Glutathione stimulates sulfated estrogen transport by multidrug resistanceprotein 1

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) tran sporter that transports a range of hydrophobic xenobiotics, as well as rela tively 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 xe nobiotics, but potential endobiotic substrates in this organ have not been identified. Previously, we have shown certain D-ring, but not A-ring, estro gen glucuronides can act as competitive inhibitors of MRP1 mediated transpo rt, suggesting that they are potential substrates for the protein. In the c ase of 17 beta -estradiol-17 beta -D-glucuronide, this has been confirmed b y direct transport studies. The Leydig cell is the major site of estrogen c onjugation in the testis. However, the principal products of conjugation ar e A-ring estrogen sulfates, which are then effluxed from the cell by an unk nown transporter. To determine whether MRP1/mrp1 could fulfill this functio n, we used membrane vesicles from MRP1-transfected HeLa cells to assess thi s possibility. We found that estradiol and estrone 3-sulfate alone were poo r competitors of MRP1-mediated transport of the cysteinyl leukotriene, leuk otriene C-4. However, in the presence of reduced glutathione (GSH), their i nhibitory potency was markedly increased. Direct transport studies using [H -3]estrone 3-sulfate confirmed that the conjugated estrogen could be effici ently transported (K-m = 0.73 muM, V-max = 440 pmol mg(-1) protein min(-1)) , but only in the presence of either GSH or the nonreducing alkyl derivativ e, S-methyl GSH. In contrast to previous studies using vincristine as a sub strate, we detected no reciprocal increase in MRP1-mediated GSH transport. These results provide the first example of GSH-stimulated, MRP1-mediated tr ansport of a potential endogenous substrate and expand the range of MRP1 su bstrates whose transport is stimulated by GSH to include certain hydrophili c conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.