Pacemaker Potentials In Interstitial Cells Of Cajal Research Articles

Grape seed powder increases gastrointestinal motility.

Grape seed is an important natural bioactive product with various health benefits. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal (GI) tract. The present study investigated the effects of grape seed powder (GSP) on ICC properties and GI motility. GSP depolarized the pacemaker potentials of ICCs in a dose‑dependent manner. Y25130 or SB269970 slightly inhibited GSP‑induced effects. However, Y25130 and SB269970 together completely blocked GSP-induced effects. In the presence of inhibitors of protein kinase C, protein kinase A, or mitogen-activated protein kinase, GSP‑induced ICC depolarization was inhibited. GSP increased the intestinal transit rate in normal mice and in mice with acetic acid-induced GI motility disorder. In addition, the levels of motilin and substance P were elevated after GSP dosing. These results demonstrate that GSP can regulate GI motility, and therefore, it is a potential therapeutic agent for treating GI motility disorders.

Salviamiltiorrhiza induces depolarization of pacemaker potentials in murine small intestinal interstitial cells of Cajal via extracellular Ca2+ and Na+ influx.

Interstitial cells of Cajal (ICCs) are pacemaker cells that control smooth muscle contraction in the gastrointestinal(GI) tract. The present study investigated the effects of Salviamiltiorrhiza (SM) on the pacemaker potentials of ICCs from the mouse small intestine invitro and on GI motility invivo. The whole‑cell patch‑clamp configuration was used to record pacemaker potential in ICCs invitro, and GI motility was investigated invivo by recording intestinal transit rate (ITR). Using the whole‑cell patch‑clamp configuration, SM depolarized the pacemaker potentials of ICCs in a dose‑dependent manner. Fulvestrant blocked SM‑induced effects but 1,3‑dihydro‑3,3‑bis(4‑hydroxyphenyl)-7-methyl‑2H‑indol‑2‑one did not. Additionally, 4‑[2‑phenyl-5,7‑bis(trifluoromethyl) pyrazolo[1,5‑a]pyrimidin‑3‑yl] phenol blocked SM‑induced effects. Intracellular guanosine 5'‑O‑(2‑thiodiphosphate), and pretreatment with extracellular Ca2+‑ and Na+‑free solutions also blocked SM‑induced effects. Furthermore, ITR values were increased by SM invivo and SM elevated the levels of motilin(MTL). The SM‑induced increase in ITR was associated with increased protein expression levels of c‑kit and the transmembrane protein16A(TMEM16A) channel. In addition, SM induced pacemaker potential depolarization through estrogen receptor β in a Gprotein‑dependent manner via extracellular Ca2+ and Na+ regulation in the murine small intestine invitro. Moreover, SM increased the ITR invivo through the MTL hormone via c‑kit and TMEM16A‑dependent pathways. Taken together, these results suggested that SM may have the ability to control GI motility and could be used as a GI motility regulator.

Effects of Chaihu-Shugan-San on Small Intestinal Interstitial Cells of Cajal in Mice.

Chaihu-Shugan-San (CSS) has been widely used as an alternative treatment for gastrointestinal (GI) diseases in East Asia. Interstitial cells of Cajal (ICCs) are pacemakers in the GI tract. In the present study, we examined the action of CSS on pacemaker potentials in cultured ICCs from the mouse small intestine in vitro and on GI motility in vivo. We used the electrophysiological methods to measure the pacemaker potentials in ICCs. GI motility was investigated by measuring intestinal transit rates (ITR). CSS inhibited the pacemaker potentials in a dose-dependent manner. The capsazepine did not block the effect of CSS. However, the effects of CSS were blocked by glibenclamide. In addition, NG-nitro-L-arginine methyl ester (L-NAME) also blocked the CSS-induced effects. Pretreatment with SQ-22536 or with KT-5720 did not suppress the effects of CSS; however, pretreatment with ODQ or KT-5823 did. Furthermore, CSS significantly suppressed murine ITR enhancement by neostigmine in vivo. These results suggest that CSS exerts inhibitory effects on the pacemaker potentials of ICCs via nitric oxide (NO)/cGMP and ATP-sensitive K+ channel dependent and transient receptor potential vanilloid 1 (TRPV1) channel independent pathways. Accordingly, CSS could provide the basis for the development of new treatments for GI motility dysfunction.

Hesperidin depolarizes the pacemaker potentials through 5-HT4 receptor in murine small intestinal interstitial cells of Cajal

ABSTRACT Hesperidin, a citrus flavonoid, can exert numerous beneficial effects on human health. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract. In the present study, we investigated potential effects of hesperidin on pacemaker potential of ICC in murine small intestine and GI motility. A whole-cell patch-clamp configuration was used to record pacemaker potential in ICC, and GI motility was investigated in vivo by recording gastric emptying (GE) and intestinal transit rate (ITR). Hesperidin depolarized pacemaker potentials of ICC in a dose-dependent manner. Pre-treatment with methoctramine or 4-DAMP did not inhibit hesperidin-induced pacemaker potential depolarization. Neither a 5-HT3 receptor antagonist (Y25130) nor a 5-HT7 receptor antagonist (SB269970) reduced the effect of hesperidin on ICC pacemaker potential, whereas the 5-HT4 receptor antagonist RS39604 was found to inhibit this effect. In the presence of GDP–β–S, hesperidin-induced pacemaker potential depolarization was inhibited. Moreover, in the presence of U73122 and calphostin C, hesperidin did not depolarize pacemaker potentials. Furthermore, hesperidin accelerated GE and ITR in vivo. These results imply that hesperidin depolarized ICC pacemaker potential via 5-HT4 receptors, G protein, and PLC/PKC dependent pathways and that it increased GI motility. Therefore, hesperidin may be a promising novel drug to regulate GI motility.

Depolarization of pacemaker potentials by caffeic acid phenethyl ester in interstitial cells of Cajal from the murine small intestine.

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 Ca2+-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 Ca2+ regulation in the murine small intestine. CAPE may therefore modulate GI motility by acting on ICCs in the murine small intestine.

The Traditional Medicine Banhasasim-Tang Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through M3 Muscarinic and 5-HT3 Receptors in Murine Small Intestine

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.

The Mechanism of Action of Ghrelin and Motilin in the Pacemaker Potentials of Interstitial Cells of Cajal from 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 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.

Rikkunshito Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through Ghrelin Receptors in Murine Small Intestine

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.

Effects of Ca2+-Activated Cl- Channel ANO1inhibitors on Pacemaker Activity in Interstitial Cells of Cajal

Background/Aims: Anoctamin1 (Ca²⁺-activated Cl^- channel, ANO1) is a specific marker of the interstitial cells of Cajal (ICC) in the gastrointestinal tract, and are candidate proteins that can function as pacemaker channels. Recently, novel selective ANO1 inhibitors were discovered and used to study Ca²⁺-activated Cl^- channels. Therefore, to investigate whether ANO1 channels function as pacemaker channels, selective ANO1 inhibitors were tested with respect to the pacemaker potentials in ICC. Methods: Whole-cell patch-clamp recording, RT-PCR, and intracellular Ca²⁺ ([Ca²⁺]_i) imaging were performed in cultured ICC obtained from mice. Results: Though CaCCinh-A01 (5 µM), T16Ainh-A01 (5 µM), and MONNA (5 µM) (selective ANO1 inhibitors) blocked the generation of pacemaker potentials in colonic ICC, they did not do so in small intestinal ICC. Though nifulmic acid (10 µM) and DIDS (10 µM) (classical Ca²⁺-activated Cl^- channel inhibitors) also had no effect in small intestinal ICC, they suppressed the generation of pacemaker potentials in colonic ICC. In addition, knockdown of ANO1 reduced the pacemaker potential frequency in colonic ICC alone. Though ANO1 inhibitors suppressed [Ca²⁺]_i oscillations in colonic ICC, they did not do so in small intestinal ICC. T-type Ca²⁺ channels were expressed in the both the small intestinal and colonic ICC, but mibefradil (5 µM) and NiCl₂ (30 µM) (T-type Ca²⁺ channel inhibitors) inhibited the generation of pacemaker potentials in colonic ICC alone. Conclusion: These results indicate that though ANO1 and T-type Ca²⁺ channels participate in generating pacemaker potentials in colonic ICC, they do not do so in small intestinal ICC. Therefore, the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

The Mechanism of Action of Zingerone in the Pacemaker Potentials of Interstitial Cells of Cajal Isolated from 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.

Naringenin inhibits pacemaking activity in interstitial cells of Cajal from murine small intestine

BackgroundNaringenin (NRG) is a common dietary polyphenolic constituent of fruits. NRG has diverse pharmacological activities, and is used in traditional medicine to treat various diseases including gastrointestinal (GI) disorders. Interstitial cells of Cajal (ICCs) are pacemaker cells of the GI tract. In this study, the authors investigated the effects of NRG on ICCs and on GI motility in vitro and in vivo. MethodsICCs were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICC clusters. The effects of NRG on GI motility were investigated by calculating percent intestinal transit rates (ITR) using Evans blue in normal mice. ResultsNRG inhibited ICC pacemaker potentials in a dose-dependent manner. In the presence of tetraethylammonium chloride or iberiotoxin, NRG had no effect on pacemaker potentials, but it continued to block pacemaker potentials in the presence of glibenclamide. Preincubation with SQ-22536 had no effect on pacemaker potentials or on their inhibition by NRG. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked pacemaker potential inhibition by NRG. In addition, L-NG-nitroarginine methyl ester blocked pacemaker potential inhibition by NRG. Furthermore, NRG significantly suppressed murine ITR enhancement by neostigmine in vivo. ConclusionThis study shows NRG dose-dependently inhibits ICC pacemaker potentials via a cyclic guanosine monophosphate/nitric oxide-dependent pathway and Ca2+-activated K+ channels in vitro. In addition, NRG suppressed neostigmine enhancement of ITR in vivo.

Hwangryunhaedok-tang induces the depolarization of pacemaker potentials through 5-HT3 and 5-HT4 receptors in cultured murine small intestine interstitial cells of Cajal.

AIMTo investigate the effects of a water extract of Hwangryunhaedok-tang (HHTE) on the pacemaker potentials of mouse interstitial cells of Cajal (ICCs).METHODSWe 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 °CRESULTSHHTE dose-dependently depolarized ICC pacemaker potentials. Pretreatment with a 5-HT3 receptor antagonist (Y25130) or a 5-HT4 receptor antagonist (RS39604) blocked HHTE-induced pacemaker potential depolarizations, whereas pretreatment with a 5-HT7 receptor antagonist (SB269970) did not. Intracellular GDPβS inhibited HHTE-induced pacemaker potential depolarization and pretreatment with a Ca2+-free solution or thapsigargin abolished the pacemaker potentials. In the presence of a Ca2+-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.CONCLUSIONThese results suggest that HHTE dose-dependently depolarizes ICC pacemaker potentials through 5-HT3 and 5-HT4 receptors via external and internal Ca2+ regulation and via G protein-, PKC- and Rho kinase-independent pathways.

Magnolia Officinalis Bark Extract Induces Depolarization of Pacemaker Potentials Through M2 and M3 Muscarinic Receptors in Cultured Murine Small Intestine Interstitial Cells of Cajal

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.

Effects of the roots of Liriope Platyphylla Wang Et tang on gastrointestinal motility function

Ethnopharmacological relevanceLiriope 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 studyBecause 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 methodsEnzymatic 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. ResultsLPE dose-dependently depolarized PPTs in ICCs. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not block LPE-induced PPT depolarization. However, pretreatment with 4-DAMP (a muscarinic M3 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 GDPβS 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 Ca2+free solution or thapsigargin (a Ca2+-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 (0.01–1g/kg administered intragastrically (i.g.)). In addition, LPE (i.g.) significantly recovered GI motility dysfunctions in both animal models. ConclusionLPE dose-dependently depolarizes ICC PPTs through M3 receptors via external and internal Ca2+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.

Effects of Schisandra chinensis extract on gastrointestinal motility in mice

Ethnopharmacological relevanceSchisandra chinensis (Turcz.) Baill. (SC) continues to be used as a traditional folk medicine in Asia, especially for the treatment of gastrointestinal (GI) disorders related to gastritis, diarrhea, enterocolitis and abnormal GI motility. Aim of the studyBecause GI disorders, especially abnormal GI motility, are major lifelong problems, we investigated the effects of SC on the pacemaker activity of the interstitial cells of Cajal (ICCs) in murine small intestine and GI motility. Materials and MethodsEnzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials generated by cultured ICCs. In vivo effects of SC on GI motility were investigated by measuring the intestinal transit rate (ITR) of Evans blue in normal and GI motility dysfunction mice. ResultsSC extracts depolarized the membrane potentials of ICCs in a dose dependent manner. Pretreatment with Ca2+ free solution or thapsigargin (a Ca2+-ATPase inhibitor in the endoplasmic reticulum) abolished the generation of pacemaker potentials by ICCs, and under these conditions, SC extract did not depolarize the membrane potentials of ICCs. In addition, membrane depolarizations were inhibited by intracellular GDPβS and by U-73122 (an active phospholipase C (PLC) inhibitor). In normal mice, ITRs were significantly increased by SC extract (0.1–1g/kg, intragastrically (i.g.)) in a dose dependent manner. Also, SC extract significantly recovered the GI motility dysfunctions in acetic acid (AA)-injected and streptozotocin (STZ)-induced diabetic mice, which are the GI motility animal models. Materials and methodsSC extract modulates pacemaker potentials in ICCs in a dose dependent manner via external and internal Ca2+ regulations, and via G protein and the PLC pathway. In addition, SC extract increased ITRs in normal and abnormal GI motility mice models. This study shows that SC extract offers a basis for the development of a prokinetic agent that prevents or alleviates GI motility dysfunctions.

Inhibitory Effects of Naeso-san on Pacemaker Potentials in Interstitial Cells of Cajal of Murine Small Intestine

The purpose of this study was to investigate the effects of Naeso-san in interstitial cells of Cajal (ICCs) in murine small intestine. First, we isolated ICCs from murine small intestine. After that, we cultured these cells for 1 days. The patch-clamp technique was applied on ICCs that formed network-like structures in culture (1 days). Spontaneous rhythms were routinely recorded from cultured ICCs under current-clamp conditions, and the ICCs within networks displayed more robust electrical rhythms (pacemaker potentials). To understand the relationship between Naeso-san and pacemaker activity in ICCs, we examined the effects of Naeso-san on pacemaker potentials of ICCs. In current clamp mode (I = 0), the addition of Naeso-san (10 mg/ml ? 50 mg/ml) decreased the amplitude and frequency of the pacemaker potentials of ICCs in a dose dependent manner. However, these effects were blocked by intracellular GDPβ S, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K+ channels blocker. Pretreatment with SQ-22536, an adenylate cyclase inhibitor, did not block the Naeso-san induced effects, whereas pretreatment with ODQ, a guanylate cyclase inhibitor, or L-NAME, an inhibitor of nitric oxide (NO) synthase blocked the Naeso-san induced effects. Our findings provide insight into unraveling the modulation of Naeso-san in pacemaker potentials of ICCs and developing therapeutic agents against gastrointestinal motility disorders.

Modeling of stochastic behavior of pacemaker potential in interstitial cells of Cajal

It is widely accepted that interstitial cells of Cajal (ICCs) generate pacemaker potentials to propagate slow waves along the whole gastrointestinal tract. Previously, we constructed a biophysically based model of ICCs in mouse small intestine to explain the pacemaker mechanism. Our previous model, however, could not explain non-uniformity of pacemaker potentials and random occurrence of unitary potentials, thus we updated our model. The inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ mobilization is a key event to drive the cycle of pacemaker activity and was updated to reproduce its stochastic behavior. The stochasticity was embodied by simulating random opening and closing of individual IP3-mediated Ca2+ channel. The updated model reproduces the stochastic features of pacemaker potentials in ICCs. Reproduced pacemaker potentials are not uniform in duration and interval. The resting and peak potentials are −75.5 ± 1.1 mV and −0.8 ± 0.5 mV, respectively (n = 55). Frequency of pacemaker potential is 14.3 ± 0.4 min−1 (n = 10). Width at half-maximal amplitude of pacemaker potential is 902 ± 6 ms (n = 55). There are random events of unitary potential-like depolarization. Finally, we compared our updated model with a recently published model to speculate which ion channel is the best candidate to drive pacemaker depolarization. In conclusion, our updated mathematical model could now reproduce stochastic features of pacemaker activity in ICCs.

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 the mouse small intestine (LB746)

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 (GI) smooth muscle. The effects of Ge‐Gen‐Tang (GGT), a traditional herbal medicine, on pacemaker potentials in cultured ICCs from the mouse small intestine were investigated. The patch‐clamp technique for whole‐cell recording was used in cultured ICCs. Under the current clamping mode, ICC generated pacemaker potentials. The resting membrane potential was ‐ 52 4 mV and the amplitude was 18.3 1.6 mV. GGT (10‐50 mg/ml) decreased the frequency and amplitude of pacemaker potentials in a concentration‐dependent manner, but had no effects on membrane potentials. In the presence of 50 mg/ml GGT, the amplitude was 1.2 0.3 mV and the frequency was 0.3 0.6 cycles/min at 50 mg/ml. However, these effects were blocked by intracellular GDPβS, a G‐protein inhibitor, and glibenclamide, a specific ATP‐sensitive K+ channels blocker. Pretreatment with SQ‐22536, an inhibitor of adenylate cyclase, before the application of GGT had no effect on pacemaker potentials. Similarly, pretreatment with ODQ, an inhibitor of guanylate cyclase, did not inhibit the pacemaker potentials. However, GGT inhibited the pacemaker potentials in the presence of SQ‐22536, not in ODQ. The amplitude was 1.7 0.6 mV in the presence of SQ‐22536 and 17.1 2.6 mV in ODQ at GGT 50 mg/ml. Also, pretreatment with L‐NAME (an inhibitor of nitric oxide (NO) synthase) before the application of GGT abolished its effects on pacemaker potentials, but L‐NAME itself had no effect. In the presence of L‐NAME, the amplitude was 18.7 0.6 mV at GGT 50 mg/ml. Taken together, GGT inhibits pacemaker potentials in ICC in a G protein‐, cGMP‐ and NO‐dependent manner. Our data suggest that the gastroprokinetic effects of GGT are mediated by the induction of pacemaker potentials in ICCs.Grant Funding Source: Supported by the Traditonal Korean Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C1886, B120008)

Sphingosine and FTY720 modulate pacemaking activity in interstitial cells of Cajal from mouse small intestine.

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca(2+) 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.

Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevancePoncirus fructus (PF) has been widely used as a traditional medicine in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. Aim of the studyPoncirus fructus (PF), also known as Poncirus trifoliata (L.) Raf. (Rutaceae), is widely used as a traditional medicine in Eastern Asia mainly to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. In a previous study, a methanol extract of PF was found to have particularly potent gastroprokinetic effects. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal tract, but the action mechanisms of PF extract in mouse small intestinal ICCs have not been investigated. Therefore, in the present study, we investigated the effects of a methanol extract of PF (MPF) in mouse small intestinal ICCs. In addition, we sought to identify the receptors involved. Materials and MethodsEnzymatic digestions were used to dissociate ICCs from small intestines. The whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. In addition, we analyzed intracellular Ca2+ concentrations ([Ca2+]i). ResultsMPF decreased the amplitudes of pacemaker potentials in ICCs, and depolarized resting membrane potentials in a concentration dependent manner. Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked MPF-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. Pretreatment with Na+ or Ca2+-free solution or thapsigargin (a Ca2+-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed MPF-induced activity. [Ca2+]i analysis showed that MPF increased [Ca2+]i. Furthermore, treatments with PD 98059, SB203580, or JNK II inhibitor blocked MPF-induced membrane depolarizations in ICCs. ConclusionThese results suggest that MPF modulates pacemaker potentials through 5-HT3 and 5-HT4 receptor-mediated pathways via external Na+ and Ca2+ influx, and via Ca2+ release from internal stores in a mitogen-activated protein kinase dependent manner. The study shows MPF is a good candidate for the development of a gastroprokinetic agent. In view of the effects of MPF on ICCs, further research is required, particularly to identify the active compound(s) involved and to determine their action mechanisms.