PHYSIOLOGY AND PHARMACOLOGY OF CILIARY BODY EPITHELIUM

这项拨款建议使用膜片钳记录，钙荧光测定法 方法和组织培养，研究其生理学和药理作用。 睫状体上皮细胞。我们提议让整个- 来自有色和无色细胞的细胞和穿孔斑片记录 低电压钳位和研究电导的变化 已知对这些细胞有某种药理作用的物质 (蛙黄素、卡巴胆碱、加压素、异丙肾上腺素、苯肾上腺素、三磷酸腺苷、VIP) 或对眼压(儿茶酚胺、多巴胺、 碳酸酐酶抑制剂、佛波醇酯、福司可林)。当发生变化时 膜电导可以记录，药物的作用效果会 具有离子选择性、电压依赖性和 作用机制(G蛋白的作用，第二信使)。我们也是 建议使用荧光染料Fura-2来研究其可能的作用 无色素睫状体上皮细胞对钙离子的反应 药理药剂，包括在完整的睫状突和细胞中 原生文化。同时测量钙离子的变化 浓度和膜电导的变化，以严格测试 认为钙离子在该组织中充当第二信使的观点。我们是 建议开发一种培养无色素单层的方法 在可渗透的载体上的电池。我们的目标是生产单层和 足够高的跨皮阻力，可以用来 测量这个组织中的离子和水通量。如果我们能够 产生这样的单层，我们将用它们来研究跨上皮 电气参数(开路电压和闭路电流)， 以及离子(Na+，K+，Cl-)的通量，它们可能在 房水的分泌物。我们还希望使量化 非色素细胞单层水通量的测量 水转运机制及药理学研究进展 睫状体。

This grant proposes to use patch-clamp recording, Ca2+ fluorometric methods, and tissue culture to study the physiology and pharmacology of epithelial cells from the ciliary body. We are proposing to make whole- cell and perforated patch recordings from pigmented and nonpigmented cells under voltage clamp and to study changes in conductance produced by substances known to have some pharmacological effect on these cells (bombesin, carbachol, vasopressin, isoproterenol, phenylephrine, ATP, VIP) or to have effects on the intraocular pressure (catecholamines, dopamine, carbonic anhydrase inhibitors, phorbol esters, forskolin). When changes in membrane conductance can be recorded, the effects of the drugs will be characterized as to their ion selectivity, voltage dependence, and mechanism of action (role of G proteins, second messengers). We are also proposing to use the fluorometric dye fura-2 to study a possible role of Ca2+ in the response of the nonpigmented ciliary body epithelial cells to pharmacological agents, both in intact ciliary processes and for cells in primary culture. Simultaneous measurements will be made of changes in Ca2+ concentration and changes in membrane conductance, to test critically the notion that Ca2+ acts as a second messenger in this tissue. We are proposing to develop a method for culturing monolayers of nonpigmented cells on permeable supports. Our goal is to produce monolayers with sufficiently high transepithelial resistances that they can be used to make measurements of ion and water flux in this tissue. If we are able to produce such monolayers, we shall use them to study transepithelial electrical parameters (open circuit voltage and closed circuit current), and the flux of ions (Na+,K+,Cl-) which are likely to be important in the secretion of the aqueous humor. We hope also to make quantitative measurements of water flux across monolayers of nonpigmented cells, to investigate the mechanisms and pharmacology of water transport in the ciliary body.