It has recently been shown that the radical nitric oxide (NO) is a previously unrecognized key player in many pathophysiological conditions of relevance to surgery, shock, and many other clinically important phenomena. Many (if not most) of these conditions involve cell and tissue injury from reactive oxidative species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical. Numerous studies in diverse disciplines have shown that NO can result in either dramatic protection or enhancement of injury. The overall purpose of the work described in this proposal is to utilize one well-developed and well-defined system, the isolated rat hepatocyte, to uncover the factors which determine whether NO will be protective or damaging in oxidative injury. The major hypothesis in this work is that the outcome (protective vs. damaging) of NO/ROS interactions in cells is determined by (1) which of the many possible chemical/biochemical NO/ROS interactions in the cell predominate under specific conditions and (2) the biological defensive responses mounted by cells when pre-exposed to small nontoxic amounts of these reactive species. The two Specific Aims based on this hypothesis are: 1) Identify which specific chemical/biochemical interactions within cells determine the protective vs. damaging effects of NO on oxidative injury. By utilizing effectors of specific biochemical steps in oxidative injury, identification will be accomplished of those steps which are modulated by NO and thus result in its protective and also damaging actions in isolated hepatocytes. 2) Identify the protective responses induced by pre-exposure to small amounts of NO/ROS which cause resistance to the damaging effects of a subsequent oxidative injury. Pre-exposure of isolated hepatocytes to a small amount of NO or ROS induces resistance to a second otherwise toxic treatment, which involves upregulation of new protein(s) synthesis. By determining the phenotypic and genotypic changes in cells induced by this pre-exposure, the specific protective mechanisms which are upregulated in this defensive response will be identif d.
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