BETA ADRENERGIC RECEPTORS--REGULATION IN CILIARY BODY

β 肾上腺素能受体 - 睫状体的调节

• 批准号: 6164629
• 负责人: Sayoko E Moroi
• 金额: $ 17.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6164629
• 关键词: G protein; RNase protection assay; adenylate cyclase; beta adrenergic receptor; binding proteins; cell biology; cell type; confocal scanning microscopy; fluorescence microscopy; genetic transcription; glaucoma; human tissue; image processing; in situ hybridization; intraocular fluid; messenger RNA; molecular biology; northern blottings; phase contrast microscopy; polymerase chain reaction; receptor binding; receptor coupling; receptor expression; tissue /cell culture; uvea ciliary body; G protein; RNase protection assay; adenylate cyclase; beta adrenergic receptor; binding proteins; cell biology; cell type; confocal scanning microscopy; fluorescence microscopy; genetic transcription; glaucoma; human tissue; image processing; in situ hybridization; intraocular fluid; messenger RNA; molecular biology; northern blottings; phase contrast microscopy; polymerase chain reaction; receptor binding; receptor coupling; receptor expression; tissue /cell culture; uvea ciliary body

Glaucoma is the premier cause of blindness in African Americans and the second leading cause of blindness in the United States. While the mainstay of treatment is beta-adrenergic receptor (beta-AR) antagonists which suppress aqueous secretion, the signaling pathways regulating the ion channels and transporters coupled to aqueous secretion are appreciated primarily at a protein level. There is little understanding of the beta-AR receptors in the aqueous pathway at the molecular level. Much remains to be learned about "upstream (e.g., transcriptional, translational, and post-translational events) and "downstream" (e.g., distribution, phosphorylation, and sequestration) pathways relative to any of the G protein-coupled receptors known at the membrane level of the ciliary epithelial bilayer. The physician scientist candidate's long-term objective is to understand the molecular and cellular biology of G protein-coupled receptors mediating aqueous secretion. She proposes to learn the tools of these disciplines and to accomplish the following four specific aims: (I) The potential heterogeneous expression of the beta-AR (i.e., beta1, beta2, and beta3) in nonpigmented ciliary epithelium (NPE) will be studied by ribonuclease protection assay. (2) The correlation of transcript heterogeneity in these cells with protein expression will be assessed by radioligand binding and adenylate cyclase assays. These molecular, pharmacological, and biochemical studies will be conducted in 5V40- transformed human NPE and confirmed in nontransformed NPE cultures. (3) One of the principal mechanisms of steroid hormone action is modulating gene transcription; the 5'-flanking regions of the beta1- and beta2-AR genes have great homology to a glucocorticoid responsive element consensus sequence. A similar beta-AR regulatory process is hypothesized in NPE. Transcriptional up-regulation of these receptors may contribute to the pathophysiology of steroid-induced glaucoma. beta-AR transcript levels and protein expression will be assessed in NPE-cells exposed to dexamethasone. (4) Since the localization of the beta-AR has not yet been determined, in situ hybridization will be used to identify the cell types expressing beta-AR transcripts within the ciliary epithelial bilayer and other regions of the anterior segment. At the level of the plasma membrane, the cellular distribution of the beta-AR is expected to be polarized given the secretory nature of the ciliary bilayer. This receptor "trafficking" pattern will be examined by confocal microscopy in cells and epithelial bilayer explants labeled with fluorescein-conjugated ligands, epitope- labeled receptors, or subtype specific antibodies; such studies reflect the physiological significance in native tissue. These proposed studies will lead to a better appreciation for the fundamental mechanisms that regulate aqueous production and that may play a role in the pathophysiology of glaucoma.

青光眼是非裔美国人失明的主要原因， 美国失明的第二大原因。同时 治疗的是β-肾上腺素能受体（β-AR）拮抗剂 抑制水溶液，信号通路调节离子 对耦合到水分分泌的通道和转运蛋白表示赞赏 主要在蛋白质水平上。对beta-ar的了解很少 分子水平的水途径中的受体。剩下很多 了解“上游（例如，转录，翻译和 翻译后事件）和“下游”（例如，分布， 相对于任何G的磷酸化和隔离途径 蛋白质偶联受体在纤毛的膜水平上已知 上皮双层。 医师科学家候选人的长期目标是了解 G蛋白偶联受体的分子和细胞生物学 介导水性分泌物。她建议学习这些工具 学科并完成以下四个特定目标：（i） β-AR的潜在异质表达（即beta1，beta2和 β3）在未沾满纤毛上皮（NPE）中，将通过 核糖核酸酶保护测定法。 （2）转录本的相关性 这些具有蛋白质表达的细胞的异质性将通过 放射性聚物结合和腺苷酸环化酶测定。这些分子， 药理学和生化研究将在5v40-- 转化的人类NPE并在非转化的NPE培养物中得到了证实。 （3） 类固醇激素作用的主要机制之一是调节 基因转录； beta1-和beta2-ar的5'频乘区域 基因与糖皮质激素的响应元件共识具有极大的同源性 顺序。在NPE中假设类似的β-AR调节过程。 这些受体的转录上调可能有助于 类固醇引起的青光眼的病理生理。 beta-ar成绩单级别和 将在暴露于地塞米松的NPE细胞中评估蛋白质表达。 （4）由于尚未确定Beta-AR的定位，因此 原位杂交将用于识别表达的细胞类型 纤毛上皮双层和其他的beta-ar成绩单 前部的区域。在质膜的水平上 鉴于 睫状双层的分泌性质。这个受体“贩运” 将通过细胞和上皮的共聚焦显微镜检查模式 双层外植体标记为荧光素偶联的配体，表位 - 标记的受体或亚型特异性抗体；这样的研究反映了 天然组织的生理意义。 这些拟议的研究将导致对 调节水性生产并可能发挥的基本机制 在青光眼的病理生理学中的作用。