RETINAL ATTACHMENT AND OUTER SEGMENT TURNOVER IN VITRO

本提案中概述的研究侧重于以下机制 负责维持视觉细胞与视网膜的连接 色素上皮(RPE)。将使用两个独立的程序来 在较大范围内评估粘合剂相互作用的相对强度 生理条件。第一，试验性分离的方法 非洲爪哇视网膜及其与单层的相互作用重建 RPE的培养将被用来测量自发的和L诱导的谷氨酸 组织在体外重新结合。第二，剥离技术，它分析 顶端RPE胞质机械性分割的趋势 视网膜脱离，将被用来区分正常的视网膜粘连 从增强的粘附性在活跃的周转期间表现出来的 分段膜。总体而言，设计的实验是为了测试 提议的两个一般机制的参与情况如下 视网膜附着的决定因素：(1)活性离子产生的力- RPE对H2O的耦合传输和(2)非共价化合物的形成 相互作用的细胞表面之间的受体-配体键。这种可能性 运输诱导的经视网膜液体流量影响光感受器- 上皮交错的检查将通过相互关联的电和 RPE单分子膜的离子传输特性及其负载能力 视网膜重位。此外，我们将尝试对细胞进行调制 使用药物操作的相互作用，以前已经 建立来改变上皮液的运输。或者， 受体-配体相互作用的参与将使用High 敏感和选择性的免疫化学试剂。定向异源抗血清 在兔体内会产生抗上皮细胞表面和光感受器表面 并且，经过适当的选择程序，用作视网膜的探针 脱落的外节间盘的重新附着和吸收。这些免疫球蛋白 分子反过来将被用作可能的生化分离工具。 所涉及的细胞成分。希望所获得的信息 通过这项研究将导致更好的管理视网膜的策略 脱离和鼓励光感受器-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.