Activation of Gq proteins coupled with 5-HT2 receptors in rat cerebral cortical membranes assessed by antibody-capture scintillation proximity assay/[S]GTPγS binding

Abstract

Background/aims: Functional activation of Gq coupled with 5-HT2 receptors was investigated in rat cerebral cortical membranes.

Methods: Antibody-capture scintillation proximity assay (SPA)/[(35)S]GTPγS binding with anti-Gαq antibody was performed.

Results: The specific [(35)S]GTPγS binding to Gαq was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [(35)S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity.

Conclusion: It is concluded that by means of antibody-capture SPA/[(35)S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M1 muscarinic receptors, one of which is mediated through 5-HT2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.