Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell.

Transport of methotrexate (4amino-lo-methylpteroylglutamic acid), a structural analogue of folic acid, was studied in L1210 mouse leukemia cells grown in tissue culture. Intracellular methotrexate was present in two states. (a) There was an essentially nonexchangeable intracellular fraction probably bound to the enzyme dihydrofolate reductase. (b) The remaining intracellular methotrexate was rapidly exchangeable and likely present in the cell water in an osmotically active state. In addition, there was a small rapidly exchanging component of methotrexate associated with the cells which had the characteristics of a loose adsorption on or near to the cell membrane. Methotrexate was shown to be negligibly metabolized by this cell system. Because of the presence of tight intracellular binding sites for methotrexate, prior to saturation of these sites, free intracellular methotrexate does not appear and consequently efflux is negligible. This permits the accurate no easurement of unidirectional uptake velocities. The major Fortion of the unidirectional influx process demonstrated MichaelisMenten kinetics with a high degree of structural specificity, competitive inhibition by folic and folinic acids, and marked temperature dependence. Addition of these competing substances in excess to cells at the steady state with extracellular methotrexate leads to countertransport of intracellular methotrexate. Electrochemical potential gradients for free intracellular methotrexate were demonstrated at low levels of extracellular methotrexate and intracellular methotrexate concentrations at equilibrium exhibited Michaelis-Menten kinetics. Efilux of intracellular methotrexate was not first order and appeared to be partially saturated. The Michaelis constant and maximum velocity for the unidirectional efflux process were estimated, and when related to these parameters for the unidirectional influx, were compatible with an accumulating system. These studies indicate that methotrexate transport in the L1210 cell is carrier-mediated and uphill. Methotrexate exhibits several qualities which make it a valuable “model substrate” for the study of the transport characteristics of folic acid and its naturally occurring derivatives. The implication of these findings with respect to the development of more effective folic acid antagonists is considered.