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.

Potential role of the gaseous mediator hydrogen sulphide (H2S) in inhibition of human colonic contractility

“…In the present work, the inhibitory effects of H 2 S on spontaneous contractions (EC 50 = 329.2 M) and EFS-induced cholinergic or tachykinergic contraction (EC 50 = 2 mM and 5.7 mM respectively) have been observed when high concentrations have been added to the bath. However, despite these relatively high concentrations, our results demonstrate that H 2 S inhibitory effects are reversible, so they may not be related to an irreversible impairment of tissue function due to toxicity as it has also been shown in isolated smooth muscle cells [21]. Moreover, prior studies have reported that even if H 2 S concentration in the external medium is high, concentration inside the tissue may be much lower, or even trivial [1].…”

“…In the respiratory system, H 2 S reversibly inhibited acetylcholine (ACh)-induced calcium oscillations responsible for the contraction of airway SMCs [19]. Similar findings have been reported in guinea-pig ileum, rat jejunum and rabbit stomach, where H 2 S significantly reduced cholinergic mediated contractions [15,20,21].…”

“…According to this hypothesis, it has been demonstrated that H 2 S inhibits calcium release through IP 3 receptors [19], which might constitute another possible mechanism involved in the reduction of excitatory pathways. A recent study suggests that H 2 S relaxant effect might be related to inhibition of carbachol-induced Rho kinase or protein kinase C activities in rabbit gastric smooth muscle cells [21].…”

Inhibition of RhoA/Rho kinase pathway and smooth muscle contraction by hydrogen sulfide

“…; Nalli et al. ). Briefly, colonic circular muscle strips were incubated for 30 min at 31°C in 15 mL of HEPES medium (120 mmol/L NaCl, 4 mmol/L KCl, 2.6 mmol/L KH 2 PO 4 , 0.6 mmol/L MgCl 2 , 25 mmol/L HEPES, 14 mmol/L glucose, 2.1% (v/v) Eagle's essential amino acid mixture, 0.1% collagenase [type II], and 0.1% soybean trypsin inhibitor).…”

“…; Nalli et al. ). Smooth muscle cells were homogenized in lysis buffer containing Triton X‐100 and protease and phosphatase inhibitors.…”