Formation of all-trans retinol after visual pigment bleaching in mouse photoreceptors

To test whether the formation of all-trans retinol limits the regeneration of the visual pigment. all-trans retinol is formed after visual pigment bleaching through the reduction of all-trans retinal in a reaction involving NADPH. This reduction begins the recycling of the chromophore for the regeneration of the visual pigment.

Experiments were performed with dark-adapted, isolated retinas and isolated photoreceptor cells from wild-type and Nrl(-/-) mice. The photoreceptors of Nrl(-/-) mice are conelike and contain only cone pigments. The formation of all-trans retinol after pigment bleaching was measured by quantitative HPLC of retinoids extracted from isolated retinas and by imaging the fluorescence of retinol in photoreceptor outer segments. Experiments were performed at 37 degrees C.

In rods, the formation of all-trans retinol proceeded with first-order kinetics, with a rate constant of 0.06 +/- 0.02 minute(-1), significantly faster than the reported rate constant for rhodopsin regeneration. In Nrl(-/-) photoreceptors, the formation of all-trans retinol occurred at least 100 times faster than in rods. For both cell types, the fraction of all-trans retinal converted to all-trans retinol at equilibrium is approximately 0.8, indicating the presence of a similar fraction of reduced NADPH.

Formation of all-trans retinol does not limit the regeneration of bleached visual pigment. Formation of all-trans retinol in the cone-like Nrl(-/-) photoreceptors is much faster than in rods, consistent with a faster regeneration of the visual pigment after bleaching. Different types of photoreceptors contain a comparable fraction of reduced NADPH to drive the reduction of all-trans retinal.