Chan Guk Park

The prevalence of and risk factors for erosive oesophagitis and non-erosive reflux disease: a nationwide multicentre prospective study in Korea

Background  Prospective nationwide multicentre studies that have evaluated endoscopic findings and reflux symptoms using a well‐designed questionnaire are very rare.

The possible roles of hyperpolarization-activated cyclic nucleotide channels in regulating pacemaker activity in colonic interstitial cells of Cajal

BackgroundHyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs.MethodsWe performed whole-cell patch clamp, RT-PCR, and Ca2+-imaging in cultured ICCs from mouse mid colon.ResultsSQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca2+ [Ca2+]i oscillations, rolipram and 8-bromo-cAMP increased [Ca2+]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca2+]i oscillations.ConclusionsHCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

The Inhibitory Effects of Hydrogen Sulfide on Pacemaker Activity of Interstitial Cells of Cajal from Mouse Small Intestine

In this study, we studied whether hydrogen sulfide (H(2)S) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of H(2)S on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular Ca(2+) analysis at 30 and RT-PCR in cultured mouse intestinal ICC. Exogenously applied sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide, caused a slight tonic inward current on pacemaker activity in ICC at low concentrations (50 and 100 microM), but at high concentration (500 microM and 1 mM) it seemed to cause light tonic inward currents and then inhibited pacemaker amplitude and pacemaker frequency, and also an increase in the resting currents in the outward direction. Glibenclamide or other potassium channel blockers (TEA, BaCl(2), apamin or 4-aminopydirine) did not have an effect on NaHS-induced action in ICC. The exogenous application of carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and thapsigargin also inhibited the pacemaker activity of ICC as NaHS. Also, we found NaHS inhibited the spontaneous intracellular Ca(2+) ([Ca(2+)](i)) oscillations in cultured ICC. In doing an RT-PCR experiment, we found that ICC enriched population lacked mRNA for both CSE and CBS, but was prominently detected in unsorted muscle. In conclusion, H(2)S inhibited the pacemaker activity of ICC by modulating intracellular Ca(2+). These results can serve as evidence of the physiological action of H(2)S as acting on the ICC in gastrointestinal (GI) motility.

5-Hydroxytryptamine Generates Tonic Inward Currents on Pacemaker Activity of Interstitial Cells of Cajal from Mouse Small Intestine

In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular Ca(2+) ([Ca(2+)](i)) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-HT(2B, 3, 4, and 7) receptors in ICC. However, SDZ 205557 (a 5-HT(4) receptor antagonist), SB 269970 (a 5-HT7 receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-HT(3) antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-HT(2B) receptor antagonist). Based on [Ca(2+)](i) analysis, we found that 5-HT increased the intensity of [Ca(2+)](i). The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-HT(3, 4, and 7) receptors via [Ca(2+)](i) mobilization and regulation of mitogen-activated protein kinases.

Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-κB and MAP kinase

AIM To investigate lipopolysaccharide (LPS) related signal transduction in interstitial cells of Cajal (ICCs) from mouse small intestine. METHODS For this study, primary culture of ICCs was prepared from the small intestine of the mouse. LPS was treated to the cells prior to measurement of the membrane currents by using whole-cell patch clamp technique. Immunocytochemistry was used to examine the expression of the proteins in ICCs. RESULTS LPS suppressed the pacemaker currents of ICCs and this could be blocked by AH6809, a prostaglandin E2-EP2 receptor antagonist or NG-Nitro-L-arginine Methyl Ester, an inhibitor of nitric oxide (NO) synthase. Toll-like receptor 4, inducible NO synthase or cyclooxygenase-2 immunoreactivity by specific antibodies was detected on ICCs. Catalase (antioxidant agent) had no action on LPS-induced action in ICCs. LPS actions were blocked by nuclear factor κB (NF-κB) inhibitor, actinomycin D (a gene transcription inhibitor), PD 98059 (a p42/44 mitogen-activated protein kinases inhibitor) or SB 203580 [a p38 mitogen-activated protein kinases (MAPK) inhibitor]. SB 203580 also blocked the prostaglandin E2-induced action on pacemaker currents in ICCs but not NO. CONCLUSION LPS inhibit the pacemaker currents in ICCs via prostaglandin E2- and NO-dependent mechanism through toll-like receptor 4 and suggest that MAPK and NF-κB are implicated in these actions.

Bradykinin modulates pacemaker currents through bradykinin B2 receptors in cultured interstitial cells of Cajal from the murine small intestine.

1 We studied the modulation of pacemaker activities by bradykinin in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole‐cell patch‐clamp technique. Externally applied bradykinin produced membrane depolarization in the current‐clamp mode and increased tonic inward pacemaker currents in the voltage‐clamp mode. 2 Pretreatment with bradykinin B1 antagonist did not block the bradykinin‐induced effects on pacemaker currents. However, pretreatment with bradykinin B2 antagonist selectively blocked the bradykinin‐induced effects. Also, only externally applied selective bradykinin B2 receptor agonist produced tonic inward pacemaker currents and ICC revealed a colocalization of the bradykinin B2 receptor and c‐kit immunoreactivities, but bradykinin B1 receptors did not localize in ICC. 3 External Na+‐free solution abolished the generation of pacemaker currents and inhibited the bradykinin‐induced tonic inward current. However, a Cl− channel blocker (DIDS) did not block the bradykinin‐induced tonic inward current. 4 The pretreatment with Ca2+‐free solution and thapsigargin, a Ca2+‐ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the bradykinin‐induced action. 5 Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the bradykinin‐induced effects on pacemaker currents. 6 These results suggest that bradykinin modulates the pacemaker activities through bradykinin B2 receptor activation in ICC by external Ca2+ influx and internal Ca2+ release via protein kinase C‐ or cyclooxygenase‐independent mechanism. Therefore, the ICC are targets for bradykinin and their interaction can affect intestinal motility.

Action of Lipopolysaccharide on Interstitial Cells of Cajal from Mouse Small Intestine

Background and Purpose: Lipopolysaccharide (LPS) induces intestinal dysmotility by alteration of smooth muscle and enteric neuronal activities. However, there is no report on the modulatory effects of LPS on the interstitial cells of Cajal (ICCs). We investigated the effect of LPS and its signal transduction in ICCs. Methods: We performed whole-cell patch clamp and RT-PCR in cultured ICCs from mouse small intestine. Results: LPS suppressed the generation of pacemaker currents of ICCs. The mRNA transcripts for Toll-like receptor 4 (TLR4) were expressed in ICCs. However, the inhibitory action of LPS on pacemaker currents from TLR4+/+ mice was not present in TLR4–/– mice. The inhibitory effects of LPS on ICCs were blocked by glibenclamide (an inhibitor of ATP-sensitive K+ channels), NS-398 (a COX-2 inhibitor), AH6808 [a prostaglandin E2 (PGE2)-EP2 receptor antagonist], ODQ (an inhibitor of guanylate cyclase) and L-NAME [an inhibitor of nitric oxide synthase (NOS)]. Furthermore, genistein and herbimycin A (tyrosine kinase inhibitors) blocked the LPS-induced inhibitory action on pacemaker activity in ICCs. Conclusions: LPS can activate ICCs to release NO and PGE2 through TLR4 activation. The released NO and PGE2 inhibit pacemaker currents by activating ATP-sensitive K+ channels. The LPS actions are mediated by tyrosine kinase signaling pathways.

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na+-free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with U73122, a phospholipase C inhibitor, or thapsigargin, a Ca2+-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca2+ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca2+-release through neurotensin receptor1.

Receptor tyrosine and MAP kinase are involved in effects of H2O2 on interstitial cells of Cajal in murine intestine

Hydrogen peroxide (H2O2) is involved in intestinal motility through changes of smooth muscle activity. However, there is no report as to the modulatory effects of H2O2 on interstitial cells of Cajal (ICC). We investigated the H2O2 effects and signal transductions to determine whether the intestinal motility can be modulated through ICC. We performed whole‐cell patch clamp in cultured ICC from murine intestine and molecular analyses. H2O2 hyperpolarized the membrane and inhibited pacemaker currents. These effects were inhibited by glibenclamide, an inhibitor of ATP‐sensitive K+ (KATP) channels. The free‐radical scavenger catalase inhibited the H2O2‐induced effects. MAFP and AACOCF3 (a cytosolic phospholipase A2 inhibitors) or SC‐560 and NS‐398 (a selective COX‐1 and 2 inhibitor) or AH6809 (an EP2 receptor antagonist) inhibited the H2O2‐induced effects. PD98059 (a mitogen activated/ERK‐activating protein kinase inhibitor) inhibited the H2O2‐induced effects, though SB‐203580 (a p38 MAPK inhibitor) or a JNK inhibitor did not affect. H2O2‐induced effects could not be inhibited by LY‐294002 (an inhibitor of PI3‐kinases), calphostin C (a protein kinase C inhibitor) or SQ‐22536 (an adenylate cyclase inhibitor). Adenoviral infection analysis revealed H2O2 stimulated tyrosine kinase activity and AG 1478 (an antagonist of epidermal growth factor receptor tyrosine kinase) inhibited the H2O2‐induced effects. These results suggest H2O2 can modulate ICC pacemaker activity and this occur by the activation of KATP channels through PGE2 production via receptor tyrosine kinase‐dependent MAP kinase activation.

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3′,5′-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca2+ imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K+ channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca2+ ([Ca2+]i) oscillations revealed that ODQ and L-NAME increased [Ca2+]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca2+]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca2+ release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.