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

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Abstract
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-triazolo [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 μm 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-ethylcarboxamido- adenosine (CGS 21680) > 2-phenylaminoadenosine (CV 1808)> R-N6-phenylisopropyladenosine (R- PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopro- pyladenosine (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)-pyra- zolo [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-cyclo- pentyl-1,3-dipropylxanthine (DPCPX) ≥ 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 Ai-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.

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