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L. R. Blakeley, K. Masutomi, R. K. Crouch, K. Nakatani, Y. Koutalos; Phototoxicity of Physiologically Generated All-Trans Retinal in Isolated Living Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3259.
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To determine the phototoxic potential of physiologically generated all-trans retinal, an obligate byproduct of the detection of light by the visual pigment.
Experiments were carried out with rod outer segment (ROS) membranes purified from bovine retinas, and isolated living rod photoreceptors obtained from frog (Rana pipiens) retinas. Photooxidation was measured as the lipid peroxidation induced by 360 nm light. Lipid peroxidation in ROS membranes was measured with the thiobarbituric acid assay and from the oxidation of the fluorescent dye BODIPY C-11; the two methods displayed excellent correlation (R=0.95). Lipid peroxidation in isolated living cells was measured with fluorescence imaging from the oxidation of internalized BODIPY C-11. The oxidation of BODIPY C-11 was measured from the shift in fluorescence between the intact (Ex: 555 nm; Em: 617 nm) and oxidized (Ex: 490 nm; Em: 528 nm) forms. Photooxidation was measured before, immediately after, and 1 hour after the bleaching of rhodopsin.
Photooxidation in pre-bleached ROS membranes increased with the concentration of exogenously added all-trans retinal. With dark-adapted ROS membranes, photooxidation was highest 1 hour after the bleaching of rhodopsin. In isolated frog photoreceptors, photooxidation was highest in metabolically compromised rod outer segments and 1 hour after the bleaching of rhodopsin. Metabolically compromised rod outer segments could not efficiently convert the all-trans retinal to retinol through reduction by the retinol dehydrogenase.
All-trans retinal released from bleached rhodopsin can mediate light-induced lipid peroxidation in purified ROS membranes and in isolated living rod photoreceptors. The removal of all-trans retinal through reduction to all-trans retinol by retinol dehydrogenase protects photoreceptors against light-induced damage.
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