Production of nitric oxide (NO.) and reactive nitrogen species (RNS) derived from NO. have been shown in vivo and appear to be both beneficial and deleterious to the cell. Recent data suggest that NO. and RNS contribute to injury in a wide variety of biologically relevant situations. During physiologically relevant exposures to toxic levels of RNS, the cells' ability to mount an adaptive stress response could determine its ability to resist lethal injury. Preliminary data indicate that NO. is capable of inducing this stress response. The objective of this proposal is to rigorously characterize the NO.-induced stress response in Chinese hamster fibroblast HA1 cells, then to determine whether or not this stress response results in resistance to RNS and/or cross resistance to oxidants. Also, preliminary data and published reports have shown that glutathione, (GSH), plays a major role in protecting cells against killing caused by exposure to lethal doses of NO.. Therefore, modulation of GSH metabolism may provide an important component of the NO.-induced stress response, and the current proposal will test this idea. The magnitude of the NO.-induced stress response will be assessed via clonogenic cell survival. Oxidized and reduced GSH levels and analytes leading to synthesis and breakdown of GSH will be quantified in cells at various times during the development of resistance to NO. using HPLC methodology. GSH metabolizing enzymes will be evaluated using activity assays and molecular techniques. These studies will then be performed in the presence and absence of inhibitors of GSH synthesis and metabolizing enzymes, and protein synthesis inhibitors to determine the causal involvement of GSH metabolism and new protein synthesis in the NO.-induced stress response.
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