Project: Research project

Project Details


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.
Effective start/end date12/1/992/28/17


  • Medicine(all)