Nitric oxide (NO) and its reaction products are key players in the physiology and pathophysiology of inflammatory settings such as sepsis and shock. Since originally funded in 1989, research supported by this grant has led to the following advances: the initial characterization of NO, production by liver cells, the molecular identification of iNOS expression by hepatocytes, the initial proof that humans express iNOS, and the first cloning of the human iNOS cDNA and gene. This work established the foundation for studies characterizing the regulation of human iNOS expression in hepatocytes at the transcriptional, post-translational, and substrate levels. The consequences of the expression of inducible NO. synthase (iNOS, NOS-2) can be either protective or damaging to the liver. More recently, we have delineated two distinct hepatoprotective actions of NO.: the stimulation of cyclic guanosine monophosphate (cGMP) and the inhibition of caspases by S-nitrosation. In contrast, iNOS/NO, promotes hepatocyte death under conditions of severe redox stress, such as hemorrhagic shock or ischemia/reperfusion. Redox stress activates an unknown molecular switch that transforms NO, which is hepatoprotective under resting conditions, into an agent that induces hepatocyte death. We hypothesize that the magnitude of the redox stress is a major determinant for the effects of NO, on cell survival by controlling the chemical fate of NO. We propose to test this hypothesis in two interrelated aims: I) to determine how cellular redox status regulates the chemical fate and function of NO in hepatocytes, and II) to determine how cyclic nucleotides (cGMP, cAMP) prevent hepatocyte apoptosis. The hypothesis to be tested under Aim I is that different redox reaction products of NO are produced under settings of redox stress vs. non-stress conditions, and these reaction products of NO are either themselves toxic or reduce the anti-apoptotic efficacy of NO.
Aim I will be pursued through in vitro work in hepatocytes exposed to either normoxic or hypoxic oxidant stress, and examining the reaction products of NO, that are formed. In vivo work in a model of hepatic ischemia/reperfusion will serve to confirm selected aspects of this work. The hypothesis to be tested under Aim II is that the protective actions of cyclic nucleotides in hepatocytes are mediated through the regulation of the pro-apoptotic molecule FADD via protective pathways that involve cyclic nucleotides and protein kinase A.
Aim II will be pursued through delineation of the level at which FADD is modulated during apoptosis by cyclic nucleotides, and through the use of pharmacological inhibitors of relevant pathways. These studies will comprehensively elucidate how the redox state in hepatocytes determines the chemical fate and function of NOx as well as the downstream effects of NOx and cyclic nucleotides on the homeostatic mechanisms that control hepatocyte survival and death.
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