Characterization of Folate Transport Mediated by a Low pH Route in Mouse L1210 Leukemia Cells with Defective Reduced Folate Carrier Function

Abstract

Folate influx at low pH was characterized in MTX rA cells, an L1210 mouse leukemia cell line with a functional defect in the reduced folate carrier. Folic acid influx in MTX rA cells was negligible at pH 7.5, increased 13-fold as the pH was decreased to 6.0, and was indistinguishable from that in L1210 cells. In contrast, while methotrexate (MTX) influx in MTX rA 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 rA cells was increased at pH < 7.0, but their pH optima and profile differed substantially. Influx of MTX and 5-methyltetrahydrofolate at pH 6.0 showed saturability, with a K t of 2.65 and 0.56 μM, 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 transport buffer and affected minimally (∼20%) by Na + substitution. The anion transport inhibitors sulfobromophthalein, diisothiocyanatostilbene disulfonic acid, and acetamidoisothiocyanatostilbene disulfonic acid were not effective inhibitors of the low pH route. MTX transport at low pH did not increase in MTX rA-R16 cells, an MTX rA derivative with 10-fold overexpression of the reduced folate carrier (RFC) due to transfection with RFC1 cDNA. Inhibition of reduced folate carrier activity with acetamidoisothiocyanatostilbene disulfonic acid resulted in identical MTX influx in L1210, MTX rA, and MTX rA-R16 cells at pH 5.5. Finally, low pH-mediated MTX influx was reduced by energy inhibitors and partially inhibited by ionophores (nigericin > monensin ≫ valinomycin). The data indicate that L1210 and MTX rA cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.