July 1997
Volume 38, Issue 8
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Articles  |   July 1997
Characterization of N5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells.
Author Affiliations
  • W Huang
    Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
  • P D Prasad
    Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
  • R Kekuda
    Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
  • F H Leibach
    Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
  • V Ganapathy
    Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Investigative Ophthalmology & Visual Science July 1997, Vol.38, 1578-1587. doi:
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      W Huang, P D Prasad, R Kekuda, F H Leibach, V Ganapathy; Characterization of N5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells.. Invest. Ophthalmol. Vis. Sci. 1997;38(8):1578-1587.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

PURPOSE: To determine the identity of the transport process that is responsible for the uptake of N5-methyltetrahydrofolate, the predominant form of folate in blood, into cultured human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells were cultured on an impermeable plastic support, and the characteristics of the uptake of radiolabeled N5-methyltetrahydrofolate into the cells were investigated. The expression of the folate receptor and the reduced-folate transporter in these cells was evaluated by functional assays and by Northern blot analysis. In addition, the characteristics of N5-methyltetrahydrofolate uptake in these cells were compared with those in folate transport-defective human breast cancer cells that were manipulated to express functionally by transfection the cloned human folate receptor and the human reduced-folate transporter. RESULTS: Transport of N5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 +/- 0.01 microM. The process was specific for such reduced folates as N5-methyltetrahydrofolate and N5-formyltetrahydrofolate. Nonreduced folate interacted with this transport process only weakly. The transport process was inhibited by anion transport inhibitors. Results of Northern blot analysis indicated the presence of transcripts specific for the reduced-folate transporter in these cells. These cells expressed very small amounts of the folate receptor evidenced from the binding of folate and from the detectable presence of folate receptor-specific transcript, but there was no evidence for participation of the receptor in the observed transport of N5-methyltetrahydrofolate. There was also no evidence in these cells for expression of the folate transporter that prefers nonreduced folate as a substrate. CONCLUSIONS: Transport of N5-methyltetrahydrofolate in human retinal pigment epithelial cells occurs exclusively through the reduced-folate transporter. The folate receptor is expressed at negligible levels in these cells and does not participate in the observed transport. Because the cells were cultured on impermeable supports, making the basolateral membrane inaccessible for transport measurements, it is speculated that the observed findings are related to the transport function of the apical membrane of these polarized cells.

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