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H Fong, M Yang; Synthesis, Binding, and Photoisomerization of All-Trans-Retinal in Cultured RPE Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4581.
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Purpose: Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination in vitro, isomerizes to the 11-cis isomer. The purpose of this work is to investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Methods: ARPE-hRGR cells, which stably express human RGR, were generated by transduction of ARPE-19 cells with a recombinant lentivirus-human RGR vector. Intact ARPE-hRGR and normal bovine RPE cells, or microsomal membranes, were incubated in the dark with [3H]all-trans-retinol. [3H]-Labeled membrane proteins were analyzed by gel electrophoresis and fluorography. Retinoids were analyzed by HPLC. Results: Exogenous [3H]all-trans-retinol was incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of [3H]all-trans-retinol resulted in physiological binding to RGR of a radiolabeled retinoid, identified as [3H]all-trans-retinal. The accumulation of all-trans-retinal was dependent on the presence of RGR. We showed that ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol, but not the 11-cis isomer. Irradiation of the ARPE-hRGR cells resulted in photoisomerization of the endogenous all-trans-retinal to 11-cis-retinal. Photoisomerization to 13-cis-retinal was not detected. Conclusion: The results confirm that all-trans-retinal is synthesized in the RPE and binds to RGR opsin in the dark. The irradiation of ARPE-hRGR cells resulted in stereospecific isomerization of all-trans-retinal to 11-cis-retinal, indicating a functional recombinant RGR. With the ARPE-hRGR cells, the 11-cis retinoid metabolic pathways that lie downstream of the RGR opsin can be analyzed under a variety of culture and lighting conditions.
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