CALCIUM AND THE NORMAL LENS AND CATARACT

钙与正常晶状体和白内障

• 批准号: 2164502
• 负责人: GEORGE DUNCAN
• 金额: $ 7.28万
• 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 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2164502
• 关键词: SDS polyacrylamide gel electrophoresis; acetylcholine; calcium; calcium channel; calcium flux; calcium transporting ATPase; calpain; cataract; cytoskeletal proteins; electrophysiology; eye pharmacology; fluorescent dye /probe; homeostasis; human tissue; immunofluorescence technique; ionophores; laboratory rat; lens; light scattering; mathematical model; oxidative stress; phosphatase inhibitor; radiotracer; second messengers; tissue /cell culture; SDS polyacrylamide gel electrophoresis; acetylcholine; calcium; calcium channel; calcium flux; calcium transporting ATPase; calpain; cataract; cytoskeletal proteins; electrophysiology; eye pharmacology; fluorescent dye /probe; homeostasis; human tissue; immunofluorescence technique; ionophores; laboratory rat; lens; light scattering; mathematical model; oxidative stress; phosphatase inhibitor; radiotracer; second messengers; tissue /cell culture

Calcium plays a critical role in a variety of cell-signalling systems and is increasingly recognised to play a significant part in athe development of cortical cataract. The main aims of this project will be to investigate the mechanisms for achieving calcium homeostasis in the lens and also the sensitivity of these mechanisms to external insult. A further aim will be to elucidate the role of the calcium-activated enzyme, calpain, in the production of lens opacities. Calcium homeostasis will be studied, principally using human lens tissues, but also on athe rat lens in vitro, by a combination of elecrophysiological, radiotracer and fluorimetric dye techniques. Thapsigargin, which selectively inhibits the CaATPase of the endoplasmic reticulum, will be an invaluable tool in these investigations and mathematical modelling techniques will be used to interpret 45Ca flux data from whole human lenses. The sensitivity of calcium permeability (channel) pathways to oxidative insult will also be studied in detail in whole human and rat lenses and in tissue cultured cells. A cation channel antibody will also be used with a combination of immunofluorescence and gold-labelling technique to study the localization of cation channels in lens epithelia and fibre cells. Increases in cytosolic calcium in single lens cells and in whole rat and human lenses will be induced by calcium ionophore (A23187), external membrane SH complexation (pCMPS) and calcium signalling agonists (acetylcholine) and will be quantitatively assayed by the above technique. The effect of these agents on lens transparency will be quantified in whole lens experiments by imaging scattered light. The effect on the lens cytoskeleton (principally actin, tubulin, vimentin and spectrin) will be studied by a combination of DSS-PAGE electrophoresis and immunofluorescence localization techniques. A concomitant study of the colocalization of lens cation channels and cytoskeletal elements will be carried out both in normal and calcium overloaded lens tissues. This study will yield valuable information both on the mechanisms whereby calcium is controlled and also the mechanism responsible for calcium- induced proteolysis and opacification following loss of control. During the course of the project a combined approach will be made that aims both to reduce calcium overload by channel blocking agents and to prevent calcium-induced proteolysis by anti-calpain compounds.

钙在各种细胞信号系统中起着至关重要的作用 越来越多地认识到在Athe Development中发挥重要作用 皮质性白内障。 该项目的主要目的是调查 在镜头中实现钙稳态的机制以及 这些机制对外部侮辱的敏感性。 另一个目标是 阐明钙激活酶，钙蛋白酶在 晶状体的生产。 钙稳态将被研究， 主要是使用人透镜组织，但也使用体外大鼠晶状体， 结合元生理，放射性示例和荧光染料 技术。 Thapsigargin，它有选择地抑制了 内质网将是这些研究的宝贵工具 数学建模技术将用于解释45CA通量 来自整个人类镜头的数据。 钙渗透性的敏感性 （渠道）氧化侮辱的途径也将详细研究 整个人类和大鼠透镜以及组织培养的细胞中。 阳离子通道 抗体还将与免疫荧光和 黄金标志性技术用于研究阳离子通道的定位 晶状体上皮和纤维细胞。 单镜细胞和整个大鼠的胞质钙增加 人透镜将由钙离子载体（A23187）诱导，外部 膜sh络合物（PCMP）和钙信号传导激动剂 （乙酰胆碱），并将通过上述技术进行定量测定。 这些药物对镜头透明度的影响将在整体上进行量化 透镜通过成像散射光进行实验。 对镜头的影响 细胞骨架（主要是肌动蛋白，微管蛋白，波形蛋白和光谱）将是 通过DSS-PAGE电泳和免疫荧光的组合研究 本地化技术。 晶状体共定位的伴随研究 阳离子通道和细胞骨架元素将在 正常和钙超负荷透镜组织。 这项研究将产生有关机制的宝贵信息 钙是控制的，也是负责钙的机制 失去控制后，诱导的蛋白水解和不透明。 期间 该项目的过程将采用合并的方法，以实现这两个方法 通过通道阻断剂减少钙过载，并防止 抗钙蛋白酶化合物钙诱导的蛋白水解。