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

人眼睫状体上皮是一个很好的细胞模型 Na，K-ATPase的功能和结构。钠泵是一种关键离子 纤毛在房水形成和运输中的泵作用 开角型青光眼中的上皮细胞，导致上皮细胞升高 眼内液体压。 拟议项目的目标是调查这种差异 猪瘟病毒α和β亚基异构体基因表达的调控 人睫状体中的NAK-ATPase。为了实现这一目标，我们将 从年轻眼球供者的睫状体构建人的cDNA文库。 该文库将作为一个工具性的工具来解决基本的 关于对银行活动的不同监管的问题 正常人和青光眼患者Na，K-ATPase的α亚型这个 实现这些目标的实验设计和方法是 以下是： A)特异性全长人c DNA克隆的分离和鉴定 对于睫状体上皮的α和β亚基亚基亚型 Na，K-ATPase。 B)α和β亚基亚基异构体的mRNAs定位 人睫状体上皮Na，K-ATPase的原位杂交研究 不同阶段的发展和分化。 C)青光眼患者眼内ATPase基因表达的调节。由北方 斑点分析，原位杂交和免疫印迹我们将确定 Na，K-ATPaseα和β亚型的表达是否在 与正常眼睛相比时的转录或翻译水平。 D)研制针对人类阿尔法特定结构域的抗体 和Na，K-ATPase的β亚型。这些抗体将提供 关于蜂窝分布、地区差异的宝贵信息， 纤毛发育过程中的结构和构象变化 上皮组织。通过免疫印迹分析，将可能确定 α和β多肽的表达是否在 翻译水平和/或描述任何翻译后 修改。 E)从克隆的cDNA中表达功能性Na，K-ATPase。引言 将编码α-亚型的克隆基因导入睫状体上皮细胞 缺少三种阿尔法亚型中的一种将使我们能够研究 α亚型的不同活性。

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.