Protection against Hypoxia-Reoxygenation Injury of Hippocampal Neurons by H2S via Promoting Phosphorylation of ROCK2 at Tyr722 in Rat Model

Abstract

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.

Protection of H2S against Hypoxia/Reoxygenation Injury in Rat Hippocampal Neurons through Inhibiting Phosphorylation of ROCK2 at Thr436 and Ser575

“…The inhibition of H 2 S on the phosphorylation of His-ROCK 2 wild at Thr436 or Ser575 seems contrary to our recent report on the promotion of H 2 S on the phosphorylation of ROCK 2 at Tyr722 [ 27 ]. It was already known that the phosphorylation of different sites of ROCK 2 had different effects on its activity; the phosphorylation of some sites can enhance the activity of ROCK 2 , but the phosphorylation of other sites can inhibit its activity.…”

Neuroprotection in an Experimental Model of Multiple Sclerosis via Opening of Big Conductance, Calcium-Activated Potassium Channels

“…The neuroprotective effect, independent of immunosuppression, is consistent with the neuroprotection observed following brain trauma, spinal cord injury, and ischemia [ 20 , 21 , 22 , 44 ]. This benefit may occur via activity on multiple complementary pathways, including the blockage of excitotoxicity and the promotion of anti-oxidant properties [ 20 , 21 , 22 , 44 , 45 ].…”