PH-DEPENDENCE OF METHOTREXATE TRANSPORT BY THE REDUCED FOLATE CARRIERAND THE FOLATE RECEPTOR IN L1210 LEUKEMIA-CELLS - FURTHER EVIDENCE FOR A 3RD ROUTE MEDIATED AT LOW PH

F2-MTX(r)A is an L1210 leukemia cell line with a functional defect in the reduced folate carrier and high level expression of folate recepto r beta. The pH-dependence of methotrexate (MTX) influx by folate recep tor beta in F2-MTX(r)A cells was characterized and compared with that of the reduced folate carrier in parental L1210 cells. MTX influx by f olate receptor beta had a pH optimum of 6.5, whereas influx mediated b y the reduced folate carrier showed a pH optimum of 7.5. Increased fol ate receptor beta-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, ap proximately 24% of MTX influx in F2-MTX(r)A cells proceeded by another mechanism. This transport route became active at pH <7.5, operated op timally at pH 6.0 to 6.5, and, unlike folate receptor beta-mediated MT X influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a K-t, of 5.3 mu M and a V-max of 1.53 nmol/g dry wt/min, was energy dep endent, was inhibited by sulfobromophthalein with a K-t of 148 mu M, a nd had similar relative affinities for folic acid, leucovorin, and 5-m ethyltetrahydrofolate. Influx of 5-methyltetrahydrofolate was also med iated by this route. The data provide further confirmatory evidence fo r an MTX influx route in F2-MTX(r)A cells, optimal at low pH and disti nct from the reduced folate carrier or the folate receptor. Copyright (C) 1996 Elsevier Science Inc.