Characterization of folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective reduced folate carrier function

Esteban E. Sierra, I. David Goldman

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Abstract

Folate influx at low pH was characterized in MTX(r)rA cells, an L1210 mouse 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 indistinguishable 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 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(r)A-R16 cells, an MTX(r)A 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(r)A, and MTX(r)A-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(r)A cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.

Original languageEnglish (US)
Pages (from-to)1505-1512
Number of pages8
JournalBiochemical Pharmacology
Volume55
Issue number9
DOIs
StatePublished - May 1 1998

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Reduced Folate Carrier Protein
Leukemia L1210
Folic Acid
Methotrexate
Folic Acid Transporters
Valinomycin
Monensin
Leucovorin
Cells
Cell Line
Defects

Keywords

  • Folate
  • Methotrexate
  • pH
  • Reduced folate carrier
  • Transport

ASJC Scopus subject areas

  • Pharmacology

Cite this

@article{55c3178d5c714b62b344f37e9c8660c1,
title = "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(r)rA cells, an L1210 mouse 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 indistinguishable 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 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(r)A-R16 cells, an MTX(r)A 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(r)A, and MTX(r)A-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(r)A cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.",
keywords = "Folate, Methotrexate, pH, Reduced folate carrier, Transport",
author = "Sierra, {Esteban E.} and Goldman, {I. David}",
year = "1998",
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TY - JOUR

T1 - Characterization of folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective reduced folate carrier function

AU - Sierra, Esteban E.

AU - Goldman, I. David

PY - 1998/5/1

Y1 - 1998/5/1

N2 - Folate influx at low pH was characterized in MTX(r)rA cells, an L1210 mouse 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 indistinguishable 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 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(r)A-R16 cells, an MTX(r)A 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(r)A, and MTX(r)A-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(r)A cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.

AB - Folate influx at low pH was characterized in MTX(r)rA cells, an L1210 mouse 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 indistinguishable 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 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(r)A-R16 cells, an MTX(r)A 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(r)A, and MTX(r)A-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(r)A cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.

KW - Folate

KW - Methotrexate

KW - pH

KW - Reduced folate carrier

KW - Transport

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U2 - 10.1016/S0006-2952(97)00673-4

DO - 10.1016/S0006-2952(97)00673-4

M3 - Article

C2 - 10076544

AN - SCOPUS:0032499415

VL - 55

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