Regulation of Reduced-Folate Transporter-1 in Retinal Pigment Epithelial Cells by Folate

• Published in: Current eye research Vol. 30; no. 1; pp. 35 - 44
• Main Authors: Naggar, Hany, Van Ells, Tracy K., Ganapathy, Vadivel, Smith, Sylvia B.
• Format: Journal Article
• Language: English
• Published: England Informa UK Ltd 2005
• Subjects: Blotting, Western; Cells, Cultured; Down-Regulation; Humans; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Pigment Epithelium of Eye - drug effects; Pigment Epithelium of Eye - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Tetrahydrofolates - pharmacology
• ISSN: 0271-3683; 1460-2202
• DOI: 10.1080/02713680490894559

Purpose: Reduced-folate transporter-1 (RFT-1), a typical transport protein with 12 membrane-spanning domains, transports reduced-folates, such as N5-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 N5-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.