Role of glutaredoxin-mediated protein S-glutathionylation in cellular nitroglycerin tolerance

We hypothesize that nitroglycerin (NTG) causes direct oxidation of multiple cellular sulfhydryl (SH) proteins and that manipula-tion of SH redox status affects NTG tolerance. In LLC-PK1 cells, we found that nitrate tolerance, as indicated by cGMP accumulation toward NTG, was accompanied by increased protein [ ³⁵S]cysteine incorporation, significant S-glutathionyla-tion of multiple proteins, and decreased metabolic activity of several SH-sensitive enzymes, including creatine kinase, xan- thine oxidoreductase, and glutaredoxin (GRX). Cells overex-pressing GRX exhibited reduced cellular protein S-glutathiony- lation (PSSG) and absence of NTG tolerance, whereas those with silenced GRX showed increased extent of NTG-induced tolerance. Incubation of LLC-PK1 cells with oxidized glutathi- one led to several major observations associated with nitrate tolerance, namely, reduced cGMP accumulation, PSSG formation, superoxide accumulation, and the attenuation of these events by vitamin C. Aortic S-glutathionylated proteins increased approximately 3-fold in rats made tolerant in vivo to NTG and showed significant negative correlation with vascular responsiveness ex vivo. NTG incubation in EA.hy926 endothelial cells and LLC-PK1 cells led to increased S-glutathionylation and activity of p21 ^ras, a known mediator of cellular signaling. These results indicate that the hallmark events of NTG tolerance, such as reduced bioactivation and redox signaling, are associated with GRX-dependent protein deglutathionylation.