Journal
PHARMACEUTICAL RESEARCH
Volume 21, Issue 7, Pages 1294-1302Publisher
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1023/B:PHAM.0000033018.97745.0d
Keywords
cell culture; biliary excretion; drug transport; drug transport models; hepatocytes; P-glycoprotein
Funding
- NIGMS NIH HHS [R01 GM041935, GM41935] Funding Source: Medline
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Purpose. The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion. These studies examined repolarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes. Methods. Protein localization and expression were determined in SC hepatocytes by confocal microscopy and Western blotting, respectively. Transporter function was evaluated by measuring [D-penicillamine(2,5)] enkephalin (H-3-DPDPE) and 5 (and 6)-carboxy-2 ', 7'-dichlorofluorescein (CDF) biliary excretion in SC hepatocytes. Results. P-gp and the canalicular marker protein dipeptidyl peptidase IV(DPPIV) co-localized by Day 3 and Day 6 in SC rat hepatocytes and SC human hepatocytes, respectively, consistent with canalicular network formation visualized by light microscopy. Co-localization of multidrug resistance associated protein 2 (MRP2) and P-gp in SC human hepatocytes was observed on Day 6 in culture. Expression levels of P-gp increased slightly in both species over days in culture; similar expression was observed for MRP2 in SC human hepatocytes. Oatp1a1 expression in SC rat hepatocytes was maintained over days in culture, whereas Oatp1a4 expression decreased. OATP1B1 expression decreased slightly on Day 3 in SC human hepatocytes. OATP1B3 expression was constant in SC human hepatocytes. In vitro biliary excretion of the opioid peptide H-3-DPDPE correlated with the proper localization of canalicular proteins in both species. Excretion of CDF in SC human hepatocytes confirmed network formation and MRP2 function. Conclusions. These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain.
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