Evidence for adenosine A2b receptors in the rat pineal gland.

We describe the effects of 5'-N-ethylcarboxamidoadenosine (NECA), a mixed A2a/A2b adenosine receptor agonist and 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoaden osin e (CGS 21680), a selective A2a agonist, on cyclic AMP and N-acetylserotonin synthesis in rat pineal gland. NECA, 1 and 10 microM, increased cyclic AMP by 5- and 25-fold and N-acetylserotonin by 40- and 60-fold respectively, whereas CGS 21680 at the same concentrations was ineffective. These results argue for the presence of adenosine A2b receptors in rat pinealocytes.     

Purinergic receptors in the nucleus tractus solitarius mediate distinct cardiorespiratory response patterns

The purpose of the study was to examine the role of brainstem purinergic receptors in mechanisms of cardiorespiratory control mediated by the nucleus tractus solitarius (NTS), the major integrative site in the brainstem involved in the regulation of cardiorespiratory function and the reflex coordination of cardiorespiratory response patterns. Microinjections of selective agonists for purinergic receptor subtypes were made into the caudal NTS of anesthetized, spontaneous breathing rats. CGS 21680, a selective agonist for adenosine A_2a receptors, elicited pronounced, dose-related decreases in blood pressure, whereas cyclopentyladenosine (CPA), a selective agonist at adenosine A₁ receptors, elicited dose-related increases in blood pressure. These depressor and pressor response patterns were completely and selectively blocked, respectively, by pretreatment with CGS 15943a, a selective A_2a receptor antagonist, and by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) a selective A₁ receptor antagonist. 5′-N-ethylcarboxamidoadenosine (NECA), an analog exhibiting mixed agonist actions at adenosine receptor subtypes, elicited both depressor and pressor responses depending on the dosage. Additionally, in contrast to the findings with CGS 21680 and CPA, only NECA elicited very pronounced, dose-related decreases in respiratory rate at dosages which did not affect blood pressure. Related lines of evidence suggested that NECA-induced depression of respiration was mediated via A_2b receptors. Microinjections of α,β-MeATP, a selective agonist for P_2x receptors, elicited apnea, and these effects were blocked by suramin, a selective antagonist at P_2x receptors, but not by DMPX, a methylxanthine exhibiting mixed antagonist properties at P₁ adenosine receptors. Moreover, similar microinjections of 2-MeSATP, a selective agonist at P_2x receptors, were ineffective in eliciting cardiorespiratory response patterns and it is likely that brainstem purinergic receptors may therefore play a crucial role under both physiologic and pathophysiologic conditions. © 1993 Wiley-Liss, Inc.     

Adenosine receptor modulation of sympathetic neurotransmission in rat isolated kidney

In the present study we set out to define, using discriminatory agonists and antagonists, the adenosine receptors modulating sympathetic neurotransmission in the rat kidney. Isolated kidneys from male Wistar rats were perfused with modified Krebs-Henseleit buffer solution at constant flow. The neuronal noradrenaline stores were labeled with ³H-noradrenaline and the renal nerves stimulated electrically (2 Hz, 3 msec, 9 mA, during 20 sec at intervals of 6 min). ³H overflow was taken as an index of ³H-noradrenaline release. The A₁ receptor selective agonists N⁶-cyclopentyladenosine (CPA), N⁶-cyclohexyladenosine (CHA), and N⁶-[R(−)-1-phenyl-2-propyl]adenosine (R-PIA), and the mixed A₁/A_2A receptor agonists 5′-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) inhibited evoked ³H outflow concentration-dependently. The selective A_2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), at concentrations selective for A_2A receptors, failed to modify ³H outflow, whereas at higher concentrations it induced inhibition. The rank order of potency of agonists, CPA > CHA = R-PIA > NECA > CADO >> CGS 21680, is typical for an interaction with the A₁ receptor. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), at concentrations selective for blockade of A₁ receptors, blocked concentration-dependently the inhibitory effects of CPA and NECA; no evidence of an increase in outflow was seen with NECA in the presence of DPCPX. The selective A_2A receptor antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazol[1,5-c] quinazoline-5-amine (CGS 15943) did not influence the agonist effects at concentrations interacting selectively with A_2A receptors but antagonized them concentration-dependently at higher, non-selective concentrations. Taken together, our data establish the presence of inhibitory adenosine A₁ receptors on the terminal sympathetic neurons of rat kidney. No evidence was obtained for the presence of functional A_2A receptors in this preparation. © 1996 Wiley-Liss, Inc.     

Species-dependent effects of adenosine receptor agonists on contractile responses of vas deferens to ATP

1 Experiments were carried out to examine the postjunctional actions of adenosine receptor agonists on the smooth muscle of the vas deferens of the guinea-pig and rabbit. 2 Although they produced neither contraction nor relaxation by themselves, adenosine analogues enhanced contractions of the guinea-pig vas deferens induced by 10 μm ATP. The rank order of potency was N⁶-cyclopentyladenosine (CPA) > 5′-N-ethylcarboxamidoadenosine (NECA) > adenosine > CGS 21680. Dose–response curves for NECA were shifted to the right by the nonselective adenosine receptor antagonist 8(p-sulphophenyl)theophylline (8-SPT; 100 μm ) and by the selective A₁-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 m m ). 3 In the rabbit vas deferens, contractions induced by ATP (1 m m ) were inhibited rather than facilitated by NECA. Neither CPA, R(–)-N⁶-(2-phenyl isopropyl)-adenosine (R-PIA) nor CGS 21680 had any effect. 4 The results indicate that the smooth muscle of the guinea-pig vas deferens expresses facilitatory adenosine A₁ receptors but not adenosine A₂ receptors. In contrast, in rabbit there are postjunctional inhibitory adenosine A_2A receptors but not adenosine A₁ receptors.     

Characterization of adenosine receptors in the rat isolated aorta

• 1.1. Adenosine and its analogues relaxed the isolated rat aorta by an endothelium-dependent mechanism with an order of potency of 5′-N-ethylcarboxamidoadenosine (NECA) > 2-(p-(2-carboxyethyl)phenethylamino)-5′-N-ethylcarboxamidoadenosine (CGS 21680) > adenosine = N⁶-(2-(4-aminophenyl)ethyl)adenosine (APNEA = N⁶-cyclopentyladenosine (CPA) > 5′ - methylthioadenosine (MTA), although the maximal response achieved by CGS 21680 was less than that achieved by NECA.
• 2.2. Both 8-sulphophenyltheophylline (8-SPT) and MTA antagonized responses to the adenosine analogues, but there were some anomolous features of this antagonism and NECA was inhibited more powerfully than the other agonists. This suggests that as well as A_2a receptors mediating relaxation, the rat aorta may relax to adenosine analogues by other mechanisms.

     

Adenosine receptor-induced cyclic AMP generation and inhibition of 5-hydroxytryptamine release in human platelets.

1. We have assessed the effects of adenosine receptor agonists and antagonists on collagen-induced 5-hydroxytryptamine (5-HT) release and cyclic AMP generation in human platelets. 2. 5'-N-ethylcarboxamidoadenosine (NECA) and CGS 21680 elicited accumulations of cyclic AMP with mean EC50 values of 2678 and 980 nM, respectively. The maximal response to CGS 21680 was approximately half that of the response to 10 microM NECA. 3. NECA and CGS 21680 inhibited collagen-induced 5-hydroxytryptamine release with mean EC50 values of 960 and 210 nM, respectively. The maximal response to CGS 21680 was approximately 25% of the response to 10 microM NECA. 4. The A1/A2a-selective adenosine receptor antagonist PD 115,199 was more potent as an inhibitor of NECA-elicited responses than the A1-selective antagonist DPCPX with calculated Ki values of 22-32 nM and > 10 microM, respectively. 5. In the presence of a cyclic AMP phosphodiesterase inhibitor, the effects of CGS 21680 on cyclic AMP accumulation and 5-HT release were enhanced to levels similar to those elicited by 10 microM NECA. In the absence of phosphodiesterase inhibition, CGS 21680 did not antagonise the effects of NECA. Furthermore, endogenous adenosine did not contribute to the effects of CGS 21680 when phosphodiesterase was inhibited. 6. We conclude that an A2a adenosine receptor appears to be involved in the NECA-elicited increases in cyclic AMP levels and inhibition of 5-HT release in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of three adenosine receptor types

1 The purpose of the present study was to classify adenosine receptors into A₁ and A₂ subtypes in a wide range of isolated tissues and cell types (rat adipocytes and atria, guinea-pig ileum and atria (A₁); guinea-pig aorta, dog coronary artery and human platelets and neutrophils (A₂)) using the R- and S-diastereoisomers of N-phenylisopropyladenosine (PIA), N-cyclopentyladenosine (CPA), the novel compound, N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR79236), N-[(2-methylphenyl)methyl]adenosine (metrifudil), 2-(phenylamino)adenosine (CV1808), and 2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamidoadenosine (CGS21680); N-ethylcarboxamidoadenosine (NECA) was used as a standard.2 Results obtained in all tissue preparations previously reported to contain A₁-receptors could be described by a single rank order of agonist potency: CPA ≥ GR79236, R-PIA ≥ NECA >> S-PIA ≥ metrifudil ≥ CV1808, CGS21680.3 In contrast, two distinct rank orders of agonist potency were observed in preparations previously reported to contain A₂-receptors. In dog coronary artery, human neutrophils and platelets the rank order of potency was: CV1808, CGS21680 ≥ NECA > R-PIA ≥ metrifudil ≥ CPA > GR79236, S-PIA. However, in guinea-pig aorta the rank order was: NECA > metrifudil > R-PIA, CPA > CV1808, GR79236 ≥ S-PIA, CGS21680.4 The results of this study are consistent with the existence of three types of adenosine receptor: A₁-and two subtypes of A₂-receptor. The receptor present in dog coronary artery, human platelets and neutrophils, probably corresponds to the A_2a subtype, whilst that present in the guinea-pig aorta may be of the A_2b subtype.     

Prejunctional stimulation of cholinergic nerves in rat airway smooth muscle by an adenosine analogue

The effect of 5′-N-ethylcarboxamidoadenosine (NECA) on rat bronchial smooth muscle was examined in vitro. Both the nerve mediated muscle contraction induced by electrical stimulation and the potassium evoked release of [³H]ACh were enhanced by NECA. The apparent affinity (EC₅₀) of NECA in the contraction experiments was 0.30 ± 0.06 μM. The adenosine (ADO) receptor antagonist, 8-phenyltheophylline (8-PT), inhibited the NECA induced potentiation of both the electrical induced contraction and the potassium evoked release of [³H]ACh. The EC₅₀ and intrinsic activity of exogenous ACh were not altered in the presence of NECA (1 μM) in experiments where smooth muscle contraction were measured, indicating that NECA has a prejunctional effect and not a postjunctional effect on muscarinic receptors. The new A₂ specific ADO receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) and ADO also enhanced the nerve-mediated contraction (EC₅₀ = 35 ± 8 μM and 69 ± 20 μM, respectively). 8-PT (10 μM) and enprofylline (ENPF) (10 μM) inhibited the electrically induced contraction by 55 ± 16% and 45 ± 5% respectively. The potassium evoked release, however, was stimulated 56 ± 6% and 39 ± 7% by 50 μM 8-PT and ENPF respectively. The results provide evidence for a NECA specific ADO receptor in rat bronchi that is most likely prejunctional. Stimulation of this receptor, which may be of an A₂ receptor subtype, enhances the nerve mediated release of ACh and thereby induce contraction of the bronchial smooth muscle.     

An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells

1. Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [³H]-adenine pre-labelling methodology.
2. Adenosine analogues showed the following rank order of potency (pD₂ value): 5′-N-ethylcarboxamidoadenosine (NECA, 5.24)>2-chloroadenosine (4.41) ⩾ adenosine (4.19)=N⁶-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.11). The A_2A-selective agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 μM.
3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76±8% NECA response), 2-chloroadenosine (70±6%) and APNEA (40±3%) were significantly reduced.
4. Antagonists of the NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA₂ value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93)=8FB-PTP (6.80)>KF 17837 (5.98)>3-propylxanthine (5.13).
5. Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A_2B adenosine receptor.
6. We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A_2B subtype.

     

Functional characterization of the adenosine receptor contributing to glycogenolysis and gluconeogenesis in rat hepatocytes

The adenosine receptor subtype mediating glucose production by glycogenolysis and gluconeogenesis was studied in primary cultured rat hepatocytes. Adenosine and adenosine agonists caused cyclic AMP accumulation in rat hepatocytes. The order of potency was 5'-N-ethylcarboxamidoadenosine (NECA)>R(-)-N(6)-(2-phenylisopropyl)adenosine (RPIA)>adenosine>2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Furthermore, adenosine agonists stimulated glycogenolysis and gluconeogenesis. The order of potency was NECA>RPIA>CGS21680. The rank order of potency is typical for adenosine A(2B) receptors. Glycogenolysis stimulated by NECA was fully inhibited by nonselective adenosine antagonists, 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943). However, the adenosine A(2A) receptor-selective antagonist, 8-(3-chlorostyryl)caffeine (CSC), and the adenosine A(1) receptor-selective antagonist, (+)-(R)-[(E)-3-(2-phenylpyrazolo[1,5-alpha]pyridin-3-yl)acryloyl]-2-piperidine ethanol (FK453), had a low inhibitory potency. A strong correlation was found between the inhibitory effect of adenosine antagonists on NECA-induced glucose production and that on intracellular cyclic AMP generation in rat hepatocytes. Our results suggest that adenosine stimulates cyclic AMP formation and regulates glycogenolysis and gluconeogenesis, most likely through the adenosine A(2B) receptor subtype in rat hepatocytes.     

Comparative effects of a selective adenosine A2 receptor agonist, CGS 21680, and nitroprusside in vascular smooth muscle.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethyolcarboxamidoa denosine) is an adenosine agonist that has been reported recently to bind selectively to adenosine A2 receptors in rat brain. This adenosine agonist, and the parent compound NECA (5'-N-ethylcarboxamidoadenosine), were found to be potent vasorelaxants of prostaglandin F2 alpha (PGF2 alpha) precontracted porcine coronary smooth muscle with EC50s of 4.5 and 9.7 nM, respectively. Schild analysis of the inhibition of CGS 21680, NECA and 2-chloroadenosine induced relaxation of the porcine coronary artery by CGS 15943 (9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-C]quinazolin-5-amine), an A2 receptor antagonist, yielded identical pA2 values for the antagonist (approximately 9.3). This indicates that the same receptor mediates the effects of these three adenosine agonists. NECA and CGS 21680 were equipotent in most vascular preparations except in the canine coronary artery. Porcine coronary arterial rings contracted with PGF2 alpha were relaxed by NECA or CGS 21680 as well as by nitroprusside; those contracted with KCl (40 mM) were relaxed only by nitroprusside. In rabbit aorta, contractions induced by phenylephrine or PGF2 alpha were inhibited by nitroprusside but not by NECA or CGS 21680. Thus, the adenosine A2 receptor agonists, NECA and CGS 21680, are potent vasorelaxants that display regional vascular and species variations that differ from those of nitroprusside.     

The cardiovascular effects of selective adenosine A1 and A2 receptor agonists in the pithed rat: no role for glibenclamide-sensitive potassium channels

Summary The cardiovascular effects of N⁶-cyclopentyladenosine (CPA), a selective adenosine A₁ receptor agonist and 2-[p-carboxyethyl)phenylethylamino]-5-N-ethylcarboxamidoadenosine (CGS 21680), a selective A₂ receptor agonist have been investigated in the pithed rat with blood pressure raised to normal levels with angiotensin II. Cumulative intravenous administration of CPA, 0.3–10 g/kg, induced dose-related falls in blood pressure and heart rate; over the same dose range CGS 21680 induced hypotension but no bradycardia. Pretreatment with a maximally effective dose of the A₁/A₂ receptor antagonist 8-(p-sulphophenyl) theophylline (8-SPT) blocked the bradycardiac effects of CPA (92-fold) more effectively than its hypotensive activity (5.1-fold); the vasodepressor effects of CGS 21680 were blocked 19-fold by 8-SPT. Glibenclamide, a blocker of ATP-sensitive potassium (K _infATP ^sup+ ) channels, administered intravenously at 20 mg/kg markedly attenuated the vasodepressor effects of the potassium channel opener, (–)-(3S,4R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(3-oxo-cyclopent-1-enyloxy)2-H-1-benzopyran-6-carbonitrile (SDZ PCO 400). In contrast, neither the hypotensive nor the bradycardic effects of CPA nor the fall in blood pressure following CGS 21680 was significantly affected by pretreatment with glibenclamide. These results indicate that a significant component of the blood pressure fall induced by CPA and CGS 21680 in the pithed rat with blood pressure supported by angiotensin II occurs by a mechanism which is insensitive to 8-SPT and unlikely, therefore, to be mediated by A₁ or A₂ receptors. Moreover, in contrast to the prevailing literature, the cardiovascular effects arising from adenosine receptor activation in this model are not mediated by glibenclamide-sensitive, K _infATP ^sup+ channels.Correspondence to J. R. Fozard at the above address     

Characterization of the Prejunctional Adenosine Receptors in the Rat Anococcygeus Muscle

Abstract— Adenosine receptor agonists inhibited electrically-evoked contractions of the rat isolated anococcygeus muscle. The compounds tested were: N⁶-cyclopentyladenosine (CPA), N((S, trans)-2-hydroxycyclopentyl)adenosine (GR79236), the R- and S-isomers of phenylisopropyladenosine (PIA), 5′-N-ethylcarboxamidoadenosine (NECA), ((2-(4-(2-carboxyethyl)phenyl)ethyl)amino)-N-ethylcarbox-amidoadenosine (CGS 21680) and N-((2-methylphenyl)methyl)adenosine (metrifudil). The rank order of agonist potency was: CPA = R-PIA = GR79236 = NECA » S-PIA > metrifudil > CGS 21680, which is consistent with an effect mediated by adenosine A₁ receptors. A similar rank order of potency was obtained for inhibition of electrically-evoked contractions of the guinea-pig ileum. However, there may be a lower receptor reserve in rat anococcygeus compared with the guinea-pig ileum, since higher concentrations of agonists were necessary to produce effects in the anococcygeus than in the guinea-pig ileum and S-PIA behaved as a partial agonist. The effect of NECA was antagonized in rat anococcygeus and guinea-pig ileum by the mixed A₁/A₂ receptor antagonist, 8-phenyltheophylline (pA₂ values of 6·8 and 6·9, respectively). The selective A₁-receptor antagonist, 8-cyclopentyl-l,3-dipropylxanthine (DPCPX), also blocked the inhibitory response to NECA in both tissues. Here, however, the pA₂ values (9·6 and 8·6, respectively) were slightly but significantly different. These values confirm that the prejunctional adenosine receptors of the rat anococcygeus are of the A₁ type, and suggest that they are similar but not necessarily identical to those of the guinea-pig ileum. The differing potencies of DPCPX as an antagonist of NECA between the preparations may reflect a tissue-dependent variation in sensitivity to this antagonist.     

Inotropic actions of adenosine derivatives in the mammalian heart

The effects of adenosine receptor (AR) stimulation on contractile parameters as well as inositol phosphates, cAMP content, and phospholamban-phosphorylation was studied in cardiac preparations. In guinea-pig papillary muscles adenosine increased inositol trisphosphate formation and also slightly elevated force of contraction. In the presence of isoprenaline, however, adenosine still enhanced inositol trisphosphate formation but reduced force of contraction. In isolated electrically driven guinea-pig ventricular cardiomyocytes the A₁-AR agonist R-PIA and the A₁/A₂-AR agonist NECA reduced isoprenaline-stimulated contractility but only R-PIA decreased isoprenaline-enhanced cAMP content. The selective A₂-AR agonist CGS 21680 increased isoprenaline-stimulated cAMP content. This effect of CGS 21680 was antagonized by the A₂-AR antagonist CGS 15943A and was increased after pertussis toxin-pretreatment. Furthermore, R-PIA and NECA decreased via A₁-ARs the isoprenaline-stimulated phospholamban-phosphorylation in guinea-pig ventricular cardiomyocytes. © 1993 Wiley-Liss, Inc.     

A1-and A2-PURINOCEPTORS IN THE GUINEA-PIG UTERUS

1. Radioligand binding and functional studies were undertaken to investigate the P₁-purinoceptors present in the separated myometrial layers and the endometrium of the guinea-pig uterus. 2. In preparations of endometrium-denuded circular myometrium, the A₂-selective agonists (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS 21680, 100 μmol/L) and N-ethylcarboxamido adenosine (NECA, 1–10 μmol/L) inhibited contractile responses to phenylephrine. In preparations of endometrium-intact circular myometrium, NECA (10 μmol/L) enhanced responses to phenylephrine. NECA did not modulate the spontaneous contractions of longitudinal myometrium. 3. Homogenate binding studies with circular myometrium, longitudinal myometrium and endometrium revealed saturable high affinity [³H]-NECA binding sites. The mean maximal densities of binding sites (B_max) were 2.08, 14.7 and 15.5 fmol/mg protein, and pK_D (neg. log dissociation constant) values were 9.82, 9.19 and 7.44, respectively. 4. (R-) and (S-) -N⁶-(2-phenylisopropyl)adenosine (R- and S-PIA) both competed for two [³H]-NECA binding sites in preparations of circular myometrium. CGS 21680 competed for two [³H]-NECA binding sites in preparations of endometrium and longitudinal myometrium. All other agonist competition was for one site only. The rank orders of potency of high affinity binding were S-PIA ≥ R-PIA ≥ CGS 21680 (circular myometrium), R-PIA ≥ CGS 21680 ≥ S-PIA (longitudinal myometrium) and CGS 21680 > > S-PIA ≥ R-PIA (endometrium). 5. In preparations of circular myometrium, longitudinal myometrium and endometrium the selective A₁-purinoceptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)-phenylxanthine (PACPX), competed for two [³H]-NECA binding sites, the non-selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), competed for one site only. 6. NECA increased cyclic adenosine monophosphate (CAMP) levels in preparations of both circular myometrium and endometrium. 7. These results indicate that P₁-purinoceptors of the A₂-subtype mediate the inhibitory effects of adenosine analogues on the phenylephrine-induced contractions of the circular myometrium of the guinea-pig, this effect is modified by the presence of the endometrium. There is no evidence that the [³H]-NECA binding sites of the longitudinal myometrium correlate with functional P₁-purinoceptors in this tissue.     

Adenosine A2 receptor regulation of apomorphine-induced turning in rats with unilateral striatal dopamine denervation

The ipsilateral intrastriatal administration of the specific adenosine A_2a receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarboxamido adenosine (CGS 21680), produced a dose related decrease in apomorphine-induced rotation in the unilaterally 6-hydroxydopamine-lesioned rat. This effect could be reversed by intrastriatal infusions of the A_2a antagonist, 4-amino-l-phenyl[1,2,4]triazolo[4,3-a]quinoxaline (CP 66,713). However, CP 66,713 had no significant effect when infused alone, neither did it influence the response to apomorphine in the absence of CGS 21680. The possible behavioural interactions between A_2a receptors and striatal ACh activity were also investigated using this model. Atropine administered intrastriatally in a dose that had no effect on the response to apomorphine reduced the inhibitory effects of CGS 21680 on apomorphine-induced turning. Naloxone also reduced the effects of apomorphine, an effect which could be prevented by the co-administration of atropine, or CP 66,713. These results indicate that adenosine agonists can modulate apomorphine-induced turning by an interaction with both cholinergic and opioidergic mechanisms in the striatum.     

Functional characterization of coronary vascular adenosine receptors in the mouse

Coronary responses to adenosine agonists were assessed in perfused mouse and rat hearts. The roles of nitric oxide (NO) and ATP-dependent K(+) channels (K(ATP)) were studied in the mouse. Resting coronary resistance was lower in mouse vs rat, as was minimal resistance (2.2+/-0.1 vs 3.8+/-0.2 mmHg ml(-1) min(-1) g(-1)). Peak hyperaemic flow after 20 - 60 s occlusion was greater in mouse. Adenosine agonists induced coronary dilation in mouse, with pEC(50)s of 9.4+/-0.1 for 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethyl carboxamidoadenosine (CGS21680, A(2A)-selective agonist), 9.3+/-0.1 for 5'-N-ethylcarboxamidoadenosine (NECA, A(1)/A(2) agonist), 8.4+/-0.1 for 2-chloroadenosine (A(1)/A(2) agonist), 7.7+/-0.1 for N(6)-(R)-(phenylisopropyl)adenosine (R-PIA, A(1)/A(2B) selective), and 6.8+/-0.2 for adenosine. The potency order (CGS21680=NECA>2-chloroadenosine>R-PIA>adenosine) supports A(2A) adenosine receptor-mediated dilation in mouse coronary vessels. 0.2 - 2 microM of the A(2B)-selective antagonist alloxazine failed to alter CGS21680 or 2-chloroadenosine responses. pEC(50)s in rat were 6.7+/-0.2 for CGS21680, 7.3+/-0.1 for NECA, 7.6+/-0.1 for 2-chloroadenosine, 7.2+/-0.1 for R-PIA, and 6.2+/-0.1 for adenosine (2-chloroadenosine>NECA=R-PIA>CGS21680> adenosine), supporting an A(2B) adenosine receptor response. NO-synthase antagonism with 50 microM N(G)-nitro L-arginine (L-NOARG) increased resistance by approximately 25%, and inhibited responses to CGS21680 (pEC(50)=9.0+/-0.1), 2-chloroadenosine (pEC(50)=7.3+/-0.2) and endothelial-dependent ADP, but not sodium nitroprusside (SNP). K(ATP) channel blockade with 5 microM glibenclamide increased resistance by approximately 80% and inhibited responses to CGS21680 in control (pEC(50)=8.3+/-0.1) and L-NOARG-treated hearts (pEC(50)=7.3+/-0.1), and to 2-chloroadenosine in control (pEC(50)=6.7+/-0.1) and L-NOARG-treated hearts (pEC(50)=5.9+/-0.2). In summary, mouse coronary vessels are more sensitive to adenosine than rat vessels. A(2A) adenosine receptors mediate dilation in mouse coronary vessels vs A(2B) receptors in rat. Responses in the mouse involve a sensitive NO-dependent response and K(ATP)-dependent dilation.     

[3H]5′-N-Ethylcarboxamidoadenosine selectively labels the low affinity adenosine binding protein,adenotin, on intact chinese hamster ovary cells

The ability of [³H]5′-N-ethylcarboxamidoadenosine (NECA) to specifically bind recognition sites on intact Chinese hamster ovary (CHO) cells was examined in the present study. Saturation experiments indicated that [³H]NECA bound with moderate affinity (Kd = 400 nM) and large capacity (apparent Bmax = 3.2 pmol/10⁵ cells) to intact CHO cells. No specific binding to these cells was observed with the A₁-selective agonist 20 nM [³H]cyclohexyladenosine or with the A₂-selective agonist 20 nM [³H]CGS 21680. Competition studies revealed that close structural analogs of NECA and the xanthine phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) inhibited 20 nM [³H]NECA binding with moderate affinity (IC₅₀s 0.5–15 μM). Adenosine also showed weak activity (IC₅₀ = 100 μM) for inhibiting [³H]NECA binding. However, a wide variety of prototypic adenosine receptor agonists and antagonists did not significantly interact with these [³H]NECA recognition sites on CHO cells. [³H]NECA binding to CHO cell membranes was not sensitive to guanine nucleotides and NECA did not stimulate cAMP formation. These results are consistent with the previously demonstrated ability of [³H]NECA to bind low affinity adenosine binding proteins (adenotin proteins), as well as, adenosine receptors in a variety of mammalian tissues. The present results further indicate that [³H]NECA selectively labels in adenotin-like recognition site on intact CHO cells in the absence of detectable binding to high affinity adenosine receptors. © 1993 Wiley-Liss, Inc.