5-HT1P Receptors in the Bowel: G Protein Coupling, Localization, and Function

Abstract

Although serotinin (5-HT) has been demonstrated to mediate slow exicitatory postsynaptic potentials in neurons of the myenteric plexus of the bowel, its role in the physiology of gastrointestinal motility and secretion is not well understood. Five subtypes of 5-HT receptor have been reported in the gut. The 5-HT2 receptor appears to be present on smooth muscle, while the others, 5-HT1A, 5-HT1P„ 5-HT3, and 5-HT4 are neuronal. The present experiments were undertaken in order to obtain more information about the location, action, and function of 5-HT1P receptors. GTP-γ-S, was found to inhibit the binding of 3H-5-HT by the 5-HT1P receptor (the IC50 for GTP-γ-S was 1.8 ± 0.4 μM). GTP-γ-S was more potent than GTP, while ATP and GMP (100 μM) were without effect. These observations are compatible with the hypothesis that the 5-HT1P receptor is coupled to a G protein. 5-HT (0.1–10μM) was also found to elevate levels of cAMP in preparations of isolated myenteric ganglia. Renzapride also increased cAMP, but the response neither to 5-HT nor to renzapride was inhibited by the 5-HT1P antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) or the 5-HT3/5-HT4 antagonist, ICS 205–930, even at 10 μM. The action of 5-HT on ganglionic cAMP, therefore, cannot now be attributed to 5-HT1P, or 5-HT4 receptors. Polyclonal anti-idiotypic antibodies were raised that recognize some, but not all, subtypes of 5-HT receptor. These antibodies were demonstrated to bind to 5-HT2, 5-HT1C, 5-HT1P, and 5-HT3, but not 5-HT1A receptors. When applied to enteric neurons, the anti-idiotypic antibodies transiently mimicked and then blocked the actions of 5-HT at 5-HT3 and 5-HT1P receptors. The blockage of responses to 5-HT that followed application of the antibody was specific, since neurons continued to respond as before to carbachol or substance P. The 5-HT1P agonist-like actions of the antibodies were prevented by desensitization of 5-HT receptors and by renzapride (1 μM). The anti-idiotypic antibodies were found to be useful for the immunocytochemical localization of specific 5-HT receptor subtypes, if they were applied to tissues in the presence of appropriate antagonists. Use of these antibodies indicated that 5-HT1P receptors are located, not only on neurons in myenteric and submucosal ganglia, but also on a subepithelial plexus of nerve fibers. Finally, 5-HT1P receptor antagonists (5-HTP-DP, renzapride, and anti-idiotypic antibodies), were found to prevent excitation of submucosal or myenteric neurons by mucosal application of cholera toxin. This observation, and the localization of 5-HT1P receptors, is consistent with the idea that they play a role in the excitation of enteric neurons during the peristaltic reflex.