Recent studies indicate that a variety of inflammatory disorders are associated with the up-regulation of the inducible form of nitric oxide synthase (iNOS) and the consequential enhanced production of the free radical nitric oxide (NO). Our long-term goal is to better understand how the oxidative and nitrosative chemistry emanating from high output of NO may contribute to the tissue dysfunction and/or injury associated with inflammation. More specifically, it has not yet been established to what extent functional alterations caused by NO-mediated S-nitrosation reactions (the addition of an NO+ group to a thiol residue) might contribute to cytostasis. Preliminary studies in our laboratory demonstrate that the intracellular production of NO via activation of iNOS inhibits cell proliferation, inhibits mitochondrial respiration, induces alteration in glutathione metabolism, and mediates the S-nitrosation of cellular proteins and peptides. The overall goal of this proposal is to determine how reactive nitrogen oxide species derived from NO may contribute to cytostasis and to obtain a better understanding of the intracellular nitrosative chemistry associated with NO. We propose that S-nitrosation reactions contribute to the inhibition of cell respiration and proliferation that is associated with increased NO production.
The specific aims of this project are 1) to establish a causative link between reactive nitrogen species and NO-mediated cytostasis; 2) to examine the mechanisms of cell mediated formation of S-nitrosothiols; 3) to define the cellular pathways of S-nitrosothiol decomposition; 4) to characterize the mechanisms by which reactive nitrogen oxide species inhibit cell respiration and to examine their relationship with cytostasis. To address these specific aims, we will focus on one major biological system, a murine fibroblast cell line that constitutively expresses the human form of the inducible nitric oxide synthase. We will also examine the kinetics and equilibria of intracellular S-nitrosothiol metabolism using newly developed chromatographic, spectrofluorometric and chemiluminescence techniques. These studies may have important implications for cellular proliferation and toxicity, such as observed in macrophage-induced cytostasis associated with host defense mechanisms, in the proliferation of vascular cells during angiogenesis, and in environmental as well as medical pulmonary exposure to NO.
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