Abstract

We have expanded previous studies with the 5-hydroxytryptamine (5-HT)₂ receptor agonist (±)-1-(2,5-dimethoxy-4-[¹²⁵I]iodophenyl)-2-aminopropane [(±)-[¹²⁵I]DOI] in human brain that had shown biphasic competition curves for several 5-HT_2A receptor antagonists by using new selective antagonists of 5-HT_2A (MDL100,907) and 5-HT_2C (SB242084) receptors together with ketanserin and mesulergine. Autoradiographic competition experiments were performed with these antagonists in human brain regions where (±)-[¹²⁵I]DOI labels almost exclusively 5-HT_2A receptors (frontal cortex and striosomes). Furthermore, the effect of uncoupling receptor/G protein complexes on antagonist competition was studied with guanosine-5′-(β,γ-imido)triphosphate [Gpp(NH)p]. Competition experiments with (±)-[³H]1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane [(±)-[³H]DOB] were also performed in membranes from Chinese hamster ovary cells (CHOFA4) expressing cloned human 5-HT_2A receptors. In both systems, ketanserin and MDL100,907 displayed biphasic competition profiles, whereas SB242084 and mesulergine competed monophasically. In absence of antagonist, 100 μM Gpp(NH)p decreased brain (±)-[¹²⁵I]DOI specific binding by 40 to 50% and (±)-[³H]DOB specific binding to CHOFA4 cells by 30%. The remaining agonist-labeled uncoupled sites were still displaced biphasically by ketanserin and MDL100,907, with unaltered affinities. Saturation experiments were performed in CHOFA4 cells. (±)-[³H]DOB labeled two sites (K_dh= 0.8 nM,K_dl = 31.22 nM). Addition of 100 μM Gpp(NH)p resulted in a single low-affinity (K_d = 24.44 nM) site with unchanged B_max. [³H]5-HT showed no specific binding to 5-HT_2A receptors. These results conform with the extended ternary complex model of receptor action that postulates the existence of partly activated receptor conformation(s) (R∗) in equilibrium with the ground (R) and the activated G protein-coupled (R∗G) conformations. Thus, both in human brain and CHOFA4 cells, the agonists possibly label all three conformations and ketanserin and MDL100,907 recognize with different affinities at least two of these conformations.