Use of Ca2+ Modulation to Evaluate Biliary Excretion in Sandwich-Cultured Rat Hepatocytes

• Published in: The Journal of pharmacology and experimental therapeutics 1999-06, Vol.289 (3), p.1592-1599
• Main Authors: Liu, X, LeCluyse, E L, Brouwer, K R, Lightfoot, R M, Lee, J I, Brouwer, K L
• Format: Article
• Language: English
• Subjects: Animals; Calcium - pharmacology; Calcium - physiology; Cell Membrane Permeability; Cells, Cultured; Intercellular Junctions - physiology; Intercellular Junctions - ultrastructure; Kinetics; Liver - drug effects; Liver - physiology; Liver - ultrastructure; Male; Models, Biological; Rats; Rats, Wistar; Ruthenium Red; Taurocholic Acid - metabolism
• ISSN: 0022-3565; 1521-0103

Previous work in our laboratory has indicated that biliary excretion of a substrate in sandwich-cultured hepatocytes can be quantitated by measurement of substrate accumulation in the presence and absence of extracellular Ca 2+ . The present study was designed to examine the effects of Ca 2+ on taurocholate accumulation and tight junction integrity in cultured hepatocytes. Kinetic modeling was used to characterize taurocholate disposition in the hepatocyte monolayers in the presence and absence of extracellular Ca 2+ . The accumulation of taurocholate in freshly isolated hepatocytes, which lack an intact canalicular network, was the same in the presence and absence of extracellular Ca 2+ . Electron microscopy studies showed that Ca 2+ depletion increased the permeability of the tight junctions to ruthenium red, demonstrating that tight junctions were the major diffusional barrier between the canalicular lumen and the extracellular space. Cell morphology and substrate accumulation studies in the monolayers indicated that Ca 2+ depletion disrupted the tight junctions in 1 to 2 min. The integrity of the disrupted tight junctions was not re-established completely after reincubation in the presence of Ca 2+ for 1 h. The accumulation of taurocholate was described best by a two-compartment model (cytosol and bile) with Michaelis-Menten kinetics for both uptake and biliary excretion. In summary, Ca 2+ depletion does not alter hepatocyte transport properties of taurocholate. Ca 2+ modulation may be a useful approach to study biliary excretion of substrates in sandwich-cultured hepatocytes.