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Background: Bojungikki-tang (BJIT) is a traditional formula used to treat Gastrointestinal (GI) diseases. Objectives: We investigated the GI motility functions in vivo and the pacemaker potential in interstitial cells of Cajal (ICCs) in vitro by BJIT. Materials and Methods: Intestinal transit rate (ITR) and serum levels of GI hormones were investigated in mice. ICC-induced pacemaker potential was evaluated using the electrophysiological method. Results: ITR values and the level of motilin significantly increased after treatment with BJIT. The BJIT-induced ITR increase was related to the increase in the expression of a c-kit. BJIT induced the pacemaker potential depolarizations and the frequency decrease of ICCs. Pretreatment with methoctramine resulted in the inhibition of BJIT-induced depolarization of the pacemaker potential. However, BJIT-induced effects were retained in the presence of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide. Furthermore, thapsigargin pretreatment resulted in the inhibition of BJIT-induced effects. Moreover, BJIT blocked both transient receptor potential melastatin 7 and calcium-activated chloride (transmembrane protein 16A) channels. Conclusion: These results indicate that BJIT can be considered a good medicine for controlling GI motility. [ABSTRACT FROM AUTHOR]

Background/Aims: Zingerone, a major component found in ginger root, is clinically effective for the treatment of various diseases. Interstitial cells of Cajal (ICCs) are the pacemaker cells responsible for slow waves in the gastrointestinal (GI) tract. We investigated the effects of zingerone on the pacemaker potentials of ICCs to assess its mechanisms of action and its potential as a treatment for GI tract motility disorder. Methods: We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs. Results: Under the current clamping mode, zingerone inhibited pacemaker potentials of ICCs concentration-dependently. These effects were blocked not by capsazepine, a transient receptor potential vanilloid 1 (TRPV1) channel blocker, but by glibenclamide, a specific ATP-sensitive K+ channel blocker. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor), LY294002 (a phosphoinositide 3-kinase inhibitor), and calphostin C (a protein kinase C (PKC) inhibitor) did not block the effects of zingerone on the pacemaker potentials relative to treatment with zingerone alone. However, zingerone-induced pacemaker potential inhibition was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor), KT5823 (a protein kinase G (PKG) inhibitor), and L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor). In addition, zingerone stimulated cyclic guanosine monophosphate (cGMP) production in ICCs. Finally, pretreatment with PD98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and SP600125 (c–Jun N–terminal kinases (JNK)–specific inhibitor) blocked the zingerone-induced pacemaker potential inhibition. Conclusion: These results suggest that zingerone concentration-dependently inhibits pacemaker potentials of ICCs via NO/cGMP-dependent ATP-sensitive K+ channels through MAPK-dependent pathways. Taken together, this study shows that zingerone may have the potential for development as a GI regulation agent.

Background: Banhasasim-tang (BHSST) is a classic herbal formulation in traditional Chinese medicine widely used for gastrointestinal (GI) tract motility disorder. We investigated the effects of BHSST on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in small intestine in vitro and its effects on GI motor functions in vivo. Methods: We isolated ICCs from the small intestines and recorded pacemaker potentials in cultured ICCs with the whole-cell patch-clamp configuration in vitro. Intestinal transit rates (ITR%) were investigated in normal mice and GI motility dysfunction (GMD) mouse models in vivo. Results: BHSST (20–50 mg/mL) depolarized pacemaker potentials and decreased their amplitudes in a concentration-dependent manner. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not inhibit BHSST-induced pacemaker potential depolarization. However, when we applied 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a muscarinic M3 receptor antagonist), BHSST-induced effects were blocked. Pretreatment with Y25130 (a 5-HT3 receptor antagonist) blocked BHSST-induced effects in ICCs. In addition, when we applied 4-DAMP and Y25130 together, BHSST-induced effects were completely blocked. Pretreatment with Ca2+-free solution or thapsigargin inhibited BHSST-induced effects. Moreover, BHSST blocked both the transient receptor potential melastatin (TRPM) 7 and voltage-sensitive calcium-activated chloride (anoctamin-1, ANO1) channels. In normal mice, ITR% values were significantly increased by BHSST in a dose-dependent manner. The ITR% of GMD mice was significantly reduced relative to those of normal mice, which were significantly reversed by BHSST in a dose-dependent manner. Conclusion: These results suggested that BHSST depolarizes the pacemaker potentials of ICCs in a dose-dependent manner through the M3 and 5-HT3 receptors via internal and external Ca2+-dependent and TRPM7- and ANO1-independent pathways in vitro. Moreover, BHSST increased ITR% in vivo in normal mice and GMD mouse models. Taken together, the results of this study showed that BHSST had the potential for development as a prokinetic agent in GI motility function. [ABSTRACT FROM AUTHOR]

The effect of acetylcholine (ACh) on pacemaking and spontaneous contractions in the gastrointestinal tract is not well characterized. The current study aims to profile the effect of several muscarinic and nicotinic receptor agonists and antagonists on pacemaker potentials in the ICR mouse ileum. Pacemaker potentials of whole thickness mouse ileal segments were recorded extracellularly using a 60-channel microelectrode array (MEA) platform. A spatiotemporal analysis integrated the frequency, amplitude, and velocity measurements of pacemaker currents. Comparative data were obtained by recording spontaneous smooth muscle tone in a conventional organ bath. On the MEA, ACh (0.3–300 μM) and bethanechol (0.3–300 μM) significantly reduced ileal pacemaker potentials. The inhibitory effect of ACh was mimicked by donepezil (300 μM) but not nicotine (0.3–7 mM). Atropine (300 μM), but not hexamethonium (300 μM), reversed the inhibitory actions of ACh and bethanechol and revealed excitatory properties manifested as increases in pacemaker frequency. A spatial analysis also revealed that atropine, but not hexamethonium, reversed the ACh-induced distortion of pacemaker propagation activity. Atropine (0.001–3 mM) and hexamethonium (0.3–7 mM) alone were inactive. In the organ bath, ACh (300 nM) and bethanechol (30 μM) induced ileal tonic contractions, while inhibiting basal spontaneous contractions at 300 μM. Atropine (1 μM), but not hexamethonium (1–300 μM), reversed both the tonic contractions and the inhibition of the spontaneous contractions of ACh and bethanechol and revealed an excitatory effect manifested as an increasing in the frequency of contractions. Muscarinic, but not nicotinic, receptors appear to mediate the inhibitory actions of ACh on mouse ileal pacemaker potentials. NEW & NOTEWORTHY The study discovered an acute action of acetylcholine on pacemaker potentials that is mediated by muscarinic receptors on the mouse ileum. Bethanechol, but not nicotine, mimicked the inhibitory actions of acetylcholine on pacemaker potentials. Atropine, but not hexamethonium, reversed the inhibitory actions of acetylcholine. When introduced after acetylcholine, atropine exhibited excitatory actions that increased the pacemaker frequency. Acetylcholine and bethanechol distorted the propagation activity and pattern, and this was also reversed by atropine. These actions of acetylcholine on pacemaker potentials may contribute to pathophysiology in bowel diseases. [ABSTRACT FROM AUTHOR]

Background: Rikkunshito has been used to treat gastrointestinal (GI) disorders. The purpose of this study was to investigate the effects of Rikkunshito, a traditional Japanese herbal medicine, on the pacemaker potentials of interstitial cells of Cajal (ICCs) from the small intestines of mice. Methods: We isolated ICCs from the small intestines of mice, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs and membrane currents. Results: Rikkunshito depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with GSK1614343 or (D-Lys3)-growth hormone-releasing peptide-6 inhibited Rikkunshito-induced depolarization of pacemaker potentials. Intracellular GDP-β-S inhibited Rikkunshito-induced effects. In Ca2+-free solution or in the presence of thapsigargin, Rikkunshito did not depolarize pacemaker potentials. Moreover, in the presence of U-73122 or xestospongin C, Rikkunshito-induced effects were inhibited. However, in the presence of staurosporine, Go6976 or Rottlerin, Rikkunshito depolarized pacemaker potentials. Furthermore, Rikkunshito inhibited both transient receptor potentials melastatin 7 (TRPM7) and Ca2+-activated Cl– channels (ANO1) currents. Conclusion: Rikkunshito depolarized pacemaker potentials of ICCs via ghrelin receptor and G protein through internal or external Ca2+-, phospholipase C-, and inositol triphosphate-dependent and protein kinase C-, TRPM7-, and ANO1-independent pathways. The study shows that Rikkunshito may alleviate GI motility disorders through its depolarizing effects on ICCs. [ABSTRACT FROM AUTHOR]

Background: Magnolia officinalis Rehder and EH Wilson (M. officinalis) are traditional Chinese medicines widely used for gastrointestinal (GI) tract motility disorder in Asian countries. We investigated the effects of an ethanol extract of M. officinalis (MOE) on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in vitro and its effects on GI motor functions in vivo. Methods: We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs in vitro. Both gastric emptying (GE) and intestinal transit rates (ITRs) were investigated in normal and GI motility dysfunction (GMD) mice models in vivo. Results: MOE depolarized ICC pacemaker potentials dose-dependently. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) and 4-DAMP (a muscarinic M3 receptor antagonist) inhibited the effects of MOE on the pacemaker potential relative to treatment with MOE alone. In addition, MOE depolarized pacemaker potentials after pretreatment with Y25130 (a 5-HT3 receptor antagonist), GR113808 (a 5-HT4 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist). However, pretreatment with RS39604 (a 5-HT4 receptor antagonist) blocked MOE-induced pacemaker potential depolarizations. Intracellular GDPβS inhibited MOE-induced pacemaker potential depolarization, as did pretreatment with Ca2+ free solution or thapsigargin. In normal mice, the GE and ITR values were significantly and dose-dependently increased by MOE. In loperamide-and cisplatin-induced GE delay models, MOE administration reversed the GE deficits. The ITRs of the GMD mice were significantly reduced relative to those of normal mice, which were significantly and dose-dependently reversed by MOE. Conclusion: These results suggest that MOE dose-dependently depolarizes ICCs pacemaker potentials through M2 and M3 receptors via internal and external Ca2+ regulation through G protein pathways in vitro. Moreover, MOE increased GE and ITRs in vivo in normal and GMD mouse models. Taken together, the results of this study show that MOE have the potential for development as a gastroprokinetic agent in GI motility function.

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]

New Findings What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate., What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl− homeostasis in ICCs can alter the smooth muscle membrane potential., Intracellular Cl− homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl− (Cl−o) with the impermeant anions gluconate and isethionate. On reducing Cl−o, effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage ( Em). Restoration of Cl− hyperpolarized smooth muscle Em proportional to the change in Cl−o concentration. Replacement of 90% of Cl−o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 m m. In Krebs solutions containing normal Cl− and 0.13 m m free Ca2+, slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl−o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl− homeostasis in ICCs can alter smooth muscle Em. [ABSTRACT FROM AUTHOR]

Background: Magnolia officinalis Rehder and EH Wilson (M. officinalis) are traditional Chinese medicines widely used for gastrointestinal (GI) tract motility disorder in Asian countries. We investigated the effects of an ethanol extract of M. officinalis (MOE) on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in vitro and its effects on GI motor functions in vivo. Methods: We isolated ICCs from small intestines, and the whole-cell patchclamp configuration was used to record the pacemaker potentials in cultured ICCs in vitro. Both gastric emptying (GE) and intestinal transit rates (ITRs) were investigated in normal and GI motility dysfunction (GMD) mice models in vivo. Results: MOE depolarized ICC pacemaker potentials dose-dependently. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) and 4-DAMP (a muscarinic M3 receptor antagonist) inhibited the effects of MOE on the pacemaker potential relative to treatment with MOE alone. In addition, MOE depolarized pacemaker potentials after pretreatment with Y25130 (a 5-HT3 receptor antagonist), GR113808 (a 5-HT4 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist). However, pretreatment with RS39604 (a 5-HT4 receptor antagonist) blocked MOE-induced pacemaker potential depolarizations. Intracellular GDPßS inhibited MOE-induced pacemaker potential depolarization, as did pretreatment with Ca2+ free solution or thapsigargin. In normal mice, the GE and ITR values were significantly and dose-dependently increased by MOE. In loperamide- and cisplatin-induced GE delay models, MOE administration reversed the GE deficits. The ITRs of the GMD mice were significantly reduced relative to those of normal mice, which were significantly and dose-dependently reversed by MOE. Conclusion: These results suggest that MOE dose-dependently depolarizes ICCs pacemaker potentials through M2 and M3 receptors via internal and external Ca2+ regulation through G protein pathways in vitro. Moreover, MOE increased GE and ITRs in vivo in normal and GMD mouse models. Taken together, the results of this study show that MOE have the potential for development as a gastroprokinetic agent in GI motility function. [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]

Background: Daikenchuto (DKT; TJ-100, TU-100), a traditional herbal medicine, is used in modern medicine to treat gastrointestinal (GI) functional disorders. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the GI tract and play important roles in the regulation of GI motility. Objective: The objective of this study was to investigate the effects of DKT on the pacemaker potentials (PPs) of cultured ICCs from murine small intestine. Materials and Methods: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record ICC PPs (current clamp mode). All experiments were performed at 30-32°C. Results: In current-clamp mode, DKT depolarized and concentration-dependently decreased the amplitudes of PPs. Y25130 (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT4 receptor antagonist) did. Methoctramine (a muscarinic M2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-diphenylacetoxy-N-methylpiperidine methiodide (a muscarinic M3 receptor antagonist) facilitated blockade of DKT-induced PP depolarization. Pretreatment with an external Ca2+-free solution or thapsigargin abolished PPs, and under these conditions, DKT did not induce PP depolarization. Furthermore, Ginseng radix and Zingiberis rhizomes depolarized PPs, whereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Conclusion: These results suggest that DKT depolarizes ICC PPs in an internal or external Ca2+-dependent manner by stimulating 5-HT4 and M3 receptors. Furthermore, the authors suspect that the component in DKT largely responsible for depolarization is probably also a component of Ginseng radix and Zingiberis rhizomes. [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]

The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoo-san (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The whole-cell patch-clamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+ i) was analyzed in cultured ICCs using fura-2-acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4-diphenylacetoxy-N-methyl-piperidine methiodide muscarinic M3 receptor antagonist inhibited DS-induced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'-[β-thio] diphosphate (GDP-β-S; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DS-induced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DS-induced pacemaker depolarizations. Pretreatment with Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DS-induced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G protein-dependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs. [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]

Lubiprostone is a chloride (Cl-) channel activator derived from prostaglandin E1 and used for managing constipation. In addition, lubiprostone affects the activity of gastrointestinal smooth muscles. Interstitial cells of Cajal (ICCs) are pacemaker cells that generate slow-wave activity in smooth muscles. We studied the effects of lubiprostone on the pacemaker potentials of colonic ICCs. We used the whole-cell patch-clamp technique to determine the pacemaker activity in cultured colonic ICCs obtained from mice. Lubiprostone hyperpolarized the membrane and inhibited the generation of pacemaker potentials. Prostanoid EP1, EP2, EP3, and EP4 antagonists (SC-19220, PF-04418948, 6-methoxypyridine-2-boronc acid N-phenyldiethanolamine ester, and GW627368, respectively) did not block the response to lubiprostone. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) did not block the response to lubiprostone. In addition, tetraethylammonium (TEA, a voltage-dependent potassium [K+] channel blocker) and apamin (a calcium [Ca2+]-dependent K+ channel blocker) did not block the response to lubiprostone. However, glibenclamide (an ATP-sensitive K+ channel blocker) blocked the response to lubiprostone. Similar to lubiprostone, pinacidil (an opener of ATP-sensitive K+ channel) hyperpolarized the membrane and inhibited the generation of pacemaker potentials, and these effects were inhibited by glibenclamide. These results suggest that lubiprostone can modulate the pacemaker potentials of colonic ICCs via activation of ATP-sensitive K+ channel through a prostanoid EP receptor-independent mechanism. [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]

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ETA receptor antagonist) or BQ-788 (ETB receptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ETB receptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ETA and ETB receptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ETA and ETB receptor activation in murine small intestinal ICCs and ETA receptor activation in murine large intestinal ICCs by external Ca2+ influx and internal Ca2+ release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Pilocarpine is a nonspecific agonist of muscarinic receptors which was recently found to activate the M receptor subtype in a voltage-dependent manner. The purpose of our study was to investigate the role of the acetylcholine (muscarinic)-activated K current ( I) on the negative chronotropic effect of pilocarpine in rabbit sinoatrial node. In multicellular preparations, we studied the effect of pilocarpine on spontaneous action potentials. In isolated myocytes, using the patch clamp technique, we studied the effects of pilocarpine on I. Pilocarpine produced a decrease in spontaneous frequency, hyperpolarization of the maximum diastolic potential, and a decrease in the diastolic depolarization rate. These effects were partially antagonized by tertiapin Q. Cesium and calyculin A in the presence of tertiapin Q partially prevented the effects of pilocarpine. In isolated myocytes, pilocarpine activated the muscarinic potassium current, I in a voltage-dependent manner. In conclusion, the negative chronotropic effects of pilocarpine on the sinatrial node could be mainly explained by activation of I. [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]

Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of caffeic acid phenethyl ester (CAPE) on the pacemaker potentials of ICCs from the mouse small or large intestine. Using the whole-cell patch-clamp configuration, we found that CAPE depolarized the pacemaker potentials of cultured ICCs from the murine small intestine in a dose-dependent manner. The estrogen receptor (ER) β antagonist PHTPP completely inhibited CAPE-induced depolarization, but the ERα antagonist BHPI did not. Intracellular GDP-β-S and pretreatment with Ca 2+ -free solution or thapsigargin also blocked CAPE-induced depolarization. To investigate the mechanisms of CAPE-mediated depolarization of ICCs, we used the nonselective cation channel (NSCC) inhibitor flufenamic acid, the Cl - channel blocker, mitogen-activated protein kinase (MAPK) inhibitors PD98059, SB203580, or SP600125, and PI3 kinase inhibitor LY294002. All inhibitors blocked the CAPE-induced pacemaker potential depolarization of ICCs. These results suggest that CAPE induces pacemaker potential depolarization through ERβ in a G protein, NSCC, Cl - channel, MAPK- and PI3 kinase dependent manner via intracellular and extracellular Ca 2+ regulation in the murine small intestine. CAPE may therefore modulate GI motility by acting on ICCs in the murine small intestine.

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 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-, 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.

Background/aims: Zingerone, a major component found in ginger root, is clinically effective for the treatment of various diseases. Interstitial cells of Cajal (ICCs) are the pacemaker cells responsible for slow waves in the gastrointestinal (GI) tract. We investigated the effects of zingerone on the pacemaker potentials of ICCs to assess its mechanisms of action and its potential as a treatment for GI tract motility disorder., Methods: We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs., Results: Under the current clamping mode, zingerone inhibited pacemaker potentials of ICCs concentration-dependently. These effects were blocked not by capsazepine, a transient receptor potential vanilloid 1 (TRPV1) channel blocker, but by glibenclamide, a specific ATP-sensitive K+ channel blocker. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor), LY294002 (a phosphoinositide 3-kinase inhibitor), and calphostin C (a protein kinase C (PKC) inhibitor) did not block the effects of zingerone on the pacemaker potentials relative to treatment with zingerone alone. However, zingerone-induced pacemaker potential inhibition was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor), KT5823 (a protein kinase G (PKG) inhibitor), and L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor). In addition, zingerone stimulated cyclic guanosine monophosphate (cGMP) production in ICCs. Finally, pretreatment with PD98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and SP600125 (c-Jun N-terminal kinases (JNK)-specific inhibitor) blocked the zingerone-induced pacemaker potential inhibition., Conclusion: These results suggest that zingerone concentration-dependently inhibits pacemaker potentials of ICCs via NO/cGMP-dependent ATP-sensitive K+ channels through MAPK-dependent pathways. Taken together, this study shows that zingerone may have the potential for development as a GI regulation agent., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

New Findings: What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl - conductance is low, whereas transmembrane Cl - movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl - homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl - homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl - (Cl - o ) with the impermeant anions gluconate and isethionate. On reducing Cl - o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (E m ). Restoration of Cl - hyperpolarized smooth muscle E m proportional to the change in Cl - o concentration. Replacement of 90% of Cl - o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca 2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl - and 0.13 mm free Ca 2+ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl - o was not observed in W/W v mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl - conductance is low, whereas transmembrane Cl - movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle E m and that altering Cl - homeostasis in ICCs can alter smooth muscle E m ., (© 2017 Mayo Clinic. Experimental Physiology © 2017 The Physiological Society.)

Aim: To investigate the effects of a water extract of Hwangryunhaedok-tang (HHTE) on the pacemaker potentials of mouse interstitial cells of Cajal (ICCs)., Methods: We dissociated ICCs from small intestines and cultured. ICCs were immunologically identified using an anti-c-kit antibody. We used the whole-cell patch-clamp configuration to record the pacemaker potentials generated by cultured ICCs under the current clamp mode ( I = 0). All experiments were performed at 30 °C-32 °C., Results: HHTE dose-dependently depolarized ICC pacemaker potentials. Pretreatment with a 5-HT 3 receptor antagonist (Y25130) or a 5-HT 4 receptor antagonist (RS39604) blocked HHTE-induced pacemaker potential depolarizations, whereas pretreatment with a 5-HT 7 receptor antagonist (SB269970) did not. Intracellular GDPβS inhibited HHTE-induced pacemaker potential depolarization and pretreatment with a Ca 2+ -free solution or thapsigargin abolished the pacemaker potentials. In the presence of a Ca 2+ -free solution or thapsigargin, HHTE did not depolarize ICC pacemaker potentials. In addition, HHTE-induced pacemaker potential depolarization was unaffected by a PKC inhibitor (calphostin C) or a Rho kinase inhibitor (Y27632). Of the four ingredients of HHT, Coptidis Rhizoma and Gardeniae Fructus more effectively inhibited pacemaker potential depolarization., Conclusion: These results suggest that HHTE dose-dependently depolarizes ICC pacemaker potentials through 5-HT 3 and 5-HT 4 receptors via external and internal Ca 2+ regulation and via G protein-, PKC- and Rho kinase-independent pathways., Competing Interests: Conflict-of-interest statement: The authors have no disclosures to report.

The prostate is a gland whose secretions contribute to the seminal fluids ejaculated upon activation of autonomic sympathetic nerves. In elder males, the prostate undergoes an increase in stroma mass and myogenic tone, leading to benign prostatic hyperplasia that occludes the proximal urethra and the presentation of various lower urinary tract symptoms that decrease their quality of life. This review summarises the role of prostatic interstitial cells (PICs) in the generation of the spontaneous tone in the prostate. It presents current knowledge of the role of Ca 2+ plays in PIC pacemaking, as well as the mechanisms by which this spontaneous activity triggers slow wave generation and stromal contraction. PICs display a small T-type Ca 2+ current (I CaT ) and a large L-type Ca 2+ current (I CaL ). In contrast to other interstitial cells in the urinary and gastrointestinal tracts, spontaneous Ca 2+ signalling in PICs is uniquely dependent on Ca 2+ influx through I CaL channels. A model of prostatic pacemaking is presented describing how I CaL can be triggered by an initial membrane depolarization evoked upon the selective opening of Ca 2+ -activated Cl - channels by Ca 2+ flowing only through I CaT channels. The resulting current flow through I CaL results in release of Ca 2+ from internal stores and the summation of Cl - -selective spontaneous transient depolarizations (STDs) to form pacemaker potentials that propagate passively into the prostatic stroma to evoke regenerative action potentials and excitation-contraction coupling.

Background: Daikenchuto (DKT; TJ-100, TU-100), a traditional herbal medicineis used in modern medicine to treat gastrointestinal (GI) functional disorders. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the GI tract and play important roles in the regulation of GI motility., Objective: The objective of this study was to investigate the effects of DKT on the pacemaker potentials (PPs) of cultured ICCs from murine small intestine., Materials and Methods: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record ICC PPs (current clamp mode). All experiments were performed at 30-32°C., Results: In current-clamp modeDKT depolarized and concentration-dependently decreased the amplitudes of PPs. Y25130 (a 5-HT 3 receptor antagonist) or SB269970 (a 5-HT 7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT 4 receptor antagonist) did. Methoctramine (a muscarinic M 2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-diphenylacetoxy-N-methylpiperidine methiodide (a muscarinic M 3 receptor antagonist) facilitated blockade of DKT-induced PP depolarization. Pretreatment with an external Ca 2+ -free solution or thapsigargin abolished PPsand under these conditions, DKT did not induce PP depolarization. Furthermore Ginseng radix and Zingiberis rhizomes depolarized PPs, whereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs., Conclusion: These results suggest that DKT depolarizes ICC PPs in an internal or external Ca 2+ -dependent manner by stimulating 5-HT 4 and M 3 receptors. Furthermore, the authors suspect that the component in DKT largely responsible for depolarization is probably also a component of Ginseng radix and Zingiberis rhizomes., Summary: Daikenchuto (DKT) depolarized and concentration-dependently decreased the amplitudes of pacemaker potentials (PPs)Y25130 (a 5-HT 3 receptor antagonist) or SB269970 (a 5-HT 7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT 4 receptor antagonist) didMethoctramine (a muscarinic M 2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-DAMP (a muscarinic M 3 receptor antagonist) facilitated blockade of DKT-induced PP depolarizationGinseng radix and Zingiberis rhizomes depolarized PPswhereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Abbreviation used: DKT: Daikenchuto, GI: Gastrointestinal, ICCs: Interstitial cells of Cajal, PPs: Pacemaker Potentials., Competing Interests: There are no conflicts of interest.

Lubiprostone is a chloride (Cl(-)) channel activator derived from prostaglandin E1 and used for managing constipation. In addition, lubiprostone affects the activity of gastrointestinal smooth muscles. Interstitial cells of Cajal (ICCs) are pacemaker cells that generate slow-wave activity in smooth muscles. We studied the effects of lubiprostone on the pacemaker potentials of colonic ICCs. We used the whole-cell patch-clamp technique to determine the pacemaker activity in cultured colonic ICCs obtained from mice. Lubiprostone hyperpolarized the membrane and inhibited the generation of pacemaker potentials. Prostanoid EP1, EP2, EP3, and EP4 antagonists (SC-19220, PF-04418948, 6-methoxypyridine-2-boronc acid N-phenyldiethanolamine ester, and GW627368, respectively) did not block the response to lubiprostone. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) did not block the response to lubiprostone. In addition, tetraethylammonium (TEA, a voltage-dependent potassium [K(+)] channel blocker) and apamin (a calcium [Ca(2+)]-dependent K(+) channel blocker) did not block the response to lubiprostone. However, glibenclamide (an ATP-sensitive K(+) channel blocker) blocked the response to lubiprostone. Similar to lubiprostone, pinacidil (an opener of ATP-sensitive K(+) channel) hyperpolarized the membrane and inhibited the generation of pacemaker potentials, and these effects were inhibited by glibenclamide. These results suggest that lubiprostone can modulate the pacemaker potentials of colonic ICCs via activation of ATP-sensitive K(+) channel through a prostanoid EP receptor-independent mechanism.