Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pHi and [Cl-]i in lung airway epithelial cells of mice

doi:10.21203/rs.3.rs-3409150/v1

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Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pH_i and [Cl^-]_i in lung airway epithelial cells of mice

https://doi.org/10.21203/rs.3.rs-3409150/v1

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Ambroxol (ABX), a frequently prescribed secretolytic agent that stimulates Ca²⁺ release from acidic stores and Ca²⁺ entry through Ca_V1.2, enhanced the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30% in ciliated lung airway epithelial cells (c-LAECs) of mice. An increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) stimulated by ABX triggers two signals in c-LAECs; an increase in pH_i (pH pathway) and a decrease in [Cl^-]_i (Cl^- pathway). The pH pathway, which was activated by the HCO₃^- entry through Na⁺-HCO₃^- cotransporter (NBC) and inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), enhanced the CBF (by 30%) and CBA (by 15-20%) and the Cl^- pathway, which was activated by Cl^- secretion through anoctamine 1 (ANO1) and inhibited by Cl^- channel blockers (NPPB and T16Ainh), enhanced the CBA (by 10-15%). The enhancement of the CBF and CBA stimulated by ABX was decreased to 50% by a Ca²⁺-free solution or nifedipine (an inhibitor of L-type Ca²⁺ channels) and was abolished by BAPTA-AM in c-LAECs, indicating that an increase in [Ca²⁺]_i stimulated by ABX is essential for activating the pH pathway and the Cl^- pathway. The enhancement of CBF and CBA enhanced by ABX was mimicked by applying a CO₂/HCO₃^--free Cl^--free solution, which increased the pH_i and decreased the [Cl^-]_i. In conclusion, ABX increased the CBF and CBA by 30% via the pH pathway and the Cl^- pathway triggered by the [Ca²⁺]_i increase in c-LAECs of mice.

[Ca2+]i

intracellular pH

intracellular Cl- concentration

cell volume

CaV1.2

Mucociliary clearance (MC), which is a host-defence mechanism of the lungs, consists of a surface fluid layer (surface mucous layer (SML) and periciliary layer (PCL)) and beating cilia lining the airway surface [1, 5, 44]. Inhaled small particles, such as bacteria, viruses and chemicals, are entrapped by the SML and swept away towards the oropharynx by the beating cilia in the PCL. The MC is compared to a belt conveyor for extruding inhaled small particles from the airways and the beating cilia are the engine that drives MC [1, 7, 44]. Impairment in the beating cilia, such as Kartagener syndrome or primary ciliary dyskinesia, causes serious respiratory diseases [1, 44]. Therefore, the drugs activating beating cilia are of particular importance to prevent or improve respiratory diseases. Ambroxol (ABX), a frequently prescribed secretolytic agent for respiratory diseases, has been shown to increase the CBF (ciliary beat frequency) and CBA (ciliary bend angle, an index of amplitude) in ciliated airway epithelial cells [33, 37]. However, the ABX-stimulated mechanism enhancing the CBF and CBA is still controversial.

Beating cilia in airways are activated by many substances such as cAMP, Ca²⁺, ATP, β₂-agonists, Cl⁻, and H⁺ (intracellular pH (pH_i)) [7, 16–20, 22–25, 38, 40, 44, 47]. Among them, Ca²⁺ is an important ion that activates beating cilia [1, 7, 44]. ABX, which acts as a weak base, stimulates pH-dependent Ca²⁺ release from acidic stores [9, 37] and voltage-dependent Ca²⁺ channels (Ca_V1.2) to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in c-LAECs [37]. Moreover, ABX has been shown to stimulate anion secretion in lung epithelial cell lines [12, 43]. These observations suggest that an increase in [Ca²⁺]_i stimulated by ABX activates Cl⁻ secretion in c-LAECs leading to a decrease in intracellular Cl⁻ concentration ([Cl⁻]_i). A decrease in [Cl⁻]_i appears to enhance the CBA in c-LAECs [16, 18–20, 47]. Moreover, Saito et al. (2023) suggested that ABX stimulates a pH_i increase in c-LAECs [37]. Thus, an increase in [Ca²⁺]_i stimulated by ABX activates a pH_i increase and an [Cl⁻]_i decrease in c-LAECs, which appear to enhance the CBF and CBA.

Previous studies in Chlamydomonas revealed that cilia have two functionally distinct molecular motors, namely outer dynein arms (ODAs) and inner dynein arms (IDAs), which regulate the ciliary beat frequency (CBF) and ciliary bend angle (CBA), respectively [4, 10]. The signalling pathways regulating the CBF and CBA have been shown to be different [18–20, 22–25]. However, the CBF has been used to assess the activity of ciliary beating. Previous studies demonstrated that a CBA increase, in addition to the CBF increase, enhances ciliary transport in the airway [18–20, 22–25, 47].

In this study, we examined the effects of ABX on the CBF and CBA in c-LAECs of mice. In the course of experiments using a CO₂/HCO₃⁻-free solution, we found that ABX stimulates two signals to increase the CBF and CBA, CO₂/HCO₃⁻ -dependent and CO₂/HCO₃⁻-independent. This study is designed to clarify the two signals increasing the CBF and CBA activated by ABX in c-LAECs.

Ethical approval

The experiments were approved by the Committees for Animal Research of Kyoto Prefectural University of Medicine (No. 26-263, April 2017) and Ritsumeikan University (BKC-HM-2017-050). The animals were cared for and the experiments were carried out according to the guidelines of these committees. Female mice (C57BL/6J, 5 weeks of age) were purchased from Shimizu Experimental Animals (Kyoto, Japan), fed standard pellet food and water ad libitum, and were used for experiments at 6-10 weeks of age. The mice were, first, anaesthetized by inhalational isoflurane (3%) and were further anesthetized by an intraperitoneal injection (ip) of pentobarbital sodium (70 mg/kg) and heparinized (1000 units/kg) for 15 min. Then, the mice were sacrificed by a high dose of pentobarbital sodium (100 mg/kg, ip).

Solutions and chemicals

The control solution containing CO₂/HCO₃^- contained (in mM): NaCl, 121; KCl, 4.5; NaHCO₃, 25; MgCl₂, 1; CaCl₂, 1.5; Na-HEPES, 5; H-HEPES, 5; and glucose, 5. To prepare the CO₂/HCO₃^--free solution, HCO₃^- was replaced with Cl^-. To prepare the Cl^--free NO₃^- solution, Cl^- was replaced with NO₃^-, and to prepare the Ca²⁺-free solution, CaCl₂ was removed from the solution. The CO₂/HCO₃^--containing solutions were aerated with 95% O₂and 5% CO₂ and the CO₂/HCO₃^--free solutions were aerated with 100% O₂. The pH of the solutions was adjusted to 7.4 by adding 1 N HCl or 1 N HNO₃, as appropriate. The experiments were carried out at 37 °C. Ambroxol (ABX), nifedipine, NPPB, DIDS and DMSO were purchased from Sigma (St. Louis, USA), and heparin, elastase, and bovine serum albumin (BSA) were purchased from Fujifilm-Wako (Osaka, Japan). All reagents were dissolved in dimethyl sulfoxide (DMSO) and prepared to their final concentrations immediately before the experiments. The DMSO, the concentration of which did not exceed 0.1%, had no effect on the CBF or CBA [22, 25, 38].

Cell preparation:

Ciliated LAECs were isolated from the lungs as previously described [16, 22, 25, 36, 37]. After sacrifice, the lungs were cleared of blood by perfusion via the pulmonary artery, and the lungs with the trachea and heart were removed from the animal en bloc. The lung cavity was washed with a Ca²⁺-free solution and then with the control solution via the tracheal cannula. After washing, the control solution containing elastase (0.2 mg/ml) and DNase I (0.02 mg/ml) was instilled into the lung cavity and the airway epithelium was digested for 40 min at 37 °C. Following this incubation, the lungs were minced using fine forceps in the control solution containing DNase I (0.02 mg/ml) and BSA (5%). The minced tissue was filtered through a nylon mesh (a sieve with 300 µm openings). The cells were washed three times with centrifugation (160×g for 5 min) and then suspended in the control solution. The cell suspension was stored at 4 °C and the cells were used within 5 hours after the isolation.

CBA and CBF measurements

Cells were placed on a coverslip precoated with Cell-Tak (Becton Dickinson Labware, Bedford, MA, USA). A coverslip with cells was set in a microperfusion chamber (20 μl) mounted on an inverted light microscope (Eclipse Ti, NIKON, Tokyo, Japan) connected to a high-speed camera (FASTCAM-1024PCI, Photron Ltd., Tokyo, Japan). The stage of the microscope was heated to 37°C, since the CBF is temperature-dependent [7, 18]. The rate of perfusion was 200 μl/min. Ciliated LAECs, which were distinguished from other cells by their beating cilia, were accounted for 5-10% of the isolated lung cells. For the CBF and CBA measurements, video images were recorded for 2 s at 500 fps (frame per second) [25]. Before the start of experiments, the cells were perfused with the control solution for 5 min. After the experiments, the CBF and CBA were measured using an image analysis program (DippMotion 2D, Ditect, Tokyo, Japan) [16-29, 22-25]. The CBF and CBA ratios (CBF_t/CBF₀ and CBA_t/CBA₀), the values of which were normalized by the control values, were used to make a comparison among the experiments. CBFs or CBAs measured every 1 min during control perfusion (5 min) were averaged and the averaged value was used as CBF₀ or CBA₀. The subscript “0” or “t” indicates the time before or after the start of experiments, respectively. Each experiment was carried out using 4-10 cover slips with cells obtained from 2-5 animals. In each coverslip, we selected a visual field with 1-2 cells or a cell block and measured their CBFs and CBAs. The normalized CBF and CBA (CBF ratio and CBA ratio) calculated from 4-12 cells were plotted and “n” shows the number of cells.

Measurement of the cell volume

The outline of a c-LAEC was traced on a video image, and the area of the cell (A) was measured by the image analysis program. The index of the cell volume (V_t/V₀ = (A_t/A₀)^1.5) was calculated [31]. The indices of the cell volume measured every 1 min during the control perfusion (5 min) were averaged and the averaged value was used as V₀. Each experiment was carried out using 4-6 cover slips obtained from 2-3 animals. The V/V₀ values calculated from 4-6 cells were plotted and “n” shows the number of cells.

Measurement of pH_i, [Cl^-]_i and [Ca²⁺]_i

Changes in intracellular pH (pH_i) were monitored by SNARF1 fluorescence (a pH dye). The c-LAECs were incubated with 10 μM carboxy SNARF1-AM for 45 min at 37 °C and the cells were set on the heated stage (37 °C) of an inverted confocal laser microscope (model LSM510 META, Carl Zeiss, Jena, Germany) [14]. The excitation was 515 nm and the emissions were 645 nm and 592 nm. The fluorescence ratio (F645/F592) was calculated to measure the pH_i. The calibration was carried out using c-LAECs perfused with a calibration solution. The calibration solution, composed of 110 mM KCl, 25 mM KHCO₃, 11 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES and 10 μM nigericin, was aerated with 95% air and 5% CO₂. The pH of the calibration solution was adjusted to 6.8, 7.2, 7.4, 7.6 or 7.8 by adding 1 M CsOH at 37 °C. The ratios of F645/F592 were plotted against the pH values of the calibration solution and the pH_i of the c-LAECs was calculated from the calibration line.

Changes in [Cl^-]_i were monitored by MQAE fluorescence (a chloride dye) [15, 16, 18-20]. Cells were incubated with 10 mM MQAE for 45 min at 37 °C. MQAE was excited at 780 nm using a 2-photon excitation laser system (MaiTai®, Spectra-Physics), and the emission was 510 nm. The fluorescence intensity ratio (F₀/F_t) was calculated.

Changes in [Ca²⁺]_i were monitored by fura-2 fluorescence (a Ca²⁺ dye). Cells were incubated with 5 μM fura-2-AM for 30 min at 37 °C. Fura-2 was excited at 340 and 380 nm, and the emission was 510 nm at 37 °C, and the ratio of fura-2 fluorescence (F340/F380) was calculated by an image analysis system (MetaFluor, Molecular Device, USA) [36, 37]. The experiments were carried out using 5 coverslips obtained from 3 animals.

Western blotting

The procedure for western blotting has already been described in a previous report [36, 17]. Protein (5-20 µg) obtained from isolated lung cells was loaded into each lane for Laemmli SDS-polyacrylamide gel electrophoresis (10%) and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked for 1 h using 5% skim milk powder in TBS-T (150 mM NaCl, 10 mM Tris-HCl, pH 8.5 and 0.1% Tween 20) and exposed to primary anti-anoctamin-1 antibody (1:200) (ABN1669 Sigma-Aldrich Merck, Darmstadt, Germany) diluted with Solution 1 (Can Get Signal, TOYOBO, Osaka, Japan) at 4 °C overnight. After rinsing with TBS-T, the secondary antibody diluted with Solution 2 (Can Get Signal, TOYOBO) was applied to the membrane for 1 h at room temperature. After rinsing with TBS-T, antigen-antibody complexes were visualized with a chemiluminescence system (Immobilon, Merck Millipore, Darmstadt, Germany).

Immunofluorescence examination

The cell suspension containing isolated lung cells (0.5 mL) was dropped and dried on the coverslip for attaching cells [16, 17, 36; 37]. The cells on the coverslip were fixed with 4 % paraformaldehyde for 30 min at room temperature and washed with ice-cold PBS containing 10 mM glycine. Then, the cells were permeabilized with PBS containing 0.1% Triton-X 100 and washed. For immunostaining, cells were incubated with an anoctamin-1 antibody and an anti-acetylated tubulin antibody (T6793, Sigma-Aldrich, St Louis, MO) at 4 °C overnight. Cells were washed with PBS containing 0.1% BSA to remove unbound antibodies. Finally, the cells were stained with Alexa 488-conjugated anti-rabbit antibody and Alexa 594-conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA). The cells were observed using confocal laser microscopy (FV10i, Olympus).

Statistical Analysis

Data are expressed as the means ± SEMs. Statistical significance was assessed by one-way analysis of variance (one-way ANOVA). Differences were considered significant at p < 0.05.

ABX-stimulated increases in CBF and CBA

In unstimulated c-LAECs perfused with the control solution, the CBF and CBA were 8-10 Hz and 70° - 90°, respectively [25]. Saito et al. (2023) demonstrated that ABX increases the CBF and CBA in a concentration-dependent manner and that ABX maximally increases the CBF and CBA at 10 µM [37]. In this study, the concentration of ABX used was 10 µM throughout the experiments. ABX gradually increased the CBF and CBA by 30% within 10 min and the ratios of the CBF and CBA at 10 min after stimulation were 1.25 (n=13) and 1.27 (n=8), respectively (Fig. 1a). We examined the effects of Ca²⁺ on the CBF and CBA stimulated by ABX. In this study, we used a nominally Ca²⁺-free solution, not EGTA-containing Ca²⁺-free solution, because the EGTA-containing Ca²⁺-free solution increases the CBF by inhibiting Ca²⁺-dependent PDE1A located in the CBF-regulating metabolon in the cilia [22]. The switch to a nominally Ca²⁺-free solution decreased the CBF and CBA by 5% within 5 min, and then stimulation with ABX increased the CBF and CBA by 10-13% within 5 min. The ratios of the CBF and CBA at 5 min after the switch were 0.96 (n=8) and 0.96 (n=6) and those 5 min after the addition of ABX were 1.04 and 1.09, respectively (Fig. 1b). Similar experiments were carried out using nifedipine (20 µM). The addition of nifedipine decreased the ratios of the CBF and CBA by 5% and then ABX stimulation increased them by 10%. Similar results have been shown in a previous report [37]. Experiments were also carried out in the absence of CO₂/HCO₃^- (Fig. 1c and 1d). The switch to the CO₂/HCO₃^--free solution immediately increased the CBF and CBA and the ratios of the CBF and CBA 5 min after the switch were 1.32 (n=8) and 1.21 (n=6), respectively. The application of CO₂/HCO₃^--free solution is a well-known procedure to increase pH_i, which enhances the CBF and CBA [16, 40]. Further ABX stimulation increased only the CBA, but not the CBF. The ratios of the CBF and CBA 5 min after ABX stimulation were 1.32 (n=6) and 1.30 (n=8), respectively. Similar experiments were carried out in the presence of nifedipine (10 µM) (Fig. 1d). The addition of nifedipine decreased the CBF and CBA by 5%. Then, the switch to CO₂/HCO₃^--free solution immediately increased the CBF and CBA. The ratios of the CBF and CBA 5 min after the switch were 1.28 (n=4) and 1.20 (n=4), respectively. Further ABX stimulation did not increase the CBF or CBA (Fig. 1d). Similar results were obtained by using a Ca²⁺-free solution. Prior addition of BAPTA-AM (10 µM, a membrane permeable analogue of BAPTA (a Ca²⁺ chelator) that binds intracellular calcium after the acetoxymethyl group is removed by the cytoplasmic esterase) completely inhibited the increases in the CBF and CBA stimulated by ABX in a Ca²⁺ solution [37]. Thus, ABX activates two signalling pathways, that is, CO₂/HCO₃^--dependent and CO₂/HCO₃^—independent signaling pathways.

[Ca²⁺]_i stimulated by ABX in c-LAECs

Changes in [Ca²⁺]_i were monitored by the ratio of fura-2 fluorescence (F340/F380) in c-LAECs. In the control solution, stimulation with ABX gradually increased F340/F380, which reached a plateau within 15 min, and then, the addition of nifedipine decreased F340/F380 to the level before ABX stimulation within 10 min (Fig. 2a). Changes in F340/F380 stimulated by ABX were measured in a Ca²⁺-free solution. ABX transiently increased F340/F380 in c-LAECs (Fig. 2b). Similar results have been shown in a previous report [37]. ABX has been shown to stimulate Ca²⁺ release from acidic stores in alveolar type II cells (ATII cells) and Ca²⁺ release was inhibited by an increase in pH_i [9, 37]. Experiments were carried out in a CO₂/HCO₃^--free solution. The switch from the control solution to the CO₂/HCO₃^--free solution did not change F340/F380, and ABX stimulation did not increase F340/F380. The addition of nifedipine did not change F340/F380 (Fig. 2c). An increase in pH_i induced by the application of CO₂/HCO₃^--free solution suppressed ABX-stimulated Ca²⁺ release from acidic stores as shown in a previous report [37]. Moreover, the CO₂/HCO₃^--free solution may induce hyperpolarization, which may decrease Ca²⁺ influx through Ca_V1.2 channels. The application of CO₂/HCO₃^--free solution inhibits Na⁺ entry via the NBC, maintaining K⁺ uptake by Na⁺/K⁺ ATPase, which induces hyperpolarization by increasing [K⁺]_i.

Changes in pH_i stimulated by ABX

Changes in pH_i were measured by the ratio of SNARF1 fluorescence (F645/F592). In the control solution, ABX stimulation gradually increased pH_i from 7.49 (just before ABX stimulation) to 7.65 (15 min after ABX stimulation, n=8) (Fig. 3a). Cells were also stimulated by ABX in a Ca²⁺-free solution (Fig. 3b) or in the presence of nifedipine (Fig. 3c). ABX increased pH_i from 7.51 to 7.58 in the Ca²⁺-free solution (n=4) or from 7.52 to 7.60 in the presence of nifedipine (n=5), although the final pH_i values were lower than those in the control solution containing Ca²⁺ (Fig. 3a-c). Changes in pH_i were measured upon applying the CO₂/HCO₃^--free solution. The switch to the CO₂/HCO₃^--free solution transiently increased and then sustained pH_i. The pH_i at 2 min after the switch was 7.87 and that at 10 min after the switch was 7.64 (n=6). Further stimulation with ABX did not change pH_i (pH_i at 10 min after ABX stimulation = 7.65) (Fig. 3d).

ABX evoked cell shrinkage and a decrease in [Cl^-]_i

ABX stimulation evoked cell shrinkage in c-LAECs. Fig. 4 shows the images of a c-LAEC perfused with the control solution. Fig. 4a and 4b show the phase contrast images of a c-LAEC just before and at 15 min after ABX stimulation, respectively. The black line in Fig. 4a shows the outline of a c-LAEC before ABX stimulation and was superimposed in Fig. 4b. The c-LAECs stimulated by ABX were smaller than those before ABX stimulation. (Fig. 4a and 4b). Thus, ABX stimulation decreased the cell volume. Cell shrinkage is known to decrease [Cl^-]_i [16, 29, 47]. We monitored [Cl^-]_i in c-LAECs, using the fluorescence of MQAE (a Cl^- sensitive fluorescence dye) [15]. ABX stimulation increased the intensity of MQAE fluorescence in a c-LAEC (Fig. 4c and 4d), indicating that ABX stimulation decreases [Cl^-]_i.

Changes in cell volume (V/V₀, index of cell volume) and [Cl^-]_i stimulated by ABX were measured in c-LAECs (Fig. 5). In the control solution, ABX stimulation decreased V/V₀ by 20%. The V/V₀ at 6 min after ABX stimulation was 0.81 (n=5). Further addition of nifedipine immediately increased V/V₀ to the level before ABX stimulation (V/V₀ at 5 min after nifedipine addition = 1.03) (Fig. 5a). Changes in [Cl^-]_i were monitored using the MQAE fluorescence ratio (F₀/F). ABX stimulation decreased F₀/F, the value of which was 0.70 at 10 min after the ABX stimulation (n=4) (Fig. 5b). Experiments were also carried out using the CO₂/HCO₃^--free solution. The switch to the CO₂/HCO₃^--free solution decreased V/V₀ to 0.90, (n=5, 5 min after the switch). Further ABX stimulation decreased V/V₀ to 0.78 (n=5, 10 min after ABX stimulation) (Fig. 5c). Changes in the MQAE fluorescence ratio (F₀/F) were also measured (Fig. 5d). The switch to the CO₂/HCO₃^--free control solution decreased F₀/F to 0.84 (n=4, 5 min after the switch), and then, ABX stimulation decreased F₀/F to 0.69 (10 min after ABX stimulation) (Fig. 5d). Similar experiments were carried out in the presence of nifedipine. In the control solution, the addition of nifedipine increased V/V₀ to 1.12 (n=4, 5 min after nifedipine addition). The switch to the CO₂/HCO₃^--free solution decreased V/V₀ to 0.93, and then ABX stimulation did not change V/V₀ (0.91 at 10 min after ABX stimulation) (Fig. 5e). Changes in [Cl^-]_i were monitored by the MQAE fluorescence ratio (F₀/F). The addition of nifedipine increased F₀/F to 1.15 (n=5, 5 min after nifedipine addition). Then, the switch to the CO₂/HCO₃^--free solution decreased F₀/F to 0.82 and ABX stimulation did not change F₀/F (0.82 at 10 min after ABX stimulation) (Fig. 5f).

CO₂/HCO₃^- -dependent pathway (pH_i pathway)

In the control solution, ABX may stimulate HCO₃^- entry via the NBC to increase pH_i. The effects of DIDS (200 µM, an inhibitor of the NBC and an anion exchanger (AE, Cl^-/HCO₃^- exchanger)) on the CBF, CBA and pH_i were examined (Fig. 6). The addition of DIDS (100 µM) gradually increased the CBF, but not the CBA (Fig. 6a). CBF reached a plateau within 15 min. The CBF and CBA ratios 15 min after DIDS addition were 1.12 (n=6) and 1.01 (n=4), respectively. The addition of DIDS increased pH_i, and the pH_i values just before and 5 min after DIDS addition were 7.45 and 7.49 (n=4), respectively (Fig. 5b). The effects of ABX on CBA and CBF were examined in the presence of DIDS (Fig. 6c). The addition of DIDS gradually increased the CBF, but not the CBA. Then, stimulation with ABX immediately increased only the CBA, but not the CBF (Fig. 5c). The CBA ratio just before ABX stimulation was 1.00 (n=4), and that at 5 min after ABX stimulation was 1.17. Changes in pH_i stimulated by ABX were measured in the presence of DIDS. The addition of DIDS alone increased pH_i. Further ABX stimulation did not affect the gradual pH_i increase induced by DIDS. The pH_i at 10 min after ABX stimulation was 7.52 (Fig. 5b). DIDS abolished the increases in the CBF and pH_i stimulated by ABX in c-LAECs.

Changes in the MQAE fluorescence ratio induced by ABX were measured in the presence of DIDS. DIDS did not affect F₀/F. Stimulation with ABX decreased F₀/F (Fig. 6d). DIDS did not affect the [Cl^-]_i decrease stimulated by ABX, and the increase in the CBA stimulated by ABX appears to be induced by an [Cl^-]_i decrease in c-LAECs.

To confirm HCO₃^- entry via the NBC, experiments were carried out using a HCO₃^--containing Cl^--free NO₃^- solution, in which the NBC is functional, but not the AE (Fig. 6e and 6f). The switch to the HCO₃^--containing Cl^--free NO₃^- solution immediately increased the CBF and CBA, and then stimulation with ABX increased both. The CBF and CBA ratios at 10 min after the switch were 1.24 (n=7) and 1.20 (n=8), and those at 10 min after the ABX stimulation were 1.33 and 1.33, respectively (Fig. 6e). Changes in pH_i were also measured. The switch to the HCO₃^--containing Cl^--free NO₃^- solution immediately increased pH_i to 7.65 (n=5) and then, the ABX stimulation increased the pH_i to 7.91. The HCO₃^--containing Cl^--free NO₃^- solution potentiated the CBF, CBA and pH_i stimulated by ABX. The HCO₃^--containing Cl^--free NO₃^- solution, which inhibits HCO₃^- extrusion via the AE while maintaining HCO₃^- influx via the NBC, may increase the concentration of HCO₃^- in c-LAECs leading to an increase in pH_i. ABX appears to stimulate HCO₃^- influx via the NBC to increase pH_i.

CO₂/HCO₃^--independent pathway (Cl^- pathway)

We examined the effects of NPPB (20 µM, a Cl^- channel blocker) on the CBF and CBA stimulated by ABX. The addition of NPPB decreased the CBA and CBF. Then, the ABX stimulation increased both. The CBF and CBA ratios at 5 min after NPPB addition were 0.92 (n= 6) and 0.96 (n=6), respectively, and those at 10 min after the ABX stimulation were 1.06 and 1.10, respectively (Fig. 7a). The addition of NPPB increased the MQAE fluorescence ratio (F₀/F) from 0.99 (n=6, before the addition) to 1.14 (5 min after the addition). Then, the ABX stimulation gradually increased F₀/F. The F₀/F at 15 min after ABX stimulation was 1.25 (Fig. 7b). The effects of NPPB on the CBF and CBA with or without ABX were similar to those of the Ca²⁺-free solution or nifedipine on them (Fig. 1b). The experiments were also carried out in CO₂/HCO₃^--free solution. The addition of NPPB decreased the CBF and CBA. The switch to the CO₂/HCO₃^--free solution increased the CBF and CBA, and then ABX stimulation did not increase the CBF or CBA (Fig. 7c). The CBF and CBA ratios were 0.95 (n=4) and 0.90 (n=6) at 5 min after NPPB addition, 1.03 and 1.11 at 5 min after the switch to the CO₂/HCO₃^--free solution, and 1.03 and 1.06 at 15 min after ABX stimulation. Changes in [Cl^-]_i were monitored by the MQAE fluorescence ratio (F₀/F) (Fig. 7d). The addition of NPPB increased F₀/F. The switch to CO₂/HCO₃^- -free solution decreased F₀/F and stimulation with ABX did not change F₀/F. The F₀/F was 1.15 (n=5) at 5 min after the NPPB addition, 0.88 at 5 min after the switch to CO₂/HCO₃^- -free solution, and 0.86 at 15 min after ABX stimulation.

A previous study demonstrated that anoctamin-1 (ANO1), a Ca²⁺-activated Cl^- channel, functions in ciliated nasal epithelial cells [20]. ABX, which increases [Ca²⁺]_i, may activate ANO1 [46]. The addition of T16Ainh (10 µM, an inhibitor of ANO1) did not change the CBF and CBA, and stimulation with ABX increased the CBF and CBA. The CBF and CBA ratios were 1.02 (n=7) and 0.95 (n=5) at 5 min after T16Ainh addition, and 1.12 and 1.12 at 10 min after the ABX stimulation (Fig. 8a). Experiments were carried out in CO₂/HCO₃^--free solution. The switch to the CO₂/HCO₃^- -free solution increased the CBF and CBA. Then, the addition of T16Ainh and further ABX stimulation did not change the CBF or CBA. The CBF and CBA ratios were 1.21 (n=5) and 1.28 (n=5) at 5 min after the switch to the CO₂/HCO₃^- -free solution, 1.23 (n=7) and 1.18 (n=5) at 5 min after the T16Ainh addition, and 1.23 and 1.20 at 10 min after the ABX stimulation (Fig. 8b).

Effects of a CO₂/HCO₃^--free Cl^--free NO₃^- solution on CBF, CBA and [Cl^-]_i

To increase pH_i and decrease [Cl^-]_i, we used the CO₂/HCO₃^--free Cl^--free NO₃^- solution, in which Cl^- in the CO₂/HCO₃^--free solution was replaced with NO₃^- [16]. The switch to the CO₂/HCO₃^--free solution increased the CBF and CBA. The CBF and CBA ratios at 5 min after the switch were 1.25 (n=5) and 1.16 (n=5), respectively. Then, the second switch to the CO₂/HCO₃^--free Cl^--free NO₃^- solution increased the CBA without any CBF increase. The CBF and CBA ratios at 5 min after the second switch were 1.27 (n=5) and 1.28 (n=5), respectively. Further ABX stimulation did not increase the CBA or CBF. The CBF and CBA ratios at 5 min after ABX stimulation were 1.31 (n=5) and 1.29 (n=5), respectively (Fig. 9a). Changes in [Cl^-]_i were also monitored by the MQAE fluorescence ratio using the same protocol. The CO₂/HCO₃^--free solution decreased F₀/F to 0.90 (5 min after the switch, n=4) and the second switch further decreased F₀/F to 0.73 (5 min after the second switch, n=4). The ABX stimulation did not decrease F₀/F (0.69 at 10 min after the ABX stimulation, n=4) (Fig. 9b). Thus, ABX actions on the CBA and CBF were mimicked by the CO₂/HCO₃^--free Cl^--free NO₃^- solution.

Expression of anoctamin-1 in c-LAECs

The expression of ANO1 (TMEM16A) was examined by the western blotting and the immunofluorescence. In western blotting, a single band of ANO1 was detected at 110 kDa (Fig. 10). Immunofluorescence analysis ofr ANO1 revealed that cilia and cell bodies were positively stained for ANO1 in c-LAECs (Fig. 11a), and cilia were positively stained for acetylated tubulin (a-tubulin, a marker of cilia) (Fig. 11b). The merged image in Fig. 11a and Fig. 11b shows that ANO1 exists in cilia (Fig. 11c). The phase contrast image of c-LAECs is shown in Fig. 11d.

This study demonstrated that ABX increases both the CBF and CBA by 30% in c-LAECs mediated via an increase in [Ca²⁺]_i, which activates two signalling pathways; the pH pathway (an increase in pH_i) and the Cl^- pathway (a decrease in [Cl^-]_i). The pHpathway increased the CBF by 30 % and the CBA by 15-20%, and the Cl^- pathway increased the CBA by 10-15%. Previous studies revealed that ABX stimulates Ca²⁺ release from acidic stores [9, 37] and Ca²⁺ entry through voltage-dependent Ca²⁺ channels (Ca_V1.2) in c-LAECs [36, 37]. Treatment with BAPTA-AM, which does not increase [Ca²⁺]_i by chelating intracellular Ca²⁺, abolished the CBF and CBA enhancement [37].

An increase in pH_i has been shown to enhance the CBF and CBA in airway ciliary cells [16, 40] and to enhance the CBF in sperm flagella [21, 32]. The present study demonstrated that the pH pathway is activated by HCO₃^- entry and is inhibited by DIDS (an inhibitor of bicarbonate transporters) in c-LAECs. There are two bicarbonate transporters in c-LAECs, NBC and AE [28]. The DIDS-sensitive AE is detected in the apical membrane and mediates HCO₃^- secretion in bronchiole epithelial cells [28]. In the HCO₃^--containing Cl^--free solution which inhibits HCO₃^- secretion via the AE because there is no extracellular Cl^-, ABX enhanced the increase in pH_i, suggesting an increase in intracellular HCO₃^- concentration by inhibiting HCO₃^- extrusion via the AE. These results indicate that the AE does not transport HCO₃^- into the cell. Based on these results, we concluded that ABX stimulated the NBC mediated by an [Ca²⁺]_i increase, the activation of which enhanced the pH_i increase by stimulating HCO₃^- influx. ABX has already been shown to increase pH_i in type II pneumocytes [9]. The expression of messenger RNA of all NBC isoforms has been identified in airway epithelial cells [28], but the membrane localization of NBC isoforms has not been identified, although NBCe1 and NBCe2 have been identified in the basolateral membrane of Calu-3 cells [26]. Moreover, DIDS did not affect the decrease in [Cl^-]_i, suggesting that the [Cl^-]_i decrease is induced by the activation of DIDS-insensitive Cl^- channels, such as ANO1.

The pH pathway was still activated by ABX even in a Ca²⁺-free solution or in the presence of nifedipine. This finding indicates that the pH pathway is activated by the Ca²⁺ release from acidic stores without any Ca²⁺ entry, although the increase in [Ca²⁺]_i is small and transient [37]. Lieb et al. (2002) reported that a transient [Ca²⁺]_i increase induced by short-term ATP stimulation induces a prolonged CBF increase in human airway ciliary cells, and they suggested that a transient [Ca²⁺]_i increase phosphorylates target proteins to induce a prolonged CBF increase [27]. The ABX-stimulated small transient increase in [Ca²⁺]_i appears to phosphorylate target proteins including the NBC to activate the pH pathway.

This study also demonstrated that an increase in the pH_i enhances not only the CBF but also the CBA in airway ciliary cells. Cilia have two functionally distinct molecular motors, the outer dynein arm (ODA) and the inner dynein arm (IDA). The ODAs control the CBF and the IDAs control the waveform including the CBA [4]. An increase in the pH_i is suggested to directly act on ODAs in sperm flagella [21]. Although there is no report showing that pH_i affects IDAs, a pH_i increase may activate IDAs, similar to ODAs, to increase the CBA. The mechanisms for regulating ODAs or IDAs mediated by pH are unknown. Previous reports have suggested that pH_i-induced changes in the histidine charge affect the activity of dynein ATPase [3]. A study of sperm flagella suggests that an increase in the pH_i activates dynein through the pH-dependent and cAMP-independent phosphorylation of dynein components and/or other axonemal proteins [32].

Previous studies have demonstrated that a decrease in [Cl^-]_i enhances the CBA at 37 °C, but not the CBF [16, 18-20, 47]. A decrease in [Cl^-]_i is coupled with cell shrinkage under iso-osmotic conditions [29, 47]. In this study, ABX decreased [Cl^-]_i coupled with cell shrinkage in c-LAECs. Previous studies demonstrated that the Ca_V1.2 channels are expressed in the cilia of c-LAECs [36, 37] and are activated by ABX [37]. The Ca²⁺ influx through Ca_V1.2 channels stimulated by ABX activated the Cl^- pathway in c-LAECs, but Ca²⁺ release from acidic stores had little effect on the Cl^- pathway.

The Cl^- pathway, which was independent of HCO₃^- (pH_i pathway), was activated by a Cl^--free NO₃^- solution and inhibited by Cl^- channel blockers (NPPB and T16Ainh) in c-LAECs. ANO1, which is a Ca²⁺-activated Cl^- channel [46] and exists in the cilia of c-LAECS, is activated by ABX mediated by Ca²⁺ entered through C_av1.2 in the cilia of c-LAECs, and it plays a key role in activating the Cl^- pathway.

The Cl^- pathway increased the CBA, but not the CBF. However, an increase in [Cl^-]_i induced by NPPB decreased both the CBF and CBA. Thus, the effects of [Cl^-]_i on the CBA and CBF are different. Yasuda et al. (2020) suggested that the concentration‒response curve of the CBA to [Cl^-]_i shifts to a lower concentration than that of the CBF [47]. An [Cl^-]_i under the control condition may maintain the IDA activity controlling CBA at the lowest level, but it may maintain the ODA-activity controlling CBF at the highest level.

There are many reports showing that a decreased [Cl^-]_i enhances cellular functions in many cell types, including airway ciliary cells [14, 38, 41]. These observations suggest that a Cl^- sensor exists in the cell, including c-LAECs. The with-no-lysine kinase1 (WNK1) or WNK4 has been shown to be an intracellular Cl^- sensor [2, 6]. Piala et al. (2014) demonstrated that the Cl^- binds to the kinase domain of WNK1 and suppresses its activity by inhibiting autophosphorylation [34]. The actions of WNK1 and WNK4 were studied in the NaCl cotransporter of distal nephrons [2]. There are four subtypes of WNK, WNK1-4, and the chloride binding site of the kinase domain is conserved among them, but the activities of the WNK subtypes are inhibited by different Cl^- concentrations, that is, WNK1 by 60-150 mM, WNK3 by 100-150 mM, and WNK4 by 0-40 mM [30, 42]. WNK1 and WNK4 have been shown to regulate epithelial Na⁺ channels [8, 35], CFTR [45], NKCC1 [13] and NCC [2, 11]. Based on these observations, our hypothesis is that WNK1 controls ODAs and WNK4 controls IDAs in c-LAECs. Further experiments are needed toverify this hypothesis.

The ABX actions (CBF and CBA increases) in c-LAECs are summarized in Fig. 12. ABX increases [Ca²⁺]_i by stimulating Ca²⁺ release from acidic stores and Ca²⁺ influx via Ca_V1.2 channels in c-LAECs. The increase in [Ca²⁺]_i activates two signaling pathways, the pH_i pathway and the Cl^- pathway. The pH_i pathway is activated by HCO₃^- entry via the NBC, which elevates pH_i. This pH_i elevation increases both the CBF and CBA. The Cl^- pathway is activated by the Cl^- secretion through ANO1 in cilia, which decreases [Cl^-]_i. ANO1 is activated by Ca²⁺ entered through nifedipine-sensitive Ca_V1.2 channels activated by ABX. The [Cl^-]_i decrease enhances the CBA.

Acknowledgements.

We thank Osaka Medical College for giving us an opportunity to perform the experiments using a video microscope equipped with a high-speed camera.

Author contributions

CS, ST, SH and TN measured ciliary activities, pH_i, [Cl^-]_i and [Ca²⁺]_i. KK and SA performed WB and IHC. TI and YM contributed to data interpretation and discussion. TN contributed the experimental design, and data analysis and data interpretation and prepared the draft of the manuscript. All authors have read and approved the final version of the manuscript and agree to be accountable for all aspects of the work.

Ethical approval

The experiments were approved by the Committees for Animal Research of Kyoto Prefectural University of Medicine (No. 26-263, April 2017) and Ritsumeikan University (BKC-HM-2017-050).

Competing interests

This study was funded by Teijin Pharma Limited to TN.

Data availability statement

The data that support the findings of this study are in the paper itself, and no shared data are used in the paper.

Funding

The Japan Society for the Promotion of Science (No. JP18H03182, to YM).

A grant fromTeijin Pharma Limited.

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Competing interest reported. This study was funded by Teijin Pharma Limited.

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Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pH_i and [Cl^-]_i in lung airway epithelial cells of mice

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