Transport of monoglucuronosyl and bisglucuronosyl bilirubin by recombinanthuman and rat multidrug resistance protein 2

The secretion of bilirubin conjugates from hepatocytes into bile represents a decisive step in the prevention of hyperbilirubinemia. The bilirubin con jugates, monoglucuronosyl bilirubin (MGB) and bisglucuronosyl bilirubin (BG B), were previously suggested to be endogenous substrates for the apical mu ltidrug resistance protein (MRP2), a member of the adenosine triphosphate ( ATP)-binding cassette family of transporters (symbol ABCC2), also termed ca nalicular multispecific organic anion transporter. We have characterized th is ATP-dependent transport using membrane vesicles from human embryonic kid ney (HEK) cells expressing recombinant rat as well as human MRP2, MGB and B GB, H-3-labeled in the glucuronosyl moiety, were synthesized enzymatically with recombinant UDP-glucuronosyltransferase 1A1, and stabilized with ascor bate, Rates for ATP-dependent transport of MGB and BGB (0.5 mu mol/L each) by human MRP2 were 183 and 104 pmol x mg protein(-1) x min(-1), respectivel y. K-m values were 0.7 and 0.9 mu mol/L for human MRP2, and 0.8 and 0.5 mu mol/L for rat MRP2, with MGB and BGB as substrates, respectively. Leukotrie ne C-4 and 17 beta-glucuronosyl estradiol, which are both known high-affini ty substrates for human MRP2, inhibited [H-3]MGB transport with IC50 values of 2.3 and 30 mu mol/L, respectively. Cyclosporin A competitively inhibite d human and rat MRP2-mediated transport of [H-3]MGB, with K-i values of 21 and 10 mu mol/L, respectively, Our results provide direct evidence that rec ombinant MRP2, cloned from rat as well as human liver, mediates the primary -active ATP-dependent transport of the bilirubin conjugates MGB and BGB.