BETA ADRENERGIC RECEPTORS--REGULATION IN CILIARY BODY

β 肾上腺素能受体 - 睫状体的调节

• 批准号: 2378030
• 负责人: Sayoko E Moroi
• 金额: $ 11.04万
• 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 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378030
• 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

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和非裔美国人失明的首要原因 在美国是导致失明的第二大原因。虽然是中流砥柱 治疗的主要药物是β-肾上腺素能受体（β-AR）拮抗剂 抑制房水分泌，调节离子的信号通路 与水性分泌物偶联的通道和转运蛋白受到赞赏 主要是在蛋白质水平。对 beta-AR 知之甚少 分子水平上水通路中的受体。还有很多工作要做 了解“上游”（例如转录、翻译和 翻译后事件）和“下游”（例如，分布， 与任何 G 相关的磷酸化和隔离）途径 睫状体膜水平已知的蛋白质偶联受体 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 介导房水分泌。她建议学习这些工具 学科建设，实现以下四个具体目标： β-AR 的潜在异质表达（即 beta1、beta2 和 beta3）在非色素性睫状上皮（NPE）中的研究将通过 核糖核酸酶保护测定。 (2) 转录本的相关性 这些细胞中蛋白质表达的异质性将通过以下方式进行评估 放射性配体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-中进行 转化的人类 NPE 并在非转化的 NPE 培养物中得到证实。 (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' 侧翼区域 基因与糖皮质激素反应元件共识具有很大的同源性 顺序。 NPE 中假设有类似的 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学。 β-AR 转录水平和 将评估暴露于地塞米松的 NPE 细胞中的蛋白质表达。 (4) 由于β-AR的定位尚未确定， 原位杂交将用于鉴定表达的细胞类型 睫状上皮双层和其他区域内的β-AR转录物 眼前节区域。在质膜水平上， 考虑到 β-AR 的细胞分布预计会出现两极分化 睫状双层的分泌性质。这种受体“贩卖” 将通过共聚焦显微镜检查细胞和上皮细胞的模式 用荧光素缀合的配体、表位标记的双层外植体 标记受体，或亚型特异性抗体；此类研究反映 在天然组织中的生理意义。 这些拟议的研究将导致人们更好地认识 调节水生产的基本机制，可能发挥作用 在青光眼的病理生理学中发挥作用。