Protection against Hypoxia-Reoxygenation Injury of Hippocampal Neurons by H(2)S via Promoting Phosphorylation of ROCK(2) at Tyr722 in Rat Model

The RhoA-ROCK signaling pathway is associated with the protective effects of hydrogen sulfide (H(2)S) against cerebral ischemia. H(2)S protects rat hippocampal neurons (RHNs) against hypoxia-reoxygenation (H/R) injury by promoting phosphorylation of RhoA at Ser188. However, effect of H(2)S on the phosphorylation of ROCK(2)-related sites is unclear. The present study was designed to investigate whether H(2)S can play a role in the phosphorylation of ROCK(2) at Tyr722, and explore whether this role mediates the protective effect of H/R injury in RHNs. Prokaryotic recombinant plasmids ROCK(2)(wild)-pGEX-6P-1 and ROCK(2)(Y722F)-pGEX-6P-1 were constructed and transfected into E. coli in vitro, and the expressed protein, GST-ROCK(2)(wild) and GST-ROCK(2)(Y722F) were used for phosphorylation assay in vitro. Eukaryotic recombinant plasmids ROCK(2)(Y722)-pEGFP-N1 and ROCK(2)(Y722F)-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 ROCK(2) at Tyr722 and BK(Ca) 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 H(2)S donor, significantly promoted phosphorylation of GST-ROCK(2)(wild) at Tyr722, while no phosphorylation of GST-ROCK(2)(Y722F) was detected. The phosphorylation of ROCK(2)(wild) promoted by NaHS was also observed in RHNs. NaHS induced more potent effects on protection against H/R injury, phosphorylation of ROCK(2) at Tyr722, inhibition of ROCK(2) activity, as well as increase of the BK(Ca) current in the ROCK(2)(Y722)-pEGFP-N1-transfected RHNs. Our results revealed that H(2)S protects the RHNs from H/R injury through promoting phosphorylation of ROCK(2) at Tyr722 to inhibit ROCK(2) activity and potentially by opening channel currents.