Abstract
The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 ± 2.1 nmol/cm to 18.1 ± 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.
Footnotes
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This study was supported in part by a grant-in-aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan; and by the Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment), MEXT, Japan.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.024836.
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ABBREVIATIONS: ABC transporter, ATP-binding cassette transporter; P-gp, P-glycoprotein; Mdr1, multidrug resistance protein 1; Abcb1, ATP-binding cassette transporter family B1; Bcrp/Abcg2, breast cancer resistant protein/ATP-binding cassette transporter family G2; Mrp2/Abcc2, multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2; BBM, brush-border membrane; ERM, ezrin/radixin/moesin; PKC, protein kinase C; nPKC, novel protein kinase C; cPKC, conventional protein kinase C; Thx, thymeleatoxin; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; MCB, monochlorobimane; p-ERM, phosphorylated ezrin/radixin/moesin; SD, Sprague-Dawley; EHBR, Eisai hyperbilirubinemic rat; mKRBB, modified Krebs-Ringer bicarbonate buffer; GS-B, glutathione S-bimane; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; TTBS, Tris-buffered saline with 0.05% Tween 20; PBS, phosphate-buffered saline; p-ezrin, phosphorylated ezrin; TCA, trichloroacetic acid; G-PBS, PBS containing 30 mM glycine.
- Accepted March 26, 2009.
- Received September 26, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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