Original Contribution
Oxidative stress and Mrp2 internalization

https://doi.org/10.1016/j.freeradbiomed.2006.02.015Get rights and content

Abstract

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 Ca2+ chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca2+-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 μM). Moreover, an increase in the intracellular Ca2+ 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, Ca2+ elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

Introduction

The multidrug resistance-associated protein 2 (Mrp2) is localized on the canalicular membrane of hepatocytes and contributes to bile flow by extensively excreting glutathione (GSH) and a variety of organic anions into bile [1]. Defective expression of MRP2 underlies the jaundice in the human Dubin-Johnson syndrome [1]. In addition to the inherited deficiency of MRP2 expression in these patients, the transporter expression is regulated on a short- and long-term basis in normal subjects, with important implications for the pathogenesis of cholestatic syndromes [1], [2]. Short-term regulation includes rapid retrieval of the transporter from the canalicular membrane of hepatocytes and its translocation into the cytosol in putative vesicles under the influence of hyperosmolarity [3], endotoxin [3], phalloidin [4], bile acids [5], [6], and oxidative stress [7], [8], [9]. On the other hand, long-term regulation includes mrp2 gene expression, which is affected by lipopolysaccharide [3], bile duct ligation [10], aniso-osmolarity [11], dexamethasone [11], or a variety of drugs [12]. As far as the putative regulatory molecules involved in the membrane surface expression of Mrp2 are concerned, several cytosolic proteins including Radixin and PDZ-K1 have been reported [13], [14], [15], [16]. Importantly, impaired steady-state canalicular surface expression of Mrp2 and jaundice have been observed in Radixin knockout mice [14], and so Radixin is now considered as the primary molecule anchoring Mrp2 to the filamentous actin (F-actin). In addition to the static expression of Mrp2 on the canalicular surface, its dynamic insertion and internalization processes are of great importance because the steady-state expression level is directly dependent on these turnover rates.

We have recently reported that Mrp2 is rapidly internalized in rat liver perfused with ethacrynic acid (EA), a highly electrophilic loop diuretic [8]. EA is conjugated with GSH and excreted into bile via Mrp2 and so an excess of EA induces acute oxidative stress by such a futile cycle [8]. As both the GSH itself and the resulting glutathione conjugates, EA-SG, are substrates of Mrp2 [8], [17], rapid down regulation of Mrp2 under GSH-depleted conditions seems a favorable feedback mechanism for hepatocytes to retain their intracellular GSH. Although it is not known whether the down regulation of Mrp2 actually contributes to cyto-protection, constitutive overexpression of human MRP2 in MDCKII cells accelerates 4-hydroxynonenal-induced GSH depletion and necrosis compared with control MDCKII cells [7]. Moreover, a well-regulated molecular mechanism rather than nonspecific cellular damage might be involved in the internalization pathway [8], because the internalization of Mrp2 induced by EA preceded the nonspecific breakdown of tight junction formation and canalicular structure.

Here, we propose the hypothesis that the presence of a specific intracellular signaling pathway produced by EA-induced oxidative stress finally leads to Mrp2 internalization. We used isolated rat hepatocyte couplets (IRHCs) and perfused rat livers to examine this hypothesis in detail. Our results showed that depletion of intracellular GSH is the trigger of this signaling pathway and novel protein kinase C (nPKC) isoforms are translocated to the membranous fraction. Elevation of intracellular Ca2+ and production of nitric oxide (NO) also play important roles in the signaling pathway of nPKC activation. Sequential propagation of these steps finally leads to specific internalization of Mrp2 from the canalicular surface.

Section snippets

Chemicals

EA, Griess reagent, nitric reductase, and rabbit anti-protein kinase Cα, δ, and ε antibodies were obtained from Sigma-Aldrich Chemical (St. Louis, MO). Collagenase S-1, 2-mercaptoethanol, and o-phthalaldehyde were from Wako Pure Chemical Industries (Osaka, Japan). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine/2HCl (H7) was obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). PKG 1a inhibitor and PKA inhibitor were from Merck (Darmstadt, Germany). Rabbit anti-Mrp2 antiserum

Effect of EA on the localization of Mrp2 in IRHCs

We have previously demonstrated that Mrp2 is internalized in the liver perfused with EA and that this is possibly related to the intracellular GSH content [8]. To investigate the molecular mechanism of the internalization, an in vitro assay system with qualitative and quantitative characteristics similar to in situ liver conditions is required. IRHCs is an established experimental system especially useful for the study of canalicular ATP-binding cassette (ABC) transporters including Mrp2 and

Discussion

The importance of the intracellular GSH content in regulating Mrp2 internalization was suggested from in situ rat liver perfusion studies using EA [8], tertiary-butyl hydroperoxide (t-BHP) and chlorodinitrobenzene (CDNB) [9]. The signaling process has now been investigated in detail using IRHCs. Severe GSH depletion induced by t-BHP and CDNB (lower than 10% of control) caused Mrp2 internalization in rat liver perfusion experiments as reported previously [9]. In our IRHCs system using EA as a

References (48)

  • K. Yamamoto et al.

    Toxicity of ethacrynic acid in isolated rat hepatocytes

    Toxicol. In Vitro

    (2002)
  • R. Kubitz et al.

    Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells

    Hepatology

    (2001)
  • J. Quick et al.

    The structure and biological activities of the widely used protein kinase inhibitor, H7, differ depending on the commercial source

    Biochem. Biophys. Res. Commun.

    (1992)
  • R. Kubitz et al.

    Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver

    J. Biol. Chem.

    (2004)
  • V. Stone et al.

    Effect of oxidative stress and disruption of Ca2+ homeostasis on hepatocyte canalicular function in vitro

    Biochem. Pharmacol.

    (1994)
  • J.F. Dufour et al.

    Nitric oxide blocks bile canalicular contraction by inhibiting inositol trisphosphate-dependent calcium mobilization

    Gastroenterology

    (1995)
  • T. Takeuchi et al.

    Nonadrenergic, noncholinergic relaxation mediated by cnitric oxide with concomitant change in Ca2+ level in rectal circular muscle of rats

    Eur. J. Pharmacol.

    (1998)
  • R. Kubitz et al.

    Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane

    Gastroenterology

    (1997)
  • M. Schmitt et al.

    Regulation of the dynamic localization of the rat Bsep gene-encoded bile salt export pump by anisoosmolarity

    Hepatology

    (2001)
  • R. Kubitz et al.

    Trafficking of the bile salt export pump from the Golgi to the canalicular membrane is regulated by the p38 MAP kinase

    Gastroenterology

    (2004)
  • M.G. Roma et al.

    Control by signaling modulators of the sorting of canalicular transporters in rat hepatocyte couplets: role of the cytoskeleton

    Hepatology

    (2000)
  • R. Prekeris et al.

    Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin

    J. Biol. Chem.

    (1998)
  • G.D. Kruh et al.

    MRP subfamily transporters and resistance to anticancer agents

    J. Bioenerg. Biomembr.

    (2001)
  • D. Rost et al.

    Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis

    Hepatology

    (1999)
  • Cited by (53)

    • Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver

      2018, Biochimica et Biophysica Acta - Molecular Basis of Disease
      Citation Excerpt :

      In line with this, a phosphorylation site for PKC, at the C-terminal of MRP2 (Ser1542), has been reported [37]. Interestingly, different PKC isoforms have been shown by our group and others to be involved in the endocytic internalization of MRP2 in different models of cholestasis, including those induced by E17G [7], oxidative stress [38], and taurolithocholate [39]. In addition, our group has also provided evidence for the participation of p38MAPK in the endocytosis of canalicular transporters in E17G-induced cholestasis [9].

    View all citing articles on Scopus
    View full text