Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl−]i Decrease in the Lung Airway Epithelial Cells of Mice

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl−]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3− cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15–20%), and a decrease in [Cl−]i, which was induced by the Cl− release via anoctamine 1 (ANO1), enhanced the CBA (by 10–15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl−]i in the c-LAECs. In conclusion, the pHi increase and the [Cl−]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.


Introduction
Mucociliary clearance (MC), which is a host defense 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][2][3].Inhaled small particles, such as bacteria, viruses and chemicals, are entrapped by the SML and swept away toward the oropharynx by the beating cilia in the PCL.The MC is compared to a belt conveyor system in sweeping away inhaled small particles from the airways, and the beating cilia are the engine that drives MC [1,3,4].Impairment of the beating cilia, such as Kartagener syndrome or primary ciliary dyskinesia, causes serious respiratory diseases [1,3].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, which increases [Ca 2+ ] i by activating a voltage-gated Ca 2+ channel, Ca V 1.2, in the ciliated lung airway epithelial cells (c-LAECs), has been shown to increase the CBF (ciliary beat frequency) and CBA (ciliary bend angle, an index of amplitude) [5,6].However, the Ca 2+ -regulated signals following Ca V 1.2 activation, which may enhance CBF and CBA, remain uncertain.
The beating cilia in the airways express many ion channels, including Ca 2+ -permeable channels, such as Ca V 1.2, transient receptor potential (TRP) V4, TRPA1 and TRPM8 [6][7][8].However, it is still controversial how these channels enhance the ciliary beating.The beating cilia in the airways are activated by many substances, such as cAMP, Ca 2+ , ATP, β 2agonists, Cl − and H + (intracellular pH (pH i )) [4,[6][7][8][9][10][11][12].Among them, Ca 2+ is an important ion that activates the beating cilia [3,4,[6][7][8].ABX stimulates Ca V 1.2 and a small transient Ca 2+ release from the acidic stores in the c-LAECs [6,13].These observations suggest that Ca V 1.2 plays an important role in the activation of ciliary beating during ABX stimulation.Moreover, ABX has been shown to stimulate anion secretion in lung epithelial cell lines [14].The activation of Ca V 1.2 may stimulate Cl − secretion in the c-LAECs, leading to a decrease in the intracellular Cl − concentration ([Cl − ] i ), since anoctamine 1 (ANO1) exists in nasal ciliated epithelial cells [12].Moreover, Saito et al. suggested that ABX stimulates a pH i increase in c-LAECs [6].Thus, Ca V 1.2 activation stimulated via ABX may increase the pH i and decrease the [Cl − ] i in c-LAECs.Previous studies have demonstrated that a pH i increase and an [Cl − ] i decrease enhance the CBF and CBA in airway ciliary cells [9,10,12].The activation of Ca V 1.2 existing in the cilia may increase pH i to enhance the CBF and CBA and decrease [Cl − ] i to enhance the CBA.
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 [15,16].Previous studies have demonstrated that a CBA increase, in addition to a CBF increase, enhances ciliary transport in the airway [12], although the CBF has been used to assess the activity of ciliary beating [3,4].The signaling pathways regulating the CBF and CBA have been shown to be different [9,11,12].ABX has been shown to enhance the CBF and CBA [6], suggesting that the activation of Ca V 1.2 may trigger two signaling pathways, increasing the CBF and CBA.
In this study, we examined the effects of Ca V 1.2 on the enhancement of the CBF and CBA in the ABX-stimulated c-LAECs of mice.In experiments using two solutions with and without CO 2 /HCO 3 − , we found that the Ca V 1.2 activated using ABX stimulated two signaling pathways to increase the CBF and CBA, CO 2 /HCO 3 − -dependent and CO 2 /HCO 3 − -independent.This study is designed to clarify how Ca V 1.2 activates the two signalling pathways increasing the CBF and CBA in c-LAECs.

Results
In unstimulated c-LAECs perfused with the HCO 3 − -containing control solution, the CBF and CBA were 8-10 Hz and 70 • -90 • , respectively [9,11].Saito et al. demonstrated that ABX increases the CBF and CBA in a concentration-dependent manner and it maximally increases the CBF and CBA at 10 µM [6].In this study, the concentration of ABX used was 10 µM.In this study, we used a nominally Ca 2+ -free solution.Because an EGTAcontaining Ca 2+ -free solution increases the CBF by inhibiting the Ca 2+ -dependent PDE1A existing in the metabolon, regulating the ODAs (CBF) in the cilia [11].To inhibit Ca V 1.2, we used nifedipine [6].To increase the pH i , we applied a CO 2 /HCO 3 − -free solution, which enhances the CBF and CBA [9,10].The application of a CO 2 /HCO 3 − -free solution is a well-known procedure to increase pH i ,.
2.1.ABX-Stimulated Cellular Events 2.1.1.ABX-Stimulated Increases in the CBF and CBA Supplementary Videos S1 and S2 show video images of a c-LAEC before and 15 min after ABX stimulation.ABX gradually increased the ratio of the CBF and CBA (normalized 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 (Figure 1A).We examined the effects of Ca 2+ on the CBF and CBA stimulated by ABX.The switch to a nominally Ca 2+ -free solution decreased the ratios of CBF and CBA by 5% within 5 min, and then stimulation using ABX increased the ratios of 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 (Figure 1B).The same experiments were carried out using nifedipine (20 µM) instead of the Ca 2+ -free solution.The addition of nifedipine decreased the ratios of the CBF and CBA by 5% and then ABX stimulation increased them by 10%.A previous study has shown similar results in c-LAECs [6].Experiments were also carried out in the absence of CO 2 /HCO 3 − (Figure 1C,D).The switch to the CO 2 /HCO 3 − -free control 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.Further ABX stimulation increased only the CBA, but not the CBF.The ratios of the CBF and CBA at 5 min after ABX stimulation were 1.32 (n = 6) and 1.30 (n = 8), respectively.The effects of nifedipine (10 µM) on the CBA increase were examined (Figure 1D).The addition of nifedipine decreased the CBF and CBA by 5%.Then, the switch to a CO 2 /HCO 3 − -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 CBA (Figure 1D).Similar results were obtained using a Ca 2+ -free solution.The CBA increase stimulated by ABX in the application of the CO 2 /HCO 3 − -free solution appears to be controlled by a [Ca 2+ ] i increase via the Ca V 1.2 activation.
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 (Figure 1A).We examined the effects of Ca 2+ on the CBF and CBA stimulated by ABX.The switch to a nominally Ca 2+free solution decreased the ratios of CBF and CBA by 5% within 5 min, and then stimulation using ABX increased the ratios of 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 (Figure 1B).The same experiments were carried out using nifedipine (20 μM) instead of the Ca 2+ -free solution.The addition of nifedipine decreased the ratios of the CBF and CBA by 5% and then ABX stimulation increased them by 10%.A previous study has shown similar results in c-LAECs [6].Experiments were also carried out in the absence of CO2/HCO3 − (Figure 1C,D).The switch to the CO2/HCO3 − -free control 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.Further ABX stimulation increased only the CBA, but not the CBF.The ratios of the CBF and CBA at 5 min after ABX stimulation were 1.32 (n = 6) and 1.30 (n = 8), respectively.The effects of nifedipine (10 μM) on the CBA increase were examined (Figure 1D).The addition of nifedipine decreased the CBF and CBA by 5%.Then, the switch to a 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 CBA (Figure 1D).Similar results were obtained using a Ca 2+ -free solution.The CBA increase stimulated by ABX in the application of the CO2/HCO3 − -free solution appears to be controlled by a [Ca 2+ ]i increase via the CaV1.2 activation.Prior addition of BAPTA-AM (10 µM, a membrane permeable analog of BAPTA, a Ca 2+ chelator, that binds the 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 Ca 2+ -free solution [6].Thus, Ca 2+ entry via the ABX-activated Ca V 1.2 triggers two signal pathways, CO 2 /HCO 3 − -dependent and CO 2 /HCO 3 − -independent pathways.
Changes in [Ca 2+ ] i were monitored via the fura-2 fluorescence ratio (F340/F380) in the c-LAECs.In the control solution, ABX stimulation gradually increased the F340/F380, which reached a plateau within 15 min, and then, the addition of nifedipine decreased the F340/F380 to the pre-stimulation level within 10 min (Figure 2A).Changes in the F340/F380 stimulated by ABX were measured in the c-LAECs perfused with a Ca 2+ -free solution.ABX induced a small transient increase in the F340/F380 (Figure 2B), as shown in a previous report [6].ABX has been shown to stimulate the Ca 2+ release from acidic stores in alveolar type II cells (ATII cells), which was inhibited by an increase in pH i [6,13].Experiments were then carried out in a CO 2 /HCO 3 − -free solution.The switch from the control solution to the CO 2 /HCO 3 − -free solution did not change the F340/F380, and ABX stimulation did not increase the F340/F380.The addition of nifedipine did not change the F340/F380 (Figure 2C).An increase in pH i induced by the application of the CO 2 /HCO 3 −free solution inhibited ABX-stimulated Ca 2+ release from the acidic stores, as shown in a previous report [6].Moreover, the CO 2 /HCO 3 − -free solution may hyperpolarize the membrane potential by inhibiting the NBC [6], which decreases the [Ca 2+ ] i to a low level.
CO2/HCO3 − .The addition of nifedipine (20 μM) decreased the CBF and CBA ratios by 5%.The to the CO2/HCO3 − -free solution immediately increased the CBF ratio by 30% and the CBA ra 20%.Further ABX stimulation did not induce any increase in the CBF ratio or the CBA ratio.
Prior addition of BAPTA-AM (10 μM, a membrane permeable analog of BAP Ca 2+ chelator, that binds the intracellular calcium after the acetoxymethyl group moved by the cytoplasmic esterase) completely inhibited the increases in the CB CBA stimulated by ABX in a Ca 2+ -free solution [6].Thus, Ca 2+ entry via the ABX-acti CaV1.2 triggers two signal pathways, CO2/HCO3 − -dependent and CO2/HCO3 − -inde ent pathways.

ABX-Stimulated Increases in [Ca 2+ ]i
Changes in [Ca 2+ ]i were monitored via the fura-2 fluorescence ratio (F340/F380) c-LAECs.In the control solution, ABX stimulation gradually increased the F340 which reached a plateau within 15 min, and then, the addition of nifedipine decrease F340/F380 to the pre-stimulation level within 10 min (Figure 2A).Changes i F340/F380 stimulated by ABX were measured in the c-LAECs perfused with a Ca solution.ABX induced a small transient increase in the F340/F380 (Figure 2B), as s in a previous report [6].ABX has been shown to stimulate the Ca 2+ release from stores in alveolar type II cells (ATII cells), which was inhibited by an increase in pHi [ Experiments were then carried out in a CO2/HCO3 − -free solution.The switch from th trol solution to the CO2/HCO3 − -free solution did not change the F340/F380, and ABX ulation did not increase the F340/F380.The addition of nifedipine did not chang F340/F380 (Figure 2C).An increase in pHi induced by the application of the CO2/H free solution inhibited ABX-stimulated Ca 2+ release from the acidic stores, as show previous report [6].Moreover, the CO2/HCO3 − -free solution may hyperpolarize the brane potential by inhibiting the NBC [6], which decreases the [Ca 2+ ]i to a low level.

ABX-Stimulated Changes in pHi
Changes in pHi were measured via the ratio of SNARF1 fluorescence (F645/F59 the control solution, ABX stimulation gradually increased the pHi from 7.49 to 7.65 ( within 15 min (Figure 3A).The cells were also stimulated by ABX in a Ca 2+ -free sol increased pH i , which inhibits Ca 2+ release from acidic stores, and it inhibits Na + entry via the NBC.The CO 2 /HCO 3 − -free solution decreases Ca V 1.2 channel activity.

ABX-Stimulated Changes in pH i
Changes in pH i were measured via the ratio of SNARF1 fluorescence (F645/F592).In the control solution, ABX stimulation gradually increased the pH i from 7.49 to 7.65 (n = 8) within 15 min (Figure 3A).The cells were also stimulated by ABX in a Ca 2+ -free solution (Figure 3B) or in the presence of nifedipine (Figure 3C).ABX increased the pH i from 7.51 to 7.58 in the Ca 2+ -free solution (n = 4, Figure 3B) and from 7.52 to 7.60 in the presence of nifedipine (n = 5, Figure 3C).Thus, the activation of Ca V 1.2 significantly increased the pH i in the ABX-stimulated c-LAECs.However, the Ca 2+ release from the acidic stores also increased the pH i , but its extent was small (Figure 3B,C).Changes in pH i were measured upon applying the CO 2 /HCO 3 − -free solution.An application of the CO 2 /HCO 3 − -free solution transiently increased and sustained the pH i .The further ABX stimulation did not change the pH i (Figure 3D).
(Figure 3B) or in the presence of nifedipine (Figure 3C).ABX increased the pHi from 7.51 to 7.58 in the Ca 2+ -free solution (n = 4, Figure 3B) and from 7.52 to 7.60 in the presence of nifedipine (n = 5, Figure 3C).Thus, the activation of CaV1.2 significantly increased the pHi in the ABX-stimulated c-LAECs.However, the Ca 2+ release from the acidic stores also increased the pHi, but its extent was small (Figure 3B,C).Changes in pHi were measured upon applying the CO2/HCO3 − -free solution.An application of the CO2/HCO3 − -free solution transiently increased and sustained the pHi.The further ABX stimulation did not change the pHi (Figure 3D). Figure 4A shows a phase contrast video image of a typical c-LAEC perfused with the control solution before ABX stimulation (Figure 4A) and that at 15 min after ABX stimulation (Figure 4B).The outline of a c-LAEC before ABX stimulation is shown in Figure 4A and was superimposed in Figure 4B.The c-LAECs stimulated by ABX were smaller than those before ABX stimulation, indicating that ABX decreased the cell volume.Cell shrinkage is known to decrease [Cl − ]i [9,12].We monitored the [Cl − ]i using a Cl − -sensitive fluorescence dye in the c-LAECs (MQAE) [9,12].ABX stimulation increased the intensity of MQAE fluorescence in one of the c-LAECs (Figure 4C,D), indicating that ABX stimulation decreases [Cl − ]i. Figure 4A shows a phase contrast video image of a typical c-LAEC perfused with the control solution before ABX stimulation (Figure 4A) and that at 15 min after ABX stimulation (Figure 4B).The outline of a c-LAEC before ABX stimulation is shown in Figure 4A and was superimposed in Figure 4B.The c-LAECs stimulated by ABX were smaller than those before ABX stimulation, indicating that ABX decreased the cell volume.Cell shrinkage is known to decrease [Cl − ] i [9,12].We monitored the [Cl − ] i using a Cl −sensitive fluorescence dye in the c-LAECs (MQAE) [9,12].ABX stimulation increased the intensity of MQAE fluorescence in one of the c-LAECs (Figure 4C

ABX-Stimulated Decreases in Cell Volume and [Cl − ]i
In the control solution, the changes in cell volume (V/V0, index of cell volume) and [Cl − ]i (F0/F, MQAE fluorescence ratio) stimulated by ABX were measured in the c-LAECs (Figure 5).ABX stimulation decreased the V/V0 to 0.81 (n = 5, 6 min after ABX stimulation) (Figure 5A) and the F0/F to 0.70 (n = 4, 10 min after the ABX stimulation) (Figure 5B).Then, the addition of nifedipine immediately recovered the V/V0 to the level before ABX stimulation (V/V0 at 5 min after nifedipine addition = 1.03) (Figure 5A) Thus, ABX stimulation decreased the cell volume (V/V0) and MQAE fluorescence ratio (F0/F) by activating CaV1.2.
To examine the relationship between the CBA and V/V0 (or [Cl − ]i) without a pHi change, experiments were also carried out using the CO2/HCO3 − -free solution, in which ABX stimulation increased only the CBA.The switch to the CO2/HCO3 − -free solution decreased the V/V0 and F0/F to 0.90 (n = 5, 5 min after the switch) and 0.84 (n = 4, 5 min after the switch), respectively.Further ABX stimulation decreased the V/V0 and F0/F to 0.78 (n = 5, 10 min after ABX stimulation, Figure 5C) and 0.69 (10 min after ABX stimulation, Figure 5D), respectively.Thus, in the CO2/HCO3 − -free solution, ABX still induced cell shrinkage, leading to [Cl − ]i decrease, although ABX did not increase the [Ca 2+ ]i (Figure 2C).The question is whether or not the cell shrinkage or [Cl − ]i decrease was CaV1.2-dependent.The effects of nifedipine on ABX-stimulated cell shrinkage and [Cl − ]i decrease were examined in the CO2/HCO3 − -free solution (Figure 5E,F).In the control solution, the addition of nifedipine increased the V/V0 and F0/F to 1.12 (n = 4, 5 min after nifedipine addition) and 1.15 (n = 5, 5 min after nifedipine addition), respectively.The switch to the CO2/HCO3 −free solution decreased the V/V0 and F0/F to 0.93 and 0.82, and then ABX stimulation did not change the V/V0 (0.91 at 10 min after ABX stimulation) (Figure 5E) and F0/F (0.82 at 10 In the control solution, the changes in cell volume (V/V 0 , index of cell volume) and [Cl − ] i (F 0 /F, MQAE fluorescence ratio) stimulated by ABX were measured in the c-LAECs (Figure 5).ABX stimulation decreased the V/V 0 to 0.81 (n = 5, 6 min after ABX stimulation) (Figure 5A) and the F 0 /F to 0.70 (n = 4, 10 min after the ABX stimulation) (Figure 5B).Then, the addition of nifedipine immediately recovered the V/V 0 to the level before ABX stimulation (V/V 0 at 5 min after nifedipine addition = 1.03) (Figure 5A) Thus, ABX stimulation decreased the cell volume (V/V 0 ) and MQAE fluorescence ratio (F 0 /F) by activating Ca V 1.2.
To examine the relationship between the CBA and V/V 0 (or [Cl − ] i ) without a pH i change, experiments were also carried out using the CO 2 /HCO 3 − -free solution, in which ABX stimulation increased only the CBA.The switch to the CO 2 /HCO 3 − -free solution decreased the V/V 0 and F 0 /F to 0.90 (n = 5, 5 min after the switch) and 0.84 (n = 4, 5 min after the switch), respectively.Further ABX stimulation decreased the V/V 0 and F 0 /F to 0.78 (n = 5, 10 min after ABX stimulation, Figure 5C) and 0.69 (10 min after ABX stimulation, Figure 5D), respectively.Thus, in the CO 2 /HCO 3 − -free solution, ABX still induced cell shrinkage, leading to [Cl − ] i decrease, although ABX did not increase the [Ca 2+ ] i (Figure 2C).The question is whether or not the cell shrinkage or [Cl − ] i decrease was Ca V 1.2-dependent.The effects of nifedipine on ABX-stimulated cell shrinkage and [Cl − ] i decrease were examined in the CO 2 /HCO 3 − -free solution (Figure 5E,F).In the control solution, the addition of nifedipine increased the V/V 0 and F 0 /F to 1.12 (n = 4, 5 min after nifedipine addition) and 1.15 (n = 5, 5 min after nifedipine addition), respectively.The switch to the CO 2 /HCO 3 − -free solution decreased the V/V 0 and F 0 /F to 0.93 and 0.82, and then ABX stimulation did not change the V/V 0 (0.91 at 10 min after ABX stimulation) (Figure 5E) and F 0 /F (0.82 at 10 min after ABX stimulation) (Figure 5F).The ABX-stimulated cell shrinkage and [Cl − ] i decrease in the CO 2 /HCO 3 − -free solution are likely to be induced by the activation of Ca V 1.2.

Effects of DIDS on the CBF, CBA and pH i
The effects of 4,4 -Diisothiocyano-2,2 -stilbenedisulfonic acid (DIDS, 100 µM, an inhibitor of the NBC and AE) on the CBF, CBA and pH i were examined (Figure 6).The addition of DIDS (100 µM) gradually increased the CBF, but not the CBA (Figure 6A).The 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 also gradually increased the pH i from 7.45 to 7.52 (n = 5, 10 min after DIDS addition), and ABX stimulation did not affect the gradual pH i increase induced by DIDS (7.55 (n = 4), 10 min after ABX stimulation, Figure 6B).The effects of ABX on the CBA and CBF were examined in the presence of DIDS (Figure 6C).The addition of DIDS gradually increased only the CBF.Then, stimulation using ABX immediately increased only the CBA by 17% without any increase in the CBF (Figure 6C).In the DIDS-treated c-LAECs, ABX did not increase the CBF and pH i , but it increased the CBA (Figure 6C).Changes in the MQAE fluorescence ratio induced by ABX were measured in the presence of DIDS.DIDS alone did not change the F 0 /F, but stimulation with ABX decreased the F 0 /F (Figure 6D).DIDS did not affect the [Cl − ] i decrease stimulated by ABX, suggesting that a [Cl − ] i decrease increased the CBA.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 19 addition of DIDS (100 μM) gradually increased the CBF, but not the CBA (Figure 6A).The 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 also gradually increased the pHi from 7.45 to 7.52 (n = 5, 10 min after DIDS addition), and ABX stimulation did not affect the gradual pHi increase induced by DIDS (7.55 (n = 4), 10 min after ABX stimulation, Figure 6B).The effects of ABX on the CBA and CBF were examined in the presence of DIDS (Figure 6C).The addition of DIDS gradually increased only the CBF.Then, stimulation using ABX immediately increased only the CBA by 17% without any increase in the CBF (Figure 6C).In the DIDS-treated c-LAECs, ABX did not increase the CBF and pHi, but it increased the CBA (Figure 6C).Changes in the MQAE fluorescence ratio induced by ABX were measured in the presence of DIDS.DIDS alone did not change the F0/F, but stimulation with ABX decreased the F0/F (Figure 6D).DIDS did not affect the [Cl − ]i decrease stimulated by ABX, suggesting that a [Cl − ]i decrease increased the CBA.− solution immediately increased the CBF and CBA, and then stimulation using 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 (Figure 6E).The HCO 3 − -containing Cl − -free NO 3 − solution alone increased the pH i from 7.43 to 7.65 (n = 5) and then, the ABX stimulation increased the pH i to 7.91.The HCO 3 − -containing Cl − -free NO 3 − solution potentiated the CBF, CBA and pH i being increased by ABX (Figure 6F).The HCO 3 − -containing Cl − -free NO 3 − solution, which inhibits HCO 3 − extrusion via the AE while maintaining HCO 3 − influx via the NBC, may increase the concentration of HCO 3 − in the c-LAECs, leading to an increase in pH i .ABX appears to stimulate the NBC to increase the pH i .

Effects of NPPB on CBF, CBA and [Cl − ] i
The addition of NPPB decreased the CBF and CBA ratios to 0.92 and 0.96 within 5 min, respectively.Then, the ABX stimulation immediately increased the CBF and CBA ratios to 1.06 and 1.10, respectively (Figure 7A).The addition of NPPB increased the MQAE fluorescence ratio (F 0 /F) from 0.99 (n = 6) to 1.14 (5 min after the addition).Then, the ABX stimulation gradually increased the F 0 /F (F 0 /F at 15 min after ABX stimulation = 1.25) (Figure 7B).In the experiments using the CO 2 /HCO 3 − -free solution, NPPB did not affect the increases in the CBF and CBA induced by the switch to the CO 2 /HCO 3 − -free solution.However, ABX stimulation did not increase the CBF or CBA (Figure 7C).The CBF and CBA ratios before and 15 min after ABX stimulation were 1.03 (n = 4) and 1.11 (n = 6) and 1.03 and 1.06 in the CO 2 /HCO 3 − -free solution with NPPB, respectively.Changes in [Cl − ] i were monitored via the MQAE fluorescence ratio (F 0 /F) (Figure 7D).The addition of NPPB increased the F 0 /F by 15%.The switch to a CO 2 /HCO 3 − -free solution, in which there was decreased Na + entry due to inhibition of the NBC, decreased the F 0 /F to 0.88 (n = 5) at 5 min after the switch, and stimulation with ABX did not change the F 0 /F (0.86 at 15 min after ABX stimulation).An increase in [Cl − ] i appears to inhibit the CBF and CBA increase stimulated by ABX [12].No decrease in [Cl − ] i induced no further increase in the CBA during ABX stimulation in the CO 2 /HCO 3 − -free solution.The addition of T16Ainh (10 μM, an inhibitor of ANO1) decreased the CBA (0.95 (n = 5) at 5 min after the addition), but not the CBF (1.02 (n = 7) at 5 min after the addition).ABX stimulation increased the CBF and CBA to 1.12 and 1.12 (at 10 min after the ABX stimulation), respectively (Figure 8A).Experiments were then carried out in a CO2/HCO3 −free solution.T16Ainh did not affect the CBF and CBA increased by the switch to the CO2/HCO3 − -free solution.Then, further ABX stimulation did not change the CBF or CBA.The CBF and CBA ratios before and after ABX stimulation were 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, respectively (Figure 8B).These results indicate that the activation of ANO1 decreases the [Cl − ]i in ABX-stimulated c-LAECs.The addition of T16Ainh (10 µM, an inhibitor of ANO1) decreased the CBA (0.95 (n = 5) at 5 min after the addition), but not the CBF (1.02 (n = 7) at 5 min after the addition).ABX stimulation increased the CBF and CBA to 1.12 and 1.12 (at 10 min after the ABX stimulation), respectively (Figure 8A).Experiments were then carried out in a CO 2 /HCO 3 −free solution.T16Ainh did not affect the CBF and CBA increased by the switch to the CO 2 /HCO 3 − -free solution.Then, further ABX stimulation did not change the CBF or CBA.The CBF and CBA ratios before and after ABX stimulation were 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, respectively (Figure 8B).These results indicate that the activation of ANO1 decreases the [Cl − ] i in ABX-stimulated c-LAECs.These results suggest that an increase in pH i and a decrease in [Cl − ] i stimulated by ABX increased the CBF and CBA.We used a CO 2 /HCO 3 − -free Cl − -free NO 3 − solution, which increases the pH i and decreases the [Cl − ] i [9].The switch to the CO 2 /HCO 3 − -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 CO 2 /HCO 3 − -free Cl − -free NO 3 − 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 (Figure 9A).Changes in [Cl − ] i were also monitored via the MQAE fluorescence ratio using the same protocol.The CO 2 /HCO 3 − -free solution decreased the F 0 /F to 0.90 (5 min after the switch, n = 4) and the second switch further decreased the F 0 /F to 0.73 (5 min after the second switch, n = 4).ABX stimulation did not decrease the F 0 /F (0.69 at 10 min after the ABX stimulation, n = 4) (Figure 9B).Thus, the effects of ABX on the CBA and CBF were mimicked by the CO 2 /HCO 3 − -free Cl − -free NO 3 − solution.
2.3.2.Effects of an ANO1 Inhibitor (T16Ainh) on CBF, CBA and [Cl − ]i The addition of T16Ainh (10 μM, an inhibitor of ANO1) decreased the CBA (0.95 (n = 5) at 5 min after the addition), but not the CBF (1.02 (n = 7) at 5 min after the addition).ABX stimulation increased the CBF and CBA to 1.12 and 1.12 (at 10 min after the ABX stimulation), respectively (Figure 8A).Experiments were then carried out in a CO2/HCO3 −free solution.T16Ainh did not affect the CBF and CBA increased by the switch to the CO2/HCO3 − -free solution.Then, further ABX stimulation did not change the CBF or CBA.The CBF and CBA ratios before and after ABX stimulation were 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, respectively (Figure 8B).These results indicate that the activation of ANO1 decreases the [Cl − ]i in ABX-stimulated c-LAECs.These results suggest that an increase in pHi and a decrease in [Cl − ]i stimulated by ABX increased the CBF and CBA.We used a CO2/HCO3 − -free Cl − -free NO3 − solution, which increases the pHi and decreases the [Cl − ]i [9].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 (Figure 9A).Changes in [Cl − ]i were also monitored via the MQAE fluorescence ratio using the same protocol.The CO2/HCO3 − -free solution decreased the F0/F to 0.90 (5 min after the switch, n = 4) and the second switch further decreased the F0/F to 0.73 (5 min after the second switch, n = 4).ABX stimulation did not decrease the F0/F (0.69 at 10 min after the ABX stimulation, n = 4) (Figure 9B).Thus, the effects of ABX 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 using Western blotting and immunofluorescence.In the Western blotting, a single band of ANO1 was detected at 110 kDa (Figure 10).The immunofluorescence analysis of ANO1 revealed that the cilia and cell bodies were positively stained for ANO1 in the c-LAECs (Figure 11A), and the cilia were positively stained for acetylated tubulin (α-tubulin, a marker of cilia) (Figure 11B).The merged image shows that ANO1 exists in the cilia (Figure 11C).A phase contrast image of the c-LAECs is shown in Figure 11D.

Expression of Anoctamin-1 in c-LAECs
The expression of ANO1 (TMEM16A) was examined using Western blotting and immunofluorescence.In the Western blotting, a single band of ANO1 was detected at 110 kDa (Figure 10).The immunofluorescence analysis of ANO1 revealed that the cilia and cell bodies were positively stained for ANO1 in the c-LAECs (Figure 11A), and the cilia were positively stained for acetylated tubulin (α-tubulin, a marker of cilia) (Figure 11B).The merged image shows that ANO1 exists in the cilia (Figure 11C).A phase contrast image of the c-LAECs is shown in Figure 11D.

Discussion
The activation of CaV1.2, a voltage-dependent Ca 2+ channel, maintained the sustained increase in [Ca 2+ ]i in ABX-stimulated-cLAECs, in which the contribution of Ca 2+ release from the stores was small [6].This study demonstrated that the [Ca 2+ ]i increase using ABX triggers CBF and CBA increases in the c-LAECs by activating two signaling pathways: the pH pathway (an increase in pHi) and the Cl − pathway (a decrease in [Cl − ]i).The pH pathway increased the CBF by 30% and the CBA by 15-20%, and the Cl − pathway increased the CBA by 10-15%.
The [Ca 2+ ]i increase is essential to increase the CBF and CBA in the ABX-stimulated c-LAECs, since prior treatment with BAPTA-AM abolished their increases.This suggests that the non-specific actions of ABX, which are not mediated by an [Ca 2+ ]i increase, are negligibly small in the regulation of the CBF and CBA.DIDS, which inhibited the pH pathway, did not affect the ABX-stimulated increase in [Ca 2+ ]i, since ABX stimulation increased only the CBA, but not the CBF, although the [Ca 2+ ]i increase appears to be sufficient to increase the CBF.Moreover, in the presence of NPPB or T116Ainh, ABX stimulation induced small increases in the CBF and CBA, suggesting the activation of the pH pathway.ABX is likely to increase [Ca 2+ ]i to a sufficient level to activate the pH pathway.A [Cl − ]i increase on the part of the inhibitors of the Cl − channels appears to decrease the CBF and CBA enhanced by the pH pathway.Yasuda et al. showed that an increase in [Cl − ]i

Discussion
The activation of CaV1.2, a voltage-dependent Ca 2+ channel, maintained the sustained increase in [Ca 2+ ]i in ABX-stimulated-cLAECs, in which the contribution of Ca 2+ release from the stores was small [6].This study demonstrated that the [Ca 2+ ]i increase using ABX triggers CBF and CBA increases in the c-LAECs by activating two signaling pathways: the pH pathway (an increase in pHi) and the Cl − pathway (a decrease in [Cl − ]i).The pH pathway increased the CBF by 30% and the CBA by 15-20%, and the Cl − pathway increased the CBA by 10-15%.
The [Ca 2+ ]i increase is essential to increase the CBF and CBA in the ABX-stimulated c-LAECs, since prior treatment with BAPTA-AM abolished their increases.This suggests that the non-specific actions of ABX, which are not mediated by an [Ca 2+ ]i increase, are negligibly small in the regulation of the CBF and CBA.DIDS, which inhibited the pH pathway, did not affect the ABX-stimulated increase in [Ca 2+ ]i, since ABX stimulation increased only the CBA, but not the CBF, although the [Ca 2+ ]i increase appears to be sufficient to increase the CBF.Moreover, in the presence of NPPB or T116Ainh, ABX stimulation induced small increases in the CBF and CBA, suggesting the activation of the pH pathway.ABX is likely to increase [Ca 2+ ]i to a sufficient level to activate the pH pathway.A [Cl − ]i increase on the part of the inhibitors of the Cl − channels appears to decrease the CBF and CBA enhanced by the pH pathway.Yasuda et al. showed that an increase in [Cl − ]i

Discussion
The activation of Ca V 1.2, a voltage-dependent Ca 2+ channel, maintained the sustained increase in [Ca 2+ ] i in ABX-stimulated-cLAECs, in which the contribution of Ca 2+ release from the stores was small [6].This study demonstrated that the [Ca 2+ ] i increase using ABX triggers CBF and CBA increases in the c-LAECs by activating two signaling pathways: the pH pathway (an increase in pH i ) and the Cl − pathway (a decrease in [Cl − ] i ).The pH pathway increased the CBF by 30% and the CBA by 15-20%, and the Cl − pathway increased the CBA by 10-15%.
The [Ca 2+ ] i increase is essential to increase the CBF and CBA in the ABX-stimulated c-LAECs, since prior treatment with BAPTA-AM abolished their increases.This suggests that the non-specific actions of ABX, which are not mediated by an [Ca 2+ ] i increase, are negligibly small in the regulation of the CBF and CBA.DIDS, which inhibited the pH pathway, did not affect the ABX-stimulated increase in [Ca 2+ ] i , since ABX stimulation increased only the CBA, but not the CBF, although the [Ca 2+ ] i increase appears to be sufficient to increase the CBF.Moreover, in the presence of NPPB or T116Ainh, ABX stimulation induced small increases in the CBF and CBA, suggesting the activation of the pH pathway.ABX is likely to increase [Ca 2+ ] i to a sufficient level to activate the pH pathway.A [Cl − ] i increase on the part of the inhibitors of the Cl − channels appears to decrease the CBF and CBA enhanced by the pH pathway.Yasuda et al. showed that an increase in [Cl − ] i decreases the CBF and CBA [12].Moreover, in the CO 2 /HCO 3 − -free solution, ABX increased only the CBA and decreased the cell volume and [Cl − ] i , effects that were eliminated using a Ca 2+ -free solution or nifedipine.In a CO 2 /HCO 3 − -free solution, ABX may increase the [Ca 2+ ] i to a level activating ANO1 in the cilia.Saito et al. suggest that ABX increases the [Ca 2+ ] i only in the cilia, although no increase in the [Ca 2+ ] i of the cell body was detected in the CO 2 /HCO 3 − -free solution [6].In the olfactory cilia, the [Ca 2+ ] i increase is limited in the same vicinity for a long time [19].Moreover, we measured the [Ca 2+ ] i in c-LAECs stimulated by ABX and ionomycin (IM, 1 µM), using fluo4 fluorescence.The increases in the fluorescence ratio (F/F 0 ) stimulated by ABX were 10-15% of those stimulated by IM (1 µM) (Supplementary Figure S1).The direct activation of the CBF by a [Ca 2+ ] i increase has been established in the beating cilia of the airways [3,4,20].However, in the ABX-stimulated c-LAECs, increases in [Ca 2+ ] i may have been too small to directly increase the CBF and CBA.
An increase in pH i enhances the CBF and CBA in airway ciliary cells [9,10] and enhances the CBF in sperm flagella [21,22].In the c-LAECs, the pH pathway is activated by HCO 3 − entry via the NBC, which is inhibited by DIDS.There are two bicarbonate transporters in the c-LAECs: the NBC and AE [17].The DIDS-sensitive AE exists in the apical membrane and mediates HCO 3 − secretion in the bronchiole epithelial cells [17].Moreover, in the HCO 3 − -containing Cl − -free solution, ABX enhanced the increase in pH i , indicating that the AE does not transport HCO 3 − into the cell in c-LAECs.In this solution, no HCO 3 − secretion via the AE occurs because of no extracellular Cl − .The inhibition of AE (no HCO 3 − extrusion via the AE retaining HCO 3 − uptake via the NBC) increases the intracellular HCO 3 − concentration to elevate the pH i .Based on these results, we concluded that ABX stimulated the NBC, mediated by a [Ca 2+ ] i increase, leading to an increase in pH i .Fois et al. demonstrated that ABX increases pH i in type II pneumocytes [13].The messenger RNAs of all NBC isoforms are expressed in the airway epithelial cells [17], but the membrane localization of the NBC isoforms has not been identified, although NBCe1 and NBCe2 have been identified in the basolateral membrane of Calu-3 cells [23].Moreover, DIDS did not affect the decrease in [Cl − ] i , indicating that the [Cl − ] i decrease is not induced by DIDS-sensitive Cl − channels in the c-LAECs.
The pH pathway was still activated by ABX in the presence of nifedipine or in a Ca 2+ -free solution.This finding indicates that the ABX-stimulated small transient Ca 2+ release from the acidic stores activates the NBC in the c-LAECs, leading to a pH i increase.Lieb et al. reported that a transient [Ca 2+ ] i increase induced by short-term ATP stimulation has been shown to induce a prolonged CBF increase in human airway ciliary cells, and they suggested that a transient [Ca 2+ ] i increase phosphorylates target proteins to induce a prolonged CBF increase [20].The ABX-stimulated small transient [Ca 2+ ] i increase appears to phosphorylate target proteins, including the NBC, to activate the pH pathway.
This study also demonstrated that a pH i increase enhances not only the CBF but also the CBA in the c-LAECs.Cilia have two functionally distinct molecular motors: the ODAs and the IDAs.The ODAs control the CBF and the IDAs control the waveform, including the CBA [15].An increase in the pH i is suggested to directly act on the ODAs in sperm flagella [21,22].Although there is no report showing that pH i affects the IDAs, a pH i increase may activate the IDAs, in a similar mechanism to activating the ODAs, to increase the CBA.The activation mechanisms of ODAs or IDAs due to a pH i increase remain uncertain.Previous reports have suggested that pH i -induced changes in the histidine charge may affect the activity of dynein ATPase [24].A study on sperm flagella suggests that an increase in the pH i activates dynein via the pH-dependent and cAMP-independent phosphorylation of dynein components and/or other axonemal proteins [22].
Previous studies have demonstrated that a decrease in [Cl − ] i enhances the CBA at 37 • C, but not the CBF [9,12].[Cl − ] i is decreased by the cell shrinkage under iso-osmotic conditions [12].ABX stimulated a cell shrinkage and a [Cl − ] i decrease in c-LAECs.Previous studies have demonstrated that Ca V 1.2 is expressed in the cilia and is activated by ABX [6].The Ca 2+ influx via Ca V 1.2 activated the Cl − pathway in the c-LAECs, but the Ca 2+ release from the acidic stores had little effect on the Cl − pathway, because ABX still decreases [Cl − ] i without a Ca 2+ release in a CO 2 /HCO 3 − -free solution.The Cl − pathway, which was independent of HCO 3 − (pH i pathway), was activated by a Cl − -free NO 3 − solution and inhibited by Cl − channel blockers (NPPB and T16Ainh) in the c-LAECs.This study demonstrated that ANO1 (a Ca 2+ -activated Cl − channel [18]) is activated by Ca v 1.2.Moreover, both channels are expressed in the cilia of the c-LAECs [6,12].The coupling of ANO1 and Ca V 1.2 in the cilia plays a key role in activating the Cl − pathway in ABX-stimulated c-LAECs.However, ANO1 was also expressed in the cell body.The ANO1 expressed in the cell body (ANO1 cell body ) may be activated by the [Ca 2+ ] i increase caused by Ca V 1.2 during ABX stimulation.The activation of ANO1 in the cell body stimulates the Cl − release from the basolateral membrane and enhances cell shrinkage in c-LAECs.However, increases in [Ca 2+ ] i are small in the cell body during ABX stimulation (Supplementary Figure S1).In a previous report, no [Ca 2+ ] i increase was detected in an airway cell line during ABX stimulation [14].Moreover, in this study, no [Ca 2+ ] i increase was detected in the CO 2 /HCO 3 − -free solution [6].However, experiments using nifedipine or a Ca 2+ -free solution revealed that ABX still activated both Ca V 1.2 and ANO1 in this solution.In the cilia, the activation of Ca V 1.2 may increase [Ca 2+ ] i to a significant level, activating ANO1 in the CO 2 /HCO 3 − -free solution.Based on these observations, the activities of ANO1 in the cell body may be low in ABX-stimulated c-LAECs, although we cannot neglect the contribution of the ANO1 in the cell body to decreased [Cl − ] i .
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. suggested that the concentration-response curve of the CBA to [Cl − ] i shifts to a lower concentration than that of the CBF [12].A level of [Cl − ] i under the control conditions may maintain the IDA activity, controlling the CBA at the lowest level, but it may maintain the ODA activity, controlling the CBF at the highest level.
There are many reports showing that decreased [Cl − ] i enhances cellular functions in many cell types, including the airway ciliary cells [25][26][27][28][29].These observations suggest that a Cl − sensor exists in these cells.With-no-lysine kinase1 (WNK1) and WNK4 have been shown to be intracellular Cl − sensors [27][28][29].Piala et al. demonstrated that the Cl − binds to the kinase domain of WNK1 and suppresses its activity by inhibiting autophosphorylation [28].The actions of WNK1 and WNK4 were studied in the NaCl cotransporter of distal nephrons [27][28][29].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 [27,28].WNK1 and WNK4 have been shown to regulate epithelial Na + channels [30,31], CFTR [32], NKCC1 [33] and NCC [26,34].Based on these observations, our hypothesis is that WNK1 controls the ODAs and WNK4 controls the IDAs in the c-LAECs.Further experiments are needed to verify this hypothesis.
ABX at a high concentration, such as 100 µM, inhibits voltage-gated Na + channels (Na V s) [35].ABX at 1 µM is unlikely to inhibit Na V s.Ciliated LAECs express epithelial Na + channels, the activation of which induces a dry airway surface by accelerating the fluid absorption observed in cystic fibrosis patients.An increase in Na + influx induces cell swelling to increase [Cl − ] i .An increase in [Cl − ] i decreases the CBF and CBA [12].In healthy airways, the c-LAECs secrete Cl − [14].The impairment of Cl − secretion, such as in cystic fibrosis, may increase [Na + ] I , leading to an [Cl − ] i increase, which may decrease the CBF and CBA [12].The inhibition of the Na V s using 10 µM ABX appears to have little effect on the CBF and CBA in c-LAECs.
Figure 12 shows a schematic diagram of the ABX-stimulated c-LAECs.ABX increases [Ca 2+ ] i by stimulating the nifedipine-sensitive Ca V 1.2 in the c-LAECs.The increases in [Ca 2+ ] i activate two signaling pathways: the pH i pathway and the Cl − pathway.The activation of Cav1.2 maintains most of the increase in [Ca 2+ ] i .The [Ca 2+ ] i increase activates NBC-entering HCO 3 − to increase the pH i .The pH i pathway (an elevation in pH i ) increases the CBF and CBA by 30% and 15-20%, respectively, in ABX-stimulated c-LAECs.The

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 using inhalational isoflurane (3%) and were further anesthetized using an intraperitoneal injection (ip) of pentobarbital sodium (70 mg/kg) and heparinized (1000 units/kg) for 15 min.Then, the mice were sacrificed with a high dose of pentobarbital sodium (100 mg/kg, ip).

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 using inhalational isoflurane (3%) and were further anesthetized using an intraperitoneal injection (ip) of pentobarbital sodium (70 mg/kg) and heparinized (1000 units/kg) for 15 min.Then, the mice were sacrificed with a high dose of pentobarbital sodium (100 mg/kg, ip).

Cell Preparation
The ciliated LAECs were isolated from the lungs using an elastase treatment [6,9,11].Following the elastase treatment, the lungs were minced using fine forceps in a 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 isolated cells were washed and then suspended in the control solution.The cell suspension was stored at 4 • C and the cells were used within 5 h after the isolation.

CBF and CBA Measurements
The cells were set in a micro perfusion chamber (20 µL) mounted on an inverted light microscope (ECLIPSE Ti, Nikon, Tokyo, Japan) connected to a high-speed camera (Photron Ltd., Tokyo, Japan) and video images were recorded for 2 s at 500 fps (frame per second) [6,9,11].The stage of the microscope was heated to 37 • C [4].After the experiments, the CBF and CBA were measured using an image analysis program (DippMotion 2D, Ditect, Tokyo, Japan) [6,9,11].The normalized CBF and CBA values and CBF and CBA ratios (CBF t /CBF 0 and CBA t /CBA 0 ), calculated from 4 to 12 cells, were used to make a comparison among the experiments.Each experiment was carried out using 4-10 cover slips with cells obtained from 2 to 5 animals."n" shows the number of cells.

Measurement of the Cell Volume
The outline of a c-LAEC was traced onto a video image, and the area of the cell (A) was measured using the image analysis program.The index of the cell volume (V t /V 0 = (A t /A 0 ) 1.5 ) was calculated [9].Each experiment was carried out using 4-6 cover slips obtained from 2 to 3 animals.The V/V 0 values calculated from 4 to 6 cells were plotted and "n" shows the number of cells.

Measurement of pH i , [Cl − ] i and [Ca 2+ ] i
Changes in intracellular pH (pH i ) were monitored via SNARF1 fluorescence (a pH dye) at 37 • C and the cells were set on the heated stage (37 • C) of an inverted confocal laser microscope (model LSM 510-META, Carl Zeiss, Jena, Germany).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 pH i of the c-LAECs was calculated using the calibration line.
Changes in [Cl − ] i were monitored via MQAE fluorescence (a chloride sensitive dye) [9,12].The cells were incubated with 10 mM MQAE for 45 min at 37 • C. The MQAE was excited at 780 nm using a 2-photon excitation laser system (Mai Tai ®® , Spectra-Physics, Santa Clara, CA, USA), and the emission was 510 nm.The ratio of MQAE fluorescence intensity (F 0 /F t ) was calculated.

Western Blotting
The procedure for Western blotting has already been described in previous reports [6].Protein (5-20 µg) obtained from the isolated lung cells was loaded into each lane.After blocking with 5% skim milk powder, the membrane with the protein was exposed to a primary anti-anoctamin-1 antibody (1:200) (ABN1669 Sigma-Aldrich Merck, Darmstadt, Germany) diluted with solution 1 (Can Get Signal, TOYOBO, Osaka, Japan), and the secondary antibody was applied to the membrane.Then, the antigen-antibody com-plexes were visualized using a chemiluminescence system (Immobilon, Merck Millipore, Darmstadt, Germany).

Immunofluorescence Examination
The cell suspension containing the isolated lung cells (0.5 mL) was dropped and dried on the coverslip for attaching the cells [6].After fixation with 4% paraformaldehyde, the cells were stained with an anoctamin-1 antibody and an anti-acetylated tubulin antibody (T6793, Sigma-Aldrich, St. Louis, MO, USA).The cells were washed with PBS containing 0.1% BSA to remove unbound antibodies.Finally, the cells were stained with Alexa 488conjugated anti-rabbit antibody and Alexa 594-conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA, USA).The cells were observed using confocal laser microscopy (FV10i, Olympus, Tokyo, Japan).

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

Figure 1 .
Figure 1.The effect of ABX on the ratios of the CBF and CBA in c-LAECs.(A): ABX (10 μM) stimulation in the presence of CO2/HCO3 − .In the CO2/HCO3 − -containing control solution, ABX stimulation (10 μM) gradually increased the ratios of the CBF and CBA by 30% within 10 min.(B): Effects of Ca 2+ -free solution on the ABX-stimulated CBF and CBA in the presence of CO2/HCO3 − .The switch to a nominally Ca 2+ -free solution decreased the CBF and CBA ratios by 5% within 5 min.Then, ABX stimulation gradually increased the CBF and CBA ratios by 10%.(C): ABX stimulation in the absence of CO2/HCO3 − .The switch to the CO2/HCO3 − -free solution immediately increased the CBF ratio by 30% and the CBA ratio by 20%.Then, ABX stimulation increased the CBA ratio by 10%, but not the CBF ratio.(D): Effects of nifedipine on the ABX-stimulated CBF and CBA ratios in the absence of

Figure 1 .
Figure 1.The effect of ABX on the ratios of the CBF and CBA in c-LAECs.(A): ABX (10 µM) stimulation in the presence of CO 2 /HCO 3 − .In the CO 2 /HCO 3 − -containing control solution, ABX stimulation (10 µM) gradually increased the ratios of the CBF and CBA by 30% within 10 min.(B): Effects of Ca 2+ -free solution on the ABX-stimulated CBF and CBA in the presence of CO 2 /HCO 3 − .The switch to a nominally Ca 2+ -free solution decreased the CBF and CBA ratios by 5% within 5 min.Then, ABX stimulation gradually increased the CBF and CBA ratios by 10%.(C): ABX stimulation in the absence of CO 2 /HCO 3 − .The switch to the CO 2 /HCO 3 − -free solution immediately increased the CBF ratio by 30% and the CBA ratio by 20%.Then, ABX stimulation increased the CBA ratio by 10%, but not the CBF ratio.(D): Effects of nifedipine on the ABX-stimulated CBF and CBA ratios in the absence of CO 2 /HCO 3 − .The addition of nifedipine (20 µM) decreased the CBF and CBA ratios by 5%.The switch to the CO 2 /HCO 3 − -free solution immediately increased the CBF ratio by 30% and the CBA ratio by 20%.Further ABX stimulation did not induce any increase in the CBF ratio or the CBA ratio.

Figure 2 .
Figure 2. Effects of ABX on [Ca 2+ ]i in c-LAECs.Changes in [Ca 2+ ]i were monitored via the fluorescence ratio (F340/F380).(A): ABX stimulation gradually increased F340/F380, which re a plateau within 15 min.Then, the addition of nifedipine gradually decreased F340/F380 to th trol level before ABX stimulation within 10 min.(B): Effects of the Ca 2+ -free solution on the stimulated [Ca 2+ ]i.In the Ca 2+ -free solution, ABX stimulation induced a small transient incre F340/F380.(C): Effects of ABX on [Ca 2+ ]i in the absence of CO2/HCO3 − .The switch to the CO2/H free solution did not change F340/F380 and then ABX stimulation did not change F340/F380.F addition of nifedipine did not change F340/F380.The CO2/HCO3 − -free solution increased pHi, inhibits Ca 2+ release from acidic stores, and it inhibits Na + entry via the NBC.The CO2/HCO solution decreases CaV1.2 channel activity.

Figure 2 .
Figure 2. Effects of ABX on [Ca 2+ ] i in c-LAECs.Changes in [Ca 2+ ] i were monitored via the fura-2 fluorescence ratio (F340/F380).(A): ABX stimulation gradually increased F340/F380, which reached a plateau within 15 min.Then, the addition of nifedipine gradually decreased F340/F380 to the control level before ABX stimulation within 10 min.(B): Effects of the Ca 2+ -free solution on the ABX-stimulated [Ca 2+ ] i .In the Ca 2+ -free solution, ABX stimulation induced a small transient increase in F340/F380.(C): Effects of ABX on [Ca 2+ ] i in the absence of CO 2 /HCO 3 − .The switch to the CO 2 /HCO 3 − -free solution did not change F340/F380 and then ABX stimulation did not change F340/F380.Further addition of nifedipine did not change F340/F380.The CO 2 /HCO 3 − -free solution

Figure 3 .
Figure 3. Effects of ABX on pHi in c-LAECs.Changes in pHi were measured using the SNARF1 fluorescence ratio (F645/F592).(A): ABX stimulation gradually increased pHi from 7.49 to 7.65 within 15 min (n = 8).(B): Effects of a Ca 2+ -free solution on the ABX-stimulated pHi.The switch to a Ca 2+ -free solution did not change pHi.Then, ABX stimulation gradually increased pHi from 7.51 to 7.58 (n = 4) (C): Effects of nifedipine on the ABX-stimulated pHi.The addition of nifedipine did not change pHi.Then, ABX stimulation gradually increased pHi from 7.52 to 7.60 (n = 5).(D): Effects of CO2/HCO3 − -free solution on the ABX-stimulated pHi.The switch to a CO2/HCO3 − -free solution induced a transient increase, followed by a sustained increase in pHi.The values of pHi before, 2 min after and 10 min after the switch were 7.45, 7.87 and 7.65 (n = 6), respectively.Further ABX stimulation did not change pHi.2.1.4.ABX-Stimulated Cell Shrinkage and a Decrease in [Cl − ]i Video Images of ABX-Stimulated Cell Shrinkage and Enhancement of MQAE Fluorescence in c-LAECs

Figure 3 .
Figure 3. Effects of ABX on pH i in c-LAECs.Changes in pH i were measured using the SNARF1 fluorescence ratio (F645/F592).(A): ABX stimulation gradually increased pH i from 7.49 to 7.65 within 15 min (n = 8).(B): Effects of a Ca 2+ -free solution on the ABX-stimulated pH i .The switch to a Ca 2+ -free solution did not change pH i .Then, ABX stimulation gradually increased pH i from 7.51 to 7.58 (n = 4) (C): Effects of nifedipine on the ABX-stimulated pH i .The addition of nifedipine did not change pH i .Then, ABX stimulation gradually increased pH i from 7.52 to 7.60 (n = 5).(D): Effects of CO 2 /HCO 3 − -free solution on the ABX-stimulated pH i .The switch to a CO 2 /HCO 3 − -free solution induced a transient increase, followed by a sustained increase in pH i .The values of pH i before, 2 min after and 10 min after the switch were 7.45, 7.87 and 7.65 (n = 6), respectively.Further ABX stimulation did not change pH i .2.1.4.ABX-Stimulated Cell Shrinkage and a Decrease in [Cl − ] i Video Images of ABX-Stimulated Cell Shrinkage and Enhancement of MQAE Fluorescence in c-LAECs Figure4Ashows a phase contrast video image of a typical c-LAEC perfused with the control solution before ABX stimulation (Figure4A) and that at 15 min after ABX stimulation (Figure4B).The outline of a c-LAEC before ABX stimulation is shown in Figure4Aand was superimposed in Figure4B.The c-LAECs stimulated by ABX were smaller than those before ABX stimulation, indicating that ABX decreased the cell volume.Cell shrinkage is known to decrease [Cl − ] i[9,12].We monitored the [Cl − ] i using a Cl −sensitive fluorescence dye in the c-LAECs (MQAE)[9,12].ABX stimulation increased the intensity of MQAE fluorescence in one of the c-LAECs (Figure4C,D), indicating that ABX stimulation decreases [Cl − ] i .

Figure 4 .
Figure 4. Video frame images and MQAE fluorescence of a c-LAEC.(A,B): Video frame images of c-LAECs before (A) and at 15 min after ABX stimulation (B).The outline of a c-LAEC before ABX stimulation is traced by the black line (A).ABX stimulation induced cell shrinkage.The traced outline of the c-LAECs in the panel (A) was superimposed onto the c-LAEC in the panel (B).The panel (B) shows that the outline of the c-LAEC stimulated by ABX was smaller than that before ABX stimulation.(C,D): Changes in MQAE fluorescence of a c-LAEC before (C) and at 15 min after ABX stimulation (D).ABX stimulation increased the intensity of MQAE fluorescence, indicating that ABX decreased [Cl − ]i in the c-LAEC.

Figure 4 .
Figure 4. Video frame images and MQAE fluorescence of a c-LAEC.(A,B): Video frame images of c-LAECs before (A) and at 15 min after ABX stimulation (B).The outline of a c-LAEC before ABX stimulation is traced by the black line (A).ABX stimulation induced cell shrinkage.The traced outline of the c-LAECs in the panel (A) was superimposed onto the c-LAEC in the panel (B).The panel (B) shows that the outline of the c-LAEC stimulated by ABX was smaller than that before ABX stimulation.(C,D): Changes in MQAE fluorescence of a c-LAEC before (C) and at 15 min after ABX stimulation (D).ABX stimulation increased the intensity of MQAE fluorescence, indicating that ABX decreased [Cl − ] i in the c-LAEC.ABX-Stimulated Decreases in Cell Volume and [Cl − ] i Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 7 of 19 min after ABX stimulation) (Figure5F).The ABX-stimulated cell shrinkage and [Cl − ]i decrease in the CO2/HCO3 − -free solution are likely to be induced by the activation of CaV1.2.

Figure 5 .
Figure 5. Changes in the cell volume and [Cl − ] i induced by ABX stimulation in c-LAECs.(A): In the CO 2 /HCO 3 − -containing control solution, ABX stimulation decreased V/V 0 by 20%.Then, the addition of nifedipine immediately increased V/V 0 to the prestimulation level.(B): In the CO 2 /HCO 3 − -containing control solution, ABX stimulation decreased the ratio of MQAE fluorescence (F 0 /F) by 30%.(C): The switch to the CO 2 /HCO 3 − -free control solution decreased V/V 0 by 10%.Then, ABX stimulation gradually decreased V/V 0 .In the CO 2 /HCO 3 − -free control solution, ABX still activates Ca V 1.2 channels.(D): The switch to the CO 2 /HCO 3 − -free control solution decreased F 0 /F by 15%, and then, ABX stimulation decreased F 0 /F by 20%.(E): Effects of nifedipine on V/V 0 stimulated using ABX.The addition of nifedipine increased V/V 0 by 10%.Then, the switch to the CO 2 /HCO 3 − -free solution decreased V/V 0 by 20%.Further ABX stimulation did not decrease V/V 0 .(F): The addition of nifedipine increased F 0 /F, and the switch to the CO 2 /HCO 3 − -free control solution decreased F 0 /F.Further ABX stimulation did not decrease F 0 /F.2.2.CO 2 /HCO 3 − -Dependent Pathway (pH i Pathway)

Figure 6 .
Figure 6.Effects of DIDS on the CBF, CBA, pHi and [Cl − ]i stimulated by ABX in c-LAECs.(A): Effects of DIDS on CBF and CBD.The addition of DIDS gradually decreased the CBF, but not the CBA in c-LAECs.(B): Effects of DIDS on pHis with or without ABX.The addition of DIDS gradually increased pHi (J) The ABX stimulation did not affect the gradual pHi increase induced by DIDS (E).(C): Changes in the CBF and CBA ratios stimulated by ABX in the presence of DIDS.The addition of DIDS gradually increased the CBF ratio by 15%, but not the CBA ratio.Then, ABX stimulation increased the CBA ratio but not the CBF ratio.(D): Effects of ABX on [Cl − ]i of c-LAECs in the presence of DIDS.The addition of DIDS slightly decreased F0/F and then ABX stimulation decreased F0/F.(E): Effects of ABX on the CBF and CBA ratios in the HCO3 − -containing NO3 − solution.The switch to the HCO3 − -containing NO3 -solution increased the CBF and CBA ratios.Then, ABX stimulation further increased the CBF and CBA ratios.(F): Effects of ABX on pHi in the HCO3 − -containing NO3 -solution.The switch to the HCO3 − -containing NO3 − solution increased pHi.Then, the ABX stimulation further increased pHi.The HCO3 − -containing NO3 − solution enhanced the pHi increase stimulated by ABX.

Figure 6 . 3 −
Figure 6.Effects of DIDS on the CBF, CBA, pH i and [Cl − ] i stimulated by ABX in c-LAECs.(A): Effects of DIDS on CBF and CBD.The addition of DIDS gradually decreased the CBF, but not the CBA in c-LAECs.(B): Effects of DIDS on pH i s with or without ABX.The addition of DIDS gradually increased pH i (J) The ABX stimulation did not affect the gradual pH i increase induced by DIDS (E).(C): Changes in the CBF and CBA ratios stimulated by ABX in the presence of DIDS.The addition of DIDS gradually increased the CBF ratio by 15%, but not the CBA ratio.Then, ABX stimulation increased the CBA ratio but not the CBF ratio.(D): Effects of ABX on [Cl − ] i of c-LAECs in the presence of DIDS.The addition of DIDS slightly decreased F 0 /F and then ABX stimulation decreased

Figure 7 .
Figure 7. Effects of NPPB (a Cl − channel blocker) on the CBF, CBA and [Cl − ]i stimulated by ABX.(A): In the CO2/HCO3 − -containing control solution, the addition of NPPB decreased the CBF and CBA ratios by 5%.Further ABX stimulation increased the CBF and CBA ratios by 10%.(B): The addition of NPPB increased F0/F.Then, ABX stimulation gradually increased F0/F.(C): The addition of NPPB decreased the CBF and CBA ratios by 5% and then the switch to the CO2/HCO3 − -free solution increased the CBF and CBA ratios.Further ABX stimulation did not change the CBF and CBA ratios.(D): The addition of NPPB increased F0/F., and then, the switch to the CO2/HCO3 − -free control solution decreased F0/F.Further ABX stimulation did not change F0/F.2.3.2.Effects of an ANO1 Inhibitor (T16Ainh) on CBF, CBA and [Cl − ]i

Figure 7 .
Figure 7. Effects of NPPB (a Cl − channel blocker) on the CBF, CBA and [Cl − ] i stimulated by ABX.(A): In the CO 2 /HCO 3 − -containing control solution, the addition of NPPB decreased the CBF and CBA ratios by 5%.Further ABX stimulation increased the CBF and CBA ratios by 10%.(B): The addition of NPPB increased F 0 /F.Then, ABX stimulation gradually increased F 0 /F.(C): The addition of NPPB decreased the CBF and CBA ratios by 5% and then the switch to the CO 2 /HCO 3 − -free solution increased the CBF and CBA ratios.Further ABX stimulation did not change the CBF and CBA ratios.(D): The addition of NPPB increased F 0 /F., and then, the switch to the CO 2 /HCO 3 − -free control solution decreased F 0 /F.Further ABX stimulation did not change F 0 /F.2.3.2.Effects of an ANO1 Inhibitor (T16Ainh) on CBF, CBA and [Cl − ] i

Figure 8 .
Figure 8. Effects of T16Ainh (an inhibitor of ANO1) on the CBF and CBA stimulated by ABX.(A): In the CO 2 /HCO 3 − -containing control solution, the addition of T16Ainh (10 µM) decreased the CBA ratio by 5%, but not the CBF ratio.Further ABX stimulation increased the CBF and CBA ratios by 10%.(B): The switch to the CO 2 /HCO 3 − -free control solution increased the CBF ratio by 30% and the CBA ratio by 20%, and then, the addition of T16Ainh did not change the CBF and CBA ratios.The addition of ABX did not change the CBF and CBA ratios.

Figure 8 .
Figure 8. Effects of T16Ainh (an inhibitor of ANO1) on the CBF and CBA stimulated by ABX.(A):In the CO2/HCO3 − -containing control solution, the addition of T16Ainh (10 μM) decreased the CBA ratio by 5%, but not the CBF ratio.Further ABX stimulation increased the CBF and CBA ratios by 10%.(B): The switch to the CO2/HCO3 − -free control solution increased the CBF ratio by 30% and the CBA ratio by 20%, and then, the addition of T16Ainh did not change the CBF and CBA ratios.The addition of ABX did not change the CBF and CBA ratios.

Figure 9 .
Figure 9. Effects of CO2/HCO3 − -free Cl − -free NO3 − solution on the CBF and CBA.(A): The switch to the CO2/HCO3 − -free solution increased the CBF and CBA ratios and then the switch to the CO2/HCO3 − -free Cl − -free NO3 − solution increased the CBA ratio, but not the CBF ratio.Further ABX stimulation did not increase the CBF and CBA ratios.Thus, the increases in CBA and CBF stimulated by ABX were mimicked by the CO2/HCO3 − -free Cl − -free NO3 − solution.(B): Changes in [Cl − ]i monitored via the MQAE fluorescence ratio (F0/F).The switch to the CO2/HCO3 − -free solution decreased F0/F and then the second switch to the CO2/HCO3 − -free Cl − -free NO3 − solution decreased F0/F.Further ABX stimulation did not change F0/F.

Figure 9 .
Figure 9. Effects of CO 2 /HCO 3 − -free Cl − -free NO 3 − solution on the CBF and CBA.(A): The switch to the CO 2 /HCO 3 − -free solution increased the CBF and CBA ratios and then the switch to the CO 2 /HCO 3 − -free Cl − -free NO 3 − solution increased the CBA ratio, but not the CBF ratio.Further ABX stimulation did not increase the CBF and CBA ratios.Thus, the increases in CBA and CBF stimulated by ABX were mimicked by the CO 2 /HCO 3 − -free Cl − -free NO 3 − solution.(B): Changes in [Cl − ] i monitored via the MQAE fluorescence ratio (F 0 /F).The switch to the CO 2 /HCO 3 − -free solution decreased F 0 /F and then the second switch to the CO 2 /HCO 3 − -free Cl − -free NO 3 − solution decreased F 0 /F.Further ABX stimulation did not change F 0 /F.

Figure 10 .
Figure 10.Western blotting for ANO1 in isolated lung cells.A single band for ANO1 was detected at 110 kDa in isolated lung cells.

Figure 10 .
Figure 10.Western blotting for ANO1 in isolated lung cells.A single band for ANO1 was detected at 110 kDa in isolated lung cells.

Figure 10 .
Figure 10.Western blotting for ANO1 in isolated lung cells.A single band for ANO1 was detected at 110 kDa in isolated lung cells.

Figure 12 .
Figure 12.Schematic diagram of the ABX-stimulated c-LAECs.ABX stimulates the Ca 2+ entry via CaV1.2 and increases [Ca 2+ ]i.The [Ca 2+ ]i increase stimulates the NBC to accelerate HCO3 − entry.The HCO3 − entering via NBC binds H + to increase pHi.The Ca 2+ entering via CaV1.2 directly stimulates ANO1 in cilia to activate Cl − secretion, which decreases [Cl − ]i.The pHi elevation enhances the CBF and CBA, and the [Cl − ]i decrease enhances the CBA.A small and transient Ca 2+ release from the acidic stores increases [Ca 2+ ]I and activates NBC in ABX-stimulated c-LAECs.

Figure 12 .
Figure 12.Schematic diagram of the ABX-stimulated c-LAECs.ABX stimulates the Ca 2+ entry via Ca V 1.2 and increases [Ca 2+ ] i .The [Ca 2+ ] i increase stimulates the NBC to accelerate HCO 3 − entry.The HCO 3 − entering via NBC binds H + to increase pH i .The Ca 2+ entering via Ca V 1.2 directly stimulates ANO1 in cilia to activate Cl − secretion, which decreases [Cl − ] i .The pH i elevation enhances the CBF and CBA, and the [Cl − ] i decrease enhances the CBA.A small and transient Ca 2+ release from the acidic stores increases [Ca 2+ ] I and activates NBC in ABX-stimulated c-LAECs.