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P. Ala–Laurila, A.V. Kolesnikov, R.K. Crouch, S.A. Shukolyukov, V.I. Govardovskii, M.E. Estevez, M.C. Cornwall; Characterization of the Early Steps of Rod and Cone Visual Cycle: Retinol Formation Rate Is Dependent on Metapigment Decay and RDH . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2037.
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To determine the rate–limiting steps of retinol production in rod and cone photoreceptors.
Measurements were performed on photoreceptors isolated from the dark–adapted retinae of the tiger salamander (Ambystoma tigrinum) and the nocturnal gecko (Gekko gekko). The appearance of all–trans retinol within photoreceptor outer segments (OS) was monitored by microfluorometric measurements after bleaching (>90%) of the visual pigments. The photoproducts of photolysis (i.e. metapigments I/II/III, retinal and retinol) were followed under the identical experimental conditions using a fast–scanning dichroic microspectrophotometer.
Metapigment decay rate (retinal release) was spatially uniform within all outer segments measured, whereas the rate of retinol production was strongly position–dependent, being fastest at the base of OS. Metapigment decay rate was 50–90 times faster in both rod and cone cells containing cone pigments (SWS2, M/LWS) compared to cells with rod pigment (RH1). However, the rate of reduction of retinal to retinol was only ca. 10–20 times faster in cones with cone pigments (SWS2, M/LWS) and ca. 3–5 times faster in rods containing SWS2 or M/LWS cone pigments than in the basal OS of rods containing RH1. We created a kinetic model of retinal–to–retinol conversion and subsequent clearance of retinol.
The rate of retinol production is defined by the metapigment decay rate and the RDH reaction rate. Either of these can be the rate–limiting step depending on the cell type and the OS region. In the basal region of the OS, high rates of retinol production correlate with fast metarhodopsin decay rates, whereas in the distal part the RDH reaction becomes always rate–limiting. We hypothesize that the varying rate of retinol production along the OS is due to the delivery of the reducing agent, NADPH, required for the RDH reaction.
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