CHARACTERIZATION OF FOLATE TRANSPORT MEDIATED BY A LOW PH ROUTE IN MOUSE L1210 LEUKEMIA-CELLS WITH DEFECTIVE REDUCED FOLATE CARRIER FUNCTION

Folate influx at low pH was characterized in MTX(r)A cells, an L1210 m ouse leukemia cell line with a functional defect in the reduced folate carrier. Folic acid influx in MTX(r)A cells was negligible at pH 7.5, increased 13-fold as the pH was decreased to 6.0, and was indistingui shable from that in L1210 cells. In contrast, while methotrexate (MTX) influx in MTX(r)A cells at pH 6.0 was 15-fold higher than at pH 7.5, in L1210 cells it was decreased by half. Influx of MTX, folic acid, 5- methyltetrahydrofolate and 5-formyltetrahydrofolate in MTX(r)A cells w as increased at pH < 7.0, but their pH optima and profile differed sub stantially. Influx of MTX and 5-methyltetrahydrofolate at pH 6.0 showe d saturability, with a K-r of 2.65 and 0.56 mu M, and a V-max of 0.45 and 0.083 nmol/g dry wt/min, respectively. MTX influx mediated by the low pH transporter was insensitive to the anionic composition of the t ransport buffer and affected minimally (similar to 20%) by Na+ substit ution. The anion transport inhibitors sulfobromophthalein, diisothiocy anatostilbene disulfonic acid, and acetamidoisothiocyanatostilbene dis ulfonic acid were not effective inhibitors of the low pH route. MTX tr ansport at low pH did not increase in MTX(r)A-R16 cells, an MTX(r)A de rivative with 10-fold overexpression of the reduced folate carrier (RF C) due to transfection with RFC1 cDNA. Inhibition of reduced folate ca rrier activity with acetamidoisothiocyanatostilbene disulfonic acid re sulted in identical MTX influx in L1210, MTX(r)A, and MTX(r)A-R16 cell s at pH 5.5. Finally, low PH-mediated MTX influx was reduced by energy inhibitors and partially inhibited by ionophores (nigericin > monensi n >> valinomycin). The data indicate that L1210 and MTX(r)A cells expr ess similar activities of a low pH folate transporter that has propert ies distinct from, and independent of, the reduced folate carrier. (C) 1998 Elsevier Science Inc.