Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger

Xiaopeng Bai; Eikichi Ihara; Katsuya Hirano; Yoshimasa Tanaka; Kayoko Nakano; Satomi Kita; Takahiro Iwamoto; Haruei Ogino; Mayumi Hirano; Yoshinao Oda; Kazuhiko Nakamura; Yoshihiro Ogawa

doi:10.1016/j.jcmgh.2017.11.004

Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na⁺/Ca²⁺ Exchanger

Cell Mol Gastroenterol Hepatol. 2017 Nov 22;5(3):209-221. doi: 10.1016/j.jcmgh.2017.11.004. eCollection 2018 Mar.

Authors

Affiliations

¹ Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
² Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Kagawa Prefecture, Japan.
³ Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
⁴ Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
⁵ Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan.
⁶ Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
⁷ Department of Molecular and Cellular Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

Abstract

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.

Keywords: 3MST, 3-mercaptopyruvate sulfurtransferase; AOA, amino-oxyacetic acid; CBS, cystathionine-β-synthase; CCh, carbachol; CSE, cystathionine-γ-lyase; ES, extracellular solution; H2S, hydrogen sulfide; Hydrogen Sulfate; KATP channels, ATP-sensitive K+ channels; KES, K+ extracellular solution; L-Asp, L-aspartic acid; L-Cys, L-cysteine; L-NAME, Nω-nitro-L-arginine methyl ester; LES, lower esophageal sphincter; Lower Esophageal Sphincter; Myogenic Tone Regulation; NCX, Na+/Ca2+ exchanger; NES, normal extracellular solution; Na+/Ca2+ Exchanger; PAG, propargylglycine; TEA, tetraethylammonium; TG, transgenic; TTX, tetrodotoxin; [Ca2+]i, cytosolic Ca2+ concentration; [Na+]o, extracellular Na+ concentration.