Intracellular Cl− Regulation of Ciliary Beating in Ciliated Human Nasal Epithelial Cells: Frequency and Distance of Ciliary Beating Observed by High-Speed Video Microscopy

Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca2+, H+ and Cl−), and is enhanced by a decreased concentration of intracellular Cl− ([Cl−]i) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl−]i increased CBD, but not CBF, and an increase in [Cl−]i decreased both CBD and CBF. Thus, [Cl−]i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl− sensors and be regulated by [Cl−]i. These observations indicate that the activation of Cl− secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl−]i in cHNECs. Thus, [Cl−]i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl− regulation of ciliary beating in cHNECs.


Introduction
The nasal and sinonasal epithelia are exposed to small inhaled airborne particles, such as allergens, chemicals, viruses and bacteria, which are removed via mucociliary clearance. Mucociliary clearance is a host defence mechanism of the respiratory tracts that comprises a thin mucous film and beating cilia [1][2][3][4][5]. In this process, small inhaled particles are entrapped by the thin mucous film (surface 2. cHNECs cHNECs isolated from nasal samples obtained during surgery or by brushing are cultured at the air-liquid interface (ALI) to differentiate into ciliated cells. cHNECs are widely used for cilia research [33][34][35][36][37]. Recent studies have demonstrated that cHNECs have some characteristic features that differ from ciliated airway cells of the trachea or lung. The ciliated tracheal and lung cells are sensitive to intracellular pH (pH i ); a high pH i increases CBF, and a low pH i decreases CBF [15,16,20,21,38]. The pH i can be changed by switching from a CO 2 /HCO 3 − -containing to a CO 2 /HCO 3 − -free solution, although the changes in pH i are small. H + is produced from CO 2 by carbonic anhydrase. Therefore, the switch to a CO 2 /HCO 3 − -free solution increases pH i , and the return to a CO 2 /HCO 3 − -containing solution decreases it [15,16,20,21,38,39]. In tracheal ciliary cells, the switch to a CO 2 /HCO 3 − -free solution has been shown to increase CBF, mediated via an increase in pH i [38]. However, in cHNECs, the switch to a CO 2 /HCO 3 − -free solution induced only a small and transient increase in pH i , leading to a slight or sometimes no increase in CBF [15,16,21]. We also applied the NH 4 + pulse to change pH i . An application of the NH 4 + pulse (an addition of NH 4 Cl, such as 25 mM) in extracellular fluid releases a small amount of NH 3 , which enters cells and traps H + to produce NH 4 + leading to an increase in pH i [21,38,39], and it induced a larger increase in pH i independent of CO 2 than that induced by the CO 2 /HCO 3 − -free solution. In cHNECs, the NH 4 + pulse induced transient CBF and pH i increases. However, in the presence of acetazolamide (an inhibitor of carbonic anhydrase, which inhibits H + production from CO 2 ), it induced sustained increases, not transient, in pHi and CBF. These results indicate that cHNECs produce a large amount of H + , even under CO 2 /HCO 3 − -free conditions. A high level of H + production suggests a high CO 2 production or a high carbonic anhydrase activity in cHNECs. Moreover, mouse nasal ciliary cells, unlike airway ciliary cells, have shown spontaneous CBF oscillation induced by periodic intracellular Ca 2+ ([Ca 2+ ] i ) spikes and no increase in CBF during isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) stimulation [40]. Thus, cHNECs possess characteristic features distinct from tracheal or lung airway ciliary cells.
The characteristic features of cHNECs distinct from trachea and lung airway ciliary cells appear to be caused by the different embryological origins. The cHNECs are derived from the surface ectoderm, while ciliary cells of trachea and lung are from the endoderm. These characteristic features appear to be beneficial for cHNECs, which are exposed to air directly. In particular, a high H + production in cHNECs prevents pH i changes induced by fluctuations in CO 2 concentrations during inspiration and expiration (0. .
A decrease in [Cl − ] i increases the amplitude, CBA or CBD, but not CBF, in cHNECs [15,16,21], similar to airway ciliary cells [20]. These observations suggest that [Cl − ] i is an important ion involved in regulating ciliary beating in airway ciliary cells, including cHNECs.

Analysis of Ciliary Beating in cHNECs
Primary cHNEC cultures are grown at the ALI and form a cell sheet similar to nasal or sinonasal epithelia. The ciliary beating of cHNECs can be observed from the apical side using high-speed video microscopy (HSVM). Since CBA measurement is difficult in the apical view, a new parameter is required to assess the amplitude of ciliary beating. Recently, CBD has been proposed as a new parameter for assessing the amplitude using HSVM and an image analysis programme.

HSVM
Recent developments in HSVM have allowed the observation of fine movements of airway ciliary beating, the analysis of which has enabled the measurement of the functional parameters of ciliary beating, not only the CBF but also the waveform or the beating pattern, including amplitude [9][10][11]. HSVM has been shown to be an effective tool for PCD diagnosis [11,41].
CBF has been established as the functional parameter of ciliary beating. However, there is no standard definition or consistency in the evaluation of ciliary waveforms, and the quantitative assessment of the waveform or of the pattern of ciliary beating remains controversial because of the waveform's complexity [11]. In our studies, the amplitude measured as CBD or CBA was shown to be an important factor in stimulating airway ciliary transport [4][5][6][7][8][9][10][11][12] and is proposed as a parameter for assessing the ciliary waveform.
Fine images of beating cilia with a high resolution in space and time are essential to measure the frequency and amplitude, and HSVM is a useful tool for this purpose. To observe ciliary beating, an inverted microscope with phase-contrast or differential interference contrast (DIC) optics as well as a high magnification objective lens (i.e., 60× or 100×) are suitable. However, the level of magnification should be selected according to the experimental purpose. The beating cilia move at a depth 5-20 µm above the apical surface of ciliary cells, and the focal plane depth of a high magnification lens with phase-contrast or DIC optics is <5 µm. In our experiments, we sometimes used HSVM equipped with a 60× objective lens without DIC or phase-contrast equipment for observing whole ciliary movements. Moreover, considering that a microscope light source may occasionally be insufficient for HSVM, especially when using DIC optics, a suitable optic, such as phase-contrast, was required. Given that CBF is highly temperature-dependent [3], a temperature-controlled micro-perfusion chamber was also necessary.
HSVM allows beating cilia from isolated ciliary cells to be viewed from three directions-a sideways profile, beating toward the lens (vertical direction), and from above [12]. However, in a sheet of cultured cells, such as cHNECs, most cells are viewed from the apical side ( Figure 1A,B). CBF can be The distance between two lines marked by ←→ represents the ciliary beat distance (CBD), while the number of peaks marked by ↓ represents ciliary beat frequency (CBF, 12.5 Hz). (D) Light intensity changes of the ciliary beating in cHNECs during stimulation with 100 µM daidzein (13 Hz). The line was placed on the same position of a cHNEC as shown in panels A and B. The traces of light intensity changes clearly show that daidzein increases CBD, but not CBF. To assess the amplitude of ciliary beating in the apical view, the distance between the start and the end of the effective stroke was measured. The measured distance is the ciliary beat distance (CBD, another index of ciliary beat amplitude). CBF, ciliary beat frequency.

Digital High-Speed Camera
Given their high frequency of 8-25 Hz at 37 °C, it is difficult to observe the fine movements of beating airway cilia at the National Television System Committee frame rate (30 fps). To visualise the complete cycle of ciliary beating, a high video recording rate (i.e., 500 Hz) is essential.

CBF Measurement
Available image analysis software for CBF measurement has been based on light intensity changes in the pixels of the recorded images over time ( Figure 1). A semiautomated programme can determine CBF in selected lines of measured cilia [11]. At present, sample movements during perfusion occasionally introduce artefacts in the CBF obtained from the programme. Thus, while semiautomated CBF analysis has some advantages, such as a shorter time requirement, certain limitations are also present. When calculating CBF, light intensity peaks in the image obtained from the programme are counted (Figures 1 and 2). To assess the amplitude of ciliary beating in the apical view, the distance between the start and the end of the effective stroke was measured. The measured distance is the ciliary beat distance (CBD, another index of ciliary beat amplitude). CBF, ciliary beat frequency.

Digital High-Speed Camera
Given their high frequency of 8-25 Hz at 37 • C, it is difficult to observe the fine movements of beating airway cilia at the National Television System Committee frame rate (30 fps). To visualise the complete cycle of ciliary beating, a high video recording rate (i.e., 500 Hz) is essential.

CBF Measurement
Available image analysis software for CBF measurement has been based on light intensity changes in the pixels of the recorded images over time ( Figure 1). A semiautomated programme can determine CBF in selected lines of measured cilia [11]. At present, sample movements during perfusion occasionally introduce artefacts in the CBF obtained from the programme. Thus, while semiautomated CBF analysis has some advantages, such as a shorter time requirement, certain limitations are also present. When calculating CBF, light intensity peaks in the image obtained from the programme are counted (Figures 1 and 2).

CBD Measurement
Ciliary beating is coordinated with metachronal waves, which exhibit a whip-like movement. In the effective stroke of ciliary beating, the cilium tip makes an arc with a maximum speed in the plane perpendicular to the cell surface, while in the recovery stroke, the cilium swings back close to the cell surface ( Figure 2). Studies have demonstrated that an abnormal waveform of ciliary beating induces ciliary dysfunction, leading to PCD, which is characterised by situs inversus totalis, sinusitis, and bronchiectasis [29]. The quantitative evaluation of ciliary waveform or ciliary wave pattern has been proposed to diagnose PCD based on an abnormal ciliary waveform or ciliary beat pattern [11,41]. Among the waveform evaluation parameters, we previously used CBD or CBA as an index of ciliary beat amplitude [4][5][6][7][8][9][10]24]. CBD has been characterised as the distance between the maximum forward and backward movements of the cilia tip [8- 10,24,30,42] and CBA as the angle between the start and the end cilium positions in the effective stroke [4][5][6][7][8]24]. As shown in Figures 1 and 2, CBD and CBA are indices of ciliary beating amplitude and are thus the parameters for evaluating the ciliary beating waveform. It is certain that CBD or CBA are, at least, two of the most important parameters for evaluating the waveform of ciliary beating. Previous studies have demonstrated that an increase in CBA or CBD enhanced the transport of microbeads in the airway surface [4] or on the surface of cHNECs [9,10,24].
Moreover, in some cases, increases in CBD or CBA occurred without any increase in CBF. As mentioned above, a CBD increase alone enhanced microbead transport [9,10,24], although increases in CBF can also have this effect. Thus, CBD measurement is essential for evaluating the ciliary function in addition to CBF measurement.
Although various abnormal waveforms have been reported in PCD, the amplitude of ciliary beating, CBD and CBA, remain controversial as a parameter for PCD diagnosis [11]. However, CBD and CBA are important factors for evaluating normal airway cilia activity because an increase in CBD or CBA was shown to enhance microbead transport [14][15][16].

Changes in [Cl − ] i
[Cl − ] i has been shown to modulate cellular functions in many cell types [22][23][24][25][26][27][28][29][30][31][32] and to be affected by cell volume changes. Many agonists activating ion transport, such as Cl − secretion and K + release, have been demonstrated to evoke cell shrinkage under the isosmotic condition in many cell types [20,22,[43][44][45][46]. This occurs when an increase in [Ca 2+ ] i or cyclic adenosine monophosphate accumulation stimulated by an agonist activates K + and Cl − channels, leading to the cellular release of KCl [20,22,32,46]. The KCl release generates an osmotic gradient between the intracellular and extracellular space and is followed by a fluid efflux. Finally, the cell volume decreases to an equilibrium condition (isosmotic cell shrinkage). In airway epithelia, the activation of Cl − secretion (Cl − release from cells) also accompanies K + release for the maintenance of the intracellular electroneutrality. The KCl release, which generates a hypoosmotic condition in intracellular space, induces fluid efflux to evoke cell shrinkage [46]. Agonists, such as procaterol, CCis and daidzein, have already been shown to stimulate cell shrinkage in airway ciliary cells and cHNECs [15,16,20,21]. Furthermore, this isosmotic cell shrinkage has been demonstrated to decrease [Cl − ] i in airway ciliary cells and cHNECs [15,16,21].
In general, K + and Cl − , which are the main intracellular cation and anion, respectively, are membrane-permeable ions, because the cell membrane has ion transporters and channels for K + and Cl − . The isosmotic cell shrinkage, which is caused by the KCl release, decreases [Cl − ] i . and MQAE fluorescence before and after daidzein stimulation. Daidzein stimulates cell shrinkage and increases the fluorescence intensity of MQAE. Relative changes in cell volume and MQAE fluorescence ratio (F0/F) are shown in Figure 4C, where subscript "0" refers to the time when the stimulation was initiated. Accordingly, daidzein decreased cell volume to 81% and F0/F to 78%. In cHNECs, a decrease in [Cl − ]i (i.e., decreased cell volume) increased only CBD but not CBF ( Figure  4D).   Figure 4A,B) [15,16,20,21,47]. Daidzein, an agonist that stimulates Cl − channels, has been shown to induce an [Cl − ] i decrease in cHNECs [15]. Figure 4A,B, in which cellular shapes have been superimposed on the MQAE fluorescence images, show changes in the cellular outline and MQAE fluorescence before and after daidzein stimulation. Daidzein stimulates cell shrinkage and increases the fluorescence intensity of MQAE. Relative changes in cell volume and MQAE fluorescence ratio (F 0 /F) are shown in Figure 4C, where subscript "0" refers to the time when the stimulation was initiated.
Accordingly, daidzein decreased cell volume to 81% and F 0 /F to 78%. In cHNECs, a decrease in [Cl − ] i (i.e., decreased cell volume) increased only CBD but not CBF ( Figure 4D).

Effects of Decreased [Cl − ]i on CBF and CBD
The effects of decreased [Cl − ]i on CBF and CBD are presented in Figure 5. A switch to a CO2/HCO3 − -free solution alone decreased [Cl − ]i [15,16,21]. This [Cl − ]i decrease is explained by the inhibition of NaCl entry via Na + -HCO3 − cotransporter (NBC) and Cl − /HCO3 − exchange (anion

Effects of Decreased [Cl − ] i on CBF and CBD
The effects of decreased [Cl − ] i on CBF and CBD are presented in Figure 5.     Figure 5A). The effects of bumetanide (bum, an inhibitor of Na+/K+/2Cl-cotransporter (NKCC)), daidzein (an activator of Clchannels) and carbocisteine (CCis, an activator of Cl − channels including cystic fibrosis transmembrane conductance regulator (CFTR)) on CBF and CBD under CO 2 /HCO 3 − -free conditions are shown in Figure 5B. The switch to CO 2 /HCO 3 − -free solution decreased [Cl − ] i as explained previously. Bum, which inhibits Na + , K + and 2Cl − entry, decreased F 0 /F ([Cl − ] i ) ( Figure 5B) [16,21]. An activation of Cl − channels by daidzein or CCis also decreased [Cl − ] i ( Figure 5B) [15,16]. Decreases in [Cl − ] i induced by NO 3 − solution, bum, daidzein and CCis increased CBD but not CBF.   [16,20]. These results appear to suggest that [Cl − ] i may inhibit CBF and CBD and that the Cl − concentration-response curve of CBF may shift to a higher concentration than that of CBD, indicating that the [Cl − ] i affects both CBF and CBD. The Cl − -binding affinity for the structures controlling CBD and CBF may depend on different temperatures.

Effects of Increased CBD on the Microbead Movement in cHNECs
To examine the effects of an increase in CBD on ciliary transport, microbeads were applied to cHNECs [14][15][16]. Microbeads reaching the surface of cHNECs were moved by the surface fluid flow driven by beating cilia. Microbead movement was observed using HSVM (60 fps). Figure 6A shows the movement of a microbead that reached the surface of a cHNEC (before daidzein addition). The large arrows show the positions of a microbead reaching the cell surface, while small arrows show the distance moved in 33 ms (over two frames). Figure 6B shows video images 5 min after daidzein addition, which enhanced the distance that a microbead moved. Figure 6C shows the CBD ratio and microbead movement before and after the daidzein addition. Accordingly, the increase in CBD enhanced microbead movement. Figure 6D shows the CBD ratio and microbead movement before and after CCis addition. Similarly, CCis addition enhanced CBD and microbead movement. CCis has already been shown to increase only CBD, without increasing CBF. The aforementioned results show that an increase in CBD alone enhances microbead movement, suggesting that CBD, in addition to CBF, is an important parameter for assessing the functions of ciliary beating.

[Cl − ]i Modulation of CBF and CBD in cHNECs
Ciliary beating is maintained by force generated by molecular motors called dyneins [51]. Motile cilia can have two functionally distinct dyneins, outer dynein arms (ODAs), which control frequency, and inner dynein arms (IDAs), which control waveform, including CBD [52,53]. This suggests that a decrease in [Cl − ]i stimulates both ODA and IDA activity, whereas an increase in [Cl − ]i inhibits both. However, an [Cl − ]i decrease stimulates ODAs to increase CBF at 25 °C, but not at 37 °C. Based on these observations, axonemal structures controlling ODAs and IDAs might have a sensor to which [Cl − ]i binds. The binding of Cl − to this sensor would decrease ODA and IDA activities, consequently decreasing CBF and CBD. The Cl − concentration-response curve for CBF might shift to a higher concentration than that for CBD. The Cl − -binding affinity for the structures controlling the ODAs might become decreased at a low temperature, and if no Cl − binds to the structures, ODA and CBF activity may increase. The Cl − -binding affinity for the structures controlling CBF might be higher than that controlling CBD.

[Cl − ] i Modulation of CBF and CBD in cHNECs
Ciliary beating is maintained by force generated by molecular motors called dyneins [51]. Motile cilia can have two functionally distinct dyneins, outer dynein arms (ODAs), which control frequency, and inner dynein arms (IDAs), which control waveform, including CBD [52,53]. This suggests that a decrease in [Cl − ] i stimulates both ODA and IDA activity, whereas an increase in [Cl − ] i inhibits both. However, an [Cl − ] i decrease stimulates ODAs to increase CBF at 25 • C, but not at 37 • C. Based on these observations, axonemal structures controlling ODAs and IDAs might have a sensor to which [Cl − ] i binds. The binding of Cl − to this sensor would decrease ODA and IDA activities, consequently decreasing CBF and CBD. The Cl − concentration-response curve for CBF might shift to a higher concentration than that for CBD. The Cl − -binding affinity for the structures controlling the ODAs might become decreased at a low temperature, and if no Cl − binds to the structures, ODA and CBF activity may increase. The Cl − -binding affinity for the structures controlling CBF might be higher than that controlling CBD.
There are numerous reports showing that [Cl − ] i modulates cellular functions in many cell types [22][23][24][25][26][27]31,54]. These observations suggest that cells have chloride sensors transducing this signal to the targets. Piala et al. (2014) showed that [Cl − ] i binds to the kinase domain of with-no-lysine kinase (WNK) 1 and inhibits WNK1 autophosphorylation, inhibiting its activity [55]. WNK1-4 are serine/threonine protein kinases lacking a lysine residue within subdomain II of the kinase domain. They share a common structure with >80% identity in their kinase domain [56,57], indicating that the chloride-binding site is conserved among the WNKs. Kinase studies have demonstrated that the affinities for Cl − differ among WNK1, WNK3, and WNK4 [57,58]. WNK4 activity is inhibited when [Cl − ] i is 0-40 mM, while those of WNK1 and WNK3 become inhibited at 60-150 mM and 100-150 mM, respectively [57]. WNK1 and WNK4 have been shown to regulate CFTR [59] and NKCC1 of olfactory sensory cilia [60]. WNK4 has also been shown to regulate epithelial Na + channels (ENaC) in various tissues including airway [61,62]. CFTR and ENaC exist in nasal epithelia [15,16,63]. Moreover, WNK1 and WNK4 have been demonstrated to exist in olfactory sensory cilia [60]. Although the presence of WNK1 and WNK4 in motile cilia of cHNECs still remains uncertain, these observations may suggest that WNK1 and WNK4 are physiological [Cl − ] i sensors in cHNECs, as shown in an NCC study using WNK4 K/O mice [64]. Further studies are necessary.
To examine the effects of [Cl − ] i on CBD and CBF in cHNECs, the relative changes in CBD and CBF (CBD ratio and CBF ratio), which were reported in previous studies [15,16,21], were plotted against the MQAE fluorescence ratio (F 0 /F, an index of [Cl − ] i ) (Figure 7). CBD (red marks) was inhibited at a lower [Cl − ] i than CBF (blue marks). The inhibition of CBD and CBF by increasing [Cl − ] i seems to be similar to the inhibition of WNK4 and WNK1 obtained by increasing [Cl − ] i in the previous studies in vitro [55,57,58].
These studies may suggest that WNK4 exists in axonemal structures controlling IDAs and WNK1 exists in those controlling ODAs. The different subtypes of WNK, such as WNK4 or WNK1, may induce different actions in response to changes in [Cl − ] i . Unfortunately, whether the axonemal structures controlling IDA and ODA possess WNK4 and WNK1 and the exact [Cl − ] i in cilia of cHNECs is unknown. If WNK4 and WNK1 exist in the axonemal structure controlling IDA and ODA, respectively, the effects of [Cl − ] i on CBF and CBA may be explained. For example, an [Cl − ] i decrease stimulated by cell shrinkage may activate WNK4, leading to the activation of IDA-controlling structures (CBD increase), but not WNK1, resulting in the continuing activation of the ODA-controlling structures (no CBF increase). A low temperature, such as room temperature, may decrease the affinities of WNK4 and WNK1 for Cl − , leading to both WNK4 and WNK1 activation, which may increase CBD and CBF.
PCD patients have mutations in microtubule motor proteins and other structural proteins controlling ciliary beating. These proteins may have Cl − sensors, such as WNK1and WNK4, and their activities may be affected by [Cl − ] i . Their mutation may abolish activations of CBD and CBF in response to a decrease in [Cl − ] i and may make ciliary activities worse. Further studies are required.
A previous study showed that a decrease in [Cl − ] i increased the dynein affinity for microtubules [68]. Fluctuations in [Cl − ] i , which modulate G proteins and GTPase [28,30,69], may also affect the interactions between tubulin and dyneins via G proteins and GTPase, which may change the sliding speed or force generation [70], leading to changes in CBF and CBD. These actions may also be mediated by WNK4 and WNK1 or be independent from them.

Conclusions
[Cl − ]i is an important signal ion that modulates ciliary beating in cHNECs. Nasal epithelia in primary culture have already shown to secrete Cl − [63,71], although nasal mucosal glands including goblet cells also secrete Cland mucins maintaining the nasal mucous layer. Under physiological conditions, Clsecretion in nasal epithelia maintains the surface serous fluid layer just below the mucous film, in which the cilia beat. Thus, Cl − secretion in cHNECs maintains the mucociliary clearance in nasal and sinonasal epithelia. Our studies in cHNECs demonstrated that an activation of Cl − secretion induces a decrease in [Cl − ]i, which increases CBD. Moreover, airway cilia express CFTR [20]. Microdomains in the cilia are suggested to be isolated from the cell body, and their circumstances may be different from those of the cell body [17]. This may suggest that the activation or inhibition of CFTR induces a larger change in [Cl − ]i in the cilia than in the cell body. The summary of Clregulation of ciliary beating in cHNECs is shown in Figure 8.
The drugs stimulating Cl − secretion, such as CCis, improve the symptoms of nasal and sinonasal diseases by increasing CBD. Moreover, a decrease in [Cl − ]i may be a new therapeutic tool for improving nasal and sinonasal problems. However, the [Cl − ]i sensor of cHNECs remains unknown. We speculate WNK1 and WNK4 as candidates of the [Cl − ]i sensor controlling CBF and CBD. Nevertheless, the response of other kinases to an [Cl − ]i decrease must not be neglected. Further studies should provide elucidation.

Conclusions
[Cl − ] i is an important signal ion that modulates ciliary beating in cHNECs. Nasal epithelia in primary culture have already shown to secrete Cl − [63,71], although nasal mucosal glands including goblet cells also secrete Cland mucins maintaining the nasal mucous layer. Under physiological conditions, Clsecretion in nasal epithelia maintains the surface serous fluid layer just below the mucous film, in which the cilia beat. Thus, Cl − secretion in cHNECs maintains the mucociliary clearance in nasal and sinonasal epithelia. Our studies in cHNECs demonstrated that an activation of Cl − secretion induces a decrease in [Cl − ] i , which increases CBD. Moreover, airway cilia express CFTR [20]. Microdomains in the cilia are suggested to be isolated from the cell body, and their circumstances may be different from those of the cell body [17]. This may suggest that the activation or inhibition of CFTR induces a larger change in [Cl − ] i in the cilia than in the cell body. The summary of Clregulation of ciliary beating in cHNECs is shown in Figure 8.
The drugs stimulating Cl − secretion, such as CCis, improve the symptoms of nasal and sinonasal diseases by increasing CBD. Moreover, a decrease in [Cl − ] i may be a new therapeutic tool for improving nasal and sinonasal problems. However, the [Cl − ] i sensor of cHNECs remains unknown. We speculate WNK1 and WNK4 as candidates of the [Cl − ] i sensor controlling CBF and CBD. Nevertheless, the response of other kinases to an [Cl − ] i decrease must not be neglected. Further studies should provide elucidation.