Smith S B, Kekuda R, Gu X, Chancy C, Conway S J, Ganapathy V
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta 30912-2000, USA.
Invest Ophthalmol Vis Sci. 1999 Apr;40(5):840-8.
Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE.
The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy.
RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region.
The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina.
叶酸对于DNA、RNA及蛋白质合成至关重要，叶酸缺乏可导致营养性弱视和视神经病变。目前，叶酸从脉络膜血供向视网膜的转运机制才刚刚开始被了解。最近有报道称，还原型叶酸转运体存在于培养的人视网膜色素上皮（RPE）细胞中，且被认为定位于这些细胞的顶端区域。作者推测，RPE在叶酸从脉络膜血向神经视网膜的向量转运中发挥作用，并且在介导这种转运过程中不仅使用还原型叶酸转运体，还使用叶酸受体α。本研究的目的是确定叶酸受体α是否存在于RPE中，如果存在，它是否沿RPE的基底外侧膜分布，从而支持该蛋白在叶酸转运至RPE初始步骤中的作用。
通过逆转录-聚合酶链反应（RT-PCR）、功能测定、原位杂交、免疫组织化学及激光扫描共聚焦显微镜分析小鼠RPE中叶酸受体α的表达。
RT-PCR分析、RT-PCR产物克隆及后续测序证实，叶酸受体α mRNA转录本在小鼠RPE/脉络膜中表达，在神经视网膜中也有表达。使用MTX(R)-ZR-75-1细胞进行的异源功能表达测定表明，通过RT-PCR从RPE/脉络膜复合体和神经视网膜获得的叶酸受体α cDNA具有功能，这通过叶酸结合及N5-甲基四氢叶酸摄取得以评估。原位杂交将叶酸受体α mRNA定位于小鼠RPE细胞和神经视网膜细胞。免疫组织化学检测到小鼠和大鼠RPE以及神经视网膜的多层中有叶酸受体α。激光扫描共聚焦显微镜显示叶酸受体α沿RPE的基底外侧区域而非顶端区域分布。
本研究首次分析了完整哺乳动物视网膜中叶酸受体α的表达。该受体在小鼠RPE中存在且具有功能。它特异性地分布于RPE的基底外侧表面，并且被认为在叶酸从脉络膜血向神经视网膜的向量转运中与还原型叶酸转运体协同发挥作用。
