RETINAL PIGMENT EPITHELIUM: VECTORIAL TRANSPORT FUNCTION

描述(摘自申请者摘要)：视网膜色素上皮 (RPE)在离子、液体和蛋白质的载体运输中起着重要作用。 从脉络膜血到感光细胞的代谢物。这样做的目的是 这个项目是第一次分析细胞和分子 RPE用来运输叶酸的机制，这对 光感受器所需的DNA、RNA和某些氨基酸的合成 细胞存活。已知其他细胞使用两种主要机制来采取 向上叶酸：叶酸受体α和还原叶酸转运体。我们将其称为 晚期还原叶酸转运蛋白1，RFT-1，以区别于其他转运蛋白 Transporter，RFT-2，是我们最近从RPE克隆的。我们建议 以下三个假设：(1)叶酸受体α和RFT-1是定位的 分别与RPE基底膜和根尖膜形成对比。 它们在所有其他叶酸运输细胞中的已知定位。我们会 通过比较免疫定位、原位杂交和 正常小鼠视网膜组织和培养的人ARPE-19细胞的功能分析 细胞与其他叶酸运输细胞。(2)RPE使用两种叶酸受体 Alpha和RFT-1实现脉络膜血中叶酸的载体转运 到感觉性视网膜。我们将通过分析叶酸的运输来测试这一点 使用在渗透性过滤器上培养的人ARPE-19细胞，通过测定 在ARPE-19细胞中对这种转运的调节，特别是对一氧化氮的调节 氧化物、抗坏血酸、谷胱甘肽和高血糖水平，并通过测量 牛RPE根尖膜RFT-1的作用机制及能量学研究 水泡。(3)RFT-2是一种参与将叶酸从 内体通过叶酸进入细胞后进入RPE细胞质 受体介导的内吞作用。我们将使用EM免疫定位对此进行测试 RFT-2和RFT-2共转染细胞的方法和叶酸转运分析 叶酸受体αcDNA。从这些研究中获得的结果如下 可获得的关于细菌向量运输的机制的第一批数据 跨RPE的叶酸。

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.