Characteristics of Gintonin-Mediated Membrane Depolarization of Pacemaker Activity in Cultured Interstitial Cells of Cajal

Abstract

BACKGROUND/AIMS: Ginseng regulates gastrointestinal (GI) motor activity but the underlying components and molecular mechanisms are unknown. We investigated the effect of gintonin, a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, on the pacemaker activity of the interstitial cells of Cajal (ICC) in murine small intestine and GI motility. MATERIALS AND METHODS: Enzymatic digestion was used to dissociate ICC from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials and currents from cultured ICC in the absence or presence of gintonin. In vivo effects of gintonin on gastrointestinal (GI) motility were investigated by measuring the intestinal transit rate (ITR) of Evans blue in normal and streptozotocin (STZ)-induced diabetic mice. RESULTS: We investigated the effects of gintonin on pacemaker potentials and currents in cultured ICC from mouse small intestine. Gintonin caused membrane depolarization in current clamp mode but this action was blocked by Ki16425, an LPA1/3 receptor antagonist, and by the addition of GDPβS, a GTP-binding protein inhibitor, into the ICC. To study the gintonin signaling pathway, we examined the effects of U-73122, an active PLC inhibitor, and chelerythrine and calphostin, which inhibit PKC. All inhibitors blocked gintonin actions on pacemaker potentials, but not completely. Gintonin-mediated depolarization was lower in Ca(2+)-free than in Ca(2+)-containing external solutions and was blocked by thapsigargin. We found that, in ICC, gintonin also activated Ca(2+)-activated Cl(-) channels (TMEM16A, ANO1), but not TRPM7 channels. In vivo, gintonin (10-100 mg/kg, p.o.) not only significantly increased the ITR in normal mice but also ameliorated STZ-induced diabetic GI motility retardation in a dose-dependent manner. CONCLUSIONS: Gintonin-mediated membrane depolarization of pacemaker activity and ANO1 activation are coupled to the stimulation of GI contractility through LPA1/3 receptor signaling pathways in cultured murine ICC. Gintonin might be a ingredient responsible for ginseng-mediated GI tract modulations, and could be a novel candidate for development as a prokinetic agent that may prevent or alleviate GI motility dysfunctions in human patients.