Interactions of the Human Multidrug Resistance Proteins MRP1 and MRP2 with Organic Anions ´ EVA BAKOS, RAYMOND EVERS, 1 EMESE SINK ´ O, ANDR ´ AS V ´ ARADI, PIET BORST, and BAL ´ AZS SARKADI National Institute of Haematology and Immunology, Research Group of the Hungarian Academy of Sciences, Budapest, Hungary (E.B., E.S., B.S.); Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary (E.B., A.V.); and The Netherlands Cancer Institute, Amsterdam, The Netherlands (R.E., P.B.) Received July 20, 1999; accepted January 4, 2000 This paper is available online at http://www.molpharm.org ABSTRACT The human multidrug resistance protein MRP1 and its ho- molog, MRP2, are both suggested as being involved in cancer drug resistance and the transport of organic anions. We ex- pressed MRP1 and MRP2 in Spodoptera frugiperda ovarian cells and compared their ATP-dependent transport properties and vanadate-sensitive ATPase activities in isolated membrane vesicles. Both MRP1 and MRP2 actively transported leukotri- ene C 4 and N-ethylmaleimide glutathione (NEM-GS), although the relative affinity of MRP2 for these substrates was found to be significantly lower than that of MRP1. Methotrexate was actively transported by both proteins, although more efficiently by MRP2. ATP-dependent NEM-GS transport by MRP1 and MRP2 was variably modulated by organic anions. Probenecid and furosemide inhibited, whereas under certain conditions sulfinpyrazone, penicillin G, and indomethacin greatly stimu- lated, MRP2-mediated NEM-GS uptake. Vanadate-sensitive ATPase activity in isolated membranes containing MRP1 or MRP2 was significantly stimulated by NEM-GS and reduced GS, although these compounds acted only at higher concen- trations in MRP2. ATP hydrolysis by MRP2 was also effectively stimulated by methotrexate. Probenecid, sulfinpyrazone, indo- methacin, furosemide, and penicillin G all significantly in- creased MRP2-ATPase activity, whereas these compounds acted more as ATPase inhibitors on MRP1. These results indi- cate that MRP1 is a more efficient transporter of glutathione conjugates and free glutathione than MRP2, whereas several anions are preferred substrates for MRP2. Our data suggest that MRP2 may be responsible for the active secretion of phar- macologically relevant organic anions, such as diuretics and antibiotics, and indicate different modulation possibilities for MRP1 or MRP2 in drug-resistant tumor cells. The human multidrug resistance proteins 1 and 2 [MRP1 and MRP2 (multispecific organic anion transporter), respec- tively] are homologous members of a subfamily of the ATP- binding cassette transporters, and both may cause multiple drug resistance in malignant tumor cells (Cole et al., 1992; Zaman et al., 1994; Cui et al., 1999). By now, at least six members of this subfamily have been identified, and they seem to play an important role in various secretory and other transport functions, predominantly in epithelial cells (Borst et al., 1997; Kool et al., 1997; Cui et al., 1999). Both MRP1 and MRP2 were shown to perform an ATP-dependent, pri- mary active transport of the glutathione (GS) conjugate leu- kotriene C 4 (LTC 4 ) and of various GS, sulfate, and glucuro- nide conjugates (Jedlitschky et al., 1994, 1996, 1997; Mu ¨ ller et al., 1994). It is most likely that MRP1 and MRP2 can also transport hydrophobic drugs (Cole et al., 1994; Hollo ´ et al., 1996; Evers et al., 1998), although cellular GS seems to be an important modulator in these transport functions (see Za- man et al., 1995; Loe et al., 1996, 1998). The physiological role of these highly promiscuous trans- porters may cover a wide range, varying from the transport of excretory compounds and the elimination of xenobiotics, to the mediation of an inflammatory response. The widely ex- pressed MRP1 has a key function in, for example, LTC 4 - dependent tissue reactions, as well as in controlling trans- port across the blood-brain barrier (Wijnholds et al., 1997; Rao et al., 1999), and in polarized cells, this protein is sorted to the basolateral membranes (see Borst et al., 1997; Deeley and Cole, 1997). MRP2 is predominantly expressed in the canalicular (apical) membranes of hepatocytes and the epi- thelial cells of kidney proximal tubules (Schaub et al., 1997; Evers et al., 1998). This protein was shown to be the most important exporter of conjugated bile salts in the liver This work was supported by research grants from OMFB, OTKA (Grants F23662, D32847, T29921), FKFP, NWO-OTKA, and ETT, Hungary, and the Dutch Cancer Society. B.S. is a recipient of a Howard Hughes International Scholarship. 1 Present address: Georg-Speyer-Haus, Paul Ehrlich Strae 42-44, 60596 Frankfurt a. M, Germany. ABBREVIATIONS: MRP1, human multidrug resistance protein 1; MRP2, human multidrug resistance protein 2; LTC 4 , leukotriene C 4 ; GS, glutathione; GSH, reduced glutathione; MDR1, human multidrug resistance protein (P-glycoprotein); NEM, N-ethylmaleimide; Sf9, Spodoptera frugiperda 9. 0026-895X/00/040760-09$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:760 –768 (2000). 760 at ASPET Journals on June 11, 2017 molpharm.aspetjournals.org Downloaded from