Roles of the RhoA-ROCK Signaling Pathway in the Endothelial H2S Production and Vasodilation in Rat Cerebral Arteries

Cerebral endothelial H2S protects against cerebral ischemia-reperfusion injury through vasodilation, but its cerebral vasodilation mechanism and regulation of production are poorly understood. The RhoA-ROCK pathway plays important roles in vascular function. In this study, the roles of this pathway in the endothelial H2S production and vasodilation in rat cerebral arteries were investigated. Acetylcholine significantly increased H2S-generating enzyme cystathionine-gamma-Iyase (CSE) and 3-mercaptopyruvate sulfur-transferase (3-MST) protein expressions and H2S production in rat cerebrovascular endothelial cells (ECs), but the increases were markedly decreased by the M receptor blocker atropine or the CSE inhibitor DL-propargylglycine. Pretreatment with DL-propargylglycine or the 3-MST inhibitor L-aspartic acid markedly reduced the acetylcholine-increased H2S; CSE protein expression and H2S levels in the ECs were obviously attenuated by the RhoA agonist U-46619 but increased by the RhoA inhibitor C3 transferase. U(46619 )also reduced 3-MST protein expression; Acetylcholine markedly inhibited RhoA protein expression and activity, but the inhibition was obviously reversed by atropine, DL-propargylglycine, and L-aspartic acid, respectively; Acetylcholine-induced endothelium-dependent vasodilation in rat cerebral basilar artery was significantly attenuated by pretreatment with DL-propargylglycine or L-aspartic acid or RhoA inhibitor CCG-1423 or ROCK inhibitor KD025, and was further decreased by co-pretreatment with DL-propargylglycine (or L-aspartic acid) and CCG-1423 (or KD025); NaHS significantly relaxed rat cerebral basilar artery vascular smooth muscle cells and inhibited ROCK(1/2 )activities, phosphorylated myosin light chain (MLC) protein expression, and KCI-increased [Ca-2(+)](p) but these relaxation and inhibitions were markedly attenuated by pretreatment with C3 transferase or ROCK inhibitor Y27632. Our results demonstrated that endothelial H2S production is promoted by activation of the M receptor but inhibited by the RhoA-ROCK pathway in rat cerebral arteries; the endothelial H2S induces cerebral vasodilation by inhibiting this pathway to reduce phosphorylation of MLC and [Ca2+](i) in vascular smooth muscle cells.