Cynaropicrin Increases [Ca<sup>2+</sup>]<sub>i</sub> and Ciliary Beat Frequency in Human Airway Epithelial Cells by Inhibiting SERCA

Mucociliary clearance (MCC) is a host defense mechanism of the respiratory system. Beating cilia plays a crucial role in the MCC process and ciliary beat frequency (CBF) is activated by several factors including elevations of the intracellular cAMP concentration ([cAMP]_i), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and intracellular pH (pH_i). In this study, we investigated whether an artichoke-extracted component cynaropicrin could be a beneficial compound for improving MCC. We found that cynaropicrin increased [cAMP]_i using A549 cells bearing Pink Flamindo. Then, we also confirmed that cynaropicrin elevates CBF using airway epithelial ciliated cells (AECCs). We next investigated the effects of cynaropicrin on the alternation of [Ca²⁺]_i, and pH_i. Cynaropicrin increased [Ca²⁺]_i, but not pH_i. Further experiments also found that cynaropicrin increased [cAMP]_i primarily by raising [Ca²⁺]_i. To elucidate the mechanisms of cynaropicrin to increase [Ca²⁺]_i, we investigated the alternation of the effects of cynaropicrin on [Ca²⁺]_i using several compounds. BTP-2 and ruthenium red (RuR) inhibited cynaropicrin-induced [Ca²⁺]_i increase and RuR reduced also [cAMP]_i. These results suggest that cynaropicrion increased [Ca²⁺]_i by augmenting the Ca²⁺ influx and that the increase of [cAMP]_i by cynaropicrin was induced by [Ca²⁺]_i elevation. Interestingly, cynaropicrin decreased the Ca²⁺ concentration in the endoplasmic reticulum following inhibition of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA). SERCA activator CDN1163 abolished this effect. Furthermore, RuR and Ca²⁺-free conditions suppressed the increase of CBF. In conclusion, cynaropicrin inhibits SERCA, induces store-operated calcium entry, and thereby increases CBF.