Localizations of visual cycle components in retinal pigment epithelium

PURPOSE: We used immunocytochemistry and confocal microscopy to determine whether enzymes of the rod visual cycle were uniformly distributed in retinal pigment epithelium (RPE) cells. The localizations of these enzymes were compared to known localizations of retinoid-binding proteins and associated proteins. METHODS: Antibodies to proteins and enzymes associated with the rod visual cycle were used for fluorescence immunocytochemistry with frozen sections of albino mouse and rat retina. Images were obtained with a laser scanning confocal microscope. RESULTS: Components associated with the rod visual cycle were distributed in three distinct patterns in mouse and rat RPE. Three visual cycle enzymes (RDH5, LRAT, and RPE65) were restricted to the somata of RPE cells and were not detected within apical processes. Ezrin, an actin-binding protein, and ERM-binding phosphoprotein50/sodium-hydrogen exchanger regulatory factor1 (EBP50/NHERF1), an ezrin-binding PDZ-domain protein, were largely restricted to RPE apical processes. The fluorescence intensity over Müller cell apical processes was less intense. Cellular retinaldehyde-binding protein (CRALBP), which binds to EBP50/NHERF1, and cellular retinol-binding protein type 1 (CRBP1) were found throughout RPE cells and Müller cells. CONCLUSIONS: Visual cycle enzymes were confined to the somata of RPE cells and did not occur within the long apical processes, either in dark- or light-adapted animals. Other components previously linked to the visual cycle (EBP50/NHERF1 and ezrin) were largely confined to the apical processes, where they could be associated with release of 11-cis-retinal or uptake of all-trans-retinol. CRALBP and CRBP1 were distributed throughout the RPE cell, where they could mediate diffusion of retinoids between apical processes and somata.