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Notes: Abstract  The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl) phenylamino] -5′-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3H] CGS 21680, in the concentration range tested (0.2–200 nM), bound to a single site with a K d of 58 nM and a B max of 353 fmol/mg protein in the hippocampus, and with a K d of 58 nM and a B max of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a K d of 17 nM and a B max of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 μM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N 6-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10–25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl-8-{4-[(2-aminoethyl)amino]carbonylmethyloxyphenyl} xanthine (XAC), 8-(3-chlorostyryl) caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX 〉 XAC ≫ CSC ≈ KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′-N-ethylcarboxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA ≈ NECA≥CADO〉 APEC ≈ CV1808 〉 CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2–200 nM) bound to a site (A2A-like) with a K d of 20 nM and a B max of 56 fmol/mg protein in the hippocampus and with a K d of 22 nM and a B max of 63 fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680 (0.2–200 nM) bound to a second high-affinity site with a K d of 97 nM and a B max of 255 fmol/mg protein in the hippocampus and with a K d of 112 nM and a B max of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A2A receptors and the other to the second high-affinity [3H]CGS 21680 binding site. In contrast, the pharmacology of [3H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [3H]CGS 21680 binding sites in the CNS and demonstrate that the [3H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A2A adenosine receptor, which predominates in the striatum.

Notes: Abstract The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A2A 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 Gi/Go 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 Gs 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 Gi/Go 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 A2A receptors since they couple to Gi/Go proteins, as well as to Gs proteins.