RETINAL ATTACHMENT AND OUTER SEGMENT TURNOVER IN VITRO

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The studies outlined in this proposal focus upon mechanisms which may be responsible for maintaining the attachment of visual cells to the retinal pigment epithelium (RPE). Two independent procedures will be utilized to assess the relative strength of adhesive interactions under a wide range of physiological conditions. First, methods for experimentally detaching Xenopus laevis retinas and reconstituting their interaction with monolayer cultures of RPE will be used to measure spontaneous and L-glutamate-induced tissue reassociation in vitro. Second, a peeling technique, which assays the tendency of apical RPE cytoplasts to partition with mechanically detached retinas, will be utilized to distinguish normal retinal adhesion from the enhanced adhesiveness apparent during active turnover of outer segment membranes. Overall, experiments have been designed to test the involvement of two general mechanisms which have been proposed as determinants of retinal attachment: (1) force generated by active ion- coupled H2O transport by the RPE and (2) formation of non-covalent receptor-ligand bonds between interacting cell surfaces. The possibility that transport-induced transretinal fluid flux influences photoreceptor- epithelium interdigitation will be examined by correlating electrical and ion transport properties of RPE monolayers with their ability to support retinal reapposition. In addition, we will attempt to modulate cell interaction using pharmacological manipulations which previously have been established to alter epithelial fluid transport. Alternatively, involvement of receptor-ligand interactions will be evaluated using highly sensitive and selective immunochemical reagents. Heteroantisera directed against epithelial and photoreceptor surfaces will be generated in rabbits and, after appropriate selection procedures, used as probes of retinal reattachment and uptake of shed outer segment discs. These immunoglobulin molecules will in turn be used as tools for possible biochemical isolation of the cell constituents involved. It is hoped that information gained through this research will lead to better strategies for managing retinal detachment and encouraging photoreceptor-RPE attachment.