Calcium Regulation in Airway Smooth Muscle

描述(由申请者提供)：建议的研究将解决涉及调节气道平滑肌(ASM)细胞内钙离子([钙]i)的信号通路的非常基本的基本问题。这项拟议研究的长期目标是了解ACh诱导ASM细胞内[Ca~(2+)]i振荡的信号通路，以及这些振荡是如何耦合到力的产生的。以往的研究表明，肌醇1，4，5-三磷酸(IP3)和环状ADP核糖(cADPR；BetaNAD的代谢物)分别通过IP3和Ryanodine受体(RyR)通道作为SR钙释放的第二信使。我们进一步证明，肌质网钙离子释放的调节是时空动态的，因此ACh诱导了重复的局部[钙]i振荡，并通过细胞传播。[Ca~(2+)]_i振荡代表肌质网钙离子通过RyR通道的重复释放，而频率和传播速度取决于激动剂和cADPR浓度。然而，IP3的产生和通过IP3通道的钙释放也是启动[Ca~(2+)]i振荡的关键。传播的[Ca~(2+)]_j振荡突出了SR Ca~(2+)释放的动态性质及其受细胞内过程的调节。因此，在ASM中，[Ca~(2+)]i振荡是[Ca~(2+)]i调节和钙依赖过程(如力的产生)的重要基础。ASM中力的产生和松弛的时间常数慢于[Ca~(2+)]_i振荡。因此，局部作用力将取决于[Ca~(2+)]_i振荡的频率，跨心肌细胞的作用力协调将取决于[Ca~(2+)]_i振荡的传播速度。我们的中心假设是ASM细胞中传播的[Ca~(2+)]_i振荡反映了通过RyR通道的Ca~(2+)释放，ASM产生的力取决于这些[Ca~(2+)]_i振荡的频率和传播速度。该方案的具体目的是：目的1：确定IP3和RyR通道在ASM细胞中的分布；目的2；表征ASM中通过IP3和RyR通道释放的SR钙；目的3；确定ACh诱导ASM中IP3和cADPR水平变化的时间过程；目的4；确定通过IP3R和RyR通道释放的钙在ACh诱导的ASM细胞[Ca2+]i振荡中的交互作用。目的：为了确定ASM中[Ca~(2+)]_i振荡和力生成之间的偶联关系，我们将研究肌浆网钙释放机制与[Ca~(2+)]_i调节和激动剂诱导的[Ca~(2+)]_i振荡之间的特征和相互关系。

DESCRIPTION (provided by applicant): The proposed studies will address very basic fundamental questions regarding signaling pathways involved in the regulation of intracellular Ca2+ ([Ca2+]i) in airway smooth muscle (ASM). The long-term goals of the proposed research are to understand the signaling pathways underlying ACh-induced [Ca2+]i oscillations in ASM cells and how these oscillations are coupled to force generation. In previous studies, we have shown that inositol 1,4,5- trisphosphate (IP3) and cyclic ADP ribose (cADPR; a metabolite of betaNAD) are produced in response to muscarinic stimulation and act as second messengers for SR Ca2+ release through IP3 and ryanodine receptor (RyR) channels, respectively. We further demonstrated that regulation of SR Ca2+ release is spatially and temporally dynamic, such that ACh induces repetitive localized [Ca2+]i oscillations that propagate through the cell. [Ca2+]i oscillations represent repetitive release of SR Ca2+ through RyR channels, while frequency and propagation velocity depend on agonist and cADPR concentrations. However, IP3 production and Ca2+ release through IP3 channels is also essential for initiation of [Ca2+]i oscillations. Propagating [Ca2+]j oscillations highlight the dynamic nature of SR Ca2+ release and its modulation by intracellular processes. Thus in ASM, [Ca2+]i oscillations are an important foundation underlying [Ca2+]j regulation and Ca2+ dependent processes such as force generation. The time constants of force generation and relaxation in ASM are slower than [Ca2+]i oscillations. Accordingly, localized force will summate depending on the frequency of [Ca2+]i oscillations and force coordination across the myocyte will depend on propagation velocity of [Ca2+]i oscillations. Our central hypothesis is that propagating [Ca2+]i oscillations in ASM cells reflect Ca2+ release through RyR channels, and that force generation by ASM depends on the frequency and propagation velocity of these [Ca2+]i oscillations. The Specific Aims of the proposal are: Aim 1: To determine the distribution of IP3, and RyR channels in ASM cells; Aim 2; To characterize SR Ca2+ release through IP3 and RyR channels in ASM; Aim 3; To determine the time course of ACh-induced changes in IP3 and cADPR levels in ASM; Aim 4; To determine the interactive roles of Ca2+ release through IP3R and RyR channels in ACh-induced [Ca2+]i oscillations in ASM cells. Aim 5: To determine the coupling between [Ca2+ ]i oscillations and force generation in ASM, In the proposed studies, we will examine the characteristics and inter-relationships of these SR Ca2+ release mechanisms vis-a-vis [Ca2+]i regulation and agonist-induced [Ca2+]i oscillations.