Use of Ca2+ modulation to evaluate biliary excretion in sandwich-cultured rat hepatocytes

Previous work in our laboratory has indicated that biliary excretion of a s ubstrate in sandwich-cultured hepatocytes can be quantitated by measurement of substrate accumulation in the presence and absence of extracellular Ca2 +. The present study was designed to examine the effects of Ca2+ on tauroch olate accumulation and tight junction integrity in cultured hepatocytes. Ki netic modeling was used to characterize taurocholate disposition in the hep atocyte monolayers in the presence and absence of extracellular Ca2+. The a ccumulation of taurocholate in freshly isolated hepatocytes, which lack an intact canalicular network, was the same in the presence and absence of ext racellular Ca2+. Electron microscopy studies showed that Ca2+ depletion inc reased the permeability of the tight junctions to ruthenium red, demonstrat ing that tight junctions were the major diffusional barrier between the can alicular lumen and the extracellular space. Cell morphology and substrate a ccumulation studies in the monolayers indicated that Ca2+ depletion disrupt ed the tight junctions in 1 to 2 min. The integrity of the disrupted tight junctions was not re-established completely after reincubation in the prese nce of Ca2+ 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, Ca2+ depletion does not alter hepatocyte transport properties of taurocholate. Ca2+ modulation may be a useful approach to study biliary excretion of substrates in sandwich-c ultured hepatocytes.