Abstract
Hydrogen sulfide (H2S) plays an important role in smooth muscle relaxation. Here, we investigated the expression of enzymes in H2S synthesis and the mechanism regulating colonic smooth muscle function by H2S. Expression of cystathionine‐γ‐lyase (CSE), but not cystathionine‐β‐synthase (CBS), was identified in the colonic smooth muscle of rabbit, mouse, and human. Carbachol (CCh)‐induced contraction in rabbit muscle strips and isolated muscle cells was inhibited by l‐cysteine (substrate of CSE) and NaHS (an exogenous H2S donor) in a concentration‐dependent fashion. H2S induced S‐sulfhydration of RhoA that was associated with inhibition of RhoA activity. CCh‐induced Rho kinase activity also was inhibited by l‐cysteine and NaHS in a concentration‐dependent fashion. Inhibition of CCh‐induced contraction by l‐cysteine was blocked by the CSE inhibitor, dl‐propargylglycine (DL‐PPG) in dispersed muscle cells. Inhibition of CCh‐induced Rho kinase activity by l‐cysteine was blocked by CSE siRNA in cultured cells and DL‐PPG in dispersed muscle cells. Stimulation of Rho kinase activity and muscle contraction in response to CCh was also inhibited by l‐cysteine or NaHS in colonic muscle cells from mouse and human. Collectively, our studies identified the expression of CSE in colonic smooth muscle and determined that sulfhydration of RhoA by H2S leads to inhibition of RhoA and Rho kinase activities and muscle contraction. The mechanism identified may provide novel therapeutic approaches to mitigate gastrointestinal motility disorders.
Highlights
Hydrogen sulfide (H2S) is produced via both enzymatic and nonenzymatic pathways in mammalian cells, but most of the H2S levels in tissues are attributed to enzymatic synthesis (Abe and Kimura 1996; Kamoun 2004; Caliendo et al 2010; Wang 2012)
Our results demonstrate selective expression of CSE in colonic smooth muscle cells of rabbit, mouse, and human, and addition of L-cysteine or sodium hydrosulfide (NaHS) causes S-sulfhydration of RhoA that lead to inhibition of RhoA and Rho kinase activities and muscle contraction in response to contractile agonists
Cystathionine-b-synthase (CBS) and cystathionine-c-lyase (CSE) antibodies were purchased from Proteintech (Chicago, IL); [32P]ATP was purchased from PerkinElmer (Cambridge, MA); HRP-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA); PVDF membranes were obtained from Millipore (Billerica, MA); Effectene Transfection Reagent, QIAEXâII was from Qiagen (Germantown, MD); culture medium (Dulbecco’s modified Eagle’s medium) was from Fisher Scientific (Ashville, NC); L-Cysteine and DL-propargylglycine (PPG) were from Sigma
Summary
Hydrogen sulfide (H2S) is produced via both enzymatic and nonenzymatic pathways in mammalian cells, but most of the H2S levels in tissues are attributed to enzymatic synthesis (Abe and Kimura 1996; Kamoun 2004; Caliendo et al 2010; Wang 2012). The main enzymes responsible for H2S generation, cystathionine-b-synthase (CBS) and cystathionine-c-lyase (CSE), are expressed in several tissues, but the pattern of expression has been reported to be tissue-specific. The H2S levels in the gastrointestinal (GI) system include two sources: a luminal sulfate-reducing bacterial source in the large intestine and an endogenous generation by different cells within the wall of GI tract (Linden et al 2010; Farrugia and Szurszewski 2014). Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
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