December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Regulation of Reduced-Folate Transporter (RFT-1) in Retinal Pigment Epithelial (RPE) Cells by Homocysteine (HCY)
Author Affiliations & Notes
  • HA Naggar
    Med College of Georgia Augusta GA
    Cell Bio & Anatomy
  • V Ganapathy
    Med College of Georgia Augusta GA
  • SB Smith
    Cell Bio & Anatomy/Ophthalmology
    Med College of Georgia Augusta GA
  • Footnotes
    Commercial Relationships   H.A. Naggar, None; V. Ganapathy, None; S.B. Smith, None. Grant Identification: NIH EY12830, EY13089, RPB
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 710. doi:
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      HA Naggar, V Ganapathy, SB Smith; Regulation of Reduced-Folate Transporter (RFT-1) in Retinal Pigment Epithelial (RPE) Cells by Homocysteine (HCY) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):710.

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

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Abstract: : Purpose: RFT-1, a typical transport protein with twelve membrane-spanning domains, transports reduced folates, such as N5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. In RPE, RFT-1 is localized to the apical membrane and is thought to transport folate from RPE to photoreceptor cells. Hyperglycemia and nitric oxide significantly decrease RFT-1 activity in cultured RPE cells. Folate is required for DNA, RNA, protein synthesis and the conversion of Hcy to methionine. Decreased folate levels are associated with increased Hcy levels and hyperhomocysteinemia is associated with diabetes. In the present study we asked whether RFT-1 activity in RPE is altered under high Hcy conditions. Methods: ARPE-19 cells were maintained in RPMI medium. For experiments, cells were incubated in Na+ uptake buffer containing 1000µM D,L-Hcy for varying time periods. The activity of RFT-1 was assessed by determining the uptake of [3H]-MTF. mRNA levels encoding RFT-1 were determined by semiquantitative RT-PCR and protein levels by Western blot analysis. Results: Exposure of ARPE-19 cells to Hcy for as short an incubation time as 2 h resulted in a slight attenuation of MTF uptake. Cells treated as long as 8 h in 1000µM D,L-Hcy led to a 75% inhibition of MTF uptake. To determine an appropriate Hcy concentration for further experiments, cells were treated with differing concentrations of D,L-Hcy ranging from 25µM to 500µM. Concentrations of D,L-Hcy as low as 25µM decreased MTF uptake. As the concentration increased, MTF uptake was inhibited in a dose-dependent manner. Specificity of Hcy-induced attenuation of MTF uptake was examined. 8 h exposure to high Hcy (500µM) led to a marked inhibition of MTF uptake, whereas uptake of other compounds such as glutamine and glutamate were not markedly inhibited in the presence of high Hcy. Kinetic analysis showed that the Hcy-induced attenuation was associated with a decrease in the maximal velocity of the transporter with no change in the substrate affinity. Semiquantitative RT-PCR demonstrated that the mRNA levels encoding RFT-1, but not GAPDH, were decreased (∼35%) in cells exposed to high Hcy. Western blot analysis showed a decrease (∼25%) in protein levels. Conclusion: These findings demonstrate for the first time that hyperhomocysteinemic conditions reduce RFT-1 activity. The Hcy-induced attenuation of transport function is specific to uptake of MTF and appears to involve a decrease in levels of RFT-1. These data may have profound implications on RPE folate transport in conditions of hyperhomocysteinemia.

Keywords: 567 retinal pigment epithelium • 402 excitatory neurotransmitters 

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