Oxidative stress and Mrp2 internalization

Free Radic Biol Med. 2006 Jun 15;40(12):2166-74. doi: 10.1016/j.freeradbiomed.2006.02.015. Epub 2006 Mar 13.


Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

MeSH terms

  • ATP-Binding Cassette Transporters / analysis
  • ATP-Binding Cassette Transporters / metabolism*
  • Animals
  • Bile Canaliculi / chemistry
  • Calcium / metabolism
  • Cell Nucleus / chemistry
  • Cells, Cultured
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Egtazic Acid / pharmacology
  • Enzyme Activation
  • Ethacrynic Acid / toxicity
  • Glutathione / metabolism
  • Hepatocytes / chemistry
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism*
  • In Vitro Techniques
  • Indoles / pharmacology
  • Isoenzymes / analysis
  • Isoenzymes / metabolism
  • Liver / chemistry
  • Liver / drug effects
  • Liver / metabolism*
  • Male
  • Maleimides / pharmacology
  • Nitric Oxide / metabolism
  • Oxidative Stress*
  • Protein Kinase C / analysis
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Protein Transport
  • Rats
  • Rats, Sprague-Dawley


  • ATP-Binding Cassette Transporters
  • Abcc2 protein, rat
  • Indoles
  • Isoenzymes
  • Maleimides
  • Protein Kinase Inhibitors
  • Nitric Oxide
  • Egtazic Acid
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • Glutathione
  • bisindolylmaleimide I
  • Ethacrynic Acid
  • Calcium