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

Shankar Prasad Parajuli,최석,이준,김영대,박찬국,김만우,김현일,염철호,전제열 대한약리학회 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.2

In this study, we studied whether hydrogen sulfide (H2S) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of H2S on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular Ca2+ analysis at 30oC 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 100μM), but at high concentration (500μM 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, BaCl2, 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 Ca2+ ([Ca2+]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, H2S inhibited the pacemaker activity of ICC by modulating intracellular Ca2+. These results can serve as evidence of the physiological action of H2S as acting on the ICC in gastrointestinal (GI) motility.

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

Parajuli, Shankar Prasad,Choi, Seok,Lee, Jun,Kim, Young-Dae,Park, Chan-Guk,Kim, Man-Yoo,Kim, Hyun-Il,Yeum, Cheol-Ho,Jun, Jae-Yeoul The Korean Society of Pharmacology 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.2

In this study, we studied whether hydrogen sulfide ($H_2S$) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of $H_2S$ on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular $Ca^{2+}$ analysis at $30^{\circ}C$ 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 $100{\mu}m$), but at high concentration ($500{\mu}m$ 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, $H_2S$ inhibited the pacemaker activity of ICC by modulating intracellular $Ca^{2+}$. These results can serve as evidence of the physiological action of $H_2S$ as acting on the ICC in gastrointestinal (GI) motility.

Inhibition of Pacemaker Activity of Interstitial Cells of Cajal by Hydrogen Peroxide via Activating ATP-sensitive K+ Channels

최석,Shankar Prasad Parajuli,정현숙,Dilli Parasad Paudyal,염철호,윤평진,전제열 대한약리학회 2007 The Korean Journal of Physiology & Pharmacology Vol.11 No.1

To investigate whether hydrogen peroxide (H2O2) affects intestinal motility, pacemaker currents and membrane potential were recorded in cultured interstitial cells of Cajal (ICC) from murine small intestine by using a whole-cell patch clamp. In whole cell patch technique at 30oC, ICC generated spontaneous pacemaker potential under current clamp mode (I=0) and inward currents (pacemaker currents) under voltage clamp mode at a holding potential of -70 mV. When ICC were treated with H2O2 in ICC, H2O2 hyperpolarized the membrane potential under currents clamp mode and decreased both the frequency and amplitude of pacemaker currents and increased the resting currents in outward direction under voltage clamp mode. Also, H2O2 inhibited the pacemaker currents in a dose-dependent manner. Because the properties of H2O2 action on pacemaker currents were same as the effects of pinacidil (ATP-sensitive K+ channels opener), we tested the effects of glibenclamide (ATP-sensitive K+ channels blocker) on H2O2 action in ICC, and found that the effects of H2O2 on pacemaker currents were blocked by co- or pre- treatment of glibenclamide. These results suggest that H2O2 inhibits pacemaker currents of ICC by activating ATP-sensitive K+ channels.

Calcitonin Gene-related Peptide Suppresses Pacemaker Currents by Nitric Oxide/cGMP-dependent Activation of ATP-sensitive K+ Channels in Cultured Interstitial Cells of Cajal from the Mouse Small Intestine

최석,Shankar Prasad Parajuli,염철호,박찬국,김만유,김영대,차경훈,박영봉,박종성,정한성,전제열 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.2

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at 30C. Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular GDPS, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive K+ channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or L-NAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive K+ channels. Nitric oxide- and guanylate cyclase- dependent pathways are involved in these effects.

Calcitonin Gene-related Peptide Suppresses Pacemaker Currents by Nitric Oxide/cGMP-dependent Activation of ATP-sensitive K⁺ Channels in Cultured Interstitial Cells of Cajal from the Mouse Small Intestine

Choi, Seok,Parajuli, Shankar Prasad,Yeum, Cheol Ho,Park, Chan Guk,Kim, Man Yoo,Kim, Young Dae,Cha, Kyoung Hun,Park, Young Bong,Park, Jong Seong,Jeong, Han Seong,Jun, Jae Yeoul Korean Society for Molecular Biology 2008 Molecules and cells Vol.26 No.2

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at 30°C. Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular GDPβS, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive K⁺ channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or L-NAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive K⁺ channels. Nitric oxide- and guanylate cyclase- dependent pathways are involved in these effects.

Imipramine Inhibits A-type Delayed Rectifier and ATP-Sensitive $K^{+}$ Currents Independent of G-Protein and Protein Kinase C in Murine Proximal Colonic Myocytes

Choi, Seok,Parajuli, Shankar Prasad,Lim, Geon-Han,Kim, Jin-Ho,Yeum, Cheol-Ho,Yoon, Pyung-Jin,Jun, Jae-Yeoul The Pharmaceutical Society of Korea 2006 Archives of Pharmacal Research Vol.29 No.11

The effects of imipramine on A-type delayed rectifier $K^{+}$ currents and ATP-sensitive $K^{+}\;(K_{ATP)$ currents were studied in isolated murine proximal colonic myocytes using the whole-cell patch-clamp technique. Depolarizing test pulses between-80 mV and +30 mV with 10 mV increments from the holding potential of-80 mV activated voltage-dependent outward $K^{+}$ currents that peaked within 50 ms followed by slow decreasing sustained currents. Early peak currents were inhibited by the application of 4-aminopyridine, whereas sustained currents were inhibited by the application of TEA. The peak amplitude of A-type delayed rectifier $K^{+}$ currents was reduced by external application of imipramine. The half-inactivation potential and the half-recovery time of A-type delayed rectifier $K^{+}$ currents were not changed by imipramine. With 0.1 mM ATP and 140 mM $K^{+}$ in the pipette and 90 mM $K^{+}$ in the bath solution and a holding potential of -80 mV, pinacidil activated inward currents; this effect was blocked by glibenclamide. Imipramine also inhibited $K_{ATP}$ currents. The inhibitory effects of imipramine in A-type delayed rectifier $K^{+}$ currents and $K_{ATP}$ currents were not changed by guanosine 5-O-(2-thiodiphosphate) ($GDP{\beta}S$) and chelerythrine, a protein kinase C inhibitor. These results suggest that imipramine inhibits A-type delayed rectifier $K^{+}$ currents and $K_{ATP}$ currents in a manner independent of G-protein and protein kinase C.

Imipramine Inhibits A-type Delayed Rectifier and ATP-Sensitive K+ Currents Independent of G-Protein and Protein Kinase C in Murine Proximal Colonic Myocytes

Seok Choi,Shankar Prasad Parajuli,Geon Han Lim,Jin Ho Kim,Cheol Ho Yeum,Pyung Jin Yoon,Jae Yeoul Jun 대한약학회 2006 Archives of Pharmacal Research Vol.29 No.11

The effects of imipramine on A-type delayed rectifier K+ currents and ATP-sensitive K+ (KATP) currents were studied in isolated murine proximal colonic myocytes using the whole-cell patchclamp technique. Depolarizing test pulses between -80 mV and +30 mV with 10 mV increments from the holding potential of -80 mV activated voltage-dependent outward K+ currents that peaked within 50 ms followed by slow decreasing sustained currents. Early peak currents were inhibited by the application of 4-aminopyridine, whereas sustained currents were inhibited by the application of TEA. The peak amplitude of A-type delayed rectifier K+ currents was reduced by external application of imipramine. The half-inactivation potential and the halfrecovery time of A-type delayed rectifier K+ currents were not changed by imipramine. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of –80 mV, pinacidil activated inward currents; this effect was blocked by glibenclamide. Imipramine also inhibited KATP currents. The inhibitory effects of imipramine in A-type delayed rectifier K+ currents and KATP currents were not changed by guanosine 5-O-(2-thiodiphosphate) (GDPβS) and chelerythrine, a protein kinase C inhibitor. These results suggest that imipramine inhibits A-type delayed rectifier K+ currents and KATP currents in a manner independent of G-protein and protein kinase C.

Inhibition of Pacemaker Activity of Interstitial Cells of Cajal by Hydrogen Peroxide via Activating ATP-sensitive $K^+$ Channels

Choi Seok,Parajuli Shankar Prasad,Cheong Hyeon-Sook,Paudyal Dilli Parasad,Yeum Cheol-Ho,Yoon Pyung-Jin,Jun Jae-Yeoul The Korean Society of Pharmacology 2007 The Korean Journal of Physiology & Pharmacology Vol.11 No.1

To investigate whether hydrogen peroxide($H_2O_2$) affects intestinal motility, pacemaker currents and membrane potential were recorded in cultured interstitial cells of Cajal(ICC) from murine small intestine by using a whole-cell patch clamp. In whole cell patch technique at $30^{\circ}C$, ICC generated spontaneous pacemaker potential under current clamp mode(I=0) and inward currents(pacemaker currents) under voltage clamp mode at a holding potential of -70 mV. When ICC were treated with $H_2O_2$ in ICC, $H_2O_2$ hyperpolarized the membrane potential under currents clamp mode and decreased both the frequency and amplitude of pacemaker currents and increased the resting currents in outward direction under voltage clamp mode. Also, $H_2O_2$ inhibited the pacemaker currents in a dose-dependent manner. Because the properties of $H_2O_2$ action on pacemaker currents were same as the effects of pinacidil(ATP-sensitive $K^+$ channels opener), we tested the effects of glibenclamide(ATP-sensitive $K^+$ channels blocker) on $H_2O_2$ action in ICC, and found that the effects of $H_2O_2$ on pacemaker currents were blocked by co- or pre- treatment of glibenclamide. These results suggest that $H_2O_2$ inhibits pacemaker currents of ICC by activating ATP-sensitive $K^+$ channels.

Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine

박일구,김진호,박찬국,김만우,Shankar Prasad Parajuli,홍찬식,최석,전제열 전남대학교 의과학연구소 2018 전남의대학술지 Vol.54 No.1

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscleactivity. We studied the functional roles of external adenosine 5’-triphosphate (ATP)on pacemaker activity in cultured ICCs from mouse small intestines by using thewhole-cell patch clamp technique and intracellular Ca2+ ([Ca2+]i) imaging. ExternalATP dose-dependently depolarized the resting membrane and produced tonic inwardpacemaker currents, and these effects were antagonized by suramin, a purinergic P2receptor antagonist. ATP-induced effects on pacemaker currents were suppressed byan external Na+-free solution and inhibited by the nonselective cation channel blockers,flufenamic acid and niflumic acid. The removal of external Ca2+ or treatment with thapsigargin(inhibitor of Ca2+ uptake into endoplasmic reticulum) inhibited the ATP-inducedeffects on pacemaker currents. Spontaneous [Ca2+]i oscillations were enhancedby external ATP. These results suggest that external ATP modulates pacemaker activityby activating nonselective cation channels via external Ca2+ influx and [Ca2+]i releasefrom the endoplasmic reticulum. Thus, it seems that activating the purinergicP2 receptor may modulate GI motility by acting on ICCs in the small intestine.

Carbachol Regulates Pacemaker Activities in Cultured Interstitial Cells of Cajal from the Mouse Small Intestine

소금영,김상훈,손홍문,Soo Jin Choi,Shankar Prasad Parajuli,최석,염철호,Pyung Jin Yoon,전제열 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.5

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca2+-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of -70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M3 receptor antagonist, but not by methotramine, a muscarinic M2 receptor antagonist. Intracellular GDP-β-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na+-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca2+. In recording of intracellular Ca2+ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca2+ concentrations with increasing of Ca2+ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M3 receptors by a G-protein dependent intracellular Ca2+ release mechanism.