PHYSIOLOGY AND PHARMACOLOGY OF CILIARY BODY EPITHELIUM

这项补助金建议种植识别有色和非有色 睫状体上皮细胞的组织培养和研究 使用膜片钳技术研究其生理学和药理学， 荧光分光光度法 我们将尝试开发 用于可重复地产生群体的改进的组织培养方法 睫状体细胞和最终单层从兔和 人类的眼睛。 我们将分离出有色和 非色素细胞使用单克隆抗体， 针对纯化的有色人种或非有色人种群体开发， 色素细胞 这些实验的目的是开发 比如说，我们可以在培养皿中加入抗体 培养的细胞，以确定非色素细胞之前， 制作膜片钳记录。 然后，我们建议使用 切除贴片和全细胞方法，以表征 单膜通道和电压依赖性电流 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表征 渗透率 全电池方法将用于电压- 夹住这些细胞，研究电导的变化 由已知对眼内 压力（β-肾上腺素能激动剂，环AMP，多巴胺）。 最后，荧光染料方法将用于测量pH值， 游离Ca 2+浓度，无论是从组织培养中的细胞， 悬浮液，以及来自原位的非色素细胞。 细胞将负载pH敏感性染料BCECF或Ca 2 +-Ca 2+。 敏感染料Fura 2。 双激发荧光分光光度计 结合图像分析， 在这些细胞中的pH调节机制和可能的作用 Ca 2+作为第二信使。

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.