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C. N. Roybal, C. Sanfilipo, S. C. Finnemann, G. H. Travis; Contribution of Outer Segment Phagocytosis to Recycling of Visual Chromophore. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4809.
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Interaction of a photon with rhodopsin (rho) causes 11-cis to all-trans isomerization of its retinaldehyde chromophore. Photoreceptors depend on the retinal pigment epithelium (RPE) to convert the resulting all-trans-retinaldehyde back to 11-cis-retinaldehyde (11-cis-RAL) for regeneration of a new light-sensitive rho. The RPE also helps support photoreceptors by daily phagocytosing the distal 10% of the outer segment (OS). This process allows photoreceptors to replace oxidized lipids and proteins. Thus, approximately 10% of total rho in the retina is processed daily by the RPE through phagocytosis. The current study addresses the question: What is the fate of 11-cis-RAL in rho following phagocytosis by the RPE?
Bovine OS were isolated, bleached and regenerated with 9-cis-RAL to yield iso-rhodopsin (iso-rho). Regenerated iso-rho or native rho were affinity purified with a monoclonal Ab against the rho C-terminus (1D4). Purified rho or iso-rho was incorporated into proteoliposomes, creating a rho/iso-rho ‘synthetic outer segment’ (SOS). For microscopy, the SOS were fluorescently tagged with FITC and incubated with mouse eyecups to determine the amount of SOS internalized into the RPE. The effect of interphotoreceptor matrix (IPM) proteins or purified MFG-E8 on phagocytosis was determined. Iso-SOS were incubated with MFG-E8, fed to mouse eyecups and harvested for HPLC analysis of the phagocytosed retinoids.
Immunofluorescence confirmed that SOS were robustly phagocytosed by the RPE. Binding of SOS to the RPE surface peaked at one hour, and internalization was complete by two hours. Similar to native OS, the rate of SOS binding and internalization increased with addition of IPM proteins or purified MFG-E8. HPLC analysis of eyecups exposed to iso-SOS showed increased 11-cis-RAL and 11-cis-retinylpalmitate. No retinoid degradation products were observed.
We have developed a mouse explant system to determine the fate of the retinoids phagocytosed with the distal OS by RPE cells. Our initial observations suggest that visual chromophore in rho is removed from phagosomes and recycled by the RPE.
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