Cerebral endothelial H 2 S 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 H 2 S production and vasodilation in rat cerebral arteries were investigated. Acetylcholine significantly increased H 2 S-generating enzyme cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) protein expressions and H 2 S 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 H 2 S; CSE protein expression and H 2 S 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 KCl-increased [Ca 2+ ] i , but these relaxation and inhibitions were markedly attenuated by pretreatment with C3 transferase or ROCK inhibitor Y27632. Our results demonstrated that endothelial H 2 S production is promoted by activation of the M receptor but inhibited by the RhoA-ROCK pathway in rat cerebral arteries; the endothelial H 2 S induces cerebral vasodilation by inhibiting this pathway to reduce phosphorylation of MLC and [Ca 2+ ] i in vascular smooth muscle cells.

The RhoA-ROCK signaling pathway is associated with the protective effects of hydrogen sulfide (H2S) against cerebral ischemia. H2S protects rat hippocampal neurons (RHNs) against hypoxia-reoxygenation (H/R) injury by promoting phosphorylation of RhoA at Ser188. However, effect of H2S on the phosphorylation of ROCK2-related sites is unclear. The present study was designed to investigate whether H2S can play a role in the phosphorylation of ROCK2 at Tyr722, and explore whether this role mediates the protective effect of H/R injury in RHNs. Prokaryotic recombinant plasmids ROCK2wild-pGEX-6P-1 and ROCK2Y722F-pGEX-6P-1 were constructed and transfected into E. coli in vitro, and the expressed protein, GST-ROCK2wild and GST-ROCK2Y722F were used for phosphorylation assay in vitro. Eukaryotic recombinant plasmids ROCK2Y722-pEGFP-N1 and ROCK2Y722F-pEGFP-N1 as well as empty plasmid were transfected into the RHNs. Western blot assay and whole-cell patch-clamp technique were used to detect phosphorylation of ROCK2 at Tyr722 and BKCa channel current in the RHNs, respectively. Cell viability, leakages of intracellular enzymes lactate dehydrogenase (LDH), and nerve-specific enolase (NSE) were measured. The H/R injury was indicated by decrease of cell viability and leakages of intracellular LDH and NSE. The results of Western blot have shown that NaHS, a H2S donor, significantly promoted phosphorylation of GST-ROCK2wild at Tyr722, while no phosphorylation of GST-ROCK2Y722F was detected. The phosphorylation of ROCK2wild promoted by NaHS was also observed in RHNs. NaHS induced more potent effects on protection against H/R injury, phosphorylation of ROCK2 at Tyr722, inhibition of ROCK2 activity, as well as increase of the BKCa current in the ROCK2Y722-pEGFP-N1-transfected RHNs. Our results revealed that H2S protects the RHNs from H/R injury through promoting phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 activity and potentially by opening channel currents.

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