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)-pyrazolo4,3-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]-triazolo1,5-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.