These studies are aimed at understanding the physiologic significance of eicosanoids made by hepatocytes and other liver cells, especially as related to liver cell injury. Prostaglandins and other eicosanoids are involved in control of liver regeneration, metabolism, and resistance to toxic injury. Liver Kupffer and endothelial cells are extremely active in eicosanoid synthesis, but hepatocytes can synthesize a significant fraction of liver prostanoids. Hepatocyte eicosanoids may play an entirely different role than non-parenchymal cell eicosanoids. Endotoxin causes increased eicosanoid synthesis by liver and other organs, and increases sensitivity to hepatotoxins. Hepatocyte eicosanoid synthesis could play an important role in modulating response to toxic or hypoxic liver injury. The function of eicosanoid synthesis by liver cells, especially hepatocytes, will be investigated by quantifying their synthetic capacity, and by examining the control of eicosanoid synthesis by endotoxin and/or cytokines. Experiments will examine synthesis by cultured liver cells, including hepatoma and biliary epithelial cells, and isolated perfused rat livers. The spectrum of eicosanoids made by cultured hepatocytes will be identified, and methods for the study of hepatocyte eicosanoid synthesis in cell culture will be optimized, in part using moderate hypoxia to decrease degradation of eicosanoids by hepatocytes. Hepatocyte eicosanoid synthesis will also be examined by perfusion of a layer of hepatocytes one or a few cells thick to minimize degradation of eicosanoids by the cell layer. Experiments will be done to determine whether hepatocytes preferentially secrete or metabolize eicsoanoids on the basal-lateral/sinusoidal or apical/canlicular membranes. Endotoxin will be studied as a stimulus for eicosanoid synthesis in whole rats. Modulation of cyclooxygenase, lipoxygenase and other proteins involved in eicosanoid synthesis will be examined by enzyme assay, immunoassay, and measurement of mRNA. Regulation of these enzymes by endotoxin and cytokines will be examined in cell culture, including hepatoma cell lines. Hypoxic injury in the isolated perfused rat liver from endotoxin-treated rats and controls will also be studied as a model for modulation of liver injury by endogenous eicosanoids.
