Native Rat Hippocampal 5-HT1AReceptors Show Constitutive Activity

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-[ 35 S]thio)-triphosphate ([ 35 S]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 [ 35 S]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 [ 35 S]GTPγS binding to Gα o to a similar extent, raising binding to approximately 130% of basal with pEC 50 values of 7.91 and 7.87, respectively. The 5-HT 1A -selective neutral antagonist [ O -methyl-3 H ]- N -(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)- N -(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with p K b values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its p K i value at r5-HT 1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [ 35 S]GTPγS binding to Gα o in a concentration-dependent manner to 90% of basal with pIC 50 values of 7.37 and 7.98, respectively. The inhibition of basal [ 35 S]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 (p K 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.