G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

Abstract

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A_2A receptor agonist 2-[4-(2-p-carboxyethyl[³H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([³H]CGS 21680) and of the adenosine A₁ receptor agonist [³H]R-phenylisopropyladenosine ([³H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [³H]CGS 21680 (30 nM) binding in the cortex than [³H]R-PIA (2 nM) binding to cortical or striatal membranes or [³H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple G_i/G_o protein-coupled receptors, more effectively inhibited [³H]R-PIA binding to cortical or striatal membranes and [³H]CGS 21680 binding in the cortex than [³H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples G_s protein-coupled receptors, more effectively inhibited [³H]CGS 21680 binding in the striatum than [³H]CGS 21680 binding in the cortex and less effectively inhibited [³H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [³H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with G_i/G_o proteins. The K _i for displacement of [2-³H]-(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, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K _i for displacement of [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([³H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A₁ antagonist, [³H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A₁ agonist, [³H]R-PIA (2 nM), in 2 or 10 mM Mg²⁺-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A₁ receptors and that limbic CGS 21680 binding sites differ from striatal-like A_2A receptors since they couple to G_i/G_o proteins, as well as to G_s proteins.