Electrophysiologic studies of bile secretion have been limited because of inaccessibility of bile canaliculi. However, isolated rat hepatocyte preparations contain small clumps of cells and cell couplets that retain their polarity and secrete bile into a closed space. Our studies indicate that micropipettes can be introduced into canaliculi of isolated liver cells permitting a full definition of electrophysiologic events in these secretory units. This proposal examines the electrical properties of these secretory units, and defines both electrical and chemical gradients across sinusoidal and canalicular membranes, membranes resistances, specific ion conductances, and ion fluxes. These studies provide basic information about the driving forces involved in maintenance of intracellular composition and in the secretion of bile constituents. Specific studies include: I. An analysis of basic electrophysiologic properties including A) intracellular ion activities and transepithelial ion gradients B) determination of conductances of sinusoidal and canalicular membranes, paracellular pathway and intercellular communications. C) analysis of permselectivity of sinusoidal membranes and tight junctions using ion substitution experiments. D) Study of activity of Na+/K+ pump by K+-readmission and ouabain inhibition, E) Studies of the distribution of ion channels by patch clamp techniques. II. Analysis of secondary active transport systems by: A) determining effects of taurocholate acid and alanine transport, B) assessing antiport systems, C) Assessment of dependence of intracellular pH on coupled transport systems using carboxyfluorescein as a pH sensitive fluorescent probe. III. Assessment of regulatory responses and effects of pathologic (cholestatic) conditions including pH and volume regulation, and effects of hormonal and cholestatic agents. These studies provide for the first time, an electrophysiologic approach to define the role of ion transport mechanisms in bile secretion and other vital functions of liver cells.
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