The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin.
Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1). To test this hypothesis directly, [3H]UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin-Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls. As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2. [3H]UCB uptake was measured in the presence of 60 microM human serum albumin at a free (unbound) concentration of UCB (B(F)) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5'-[beta,gamma-methylene]triphosphate). MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly. [3H]UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 microM MK571 and followed saturative kinetics with K(m)=10+/-3 nM (B(F)) and V(max)=100+/-13 pmol x min(-1) x (mg of protein)(-1). UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1. Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity.
Rigato
Igino
I
Liver Research Center, Bldg. Q, AREA Science Park Basovizza, SS 14 Km 163.5, 34012 Trieste, Italy.
Pascolo
Lorella
L
Fernetti
Cristina
C
Ostrow
J Donald
JD
Tiribelli
Claudio
C
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001663:Bilirubin / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*
D000255:Adenosine Triphosphate; D001663:Bilirubin; D005978:Glutathione; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C421109:multidrug resistance-associated protein 1