5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes

Receptor-mediated guanine nucleotide-binding regulatory protein ( G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [S-35] guanosine-5'- O-(3-thio) triphosphate ([S-35] GTP gamma S) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through G(i) type G proteins. In the present study, 5-HT-stimulated [S-35] GTP gamma S binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl2 and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 mu M GDP, 5 mM MgCl2 and 100 mM NaCl. The specific [S-35] GTP gamma S binding was stimulated by several compounds that had been shown to be agonists at 5HT(1B/1D) receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC(50)) for these agonists were significantly correlated with their pK(i)'s reported in the previous study of 5-HT1B receptor binding in rat frontal cortical membranes. The increase in specific [S-35] GTP gamma S binding in response to 1 mu M 5-HT was potently inhibited by several 5-HT1B/1D receptor antagonists as well as beta-adrenoceptor antagonists such as S(-)cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl) piperazin-1-yl]- 1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT1D receptors, was inactive as an antagonist at least up to 1 mu M. Additionally, the concentration-response curve for 2-[5[ 3-( 4-methylsulphonylamino) benzyl-1,2,4-oxadiazol-5- yl]-1H-indol-3-yl] ethanamine (L694247) was shifted rightward in parallel by the addition of S(-)- cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [S-35] GTP gamma S binding was reduced by 1'-methyl-5-([2'-methyl-4'-(5-methyl- 1,2,4-oxadiazol-3-yl) biphenyl-4-yl] carbonyl)-2, 3,6,7-tetrahydrospiro( furo[2,3-f] indole-3,4'-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nMS(-)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT1B receptors in the present functional assay system. 5-HT-stimulated [S-35] GTP gamma S binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT1B receptors and their coupled G proteins in this brain region.