PHYSIOLOGY AND PHARMACOLOGY OF CILIARY BODY EPITHELIUM

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This grant proposes to grow identified pigmented and non-pigmented epithelial cells from ciliary body in tissue culture and to study their physiology and pharmacology using patch-clamp techniques and fluorescence spectrophotometry. We shall attempt to develop improved tissue culture methods for reproducibly producing groups of ciliary body cells and eventually monolayers from rabbit and from human eyes. We shall isolate markers for both pigmented and non-pigmented cells using monoclonal antibodies which have been developed against purified populations of pigmented or non- pigmented cells. The aim of these experiments is to develop methods which will allow us, for example, to add antibody to a dish of cultured cells to identify the non-pigmented cells prior to making patch-clamp recordings from them. We then proposed to use both excised-patch and whole-cell methods, in order to characterize both single membrane channels and voltage-dependent currents from cultured epithelial cells. The purpose of these experiments is to provide a complete characterization of epithelial cell ion permeabilities. Whole-cell methods will then be used to voltage- clamp these cells, in order to study changes in conductance produced by substances known to have effects on the intraocular pressure (beta-adrenergic agonists, cyclic AMP, dopamine). Finally, fluorescent dye methods will be used to measure pH and free Ca2+ concentration, both from cells in tissue culture and in suspensions, as well as from the non-pigmented cells in situ. Cells will be loaded with the pH-sensitive dye BCECF or the Ca2+- sensitive dye Fura 2. A dual excitation spectro-fluorometer together with image analysis will be used to investigate the mechanisms of pH regulation in these cells and the possible role of Ca2+ as a second messenger.