Regulation of reduced-folate transporter-1 in retinal pigment epithelial cells by folate

Hany Naggar, Tracy K. Van Ells, Vadivel Ganapathy, Sylvia B Smith

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Purpose: Reduced-folate transporter-1 (RFT-1), a typical transport protein with 12 membrane-spanning domains, transports reduced-fblates, such as N 5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. RFT-1 is localized to the RPE apical membrane and transports folate from RPE to photoreceptor cells. We asked whether RFT-1 activity in RPE is altered under high folate conditions. Materials and methods: ARPE-19 cells were cultured 24, 48, or 72 h in medium containing either 0.5 nM, 5.0 nM, or 2.26 μM MTF, and the activity of RFT-1 was assessed by determining the uptake of N 5-MTF. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis were used to study RFT-1 gene and protein expression. Results: Cells treated for 72 h with 2.26 μM MTF showed a significant (40%) decrease in MTF uptake compared to cells exposed to 0.5 nM or 5 nM MTF. The effect of high concentrations of folate on RFT-1 activity was specific. Kinetic analysis showed that folate-induced attenuation of RFT-1 activity was associated with a decrease in the maximal velocity of the transporter, but no change in the substrate affinity. Steady-state levels of RFT-1 mRNA and protein decreased significantly in the presence of excess folate. Conclusions: Excess folate levels downregulate RFT-1 in RPE. This study represents the first molecular analysis of the regulation of RFT-1 by folate in RPE and reveals attenuation of the activity and expression of a folate transport protein under conditions of high levels of folate.

Original languageEnglish (US)
Pages (from-to)35-44
Number of pages10
JournalCurrent Eye Research
Volume30
Issue number1
DOIs
StatePublished - Jan 1 2005

Fingerprint

Reduced Folate Carrier Protein
Retinal Pigments
Folic Acid
Epithelial Cells
Folic Acid Transporters
Photoreceptor Cells
Membranes
Reverse Transcription
Cultured Cells
Carrier Proteins
Proteins
Down-Regulation

Keywords

  • ARPE-19 cells
  • Cell culture
  • Folate
  • Reduced-folate transporter
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Regulation of reduced-folate transporter-1 in retinal pigment epithelial cells by folate. / Naggar, Hany; Van Ells, Tracy K.; Ganapathy, Vadivel; Smith, Sylvia B.

In: Current Eye Research, Vol. 30, No. 1, 01.01.2005, p. 35-44.

Research output: Contribution to journalArticle

Naggar, Hany ; Van Ells, Tracy K. ; Ganapathy, Vadivel ; Smith, Sylvia B. / Regulation of reduced-folate transporter-1 in retinal pigment epithelial cells by folate. In: Current Eye Research. 2005 ; Vol. 30, No. 1. pp. 35-44.
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abstract = "Purpose: Reduced-folate transporter-1 (RFT-1), a typical transport protein with 12 membrane-spanning domains, transports reduced-fblates, such as N 5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. RFT-1 is localized to the RPE apical membrane and transports folate from RPE to photoreceptor cells. We asked whether RFT-1 activity in RPE is altered under high folate conditions. Materials and methods: ARPE-19 cells were cultured 24, 48, or 72 h in medium containing either 0.5 nM, 5.0 nM, or 2.26 μM MTF, and the activity of RFT-1 was assessed by determining the uptake of N 5-MTF. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis were used to study RFT-1 gene and protein expression. Results: Cells treated for 72 h with 2.26 μM MTF showed a significant (40{\%}) decrease in MTF uptake compared to cells exposed to 0.5 nM or 5 nM MTF. The effect of high concentrations of folate on RFT-1 activity was specific. Kinetic analysis showed that folate-induced attenuation of RFT-1 activity was associated with a decrease in the maximal velocity of the transporter, but no change in the substrate affinity. Steady-state levels of RFT-1 mRNA and protein decreased significantly in the presence of excess folate. Conclusions: Excess folate levels downregulate RFT-1 in RPE. This study represents the first molecular analysis of the regulation of RFT-1 by folate in RPE and reveals attenuation of the activity and expression of a folate transport protein under conditions of high levels of folate.",
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AB - Purpose: Reduced-folate transporter-1 (RFT-1), a typical transport protein with 12 membrane-spanning domains, transports reduced-fblates, such as N 5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. RFT-1 is localized to the RPE apical membrane and transports folate from RPE to photoreceptor cells. We asked whether RFT-1 activity in RPE is altered under high folate conditions. Materials and methods: ARPE-19 cells were cultured 24, 48, or 72 h in medium containing either 0.5 nM, 5.0 nM, or 2.26 μM MTF, and the activity of RFT-1 was assessed by determining the uptake of N 5-MTF. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis were used to study RFT-1 gene and protein expression. Results: Cells treated for 72 h with 2.26 μM MTF showed a significant (40%) decrease in MTF uptake compared to cells exposed to 0.5 nM or 5 nM MTF. The effect of high concentrations of folate on RFT-1 activity was specific. Kinetic analysis showed that folate-induced attenuation of RFT-1 activity was associated with a decrease in the maximal velocity of the transporter, but no change in the substrate affinity. Steady-state levels of RFT-1 mRNA and protein decreased significantly in the presence of excess folate. Conclusions: Excess folate levels downregulate RFT-1 in RPE. This study represents the first molecular analysis of the regulation of RFT-1 by folate in RPE and reveals attenuation of the activity and expression of a folate transport protein under conditions of high levels of folate.

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