Relationship Between Reduction of All-Trans Retinal to All-Trans Retinol and Dark-Adaptation in Salamander Rods

Abstract

Abstract: : Purpose: To investigate the relationship between retinal to retinol reduction, following visual pigment bleaching and its relationship to recovery of dark current and sensitivity in rod photoreceptors. Methods: Rods were isolated from dark-adapted salamander retinae and mounted in a superfusion chamber on the stage of an inverted microscope equipped with a high sensitivity digital CCD camera. Because the cells were separated from the retinal pigment epithelium, no regeneration of visual pigment was possible. The spatial distribution of retinol fluorescence within the rod outer segments (excitation: 360 nm, emission: 480nm-540 nm) was measured before and following bleaching (520 nm). Measurement of extracellular membrane current in these same photoreceptors was made simultaneously before and following the bleaching exposure. Results: A persistent fluorescence attributable to all-trans retinol developed slowly in the outer segments of rods following light exposure that photoactivated >99% of the pigment. Fluorescence increased with a time constant of 12.0±1.4 min (SEM, N=7). The recovery of sensitivity and dark current was followed in these same cells. Following exposure to the bleaching light, a period of about 10 min elapsed, during which the response was refractory. After this period, the dark current recovered with a time constant of 12.6±1.1 min (SEM, N=7). These measurements were compared to similar measurements made on rods in the presence of 100µM interphotoreceptor retinoid-binding protein (IRBP) added to the superfusate. In this solution, the maximum fluorescence was only 20% of control, and returned close to the pre-bleach baseline within 20-30 min. The time constant for the recovery of dark current in IRBP solution was 8.5±1.0 min (SEM, N=7). Our previous measurements of bleach-induced fluorescence in salamander rod outer segments have shown that IRBP facilitates removal of all-trans retinol from rods. Conclusions: We demonstrate a positive correlation between the reduction of all-trans retinal to all-trans retinol and the recovery of sensitivity in rod photoreceptors. Furthermore, we show that the presence of IRBP both facilitates the removal of all-trans retinol from the rods and speeds up the time course of recovery of dark current.

Keywords: photoreceptors • electrophysiology: non-clinical • retinoids/retinoid binding proteins