Abstract
Abstract: :
Purpose: Regeneration of rhodopsin 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. Here, we show that at least one enzymatic step in the visual cycle is regulated by light. Methods: For in vitro studies, human fetal RPE cells were grown as monolayers on polycarbonate membranes. We added all–trans–retinol (atROL) at 5 µM to the culture media containing BSA (1%), preincubated in dark for three hours, then exposed cells to fluorescent light at 2000 lux for two hours or maintained in darkness. The light–exposed cultures were protected from UV radiation with a plexiglass shield. The cells and media were extracted and analyzed by high performance liquid chromatography (HPLC) for retinoid content. For in vivo studies, wild–type and rpe65–/– mice were dark–adapted overnight, then exposed to fluorescent light at 800 lux for two hours or maintained in darkness. Retinoids were extracted from eyecup homogenates and analyzed by HPLC. Results: Untreated RPE cells contained no endogenous retinoids by HPLC analysis. The cells accumulated all–trans–retinyl esters (atRE’s) during the three hours preincubation with atROL. Cells kept in darkness continued to accumulate atRE’s during the two hours of additional incubation. Cells exposed to light, however, had 60% lower levels of atRE’s compared to cells incubated in the dark. Also, levels of atROL were 40% lower in the light– compared to dark–incubated cells. Interestingly, levels of 11–cis–retinaldehyde in the media were nearly twice as high in the light– compared to the dark–incubated cultures. As reported, atRE levels were much higher in eyecups from rpe65 –/– compared to age–matched wild–type mice. Four–month–old rpe65 –/– mice exposed to light at 800 lux showed a 40% reduction in atRE’s compared to age–matched rpe65 –/– mice maintained in darkness. Similar reductions in atROL were also seen in light– versus dark–adapted rpe65 –/– mice. In contrast, atRE’s increased by 2.7–fold in light– versus dark–adapted wild–type mice. Conclusions: These results suggest that RPE cells are inherently light sensitive, and that RPE cells regulate the turnover of atRE’s in a light–dependent fashion. This light–dependent regulation of the visual cycle is not mediated by photoreceptors. Presumably, this novel regulatory pathway uses one of the non–photoreceptor opsins (RGR or peropsin) in RPE cells as a light detector.
Keywords: retinal pigment epithelium • retinoids/retinoid binding proteins • opsins