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-

Multidrug resistance protein 1 (MRP1) is an ATPbinding cassette (ABC) transporter that transports a range of hydrophobic xenobiotics, as well as relatively hydrophilic organic anion conjugates. The protein is present at high levels in testicular Leydig and Sertoli cells. Studies with knockout mice suggest that MRP1 may protect germ cells from exposure to some cytotoxic xenobiotics, but potential endobiotic substrates in this organ have not been identified. Previously, we have shown certain D-ring, but not A-ring, estrogen glucuronides can act as competitive inhibitors of MRP1 mediated transport, suggesting that they are potential substrates for the protein. In the case of 17␤-estradiol-17␤-D-glucuronide, this has been confirmed by direct transport studies. The Leydig cell is the major site of estrogen conjugation in the testis. However, the principal products of conjugation are A-ring estrogen sulfates, which are then effluxed from the cell by an unknown transporter. To determine whether MRP1/mrp1 could fulfill this function, we used membrane vesicles from MRP1-transfected HeLa cells to assess this possibility. We found that estradiol and estrone 3-sulfate alone were poor competitors of MRP1-mediated transport of the cysteinyl leukotriene, leukotriene C 4 . However, in the presence of reduced glutathione (GSH), their inhibitory potency was markedly increased. Direct transport studies using [ 3 H]estrone 3-sulfate confirmed that the conjugated estrogen could be efficiently transported (K m ‫؍‬ 0.73 M, V max ‫؍‬ 440 pmol mg ؊1 protein min ؊1 ), but only in the presence of either GSH or the nonreducing alkyl derivative, S-methyl GSH. In contrast to previous studies using vincristine as a substrate, we detected no reciprocal increase in MRP1-mediated GSH transport. These results provide the first example of GSH-stimulated, MRP1-mediated transport of a potential endogenous substrate and expand the range of MRP1 substrates whose transport is stimulated by GSH to include certain hydrophilic conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.