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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 Ca2+- free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate (IP3), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the IP3 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-, IP3-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular Ca2+ 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. [ABSTRACT FROM AUTHOR]

To investigate the role of ATP-sensitive K + (K ATP ) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca 2+ ([Ca 2+ ] i ) imaging were performed in cultured colonic ICC. Pinacidil (a K + channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a K ATP channel blocker). RT-PCR showed that K ir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial K ATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca 2+ ([Ca 2+ ] i ) oscillations showed that glibenclamide increased the frequency of [Ca 2+ ] i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and K ir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, K ATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Liriope platyphylla Wang et Tang continues to be used in Korea as a traditional medicine for the treatment of gastrointestinal (GI) disorders related to constipation and abnormal GI motility. Aim of the study Because GI disorders, especially GI motility dysfunctions, are major lifelong problems, the authors investigated the effects of a water extract of the roots of L . platyphylla Wang et Tang (LPE) on the pacemaker potentials (PPTs) of interstitial cells of Cajal (ICCs) and on GI motility in male ICR mice. Materials and methods Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record PPTs generated by cultured ICCs in vitro. In vivo effects of LPE on GI motility were investigated by measuring intestinal transit rates (ITRs) of Evans blue in normal mice and in acetic acid (AA) and streptozotocin (STZ)-induced diabetic mouse models of GI motility dysfunction. Results LPE dose-dependently depolarized PPTs in ICCs. Pretreatment with methoctramine (a muscarinic M 2 receptor antagonist) did not block LPE-induced PPT depolarization. However, pretreatment with 4-DAMP (a muscarinic M 3 receptor antagonist) blocked LPE-induced PPT depolarization. In addition, treatment with LY294002 (a phosphoinositide 3-kinase (PI3K) inhibitor) also blocked LPE-induced PPT depolarization. Intracellular GDP β S inhibited LPE-induced PPT depolarization, and LPE-induced PPT depolarization was found to occur in a phospholipase C (PLC)- and a protein kinase C (PKC)-dependent manner. Pretreatment with Ca 2+ free solution or thapsigargin (a Ca 2+ -ATPase inhibitor in endoplasmic reticulum) abolished PPTs, and under these conditions, LPE did not depolarize ICC PPTs. In normal mice, ITRs were significantly and dose-dependently increased by LPE (0.01–1 g/kg administered intragastrically (i.g.)). In addition, LPE (i.g.) significantly recovered GI motility dysfunctions in both animal models. Conclusion LPE dose-dependently depolarizes ICC PPTs through M 3 receptors via external and internal Ca 2+ regulation and via G protein-, PI3K-, PLC- and PKC- dependent pathways in vitro . Also, in vivo , LPE increased ITRs in treatment naïve mice and our two mouse models of GI dysfunction. Therefore, this study shows that LPE offers a basis for the development of a prokinetic agent that prevents or alleviates GI motility dysfunctions. [ABSTRACT FROM AUTHOR]

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 12 h 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-2 AM. 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 GDPβS, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K + channels blocker. Prazosin (α1-adrenoceptor antagonist) and butoxamine (β2-adrenoceptor antagonist) did not block the GGT-induced effects, whereas atenolol (β1-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 β1-adrenoceptors. [ABSTRACT FROM AUTHOR]

We investigated the presence of β 3 -adrenoceptor and its functional effects on pacemaker potentials in colonic interstitial cells of Cajal (ICCs) from mice. The whole-cell patch clamp technique was used to record pacemaker potentials in cultured ICCs and reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA transcript levels β-adrenoceptors. The β 3 -adrenoceptor agonist, BRL37344, reduced the frequency of pacemaker potentials in a concentration-dependent manner. The inhibitory effects of BRL37344 were blocked by the pretreatment of propranolol, a nonspecific β-adrenoceptor antagonist, but not by the selective β 1 -adrenoceptor antagonist atenolol and the selective β 2 -adrenoceptor antagonist butoxamine. β 3 -adrenoceptor antagonists SR59230A and L748337 blocked the inhibitory effects of BRL37344. RT-PCR revealed mRNA transcripts of β 1 - and β 3 -adrenoceptor, but not β 2 -adrenoceptor, in c-kit- and Ano-1-positive colonic ICCs. The K + channel blockers tetraethylammonium, apamin, and glibenclamide did not block the effects of BRL37344. N ω -Nitro- l -arginine methyl ester hydrochloride (L-NAME), an NO synthase inhibitor, and chelerythrine, a protein kinase C inhibitor, also did not block the effects of BRL37344. Noradrenaline mimicked the effects of BRL37344 in colonic ICCs. However, the inhibitory effects of noradrenaline on pacemaker potentials were blocked only by pretreatment with atenolol but not by butoxamine, SR59230A, or L748337. In small intestinal ICCs, BRL37344 had no effect on pacemaker potentials and mRNA transcripts of β 1 -and β 2 -adrenoceptor, but not β 3 -adrenoceptor were detected. These results suggest that β 3 -adrenoceptors are present in colonic ICCs and may play a role in regulating gastrointestinal motility by the inhibition of pacemaker potentials. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca activated Cl channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720-P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility. [ABSTRACT FROM AUTHOR]

[Display omitted] • TRPV1 and TRPA1 do not play major roles in regulating pacemaking potentials in the ileum of the mouse. • The inhibitory actions of TRPV1-specific agonists, capsaicin, and TRPV1. • antagonist, capsazepine, on pacemaking potentials is not likely to act via TRPV1 receptors. • Non-selective TRP antagonist, ruthenium red, has unique actions to increase pacemaker frequency at 300 μM. • Both TRPA1 agonists and antagonists have the ability to reduce pacemaker frequency. The roles of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and subfamily A, member 1 (TRPA1) in mechanisms of gastrointestinal motility are complex. This study aimed to clarify the effects of several TRPV1 and TRPA1 ligands on the electrical potentials generated by pacemaker cells in the mouse-isolated ileum. The pacemaker potentials of ileal segments of mice were recorded extracellularly using a 60-channel microelectrode array. The dominant frequencies, average waveform periods and propagation velocities were quantified. The effects of TRPV1 and TRPA1 agonist and antagonist were compared with the baseline recordings. The electrophysiological recordings showed that capsaicin (30 μM to 3 mM), resiniferatoxin (300 μM), capsazepine (100–300 μM), allyl isothiocyanate (300 μM), isovelleral (300 μM), icilin (300 μM), A-967,079 (10 μM), AP18 (20 μM) and HC-030,031 (50 μM) significantly reduced the pacemaker frequency and increased the waveform period relative to the baseline. Conversely, ruthenium red (300 μM) significantly increased the pacemaker frequency and reduced the waveform period. Capsaicin (3 mM) and AP18 (20 μM) also significantly reduced the propagation velocity. However, all tested antagonists failed to inhibit the effects of agonists. AMG9810 (300 μM), but not A-967,079 (300 μM), significantly inhibited the increases in pacemaker frequency caused by increased temperatures. Our findings suggest that TRPV1 and TRPA1 play a minor role in regulating pacemaker potentials and that at non-specific actions at other TRP and ion channels most likely contributed to the overall effects on the electrophysiological recordings that we observed. [ABSTRACT FROM AUTHOR]