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.
