Abstract
The multidrug resistance-associated protein Mrp2 is expressed in liver, kidney, and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells. We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat Mrp2. To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules. Both 17β-estradiol-17-β-d-glucuronide ([3H]E217βG, 50 nm) and [3H]leukotriene C4 (3 nm) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion. Uptake of [3H]E217βG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 μm). Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of [3H]E217βG transport. The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, α-naphthyl-β-d-glucuronide, andp-nitrophenyl-β-d-glucuronide did not inhibit transport. In conclusion, we show direct ATP-dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity.
Footnotes
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Send reprint requests to: Dr. F. G. M. Russel, University of Nijmegen, Department of Pharmacology 233, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail:f.russel{at}farm.kun.nl
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This work was supported in part by the Netherlands Organization for Scientific Research through Grants 805–05.041 (J.B.K.) and 900.522.132 (M.A. van K.). P.M.T.D. is an investigator of the Royal Netherlands Academy of Arts and Sciences.
- Abbreviations:
- MRP2
- multidrug resistance-associated protein 2
- cMOAT
- canalicular multispecific organic anion transporter
- TR−
- transport-deficient rat
- EHBR
- Eisai hyperbilirubinemic rat
- MRP1
- multidrug resistance-associated protein 1
- ABC
- ATP-binding cassette
- CFTR
- cystic fibrosis transmembrane conductance regulator
- LTC4
- leukotriene C4
- E217βG
- 17β-estradiol-17-β-d-glucuronide
- FL
- fluorescein
- MTX
- methotrexate
- FL-MTX
- fluorescein methotrexate
- CsA
- cyclosporin A
- MK571
- 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethyl-amino-3-oxopropyl)-thio}-methyl]thio)propanoic acid
- Received January 2, 1998.
- Accepted March 10, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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