Modeling Beta2 receptor activity in cellular environment

细胞环境中 Beta2 受体活性的建模

• 批准号: 6961771
• 负责人: THOMAS C RICH
• 金额: $ 2.13万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6961771
• 关键词: Adenoviridae; beta adrenergic receptor; biological signal transduction; biosensor device; calcium flux; cardiac myocytes; cell surface receptors; chemical kinetics; confocal scanning microscopy; cyclic AMP; diffusion; enzyme activity; enzyme inhibitors; forskolin; green fluorescent proteins; laboratory rat; manganese; mathematical model; membrane channels; newborn animals; phosphodiesterases; prostaglandin E; protein kinase A; single cell analysis; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Changes in cyclic AMP (cAMP) levels transmit information to downstream effectors including protein kinase A (PKA) and cyclic nucleotide-gated (CNG) channels. In turn, these enzymes regulate such diverse cellular responses as Ca2+ influx, excitability, and gene expression. It is accepted that the localization and frequency content of cAMP signals help to orchestrate a wide variety of cellular functions, yet little is known about either the sub-cellular localization or dynamics of these signals. The overall goal of this project is to elucidate the molecular and cellular mechanisms that localize cAMP signals, the frequency content of cAMP signals, and the potential roles of cAMP oscillations in cellular function. Addressing these issues will require an innovative approach for measuring cAMP levels in single cells and. To this end, we have developed high-resolution cAMP sensors based on genetically-engineered CNG channels. These sensors measure cAMP signals near the surface membrane with unprecedented spatial and temporal resolution. The following Specific Aims outline a plan to apply this approach to study the sub-cellular localization and frequency content of cAMP signals in neonatal cardiac myocytes. Aim 1. Determine which PDE types regulate cAMP signals triggered by different agents and how inhibition of different PDE types affects the kinetics of cAMP signals. Aim 2. Determine the relative contributions of diffusional barriers, PDE activity, and buffering by PKA in localizing cAMP signals. Aim 3. Develop mathematical models describing the spatial spread and kinetics of cAMP signals throughout the cell. Aim 4. Develop integrated mathematical models of the activation and desensitization of beta2ARs in the cellular environment. The proposed studies are particularly relevant in cardiac myocytes. The intimate relationships between beta-adrenergic signaling, cAMP production, cardiac excitability, and disease are well documented. However, there is a great deal of controversy surrounding the roles of beta1- and beta2-adrenergic receptors, 'switching', differential activation of Gs and Gi, and compartmentation of responses. Measuring single-cell, cAMP signals triggered by agents that activate specific GPCRs (e.g., beta2-adrenergic receptors) or inhibit phosphodiesterase activity will shed new light on the physiologic functions of these enzymes and their relation to cardiac function. Importantly, the development of integrated mathematical models that accurately describe beta2-adrenergic receptor desensitization will give us a better understanding of the impact of pharmacological agents such as beta-blockers, inverse agonists, and asthma drugs on signaling networks and cellular physiology.

描述（由申请人提供）： 环磷酸腺苷 (cAMP) 水平的变化将信息传递至下游效应器，包括蛋白激酶 A (PKA) 和环核苷酸门控 (CNG) 通道。反过来，这些酶调节多种细胞反应，如 Ca2+ 流入、兴奋性和基因表达。人们普遍认为，cAMP 信号的定位和频率内容有助于协调多种细胞功能，但人们对这些信号的亚细胞定位或动态知之甚少。该项目的总体目标是阐明定位 cAMP 信号的分子和细胞机制、cAMP 信号的频率内容以及 cAMP 振荡在细胞功能中的潜在作用。解决这些问题需要一种测量单细胞中 cAMP 水平的创新方法。为此，我们开发了基于基因工程 CNG 通道的高分辨率 cAMP 传感器。这些传感器以前所未有的空间和时间分辨率测量表面膜附近的 cAMP 信号。以下具体目标概述了应用这种方法来研究新生儿心肌细胞中 cAMP 信号的亚细胞定位和频率内容的计划。目标 1. 确定哪些 PDE 类型调节不同药物触发的 cAMP 信号，以及不同 PDE 类型的抑制如何影响 cAMP 信号的动力学。目标 2. 确定扩散屏障、PDE 活性和 PKA 缓冲在定位 cAMP 信号中的相对贡献。目标 3. 开发数学模型来描述 cAMP 信号在整个细胞中的空间传播和动力学。目标 4. 开发细胞环境中 β2AR 激活和脱敏的综合数学模型。拟议的研究与心肌细胞特别相关。 β-肾上腺素能信号传导、cAMP 产生、心脏兴奋性和疾病之间的密切关系已有详细记录。然而，围绕 β1 和 β2 肾上腺素能受体的作用、“转换”、Gs 和 Gi 的差异激活以及反应的划分存在很多争议。测量由激活特定 GPCR（例如 β2 肾上腺素受体）或抑制磷酸二酯酶活性的药物触发的单细胞 cAMP 信号，将为了解这些酶的生理功能及其与心脏功能的关系提供新的线索。重要的是，准确描述β2-肾上腺素能受体脱敏的综合数学模型的发展将使我们更好地了解β-受体阻滞剂、反向激动剂和哮喘药物等药物对信号网络和细胞生理学的影响。