Reactive oxygen species induce a Ca(2+)-spark increase in sensitized murine airway smooth muscle cells.

The level of reactive oxygen species (ROS) and the activity of spontaneous, transient, localized Ca(2+) increases (known as Ca(2+) sparks) in tracheal smooth muscle cells (TSMCs) in an experimental allergic asthma mouse model has not yet been investigated. We used laser confocal microscopy and fluorescent dyes to measure ROS levels and Ca(2+) sparks, and we found that both events were significantly increased in TSMCs obtained from ovalbumin (OVA)-sensitized/-challenged mice compared with control mice. ROS levels began to increase in TSMCs after the first OVA challenge, and this increase was sustained. However, this elevation and Ca(2+)-spark increase was abolished after the administration of the ROS scavenger N-acetylcysteine amide (NACA) for 5days. Furthermore, a similar inhibition was also observed following the direct perfusion of NACA into cells isolated from the (OVA)-sensitized mice that were not treated with NACA. Moreover, we used 0.1-mM caffeine treatment to increase the Ca(2+) sparks in single TSMCs and observed cell shortening. In addition, we did not find increases in the mRNA levels of ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors in the tracheal smooth muscle cells of (OVA)-sensitized mice compared with controls. We concluded that ROS and Ca(2+) sparks increased in (OVA)-sensitized TSMCs. We found that ROS induces Ca(2+) sparks, and increased Ca(2+) sparks resulted in the contraction of (OVA)-sensitized TSMCs, resulting in the generation of airway hyperresponsiveness (AHR). This effect may represent a novel mechanism for AHR pathogenesis and might provide insight into new methods for the clinical prevention and treatment of asthma and asthmatic AHR.

Methods to measure and analyze ciliary beat activity: Ca2+ influx-mediated cilia mechanosensitivity.

Airway ciliary beat activity (CBA) plays a pivotal role in protecting the body by removing mucus and pathogens from the respiratory tract. Since CBA is complicated and cannot be characterized by merely frequency, we recorded CBA using laser confocal line scanning and defined six parameters for describing CBA. The values of these parameters were all above 0 when measured in beating ciliated cells from mouse tracheae. We subsequently used 10 µM adenosine-5'-triphosphate (ATP) to stimulate ciliated cells and simultaneously recorded intracellular Ca(2+) levels and CBA. We found that intracellular Ca(2+) levels first increased, followed by an increase in CBA. Among the six parameters, frequency, amplitude, and integrated area significantly increased, whereas rise time, decay time, and full duration at half maximum markedly decreased. The results suggest that these six parameters are appropriate for assessing CBA and that increased intracellular Ca(2+) levels might enhance CBA. We next used our established methods to observe changes in mechanically stimulated cilia tips. We found that mechanical stimulation-induced changes in both intracellular Ca(2+) levels and CBA were not only similar to those induced by ATP, but were also blocked by treatment with a Ca(2+) chelator, BAPTA-AM, (10 µM) for 10 min. Moreover, while the same blockage was observed under Ca(2+)-free conditions, addition of 2 mM Ca(2+) into the chamber restored increases in both intracellular Ca(2+) levels and CBA. Taken together, we have provided a novel method for real-time measurement and complete analysis of CBA as well as demonstrated that mechanical stimulation of cilia tips resulted in Ca(2+) influx that led to increased intracellular Ca(2+) levels, which in turn triggered CBA enhancement.