Light stimulates endocytosis of IRBP by the RPE

The mechanism by which IRBP specifically enhances the delivery and release of retinol by the RPE is unknown. Purpose. Does IRBP interact with the RPE through an endocytic mechanism? Methods. Immunoelectronmicroscopy and metabolic labelling techniques were used to determine the distribution of IRBP during light, dark and cyclic conditions in adult Xenopus. Our studies utilized a polyclonal antibody against recombinant IRBP expressed in E. coli. Post embedding immunoEM was performed on retinas processed in Epon, Lowicryl and LR White resins. Metabolic labelling was carried out by intraocular injection of [³⁵S]methionine. The specific activity as a function of time was determined by SDS-PAGE followed by phosphoimaging. The distribution of IRBP was characterized by immunopreciptation and Western blot analysis. Results. Under cyclic light, IRBP is cleared from the interphotoreceptor matrix according to a double exponential decay with half lives of 0.8 and 174 days. During constant light, the clearance of IRBP rom the matrix is accelerated compared to cyclic or constant dark conditions. ImmmunoEM shows that IRBP is internalized into endosomes within the basal RPE and during ROS phagocytosis. The number of endocytic vesicles appears to increase in the light. Conclusions. IRBP is cleared from the matrix by endocytosis and phagocytosis. Our data suggests that IRBP accomplishes the delivery and or release of retinol through a pathway involving its endocytosis by the RPE.