| Nitric oxide (NO) is a labile and dynamic signaling molecule that regulates a number of essential physiological processes. The endothelial isoform of nitric oxide synthase (eNOS) in the vascular endothelium is best characterized for its role in maintaining vascular tone and blood pressure. The regulation of eNOS activity is a complex interplay between post-translational modifications, substrate/co-factor availability, and protein-protein interactions. Generally, NO signaling is mediated either by direct binding of NO to protein thiols and transition metals or through chemistries involving species that are derived from NO, via secondary reactions. Although the subcellular localization of eNOS can influence enzyme activity, the impact of the site of NO production on both localized cell signaling and global cell physiology remains largely unexplored. We hypothesize that NO will preferentially react with targets proximal to its site of formation to mediate cell signaling and hence, the subcellular localization of eNOS will significantly influence the cell signaling actions of NO. The current study examined the impact of eNOS restricted to the cytosol vs. the outer mitochondrial membrane for signaling by NO. Two general experimental strategies were used, one involving the assessment of reported NO-dependent mitochondrial functions and a second in which I undertook a broad untargeted survey of the effects of NO on metabolism using a comprehensive mass spectrometry-based metabolomics approach. Using the metabolomics strategy, a quantitative survey of global changes in small molecule expression levels led to the identification of significant and unanticipated NO-mediated actions. A complimentary approach for the study of locally-produced NO on metabolism was provided by targeted analysis of select molecules (initially identified in the unbiased approach) and closely related biochemical pathways to which they contribute. In this study, I describe the application of metabolomics as a powerful tool for the discovery and identification of metabolites that are differentially expressed as a result of compartmentalized eNOS and NO signaling. This approach confirmed anticipated changes in citrulline and revealed unanticipated changes in the transsulfuration pathway and the GSH:GSSG ratio that occurred exclusively with cytosolic eNOS, as well as enhanced expression of phosphatidylcholine with active eNOS, irrespective of subcellular domain. Characterization of citrulline metabolism and the metabolic flux through the citrulline-arginine cycle demonstrated a preferential diminishment in citrulline expression with mitochondrial eNOS, suggesting localization-dependent differences in arginine regeneration. |
