Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protei n (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investig ated the possibility that the glutathione conjugate of 13-oxooctadecadienoi c acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps . The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidatio n product of linoleic. acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent, In cultured HT-29 cell s as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,g amma -methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhib ition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both con jugates exhibits saturation with an apparent K-m of 325.5 muM and a V-max o f 0.0669 nmol/mg protein per min for 13-OXO-SG and a K-m of 169 muM and a V -max of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibitio n is observed when both conjugates are present simultaneously which is cons istent with the involvement of common pumps. The data in this report demons trate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid. (C) 2001 Elsevier Sc ience B.V. All rights reserved.