Abstract
Abstract: :
Purpose: Exposure to insecticides that result in Ch overstimulation has been associated with retinal degeneration. Ch overstimulation results in a decreased response of the IP 2nd messenger signaling pathway in retina and RPE. Studies in RCS rats suggest that dysfunction in this 2nd messenger pathway may play a role in the dysfunctional phagocytosis of rod outer segments (ROS) by the RPE, ultimately resulting in retinal degeneration. We hypothesize that a decrease in IP response due to Ch overstimulation will similarly result in decreased ROS phagocytosis. This abstract describes the initial steps in the development of this model, the effects of Ch overstimulation on IP release in cultured RPE cells. Methods: ARPE-19 cells, a human RPE cell line, were grown to confluence on cover slips in DMEM/F12 with 10% FBS (Osborne, et. al., 1991). 3H-myo-inositol was incorporated overnight with or without carbachol (10-7 to 10-1 M) to model cholinergic overstimulation as might occur in the presence of certain classes of pesticides. IP released during a 45 minute incubation with 1mM carbachol was separated by anion exchange chromatography. In addition, a carbachol dose response (0 (basal),10-5 to 10-3 M) was established for IP release in the absence of Ch overstimulation. Results: Stimulated IP release from cultured ARPE-19 cells increased with the concentration of carbachol. Pre-incubation with carbachol resulted in a dose-dependent decrease of the carbachol-stimulated response, but no change in basal release of IPs. Conclusions: ARPE-19 cells have a muscarinic-linked IP 2nd messenger pathway that responds well to carbachol stimulation. The decrease in stimulated release of IPs after cholinergic overstimulation in the absence of a change in basal release suggests that this decrease is due to downregulation at either the receptor or 2nd messenger level. Further work will investigate the effect of cholinergic overstimulation and decreased IP release on ROS phagocytosis. This abstract does not reflect USEPA policy.
Keywords: retinal pigment epithelium • retinal degenerations: cell biology • second messengers: pharmacology/physiology