RESUMO
BACKGROUND: Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney. METHODS: Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1. RESULTS: Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2. CONCLUSIONS: PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/fisiologia , Proteínas de Membrana Transportadoras , Ácido p-Aminoipúrico/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Células HeLa , Humanos , Cinética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Ocratoxinas/metabolismo , Ocratoxinas/farmacologia , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ácido p-Aminoipúrico/antagonistas & inibidoresRESUMO
Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates. We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter). In membrane vesicles from MRP1-transfected HeLa cells mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin (each at 0.5 microM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively. Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates. In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively. ATP-dependent transport of the glutathione S-conjugate -3H-leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 microM. The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/farmacologia , Bilirrubina/análogos & derivados , Fígado/metabolismo , Animais , Bilirrubina/metabolismo , Bilirrubina/farmacologia , Transporte Biológico , Proteínas de Transporte/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Cinética , Leucotrieno C4/análogos & derivados , Leucotrieno C4/metabolismo , Fígado/química , Proteínas de Membrana Transportadoras , Ratos , Ratos Wistar , Especificidade por Substrato , Transfecção/genética , Células Tumorais CultivadasRESUMO
The secretion of bilirubin conjugates from hepatocytes into bile represents a decisive step in the prevention of hyperbilirubinemia. The bilirubin conjugates, monoglucuronosyl bilirubin (MGB) and bisglucuronosyl bilirubin (BGB), were previously suggested to be endogenous substrates for the apical multidrug resistance protein (MRP2), a member of the adenosine triphosphate (ATP)-binding cassette family of transporters (symbol ABCC2), also termed canalicular multispecific organic anion transporter. We have characterized this ATP-dependent transport using membrane vesicles from human embryonic kidney (HEK) cells expressing recombinant rat as well as human MRP2. MGB and BGB, (3)H-labeled in the glucuronosyl moiety, were synthesized enzymatically with recombinant UDP-glucuronosyltransferase 1A1, and stabilized with ascorbate. Rates for ATP-dependent transport of MGB and BGB (0.5 micromol/L each) by human MRP2 were 183 and 104 pmol x mg protein(-1) x min(-1), respectively. K(m) values were 0.7 and 0.9 micromol/L for human MRP2, and 0.8 and 0.5 micromol/L for rat MRP2, with MGB and BGB as substrates, respectively. Leukotriene C(4) and 17beta-glucuronosyl estradiol, which are both known high-affinity substrates for human MRP2, inhibited [(3)H]MGB transport with IC(50) values of 2.3 and 30 micromol/L, respectively. Cyclosporin A competitively inhibited human and rat MRP2-mediated transport of [(3)H]MGB, with K(i) values of 21 and 10 micromol/L, respectively. Our results provide direct evidence that recombinant MRP2, cloned from rat as well as human liver, mediates the primary-active ATP-dependent transport of the bilirubin conjugates MGB and BGB.