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 byremoving mucus and pathogens from the respiratory tract. Since CBA is complicated andcannot be characterized by merely frequency, we recorded CBA using laser confocal linescanning and defined six parameters for describing CBA. The values of these parameters wereall above0when measured in beating ciliated cells from mouse tracheae.We subsequently used10μM adenosine-5′-triphosphate （ATP） to stimulate ciliated cellsand simultaneously recorded intracellular Ca²⁺levels and CBA. We found that intracellularCa²⁺levels firstincreased, followed by an increase in CBA. Among the sixparameters,frequency, amplitude, and integrated area significantly increased, whereas rise time, decaytime, and full duration at half maximum markedly decreased. The results suggest that thesesix parameters are appropriate for assessing CBA and that increased intracellular Ca²⁺levelsmight enhance CBA.We next used our established methods to observe changes in mechanically stimulatedcilia 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 bytreatment with a Ca²⁺chelator, BAPTA-AM,（10μM） for10min. Moreover, while the sameblockage was observed under Ca²⁺-free conditions, addition of2mM Ca²⁺into the chamberrestored increases in both intracellular Ca²⁺levels and CBA.Taken together, we have provided a novel method for real-time measurement andcomplete analysis of CBA as well as demonstrated that mechanical stimulation of cilia tipsresulted in Ca²⁺influx that led to increased intracellular Ca²⁺levels, which in turn triggeredCBA enhancement.