VISUAL PIGMENT AND PHOTORECEPTOR ADAPTATION

杆状暗适应需要失活和再生 漂白(光激活)视紫红质。视紫红质再生，即 11-顺式视网膜被视蛋白结合，已知依赖于循环 眼睛组织中的类维A酸。然而，有两个根本问题 与这一进程相关的问题仍未解决：(1) 限制体内再生的维甲酸循环事件，以及(2) 再生在停止漂白脱感中的作用。这个 拟议的项目通过生化和生物化学解决这两个问题 小鼠和蟾蜍的视网膜电信号(ERG)研究及ERG研究 关于人类受试者。一个主要的推动力是推断人体杆状物的恢复 基于与从鼠标获得的数据的相关性的机制。ERG 实验将采用一种新的、无创的、成对闪光的ERG方法 推导出活体棒对测试闪光的全部时间响应过程。 视网膜电信号衍生的人和小鼠视杆的动力学和灵敏度特性 响应将根据照明条件和 漂白后恢复时间。小鼠的平行生化实验 将决定维甲酸循环反应和视紫红质的动力学 再生。联合ERG和生化结果将用于测试 关于体内再生受以下因素限制的假说(1) 全反式视网膜的酶促还原在视杆细胞中的传递 11顺式视网膜至视杆，或它们的组合。数据将 同时解决(2，上图)再生事件是否关闭 漂白脱敏或仅仅重建最大量子 捕获效率。该项目的另一个重点是测试 最近的假说是二十二碳六烯酸(DHA)调节这种活性 光感受器间类视黄酸结合蛋白(IRBP)是一种胞外蛋白 (光感受器间基质；IPM)已知支持维甲酸的蛋白质 视网膜色素上皮(RPE)与视杆细胞之间的转移 视黄醇的视觉周期。这将通过测试效果来解决 添加DHA对IRBP类维甲酸转运活性的影响 和相关的重组制剂，并通过测试相关性 IPM内源性DHA水平与维甲酸周期的关系 蟾蜍和老鼠的眼睛。该项目的成果将从根本上促进 功能正常的视杆细胞暗适应恢复的知识。这个 ERG数据将为杆件回收研究提供直接基础 人类视网膜疾病和动物模型中的异常 转基因小鼠。

Rod dark adaptation requires the deactivation and regeneration of bleached (photoactivated) rhodopsin. Rhodopsin regeneration, i.e., the binding of 11-cis retinal by opsin, is known to depend on the recycling of retinoid within the eye tissues. However, two fundamental issues relevant to this process remain unresolved: (1) the identify of the retinoid cycle event that rate-limits regeneration in vivo, and (2) the role of regeneration in the shut-off of bleaching desensitization. The proposed project addresses these two issues through biochemical and electroretinographic (ERG) studies on mouse and toad, and in ERG studies on human subjects. A major thrust is to infer human rod recovery mechanisms based on correlations with data obtained from mouse. ERG experiments will employ a new, noninvasive, paired-flash ERG method to derive the full time course of the in vivo rod response to a test flash. Kinetic and sensitivity properties of ERG-derived human and mouse rod responses will be analyzed in relation to illumination conditions and post-bleach recovery time. Parallel biochemical experiments on mice will determine the kinetics of retinoid cycle reactions and rhodopsin regeneration. Combined ERG and biochemical results will serve to test the hypothesis (1, above) that regeneration in vivo is rate-limited by the enzymatic reduction of all-trans retinal in the rods, the delivery of 11-cis retinal to the rods, or a combination of these. The data will also resolve (2, above) whether the regeneration event shuts off bleaching desensitization or merely re-establishes maximal quantum capture efficiency. A further thrust of the project is to test the recent hypothesis that docosahexaenoic acid (DHA) regulates the activity of interphotoreceptor retinoid-binding protein (IRBP), an extracellular (interphotoreceptor matrix; IPM) protein known to support retinoid transfer between the retinal pigment epithelium (RPE) and the rods in the retinoid visual cycle. This will be addressed by testing the effect of added DHA on IRBP's retinoid-transfer activity in toad RPE-eyecups and related reconstituted preparations, and by testing for correlations between endogenous DHA level in the IPM and retinoid cycle operation in the toad and mouse eye. Results of the project will advance fundamental knowledge of dark-adaptation recovery in normally functioning rods. The ERG data will provide immediate foundation for studies of rod recovery abnormalities in human retinal disease and in animal models such as transgenic mice.