.) Hypotheses are: (a) that the oxidant-induced elevation in cellular GSH content, results from an increase in gamma-glutamyl cycle activity in most cells (or increased uptake of intact GSH in some epithelial cells); (b) that both the content and oxidation state of GSH regulate these processes; and (c) that increased gamma-glutamyl transpeptidase (GGT) activity allows greater use of extracellular GSH for de nova GSH synthesis and thereby enhances antioxidant defense. In vitro models with endothelial cells, epithelial cell lines derived from lungs, and NIH-3T3 cells will be used in the study.
The Specific Aims are to: (1) determine conditions under which GSH precursors or extracellular GSH protect against hyperoxia, menadione or paraquat; (2) determine the mechanisms for the elevation of GSH content by measuring activities of GGT and other gamma-glutamyl cycle enzymes, and the rates of uptake of amino acid precursors of GSH or intact GSH; (3) determine whether the oxidation state of GSH as well as GSH concentration regulates GSH synthesis or uptake; and (4) determine whether increased GGT expression (due to transfection with a GGT cDNA construct, induction, or differentiation) increases resistance of cells to oxidant stress. The objective of this application is to understand the mechanisms whereby increased synthesis or uptake of GSH acts in protection against oxidants. GSH is however, also of crucial importance in the metabolic transformation of numerous pharmacologic, physiologic and environmental agents. The long-term objective of this research is to achieve an understanding of the regulation of GSH synthesis and uptake in GSH-dependent defense against both oxidant and non-oxidant toxins. A rational approach to design of therapeutic assistance to detoxification relies upon such an understanding.
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