Transport of methotrexate in L1210 cells: Effect of ions on the rate and extent of uptake

Abstract

Transport of methotrexate (MTX) in L1210 cells is highly dependent upon the ionic composition of the external medium. Half-maximal rates of MTX transport ( K t values) vary from 0.9 μ m in cells suspended in potassium-Hepes buffer containing Mg 2+ (Hepes-Mg), to 10 μ m in phosphate-buffered saline (PBS). At saturating levels of substrate, however, transport rates approach the same maximum velocity ( V) regardless of the buffering medium. The increased K t value for MTX in PBS is due to the presence of the competitive inhibitors, phosphate ( K i = 0.87 mM) and Cl − ( K i = 46 mM). Concentration gradients for MTX at the steady state are also much lower (about 20-fold) in PBS than in Hepes-Mg; the components of PBS that reduce this uptake parameter are phosphate, Cl −, Ca 2+, and Na +. Ions that decrease the influx rate or the steady-state level also produce an increase in MTX efflux. Glucose (which increases ATP levels) reduces influx rates and steady-state levels of MTX, and induces efflux in both PBS and Hepes-Mg. Conversely, the combination of azide plus iodoacetate (which reduces ATP levels) stimulates MTX uptake in PBS, but has little effect on MTX transport parameters in Hepes-Mg. The unusually high sensitivity of MTX transport to various anions is consistent with the hypothesis that this system catalyzes the exchange of external MTX for an intracellular anion, and that efflux of the anion down a concentration gradient provides the driving force for active transport of MTX.