Inhibitory effects of probenecid on the individual transport routes which mediate the influx and efflux of methotrexate in L1210 cells.

L1210 cells contain a single transport system which mediates the influx of methotrexate and at least three routes for drug efflux [G. B. Henderson and E. M. Zevely, J. biol. Chem. 259, 1526 (1984)]; each of these processes is sensitive to probenecid. The influx carrier was inhibited reversibly and completely by probenecid with a Ki of 0.25 mM, while efflux via the same system was relatively unaffected by this compound (50% inhibition above 2.0 mM). The two remaining efflux routes (which do not contribute to methotrexate influx) showed a much higher sensitivity to probenecid. Efflux via these components was reduced half-maximally at probenecid concentrations of 0.08 and 0.22 mM, respectively, and a complete block was achieved with excess amounts (2.0 mM) of the inhibitor. Intracellular levels of ATP, glucose metabolism, and the membrane potential were also reduced by probenecid, indicating that the mechanism for inhibiting methotrexate efflux may involve the ability of probenecid to act as a metabolic inhibitor. Probenecid may have a broad capacity for inhibiting anion transport processes since it also reduced sulfate influx and efflux via the general anion carrier system.