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：白内障和后囊膜混浊（PCO）是主要的 消耗全世界的卫生预算，这是一个长期的问题。 该项目的目标是制定战略，防止这两种情况的发生。 条件 已知钙在皮质性白内障中起关键作用， 并且涉及蛋白酶钙蛋白酶的活化。 最近， 已经揭示钙在正常人透镜中也具有重要作用 通过内质网钙动员的细胞信号传导 通过G蛋白偶联受体储存。 这些受体的激动剂 包括ATP和乙酰胆碱，后者与白内障有关 阵 具体的目的是首先阐明激动剂诱导的位点 钙从完整的人透镜的细胞内储存中释放。 第二，定位钙库操纵的钙进入途径， 它们的激活和失活特性。 第三，本地化 其中钙的增加导致钙蛋白酶激活和 透明度，并测试钙蛋白酶拮抗剂。 最终目标是 开发和测试疏水性人工透镜涂层，以防止PCO，基于 钙细胞信号拮抗剂如毒胡萝卜素 （Tg）的温度下进行。 由于在透镜中存在相当大的种间差异， 行为项目设计的一个重要方面是开发 技术，包括长期培养，只使用人的透镜， 囊袋制剂。 技术包括共聚焦荧光（Fluo-3） 用于细胞钙动力学和电生理及放射性示踪剂的成像 表征钙进入途径的方法。 钙蛋白酶激活将 用荧光探针测量，透镜混浊定位， 通过数字成像进行定量，并通过 免疫细胞化学和凝胶电泳。 钙蛋白酶拮抗剂将是 测试以确定最好地保持透镜透明度的那些。 的 囊袋培养系统将是非常宝贵的测试， 眼内药物的生长抑制（从而抑制PCO）特性 用毒胡萝卜素包被的晶状体（IOL）。 3 H标记的Tg将用于 优化涂层工艺。 Tg涂层IOL消除 囊袋制剂中激动剂诱导的钙信号传导将作为 涂层功效的准确评估。 因此，这项研究的目的不仅是阐明钙的作用 在正常人的透镜，而且还确定预防策略， 皮质性白内障和后囊混浊