CIRCADIAN RETINAL MOTILITY ROLE OF CYCLIC NUCLEOTIDES

这项研究的主要目的是了解 光感受器和RPE细胞中的过程受光的调节， 昼夜节律和形态发生信号，即。要确定中的所有步骤 从生理信号引导的调节通路 最终达到适当的效应器系统(即，微管- 或基于肌动蛋白的细胞骨架)负责产生运动。 这项工作强调使用硬骨鱼视网膜运动作为一种 模型系统，因为有强大的定量分析可用， 允许分析细胞外和细胞内的影响 监管机构对这些动向的监管。从这些信息中获得的信息 视网膜运动研究将被用来加深我们对 对视网膜至关重要的其他光感受器和RPE运动过程 在所有物种中都有功能，如吞噬和形态发生。 上一次赠款期间的工作表明，细胞外 信使多巴胺通过诱导光适应来模拟光的启动 光感受器和RPE中的视网膜运动，以及 细胞内涉及钙离子、cAMP、 和蛋白激酶C有助于调节肌动蛋白-和 产生视网膜运动的微管依赖的过程 动静。本申请提出了以下具体方案 进一步澄清这些代理人在以下方面的作用的方法 光感受器和RPE运动的调节：1)确定 多巴胺和光是否对光感受器有类似的影响 和RPE环核苷酸水平通过确定 光照、黑暗和多巴胺对cAMP和cGMP水平的影响 光感受器片段(杆状内外节段、RIS-ROS和 视锥内外节段(CIS-COS)和分离的RPE细胞； 探讨钙离子、环磷酸腺苷和特定蛋白的作用 视杆细胞、视锥细胞和视网膜色素上皮细胞中调节视网膜运动的激酶 细胞通过进一步表征磷脂效应和通过 将裂解细胞运动模型研究扩展到视杆和RPE；3)扩展到 研究运动性RIS-ROS中蛋白质磷酸化的变化， 顺式-COS和RPE细胞对光、暗、 多巴胺、钙离子、cAMP和蛋白激酶C；4)以表征 RIS-ROS、CIS-COS和RPE细胞的细胞骨架成分 SDS PAGE、Western blotts和免疫定位；5)使用 免疫细胞化学检测细胞分布的变化 光感受器形态发生过程中的细胞骨架成分 RPE。

The primary objective of this research is to understand how motile processes in photoreceptors and RPE cells are regulated by light, circadian, and morphogenetic signals, ie. to identify all steps in the regulatory pathways which lead from the physiological signals ultimately to the appropriate effector systems (ie. microtubule- or actin-based cytoskeletons) responsible for producing movement. This work emphasizes use of teleost retinomotor movements as a model system since powerful quantitative assays are available which permit analysis of the effects of extra- and intra-cellular regulators on these movements. Information gained from these retinomotor studies will be used to further our understanding of other photoreceptor and RPE motile processes crucial to retinal function in all species, such as phagocytosis and morphogenesis. Work in the last grant period showed that the extracellular messenger dopamine mimics light onset by inducing light-adaptive retinomotor movements in photoreceptors and RPE, and that intracellular pathways involving specific interplays of Ca++, cAMP, and protein kinase C contribute to regulating the actin- and microtubule-dependent processes which produce retinomotor movements. The present application proposes the following specific approaches to further clarify the roles of these agents in regulation of photoreceptor and RPE motility: 1) To ascertain whether dopamine and light have similar effects on photoreceptor and RPE cyclic nucleotide levels by determining the effects of light, darkness, and dopamine on cAMP and cGMP levels in isolated photoreceptor fragments (rod inner-outer segments, RIS-ROS, and cone inner-outer segments, CIS-COS) and in isolated RPE cells; 2) To investigate the roles of Ca++, cAMP, and specific protein kinases in regulating retinomotor movements in rods, cones, and RPE cells by further characterization of phorbolester effects and by extending lysed cell motile model studies to rods and RPE; 3) To investigate changes in protein phosphorylation in motile RIS-ROS, CIS-COS, and RPE cells that occur in response to light, dark, dopamine, Ca++, cAMP, and protein kinase C; 4) To characterize the cytoskeletal components of RIS-ROS, CIS-COS, and RPE cells using SDS PAGE, Western Blots, and immunolocalization; 5) To use immunocytochemistry to identify changes in distribution of cytoskeletal components during morphogenesis of photoreceptors and RPE.