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Oxidative stress and Mrp2 internalization

Shuichi Sekine1, Kousei Ito, Toshiharu Horie

  • 1Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chiba, 260-8675, Japan.

Free Radical Biology & Medicine
|June 21, 2006
PubMed

Insights

Ethacrynic acid (EA) causes liver oxidative stress, leading to the internalization of the bile transporter multidrug resistance-associated protein 2 (Mrp2). This process involves calcium elevation and nitric oxide release, mediated by non-specific protein kinase C activation.

Area of Science:

  • Hepatology
  • Cell Biology
  • Biochemistry

Background:

  • Oxidative stress in the liver can lead to cholestasis.
  • The biliary transporter multidrug resistance-associated protein 2 (Mrp2/Abcc2) is crucial for bile flow.
  • Previous studies showed Mrp2 internalization during ethacrynic acid (EA)-induced oxidative stress, but the mechanism was unclear.

Purpose of the Study:

  • To investigate the signaling pathway and regulatory molecules involved in EA-induced Mrp2 internalization.
  • To elucidate the mechanism of Mrp2 internalization in rat liver.

Main Methods:

  • Isolated rat hepatocyte couplets (IRCHs) were used to study EA effects.
  • The Mrp2 index (canalicular membrane staining) was measured.
  • Involvement of protein kinases (PKC, PKA, PKG) and calcium signaling was assessed using specific inhibitors and EGTA.

Main Results:

  • EA treatment significantly reduced the Mrp2 index in IRCHs.
  • This reduction was prevented by a non-specific PKC inhibitor (Gö6850) and a calcium chelator (EGTA).
  • EA increased intracellular calcium levels and nitric oxide (NO) release, which were blocked by EGTA. Mrp2 internalization was also blocked by EGTA.

Conclusions:

  • EA induces Mrp2 internalization through a sequential pathway: reduced GSH, increased intracellular calcium, NO production, and non-specific PKC activation.
  • Calcium and NO signaling play critical roles in EA-induced Mrp2 internalization.
  • This study clarifies the mechanism underlying Mrp2 transporter dysfunction during oxidative stress.

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