Cunha R A, Constantino M D, Ribeiro J A
Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, Portugal.
Naunyn Schmiedebergs Arch Pharmacol. 1999 Apr;359(4):295-302. doi: 10.1007/pl00005355.
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'-(beta,gamma-imido)triphosphate (1-300 microM), 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 microM) or pertussis toxin (1-100 microg/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 microg/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 microg/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, 1nM) by CGS 21680 was 110 nM (95%CI: 98-122 nM) in non-treated, 230 (167-292) nM in pertussis toxin (25 microg/ml)-treated and 222 (150-295) nM in cholera toxin (50 microg/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-triazol o(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 Mg(2+)-medium, either at 25 degrees C or 37 degrees 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 G(i)/G(o) proteins, as well as to G(s) proteins.
研究了鸟嘌呤核苷酸结合蛋白（G蛋白）调节剂对腺苷A2A受体激动剂2-[4-(2-对羧乙基[3H])苯基-氨基]-5'-N-乙基羧酰胺腺苷（[3H]CGS 21680）以及腺苷A1受体激动剂[3H]R-苯异丙基腺苷（[3H]R-PIA）与大鼠皮质和纹状体膜结合的影响。能使所有G蛋白解偶联的鸟苷5'-(β,γ-亚氨基)三磷酸（1 - 300 μM），对皮质中[3H]CGS 21680（30 nM）结合的抑制作用比对皮质或纹状体膜中[3H]R-PIA（2 nM）结合以及纹状体中[3H]CGS 21680（30 nM）结合更有效。能使G(i)/G(o)蛋白偶联受体解偶联的N-乙基马来酰胺（1 - 300 μM）或百日咳毒素（1 - 100 μg/ml），对皮质或纹状体膜中[3H]R-PIA结合以及皮质中[3H]CGS 21680结合的抑制作用比对纹状体中[3H]CGS 21680结合更有效。能使G(S)蛋白偶联受体解偶联的霍乱毒素（2.5 - 250 μg/ml），对纹状体中[3H]CGS 21680结合的抑制作用比对皮质中[3H]CGS 21680结合更有效，而对皮质或纹状体膜中[3H]R-PIA结合的抑制作用较弱。用百日咳毒素（50 μg/ml）处理溶解的皮质膜后，[3H]CGS 21680（30 - 100 nM）结合减少，在用G(i)/G(o)蛋白重构后几乎完全恢复。在未处理的皮质膜中，CGS 21680对[2-3H]-(4{2-[7-氨基-2-(2-呋喃基)(1,2,4)三唑并(2,3-a)(1,3,5)三嗪 - 5-基氨基]乙基}苯酚)（[3H]ZM 241385, 1 nM）的置换K(i)为1纳摩尔（95%置信区间：9纳摩尔 - 122纳摩尔），在百日咳毒素（25 μg/ml）处理的皮质膜中为230（167 - 292）纳摩尔，在霍乱毒素（50 μg/ml）处理的皮质膜中为222（150 - 的295）纳摩尔；相比之下，在未处理的皮质膜中，CGS 21680对[3H]-5-氨基-7-(2-苯乙基)-2-(2-呋喃基)-吡唑并(4,3-e)-1,2,4-三唑并(1,5-c)嘧啶（[3H]SCH 58261, 1 nM）的置换K(i)为74（57 - 91）纳摩尔，在百日咳毒素处理的皮质膜中为71（44 - 100）纳摩尔，在霍乱毒素处理的皮质膜中为147（100 - 193）纳摩尔。最后，在25℃或37℃下，无论是在皮质还是纹状体膜中，在2或10 mM Mg(2+)介质中，CGS 21680对A1拮抗剂[3H]8-环戊基-1,3-二丙基黄嘌呤（2 nM）和A1激动剂[3H]R-PIA（2 nM）的结合呈单相置换。这些结果表明，CGS 21680不与A1受体结合，且边缘系统CGS 21680结合位点不同于纹状体样A2A受体，因为它们既能与G(i)/G(o)蛋白偶联，也能与G(s)蛋白偶联。
