Characterisation of Gs activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine
- 作者：Cussac ; Didier ; Pasteau ; Valé ; rie ; Millan ; Mark J.
- 关键词：Dopamine D1 receptor ; Gs protein ; Scintillation proximity assay ; Clozapine ; Haloperidol
- 刊名：European Journal of Pharmacology
- 出版年：2004
- 期刊代码：24_00142999
- 类别：neu
- 出版时间：February 6, 2004
- 卷：485
- 期：1-3
- 页码：111-117
- 文件大小：287 K
摘要
Herein, we examined the direct coupling of human dopamine D1 receptors to Gs 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 (
100%) in stimulating guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding to Gs 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]GTPγS binding to Gs with a pKB (9.29) close to its pKi (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated Gs activation with pKB's of 6.7 and 7.3, respectively, values close to their pKi values at these sites. In conclusion, the use of an anti-Gs 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.
100%) in stimulating guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding to Gs 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]GTPγS binding to Gs with a pKB (9.29) close to its pKi (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated Gs activation with pKB's of 6.7 and 7.3, respectively, values close to their pKi values at these sites. In conclusion, the use of an anti-Gs 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.