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Leopold Adler, IV, Chunhe Chen, Yiannis Koutalos; Generation of NADPH by Mitochondrial-Mediated Pathways in Mouse Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3360.
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To determine the contribution of different NADPH-generating metabolic pathways to the reduction of all-trans retinal to retinol in the outer segment of mouse 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.
Experiments were carried out with dark-adapted living rod photoreceptors isolated from 2-3 months old 129/sv wild type mice. The amount of all-trans retinol in the outer segment was measured at various times after bleaching from its fluorescence (excitation 360 nm; emission >420 nm). Experiments were carried out at 37 0C. A numerical model describing the formation of all-trans retinol was developed. From this model, the concentration of and production rate of NADPH were estimated from the difference in the known kinetics of all-trans retinal release and those of all-trans retinol formation.
All-trans retinol formation was suppressed in the absence of glucose, and was restored by addition of pyruvate. In the presence of 5 mM glucose, NADPH was supplied to the rod outer segment at a rate of ~0.22 mM min-1. The concentration of cytosolic outer segment NADPH present before bleaching was ~0.3 mM. In the absence of glucose, concentrations of 0.5 - 1.0 mM of pyruvate were sufficient to restore NADPH supply rates of 0.20 - 0.24 mM min-1.
Mitochondrial-mediated pathways can generate NADPH at sufficient rates to sustain the reduction of all-trans retinal to retinol following rhodopsin bleaching.
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