CALCIUM AND THE NORMAL LENS AND CATARACT

钙在各种细胞信号系统中起着关键作用， 越来越多的人认识到， 皮质性白内障 该项目的主要目的是调查 在透镜中实现钙稳态的机制以及 这些机制对外部损伤的敏感性。 另一个目标是 为了阐明钙激活酶calpain在 产生透镜混浊。 将研究钙稳态， 主要使用人的透镜组织，但也可以在体外对大鼠的透镜进行， 通过电生理学、放射性示踪剂和荧光染料的组合 技术. Thapsigargin选择性地抑制细胞膜上的CaATPase， 内质网，将是一个宝贵的工具，在这些调查 并利用数学模型技术解释~（45）Ca通量 整个人类晶状体的数据。 钙离子渗透敏感性 （通道）途径氧化损伤也将详细研究， 整个人类和大鼠晶状体和组织培养细胞中。 阳离子通道 抗体也将与免疫荧光和免疫荧光的组合一起使用。 金标记技术，以研究阳离子通道的定位， 透镜上皮细胞和纤维细胞。 单个透镜细胞和整个大鼠中的细胞溶质钙增加， 人晶状体将被钙离子载体（A23187）诱导， 膜SH络合（pCMPS）和钙信号激动剂 （乙酰胆碱），并将通过上述技术定量测定。 这些试剂对透镜透明度的影响将被整体量化 透镜实验通过成像散射光。 对透镜的影响 细胞骨架（主要是肌动蛋白、微管蛋白、波形蛋白和血影蛋白）将被 用DSS-PAGE电泳和免疫荧光相结合的方法研究 本地化技术。 透镜共定位的伴随研究 阳离子通道和细胞骨架元素将在 正常和钙超载的透镜组织。 这项研究将产生有价值的信息，无论是在机制， 钙是受控制的，也是负责钙的机制， 在失去控制后诱导蛋白水解和浑浊化。 期间 在项目过程中，将采取一种综合方法， 通过通道阻断剂减少钙超载， 抗钙蛋白酶化合物引起的钙诱导的蛋白水解。

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.