Mediation of cellular anion detoxification in leukemic cells by unidirectional efflux pumps

Methotrexate influx in L1210 cells is mediated almost entirely by a single system, whereas efflux occurs via three routes, the influx carrier functioning in reverse and two additional systems which are operationally unidirectional. The two unidirectional routes show considerable similarities in energy-dependence and inhibitor sensitivities but can be separated by their differential inhibition by bromosulfophthalein (BSP), probenecid, and vincristine. The predominant route is inhibited by BSP and vincristine, whereas the other route is sensitive to probenecid. A search for additional anion substrates for the unidirectional efflux systems for methotrexate led to the finding that cholate exits L1210 cells via the same two unidirectional efflux systems as methotrexate. Cholate efflux is carrier-mediated, energy-dependent, and unidirectional, and it can be inhibited by several compounds which inhibit the efflux of methotrexate. Moreover, the same concentration of each compound which produced a half-maximal inhibition of methotrexate efflux also inhibited the efflux of cholate by 50%. Cholate efflux occurred predominantly (85%) via the BSP-sensitive route. The observation that methotrexate and cholate share the same efflux systems in L1210 cells suggest that perhaps other large anions are also accommodated by these systems since methotrexate and cholate differ in net negative charge and have few common structural features. The hypothesis advanced from these studies has been that the function of the unidirectional efflux systems is to extrude various organic anion catabolites which might otherwise become toxic if allowed to accumulate within cells. Possible intracellular anions in this latter category include sulfonated or carboxylated steroids, bilirubin, and products of vitamin D and prostaglandin catabolism. Unidirectional efflux pumps have been identified for other anionic compounds including cyclic AMP and oxidized glutathione, and in both cases similarities were apparent with the efflux of methotrexate and cholate. The most compelling comparison was with prostaglandin A1 which inhibited the efflux of cyclic AMP, methotrexate, and cholate half-maximally at the same low concentration of 0.1 microM. Energy-dependent unidirectional efflux pumps for large neutral or cationic drugs have also been identified in cells with acquired multidrug resistance. The latter efflux activity is thought to be mediated by a membrane glycoprotein (p170) which is also sensitive to several of the various inhibitors (reserpine, verapamil, and quinidine) which reduce the efflux of methotrexate and cholate.