Regulation of the pacemaker activities in cultured interstitial cells of Cajal by Citrus unshiu peel extracts.

The Citrus unshiu peel has been widely used for the treatment of gastrointestinal (GI) disorders in Eastern traditional medicine. The present study aimed to investigate the effects of Citrus unshiu peel extract (CPE) on the pacemaker activity of the GI tract in cultured interstitial cells of Cajal (ICCs) derived from the mouse small intestine. The whole­cell patch­clamp configuration was used to record pacemaker potentials. In current clamp mode, exposure to CPE caused membrane pacemaker depolarization in a concentration­dependent manner. In the presence of the muscarinic M2 receptor antagonist, methoctramine, CPE induced membrane pacemaker depolarization, whereas treatment with the muscarinic M3 receptor antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide, inhibited CPE­induced responses. When the pipette solution contained guanosine 5'-(ß-thio) diphosphate trilithium salt (1 mM), CPE marginally induced membrane pacemaker depolarization. In addition, CPE­induced membrane pacemaker depolarization was inhibited following exposure to the active phospholipase C (PLC) inhibitor U­73122, but not the inactive PLC inhibitor U­73343. In the presence of a p42/p44 mitogen­activated protein kinase (MAPK) inhibitor (PD98059), a p38 MAPK inhibitor (SB203580) or a c­jun NH2­terminal kinase (JNK) II inhibitor, CPE failed to induce membrane pacemaker depolarization. These results suggest that CPE may affect GI motility through modulating ICC pacemaker activity by activating the muscarinic M3 receptor and inducing the G­protein dependent PLC and MAPK signaling pathways.

The traditional herbal medicine, Ge-Gen-Tang, inhibits pacemaker potentials by nitric oxide/cGMP dependent ATP-sensitive K(+) channels in cultured interstitial cells of Cajal from mouse small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Ge-Gen-Tang (GGT) is a traditional Chinese medicinal formula composed of Puerariae radix (Pueraria lobata Ohwi), Ephedrae Herba (Ephedra sinica Stapf), Cinnamomi Ramulus (Cinnamomum cassia Blume), Paeoniae Radix (Paeonia lactiflora Pallas), Glycyrrhizae Radix preparata (Glycyrrhiza uralensis Fischer), Zingiberis Rhizoma (Zingiber officinale Roscoe), and Zizyphi Fructus (Ziziphus jujuba Mill. var. inermis Rehder) and is widely used to ameoliorate the symptoms of gastrointestinal (GI) disorders related to diarrhea and intestinal mucosal immunity and for anti-cold, antipyretic and analgesic in Eastern Asia. AIM OF THE STUDY: Interstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. We investigated the effects of GGT on pacemaker potentials in cultured ICCs from the mouse small intestine, and sought to identify the receptors and the action mechanisms involved. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed on within 12h after culture. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. Intracellular Ca(2+) ([Ca(2+)]i) increase was studied in cultured ICCs using fura-2AM. All of the experiments were performed at 30-32°C. RESULTS: Under the current clamping mode, GGT decreased the amplitude and frequency of pacemaker potentials; however, these effects were blocked by intracellular GDPßS, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K(+) channels blocker. Prazosin (α1-adrenoceptor antagonist) and butoxamine (ß2-adrenoceptor antagonist) did not block the GGT-induced effects, whereas atenolol (ß1-adrenoceptor antagonist) blocked the GGT-induced effects. Also, yohimbine (α2-adrenoceptor antagonist) partially blocked the GGT-induced effects. Pretreatment with SQ-22536, an adenylate cyclase inhibitor, did not block the GGT-induced effects, whereas pretreatment with ODQ, a guanylate cyclase inhibitor, or L-NAME, an inhibitor of nitric oxide (NO) synthase, did. Additionally, [Ca(2+)]i analysis showed that GGT decreased [Ca(2+)]i. CONCLUSION: These results suggest that GGT inhibits pacemaker potentials in ICCs in a G protein-, cGMP- and NO-dependent manner through stimulation of α2 and ß1-adrenoceptors.