Effects of 2,4-Dinitrophenol and Other Metabolic Inhibitors on the Bidirectional Carrier Fluxes, Net Transport, and Intracellular Binding of Methotrexate in Ehrlich Ascites Tumor Cells

Abstract 2,4-Dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, has been frequently used to evaluate the effects of energy depletion on methotrexate (MTX) transport. The results from these studies, however, have shown a multiplicity of effects that suggest a more complicated interaction with the MTX transport system than adenosine 5′-triphosphate depletion alone. Accordingly, studies were undertaken to compare the effects of DNP with a variety of other metabolic inhibitors on influx, efflux, net uptake, and intracellular binding of MTX in Ehrlich ascites tumor cells. Low concentrations of DNP (0.1 mm) inhibited efflux and increased the intracellular steady-state concentration of MTX, both of which were totally reversed by glucose. These alterations were similar to those caused by inhibitors of the electron transport system (azide, rotenone, antimycin A, cyanide, and oligomycin) and anaerobic glycolysis (2-deoxyglucose) and are compatible with inhibition of an energy-dependent exit pump. As the concentration of DNP was increased, influx was competitively inhibited with a Ki of 336 µm. Inhibition was instantaneous and was reversed by removal of DNP but not by addition of glucose. The inhibition of MTX influx by DNP was different from the effects of other metabolic inhibitors which consistantly stimulated influx, an effect totally reversed by glucose. Likewise, as DNP concentrations were increased, inhibition of efflux was enhanced but was only partially reversed by glucose, which suggests that at high concentrations of DNP inhibition was due to both energy depletion and an additional mechanism not associated with energy metabolism. DNP, as well as all other metabolic inhibitors, increased the apparent nonexchangeable fraction of MTX; evidently, this is due to energy depletion, since this effect was totally reversed by glucose. The additional nonexchangeable MTX remained unchanged over at least 2 hr of incubation and was not diminished by repeated changes of the extracellular fluid; this suggests that it was tightly bound. Dicumarol, another uncoupler of oxidative phosphorylation, produced effects similar to those of DNP; however, arsenate had little effect on MTX transport, which suggests that these alterations were not due to uncoupling alone. The results indicate that although most metabolic inhibitors affect MTX transport by inhibition of energy metabolism, DNP affects the system in at least two ways: (a) by energy depletion similar to that of other metabolic inhibitors; and (b) by an apparent interaction with the carrier at the cell membrane which inhibits bidirectional fluxes. The results may resolve some of the variation in the literature on the effects of DNP on MTX transport by showing that observations are markedly influenced by (a) the concentration of the inhibitor, (b) whether measurements are taken during influx, net transport, or the steady state, (c) whether total intracellular or freely exchangeable MTX is determined, and (d) whether or not glucose or other energy-producing substrates are present.