Liver damage is part of the multiple organ failure (MOF) syndrome which frequently follows successful resuscitation of traumatized/septic surgical patients. We have shown that Kupffer cells (KC) have profound effects on hepatocyte (HC) metabolism in vitro. Most significantly, we showed that a mixture of IFNg and LPS plus KC cytokines (IL-1, TNF) act synergistically in vitro on HC to upregulate a unique inducible enzyme, nitric oxide (N=O) synthase, which converts arginine to N=O plus citrulline. In turn, N=O inhibits HC protein synthesis via a post-translational mechanism, upregulates guanylate cyclase, and impairs mitochondrial function. HC N=O synthase is also induced in vivo in inflammatory states and enzyme inhibition under these conditions produces hepatic necrosis. Thus, the importance of N=O synthase to hepatic dysfunction in sepsis cannot be overlooked. The goal of this proposal is to explore the factors in vitro and in vivo which regulate HC N=O synthase activity. We hypothesize that one or more of the cytokines are inductive cytokines which control N=O synthase gene expression, while others are adjuvant cytokines which control pre- and post-transcriptional events.
In AIM I, therefore, we will determine: a) the substrate and cofactor requirements of the enzyme; b) which cytokine(s) acts to express the HC gene for the enzyme; and c) which cytokine(s) regulate cofactor availability, alter cytokine receptor expression, and control second messenger systems. The possibility that endogenous competitive inhibitors, glucocorticoids, eicosanoids, and other cytokines downregulate N=O synthase activity will also be explored in vitro and in experimental models of sepsis in vivo. The experiments are simple. Purified HC will be exposed in vitro to putative regulators of N=O synthase activity. A comparison of enzyme activity in cells, cytosols, and purified protein with mRNA expression will help distinguish between those factors which regulate gene expression and various pre- or post-transcriptional events. Selected in vivo models to test the physiologic significance of the in vitro studies will be set up. In order to fully understand the cytokine regulation of N=O synthase mRNA expression, we need to purify the enzyme and clone the cDNA for HC N=O synthase (AIM II). Thus, by combining molecular techniques with the conventional techniques of cell biology and experimental in vivo modeling, we can gain a more complete understanding of both the mechanisms and the physiologic importance of N=O synthase regulation.
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