Abstract 922: Role Of Protein SH-Oxidation In Initiating Cellular Nitroglycerin Tolerance
Cellular and vascular tolerance to nitroglycerin (NTG) is accompanied by oxidative stress, but the initiating mechanism is not defined. We hypothesize that NTG causes direct oxidation of multiple cellular SH-proteins and that manipulation of SH-redox status affects NTG-tolerance. In LLC-PK1 cells, cyclic guanosine monophosphate (cGMP) accumulation upon challenge with 3.16 microM NTG was significantly reduced after pre-incubation with 1 microM NTG for 5 hrs (124±45 vs. 30.5±12.1 pmol/mg protein, p<0.05), indicating NTG tolerance. This phenomenon was accompanied by increased protein 35S-incorporation in the presence of extracellular 35S-cysteine (p<0.05), significant S-glutathionylation (S-glu) of multiple proteins (p<0.05 for proteins of 20, 25, 33, and 40 kDa, by Western blotting), increased superoxide accumulation (ethidium fluorescent intensity of 48.3±0.8 vs. 67.6±4.0, via flow cytometry, p<0.001) and decreased metabolic activity of several SH-sensitive enzymes (expressed as % activity vs control, all p<0.05), including creatine kinase (18%), aldehyde dehydrogenases (ALDH1:60%, ALDH2: 45%), xanthine dehydrogenase (33%), and glutaredoxin (GRX, 45%). Over-expression of cellular GRX via a pcDNA3-His-GRX expression vector resulted in a 3-fold increase in GRX activity in LLC-PK1 cells. When exposed to NTG under tolerance-inducing conditions, these cells exhibited reduced cellular protein S-glu and absence of NTG tolerance, as indicated by the lack of change in the NTG-cGMP dose response curve vs control (ANOVA p<0.05). GRX mRNA-silenced cells (GRX siRNA sequence: 5′-GCUCCUCAGCCAAUUGCCCUUCAAA-3′, vs control) showed 50% reduction of GRX activity, while the extent of NTG-induced tolerance increased approximately two-fold vs control (ANOVA, p<0.05). NTG tolerance in LLC-PK1 cells can be mimicked by incubation with oxidized glutathione (GSSG greater than 50 microM) in terms of reduced cGMP accumulation, protein S-glu formation, superoxide production, and the ability of vitamin C to attenuate these events. These results indicate that NTG-mediated protein SH-oxidation, including S-glu, may be responsible for initiating cellular nitrate tolerance, and possibly its downstream effects through redox signaling.