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Cole Milliken, Chunhe Chen, Patrice Goletz, Federico Gonzalez-Fernandez, Yiannis Koutalos; Formation of all-trans retinol after bleaching in isolated human cone photoreceptors. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4510.
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© ARVO (1962-2015); The Authors (2016-present)
Visual pigment is the primary light detector that allows the retina to detect light and generate the electrical signal necessary for vision. Visual pigment is present in the outer segments of photoreceptor cells and contains chromophore, 11-cis retinal. Light isomerizes 11-cis retinal to all-trans retinal, which must be reisomerized to regenerate rhodopsin. An important step in the process is the removal of all-trans retinal, which is released in the outer segments following light detection. Removal begins with the reduction of all-trans retinal to all-trans retinol. The purpose of our investigation was to measure the kinetics of retinol formation in isolated living human cone photoreceptors, the cells responsible for vision in daylight.
Interphotoreceptor Retinoid Binding Protein (IRBP) was purified from bovine retinas, and its concentration determined by absorbance spectroscopy and amino acid analysis. Single living cone photoreceptors were isolated from the maculas of human cadaver eyes (ages 85 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. The cell was then bleached and all-trans retinol formation was measured by imaging its fluorescence (λex 360 nm; λem >420 nm) in the outer segment. Excitation of outer segment fluorescence with 340 and 380 nm light confirmed that the signal was mostly due to all-trans retinol. Experiments were carried out at 37 0C.
Bleaching of regenerated isolated human cone photoreceptors resulted in an increase in all-trans retinol fluorescence in the outer segment. Fluorescence increased with a rate constant of 1.77 min-1 reaching a peak approximately 1 min after bleaching. Subsequently, outer segment fluorescence declined indicating the removal of retinol. Retinol formation in cones is several times faster than in rods, where it proceeds with a rate constant of 0.2 – 0.4 min-1 with the fluorescence reaching a peak approximately 10 min after bleaching.
Formation of all-trans retinol in the outer segments of human cone photoreceptors proceeds much faster than in rods, consistent with the faster regeneration of cone visual pigment after bleaching. The results point to the ability of the metabolic machinery of human cone photoreceptors to supply the NADPH necessary for the formation of all-trans retinol at a high rate.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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