Functional Diversity of Compartmentalized Calcium Signaling in Airway Smooth Muscle

项目摘要 在所提出的研究中，我们试图解开细胞内钙离子在调节中的复杂作用， 气道平滑肌（ASM）收缩。虽然生化事件介导的影响，钙对 过桥自行车和收缩已经被理解和接受多年，仍然令人困惑的是， ASM细胞如何分隔钙来调节收缩机制。我们最近的发现 表明细胞内钙信号传导的传统和简单的评估没有预测性， 调节平滑肌收缩状态的能力。在最近的出版物中，我们展示了 当受到刺激时，细胞内钙离子升高介导ASM松弛的能力似乎是矛盾的。 苦味受体激动剂（TAS 2 R），而几乎所有其他G蛋白偶联受体（GPCR） 能够诱导增加的细胞内钙介导的收缩。类似地，配体如天然的 类黄酮、氯离子通道拮抗剂、GABAA激动剂引起急性ASM松弛，尽管它们引起急性平滑肌松弛。 急性一过性[Ca 2 +]i升高。显然，GPCR对ASM收缩状态的不同影响反映了 细胞对钙信号进行区室化的能力长期受到重视，但了解甚少。舒张 钙信号传导的作用，如果被理解，可以在治疗上用于气道的管理 高反应性为了实现我们的目标，我们将联合收割机的专业知识和战略结合在5个主要领域： 生物学、钙生物物理学、细胞成像、蛋白质组学和数学建模。目标1研究将定义 不同浓度的钙离子对平滑肌细胞钙信号时空分布的影响 GPCR激动剂。我们将定义和定位所有的蛋白质，结构元件，和酶的活动， 调节用不同GPCR激动剂刺激的ASM细胞中的钙分布。我们将测试 这些调节元件的改变细胞内钙的特征和ASM的相关调节 语气在目标2研究中，我们将采用高光谱成像和磷酸蛋白质组学工具来辨别GPCR- 特异性激活ASM中的效应蛋白。有了这些数据，我们将开发描述性和预测性的 数学模型（目标3），定义了关键的调控功能，使区室化钙 信号并预测这种信号的功能后果。计算模型将是 使用通过使用原代人气道平滑肌细胞的研究获得的实验数据进行验证 和纸巾。我们的成功依赖于我们团队创造性地应用和协调切割的独特能力 边缘成像方法，多荧光团高光谱图像分析，并建模，以良好的定义 这些实验系统证明了细胞内免疫的不同和无法解释的功能后果， 钙这些发现不仅将推进受体生物学和钙信号传导的基础科学领域， 而且还鉴定其操作可用于开发新型支气管扩张剂疗法的靶点。

Project Summary In the proposed studies we seek to disentangle the complex effects of intracellular calcium in the regulation of airway smooth muscle (ASM) contraction. Although biochemical events mediating the effects of calcium on cross-bridge cycling and contraction have been understood and accepted for years, what remains mystifying is how the ASM cell compartmentalizes calcium to regulate the contractile machinery. Our recent findings demonstrate that conventional and simplistic assessments of intracellular calcium signaling have no predictive ability regarding regulation of smooth muscle contractile state. In recent publications we demonstrate the seemingly paradoxical ability of elevated intracellular calcium to mediate relaxation of ASM when stimulated by agonists of bitter taste receptors (TAS2Rs) whereas almost all other G protein-coupled receptors (GPCRs) capable of inducing increased intracellular calcium mediate contraction. Similarly, ligands such as natural flavonoids, chloride channel antagonists, GABAA agonists cause acute ASM relaxation despite causing an acute transient [Ca2+]i increase. Clearly, the disparate effects by GPCRs on ASM contractile state reflect the long-appreciated but poorly-understood ability of the cell to compartmentalize calcium signaling. The relaxant effect of calcium signaling, if understood, could be exploited therapeutically for the management of the airway hyperresponsiveness. To achieve our goal we will combine expertise and strategies in 5 major areas: GPCR biology, biophysics of calcium, cell imaging, proteomics and mathematical modeling. Aim 1 studies will define the mechanisms involved in spatial and temporal distribution of calcium signaling in ASM induced by different GPCR agonists. We will define and localize all proteins, structural elements, and enzymatic activities that regulate calcium distribution in ASM cells stimulated with different GPCR agonists. We will test how disruption of these regulatory elements alters the features of intracellular calcium and the associated regulation of ASM tone. In aim 2 studies, we will employ hyperspectral imaging and phosphoproteomics tools to discern GPCR- specific activation of effector proteins in ASM. With these data we will develop descriptive and predictive mathematical models (Aim 3) that define the key regulatory features enabling compartmentalized calcium signaling and predict the functional consequences of such signaling. The computational models will be validated using experimental data obtained through studies using primary human airway smooth muscle cells and tissues. Our success banks on the unique ability of our team to creatively apply and coordinate cutting edge imaging approaches, multi-fluorophore hyperspectral image analysis, and modeling to well-defined experimental systems that demonstrate disparate and unexplained functional consequences of intracellular calcium. The findings will not only advance the basic science fields of receptor biology and calcium signaling, but also identify targets whose manipulation could be exploited for developing novel bronchodilator therapies.