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 Ca²⁺-free than in Ca²⁺-containing external solutions and was blocked by thapsigargin. We found that, in ICC, gintonin also activated Ca²⁺-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.

Quercetin Inhibits Pacemaker Potentials via Nitric Oxide/cGMP-Dependent Activation and TRPM7/ANO1 Channels in Cultured Interstitial Cells of Cajal from Mouse Small Intestine

“…In a previous study, we found that ANO1 was well expressed in ICCs within 12 hours of culture, but subsequently decreased with time [40]. Therefore, in the present study, we used ICCs that had been cultured for least than 12 hours (Fig.…”

“…Furthermore, TRPM7 and ANO1 mRNA have been reported in ICCs and TRPM7 and ANO1 currents have been described [24,35,40,41]. Thus, we examined the effects of quercetin on the expressions of ANO1 and TRPM7 channels in HEK 293 cells, which overexpress both channels.…”

“…The role played by ANO1 in ICC pacemaker activity was identified in ANO1 knockout mice [37], and several other authors have since shown that ANO1 participates in pacemaker activity [38,39]. Kim et al [24,40] showed that TRPM7 mRNA and TRPM7 currents are expressed in cultured ICCs, and Shahi et al [40,41] showed that ANO1 mRNA and ANO1 currents are also expressed in cultured ICCs. Accordingly, it has been proposed that TRPM7 and ANO1 be considered potential targets for the pharmacological treatment of GI motility disorders.…”

Menthol Modulates Pacemaker Potentials through TRPA1 Channels in Cultured Interstitial Cells of Cajal from Murine Small Intestine

“…Slow waves play an important role in the regulation of GI motility by determining the frequency and timing of smooth muscle contractions, and ICCs, which are connected to smooth muscle cells by gap junctions to form a network, are the pacemaker cells that generate slow waves by producing spontaneous PPs. Accordingly, PPs generated by ICCs are directly transmitted to smooth muscles through gap junctions [13][14][15]. In addition, ICCs mediate inhibitory and excitatory signals from the enteric nervous system to smooth muscles, and thus, importantly regulate GI motility.…”

Effects of Prunus mume Siebold & Zucc. in the pacemaking activity of interstitial cells of Cajal in murine small intestine

“…It has been determined that Poncirus fructus (PF) is able to modulate pacemaker electrical potentials via the 5-hydroxytryptamine (5-HT) 3 and 5-HT 4 receptor pathways in a MAPK-dependent manner (24) and gintonin-mediated membrane depolarization and Ca 2+ -activated Cl -channel activation have been observed in cultured murine ICCs via a lysophosphatidic acid 1/3 receptor signaling pathway (41). Additionally, it was determined that San-Huang-Xie-Xin-tang (SHXXT) is able to modulate pacemaker electrical potentials (42).…”