Functional implications from the effects of 1-chloro-2,4-dinitrobenzene and ethacrynic acid on efflux routes for methotrexate and cholate in L1210 cells.

Abstract

1-Chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were examined for the ability to inhibit unidirectional efflux routes in L1210 cells that extrude both methotrexate and cholate (system I) and methotrexate alone (system II). These electrophiles were selected for study because of their known ability to undergo rapid intracellular conversion to glutathione conjugates. CDNB produced typical inhibitor kinetics and was a moderate inhibitor of both system I (IC50 = 4.8 microM) and system II (IC50 = 7.5 microM) with methotrexate as the substrate. However, a complex response was observed when cholate was employed as an alternative substrate for system I. Cholate efflux was stimulated initially at low levels of CDNB, but then slowed to a net inhibition as CDNB concentrations exceeded 10 microM. The latter characteristics for CDNB were not observed with ethacrynic acid, which produced a comparable inhibition of efflux system I regardless of the substrate employed (IC50 = 4.6 microM). Efflux measurements in an L1210/C7 variant which lacks system I confirmed that CDNB stimulates the activity of a substantial and unique efflux activity for cholate (system III). The inhibition of system I and II by CDNB and ethacrynic acid was not reversed by a wash step but required inhibitor removal and subsequent incubation at 37 degrees C. This slow reversal was attributed to a time-dependent clearance of inhibitory glutathione conjugates. A correlation between efflux systems for anions and anionic glutathione conjugates was demonstrated further by the ability of prostaglandin A1 and indomethacin, two potent inhibitors of methotrexate and cholate efflux, to inhibit the efflux of 2,4-dinitrophenyl-S-glutathione. These results support the hypothesis that efflux systems for methotrexate and cholate in L1210 cells are part of a family of efflux pumps which function in vivo to extrude various anions and anionic glutathione conjugates.