SCH 58261 and ZM 241385 differentially prevent the motor effects of CGS 21680 in mice: evidence for a functional 'atypical' adenosine A(2A) receptor

The acute motor effects elicited by drugs acting upon adenosine A(2A) receptors, namely the highly selective agonist CGS 21680 or the antagonists SCH 58261 and ZM 241385, were investigated in mice. CGS 21680 dose-dependently (0.1-2.5 mg/kg i.p.) decreased horizontal and vertical motor activities. The depressant effect of CGS 21680 (0. 5 mg/kg i.p.) was maintained in mice pretreated by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (10-30 mg/kg i.p. ), which poorly penetrates the blood-brain barrier, but was completely lost in adenosine A(2A) receptor knockout mice. Thus, the adenosine A(2A) receptor is critically involved in motor activity. SCH 58261 (1-10 mg/kg i.p.) increased locomotion and rearing with a quick onset, but for a shorter period in mice habituated to the environment than in mice unfamiliar to it. ZM 241385 (7.5-60 mg/kg i. p.) stimulated horizontal and vertical activities with a slow onset at the two highest tested doses, similarly in naive and in habituated mice. The increase in locomotion elicited by ZM 241385 (15-30 mg/kg i.p. and 10-20 nM i.c.v.) was retained in mice treated by CGS 21680 (0.5 mg/kg i.p.) but that elicited by SCH 58261 (1-3-10 mg/kg i.p. and 10-20 nM i.c.v.) partially subsided. In conclusion, both 'striatal-like'/'SCH 58261-sensitive' adenosine A(2A) receptors and 'ZM 241385-sensitive'/'atypical' CGS 21680 binding sites may mediate CGS 21680-induced motor effects. Moreover, our results suggest that 'atypical' CGS 21680 binding sites could be adenosine A(2A) receptors with a peculiar pharmacological profile.     

Antagonism of coronary artery relaxation by adenosine A2A-receptor antagonist ZM241385

We have tested the existence of functional A2A adenosine receptor in porcine coronary artery using, for the first time, the new A2A antagonist ZM241385. Nonselective agonist NECA and A2A-selective agonist CGS21680 produced concentration-dependent relaxation of prostaglandin F2alpha (PGF2alpha)-precontracted endothelium intact (E+) and denuded (E-) rings. Relaxation was significantly greater in E+ rings than in E-rings. A2A adenosine receptor-selective antagonist, ZM241385 (10(-6) M), significantly attenuated the relaxation responses. The antagonism of ZM241385 was compared with that of SCH58261 (10(-6)M), another A2A adenosine receptor-selective antagonist, which also significantly attenuated the relaxation response to both agonists. However, ZM241385 produced a significantly greater shift of the relaxation-response curves to the right compared with SCH58261 both in E+ and E- rings. The data show for the first time that ZM241385 is a potent A2A-receptor antagonist in porcine coronary artery and a useful tool to study A2A-receptor function.     

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.     

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.     

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.     

Allosteric modulation of A(2A) adenosine receptors by amiloride analogues and sodium ions

Allosteric regulation of rat A(2A) adenosine receptors by amiloride, amiloride analogues, and sodium ions was studied by investigating their ability to influence the dissociation of [(3)H]4-2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3, 5]triazin-5-yl-amino]ethylphenol ([(3)H]ZM241385) from receptors in rat striatal membranes. Both amiloride and its analogues accelerated the dissociation, the analogues being more potent than amiloride itself. In contrast, sodium ions decreased the rate of [(3)H]ZM241385 dissociation in a concentration-dependent manner, and this rate was not influenced by guanosine triphosphate, N-ethylmaleimide, suramin, or the selective A(2A) adenosine receptor antagonist, 5-amino-2-(2-furyl)-7(2-phenylethyl)pyrazolo[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine (SCH58261). The effect of competition between the amiloride analogue 5-(N,N-hexamethylene)amiloride (HMA) and sodium ions on [(3)H]ZM241385 dissociation was also explored. The addition of sodium ions resulted in a concentration-dependent rightward shift of the HMA response curve. The slopes of the HMA concentration-response curves in the presence and absence of sodium ions were not significantly different, which suggests that sodium ions and amiloride analogues act at a common allosteric site on the A(2A) adenosine receptor. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the amiloride analogues.     

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.     

ZM 241385, an adenosine A(2A) receptor antagonist, inhibits hippocampal A(1) receptor responses

4-(2-[7-amino-2-(2-furyl?1,2,4?-triazolo?2,3a?-?1,3, 5?triazin-5-yl-amino]ethyl)phenol (ZM 241385) has been used as an antagonist of adenosine A(2A) receptors, exhibiting high selectivity over adenosine A(1) receptors. We now report that ZM 241385 (10-50 nM) attenuated the inhibitory action of N(6)-cyclopentyladenosine (10 nM) and R(-)-N(6)-phenylisopropyladenosine (R-PIA, 20 nM), two selective adenosine A(1) receptor agonists, on hippocampal population spike amplitude. This effect is unlikely to be a direct antagonism of adenosine A(1) receptor since the K(i) of ZM 241385 to displace [3H]PIA (2 nM) binding, from hippocampal membranes ranged from 0.8 to 1.9 microM. These results question the usefulness of ZM 241385 to define adenosine A(2A) receptors actions in functional studies.     

Tonic adenosine A1 and A2A receptor activation is required for the excitatory action of VIP on synaptic transmission in the CA1 area of the hippocampus

Adenosine can regulate synaptic transmission through modulation of the action of other neurotransmitters. The influence of adenosine on VIP enhancement of synaptic transmission in hippocampal slices was investigated. Facilitation of fEPSP slope by 1 nM VIP (23.3+/-1.3%) was turned into an inhibition (-12.1+/-3.4%) when extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1U/ml). Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A(2A) receptors with ZM241385 (20 nM) attenuated the effect of VIP. When both DPCPX and ZM241385 were present the effect of VIP was abolished. In the presence of ADA, selective A(1) receptor activation with N(6)-cyclopentyladenosine (CPA, 15 nM) or A(2A) receptor-activation with CGS21680 (10 nM) partially readmitted the excitatory effect of VIP on fEPSPs. In contrast, facilitation of PS amplitude by 1 nM VIP (19.1+/-1.2%) was attenuated in the presence of ADA or DPCPX but was not changed by ZM241385. CPA, in the presence of ADA, fully restored the effect of VIP on PS amplitude. In conclusion, VIP facilitation of synaptic transmission to hippocampal pyramidal cell dendrites is dependent on both A(1) and A(2A) receptor activation by endogenous adenosine. VIP effects on PS amplitude are only dependent on A(1) adenosine receptor activation. This differential sensitivity to adenosine modulation might be due to the different VIP circuits contributing to VIP effects on pyramidal cell dendrites and pyramidal cell bodies.     

The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosine receptor antagonist.

1. This paper describes the in vitro pharmacology of ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin- 5-yl amino]ethyl) phenol), a novel non-xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype. 2. ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5'-N-ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02-10.02). In guinea-pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2-chloroadenosine (2-CADO) and 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45-8.68) and 9.02 (c.l., 8.79-9.24) respectively. 3. ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea-pig aorta with a pA2 of 7.06, (c.l., 6.92-7.19). 4. ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R-phenylisopropyladenosine (R-PIA) with a pIC50 of 5.69 (c.l., 5.57-5.81). ZM 241385 antagonized the bradycardic action of 2-CADO in guinea-pig atria with a pA2 of 5.95 (c.l., 5.72-6.18). 5. ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in chinese hamster ovary cells, it displaced iodinated aminobenzyl-5'-N-methylcarboxamido adenosine (AB-MECA) with a pIC50 of 3.82 (c.l., 3.67-4.06). 6. ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of A2A adenosine receptors and associated Galphas proteins by ZM 241385 treatment of porcine coronary artery

Functional regulation and expression of the adenosine A2A receptor and associated G-protein were investigated in porcine coronary artery exposed to an A2A receptor antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). The arteries were incubated for 3 days in culture medium in the absence (control) and presence (treated) of 10 microM ZM 241385. Changes in isometric tension by adenosine receptor agonists were evaluated in endothelium-free tissues. ZM 241385-treatment produced a statistically significant rightward displacement of CGS-21680, NECA, and CAD concentration-response curves compared with the respective controls (P < 0.05). The EC50, expressed in nM, values in treated and control tissues were: 617.3 +/- 23 versus 24.9 +/- 1.5 for CGS-21680 (2-(p-(2-carboxyethyl)phenethylamino)-5'N-ethylcarboxamidoadenosine), 27.4 +/- 6.3 versus 3.06 +/- 0.8 for NECA (5'-N-ethylcarboxamidoadenosine), and 5786.2 +/- 160 versus 89.2 +/- 24.1 for CAD (chloroadenosine). However, the relaxing effect of forskolin remained unchanged in treated and control tissues. The concentration-response curves for NECA, CAD, and CGS-21680 were also displaced to the right when cAMP levels were measured in treated and control smooth muscle cells while no differences were observed with forskolin. Quantitative Western blot analysis demonstrated that the density of A2A receptors increased in ZM 241385-treated artery. We also showed a significant decrease in Galphas protein levels after ZM 241385 treatment compared with control. Taken together, these data indicate that prolonged blockade of A2A receptors in the coronary artery leads to desensitization of the functional effects of adenosine agonists by a mechanism that involves decreases in cAMP production. This was associated with an up-regulation of A2A receptors and a decrease in Galphas protein expression.     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist,to rat striatal membranes.

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5''-N-ethyl carboxamidoadenosine (2HE-NECA) > 5''-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5''-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

A(2A) adenosine receptor mediated potassium channel activation in rat epididymal smooth muscle

The effects of A(2) adenosine receptor agonists upon phenylephrine-stimulated contractility in preparations of rat epididymis were investigated. Preparations responded to phenylephrine (3 microM) with submaximal contractions. Adenosine and the stable agonists 5'-N-ethylcarboxamido-adenosine (NECA) and 2-p-(2-carboxyethyl) phenethylamino-N-ethylcarboxamide adenosine (CGS 21680) inhibited phenylephrine-induced contractions (potency order, NECA>CGS 21680>adenosine). The A(2A) receptor-selective 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, 30 microM) blocked the response to NECA. The A(2A) adenosine receptor-mediated inhibitory responses to NECA were reduced by the K(ATP) channel blocker, glibenclamide (3 microM) and abolished by charybdotoxin (100 nM). The diterpene forskolin elicited a concentration-dependent inhibition of phenylephrine (3 microM)-stimulated contractility (by 62+/-8% of control at 100 microM). Charybdotoxin (100 nM), but not glibenclamide (3 microM) blocked the forskolin (10 microM) inhibition of phenylephrine-stimulated contractility. NECA elicited concentration-dependent increases in both cyclic AMP and cyclic GMP accumulation which were antagonized by ZM 241385 (30 nM). The protein kinase G activator, APT-cyclic GMP (8-(-Aminophenylthio) guanosine-3',5'-cyclic monophosphate) and the protein kinase A activator (Sp)-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Sp-8-Br-cyclic AMPs), inhibited phenylephrine (3 microM) induced contractions of rat epididymis. Glibenclamide (3 microM), but not charybdotoxin (100 nM), inhibited ATP-cyclic GMP responses. Charybdotoxin (100 nM), but not glibenclamide (3 microM) reduced the effect of Sp-8-Br-cyclic AMPs. This study shows that the A(2A) adenosine receptor inhibition of epididymal contractility may be mediated through the activation of charybdotoxin- and glibenclamide-sensitive potassium channels and may involve the activation of both protein kinases A and G.     

Characterization of [3H]ZM 241385 binding in wild-type and adenosine A2A receptor knockout mice

The binding of the adenosine A(2A) receptor antagonist [3H] 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol ([3H]ZM 241385) to mouse brain and spinal cord was investigated. In brain homogenates, single-site binding was observed with a Bmax of 299+/-28 fmol mg(-1) protein and a Kd of 0.75+/-0.08 nM. In autoradiographic studies, there was a high density of specific binding of [3H]ZM 241385 in the striatum, with a very low density in the cortex and no binding elsewhere in the brain or in the spinal cord. All specific binding of [3H]ZM 241385 was lost in genetically modified mice lacking the adenosine A(2A) receptor, confirming the selectivity of this radioligand.     

Adenosine receptors involved in modulation of noradrenaline release in isolated rat tail artery

Adenosine receptors involved in the modulation of noradrenaline release from postganglionic sympathetic nerves in rat tail artery were characterized by studying the effects of adenosine-receptor agonists and antagonists on electrically evoked tritium overflow (100 pulses, 5 Hz) and by immunohistochemistry. The adenosine A1 receptor-selective agonist N6-cyclopentyladenosine (CPA; 1-100 nM) and the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA; 1-10 microM) decreased tritium overflow. These effects were blocked by the adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 30 nM). The adenosine A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine (CGS 21680; 1-100 nM) enhanced tritium overflow, an effect blocked by the adenosine A(2A) receptor-selective antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261; 20 nM) but not changed by the adenosine A(2B) receptor-selective antagonist N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl) phenoxy]acetamide (MRS 1706; 20 nM). In the presence of DPCPX (30 nM), NECA enhanced tritium overflow, an effect abolished by MRS 1706 but not influenced by SCH 58261. Immunohistochemistry revealed immunoreactivity for all adenosine-receptor subtypes. Areas of co-localization were found for neurofilament with adenosine A1, A(2A) and A(2B) but not A3 receptors. In conclusion, the present study provides functional and morphological evidence for the occurrence of multiple adenosine receptor-mediated modulation of noradrenaline release in the rat tail: inhibition mediated by adenosine A1 receptors and facilitation mediated by both adenosine A(2A) and A(2B) 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.     

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.     

咖啡因对乙醛诱导的HSC-T6中TGF-β1,CTGF信号转导通路的影响

目的探讨咖啡因(caffeine)对乙醛诱导的大鼠肝星状细胞系(Hepatic Stellate Cell-T6,HSC-T6)中转化生长因子-β1(Trans-forming Growth Factor-β1,TGF-β1),结缔组织生长因子(Connective Tissue Growth Factor,CTGF)信号转导通路的影响。方法实验设正常组(常规培养),模型组及腺苷受体(Adenosine Receptor,AR)调节剂干预组。分别给予caffeine(4 mmol.L-1)[1-2],腺苷A2A受体拮抗剂ZM241385(1μmol.L-1)[3],腺苷A2A受体激动剂CGS21680(1μmol.L-1)[3],caffeine+CGS21680,ZM241385+CGS21680与HSC-T6共同培养,1 h后加入终浓度200μmol.L-1的乙醛刺激(每12 h补充1次),继续培养48 h。采用免疫细胞化学法检测HSC-T6中α-平滑肌肌动蛋白(α-SMA)的表达,RT-PCR法检测TGF-β1和CTGF mRNA水平,Western blot方法检测各组HSC-T6中CTGF蛋白表达。结果与模型组比较,caffeine及ZM241385均显著降低HSC-T6中α-SMA,TGF-β1和CTGF的表达,而caffeine及ZM241385合用CGS21680上述作用有所逆转。结论 Caffeine能够显著降低乙醛诱导的HSC-T6活化,并且显著抑制HSC-T6中TGF-β1和CTGF的表达水平,其机制可能与拮抗腺苷A2A受体介导的信号通路有关。     

Adenosine A2A receptor antagonism increases striatal glutamate outflow in dopamine-denervated rats

The objective of the work was to study, by in vivo microdialysis, the effect of the adenosine A(2A) receptor antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261) on glutamate outflow in the striata of unilateral 6-hydroxydopamine-infused rats. Two vertical microdialysis probes were implanted bilaterally in both the denervated striatum and in the intact striatum. Glutamate concentrations in the dialysate were determined by high-performance liquid chromatography (HPLC). Infusion of the adenosine A(2A) receptor antagonist SCH 58261 (50 nM), through the microdialysis fiber, significantly increased glutamate outflow from the denervated striatum while it decreased glutamate outflow from the intact striatum. The opposite effects of SCH 58261 on glutamate outflow in the intact and 6-hydroxydopamine-lesioned striatum might be attributed to blockade of striatal adenosine A(2A) receptors located on either striatal indirect output pathways or glutamatergic terminals. These results may be relevant to our understanding of the mechanism of action of adenosine A(2A) receptor antagonists in Parkinson's disease.