The goal of this proposal is to determine how disruption of thiol metabolism by reactive nitrogen species (RNS) derived from nitric oxide (NO.) contributes to activation of oxidation/reduction (redox) sensitive signal transduction pathways leading to the induction of cytotoxic as well as cytoprotective responses. This goal was chosen on the basis of data gathered in the first funding period and data from the literature showing that NO. altered thiol status, redox sensitive kinase activation and the activation of redox sensitive transcription factors (AP-1 and NF-kappaB) leading to the induction of both cytoprotective as well as cytotoxic responses. The proposed experiments will determine how RNS derived from NO.: 1) alter intracellular soluble thiol pools (i.e., glutathione, cysteine, gamma-glutamylcysteine) as well as thiol metabolizing enzymes (i.e., gamma-glutamylcysteine synthetase) in fibroblasts and endothelial cells; 2) alter activity or levels of thiol containing proteins (ie., thioredoxin, thioredoxin reductase) known to transmit redox signals from soluble thiols to transcription factors (ie., AP-1 and NF-kappaB); 3) contribute to the activation of redox sensitive kinases (ie., mitogen activated protein kinases) thought to be involved with AP-1 and NF-kappaB activation; 4) effect the activation of redox sensitive transcription factors (ie., AP-1 and NF-kappaB) and genes thought to be regulated by AP- 1 and NF-kappaB (ie., gamma-glutamylcysteine synthetase). Finally, the experiments will determine the relative contribution of nitric oxide- induced alterations in thiol metabolism, signal transduction and transcription factor activation to biological responses seen in endothelial cells, HAl hamster fibroblasts, and nitric oxide resistant variants derived from HAl. A rigorous interdisciplinary approach using cell biology, biochemistry, molecular biology, and analytical chemistry will be used to pursue these Aims. Pharmacological agents (ie., N- acetylcysteine, and inhibitors of kinases, protease, and thioredoxin reductase) as well as molecular manipulations (ie., antisense, dominant negative, dominant positive transfections) will be used to probe causal relationships between NO-induced alterations in thiols and signal transduction pathways, and biological outcomes seen following exposure to RNS. The long term goal is identification of basic mechanisms by which RNS after biological outcomes during oxidative stress.
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