During severe infection, the liver becomes the principle organ of amino acid uptake, an adaptive response which supports acute-phase protein synthesis and confers a survival advantage to the host. Increased hepatic amino acid extraction during sepsis is largely attributable to augmented plasma membrane transport activity. The focus of the proposed research is to examine the role of non- parenchymal liver cells as the source of plasma-borne mediators that trigger increased plasma membrane amino acid uptake in parenchymal cells. We are particularly interested in the amino acid uptake in parenchymal cells. We are particularly interested in the amino acid glutamine because it has been shown to be """"""""conditionally-essential"""""""" in some catabolic states, and transport across the plasma membrane may represent a rate-limiting step in its metabolism. Preliminary data from our laboratory indicate that the ability to enhance hepatic glutamine transport is cytokine-mediated and is essential for survival. Isolated rat hepatocytes will be utilized to examine the effects of individual cytokines, hormones, conditioned media from non-parenchymal cells, and serum from endotoxin- treated animals on amino acid transport. To examine the role of inflammatory cytokines/hormones, isolated rat parenchymal cells will be incubated with IL-1, TNF, IL-6, PGE2, and dexamethasone, alone and in combination, followed by assay of glutamine and alanine transport activity. To elucidate the role of sinusoidal cells in the response, non-parenchymal cells (NPC's: Kupffer cells, endothelial cells) will be isolated and cultured L+ endotoxin or TNF- alpha in the presence and absence of neutralizing antibodies to effective cytokines, and in the presence and absence of a cyclooxygenase inhibitor. Conditioned media from these cells will be used for the culture of parenchymal cells prior to transport analysis. Similar experiments will be conducted with serum and non- parenchymal cells from rats injected with saline (control) or endotoxin. To determine if peripheral inflammatory cell infiltrates contribute to cytokine elaboration during the hepatic septic response, rats will be injected with endotoxin and sacrificed at specific times thereafter. The livers from these animals will be processed for histological examination (core facility), and infiltrate appearance compared with that of enhanced amino acid uptake. If necessary, identification of inflammatory cell infiltrates will be performed by use of antibodies to known surface markers on inflammatory cells. Results from these studies will be important because a better understanding of this adaptive response will lead to improved nutritional/hormonal therapies targeted to benefit the patient.
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