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Retinoids pp 55-73 | Cite as

Culture of Highly Differentiated Human Retinal Pigment Epithelium for Analysis of the Polarized Uptake, Processing, and Secretion of Retinoids

  • Jane Hu
  • Dean Bok
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 652)

Abstract

The retinal pigment epithelium (RPE) occupies a strategic position within the eye, given its location between the neurosensory retina and the vascular bed (choroid) that nourishes the photoreceptor cells (rods and cones). Among the many attributes of this versatile monolayer of cells is its unique ability to convert vitamin A (retinol) into the prosthetic group (11-cis-retinal) for the rod and cone opsins, the photopigments essential for vision. It does so by absorbing retinol via a receptor-mediated process that involves the interaction of a carrier protein secreted by the liver, retinol-binding protein (RBP), and a receptor/channel that is the gene product of STRA6 (stimulated by retinoic acid 6). Following its uptake through the basolateral plasma membrane of the RPE, retinol encounters a brigade of binding proteins, membrane-bound receptors, and enzymes that mediate its multi-step conversion to 11-cis-retinal and the transport of this visual chromophore to the light-sensitive photoreceptor cell outer segment, the portion of the cell that houses the phototransduction cascade. This process is iterative, repeating itself via the retinoid visual cycle. Most of the human genes that code for this cohort of proteins carry disease-causing mutations in humans. The consequences of these mutations range in severity from relatively mild dysfunction such as congenital stationary night blindness to total blindness. The RPE, although post-mitotic in situ, is capable of proliferation when removed from its native milieu. This offers one the opportunity to study the retinoid visual cycle in modular form, providing insights into this intriguing process in health and disease. This chapter describes a cell culture method whereby the entire visual cycle can be created in vitro.

Key words

Retinoid visual cycle retinal pigment epithelium 11-cis-retinal all-trans-retinal inherited retinal disease Stargardt macular dystrophy Leber congenital amaurosis age-related macular degeneration 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Jane Hu
    • 1
  • Dean Bok
    • 1
  1. 1.Jules Stein Eye Institute and Department of Neurobiology, David Geffen School of MedicineUniversity of CaliforniaLos AngelesUSA

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