Huang W, Prasad P D, Kekuda R, Leibach F H, Ganapathy V
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Invest Ophthalmol Vis Sci. 1997 Jul;38(8):1578-87.
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.
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.
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.
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.
确定负责将血液中叶酸的主要形式N5-甲基四氢叶酸摄取到培养的人视网膜色素上皮细胞中的转运过程的特性。
将人视网膜色素上皮细胞培养在不可渗透的塑料支持物上，研究放射性标记的N5-甲基四氢叶酸摄取到细胞中的特性。通过功能测定和Northern印迹分析评估这些细胞中叶酸受体和还原型叶酸转运体的表达。此外，将这些细胞中N5-甲基四氢叶酸摄取的特性与叶酸转运缺陷型人乳腺癌细胞中的特性进行比较，这些乳腺癌细胞通过转染克隆的人叶酸受体和人还原型叶酸转运体进行功能操作。
N5-甲基四氢叶酸进入这些细胞的转运通过单一的可饱和过程发生，米氏常数为0.13±0.01微摩尔。该过程对N5-甲基四氢叶酸和N5-甲酰四氢叶酸等还原型叶酸具有特异性。非还原型叶酸与该转运过程的相互作用较弱。该转运过程受到阴离子转运抑制剂的抑制。Northern印迹分析结果表明这些细胞中存在还原型叶酸转运体特异性转录本。从叶酸结合和叶酸受体特异性转录本的可检测存在证明，这些细胞表达极少量的叶酸受体，但没有证据表明该受体参与观察到的N5-甲基四氢叶酸的转运。这些细胞中也没有证据表明存在以非还原型叶酸为底物的叶酸转运体的表达。
人视网膜色素上皮细胞中N5-甲基四氢叶酸的转运仅通过还原型叶酸转运体发生。这些细胞中叶酸受体的表达水平可忽略不计，不参与观察到的转运。由于细胞培养在不可渗透的支持物上，使得基底外侧膜无法进行转运测量，推测观察到的结果与这些极化细胞顶端膜的转运功能有关。
