Bile secretion is a major hepatic function, frequently impaired in diseases of the liver resulting in the syndrome of cholestasis and occasionally resulting in progressive liver injury and death. Since 1974, this grant has focused on the development of new techniques to understand the basic mechanisms of this secretory process. I. Utilizing isolated hepatocyte couplets (a novel primary secretory unit that transports solutes and secretes bile in short term cultures), and subconfluent hepatocyte monolayers, we will examine the functional role of previously identified ion transports systems (Na+/H+exchange, HC03/Cl-exchange Na+/HC03 symport) and ion channels (K+, CL-), in the regulation of cell pH and cell volume and the relationship of these transport events to bile secretion (bile acid independent secretion). Agents that stimulate (DBcAMP) and inhibit (phorbol esters) this secretory process will be examined for an effect on these events. These studies will be facilitated by fluorescent techniques for measurement of intracellular pH and Ca++, and intracanalicular pH, and confocal imaging equipment for intracellular and canalicular resolution. DIC optical systems with image enhancement equipment will permit measurements of canalicular volume to be made to assess secretory rates in the isolated cells. II. The transcytotic vesicle pathway, and its mechanism of regulation by agents such as DBcAMP and phorbol esters will be assessed in isolated perfused rat livers using fluid phase markers (HRP, dextran). Techniques will also be explored for developing an in-vitro fusion assay between intracellular vesicles and canalicular membranes as a model for examining both stimulation and inhibition of this pathway. III. Finally, techniques will be developed for the isolation of bile duct epithelial cells and establishment of monolayers in order to characterize the membrane transport systems used for maintaining pHi, cell volume and secretory and absorptive functions, using techniques developed for the study of isolated hepatocytes.
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