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Y. Koutalos, C. Chen, L. Blakeley, R. K. Crouch, G. H. Travis, J. Chen, C.-K. Chen; Formation of All-Trans Retinol in the Rod Outer Segments of Wild Type, ABCR Knockout, Rhodopsin Kinase Knockout, and Arrestin Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5635.
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To examine the reduction of all-trans retinal to retinol in living mouse rod photoreceptors following visual pigment bleaching. To examine the role of enzymatic defects that can influence the release of all-trans retinal from bleached rhodopsin and its access to retinol dehydrogenase on retinol formation.
Experiments were carried out with isolated dark-adapted retinas from wild type, ABCR knockout, rhodopsin kinase knockout, and arrestin knockout mice. Animals were 8-10 weeks old. All-trans retinol formation after rhodopsin bleaching was measured by quantitative HPLC of retinoids extracted from isolated retinas and by imaging the fluorescence of retinol (Ex: 360 nm; Em: >420 nm) in the rod outer segments of living retinal slices. Experiments were carried out at room temperature and at 37 0C.
The fraction of all-trans retinal converted to retinol was 2-3 times higher at 37 0C than at room temperature. In the presence of 5 mM glucose, the formation of retinol was not affected by the O2 tension. Pyruvate (1 mM) could partially support the formation of retinol in the absence of glucose. The overall kinetics and levels of retinol formation were similar for wild type and the three types of knockout mice.
In mouse rod photoreceptors, metabolic activity plays a critical role in the formation of retinol. The NADPH necessary for the reduction of all-trans retinal to retinol can be supplied from outside the rod outer segment. The enzymatic defects present in the three types of knockout animals do not affect the formation of retinol.
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