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

Shankar Prasad Parajuli; Seok Choi; Jun Lee; Young Dae Kim; Chan Guk Park; Man Yoo Kim; Hyun Il Kim; Cheol Ho Yeum; Jae Yeoul Jun

doi:10.4196/kjpp.2010.14.2.83

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Parajuli, Choi, Lee, Kim, Park, Kim, Kim, Yeum, and Jun: The Inhibitory Effects of Hydrogen Sulfide on Pacemaker Activity of Interstitial Cells of Cajal from Mouse Small Intestine

Original Article

Korean J Physiol Pharmacol 2010;14(2):83-89.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.2.83

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

Shankar Prasad Parajuli^1,^∗, Seok Choi^1,^∗, Jun Lee², Young Dae Kim², Chan Guk Park², Man Yoo Kim², Hyun Il Kim¹, Cheol Ho Yeum¹, Jae Yeoul Jun^1,

¹Department of Physiology, College of Medicine, Chosun University, Gwangju 501-759, Korea

²Department of Internal Medicine, College of Medicine, Chosun University, Gwangju 501-759, Korea

Corresponding to: Jae Yeoul Jun, Department of Physiology, College of Medicine, Chosun University, 375, Seosuk-dong, Dong-gu, Gwangju 501-759, Korea. (Tel) 82-62-230-6412, (Fax) 82-62-232-4943, (E-mail) jyjun@chosun.ac.kr

^∗ These authors contributed equally to this work.

Received 15 March 2010 Revised 29 March 2010 Accepted 30 March 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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 30°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 μM), but at high concentration (500 μM 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.

Keywords: Interstitial cells of Cajal (ICC), Intestinal motility, Pacemaker currents, Sodium hydrogen sufide (NaHS)

References

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Fig. 1.

Effects of NaHS on pacemaker potentials recorded in cultured ICC from mouse small intestine. (A) shows the pacemaker potentials of ICC exposed to NaHS (1 mM) in the current clamping mode (I=0). NaHS induced membrane hyperpolarization and inhibited the amplitude and frequency of pacemaker potential in ICC. The dot lines indicate the control resting membrane potentials levels. Responses to NaHS are summarized in (B) and (C). The bars represent means±SE. ^∗Asterisks mean significantly different from the controls (p<0.05).

Fig. 2.

Effects of NaHS on pacemaker currents recorded in cultured ICC from mouse small intestine. (A∼C), and (D) show pacemaker currents of ICC exposed to NaHS (50, 100, 500 μM or 1 mM respectively) at a holding potential of – 70 mV. Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. The dot lines indicate the control resting current levels. (E, F), and (G) summarize the inhibitory effects of NaHS on pacemaker currents in ICC. Bars represent means±SE. ^∗Asterisks mean significantly different from the controls (p<0.05).

Fig. 3.

Effects of TEA, BaCl₂, apamin, 4-aminopyridine and glibenclamide on NaHS-induced response in cultured ICC from mouse small intestine. Pretreatment with (A) TEA (20 mM), (B) BaCl₂ (50 μM), (C) apamin (100 μM), (D) 4-aminopyridine (5 mM) or (E) glibenclamide (10 μM) did not affect the inhibitory effects of NaHS (1 mM). Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. The dot lines indicate the control resting current levels. TEA: tetraethylammonium chloride.

Fig. 4.

Effects of thapsigargin or FCCP on pacemaker currents and effects of NaHS on [Ca²⁺]_i oscillation in cultured ICC from mouse small intestine. (A) Treatment with thapsigargin (5 μM) inhibited the pacemaker currents of ICC. (B) Treatment with FCCP (10 μM) in ICC also inhibited the pacemaker currents. (C) Sequential fluorescence intensity images of fluo-4-loaded cultured ICC in normal condition or in presence of NaHS (1 mM). The interval of representative frame was 1 second and the exposure time of each frame was 500 ms. (D) Fluorescence intensity changes plotted in (C) white marker. (Inset) Graphic representation of the inhibition of intensity at peak point of Ca²⁺ oscillations by NaHS. ^∗(p<0.05) Significantly different from the untreated control. Con, control.

Fig. 5.

Agarose gels of the RT-PCR products of CSE and CBS enzymes using cultured cells and separated ICC. (A) Amplified cDNA prepared from pure ICC separated by magnetic cell separation and visualized in 2% gel. (B) Amplified cDNA from unsorted muscle cells and visualized in 2% gel (myosin-smooth muscle cells marker, PGP9.5-neuronal cell marker, CSE-cystathionine gama lyase, CBS-cystathionine beta synthase).

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