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

doi:10.1053/gast.1997.v113.pm9352844

Gastroenterology

Volume 113, Issue 5, November 1997, Pages 1438-1442

https://doi.org/10.1053/gast.1997.v113.pm9352844 Get rights and content

Abstract

BACKGROUND & AIMS: Circumstantial evidence suggests a regulation of biliary secretion by transporter insertion and retrieval into and from the canalicular membrane. This study was undertaken to provide direct evidence for such a process.

METHODS: Osmosensitivity of the subcellular localization of the mrp2 gene-encoded conjugate export pump (MRP2) was studied by immunofluorescence and confocal laser scanning microscopy of isolated hepatocyte aggregates and in perfused rat liver.

RESULTS: MRP2 was localized largely in membranes of the pseudocanaliculi formed by isolated hepatocyte aggregates during hypo- osmotic exposure, whereas after hyperosmotic exposure MRP2 was also detectable in intracellular vesicles. In perfused liver, the EAG15 antibody specific for rat MRP2 and the ZO-1 antibody specific for tight junctions produced immunostaining of the canalicular membrane. However, the relative amount of MRP2 increased significantly in the pericanalicular region with increasing perfusate osmolarity, as shown by confocal microscopy of intracellular vesicles containing MRP2 (but not ZO-1) and by computed densitometry. The osmodependent distribution of MRP2 between the canalicular membrane and intracellular, pericanalicular vesicles occurred within 30 minutes and was fully reversible.

CONCLUSIONS: The findings provide direct evidence for an osmosensitive dynamic insertion and retrieval of the canalicular MRP2 transporter into and out of the canalicular membrane.

(Gastroenterology 1997 Nov;113(5):1438-42)

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Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~ 30 min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague–Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2 h without changing the hepatic protein expression of MRP2. While the 8 h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2.
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Citation Excerpt :
Hypoosmotic liver cell swelling increases within minutes the transport capacity for bile acids in a microtubule- and MAP kinase-dependent fashion, whereas hyperosmotic shrinkage is cholestatic (15, 16). These osmolarity effects on biliary secretion involve a rapid insertion/retrieval process of transporter molecules, such as Bsep and Mrp2 into/from the canalicular membrane (17, 18). Changes in liver cell hydration not only control biliary excretion but also hepatic metabolism, gene expression, and transport across the plasma membrane through activation of osmo-sensing and osmo-signaling pathways (for reviews, see Refs. 19, 20).
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Oxidative stress is a feature of cholestatic syndrome and induces multidrug resistance-associated protein 2 (Mrp2) internalization from the canalicular membrane surface. We have previously shown that the activation of a novel protein kinase C (nPKC) by oxidative stress regulates Mrp2 internalization. The internalized Mrp2 was recycled to the canalicular surface in a protein kinase A (PKA)-dependent manner after intracellular glutathione (GSH) levels were replenished. However, the putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. In this study, we investigated the effect of changing the intrahepatic redox status on the C-terminal phosphorylation status of radixin (p-radixin), which links Mrp2 to F-actin, and the interaction of p-radixin with Mrp2 in rat hepatocytes. We detected a significant decrease in the amount of p-radixin that co-immunoprecipitated with Mrp2 after tertiary-butylhydroperoxide (t-BHP) treatment. After treatment with GSH-ethylester (GSH-EE), the phosphorylation level became the same as that of the control. A PKC and protein phosphatase (PP)-1/2A inhibitor, but not a PP-2A selective inhibitor, prevented the t-BHP-induced decrease of p-radixin and subsequent canalicular Mrp2 localization. In contrast, a PKA inhibitor affected the recovery process facilitated by GSH-EE treatment. In conclusion, the interaction of p-radixin with Mrp2 was decreased by the activation of PKC and PP-1 under oxidative stress conditions which subsequently led to Mrp2 internalization, whereas the interaction of p-radixin and Mrp2 was increased by the activation of PKA during recovery from oxidative stress.

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