Multidrug Resistance Protein (MRP)-mediated Transport of Leukotriene C4 and Chemotherapeutic Agents in Membrane Vesicles

Abstract

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 C[(4)] (LTC[(4)]) 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[IMG]_21119_tex2html_wrap118.gif"> values for LTC[(4)] transport by membrane vesicles from T14 cells were 529 {+/-} 176 pmol mg[IMG]_21119_tex2html_wrap126.gif"> min[IMG]_21119_tex2html_wrap126.gif"> and 105 {+/-} 31 nM, respectively. At 50 nM LTC[(4)], the K[IMG]_21119_tex2html_wrap118.gif"> (ATP) was 70 {micro}M. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC[(4)] 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 [[^3]H]LTC[(4)] and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC[(4)] 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 [[^3]H]LTC[(4)] was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC[(4)] 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 [[^3]H]LTC[(4)] transport (K[IMG]_21119_tex2html_wrap154.gif">[IMG]_21119_tex2html_wrap156.gif"> 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 [[^3]H]LTC[(4)] transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC[(4)] transport but inhibition by these compounds was enhanced by GSH. Uptake of [[^3]H]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 LTC[(4)] transport and prevents photolabeling of MRP by LTC[(4)], 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.