Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]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)/[³⁵S]GTPγS binding with anti-Gα_q antibody was performed. Results: The specific [³⁵S]GTPγS binding to Gα_q 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 [³⁵S]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/[³⁵S]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.