December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Synthesis, Binding, and Photoisomerization of All-Trans-Retinal in Cultured RPE Cells
H Fong; M Yang
Author Affiliations & Notes
  • H Fong
    Ophthalmology and Microbiology USC/Doheny Eye Institute Los Angeles CA
  • M Yang
    Molecular Microbiology and Immunology University of Southern California Los Angeles CA
  • Footnotes
    Commercial Relationships   H. Fong, None; M. Yang, None. Grant Identification: Support: NIH Grant EY08364
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 4581. doi:
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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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Abstract

Abstract: : 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.

Keywords: 399 enzymes/enzyme inhibitors 
© 2002, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
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