Retinoid changes in the in vitro regeneration of frog visual pigments

To investigate the regeneration of visual pigment, the changes in composition and quantity of retinoids were measured by high-performance liquid chromatography (HPLC). Eye cups or eye cup sections of dark-adapted frogs were exposed to light (greater than 500 nm) and incubated in the dark for several hours (pH 7.4, 27 +/- 1 degree C). Retinoids extracted by the oxime method before and after illumination were analysed by HPLC. In the dark, every eye cup or eye cup section contained 11-cis and all-trans isomers of retinyl palmitate (the proportion of 11-cis was nearly 40%). The amount of retinyl palmitate was 1-1.5 mol equiv of visual pigment. After 80% of the visual pigment had been bleached by illumination, eye cups or eye cup sections were incubated in the dark for 10 h. During the incubation, 70% of the bleached pigment was regenerated, and the proportion of 11-cis retinyl ester decreased from 40% to 13%. These results indicate that stored 11-cis retinyl ester is used for the regeneration. The regeneration rate of A2-pigment (half time = 75 min) was faster than that of A1-pigment (half time = 90 min), consistent with the result of Tsin & Flores' (1986) in vivo experiment with goldfish.