FIGURE 5.

The ratio Fex-380/Fex-340 of the fluorescence intensities excited by 380- and 340-nm light is indicative of the retinol fraction in the outer (A) and inner (B) segments of rod photoreceptors. The Fex-380/Fex-340 ratio characteristic of retinol was obtained by loading intact cells with 50 μm retinol (ROL). The Fex-380/Fex-340 ratio characteristic of retinal was obtained by loading intact cells deficient in both Rdh8 and Rdh12 with 50 μm retinal (RAL). The fluorescence of wild-type (wt) outer and inner segments, 30 min after bleaching, has a ratio consistent with high retinol. The ratio shifts to higher values, indicative of high retinal, for both outer and inner segments of all types of cells loaded with high concentrations of retinal (50 μm). Broken off rod outer segments (ROS) of all types of cells also have a high Fex-380/Fex-340 ratio, again indicating retinal. The outer segments of rods that contain Rdh8 (wild type and Rdh12^−/−) exhibit a ratio indicative of high retinol, even when loaded with moderate concentrations of retinal (RAL 5 μm). Rdh8 deficiency results in higher outer segment Fex-380/Fex-340 ratios, indicating high retinal, even with the endogenous chromophore only. The inner segments of cells that lack Rdh8 also exhibit higher ratios as well. Rdh12 deficiency results in higher inner segment Fex-380/Fex-340 ratios, indicating higher retinal, even with the endogenous chromophore only. The data shown are the means ± S.E., with n ≥ 5 cells. The temperature was 37 °C.