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-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (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 U₄₆₆₁₉ but increased by the RhoA inhibitor C3 transferase. U₄₆₆₁₉ 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 KCl-increased [CA²⁺]_i, 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.