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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036854-03
Application #
3235389
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1986-03-01
Project End
1991-02-28
Budget Start
1988-03-01
Budget End
1989-02-28
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Weinman, S A; Graf, J; Veith, C et al. (1993) Electroneutral uptake and electrogenic secretion of a fluorescent bile salt by rat hepatocyte couplets. Am J Physiol 264:G220-30
Sellinger, M; Weinman, S A; Henderson, R M et al. (1992) Anion channels in rat liver canalicular plasma membranes reconstituted into planar lipid bilayers. Am J Physiol 262:G1027-32
Haddad, P; Beck, J S; Boyer, J L et al. (1991) Role of chloride ions in liver cell volume regulation. Am J Physiol 261:G340-8
Benedetti, A; Strazzabosco, M; Corasanti, J G et al. (1991) Cl(-)-HCO3- exchanger in isolated rat hepatocytes: role in regulation of intracellular pH. Am J Physiol 261:G512-22
Boyer, J L; Meier, P J (1990) Characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles. Methods Enzymol 192:517-33
Boyer, J L; Phillips, J M; Graf, J (1990) Preparation and specific applications of isolated hepatocyte couplets. Methods Enzymol 192:501-16
Meier, P J; Boyer, J L (1990) Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes. Methods Enzymol 192:534-45
Graf, J; Boyer, J L (1990) The use of isolated rat hepatocyte couplets in hepatobiliary physiology. J Hepatol 10:387-94
Weinman, S A; Graf, J; Boyer, J L (1989) Voltage-driven, taurocholate-dependent secretion in isolated hepatocyte couplets. Am J Physiol 256:G826-32
Henderson, R M; Graf, J; Boyer, J L (1989) Inward-rectifying potassium channels in rat hepatocytes. Am J Physiol 256:G1028-35

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