Ambroxol-enhanced ciliary beating via voltage-gated Ca²⁺ channels in mouse airway ciliated cells

Ambroxol (ABX) facilitates the mucociliary clearance (MC) by enhancing ciliary beating in airways. In this study, we focused on airway ciliary beating enhanced by ABX. However, little is known about the ABX-stimulated Ca²⁺ signalling activating airway ciliary beating. Airway ciliated cells isolated from mice lungs were observed by a high-speed video microscope, and the activities of beating cilia were assessed by CBF (ciliary beat frequency) and CBD (ciliary bend distance, an index of amplitude). ABX (10 μM) enhanced the CBF and CBD by 30%, and the enhancement was inhibited by nifedipine (20 μM, a L-type voltage-gated Ca²⁺ channel (Ca_V) inhibitor), or a Ca²⁺-free solution (approximately 50%). Pre-treatment with BAPTA-AM (10 μM, a chelator of intracellular Ca²⁺) abolished ABX-stimulated increases in CBF, CBD and [Ca²⁺]_i. Thus, ABX increases [Ca²⁺]_i (intracellular Ca²⁺ concentration) by stimulating Ca²⁺ release from the internal stores and nifedipine-sensitive Ca²⁺ entry. A previous study demonstrated the expression of Ca_V1.2 in airway cilia. ABX enhanced CBF, CBD and [Ca²⁺]_i even in a high extracellular K⁺ concentration (155.5 mM), suggesting that it activates Ca_V1.2 except by depolarization. These enhancements were inhibited by nifedipine. In conclusion, ABX, which increases [Ca²⁺]_i by stimulating Ca²⁺ release from internal stores and Ca²⁺ entry through Ca_V1.2s, enhanced CBF and CBD in airway ciliated cells. ABX is a novel agonist that modulates Ca_V1.2 of airway beating cilia to enhance CBF and CBD.