OCULAR ACTIONS OF ENDOTHELINS

描述(改编自申请人的摘要)： 这项提案的总体目标是勾勒出 参与调节内皮素的合成和释放 (ETS)在眼组织中的表达，并确定内皮素的靶点和 细胞的作用机制。要检验的假设是外星人是 在睫状上皮和其他组织中合成和储存，并 由多种信号释放，包括细胞因子发挥旁分泌作用 对睫状肌和小梁网的影响以增强房水 体液流出，降低眼压。初步数据来自 申请人的实验室表明促炎症细胞因子 肿瘤坏死因子-α刺激睫状体合成和释放ET-1 上皮细胞和ET-1通过ETA受体作用于人睫状肌 增强PLC活性和钙动员。后一种效应 被认为与ET引起的肌肉收缩有关。 尽管内皮素对眼压和眼球收缩的作用 血管平滑肌的作用机制已经得到了很好的证明 负责调节眼部ET的合成、释放和释放 内皮素的作用还不完全清楚。以下是具体的 AIMS计划解决这些机制：(1)确定ET是否， 它的前体和合成酶存在于人的睫毛中 上皮、睫状肌和小梁网细胞和组织 应用免疫荧光显微镜、放射免疫分析和Western Blot 分析；(2)确定负责的信号和机制 通过对信号的研究调节ET的合成和释放 由肿瘤坏死因子-α和自主神经激动剂激活的转导通路， 包括蛋白激酶C(PKC)亚型、一氧化氮的作用 (3)测定组织中ET的含量 利用RT-PCR技术研究ETS的部位受体和细胞作用机制 聚合酶链式反应、核糖核酸酶保护分析和原位杂交组织化学 在正常细胞因子和肾上腺素能/胆碱能刺激条件下， 并描述了与ET相关的信号转导途径 受体激活；以及(4)确定受体的功能作用 通过将信号转导机制连接到 测量睫状肌和小梁网的收缩 单细胞收缩，肌球蛋白轻链磷酸化，改变 流出设施使用隔离的眼前段灌流人眼。 离体毛状体上皮细胞离子转运过程的改变 也要接受检查。这些具体目标旨在确定 眼组织中ET释放和合成的机制 并确定ET靶点。这项研究将提供信息 关于ETS在眼压稳态中可能起到的作用。

DESCRIPTION (Adapted from applicant's abstract): The overall goals of this proposal is to delineate the processes involved in the regulation of the synthesis and release of endothelins (ETs) in ocular tissues and to determine endothelin's target sites and cellular mechanism of action. The hypothesis to test is that ET is synthesized and stored in ciliary epithelium and other tissues and is released by a variety of signals, including cytokines to exert paracrine effects on the ciliary muscle and trabecular meshwork to enhance aqueous humor outflow and decrease intraocular pressure. Preliminary data from the applicant's laboratory indicates that the proinflammatory cytokine TNF-alpha stimulates the synthesis and release of ET-1 from ciliary epithelium and ET-1 acts on human ciliary muscle through an ETA receptor to enhance PLC activity and calcium mobilization. This latter effect is thought to be responsible for the muscle contraction induced by ET. Although endothelins' actions on intraocular pressure and contraction of the vascular smooth muscle are well documented, the mechanisms responsible for the regulation of ocular ET synthesis, release and actions of endothelins are not fully understood. The following specific aims are planned to address these mechanisms: (1) to determine if ET, its precursors and synthetic enzymes are present in human ciliary epithelium, ciliary muscle and trabecular meshwork cells and tissues using immunofluorescent microscopy, radioimmunoassay and Western Blot analysis; (2) to determine the signals and mechanisms responsible for the regulation of ET's synthesis and release by investigating the signal transduction pathways activated by TNF-alpha and autonomic agonists, including the role of protein kinase C (PKC) isoforms, nitric oxide (NO), and other messengers on the release of ET; (3) to determine tissue site receptors and cellular mechanism of action released ETs using RT- PCR, RNase protection analysis and in situ hybridization histochemistry under normal cytokine, and adrenergic/cholinergic stimulated conditions, and characterize the signal transduction pathways associated with ET receptor activation; and (4) to determine the functional role of released endothelins by linking the signal transduction mechanisms to contraction in the ciliary muscle and trabecular meshwork by measuring single cell contraction, myosin light chain phosphorylation, changes in outflow facility using the isolated anterior segment perfused human eye. Changes in ion transport process of the isolated ciliary epithelium will also be examined. These specific aims are designed to determine the mechanisms involved in the release and synthesis of ET in ocular tissues and to identify ET target sites. This study will provide information on the role ETs may play in intraocular pressure (IOP) homeostasis.