ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 349, No. 1, January 1, pp. 113–121, 1998 Article No. BB970445 Uncouplers of Mitochondrial Oxidative Phosphorylation Are Not Substrates of the Erythrocyte Glutathione- S -Conjugate Pump 1 Adam Sokal 2 and Grzegorz Bartosz Department of Molecular Biophysics, University of L Q o ´dz ´, Banacha 12/16, 90-237 L Q o ´dz ´, Poland Received June 16, 1997, and in revised form September 24, 1997 nitrophenyl-S-glutathione (DNP-SG) 3 transport into Uncouplers of mitochondrial oxidative phosphoryla- inside-out vesicles of erythrocyte membranes (2). A va- tion, dinitrophenol (DNP) and carbonyl cyanide p- riety of glucuronides and sulfates were found to inhibit trifluoromethoxyphenylhydrazone (FCCP), were the high-K m DNP-SG transporter (3). The ability of found to stimulate Mg 2/ -ATPase activity of human DNP-SG to stimulate the Mg 2/ -ATPase activity of erythrocyte membranes in a manner competitive with erythrocyte membranes is interpreted as a proof that respect to 2,4-dinitrophenyl-S-glutathione (DNP-SG) glutathione-S conjugates are transported by a specific which suggested that these compounds may also be active transport system (4). Recently the multidrug re- substrates of the glutathione-S-conjugate pump. We sistance-associated protein (MRP1) was demonstrated confirm that the stimulation of erythrocyte membrane to transport glutathione-S conjugates (5, 6). This 190- ATPase activity by DNP and by another uncoupler, kDa membrane protein was originally described as a carbonyl cyanide m-chlorophenylhydrazone (CCCP), membrane protein different from glycoprotein P which is competitive with respect to DNP-SG. However, we mediates the multidrug resistance of tumor cells. It found no evidence for active transport of DNP and was shown that MRP1-overexpressing cell lines are re- CCCP out of erythrocytes and demonstrate that they sistant against anthracyclines, Vinca alkaloids, and inhibit the low-affinity component of DNP-SG trans- etoposide (7, 8). The structural similarity between port noncompetitively while stimulating the high-af- MRP1 and glycoprotein P suggests that MRP and gly- finity DNP-SG transport (mediated by multidrug resis- coprotein P represent a similar mechanism of drug re- tance-associated protein, MRP1). Implications of these sistance and that MRP1 acts as an ATP-dependent findings may indicate the electrogenic nature of transporter of these drugs. It was suggested that gluta- MRP1-mediated transport of glutathione-S conjugates thione-S-conjugate transporter can actively export glu- and stimulation of aminophospholipid translocase tathione – platinum complexes (9). All these observa- (flippase) rather than the glutathione-S-conjugate tions show that MRP1 has a broad substrate specificity pump by the uncouplers.  1998 Academic Press Key Words: erythrocyte; glutathione; glutathione S- encompassing amphipathic cations, anions, and metal conjugates; multidrug resistance-associated protein; complexes of glutathione. Additionally, Winter et al. membrane; transport. showed that oxidative phosphorylation uncouplers DNP and FCCP are able to stimulate the ATPase activ- ity of erythrocyte membranes and that these agents compete with S-(2,4-dinitrophenyl)glutathione for acti- Transport of glutathione-S conjugates out the cells vation of erythrocyte membranes ATPase. They con- is an important part of cellular detoxification mecha- cluded that uncouplers of mitochondrial oxidative nisms (1). It has been shown that there are two high- phosphorylation could be potential substrates for the and low-affinity components of ATP-dependent 2,4-di- 3 Abbreviations used: DNP-SG, 2,4-dinitrophenyl-S-glutathione; 1 This work was supported by the Grant 6P04A 05309 of the Polish Committee for Scientific Research. MRP1, multidrug resistance-associated protein; DNP, dinitrophenol; FCCP, p-trifluoromethoxyphenylhydrazone; CCCP, carbonyl cyanide 2 To whom correspondence should be addressed. Fax (/48) 42 354473. E-mail: asokal@biol.uni.lodz.pl or gbartosz@biol.uni.lodz.pl. m-chlorophenylhydrazone. 113 0003-9861/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.