We continue to test the hypothesis that exocytosis of lysosomal contents into biliary canaliculi is a major excretory route for hepatocyte lysosomes. We propose to: (1) Relate biliary excretion of lysosomal contents to turnover of lysosomal constituents by studying: a) turnover rates of hepatic lysosomal enzymes relative to their biliary excretion; b) amount of lysosomal enzymes released by isolated hepatocytes relative to their cellular content; c) effects of alterations in hepatic autophagy and heterophagy on biliary excretion of lysosomal enzymes. (2) Study regulation of hepatic lysosomal enzyme activities by measuring activity and content of lysosomal hydrolases by enzymatic assays and radioimmunoassays, respectively. (3) Study hepatic handling of chole-cystokinin (CCK) and renin, proteins that may be processed in hepatocyte lysosomes, by: a) determining hepatic sites of cellular and subcellular metabolism of labeled CCK peptides; b) purifying and labeling rat renal renin and studying hepatic processing of labeled ligand. (4) Characterize the role of hepatocyte lysosomes in biliary metal excretion using animal models of hepatic iron and copper overload by assessing: a) effects of lysosomotropic agents on biliary metal secretion; b) the form of iron and copper in bile; c) metal-induced changes in hepatic lysosomal enzyme content, lipid and protein composition of lysosomal membranes, and appearance of pericanalicular lysosomes; d) the fate of biliary iron in the intestine; and e) the effect on hepatic and biliary metal concentrations of chronic administration of agents which accelerate release of lysosomal contents into bile. (5) Explore the role of hepatocyte lysosomes in lipoprotein processing and in biliary excretion of lipids and apolipoproteins by: a) characterizing molecular forms of apolipoproteins B, A-I and A-II in human bile; b) studying the relationship between biliary lipid secretion and hepatic uptake of LDL; c) assessing binding to liver plasma membranes of labeled LDL and HDL3 after bile acid administration; and d) studying effects of lysosomotropic agents on biliary lipid secretion. We will utilize intact rats and isolated livers, cells, and organelles, and apply novel biochemical and morphologic techniques (radioimmunoassays, immunocytochemistry). Our long-term objectives are to define the physiological and biochemical bases for and the functional significance of the release of lysosomal contents into bile, identify disturbances of this pathway relevant to disease, and develop therapies which accelerate biliary excretion of toxic materials that accumulate in hepatocyte lysosomes.
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