P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide

Pharm Res. 2004 Jul;21(7):1294-302. doi: 10.1023/b:pham.0000033018.97745.0d.

Abstract

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-penicillamine2,5]enkephalin (3H-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 3H-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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • Animals
  • Bile / drug effects
  • Bile / metabolism*
  • Biological Transport
  • Blotting, Western
  • Cells, Cultured
  • Dipeptidyl Peptidase 4 / metabolism
  • Enkephalin, D-Penicillamine (2,5)- / pharmacokinetics*
  • Fluorescent Antibody Technique
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism*
  • Humans
  • Male
  • Membrane Transport Proteins / metabolism
  • Microscopy, Confocal
  • Models, Biological
  • Multidrug Resistance-Associated Proteins / metabolism
  • Organic Anion Transporters / metabolism
  • Rats
  • Rats, Wistar
  • Species Specificity

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Membrane Transport Proteins
  • Multidrug Resistance-Associated Proteins
  • Organic Anion Transporters
  • multidrug resistance-associated protein 2
  • Enkephalin, D-Penicillamine (2,5)-
  • Dipeptidyl Peptidase 4