title: Inhibition of RhoA-dependent pathway and contraction by endogenous hydrogen sulfide in rabbit gastric smooth muscle cells.
abstract: Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, increase cyclic nucleotide levels and inhibit muscle contraction via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase (MLCP). H2S produced as an endogenous signaling molecule synthesized mainly from l-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) regulates muscle contraction. The aim of this study was to analyze the expression of CSE and H2S function in the regulation of MLCP activity, 20-kDa regulatory light chain of myosin II (MLC20) phosphorylation, and contraction in isolated gastric smooth muscle cells. Both mRNA expression and protein expression of CSE, but not CBS, were detected in smooth muscle cells of rabbit, human, and mouse stomach. l-cysteine, an activator of CSE, and NaHS, a donor of H2S, inhibited carbachol-induced Rho kinase and PKC activity, Rho kinase-sensitive phosphorylation of MYPT1, PKC-sensitive phosphorylation of CPI-17, and MLC20 phosphorylation and sustained muscle contraction. The inhibitory effects of l-cysteine, but not NaHS, were blocked upon suppression of CSE expression by siRNA or inhibition of its activity by dl-propargylglycine (PPG) suggesting that the effect of l-cysteine is mediated via activation of CSE. Glibenclamide, an inhibitor of KATP channels, had no effect on the inhibition of contraction by H2S. Both l-cysteine and NaHS had no effect on basal cAMP and cGMP levels but augmented forskolin-induced cAMP and SNP-induced cGMP formation. We conclude that both endogenous and exogenous H2S inhibit muscle contraction, and the mechanism involves inhibition of Rho kinase and PKC activities and stimulation of MLCP activity leading to MLC20 dephosphorylation and inhibition of muscle contraction.
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Ancy D
authors.initials: AD
authors.lastname: Nalli
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Rajagopal
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: John R
authors.initials: JR
authors.lastname: Grider
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia skarnam@vcu.edu.
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003542:Cystathionine gamma-Lyase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006862:Hydrogen Sulfide / Q000378:metabolism* / Q000494:pharmacology; D008810:Mice, Inbred C57BL; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D013270:Stomach / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D014162:Transfection; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: C491521:CPI-17 protein, Oryctolagus cuniculus; D009124:Muscle Proteins; D018994:Myosin Light Chains; D020030:Nitric Oxide Donors; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D003542:Cystathionine gamma-Lyase; D006152:Cyclic GMP; D006862:Hydrogen Sulfide
keywords: H2S; Rho kinase; muscle contraction; muscle relaxation; protein kinase C