Studies of vitamin A metabolism in the retinal pigment epithelium

The retinoid visual cycle is a sequence of metabolic and transport reactions of retinoids in the retina and the retinal pigment epithelium (RPE). Many proteins are involved in this cycle and play critical roles in the generation of 11-cis retinal, the chromophore that binds opsin to form the visual pigments. Mutations of these proteins cause eye diseases that can eventually lead to blindness.; RPE65 and LRAT are essential proteins involved in the visual dark cycle to regenerate visual pigments, while RDH10 is thought to produce all- trans retinal, the substrate for the retinal G protein-coupled receptor (RGR) in the photic cycle to regenerate visual pigments. This research is focused on studying the visual cycle proteins (RPE65, LRAT and RDH10) and tested the hypothesis that the HEK293 cells can be used as an in vitro model to study visual vitamin A metabolism.; The protein RPE65 was purified from native bovine RPE and the molecular mass of this protein was determined. Retinoic acid (RA) was found to down-regulate RPE65 expression and promoter activity in the HEK293 cells, and this regulation is mediated through the retinoic acid (RA) receptors. The RPE65 expressing HEK293 cell line also expresses functional lecithin:retinol acyltransferase (LRAT) and other retinoid binding proteins, which made the HEK293 cell line a good model system to study the visual cycle in vitro. However, RDH10 is not expressed in the HEK293 cells. The recombinant human RDH10 protein was expressed in COS cells (CV1 monkey cells transformed with SV40 [simian virus 40] possessing a defective origin of replication) and RDH10 was found to oxidize all-trans retinol to all-trans retinal, 17-beta estradiol to estrone, and prefers NADP as its cofactor. All the above findings support the hypothesis that HEK293 cells can be used as a model to study the visual dark cycle.