Binding of the radioligand [H-3]-SCH 58261, a new non-xanthine A(2A) adenosine receptor antagonist, to rat striatal membranes

1 The present study describes the binding to rat striatal A(2A) adenosine receptors of the new potent and selective antagonist radioligand, [H-3]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine, [H-3]-SCH 58261. 2 [H-3]-SCH 58261 specific binding to rat striatal membranes (>90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [H-3]-SCH 58261 labelled a single class of recognition sites with high affinity (K-d=0.70 nM) and limited capacity (apparent B-max=971 fmol mg(-1) of protein). The presence of 100 mu M GTP in the incubation mixture did not modify [H-3]-SCH 58261 binding parameters. 3 Competition experiments showed that [H-3]-SCH 58261 binding is consistent with the labelling of A(2A) striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [H-3]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA)>5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(2-carboxyerhyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680)>2-phenylaminoadenosine (CV 1808)>R-N-6-phenylisopropyiadenosine (R-PIA)>N-6-cyclohexyladenosine (CHA)=2-chloro-N-6-cyclopentyladenosine (CCPA)>S-N-6-phenylisopropyladenosine (S-PIA). 4 Adenosine antagonists inhibited [H-3]-SCH 55261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazoio[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 5826l>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)greater than or equal to 8-phenyltheophylline (8-PT). 5 The K-i values for adenosine antagonists were similar to those labelled with the A(2A) agonist [H-3]-CGS 21680. Affinities of agonists were generally lower. The A(1)-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [K-3]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [H-3]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6 The present results indicate that [H-3]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A(2A) Striatal receptors. High receptor affinity. good selectivity and very low non-specific binding make [H-3]-SCH 5826] an excellent probe for studying the A(2A) adenosine receptor subtype in mammalian brain.