Abstract
Abstract: :
Purpose: Rhodopsin regeneration following a photobleach requires all–trans to 11–cis re–isomerization of the retinaldehyde chromophore. This process is called the visual cycle and takes place predominantly in cells of the retinal pigment epithelium (RPE). Little is known about how the visual cycle is regulated. We hypothesize that the visual cycle is regulated by light, and RGR is the sensor molecule in the RPE. Two modified systems that lack any input from photoreceptors are being used: human fetal RPE (hRPE) cultured cells and visual chromophore deficient rpe65–/– mice. Methods: For in vitro studies, hRPE cells with no detectable retinoids were grown as monolayers. The RNA interfering (RNAi) technique was used to achieve ∼75% suppression of RGR expression in the cells. Control and RGR siRNA–transfected cells were treated with physiological concentrations of vitamin A (atROL), preincubated overnight in the dark, and then exposed to light for two hours or maintained in darkness. The cells and media were extracted and analyzed by HPLC for retinoid content. For in vivo studies, we have generated rpe65–/–rgr–/– double knockout mice. Three month old rpe65–/–, rgr–/–, wild–type and rpe65–/–rgr–/– were analyzed under three conditions: (1) overnight dark–adaptation (DA), (2) two hours exposure to light and (3) two hours DA following light exposure. Retinoids were extracted from eyecup homogenates and analyzed by HPLC. Results: The RPE cells accumulated atRE’s during preincubation with atROL. Control cells show a light–dependent depletion of atRE’s. In contrast, the mobilization of atRE’s was diminished in RGR siRNA–transfected cells. Interestingly, similar levels of 11–cis–retinaldehyde (11cRAL) were detected in the apical media of RGR siRNA–transfected and control cells under both light exposure conditions. The levels of 11cRAL were twice as high in the light– compared to the dark–incubated cultures. As expected, the double knockout mice have similar atRE levels to rpe65 –/– and ∼20–fold more compared to WT mice. We observed dramatic mobilization (>250 pmol/eye) of atRE’s in rpe65 –/– mice exposed to light compared to mice maintained in darkness. Significantly, light–dependent mobilization of atRE’s was abolished in rpe65–/–rgr–/– mice. Conclusions: The RPE has never been shown to be capable of detecting light. Collectively, RPE–culture and mouse data suggest that RPE cells are inherently light sensitive. RGR seems to be the light–detecting pigment in a new signaling pathway that modulates the turnover of atRE’s in the RPE cells.
Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • opsins