Zinc is an integral component of a large number of gene products including enzymes, transcription factors, and structural proteins. The intracellular free concentration of zinc is maintained at extraordinarily low levels by the actions of various zinc transporters, vesicular storage sites, and metal binding proteins of which metallothionein (MT) is a major contributor. Little is known regarding the mechanisms of zinc homeostasis in endothelial cells. Endothelium has been the locus of early structural and functional changes in a variety of pro-oxidative conditions many of which are affected by the simultaneous production of NO. The applicant hypothesizes that S-nitrosylation of zinc thiolate clusters in MT is a critical component of cellular redox sensitivity linking NO to zinc homeostasis in endothelial cells. The resultant NO-induced changes in labile zinc are hypothesized to inhibit pro-apoptotic pathways but may contribute alternatively to non-apoptotic oxidant-induced endothelial cell injury and necrosis. The applicant proposes to: 1) determine a role for zinc in intracellular signaling of NO in lung endothelial cells and determine the molecular determinants of zinc homeostasis in these cells, 2) identify mechanisms by which Zn affects endothelial cell sensitivity to apoptotic (LPS) or necrotic (tert butylhydroperoxide) stimuli and evaluate the contribution of Zn to NO-induced changes in sensitivity to these stimuli, and 3) determine the role of pulmonary endothelial cell Zn-MT in the resistance of intact mice (wt and MT over expressers) due to conditioning with NO.
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