Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdo wn in skeletal muscle cells by inhibiting calpain cleavage of talin. The ni tric oxide donor sodium nitroprusside prevented many of the effects of calc ium ionophore on C2C12 muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachmen t, and loss of cellular protein. These results indicate that nitric oxide i nhibition of calpain protected the cells from ionophore-induced proteolysis . Calpain inhibitor I and a cell-permeable calpastatin peptide also protect ed the cells from proteolysis, confirming that ionophore-induced proteolysi s was primarily calpain mediated. The activity of m-calpain in a casein zym ogram was inhibited by sodium nitroprusside, and this inhibition was revers ed by dithiothreitol. Previous incubation with the active site-targeted cal pain inhibitor I prevented most of the sodium nitroprusside-induced inhibit ion of m-calpain activity. These data suggest that nitric oxide inhibited m -calpain activity via S-nitrosylation of the active site cysteine. The resu lts of this study indicate that nitric oxide produced endogenously by skele tal muscle and other cell types has the potential to inhibit m-calpain acti vity and cytoskeletal proteolysis.