RETINAL PIGMENT EPITHELIUM: VECTORIAL TRANSPORT FUNCTION

描述（改编自申请人摘要）：视网膜色素上皮 (RPE)在离子、流体和流体的矢量传输中起着至关重要的作用， 从脉络膜血液到感光细胞的代谢物。这样做的目的 该项目是第一次分析细胞和分子 RPE用于运输叶酸的机制，这是至关重要的， 合成DNA，RNA和一些氨基酸，因此需要感光体 细胞存活已知其他细胞使用两种主要机制来摄取 上叶酸：叶酸受体α和还原叶酸转运蛋白。我们称之为 后者还原叶酸转运蛋白1，RFT-1，以区分它与另一个 RFT-2是我们最近从视网膜色素上皮中克隆出来的。我们建议 以下三个假设：（1）叶酸受体α和RFT-1定位于 基底外侧和顶端RPE膜，分别，这是在对比 它们在所有其他叶酸转运细胞中的已知定位。我们将 通过比较免疫定位、原位杂交和 在完整小鼠视网膜组织和培养的人ARPE-19中的功能测定 细胞与其他叶酸转运细胞。(2)RPE同时使用叶酸受体 α和RFT-1以实现叶酸从脉络膜血液的载体转运 到视网膜上。我们将通过分析叶酸的转运来测试这一点 使用在可渗透过滤器上培养的人ARPE-19细胞，通过测定 ARPE-19细胞中这种转运的调节，特别强调一氧化氮 氧化物，抗坏血酸，谷胱甘肽和高葡萄糖水平，并通过测量 RFT-1对牛RPE细胞顶膜的作用机制和能量学 囊泡(3)RFT-2是一种蛋白质，参与将叶酸从胃肠道递送到胃肠道。 在通过叶酸进入细胞后，内体进入RPE细胞质 受体介导的内吞作用。我们将使用EM免疫定位来测试这一点 方法和用RFT-2和 叶酸受体α cDNA。这些研究的结果将 第一个可用的数据的机制，涉及矢量运输的 叶酸穿过RPE。

DESCRIPTION (Adapted from applicant's abstract): Retinal pigment epithelium (RPE) plays an essential role in vectorial transport of ions, fluid and metabolites from choroidal blood to photoreceptor cells. The purpose of this project is to analyze, for the first time, the cellular and molecular mechanisms used by RPE to transport folate, which is essential for the synthesis of DNA, RNA and some amino acids and thus required for photoreceptor cell survival. Two major mechanisms are known to be used by other cells to take up folate: folate receptor alpha and reduced-folate transporter. We call the latter reduced-folate transporter 1, RFT-1 to differentiate it from another transporter, RFT-2 that we have cloned recently from RPE. We propose the following three hypotheses: (1) Folate receptor alpha and RFT-1 are localized to the basolateral and apical RPE membranes, respectively, which is in contrast to their known localization in all other folate-transporting cells. We will test this hypothesis by comparing immunolocalization, in situ hybridization and functional assays in intact mouse retinal tissue and cultured human ARPE-19 cells with other folate-transporting cells. (2) RPE uses both folate receptor alpha and RFT-1 to achieve vectorial transport of folate from choroidal blood to the sensory retina. We will test this by analyzing the transport of folate using human ARPE-19 cells cultured on permeable filters, by determining the regulation of this transport in ARPE-19 cells with special emphasis on nitric oxide, ascorbic acid, glutathione and high glucose levels, and by measuring the operational mechanism and energetics of RFT-1 using bovine RPE apical membrane vesicles. (3) RFT-2 is the protein involved in delivering folate from the endosome to the RPE cytoplasm subsequent to this entry into the cell via folate receptor-mediated endocytosis. We will test this using EM immunolocalization methods and folate transport assays in cells co-transfected with RFT-2 and folate receptor alpha cDNAs. The results obtained from these studies will be the first data available on the mechanisms involved in vectorial transport of folate across RPE.