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

Abstract

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 Ca²⁺ levels and CBA. We found that intracellular Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ levels and CBA were not only similar to those induced by ATP, but were also blocked by treatment with a Ca²⁺ chelator, BAPTA-AM, (10 μM) for 10 min. Moreover, while the same blockage was observed under Ca²⁺-free conditions, addition of 2 mM Ca²⁺ into the chamber restored increases in both intracellular Ca²⁺ 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 Ca²⁺ influx that led to increased intracellular Ca²⁺ levels, which in turn triggered CBA enhancement.