RETINAL ATTACHMENT AND OUTER SEGMENT TURNOVER IN VITRO

本提案中概述的研究侧重于可能 负责维持视细胞与视网膜的附着 色素上皮（RPE）。 将使用两个独立的程序， 评估在宽范围内粘合剂相互作用的相对强度， 生理条件。 第一，实验性分离的方法 非洲爪蟾视网膜及其与单层膜相互作用的重建 RPE的培养物将用于测量自发的和L-谷氨酸诱导的 体外组织再结合。 第二，一种剥皮技术， 顶端RPE细胞质的机械分配倾向 视网膜脱离，将用于区分正常的视网膜粘连 从外部积极周转期间明显的增强 节膜。 总的来说，已经设计了实验来测试 两个一般性机制的参与， 视网膜附着的决定因素：（1）活性离子产生的力- 通过RPE的耦合H2O运输和（2）形成非共价的 相互作用的细胞表面之间的受体-配体键。 的可能性 运输诱导的跨视网膜液体流量影响光感受器， 将通过相关的电学和生物学特性来检查上皮交错， RPE单分子膜的离子输运性质及其支持能力 视网膜复位 此外，我们将尝试调节细胞 使用药理学操作的相互作用， 改变上皮细胞的液体运输。 可选择地， 受体-配体相互作用的参与将使用高度 敏感和选择性免疫化学试剂。 定向异源抗血清 将在兔中产生针对上皮和感光器表面的 并在适当的选择程序后，用作视网膜的探针， 脱落的外节盘的再附着和吸收。 这些免疫球蛋白 分子将反过来被用作可能的生化分离的工具， 所涉及的细胞成分。 希望获得的信息 通过这项研究将导致更好的战略，管理视网膜 脱离并促进光感受器-RPE附着。

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.