Endogenous Hydrogen Sulfide in Rabbit Gastric Smooth Muscle Cells

42 43 Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, 44 increase cyclic nucleotide levels and inhibit muscle contraction via inhibition of MLC 45 kinase and activation of MLC phosphatase. H2S produced as an endogenous signalling 46 molecule synthesized mainly from L-cysteine via cystathionine-γ-lyase (CSE) and 47 cystathionine-β-synthase (CBS) regulate muscle contraction. The aim of this study was 48 to analyse the expression of CSE and H2S function in the regulation of MLCP activity, 49 MLC20 phosphorylation and contraction in isolated gastric smooth muscle cells. Both 50 mRNA and protein expression of CSE, but not CBS was detected in smooth muscle 51 cells of rabbit, human and mouse stomach. L-cysteine, an activator of CSE, and NaHS, 52 a donor of H2S, inhibited carbachol-induced Rho kinase and PKC activity, Rho kinase53 sensitive phosphorylation of MYPT1 and PKC-sensitive phosphorylation of CPI-17, 54 MLC20 phosphorylation and sustained muscle contraction. Inhibitory effects of L55 cysteine, but not NaHS were blocked upon suppression of CSE expression by siRNA or 56 inhibition of its activity by DL-propargylglycine (PPG) suggesting that the effect of L57 cysteine is mediated via activation of CSE. Glibenclamide, an inhibitor of KATP channels 58 and a known target of H2S, had no effect on the inhibition of contraction by H2S. Both L59 cysteine and NaHS had no effect on basal cAMP and cGMP levels, but augmented 60 forskolin-induced cAMP and SNP-induced cGMP formation. We conclude that both 61 endogenous and exogenous H2S inhibit muscle contraction, and the mechanism 62 involves inhibition of Rho kinase and PKC activities and stimulation of MLC 63 phosphatase activity leading to MLC20 dephosphorylation and inhibition of muscle 64