BETA ADRENERGIC RECEPTORS--REGULATION IN CILIARY BODY

青光眼是非洲裔美国人失明的首要原因， 美国第二大致盲原因虽然中流砥柱 β-肾上腺素能受体（β-AR）拮抗剂， 抑制水分泌，调节离子的信号通路 通道和转运蛋白偶联到水性分泌物中， 主要是蛋白质水平。对β-AR的了解很少 在分子水平上的水通道中的受体。仍有许多工作要 了解“上游”（例如，转录、翻译和 翻译后事件）和“下游”（例如，分发， 磷酸化和螯合）途径相对于任何G 睫状体细胞膜水平的蛋白偶联受体 上皮双层 医生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。她建议学习这些工具， （一）有下列情形之一的; β-AR的潜在异质表达（即，beta1，beta2， 无色素睫状上皮（NPE）中的β 3）将通过以下方法进行研究： 核糖核酸酶保护测定。(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5V 40- 转化的人NPE，并在非转化的NPE培养物中确认。（三） 类固醇激素作用的主要机制之一是调节 基因转录; β 1-和β 2-AR的5 '侧翼区 基因与糖皮质激素应答元件共有序列具有很大的同源性， 顺序假设NPE中存在类似的β-AR调节过程。 这些受体的转录上调可能有助于 类固醇诱发青光眼的病理生理学β-AR转录水平， 在暴露于地塞米松的NPE细胞中评估蛋白质表达。 (4)由于β-AR的定位尚未确定， 将使用原位杂交来鉴定表达 睫状体上皮双层内的β-AR转录物和其他 前节的区域。在质膜水平， β-AR的细胞分布预计将被极化， 睫状体双层的分泌性质。这种受体“贩运” 将通过共聚焦显微镜检查细胞和上皮细胞中的模式 用荧光素缀合的配体、表位- 标记受体或亚型特异性抗体;这些研究反映了 在自然组织中的生理意义。 这些拟议的研究将有助于更好地了解 调节水生产的基本机制， 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.