Relaxation of mouse isolated aorta to adenosine and its analogues does not involve adenosine A(1), A(2) or A(3) receptors

Relaxations to adenosine and analogues were investigated in the mouse aorta in the presence of the adenosine A(1) receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 30 nM), which did not affect relaxations to adenosine or its analogue N(6)-R-phenylisopropyladenosine (R-PIA) but abolished contractile adenosine A(1) receptor-mediated responses to these agonists. Relaxations to adenosine, 5'-N-ethylcarboxamidoadenosine, R-PIA, 2-[p-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), and N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were unaffected by the adenosine A(1)/A(2) receptor antagonist 8-sulphophenyltheophylline (100 microM). IB-MECA relaxations were unaffected by the adenosine A(3) receptor-selective antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191, 30 microM) and R-PIA relaxations were unaffected by N(G)-nitro-L-arginine methyl ester (100 microM) and endothelium removal. In conclusion, relaxant responses to adenosine and analogues do not involve adenosine A(1), A(2) or A(3) receptors and are endothelium- and nitric oxide-independent.     

Characterisation of adenosine receptors mediating relaxation in hamster isolated aorta

The aim of this study was to characterise the receptor(s) mediating relaxations to adenosine and its analogues in the hamster isolated aorta. Adenosine relaxed the aorta but there was no significant difference between pIC20 values in the absence and presence of 8-sulphophenyltheophylline (8-SPT, 50 microM), although there was a small right-shift (approximately threefold) of the lower portion of the curve in the presence of 8-SPT. However, in the presence of the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1 microM), curves to adenosine were left-shifted by approximately 100-fold and an apparent pK(B) for 8-SPT of 5.79+/-0.05 was obtained. Likewise, 5'-N-ethylcarboxamidoadenosine (NECA) relaxed the aorta but curves were biphasic. The first phase of the curve was blocked by 8-SPT (10-100 microM, pA2 = 5.75+/-0.14) and the A2A-selective antagonist 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylaminolethyl) phenol (ZM 241385, 3 nM-1 microM, pK(B)=9.17+/-0.10). Similarly, the A2A-selective agonist 2-[p)-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxam idoadenosine (CGS 21680) relaxed the tissues but curves were biphasic and the first phase was again blocked by ZM 241385 (10 nM, apparent pK(B)=9.06+/-0.34). In contrast, relaxations to N6-R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), 2-chloroadenosine (2-CADO) and N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were not blocked by 8-SPT (50 microM). Responses to IB-MECA were also not blocked by the A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihyd ropyridine-3,5-dicarboxylate (MRS 1191, 30 microM). The asymptote of the first phase of curves to NECA was markedly reduced (and in some preparations the first phase was completely abolished) both in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), and in the absence of endothelium. Likewise, the first phase of curves to CGS 21680 was abolished both in the presence of L-NAME (0.1 mM) and in the absence of endothelium. In contrast, there were only relatively small shifts to the right of curves to adenosine and the other analogues in the presence of L-NAME or the absence of endothelium (between three- and fivefold). The data suggest the presence of A2A receptors which are located on the endothelium and mediate release of nitric oxide. These receptors are activated by NECA, CGS 21680 and adenosine (in the presence of uptake blockade). The resistance to blockade of relaxations to adenosine (in the absence of uptake inhibitor), CPA, R-PIA, 2-CADO, IB-MECA and high concentrations of NECA and CGS 21680 by 8-SPT or ZM 241385 suggests the presence of an additional mechanism(s). Data obtained with adenosine in the absence and presence of NBTI suggest that the endogenous ligand may cause relaxation via an intracellular mechanism.     

Effects of adenosine receptor agonists on guinea-pig isolated working hearts and the role of endothelium and NO

The hypothesis that the coronary vasodilator effects of adenosine receptor agonists are independent of the vascular endothelium or mediators derived therefrom was examined in guinea-pig isolated working hearts. Adenosine receptor agonists, 5'-(N-ethylcarboxamido)-adenosine (NECA; two-fold selective for A2 over A1 receptors), 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; A2A selective), N6-cyclopentyl-adenosine (CPA; A1 selective) and N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; A3 selective), were infused (3 x 10(-7) M) after endothelium removal by passing oxygen through the coronary circulation. In spontaneously beating hearts, CGS21680 and NECA increased, while CPA decreased, coronary flow. NECA and CPA reduced heart rate, left ventricular pressure and aortic output. The nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NOARG; 3 x 10(-5) M) abolished the vasodilatation by NECA but not CGS21680, indicating that nitric oxide (NO) of a non-endothelial source mediated the NECA response. Coronary vasodilatation by CGS21680 was inhibited bythe A2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). Indometacin (10(-6) M) attenuated the coronary vasodilatation to CGS21680, suggesting a partial role for cyclooxygenase products. IB-MECA had no effect, indicating no A3 receptor involvement. In paced working hearts, the responses were similar except CPA had no effect on coronary flow or aortic output and CGS21680 increased left ventricular pressure and the maximum rate of ventricular pressure rise. This study has demonstrated functionally effective removal of the endothelium by a novel method of passing oxygen through the coronary vasculature. A coronary vasodilator action of adenosine receptor agonists mediated via A2A receptors is endothelium- and NO-independent, but partially involves cyclooxygenase products.     

Purinergic modulation of [(3)H]GABA release from rat hippocampal nerve terminals

The hippocampal GABAergic system is assumed not to be a target for purine modulation. We have now confirmed that neither adenosine A(1) and A(3) receptor nor nucleotide P(2) or P(4) receptor activation modified the K(+)-evoked [(3)H]GABA release from hippocampal synaptosomes. However, activation of adenosine A(2A) receptors with CGS 21680 (10 nM) or HENECA (30 nM) facilitated GABA release by 32% and 21%, respectively. These effects were prevented by the A(2A) antagonist, ZM 241385 (20 nM). A(2A) receptors may activate adenylate cyclase and protein kinase A since CGS 21680 (10 nM) facilitation was partially prevented by 8-bromo-cAMP (1 mM), forskolin (10 microM) and HA-1004 (10 microM). Protein kinase C may also be recruited, since chelerythrine (6 microM) and phorbol-12, 13-didecanoate (250 nM) attenuated CGS 21680 (10 nM) facilitation of [(3)H]GABA release. Omega-agatoxin-IVA (200 nM) occluded CGS 21680 facilitation suggesting the involvement of P-type calcium channels. Thus, the adenosine A(2A) receptor system appears to be one of the first presynaptic neuromodulatory systems able to enhance the evoked release of GABA from hippocampal nerve terminals.     

Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line.

1. The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 2. Adenosine receptors were detected by RT - PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9+/-0.2 nM and Bmax of 23+/-7 fmol x mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1+/-0.2 nM and a Bmax of 220+/-7 fmol x mg(-1) of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3+/-0.7 nM and Bmax of 291+/-50 fmol x mg(-1) of protein. 3. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. 4. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. 5. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A - A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.     

Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis.

1. The effect of the A2A adenosine receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]-gamma-aminobutyric acid ([3H]-GABA) from nerve terminals derived from the caudate-putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nM) inhibited the [3H]-GABA release evoked by 15 mM KCl but had no effect on that evoked by 30 mM KCl. 2. The ability of CGS 21680 (1 nM) to inhibit the release of [3H]-GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 microM), phaclofen (100 microM) and 2-hydroxysaclofen (100 microM). Similarly the opioid receptor antagonist, naloxone (10 microM), the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 40 nM), and the cholinoceptor antagonists, mecamylamine (10 microM) and atropine (100 nM) had no effect on this inhibition. 3. The ability of CGS 21680 (0.1 nM) to stimulate the release of [3H]-acetylcholine ([3H]-ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 microM), 2-hydroxysaclofen (100 microM), phaclofen (100 microM), naloxone (10 microM) and DPCPX (4 nM). 4. The novel A2A receptor antagonist, (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837), blocked the CGS 21680 (1 nM)-induced inhibition of [3H]-GABA efflux with an EC50 of approximately 30 nM and also antagonized the CGS 21680 (0.1 nM)-induced stimulation of [3H]-ACh release with an EC50 of approximately 0.3 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride), a new potent and selective adenosine A3 receptor antagonist

We investigated the biochemical and pharmacological properties of a new adenosine A(3) receptor antagonist, KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride). This compound was characterized using N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]AB-MECA) or [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to membranes from human embryonic kidney 293 (HEK293) cells expressing human adenosine A(3) receptors. KF26777 showed a K(i) value of 0.20+/-0.038 nM for human adenosine A(3) receptors labeled with [125I]AB-MECA and possessed 9000-, 2350- and 3100-fold selectivity vs. human adenosine A(1), A(2A) and A(2B) receptors, respectively. The inhibitory mode of binding was competitive. KF26777 inhibited the binding of [35S]GTPgammaS stimulated by 1 microM 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA). The IC(50) value was 270+/-85 nM; the compound had no effect on basal activity. Dexamethasone treatment for HL-60 cells, human promyelocytic leukemia, up-regulated functional adenosine A(3) receptors expression, and resulted in the enhanced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) via the adenosine A(3) receptor. KF26777 antagonized this [Ca(2+)](i) mobilization induced by Cl-IB-MECA, with a K(B) value of 0.42+/-0.14 nM. These results indicate that KF26777 is a highly potent and selective antagonist of the human adenosine A(3) receptor.     

Adenosine A(2A) receptors in portal hypertension: their role in the abnormal response to adenosine of the cranial mesenteric artery in rabbits

1. Adenosine is a regulator of mesenteric vasodilation involved in auto-regulation and post-prandial hyperemia, but the adenosine receptor subtype involved in this relaxant effect is poorly characterized. We have now pharmacologically characterized this receptor in rabbit mesenteric arteries and investigated how this adenosine receptor response changes in portal hypertensive animals since the adenosine response is decreased. 2. The closest non-metabolisable adenosine analogue, 2-chloroadenosine (CADO), the mixed A(1)/A(2) receptor agonist, 5'-ethylcarboxamidoadenosine (NECA), and the selective A(2A) receptor agonist, 2-[4-(2-p-carbonyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) (1 pM -- 1 mM) relaxed noradrenaline pre-contracted arteries with a rank order of potency of CGS 21680 (EC(50)=20 nM) > or = NECA (60 nM)>CADO (640 nM). 3. The selective A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)-[1,2,4]-triazolo[2,3-a][1,3,5]-triazin-5-ylamino]ethyl)phenol (ZM 241385, 100 nM), shifted to the right the CADO concentration-response curve. 4. In portal hypertensive animals, there was mainly a decreased potency but also a decreased efficacy of all tested adenosine agonists compared to normal animals. Concomitantly, there was a decreased adenosine plasma level and a decreased binding density of [(3)H]-CGS 21680 and [(3)H]-ZM 241385 to mesenteric artery membranes from portal hypertensive compared to normal rabbits. 5. These results indicate that A(2A) receptor activation is required for the adenosine-induced mesenteric relaxation and that the decreased density of A(2A) receptors may contribute to the decreased relaxation induced by adenosine of mesenteric arteries in portal hypertensive animals.     

Apparent involvement of the A(2A) subtype adenosine receptor in the anti-inflammatory interactions of CGS 21680, cyclopentyladenosine, and IB-MECA with human neutrophils

This study was undertaken to identify the adenosine receptor (AR) subtypes which down-regulate the proinflammatory activities of human neutrophils, as well as the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) and its relationship to cellular handling of Ca(2+) in mediating these effects. Neutrophils were treated with varying concentrations (0.01-1 microM) of AR agonists operative at A(1) (N(6)-cyclopentyladenosine, CPA), A(2A) (2(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosi ne, CGS 21680), and A(3) (N(6)-(3-iodobenzyl-5'-N-methylcarbamoyladenosine, IB-MECA) receptors, after which they were activated with the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 microM). Intracellular cAMP, superoxide, and elastase were assayed using radioimmunoassay, lucigenin-enhanced chemiluminescence (LECL), and colorimetric procedures, respectively, while changes in the concentrations of cytosolic Ca(2+) were monitored by fura-2-based spectrofluorimetry. CGS 21680, at all concentrations tested, inhibited superoxide production in a dose-related manner, while CPA and IB-MECA were effective only at the highest concentrations tested (0.5-1 microM). The release of elastase from activated neutrophils was also inhibited by all three AR agonists, but was more sensitive to CGS 21680 and IB-MECA than was superoxide production. The inhibitory effects of all 3 agonists on superoxide production and elastase release were associated with accelerated clearance of Ca(2+) from the cytosol of activated neutrophils, and were effectively neutralized by pretreatment of the cells with the highly selective A(2A)R antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1, 2,4]triazolo[2,3-a][1,3,5]triazin-5yl amino]ethyl)phenol). Increased cAMP was detected in neutrophils treated with CGS 21680 and IB-MECA (1 microM). These data support the involvement of the A(2A)R subtype in the suppression of superoxide production and degranulation by activated human neutrophils, probably by cAMP-mediated alterations in Ca(2+) handling.     

Effects of adenosine A1 and A2A receptor activation on the evoked release of glutamate from rat cerebrocortical synaptosomes

1. The effects of adenosine A2A and A1 receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands. 2. Adenosine (0.1 microM) produced a significant potentiation of the Ca2+-dependent K+ (15 mM)-evoked [3H]-D-aspartate overflow (20.4+/-3.5%), which was blocked by A2A blocker SCH58261 (0.1 microM). At higher concentrations (10 - 1000 microM) adenosine inhibited in a DPCPX-sensitive manner the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow. The inhibitory effect of adenosine at 1000 microM was significantly increased by SCH58261. This inhibition was antagonized by 1 microM DPCPX. Adenosine did not produce any effect on basal release. 3. The A2A receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca2+-dependent K+-evoked overflow of [3H]-D-aspartate (EC50 approximately 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A2A receptor antagonist SCH58261 (IC50 approximately 5 nM). 4. The A1 receptor agonist CCPA inhibited the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow (EC50 approximately 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A1 receptor antagonist DPCPX. 5. The K+ (15 mM)-evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 microM). The effect of CGS 21680 was abolished by SCH58261 (0.1 microM) and that of CCPA by DPCPX (0.1 microM). 6. It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A1 and excitatory A2A receptors present on glutamatergic terminals of the rat cerebral cortex.     

Allosteric modulation of A(3) adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives

Allosteric modulators of A(1) and A(2A) adenosine receptors have been described; however, for the A(3) adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A(3) adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [(125)I]N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [(125)I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [(3)H]PSB-11 from the A(3) adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A(1) receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [(125)I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [(3)H]N(6)-[(R)-phenylisopropyl]adenosine from the A(1) adenosine receptor or [(3)H]CGS21680 from the A(2A) adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.     

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.     

Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; 100 nM) and the adenosine A3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A2A and A3 receptors, which are capable of modulating TNF-alpha release.     

A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase.

Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.     

Adenosine receptor-mediated modulation of acetylcholine release from rat striatal synaptosomes.

1. The effects of A1 and A2a adenosine receptor agonists on the veratridine-evoked release of [3H]-acetylcholine ([3H]-ACh) from rat striatal synaptosomes was investigated by use of the A1-selective agonist, R-PIA and the 185 fold selective A2a agonist, CGS 21680. The effects of NECA, which is equipotent at both receptor subtypes, were also studied. 2. The evoked release of [3H]-ACh was significantly enhanced by the A2a agonist CGS 21680 but decreased by the A1 agonist, R-PIA. The effects of NECA were dependent on the concentration used, with high concentrations inhibiting and low concentrations enhancing the evoked release of [3H]-ACh. In the absence of any antagonists, the rank order of potency for these three drugs on increasing [3H]-ACh release was CGS 21680 > NECA > R-PIA. 3. The stimulatory effects of CGS 21680 and low NECA concentrations on evoked [3H]-ACh release were antagonized by the A2a receptor antagonists, CP66,713 (300 nM) and CGS 15943A (50 nM) whilst the inhibitory effects of R-PIA were reversed by the selective A1 antagonist, DPCPX (4 nM). In the presence of DPCPX, NECA greatly enhanced the evoked release of [3H]-ACh at all concentrations studied when, during such A1 receptor blockade, the rank order of potency was NECA >> CGS 21680 > R-PIA. 4. These results demonstrate that both A1 and A2a adenosine receptors modulate the veratridine-evoked release of [3H]-ACh from rat striatal synaptosomes.     

Binding of the prototypical adenosine A(2A) receptor agonist CGS 21680 to the cerebral cortex of adenosine A(1) and A(2A) receptor knockout mice

1. 2-p-(2-carboxyethylphenethylamino-5'-ethylcarboxamidoadenosine) (CGS 21680) is considered the reference compound to study adenosine A(2A) receptors. However, CGS 21680 binding in the cerebral cortex, where adenosine A(1) receptors are predominant, displays a mixed A(2A)/A(1) receptor pharmacology. We now use adenosine A(1) and A(2A) receptor knockout mice to investigate the characteristics of cortical [(3)H]CGS 21680 binding. 2. [(3)H]CGS 21680 binding to the cerebral cortex was strongly reduced in adenosine A(1) receptor knockout mice, but only slightly reduced in A(2A) receptor knockout mice compared with the corresponding wild-type littermates. 3. Another selective A(2A) receptor ligand, [(3)H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), displayed a saturable binding to mouse cortical membranes, albeit with a binding density 20 times lower than that of striatal membranes, and this [(3)H]SCH58261 binding was abolished in both striatal and cortical membranes of A(2A) receptor knockout mice and unchanged in A(1) receptor knockout mice. 4. The presence of A(2A) receptors in cortical neurons was further confirmed by Western blot in mouse cortical nerve terminal membranes. 5. It is concluded that, although A(2A) receptors are present in the cerebral cortex, the purportedly selective A(2A) receptor agonist [(3)H]CGS 21680 binds in the cerebral cortex to an entity that requires the presence of adenosine A(1) receptors. Thus, CGS 21680 should be used with care in all preparations where adenosine A(1) receptors out-number A(2A) receptors.     

Characterization of adenosine receptors mediating the vasodilator effects of adenosine receptor agonists in the microvasculature of the hamster cheek pouch in vivo

1 The aim of this study was to characterize the adenosine receptor mediating vasodilation in the microvasculature of the hamster cheek pouch in vivo. A range of adenosine agonists was used including N6-cyclopentyladenosine (CPA) (A1 agonist), 5'-N-ethylcarboxamidoadenosine (NECA) (non-selective), 2-chloroadenosine (2CADO) (non-selective), 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) (A2A agonist), N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IBMECA) (A3 agonist) and adenosine, as well as the adenosine antagonists 8-sulphophenyltheophylline (8-SPT) (A1/A2 antagonist), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (A1 antagonist) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) (A2A antagonist). 2 All the adenosine analogues used induced vasodilation at concentrations between 10 nm and 1 microm, and the potency order was NECA > CGS 21680 > 2CADO > CPA=IBMECA > adenosine, indicating an action at A2A receptors. 8-SPT (50 microm) antagonized vasodilator responses to NECA with an apparent pKB of 5.4, consistent with an action at A1 or A2 receptors and confirming that A3 receptors are not involved in this response. 3 DPCPX (10 nm) had no effect on vasodilation evoked by NECA, suggesting that this response was not mediated via A1 receptors, while ZM 241385 (10 nm) antagonized dilator responses to NECA with an apparent pKB of 8.9 consistent with an action via A2A receptors. 4 Overall these results suggest that adenosine A2A receptors mediate vasodilation in the hamster cheek pouch in vivo.     

Adenosine mediates relaxation of human small resistance-like coronary arteries via A2B receptors.

1. The receptor subtype and mechanisms underlying relaxation to adenosine were examined in human isolated small coronary arteries contracted with the thromboxane A2 mimetic, 1,5,5-hydroxy-11alpha, 9alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) to approximately 50% of their maximum contraction to K+ (125 mM) depolarization (Fmax). Relaxations were normalized as percentages of the 50% Fmax contraction. 2. Adenosine caused concentration-dependent relaxations (pEC50, 5.95+/-0.20; maximum relaxation (Rmax), 96.7+/-1.4%) that were unaffected by either combined treatment with the nitric oxide inhibitors, NG-nitro-L-arginine (L-NOARG; 100 microM) and oxyhaemoglobin (HbO; 20 microM) or the ATP-dependent K+ channel (KATP) inhibitor, glibenclamide (10 microM). The pEC50 but not Rmax to adenosine was significantly reduced by high extracellular K+ (30 mM). Relaxations to the adenylate cyclase activator, forskolin, however, were unaffected by high K+ (30 mM). 3. Adenosine and a range of adenosine analogues, adenosine, 2-chloroadenosine (2-CADO), 5'-N-ethyl-carboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA), S(+)-N6-(2-phenylisopropyl)-adenosine (S-PIA), N6-cyclopentyladenosine (CPA), 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta- D-ribofuranuronamide (IB-MECA), 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680), relaxed arteries with a rank order of potency of NECA= 2-CADO >adenosine= IB-MECA = R-PIA= CPA > S-PIA)> CGS 21680. 4. Sensitivity but not Rmax to adenosine was significantly reduced approximately 80 and 20 fold by the non-selective adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT) and the A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). By contrast, the A1-selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) had no effect on pEC50 or Rmax to adenosine. 5. These results suggest that A2B receptors mediate relaxation to adenosine in human small coronary arteries which is independent of NO but dependent in part on a K+-sensitive mechanism.     

Adenosine-mediated cardioprotection in ischemic-reperfused mouse heart.

We investigated the roles of A1, A2A, or A3 receptors and purine salvage in cardioprotection with exogenous adenosine, and tested whether A2A -mediated reductions in perfusion pressure modify post-ischemic recovery. Treatment with 10(-5) or 5 x 10(-5) M adenosine improved contractile recovery from 20 min ischemia 45 min reperfusion in isolated mouse hearts. Protection was attenuated by adenosine kinase inhibition (10(-5) M iodotubercidin) and receptor antagonism (5 x 10(-5) M 8-rho-sulfophenyltheophylline, 8-SPT). Enzyme efflux mirrored contractile recoveries. A 3 agonism with 10(-7) M 2-chloro- N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) improved ischemic tolerance whereas A1 agonism with 5 x 10(-8) M N6-cyclopentyladenosine (CPA) and A2A agonism with 10(-9) M 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) or 2 x 10(-8) M methyl-4-(3-[9-[4S,5S,2R,3R)-5-(N-ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]-6-aminopurin-2-yl)]prop-2-ynyl) cyclohexane-carboxylate (ATL-146e) were ineffective. Protection via A1 receptor overexpression was enhanced by adenosine, but unaltered by A1 or A2A agonists. Finally, post-ischemic dysfunction in hearts perfused at constant flow was dependent on coronary pressure, with A2A AR-mediated reductions in pressure reducing diastolic contracture, and elevated perfusion pressure worsening contracture. Data indicate that cardioprotection with exogenous adenosine in asanguinous hearts involves purine salvage and activation of A3 but not A1 or A2A receptors.     

Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices

1. In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia-like conditions. 2. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoade nos ine ), and of selective A2A receptor antagonists, ZM 241385 (4-(2-[7-amino-2-(2-furyl)-?1,2,4?-triazolo?2,3-a??1,3, 5?triazin-5-ylamino]ethyl)phenol) and SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode. 3. The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1, 3-dipropyl-8-cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia. 4. A prolonged application of either adenosine (100 micronM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM). 5. SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia-induced depression. 6. These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1-mediated depression of synaptic transmission induced by in vitro ischaemia.