Sepsis is a major cause of morbidity and mortality in intensive care units in the United States. Recent advances in our understanding of this complex syndrome have been based on discoveries that the induction of nitric oxide (NO) synthesis in multiple cell types plays a major role in host response during sepsis. Cellular NO synthesis is absolutely dependent on availability of arginine, which donates the nitrogen atom in NO. Thus, an NO-producing cell must obtain arginine from exogenous sources or synthesize its won supply. We have recently demonstrated that cellular arginine from exogenous sources or synthesize its own supply. We have recently demonstrated that cellular arginine biosynthetic capacity [determined by activity of argininosuccinate synthetase (AS] is induced by the same mediators of septic response--endotoxin and cytokines--that induce nitric oxide synthase (NOS), the enzyme responsible for NO synthesis. Based on these and other results, we hypothesize that endogenous arginine synthesis plays a crucial role in regulating host responses during sepsis. We therefore propose two interrelated specific aims to test this hypothesis.
These aims i nclude both in vitro and in vivo studies to elucidate the regulation and impact of arginine biosynthesis on NO production by two representative cell types which normally have low (macrophage) or high (hepatocyte) arginine synthesis rates:
AIM I. To determine the mechanisms involved in regulation of arginine biosynthesis during sepsis and in response to specific inflammatory stimuli. We will characterize AS expression in four animal models of sepsis/inflammation and in response to specific inflammatory stimuli, and determine which steps in AS expression (transcription, mRNA stability, etc.) are regulated.
AIM II. To determine the dependence of NO production on endogenous arginine synthesis during sepsis and in response to specific inflammatory stimuli. I.e., under what conditions is arginine synthesis limiting for NO production? These studies will include measurements of NO production as a function of varying extracellular amino acid levels, design and testing of a novel AS inhibitor, and independently varying NOS and AS activity in transfected cell lines. The ultimate physiologic test will be to determine the responses to sepsis and inflammatory stimuli in a novel transgenic mouse strain in which extrahepatic AS expression has been """"""""knocked out"""""""". This information will provide valuable insights into the induction and maintenance of the septic response and may provide rationales for new treatment strategies.
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