TY - JOUR
T1 - Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger
AU - Bai, Xiaopeng
AU - Ihara, Eikichi
AU - Hirano, Katsuya
AU - Tanaka, Yoshimasa
AU - Nakano, Kayoko
AU - Kita, Satomi
AU - Iwamoto, Takahiro
AU - Ogino, Haruei
AU - Hirano, Mayumi
AU - Oda, Yoshinao
AU - Nakamura, Kazuhiko
AU - Ogawa, Yoshihiro
N1 - Funding Information:
To evaluate the functional role of endogenous H2S in the generation of basal tone of LES, we examined the effects of inhibitors of 3 H2S-generating enzymes (CBS, 3MST, and CSE) on the resting level of tension (Figure 1). AOA, an inhibitor of CBS, and L-Asp, an inhibitor of 3MST, induced concentration-dependent relaxation (Figure 1A and B). The maximum relaxation obtained with 1 mM AOA and 3 mM L-Asp was −19.7% ± 3.10% (n = 4) and −19.5% ± 3.47% (n = 4) (Figure 1C). However, PAG, an inhibitor of CSE, induced no relaxation even at 3 mM (Figure 1C). The relaxant effects observed with AOA and L-Asp were partly reversed by the addition of L-Cys, which serves as a substrate for the generation of H2S (Figure 1D and E). The extent of contraction induced by 10 mM L-Cys reached 4.8% ± 1.38% (n = 4) and 12.0% ± 2.54% (n = 4), in the presence of 1 mM AOA and 3 mM L-Asp, respectively. The combination of 1 mM AOA and 3 mM L-Asp induced a significantly larger relaxation (−27.4% ± 2.55%; n = 5) than either 1 mM AOA (−19.7% ± 3.10%; n = 4) or 3 mM L-Asp (−19.5% ± 3.47%; n = 4) alone. The addition of L-Cys up to 10 mM failed to reverse the relaxation induced by combined treatment with 1 mM AOA and 3 mM L-Asp (Figure 1F). These functional experiments suggest that CSB and 3MST, but not CSE, are major enzymes that generate H2S in porcine LES. Supporting this conclusion, a histochemical analysis revealed the expression of CSB and 3MST, but not CSE, in the porcine LES (Figure 2). Both circular and longitudinal muscle layers showed positive staining for both enzymes. Effects of H2S-generating enzyme inhibitors on circular smooth muscle of esophageal body were also examined. In a similar manner to LES, both AOA and L-Asp induced concentration-dependent relaxation, whereas PAG induced no relaxation. However, the extents of maximum relaxation obtained with 1 mM AOA (−10.9% ± 1.57%; n = 4) and 3 mM L-Asp (−10.4% ± 2.02%; n = 4) in esophageal body were significantly lower than those in LES, respectively (Figure 1G). In addition, contributions of L-type Ca2+ channels and a Rho kinase signaling pathway to the basal tone of LES were examined by using the pharmacologic inhibitors including nifedipine and Y-27632. The maximum relaxations obtained with 10 μM nifedipine (−12.4% ± 1.38%; n = 4) and 10 μM Y27632 (−7.1% ± 2.76%; n = 4) were significantly lower than that (27.4% ± 2.55%; n = 5) with 1 mM AOA and 3 mM L-Asp.
Publisher Copyright:
© 2018 The Authors
PY - 2018/3
Y1 - 2018/3
N2 - Background and Aims: Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. Methods: Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. Results: Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. Conclusions: Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.
AB - Background and Aims: Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. Methods: Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. Results: Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. Conclusions: Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.
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U2 - 10.1016/j.jcmgh.2017.11.004
DO - 10.1016/j.jcmgh.2017.11.004
M3 - Article
AN - SCOPUS:85043603480
VL - 5
SP - 209
EP - 221
JO - Cellular and Molecular Gastroenterology and Hepatology
JF - Cellular and Molecular Gastroenterology and Hepatology
SN - 2352-345X
IS - 3
ER -