A unique interaction between the folate analog, methotrexate (4-amino-4-deoxy-10-methylpteroylglutamic acid), and the naturally occurring folates in L1210 leukemia and Ehrlich ascites tumor cells provides a useful model for the study of heteroexchange diffusion. The presence of intracellular binding sites with a high affinity for methotrexate but a low affinity for folic acid and its tetrahydrofolate derivatives permit the measurement of true unidirectional influx rates for methotrexate and assure that the trans-stimulation of methotrexate uptake by the intracellular presence of the other folates is due solely to a primary augmentation of this carrier influx mechanism. Further, since free methotrexate does not appear prior to saturation of the binding sites, the reaction between the folates and carrier at the inner cell membrane is undisturbed by methotrexate released from carrier as the complex enters the cell during heteroexchange, facilitating quantitation of the kinetic alterations which occur for methotrexate influx during trans-stimulation. Trans-stimulation of methotrexate influx was observed in cells preloaded with 5-formyl-, or 5-methyl-tetrahydrofolate, or folic acid. The latter was observed even when intracellular reduction of folic acid to its tetrahydrofolate derivatives was inhibited. However, initial uptake rates for methotrexate were unaltered in cells preloaded with nonlabeled methotrexate. Methotrexate influx conformed to Michaelis-Menten kinetics in L1210 cells preloaded to a single intracellular 5-formyltetrahydrofolate level and when compared to control cells, manifested an increase in the influx vmax without a significant change in the influx Kt. The initial uptake rate for 5-methyltetrahydrofolate was increased in cells preloaded with either 5-formyltetrahydrofolate or folic acid. These studies further support a carrier transport mechanism for methotrexate and the naturally occurring folates in L1210 leukemia cells and extend these findings to the Ehrlich ascites tumor cell as well.
ASJC Scopus subject areas
- Cell Biology