The Mechanism of Action of Ghrelin and Motilin in the Pacemaker Potentials of Interstitial Cells of Cajal from the Murine Small Intestine

Abstract

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5′-diphosphate-β-S and pre-treatment with Ca ²⁺-free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate (IP ₃), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the IP ₃ receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, IP ₃-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular Ca ²⁺ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.