NA, K-ATPASE GENE EXPRESSION IN THE HUMAN CILIARY BODY

人睫状体上皮是一个很好的细胞模型， Na，K-ATP酶的结构和功能。钠泵是一个关键的离子 通过睫状体泵入水状体液体的形成和运输 开角型青光眼中的上皮细胞，导致 眼内液体压力。 拟议项目的目的是调查 调节α和β亚单位同种型的基因表达 人睫状体中的NaK-ATPase。为了实现这一目标，我们将 从年轻眼供体的睫状体构建人cDNA文库。 这个cDNA文库将作为一个工具，以解决基本的 关于对联合国系统各组织的活动实行差别管制的问题 正常和青光眼眼Na，K-ATP酶α亚型。的 实现这些目标的实验设计和方法是 以下内容： A）特异性全长人cDNA克隆的分离和表征 对于睫状上皮的α和β亚单位同种型 Na，K-ATPase。 B）针对所述细胞的α和β亚基同种型的mRNA的定位。 人睫状体上皮Na，K-ATP酶的原位杂交研究 发展和分化的不同阶段。 C）青光眼眼睛中ATP酶基因表达的调节。由北方 印迹分析，原位杂交和免疫印迹，我们将确定 Na，K-ATP酶α和β亚型的表达是否在 与正常眼睛相比，转录或翻译水平。 D）开发针对人α-淀粉样蛋白的特异性结构域的抗体 和Na，K-ATP酶的β同种型。这些抗体将提供 关于细胞分布，区域差异， 纤毛发育过程中的结构和构象变化 上皮通过免疫印迹分析，可以确定 α和β多肽的表达是否在 翻译水平和/或表征任何翻译后 修改. E）从克隆的cDNA表达功能性Na，K-ATP酶。引入 将编码α亚型的克隆cDNA导入睫状上皮细胞 缺乏三种α异构体之一将使我们能够研究 α异构体的不同活性

The human ciliary epithelium is an excellent cellular model to understand the function and structure of the Na,K-ATPase. The Na-pump is a pivotal ion pump in the formation and transport of aqueous humor fluid by the ciliary epithelium which in Open Angle Glaucoma, leads to an elevation of intraocular fluid pressure in the eye. The objective of the proposed project is to investigate the differential regulation of gene expression of the alpha and beta subunit isoforms of the NaK-ATPase in the human ciliary body. To achieve this goal we will construct a human cDNA library from the ciliary body of a young eye donor. This cDNA library will serve as an instrumental tool to address basic questions with respect to the differential regulation of activity of the alpha isoforms of the Na,K-ATPase in normal and in Glaucoma eyes. The experimental design and methods for achieving these goals are the following: A) Isolation and characterization of full length human cDNA clones specific for the alpha and beta subunit isoforms of the ciliary epithelium Na,K-ATPase. B) The localization of mRNAs for alpha and beta subunit isoforms of the Na,K-ATPase by in situ hybridization in the human ciliary epithelium in various stages of development and differentiation. C) Regulation of ATPase gene expression in eyes with Glaucoma. By Northern blot analysis, in situ hybridization and immunoblotting we will determine whether the expression of Na,K-ATPase alpha and beta isoforms is altered at the transcription or translational level when compared to normal eyes. D) Development of antibodies against specific domains of the human alpha and beta isoforms of the Na,K-ATPase. These antibodies will provide valuable information on the cellular distribution, regional differences, structure and conformational changes during development of the ciliary epithelium. By immunoblotting analysis it will be possible to determine whether the expression of the alpha and beta polypeptides is regulated at the translational level and/or to characterize any post-translational modifications. E) Expression of functional Na,K-ATPase from cloned cDNAs. The introduction of cloned cDNA coding for the alpha isoforms into ciliary epithelial cells lacking one of the three alpha isoforms will allow us to study aspects of the differential activity of the alpha isoforms.