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Yiannis Koutalos, Chunhe Chen, Zsolt Ablonczy, Rosalie Crouch, Molly Sprada, Federico Gonzalez-Fernandez; Formation Of All-Trans Retinol After Bleaching In Single Isolated Human Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3348.
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To examine the formation of all-trans retinol after rhodopsin bleaching in the outer segment of single living human rod photoreceptors. All-trans retinol is formed in rod outer segments from the reduction of the all-trans retinal released from photoactivated rhodopsin via a reaction that utilizes NADPH.
Interphotoreceptor retinoid-binding protein (IRBP) was extracted from the soluble interphotoreceptor matrix fraction of bovine retinas, and purified by a combination of concanavalin-A affinity, ion exchange, and S-300 size-exclusion chromatography. The concentration of the purified IRBP was determined by absorbance spectroscopy, and amino acid analysis. Single living rod photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years) obtained from National Disease Resource Interchange. The visual pigment of isolated cells was regenerated by incubating with 11-cis retinal using IRBP as a carrier. After regeneration, the cell was bleached and all-trans retinol formation was measured by imaging its fluorescence (excitation 360 nm; emission >420 nm) in the outer segment. Experiments were carried out at 37 0C.
Bleaching of regenerated isolated human rod photoreceptors resulted in an increase in all-trans retinol fluorescence in the outer segment. Formation of all-trans retinol proceeded with a rate constant of 0.2 - 0.4 min-1, which is several times faster than in mouse rod photoreceptors. Subsequently, outer segment fluorescence declined indicating the elimination of retinol.
Formation of all-trans retinol in the outer segments of human rod photoreceptors proceeds much faster than in mouse cells. This is consistent with the faster regeneration of rhodopsin after bleaching in humans. The results point to the ability of the metabolic machinery of human rod photoreceptors to supply at a high rate the NADPH necessary for the formation of all-trans retinol.
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