The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S-conjugates. In the present study, we have examined the transport of leukotriene C4 (LTC4) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V(max) and K(m) values for LTC4 transport by membrane vesicles from T14 cells were 529 +/- 176 pmol mg(-1) min(-1) and 105 +/- 31 nM, respectively. At 50 nM LTC4, the K(m) (ATP) was 70 micron. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC4 transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC4 and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC4 transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC4 was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC4 transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC4 transport (K(i(app)) 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC4 transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC4 transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC4 transport and prevents photolabeling of MRP by LTC4, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-dependent transport of vincristine and that transport is GSH-dependent.

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Multidrug Resistance Protein 1 (MRP1) 1 is a member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that has been shown to confer resistance to a variety of natural product type drugs (1-6). The drug resistance phenotype conferred by MRP1 is similar to that resulting from overexpression of P-glycoprotein (P-gp) (reviewed in Refs. 7-9) and is typically associated with an ATP-dependent decrease in drug accumulation and an increase in drug efflux (4, 6). Although both ABC proteins can function as energy-dependent efflux pumps for a range of natural product type drugs, there is very limited primary structure similarity between them, and phylogenetic analyses suggest that they evolved from different ancestral proteins. There is also considerable evidence that the mechanisms by which MRP1 and P-gp transport drugs are different (reviewed in Ref. 8).In addition to its ability to confer multidrug resistance, MRP1, unlike P-gp, has been shown by in vitro studies using inside-out membrane vesicles to transport a structurally diverse array of organic, anionic conjugates (reviewed in Ref. 9). These include GSH-, glucuronide-, and sulfate-conjugated aliphatic, prostanoid, and heterocyclic compounds. The two highest affinity substrates identified to date are the proinflammatory cysteinyl leukotriene C 4 (LTC 4 ) (10 -12) and the GSH-

In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

Overexpression of the human multidrug-resistance protein (MRP) causes a form of multidrug resistance similar to that conferred by P-glycoprotein, although the two proteins are only distantly related. In contrast to P-glycoprotein, human MRP has also been shown to be a primary active transporter of a structurally diverse range of organic anionic conjugates, some of which may be physiological substrates. At present, the mechanism by which MRP transports these compounds and mediates multidrug resistance is not understood. With the objective of developing an animal model for studies on the normal functions of MRP and its ability to confer multidrug resistance in vivo, we recently cloned the murine ortholog of MRP (mrp). To assess the degree of functional conservation between mrp and MRP, we directly compared the drug cross-resistance profiles they confer when transfected into human embryonic kidney cells, as well as their ability to actively transport leukotriene C4, 17beta-Estradiol 17beta-(D-glucuronide), and vincristine; mrp and MRP conferred similar drug resistance profiles, with the exception that only MRP conferred resistance to the anthracyclines tested. Consistent with these findings, accumulation of [3H]vincristine and [3H]VP-16 was decreased, and efflux of [3H]vincristine was increased in both murine and human MRP-transfected cell populations, whereas only human MRP-transfected cells displayed decreased accumulation and increased efflux of [3H]daunorubicin. Membrane vesicles derived from both transfected cell populations transported leukotriene C4 in an ATP-dependent manner with comparable efficiency, although the efficiency of 17beta-estradiol 17beta-(D-glucuronide) transport was somewhat higher with MRP transfectants. ATP-dependent transport of vincristine was also observed with vesicles from mrp and MRP transfectants but only in the presence of glutathione. These studies reveal intrinsic differences between the murine and human MRP orthologs with respect to their ability to confer resistance to a major class of chemotherapeutic drugs.