Bile secretion is a major hepatic function, frequently impaired in diseases of the liver resulting in cholestasis, and occasionally resulting in progressive liver injury and death. Previous studies from this laboratory in the past 11 years relating to mechanisms of bile formation and cholestasis have led to the development of a testable model of bile secretion and two new techniques, a highly purified isolated canalicular membrane preparation and an isolated hepatocyte couplet system that now permit direct access to the excretory membrane of the hepatocyte and the canalicular lumen. These two techniques will be utilized in the present study to: 1) Define the driving forces for inorganic and organic solutes across the liver cell membrane domains utilizing fluorescent dyes and radioisotopes in membrane vesicle systems, and examining the effects of choleretic and cholestatic agents on these mechanisms. 2) Examine the effect of bile anions/(taurocholate) and cations (chlorpromazine) on membrane fluidity in separate halfs of the membrane bilayer utilizing fluoroprobes and Arrhenius plots of canalicular membrane enzyme activities. 3) Identify and characterize specific canalicular membrane proteins by affinity chromatography and preparation of specific antisera. 4) Characterize the secretory properties of the isolated hepatocyte couplet system utilizing histochemical and cytochemical assays of canalicular enzymes, morphologic assessment of tight junction permeability, and video image enhancement techniques to measure rates of secretion in this system. Specific ion requirements for bile secretion and cell volume regulation in this model will also be examined. 5) Marine elasmobranchs will be used for comparative studies of the role of organic anion transport proteins and specific ion requirements for bile secretion and cell volume regulation. Together, these different approaches, all of which complement one another, should lead to further identification of the pumps, carriers and channels on sinusoidal and canalicular membranes that determine bile acid dependent and independent secretion, and regulation of hepatocye cell volume. A further understanding of mechanisms of cholestasis is dependent on this information.
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