Effect of arginase on NO‐mediated protein modifications in endothelial cells

Abstract

It is known that arginase is able to modify Nitric Oxide (NO) production through competition with NO synthase for the common substrate L‐arginine. We hypothesize that by modifying NO production arginase may affect NO‐mediated protein modifications – S‐nitrosylation and nitration. We studied NO production and arginase activity in human endothelial cells (EC) exposed to hypoxia. We found that the exposure of EC to 1% O2 for 24h induces an increase in NO production and an elevation in the expression of arginase II (Arg II) isoform which localizes in mitochondria. To study the effect of Arg II on S‐nitrosylation and nitration we used the arginase‐specific inhibitor BEC. After exposure of EC to hypoxia with or without BEC, nitrated proteins were precipitated with nitro‐tyrosine antibody, and then nitration of individual proteins was analyzed by Western blotting. In another set of experiments S‐nitrosylated proteins in cell lysates were analyzed by biotin‐switch assay. We found that the treatment of EC with BEC induced remarkable nitration of the mitochondrial protein cytochrome c oxidase (COX) both in normoxia and hypoxia. For non‐mitochondrial protein GAPDH treatment with BEC upregulated both S‐nitrosylation and nitration only in hypoxic conditions. Hence, we report the ability of Arg II to modify NO‐mediated protein modifications in EC.This work was supported by Florida DOH (KK), VA and NIH (JMP).