RETINAL PIGMENT EPITHELIUM--VECTORIAL TRANSPORT FUNCTION

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.