IP3 GENERATION INCREASES ROD OUTER SEGMENT PHAGOCYTOSIS BY CULTURED ROYAL-COLLEGE OF SURGEONS RETINAL-PIGMENT EPITHELIUM

Purpose. To measure outer segment phagocytosis in cultures of Royal Co llege of Surgeons (RCS) rat retinal pigment epithelium (RPE) that have been treated with carbachol. Carbachol treatment of RCS RPE results i n an increase in the second messenger inositol triphosphate, which mim ics that observed in normal RPE after interaction with rod outer segme nts (ROS). Methods. Cultures of RCS RPE were phagocytically challenged with isolated rat ROS for 2 hours. Carbachol (1 mM) was added to some cultures to stimulate inositol triphosphate synthesis, and incubation continued for 30 minutes at 37 degrees C. Inositol triphosphate conce ntration was measured by radioreceptor assay. Bound and ingested outer segments were quantified by double immunofluorescent staining. Ingest ion of outer segment membranes was confirmed by electron microscopy an d immunogold staining. Results. Carbachol treatment was associated wit h a rapid and temporary increase in inositol triphosphate levels. Roya l College of Surgeons rat RPE phagocytically challenged with outer seg ments and treated with carbachol showed significantly higher ingestion (34%) compared to untreated RCS RPE (9%)(P < 0.05). Conclusions. Expo sure of cultured RCS RPE to carbachol increases the intracellular conc entration of inositol triphosphate and enhances phagocytosis of bound ROS. These results support the hypothesis that the phagocytic defect i n RCS RPE is related to an abnormality in the generation of inositol t riphosphate as a second messenger after outer segment recognition and binding.