FUNCTIONAL IMPLICATIONS FROM THE EFFECTS OF 1-CHLORO-2,4-DINITROBENZENE AND ETHACRYNIC-ACID ON EFFLUX ROUTES FOR METHOTREXATE AND CHOLATE IN L1210 CELLS

1-Chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were examined f or 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 becaus e of their known ability to undergo rapid intracellular conversion to glutathione conjugates. CDNB produced typical inhibitor kinetics and w as a moderate inhibitor of both system I (IC50 = 4.8 mu m) and system II (IC50 = 7.5 mu m) with methotrexate as the substrate. However, a co mplex response was observed when cholate was employed as an alternativ e substrate for system I. Cholate efflux was stimulated initially at l ow levels of CDNB, but then slowed to a net inhibition as CDNB concent rations exceeded 10 mu m 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 mu m). Efflux measurements in an L1210/C7 variant which lacks system I c onfirmed 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 in hibitory glutathione conjugates. A correlation between efflux systems for anions and anionic glutathione conjugates was demonstrated further by the ability of prostaglandin A, and indomethacin, two potent inhib itors of methotrexate and cholate efflux, to inhibit the efflux of 2,4 -dinitrophenyl-S-glutathione. These results support the hypothesis tha t 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.