PHYSIOLOGY AND PHARMACOLOGY OF CILIARY BODY EPITHELIUM

这项拨款建议种植经鉴定的有色和无色 睫状体上皮细胞在组织培养中的研究 它们的生理和药理学使用膜片钳技术和 荧光分光光度法。我们将努力发展 用于繁殖群的改良组织培养方法 从兔的睫状体细胞到最终的单层 从人类的眼睛里。我们将分离出带色素的标记和 使用单抗培养的无色素细胞 针对纯净的有色或非有色种群开发的 有色细胞。这些实验的目的是开发 例如，允许我们将抗体添加到培养皿中的方法 以在培养前鉴定无色素细胞 用它们制作膜片钳录音。然后我们提议使用 切除贴片和全细胞方法，以表征 单个膜通道和电压依赖电流都来自 培养的上皮细胞。这些实验的目的是 提供上皮细胞离子的完整特征 渗透率。然后将使用全电池方法来对- 夹住这些细胞，以研究电导的变化 由已知对眼内有影响的物质产生 压力(β-肾上腺素能激动剂、环磷酸腺苷、多巴胺)。 最后，将使用荧光染料方法测量pH和 组织培养和组织培养中细胞的游离钙浓度 悬浮液，以及来自原位的无色素细胞。 细胞将被装载上pH敏感的染料BCECF或钙离子 灵敏染料Fura-2.双激发光谱荧光仪 结合图像分析将被用于研究 这些细胞的pH调节机制及其可能的作用 作为第二信使的钙离子。

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.