M1686 Activation of Muscarinic Acetylcholine Receptor Subtype 4 is Essential for Carbachol- Induced Pepsinogen Secretion in Mice: Relation to D Cells/Somatostatin

Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1~M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. Although the acid response to acetylcholine is considered to be mainly mediated by the activation of M3-receptors, we recently found that carbachol (CCh)-induced acid secretion was markedly decreased in M4 knockout (KO) mice. In the present study, we demonstrated, using M1~M5 KO mice, the importance of M4 receptors in the cholinergic stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice (12-20 wks old) of wild-type (WT) and M1~M5 KO were used. Under urethane anesthesia, the abdomen was incised, and an acute fistula prepared with a polyethylene tube was provided in the stomach through a pylorus. Then, the stomach was instilled with saline (0.4 ml) through the fistula, and the solution was changed every 20 min. CCh (30 μg/kg) was given SC as a single injection. Atropine (0.3 mg/kg) or CYN154806 [somatostatin-2 receptor (SST2R) antagonist: 0.1-3mg/kg] was given SC 20 min before CCh. Expressions of D cells and M4 receptors was examined immunohistochemically by double staining with anti-somatostatin and anti-M4 receptor antibodies. Results: CCh caused an increase of acid secretion in WT mice, and the effect was completely inhibited by prior administration of atropine. The stimulatory effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KOmice, as compared toWT; especially, the response was all but completely abolished in M4 KO mice. CYN154806, the SST2R antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Somatostatin inhibited the secretion of acid under basal and CCh-stimulated conditions. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. It was also found that serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions and that the somatostatin levels in WT mice were decreased in response to CCh. Conclusion: These results suggest that under cholinergic stimulation the secretion of acid is mediated by the activation of both M3 and M4 receptors but does not involve other muscarinic receptor subtypes, and the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of somatostatin through SST2 receptors, resulting in enhancement of the acid response mediated by M3 receptors on parietal cells.