Cussac Didier, Pasteau Valérie, Millan Mark J
Psychopharmacology Department, Institut de Recherches Servier, Centre de Recherches de Croissy, 125, Chemin de Ronde, 78290 Croissy-sur-Seine, France.
Eur J Pharmacol. 2004 Feb 6;485(1-3):111-7. doi: 10.1016/j.ejphar.2003.11.077.
Herein, we examined the direct coupling of human dopamine D1 receptors to G(s) proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists ( approximately 100%) in stimulating guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding to G(s) in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [35S]GTP gamma S binding to G(s) with a pK(B) (9.29) close to its pK(i) (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated G(s) activation with pK(B)'s of 6.7 and 7.3, respectively, values close to their pK(i) values at these sites. In conclusion, the use of an anti-G(s) protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.
在此，我们使用抗体捕获测定法以及采用闪烁邻近测定微珠的检测技术，研究了人类多巴胺D1受体与G(s)蛋白的直接偶联。使用特异性抗体，多巴胺（DA）以及选择性多巴胺D1受体激动剂6-氯-7,8-二羟基-1-苯基-2,3,4,5-四氢-1H-3-苯并氮杂卓（SKF81297）和3-烯丙基-6-氯-7,8-二羟基-1-苯基-2,3,4,5-四氢-1H-3-苯并氮杂卓（SKF82958），在刺激L细胞中鸟苷-5'-O-(3-[35S]硫代)-三磷酸（[35S]GTPγS）与G(s)结合时表现为高效激动剂（约100%），而2,3,4,5-四氢-7,8-二羟基-1-苯基-1H-3-苯并氮杂卓（SKF38393）表现出部分激动剂特性（70%）。多巴胺的作用是由人类多巴胺D1受体特异性介导的，因为选择性人类多巴胺D1受体拮抗剂（R）-(+)-8-氯-2,3,4,5-四氢-3-甲基-5-苯基-1H-苯并氮杂卓-7-醇（SCH23390）阻断多巴胺诱导的[35S]GTPγS与G(s)的结合，其pK(B)（9.29）接近其pK(i)（9.33）。抗精神病药物氯氮平和氟哌啶醇单独测试时无内在活性，分别以pK(B)值6.7和7.3抑制多巴胺刺激的G(s)激活,这些值接近它们在这些位点的pK(i)值。总之，将抗G(s)蛋白免疫沉淀测定与闪烁邻近测定相结合，能够在G蛋白水平直接评估多巴胺D1受体配体的功能活性。采用这种新技术，典型和非典型抗精神病药物氯氮平和氟哌啶醇在多巴胺D1受体上均表现出拮抗剂特性。
