyocytes from adult rat and dog heart

Isolated metabolically stable cardiomyocytes from adult rats and mongrel dogs were used to characterize the mechanism underlying the antiadrenergic effect of adenosine. In a system not affected by cellular heterogeneity, isoproterenol (3×10 −9 M – 10 −5 M) in the presence of adenosine deaminase (5U/ml) dose dependently increased cellular cAMP (5 – 80 pmol/mg). The effect of isoproterenol (0.1 uM) was inhibited by various adenosine derivatives, the rank order of potency being in the rat: (-)-N 6-(R-phenyl-isopropyl)-adenosine (R-PIA) > 5′-N-ethylcarboxamidoadenosine (NECA) > S-PIA, and in the dog NECA>R-PIA>S-PIA. The cAMP increase induced by forskolin (1 μM) was attenuated in the rat by R-PIA. 8-phenyltheophylline (3 μM) antagonized the effect of R-PIA on isoproterenol-stimulated cAMP formation. Basal cAMP content was not influenced by R-PIA or NECA. Omission of adenosine deaminase from the incubation medium attenuated the isoproterenol-induced cAMP increase in the rat by about 30%. Our findings provide evidence for the presence of adenylate cyclase-coupled A 1-adenosine receptors on cardiomyocytes which may mediate the antiadrenergic effect of adenosine in the heart.