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 substrate 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 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 Ca2+. The accumulation of taurocholate in freshly isolated hepatocytes, which lack an intact canalicular network, was the same in the presence and absence of extracellular Ca2+. Electron microscopy studies showed that Ca2+ 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 Ca2+ 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 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-cultured hepatocytes.
Liu
X
X
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, USA.
LeCluyse
E L
EL
Brouwer
K R
KR
Lightfoot
R M
RM
Lee
J I
JI
Brouwer
K L
KL
D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D007365:Intercellular Junctions / Q000502:physiology / Q000648:ultrastructure; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008954:Models, Biological; D051381:Rats; D017208:Rats, Wistar; D012430:Ruthenium Red; D013656:Taurocholic Acid / Q000378:metabolism*
D012430:Ruthenium Red; D013656:Taurocholic Acid; D002118:Calcium