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

描述：本项目的长期目标是确定 透镜Na，K-ATP酶及其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， 透镜Na+浓度，在人类白内障晶状体中常见，被认为 是由于透镜的膜Na+转运系统功能障碍所致 上皮 透镜上皮细胞Na，K-ATP酶对ATP的水解是 在大多数人类白内障晶状体中发生了改变。 此外，一些 ATP水解的这些变化与系统性疾病相关， 糖尿病和高血压。 最近的研究（在进展中介绍） 报告显示了一个更复杂的画面。 至少有三种类型的Na， 透镜上皮细胞质膜K-ATP酶和一种Na，K-ATP酶 存在于透镜纤维的质膜中。 透镜纤维的Na，K-ATP酶 负责晶状体Na+：K+交换的大部分。 人 白内障晶状体透镜纤维细胞Na，K-ATPase部分或全部 压抑 Na，K-ATP酶也存在于细胞核和内质网中 培养的透镜上皮细胞的网状结构、细胞核和内质网 老鼠的肝脏 拟议研究的主要重点是表征 正常晶状体亚细胞组分的Na，K-ATP酶。 假设 I. Na，K-ATP酶亚基在质膜和亚细胞中具有活性 细胞器包括细胞核和内质网。 我的目标是， II和III旨在验证这一假设。 目的一是确定 与催化性a亚单位同种型相关的B亚单位同种型 哺乳动物透镜的质膜结合Na，K-ATP酶。 目标二是 为了确定a和B亚单位同种型的亚细胞位置， 培养透镜上皮细胞，在新鲜透镜的上皮中和在 新鲜透镜表面纤维的免疫细胞化学染色， 抗体染色的蛋白质印迹的SDSPAGE分离仔细 制备亚细胞级分。 目的III是对Na，K-ATP酶进行表征 在通过洋地黄生物碱结合研究的每个亚细胞级分中， 洋地黄生物碱敏感的ATP水解测量和洋地黄 生物碱敏感性磷酸酶形成。 假设二。 慢性 胰岛素或葡萄糖的升高改变了透镜血浆的相互作用 膜Na，K-ATP酶与其ATP连接的效应物，从而影响变化 K+运输。 目的四旨在检验这一假设。 目标四： 确定慢性血糖和胰岛素升高的影响 用~（86）Rb ~+作为示踪剂，测定了大鼠肺上皮细胞和肺纤维中K ~+内流的水平。 培养晶状体。

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.