| The optimal therapeutic use of organic nitrates is hindered by the lack of thorough understanding of their bioactivation pathways and the development of nitrate tolerance, two phenomena which appear to be highly related to each other. In this thesis work, we seek to provide a better understanding of the various determinants of both processes, using nitroglycerin (NTG) as a model nitrate. To achieve sufficient analytical sensitivity, a liquid chromatographic-mass spectrometric method for NTG and its metabolites was developed. Comparison between LLC-PK1 cells and cultured human vascular cells showed that the latter were not suitable for the in vitro examination of nitrate metabolism and tolerance, due to the lack of expression of mitochondrial aldehyde dehydrogenase (ALDH2) activity and the alpha-subunit of guanylyl cyclase. Using LLC-PK1 cells and animal models, we examined the exclusive validity of the two major hypotheses for nitrate tolerance, namely, the superoxide (O2·-)-oxidative stress hypothesis and the ALDH2 impaired biotransformation hypothesis. We found that both hypotheses were unable to explain our observed results in nitrate tolerance. We observed that O2·- accumulation was not associated with tolerance development, and that angiotensin II and NADPH oxidase were unlikely to be involved in NTG-mediated oxidative stress. Silencing of the mRNA that encoded ALDH2 suppressed its enzyme activity in LLC-PK1 cells, but did not alter NTG metabolism and tolerance. We showed that NTG-induced, --SH oxidation (including S-glutathionylation) of multiple cellular proteins occurred during tolerance development, which was accompanied by loss of the metabolic activity of many thiol-sensitive enzymes and activation of xanthine oxidoreductase to increase O2·- production. Protein S-glutathionylation, particularly of transcription factors, has been shown to regulate signal transduction and gene expression, and thus has the potential to provide a mechanistic pathway for the counter-regulatory events seen with nitrate tolerance, including withdrawal rebound and alteration of vascular gene expression. Oxidized glutathione, which induced S-glutathionylation, produced many of the signal events characteristic of NTG tolerance. Taken together, this work indicated that several existing mechanisms of nitrate tolerance can be interpreted in light of an initiating event of NTG-induced multiple --SH protein oxidation during bioactivation. This mechanism could therefore possibly provide a unifying theory to explain nitrate tolerance. |
