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Chunhe Chen, Leopold Adler, Yiannis Koutalos; Metabolic activity of single isolated mouse rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1763.
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© 2017 Association for Research in Vision and Ophthalmology.
To determine the effectiveness of different metabolites as metabolic substrates of mouse rod photoreceptors. In mouse rod photoreceptor outer segments, the all-trans retinal released from photoactivated rhodopsin is reduced to all-trans retinol via a reaction that utilizes metabolic input in the form of NADPH. We have used the conversion of all-trans retinal to all-trans retinol to measure the metabolic activity of single rod photoreceptors isolated from mouse retinas.
Experiments were carried out with dark-adapted living rod photoreceptors isolated from 2-3 month old 129/sv wild type mice. NADPH generation in a single cell 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. For an experiment, all-trans retinal was generated by exposing a single dark-adapted cell to >530 nm light for 1 min, and the extent of conversion to all-trans retinol was measured at 30 min after light exposure. The value of the Fex-340/Fex-380 ratio was used to calculate the fraction of all-trans retinal converted to all-trans retinol and the corresponding fraction of NADPH (Adler et al. 2014, J Biol Chem 289:1519). Amino acids were used as metabolic substrates at a concentration of 0.5 mM.
In the presence of 5 mM glucose as metabolic substrate, single isolated mouse rods converted ~70-80% of all-trans retinal to retinol, corresponding to an NADPH fraction of ~10-20%. From among the 20 amino acids, glutamine (0.5 mM) supported a similar level of conversion of all-trans retinal to retinol as 5 mM glucose, corresponding to similar fraction of NADPH. The rest of the amino acids (at 0.5 mM concentration) supported the conversion of all-trans retinal to retinol to a much lesser extent, indicating NADPH fractions of 1-2% at the most. With either glucose or glutamine as metabolic substrate, the presence of formic acid (5 mM) resulted in significantly lower conversion of all-trans retinal to retinol, indicating lower NADPH fractions.
Glucose and glutamine are the preferred metabolic substrates of mouse rod photoreceptors. Other amino acids can support metabolic activity only to a limited extent, perhaps due to the absence of efficient transporters or lack of the required intracellular metabolic machinery. Formic acid, the toxic metabolite of methanol, can significantly suppress rod photoreceptor metabolic activity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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