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Leopold Adler, Chunhe Chen, Federico Gonzalez-Fernandez, Yiannis Koutalos; Opsin availability prevents formation of lipofuscin precursors from 11-cis retinal in human rod photoreceptor outer segments. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):38.
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© ARVO (1962-2015); The Authors (2016-present)
In rod photoreceptor outer segments, 11-cis retinal combines with available opsin to regenerate rhodopsin. 11-cis retinal can also react with outer segments to generate precursors of lipofuscin, which eventually accumulates in RPE lysosomes. We tested whether the presence of opsin affects the formation of lipofuscin precursors from 11-cis retinal in isolated human rod photoreceptors.
Fresh human eyes (donor ages: 53-83 years) were procured through the National Disease Resource Interchange. Eyes were dissected and the retinas isolated within 48 hrs of death. Isolated retinas were bleached to remove all residual rhodopsin, and stored in physiological solution at 4°C. Metabolically active rod photoreceptors were isolated from the peripheral region of the retinas. Interphotoreceptor retinoid binding protein (IRBP) was extracted from bovine retinas and purified to homogeneity by combination of Con-A affinity, ion exchange, and size-exclusion chromatography; its concentration was determined by amino acid analysis. Isolated photoreceptors were incubated in the presence of either 10 μM IRBP with 5 μM 11-cis retinal to regenerate rhodopsin, or 10 μM IRBP alone. After incubation, cells were washed with physiological solution and placed on a fluorescence microscope stage at 37°C. Cells were then exposed to different concentrations of 11-cis retinal with bovine serum albumin as carrier. The levels of lipofuscin precursors in the rod outer segment were determined from their fluorescence (ex, 490 nm; em >520 nm).
Initial levels of lipofuscin precursors did not vary appreciably with time after dissection. Exposure to 11-cis retinal resulted in a significant increase in lipofuscin precursor levels. This increase in lipofuscin precursor levels was much higher in cells that contained rhodopsin as opposed to opsin. Exposure to higher concentrations of 11-cis retinal resulted in correspondingly greater increases in lipofuscin precursor formation.
11-cis retinal generates lipofuscin precursors in the outer segment of human rod photoreceptors. Available opsin limits the formation of lipofuscin precursors, likely by acting as a sink for 11-cis retinal.
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