C A Heth1, P A Marescalchi. 1. Berman-Gund Laboratory for the Study of Retinal Degeneration, Massachusetts Eye and Ear Infirmary, Boston 02114.
Abstract
PURPOSE: To evaluate the generation of the second messenger molecule inositol triphosphate (IP3) in response to phagocytic challenge in cultures of retinal pigment epithelium (RPE) prepared from normal Long Evans or dystrophic Royal College of Surgeons (RCS) rats. METHODS: RPE cultures were phagocytically challenged with isolated rat rod outer segments (ROS) or polystyrene latex spheres (PSL). Carbachol was added to some cultures as a positive control to stimulate the IP3 pathway. Inositol triphosphate levels in RPE cells were analyzed by high-pressure liquid chromatography and radioreceptor assay. Incorporation of 3H-myoinositol into phosphatidylinositol lipids was analyzed by thin layer chromatography and autoradiography. RESULTS: Long Evans RPE phagocytically challenged with ROS or exposed to carbachol showed a significant increase in IP3 levels compared to unchallenged control cultures or cultures phagocytically challenged with PSL. RCS RPE cells did not show an increase in IP3 levels upon phagocytic challenge with ROS or PSL. Carbachol treatment of RCS RPE produced an increase in IP3 levels, demonstrating that the components of the IP3 pathway are present and the pathway can be activated. Phagocytic challenge of Long Evans RPE with ROS was associated with a decrease in the IP3 precursor, phosphatidylinositol bisphosphate (PIP2). No decrease in PIP2 was observed in RCS RPE incubated with ROS. CONCLUSIONS: These results show that phagocytic challenge of Long Evans RPE with ROS is associated with PIP2 hydrolysis and subsequent generation of IP3 as a second messenger. In RCS RPE, ROS stimulation of the IP3 pathway is absent.
PURPOSE: To evaluate the generation of the second messenger molecule inositol triphosphate (IP3) in response to phagocytic challenge in cultures of retinal pigment epithelium (RPE) prepared from normal Long Evans or dystrophic Royal College of Surgeons (RCS) rats. METHODS: RPE cultures were phagocytically challenged with isolated rat rod outer segments (ROS) or polystyrene latex spheres (PSL). Carbachol was added to some cultures as a positive control to stimulate the IP3 pathway. Inositol triphosphate levels in RPE cells were analyzed by high-pressure liquid chromatography and radioreceptor assay. Incorporation of 3H-myoinositol into phosphatidylinositol lipids was analyzed by thin layer chromatography and autoradiography. RESULTS: Long Evans RPE phagocytically challenged with ROS or exposed to carbachol showed a significant increase in IP3 levels compared to unchallenged control cultures or cultures phagocytically challenged with PSL. RCS RPE cells did not show an increase in IP3 levels upon phagocytic challenge with ROS or PSL. Carbachol treatment of RCS RPE produced an increase in IP3 levels, demonstrating that the components of the IP3 pathway are present and the pathway can be activated. Phagocytic challenge of Long Evans RPE with ROS was associated with a decrease in the IP3 precursor, phosphatidylinositol bisphosphate (PIP2). No decrease in PIP2 was observed in RCS RPE incubated with ROS. CONCLUSIONS: These results show that phagocytic challenge of Long Evans RPE with ROS is associated with PIP2 hydrolysis and subsequent generation of IP3 as a second messenger. In RCS RPE, ROS stimulation of the IP3 pathway is absent.