Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells.
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 17beta-estradiol-17-beta-D-glucuronide ([3H]E217betaG, 50 nM) and [3H]leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion. Uptake of [3H]E217betaG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 microM). Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of [3H]E217betaG transport. The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-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.
van Aubel
R A
RA
Department of Pharmacology, University of Nijmegen, 6500 HB Nijmegen, The Netherlands.
van Kuijck
M A
MA
Koenderink
J B
JB
Deen
P M
PM
van Os
C H
CH
Russel
F G
FG
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D027321:Anion Transport Proteins; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D018432:Drug Resistance, Multiple; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate