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Chunhe Chen, Leopold Adler, Lynn J. Poole Perry, Yiannis Koutalos; NADPH generation in single isolated human rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):194.
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The reduction of all-trans retinal to retinol in rod photoreceptor outer segments requires NADPH. We have used the conversion of all-trans retinal to retinol to measure the ability of single living rod photoreceptor cells isolated from human donor eyes to generate NADPH.
Fresh human eyes without diagnosed eye disease (donor ages: 53-84 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 residual rhodopsin and stored in physiological solution at 4 °C. Metabolically active rod photoreceptors were isolated from the peripheral region of the retinas and placed on a fluorescence microscope stage at 37 °C. NADPH generation in a single cell was measured from the change in outer segment fluorescence after a 5 min exposure to 5 µM exogenous all-trans retinal supplied using 1% bovine serum albumin as carrier. The conversion of the supplied all-trans retinal to retinol was measured from the ratio Fex-340/Fex-380 of the fluorescence intensities excited by 340 and 380 nm light with emission collected >420 nm. The value of the Fex-340/Fex-380 ratio was used to calculate the fraction of all-trans retinal converted to retinol and the corresponding fraction of NADPH (Adler et al. 2014, J Biol Chem 289:1519).
In the presence of 5 mM glucose for metabolic substrate, single isolated human rods converted ~80% of supplied all-trans retinal to retinol, corresponding to an NADPH fraction of ~0.2. Conversion of all-trans retinal declined at extracellular glucose concentrations lower than ~100 µM. Conversion of all-trans retinal remained stable for rods isolated within the first 3 days following eye dissection and retina isolation and began declining thereafter.
Human rod photoreceptors isolated from donor eyes can generate NADPH, as inferred from their ability to reduce exogenously supplied all-trans retinal to retinol. NADPH generation by an isolated rod requires the presence of extracellular metabolic substrate, indicating the lack of significant intracellular metabolic stores. The ability of rod photoreceptors to generate NADPH persists for several days after donor death.
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