Calcium Regulation in Airway Smooth Muscle

描述（由申请人提供）：拟议的研究将解决涉及气道平滑肌（ASM）细胞内Ca2+ ([Ca2+]i)调节的信号通路的非常基本的基本问题。提出的研究的长期目标是了解ASM细胞中ach诱导的[Ca2+]i振荡的信号通路，以及这些振荡如何耦合到力的产生。在之前的研究中，我们已经发现肌醇1,4,5-三磷酸（IP3）和环ADP核糖（cADPR， betaNAD的代谢物）在毒蕈碱刺激下产生，并分别通过IP3和ryanodine受体（RyR）通道作为SR Ca2+释放的第二信使。我们进一步证明，SR Ca2+释放的调节在空间和时间上是动态的，因此ACh诱导通过细胞传播的重复局部[Ca2+]i振荡。[Ca2+]i振荡代表SR Ca2+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通道释放Ca2+对于[Ca2+]i振荡的起始也是必不可少的。传播[Ca2+]j振荡突出了SR Ca2+释放的动态性质及其通过细胞内过程的调节。因此，在ASM中，[Ca2+]i振荡是[Ca2+]j调控和Ca2+依赖性过程（如力产生）的重要基础。ASM中力产生和弛豫的时间常数比[Ca2+]i振荡慢。因此，局部力将取决于[Ca2+]i振荡的频率和跨肌细胞的力协调将取决于[Ca2+]i振荡的传播速度。我们的中心假设是，在ASM细胞中传播的[Ca2+]i振荡反映了Ca2+通过RyR通道释放，并且ASM产生的力取决于这些[Ca2+]i振荡的频率和传播速度。该提案的具体目的是：目的1：确定ASM细胞中IP3和RyR通道的分布；目标2;研究ASM中SR Ca2+通过IP3和RyR通道释放的特性；目标3;测定乙酰胆碱诱导急性心肌梗死IP3和cADPR水平变化的时间过程；目标4;确定通过IP3R和RyR通道释放Ca2+在ach诱导的ASM细胞[Ca2+]i振荡中的相互作用。目的5：为了确定ASM中[Ca2+]i振荡和力产生之间的耦合，在拟议的研究中，我们将研究这些SR Ca2+释放机制相对于[Ca2+]i调节和激动剂诱导的[Ca2+]i振荡的特征和相互关系。

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.