Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells

Esteban E. Sierra, Kevin E. Brigle, Michael J. Spinella, I. David Goldman

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-β (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-β and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 μM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-β exhibited an influx Kt one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO42- was inhibitory. Anions enhanced MTX binding to folate receptor-β with isethionate > SO42- > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-β-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.

Original languageEnglish (US)
Pages (from-to)1287-1294
Number of pages8
JournalBiochemical Pharmacology
Volume50
Issue number8
DOIs
StatePublished - Oct 12 1995
Externally publishedYes

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Reduced Folate Carrier Protein
Leukemia L1210
Folic Acid
Methotrexate
Transport properties
Cells
Anions
Cell Line
Sulfobromophthalein
Probenecid
Chlorides
Buffers
Substitution reactions

Keywords

  • binding
  • reduced folate carrier, folate receptor, methotrexate
  • transport

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells. / Sierra, Esteban E.; Brigle, Kevin E.; Spinella, Michael J.; Goldman, I. David.

In: Biochemical Pharmacology, Vol. 50, No. 8, 12.10.1995, p. 1287-1294.

Research output: Contribution to journalArticle

Sierra, Esteban E. ; Brigle, Kevin E. ; Spinella, Michael J. ; Goldman, I. David. / Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells. In: Biochemical Pharmacology. 1995 ; Vol. 50, No. 8. pp. 1287-1294.
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N2 - This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-β (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-β and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 μM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-β exhibited an influx Kt one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO42- was inhibitory. Anions enhanced MTX binding to folate receptor-β with isethionate > SO42- > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-β-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.

AB - This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-β (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-β and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 μM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-β exhibited an influx Kt one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO42- was inhibitory. Anions enhanced MTX binding to folate receptor-β with isethionate > SO42- > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-β-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.

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