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.
|Effective start/end date||7/1/03 → 2/28/15|