BIOCHEMICAL RESPONSES OF HUMAN CILIARY EPITHELIAL CELLS

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Knowledge of aqueous humor secretion and regulation of aqueous production in the human eye is relevant to the understanding and treatment of glaucoma. Aqueous humor is secreted by the ciliary epithelium and its composition is largely dependent on the mechanism of active transport of plasma proteins an electrolites in these cells. The rate of formation of aqueous humor is modulated by the sympathetic (adrenergic) system (Neufeld, A.H., 1984). The development of a tissue culture system for the ciliary epithelial (CE) cells, namely the nonpigmented (NPE) and pigmented (PE) epithelia is important because it permits the in vitro study of the cellular and molecular functions of these cells which are very difficult to carry out in vivo. The aim of this proposal is to develop antibodies to both membrane-bound surface proteins and internal proteins which are specific markers for NPE and PE cells using the hybridoma technology. These antibodies will serve as probes to investigate the differentiated properties of these cells in culture. In addition, it will allow for the isolation (i.e. fluorescence activated cell sorter) of NPE and PE cells based on their potential immunological differences. These antibodies will be used to facilitate the understanding of the secretory function and transport of NPE and PE cells. Secretory function will be studied by developing antibodies to a 76KDa protein secreted by CE in culture. Ion transport will be studied by immunobloting analysis of (Na+, K+)-ATPase activity by antibodies to the alpha subunit of this enzyme. Sodium pump transport activity will assessed by ouabain-sensitive 86Rb+ uptake. In addition, the mechanism of transduction by external signals into intracellular events (i.e., protein phosphorylation) will be studied.