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

Abstract

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-​triazolo[1,​5-​c]​pyrimidine, [3H]​-​SCH 58261. [3H]​-​SCH 58261 specific binding to rat striatal membranes (>90​%) was saturable, reversible and dependent upon protein concn. Satn. expts. revealed that [3H]​-​SCH 58261 labeled 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 μM GTP in the incubation mixt. did not modify [3H]​-​SCH 58261 binding parameters. Competition expts. showed that [3H]​-​SCH 58261 binding is consistent with the labeling 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'-​ethylcarboxamidoadenosine (2HE-​NECA)​>5'-​N-​ethylcarboxamidoadenosine (NECA)​>2-​[4-​(2-​carboxyethyl)​phenethylamino]​-​5'-​N-​ethylcarboxamidoadenosine (CGS 21680)​>2-​phenylaminoadenosine (CV 1808)​>R-​N6-​phenylisopropyladenosine (R-​PIA)​>N6-​cyclohexyladenosine (CCPA)​>S-​N6-​phenylisopropyladenosine (S-​PIA)​. Adenosine antagonists inhibited [3H]​-​SCH 58261 binding in the following order: 5-​amino-​9-​chloro-​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)​≥8-​phenyltheophylline (8-​PT)​. The Ki values for adenosine antagonists were similar to those labeled 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 examd. inhibited [3H]​-​SCH 58261 binding with Hill coeffs. not significantly different from unity. 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