Our OVERALL OBJECTIVES continue to be to understand how liver cells, process exogenous and endogenous substances and to identify the physiologic consequences of these processing activities. Our GENERAL HYPOTHESIS is that cellular processing (internalization/transport/structural modification/externalization) involves a coordinated series of anatomically and temporally discrete events that are integral to and selectively coupled with the absorptive, digestive, and secretory activities of hepatic epithelia--namely, bile duct epithelial cells and hepatocytes. We will employ current and complementary biochemical and quantitative morphological techniques to define regulatory and mechanistic aspects of cell processing by focusing on: (i) the role of exocytosis and endocytosis in regulating secretion by intrahepatic bile duct epithelial cells (IBDEC); and (ii) the role of lysosomal proteolysis and vesicular transport in regulating digestion and secretion by hepatocytes. The proposal has two specific aims. First, we will CHARACTERIZE THE PLASMA-MEMBRANE DEPENDENT COMPONENTS OF CELLULAR PROCESSING (i.e., endocytosis/exocytosis) IN IBDEC by: (a) testing the HYPOTHESIS that hormone-responsive ductular bile flow involves the selective recycling by coupled exocytic/endocytic insertion and retrieval of specific, transport-protein containing microdomains of the apical plasma membrane of IBDEC; (b) testing the HYPOTHESIS that secretin interacts with a receptor on IBDEC activating second messengers that stimulate exocytosis by acidic organelles; and (c) developing and applying additional novel technology (monoclonal antibodies, organelle isolation methods, polar primary cultures) for IBDEC. Second, we will DETERMINE THE MECHANISMS FOR THE CATALYTIC AND MOTILE COMPONENTS OF CELLULAR PROCESSING (lysosomal digestion/vesicular transport) IN HEPATOCYTES by: (a) examining lysosomal autophagy using an in vitro, cell-tree system of highly-purified, subfractions of hepatocyte lysosomes; and (b) characterizing the dynamics of lysosomal heterophage using both isolated hepatocytes and a liver- derived, in vitro cell-free system containing purified endosomes, lysosomes and cytoskeletal elements. The proposed studies are feasible now because we have at our disposal new experimental models (isolated/short-term cultured IBDEC and in vitro cell-free systems), novel biochemical methods (fluorescence assays for both exocytosis and heterophagy), and advanced cell biologic techniques (fluorescence spectroscopy, immunocytochemical and freeze-fracture electron microscopy, and digitized video and confocal microscopy). Our LONG-TERM GOALS are to define the biochemical basis for and the physiologic consequences of cellular processing in hepatic epithelia, to identify disturbances of the individual components of cellular processing relevant to disease, and ultimately to develop therapies for treatment of processing disorders of hepatic epithelia.
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