Characterization of N 5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells

Wei Huang, Puttur D Prasad, Ramesh Kekuda, Frederick H. Leibach, Vadivel Ganapathy

Research output: Contribution to journalArticle

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Abstract

Purpose: To determine the identity of the transport process that is responsible for the uptake of N 5-methyltetrahydrofolate, the predominant from 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 N 5-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 N 5-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 N 5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 ± 0.01 μM. The process was specific for such reduced folates as N 5- methyltetrahydrofolate and N 5-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 N 5- 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 N 5-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.

Original languageEnglish (US)
Pages (from-to)1578-1587
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number8
StatePublished - Aug 12 1997

Fingerprint

Retinal Pigments
Folic Acid
Folic Acid Transporters
Epithelial Cells
Northern Blotting
5-methyltetrahydrofolate
Leucovorin
Plastics
Anions
Transfection
Cultured Cells
Cell Membrane
Breast Neoplasms

Keywords

  • Folate receptor
  • Folate transport
  • Potocytosis
  • Reduced-folate transporter
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Characterization of N 5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells. / Huang, Wei; Prasad, Puttur D; Kekuda, Ramesh; Leibach, Frederick H.; Ganapathy, Vadivel.

In: Investigative Ophthalmology and Visual Science, Vol. 38, No. 8, 12.08.1997, p. 1578-1587.

Research output: Contribution to journalArticle

Huang, Wei ; Prasad, Puttur D ; Kekuda, Ramesh ; Leibach, Frederick H. ; Ganapathy, Vadivel. / Characterization of N 5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells. In: Investigative Ophthalmology and Visual Science. 1997 ; Vol. 38, No. 8. pp. 1578-1587.
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abstract = "Purpose: To determine the identity of the transport process that is responsible for the uptake of N 5-methyltetrahydrofolate, the predominant from 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 N 5-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 N 5-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 N 5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 ± 0.01 μM. The process was specific for such reduced folates as N 5- methyltetrahydrofolate and N 5-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 N 5- 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 N 5-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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T1 - Characterization of N 5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells

AU - Huang, Wei

AU - Prasad, Puttur D

AU - Kekuda, Ramesh

AU - Leibach, Frederick H.

AU - Ganapathy, Vadivel

PY - 1997/8/12

Y1 - 1997/8/12

N2 - Purpose: To determine the identity of the transport process that is responsible for the uptake of N 5-methyltetrahydrofolate, the predominant from 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 N 5-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 N 5-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 N 5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 ± 0.01 μM. The process was specific for such reduced folates as N 5- methyltetrahydrofolate and N 5-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 N 5- 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 N 5-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.

AB - Purpose: To determine the identity of the transport process that is responsible for the uptake of N 5-methyltetrahydrofolate, the predominant from 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 N 5-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 N 5-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 N 5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 ± 0.01 μM. The process was specific for such reduced folates as N 5- methyltetrahydrofolate and N 5-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 N 5- 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 N 5-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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KW - Potocytosis

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