Transport Energetics of the Folic Acid Analogue, Methotrexate, in L1210 Leukemia Cells

Abstract

Energy-dependent transport of the folic acid analogue, methotrexate, was studied in L1210 murine leukemia cells. A number of metabolic inhibitors were found to enhance the unidirectional influx and accumulation of methotrexate. This phenomenon was studied in detail with sodium azide. The azide-induced increase in the unidirectional influx of methotrexate was associated with a rise in both the maximum transport velocity and the influx Michaelis constant. The effect of azide on influx was not related to an increase in the passive diffusion of methotrexate. Addition of pyruvate, an energy substrate for those metabolic processes inhibited by azide, was found to depress methotrexate influx in the presence and absence of azide. The unidirectional efflux of methotrexate was slowed by azide. Efflux from cells loaded to high levels of methotrexate in the presence of azide was a two-component process with the major part of intracellular methotrexate exiting at a slow and constant velocity. The data suggested that azide reduces the maximum transport velocity and the Michaelis constant for the unidirectional efflux process. In the absence of azide the electrochemical potential for intracellular methotrexate exceeds that for extracellular methotrexate. The data indicate that azide further increases this electrochemical potential difference. These studies therefore suggest that energy-dependent transport of methotrexate is enhanced in the presence of azide in the L1210 leukemia cell.