Carbocisteine stimulated an increase in ciliary bend angle via a decrease in [Cl−]i in mouse airway cilia
Carbocisteine (CCis), a mucoactive agent, is widely used to improve respiratory diseases. This study demonstrated that CCis increases ciliary bend angle (CBA) by 30% and ciliary beat frequency (CBF) by 10% in mouse airway ciliary cells. These increases were induced by an elevation in intracellular pH (pHi; the pHi pathway) and a decrease in the intracellular Cl− concentration ([Cl−]i; the Cl− pathway) stimulated by CCis. The Cl− pathway, which is independent of CO2/HCO3−, increased CBA by 20%. This pathway activated Cl− release via activation of Cl− channels, leading to a decrease in [Cl−]i, and was inhibited by Cl− channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid and CFTR(inh)-172). Under the CO2/HCO3−-free condition, the CBA increase stimulated by CCis was mimicked by the Cl−-free NO3− solution. The pHi pathway, which depends on CO2/HCO3−, increased CBF and CBA by 10%. This pathway activated HCO3− entry via Na+/HCO3− cotransport (NBC), leading to a pHi elevation, and was inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid. The effects of CCis were not affected by a protein kinase A inhibitor (1 μM PKI-A) or Ca2+-free solution. Thus, CCis decreased [Cl−]i via activation of Cl− channels including CFTR, increasing CBA by 20%, and elevated pHi via NBC activation, increasing CBF and CBA by 10%.
KeywordsAirway cilia Intracellular Cl− concentration Ciliary beating angle Inner dynein
We thank Osaka Medical College for giving us an opportunity to perform the experiments using the video microscope equipped with a high-speed camera.
This work was partly supported by Grants-in-Aid for Scientific Research from the Japan Society of the Promotion of Science to YM (No. JP18H03182) and to SH (No. 17K08545).
Compliance with ethical standards
The procedures and protocols for the experiments were approved by the Committee for Animal Research of Kyoto Prefectural University of Medicine (No. 26-263) and Ritsumeikan University (No. BKC 2017-050). The animals were cared for, and the experiments were carried out according to the guidelines of this committee. Female mice (C57BL/6J, 6 weeks of age) were purchased from Shimizu Experimental Animals (Kyoto, Japan) and fed standard pellet food and water ad libitum.
Conflict of interest
The authors declare that they have no conflict of interest.
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