Effect of S-Nitrosothiols on Cellular Glutathione and Reactive Protein Sulfhydryls

S-Nitrosothiols may cause many of the biological effects of NO and cellular effects have been attributed to S-nitrosylation of reactive protein sulfhydryls. This report examines the effect of S-nitrosothiols on the low-molecular-weight thiols and protein thiols in NIH/3T3 cells. A low concentration of S-nitrosocysteine increased the cysteine content of the cells, with no evidence of either low-molecular-weight thiol or protein S-nitrosylation. Millimolar amounts of S-nitrosocysteine produced S-nitrosoglutathione (GSNO), cysteinyl glutathione, cysteine, and glutathione disulfide. Large amounts of protein S-nitrosylation and lesser amounts of protein S-glutathiolation and S-cysteylation were also observed. GSNO and S-nitroso-N-acetylpenicillamine (SNAP) were much less effective than S-nitrosocysteine, but a combination of cysteine and GSNO produced S-nitrosocysteine-like effects. In cultured hepatocytes, millimolar S-nitrosocysteine was significantly less effective since the cells contained three times more glutathione than NIH/3T3 cells. Results suggest that S-nitrosocysteine enters cells intact, and low concentrations do not significantly increase cellular pools of S-nitrosothiol or S-nitrosylated protein. Millimolar concentrations of S-nitrosocysteine generate S-nitrosylated, S-glutathiolated, and S-cysteylated proteins, as well as a variety of low-molecular-weight disulfides and S-nitrosothiols.