Functional Diversity of Compartmentalized Calcium Signaling in Airway Smooth Muscle

项目摘要 在拟议的研究中，我们试图解开细胞内钙在调节中的复杂作用。 气道平滑肌(ASM)收缩。尽管生物化学事件介导了钙对人体健康的影响 跨桥自行车和收缩运动多年来一直被人们理解和接受，但令人费解的是 ASM细胞如何划分钙以调节收缩机械。我们最新的发现 证明对细胞内钙信号的常规和简单化评估没有预测性 对平滑肌收缩状态的调节能力。在最近的出版物中，我们展示了 刺激时细胞内钙升高似乎自相矛盾地调节ASM的松弛 苦味受体激动剂(TAS2Rs)而几乎所有其他G蛋白偶联受体(GPCRs) 能够诱导细胞内钙离子介导的收缩增加。同样，天然等配体 黄酮类，氯通道拮抗剂，GABAA激动剂引起急性ASM松弛，尽管引起 急性一过性[钙]i升高。显然，GPCRs对ASM收缩状态的不同影响反映了 长期以来，人们对细胞划分钙信号的能力赞不绝口，但知之甚少。松弛药 如果了解钙信号的作用，可以将其用于治疗呼吸道管理。 高反应性。为了实现我们的目标，我们将结合5个主要领域的专业知识和战略： 生物学、钙的生物物理学、细胞成像、蛋白质组学和数学建模。目标1研究将定义 不同浓度的钙离子在ASM中的时空分布机制 GPCR型激动剂。我们将定义和定位所有蛋白质、结构元素和酶活性 调节不同GPCR激动剂刺激的ASM细胞内钙的分布。我们将测试如何颠覆 这些调控元件改变了细胞内钙的特性以及与ASM相关的调控 语气。在Aim 2研究中，我们将使用高光谱成像和磷酸蛋白质组学工具来识别gpr- ASM中效应蛋白的特异性激活。有了这些数据，我们将制定描述性和预测性 数学模型(目标3)，定义了允许区分钙的主要监管特征 并预测这种信令的功能后果。计算模型将是 通过使用原代人呼吸道平滑肌细胞的研究获得的实验数据进行验证 还有纸巾。我们的成功有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和清晰定义的建模 演示细胞内不同且无法解释的功能后果的实验系统 钙。这些发现不仅将推进受体生物学和钙信号的基础科学领域， 而且还确定了可以利用其操纵来开发新的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.