ATP LINKED EFFECTORS OF NA+, K+ ATPASE AND CATARACTS

描述：该项目的长期目标是确定 晶状体Na，K-ATPase及其ATP连接的效应物(H+，Na+，K+，ATP，ADP， 和洋地黄生物碱)在白内障形成中的作用。从历史上看，增加了 晶状体Na+浓度，在人类白内障晶状体中常见，被认为是 由晶状体膜钠离子转运系统功能障碍引起 上皮组织。晶状体上皮细胞Na，K-ATPase对ATP的水解作用 在大多数人类白内障晶状体中发生了改变。此外，有几个 这些ATP水解酶的变化与系统性疾病有关， 糖尿病和高血压。更新的研究(在进展中提出 报告显示了一幅更为复杂的图景。钠至少有三种类型， 质膜晶状体上皮细胞中的K-ATPase和一种Na，K-ATPase 在晶状体纤维的质膜中。晶状体纤维的Na，K-ATPase 负责大部分的透镜状Na+：K+交换。在人类中 白内障晶状体纤维细胞Na，K-ATPase部分或完全 被禁止了。Na，K-ATPase也存在于细胞核和内质中 培养的晶状体上皮细胞的网状结构及胞核和内质网 大鼠肝脏的。拟议的研究的主要焦点是 正常晶状体亚细胞组分的Na，K-ATPase假设 Na，K-ATPase亚基在质膜和亚细胞中都很活跃 细胞器包括细胞核和内质网。我的目标是， II和III旨在检验这一假说。目标一是确定 与催化a亚基异构体相关的B亚基异构体 哺乳动物晶状体质膜结合的Na，K-ATPase。AIM II是 确定A和B亚基异构体的亚细胞位置 培养晶状体上皮细胞，在新鲜晶状体上皮细胞中和在 新鲜晶状体表面纤维的免疫细胞化学研究 SDSPAGE分离的Western blotts抗体染色 准备了亚细胞部分。目的III是研究Na，K-ATPase的特性 在洋地黄生物碱结合研究的每个亚细胞部分中， 洋地黄生物碱敏感的ATP水解度测定及洋地黄 生物碱敏感的磷酸酶形成。假设二.慢性 胰岛素或葡萄糖的升高改变了晶状体血浆的相互作用 膜Na，K-ATPase与其ATP连接的效应器，从而影响变化 在钾离子转运中。AIM IV就是为了检验这一假设而设计的。目标四是 确定慢性高血糖和高胰岛素的影响 肾小管上皮和纤维中K+内流的水平(以86Rb+为示踪剂) 培养的镜片。

DESCRIPTION: The long term goal of this project is to determine the role of the lens Na,K-ATPases and their ATP-linked effectors (H+, Na+, K+, ATP, ADP, and digitalis alkaloids) in cataract formation. Historically, increased lens Na+ concentrations, common in human cataract lenses, were believed to result from the dysfunction of membrane Na+ transport systems of the lens epithelium. ATP hydrolysis by the Na, K-ATPase of the lens epithelium is altered in a majority of human cataract lenses. Furthermore, several of these changes in ATP hydrolysis correlate with the systemic diseases, diabetes and hypertension. More recent studies (presented in the progress report suggest a more complex picture. There are at least 3 types of Na, K-ATPase in the plasma membrane lens epithelium and one type of Na,K-ATPase in the plasma membrane of lens fibers. The Na,K-ATPase of the lens fibers is responsible for a majority of the lenticular Na+:K+ exchange. In human cataract lenses the lens fiber cell Na,K-ATPase is partially to completely inhibited. Na,K-ATPases are also present in cell nucleus and endoplasmic reticulum of cultured lens epithelial cells and in the cell nucleus and ER of rat liver. The primary focus of the proposed research is to characterize the Na,K-ATPases of the subcellular fractions of normal lenses. Hypothesis I. Na,K-ATPase subunits are active in the plasma membrane and subcellular organelles including the cell nucleus and endoplasmic reticulum. Aims I, II, and III are designed to test this hypothesis. Aim I is to determine the B subunit isoforms that are associated with the catalytic a subunit isoforms of the plasma membrane bound Na, K-ATPases of the mammalian lens. Aim II is to determine the subcellular locations of the a and B subunit isoforms in culture lens epithelial cells, in epithelium of fresh lens and in the superficial fibers of fresh lens using immunocytochemistry as well as antibody staining of Western blots of SDSPAGE separations of carefully prepared subcellular fractions. Aim III is to characterize the Na,K-ATPases in each subcellular fraction by the digitalis alkaloid binding studies, digitalis alkaloid sensitive ATP hydrolysis measurements and digitalis alkaloid sensitive phosphoenzyme formation. Hypothesis II. Chronic elevation of insulin or glucose change the interaction of the lens plasma membrane Na,K-ATPase with their ATP linked effectors, thus affecting changes in K+ transport. Aim IV is designed to test this hypothesis. Aim IV is to determine the effects the effects of chronic elevated glucose and insulin levels of K+ influx (86Rb+ as a tracer) in the epithelium and fibers of cultured lenses.