CALCIUM AND THE NORMAL LENS AND CATARACT

钙与正常晶状体和白内障

• 批准号: 2888446
• 负责人: GEORGE DUNCAN
• 金额: $ 8.56万
• 依托单位: UNIVERSITY OF EAST ANGLIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2888446
• 关键词: biological signal transduction; calcium flux; calpain; cataract; cell growth regulation; confocal scanning microscopy; disease /disorder prevention /control; electrophysiology; endoplasmic reticulum; enzyme activity; enzyme induction /repression; enzyme inhibitors; fluorescent dye /probe; gel electrophoresis; human tissue; immunocytochemistry; lens; lens disorder; membrane permeability; organ culture; radionuclides; surface coating; thapsigargin

DESCRIPTION: Cataract and posterior capsule opacification (PCO) are major drains on health budgets throughout the world and it is a long-term objective of this project to develop strategies to prevent both of these conditions. Calcium is known to play a critical role in cortical cataract, and activation of the protease calpain is implicated. More recently it has been revealed that calcium also has an important role in normal human lens cell signaling through calcium mobilization from endoplasmic reticulum stores, via G-protein coupled receptors. Agonists for these receptors include ATP and acetylcholine and the latter has been implicated in cataract formation. The specific aims are firstly to elucidate the sites of agonist-induced calcium release from intracellular stores in the intact human lens. Secondly to localize the store-operated calcium entry pathways and study their activation and inactivation characteristics. Thirdly to localize where a calcium increase leads to calpain activation and loss of transparency, and to test calpain antagonists. The final aim will be to develop and test hydrophobic intraocular lens coatings to prevent PCO, based on knowledge of calcium cell signaling antagonists such as thapsigargin (Tg). Since there are considerable interspecies differences in lens behaviour a significant aspect of project design has been the development of techniques, including long-term culture, employing only human lens and capsular bag preparations. Techniques include confocal fluorescent (Fluo-3) imaging for cell calcium dynamics and electrophysiological and radiotracer methods to characterize the calcium entry pathways. Calpain activation will be measured with a fluorescent probe, lens opacification localized and quantified by digital imaging, and proteolysis studied by immunocytochemistry and gel electrophoresis. Calpain antagonists will be tested to determine those which best preserve lens transparency. The capsular bag culture system will be invaluable for testing the growth-inhibiting (and hence PCO inhibiting) properties of intraocular lenses (IOLs) coated with thapsigargin. 3H-labeled Tg will be used to optimize the coating technology. The ability of Tg-coated IOLs to abolish agonist-induced calcium signaling in capsular bag preparations will serve as an accurate assessment of coating efficacy. This investigation aims therefore not only to elucidate the role of calcium in the normal human lens, but also to identify strategies for preventing both cortical cataract and PCO based on that knowledge.

描述：白内障和后胶囊不透明（PCO）是主要的 全世界的健康预算排出，这是一件长期的 该项目的目的是制定策略以防止这两种 状况。 已知钙在皮质白内障中起关键作用， 并涉及蛋白酶钙蛋白酶的激活。 最近它有 被发现钙在正常人镜头中也具有重要作用 细胞信号通过内质网的钙动员通过 商店，通过G蛋白偶联受体。 这些受体的激动剂 包括ATP和乙酰胆碱，后者与白内障有关 形成。 具体目的首先是为了阐明激动剂引起的位置 钙从完整的人透镜中的细胞内存储中释放。 其次，要定位商店经营的钙进入途径并进行研究 它们的激活和灭活特征。 第三要本地化 钙增加导致钙蛋白酶的激活和丧失 透明度，并测试钙蛋白酶拮抗剂。 最终目标是 开发和测试疏水性透镜涂料以防止PCO，基于PCO 关于钙细胞信号传导拮抗剂（例如thapsigargin）的知识 （TG）。 由于镜头有很大的种间差异 行为项目设计的一个重要方面是发展 技术，包括长期文化，仅采用人类镜头和 囊袋准备。 技术包括共聚焦荧光（Fluo-3） 细胞钙动力学以及电生理和放射性示例的成像 表征钙进入途径的方法。 钙蛋白酶的激活将 用荧光探针测量，镜头不适局部，并 通过数字成像和蛋白水解量化 免疫细胞化学和凝胶电泳。 钙蛋白酶对手会 测试以确定最能保持透明度的透明度的测试。 这 囊袋培养系统对于测试将是无价的 眼内抑制生长（因此抑制PCO）特性 透镜（IOL）涂有Thapsigargin。 3H标记的TG将用于 优化涂料技术。 TG涂层IOL废除的能力 囊袋制剂中激动剂引起的钙信号传导将作为 准确评估涂料功效。 因此，这项研究的目的不仅旨在阐明钙的作用 在正常的人类镜头中，但也确定防止的策略 基于这些知识的皮质性白内障和PCO均均可得到。