BETA ADRENERGIC RECEPTORS--REGULATION IN CILIARY BODY

青光眼是非裔美国人失明的首要原因， 在美国，失明的第二大原因。而中流砥柱 治疗的是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 连接到含水分泌物的通道和转运体是值得赞赏的 主要是在蛋白质水平上。人们对β-AR知之甚少 在分子水平上，水通道中的受体。还有很多事情要做 了解上游(例如，转录、翻译和 翻译后事件)和“下游”(例如，分发， 磷酸化和隔离)途径相对于G 纤毛膜水平已知的蛋白质偶联受体 上皮性双层。 内科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 调节房水分泌。她建议学习这些工具 并实现以下四个具体目标：(1) β-AR的潜在异质性表达(即，β1、β2和 Beta3)在无色素睫状体上皮(NPE)中的作用 核糖核酸酶保护试验。(2)成绩单的相关性 这些表达蛋白质的细胞的异质性将通过以下方法进行评估 放射配基结合和腺苷环化酶分析。这些分子， 药理和生化研究将在5V40- 转化人NPE，并在未转化的NPE培养中得到证实。(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 基因与糖皮质激素反应元件共识有很大的同源性 序列。NPE中假设了一个类似的Beta-AR监管过程。 这些受体的转录上调可能有助于 激素性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的本地化尚未确定，在 将使用原位杂交来鉴定表达的细胞类型 睫状体上皮双层和其他组织中的β-AR转录本 前段的区域。在质膜水平上， β-AR的细胞分布预计会极化，因为 纤毛双层的分泌性。这种受体“贩卖” 将通过共聚焦显微镜检查细胞和上皮中的图案 用荧光素标记的双层外植体，表位- 标记的受体或亚型特异性抗体；这种研究反映了 在自然组织中的生理意义。 这些拟议的研究将使人们更好地认识到 调节含水生产的基本机制及其可能发挥的作用 在青光眼的病理生理学中的作用。

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.