Abstract

Previous studies have shown that human 5-hydroxytryptamine (5-HT)_1A receptors stably expressed in transfected cell lines show constitutive G-protein activity, as revealed by the inhibitory effect of inverse agonists, such as spiperone, on basal guanosine-5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding. In the present study, we evaluated the constitutive activity of native rat 5-HT_1A receptors in hippocampal membranes. Using anti-Gα_o-antibody capture coupled to scintillation proximity assay under low sodium (30 mM) conditions, we observed high basal [³⁵S]GTPγS binding to Gα_o subunits (defined as 100%). Under these conditions, 5-HT and the prototypic selective 5-HT_1A agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] both stimulated [³⁵S]GTPγS binding to Gα_o to a similar extent, raising binding to approximately 130% of basal with pEC₅₀ values of 7.91 and 7.87, respectively. The 5-HT_1A-selective neutral antagonist [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with pK_b values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its pK_i value at r5-HT_1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [³⁵S]GTPγS binding to Gα_o in a concentration-dependent manner to 90% of basal with pIC₅₀ values of 7.37 and 7.98, respectively. The inhibition of basal [³⁵S]GTPγS binding induced by maximally effective concentrations of spiperone (10 μM) or methiothepin (1 μM) was antagonized by WAY100,635 in a concentration-dependent manner (pK_b, 9.52 and 8.87, respectively), thus indicating that this inverse agonism was mediated by 5-HT_1A receptors. These data provide the first demonstration that native rat serotonin 5-HT_1A receptors can exhibit constitutive activity in vitro.