Bicarbonate transport of airway surface epithelia in luminally perfused mice bronchioles

HCO3− secretion in distal airways is critical for airway mucosal defense. HCO3−/H+ transport across the apical membrane of airway surface epithelial cells was studied by measuring intracellular pH in luminally microperfused freshly dissected mice bronchioles. Functional studies demonstrated that CFTR, ENaC, Cl−–HCO3− exchange, Na+-H+ exchange, and Na+–HCO3− cotransport are involved in apical HCO3−/H+ transport. RT-PCR of isolated bronchioles detected fragments from Cftr, α, β, γ subunits of ENaC, Ae2, Ae3, NBCe1, NBCe2, NBCn1, NDCBE, NBCn2, Nhe1, Nhe2, Nhe4, Nhe5, Slc26a4, Slc26a6, and Slc26a9. We assume that continuous decline of intracellular pH following alkaline load demonstrates time course of HCO3− secretion into the lumen which is perfused with a HCO3−-free solution. Forskolin-stimulated HCO3− secretion was substantially inhibited by luminal application of CFTRinh-172 (5 μM), H2DIDS (200 μM), and amiloride (1 μM). In bronchioles from a cystic fibrosis mouse model, basal and acetylcholine-stimulated HCO3− secretion was substantially impaired, but forskolin transiently accelerated HCO3− secretion of which the magnitude was comparable to wild-type bronchioles. In conclusion, we have characterized apical HCO3−/H+ transport in native bronchioles. We have demonstrated that cAMP-mediated and Ca2+-mediated pathways are involved in HCO3− secretion and that apical HCO3− secretion is largely mediated by CFTR and H2DIDS-sensitive Cl−–HCO3− exchanger, most likely Slc26a9. The impairment of HCO3− secretion in bronchioles from a cystic fibrosis mouse model may be related to the pathogenesis of early lung disease in cystic fibrosis.


Introduction
The airway surface liquid (ASL) is a thin layer of fluid covering the luminal surface of airway epithelium. The ASL is composed of inner periciliary liquid layer (PCL) and outer single-layer thin mucus. Proper volume/depth, viscosity, and pH of ASL are required for efficient mucociliary clearance and antimicrobial activity [11,38,47].
It is widely accepted that the volume/depth of PCL is determined by Cl − secretion via cystic fibrosis transmembrane conductance regulator (CFTR) and Ca 2+ -activated and mucociliary transport in ex vivo pig trachea under acetylcholine (ACh) stimulation was more dependent on HCO 3 − secretion than Cl − secretion [13]. ASL pH in vivo newborn CF pigs was more acidic compared to wild-type and the impaired bacterial-killing activity of CF ASL was rescued by adding NaHCO 3 [38]. Cellular mechanisms for HCO 3 − transport in airways have been studied using cultured human nasal epithelial cells [36,37] and Calu-3 cells, a model of serous cells of submucosal glands [20,25,28]. However, HCO 3 − transport in distal airways/ bronchioles is not well understood. ASL pH was more alkaline in lower airways than in upper airways in human [34]. Thus, a balance of HCO 3 − and H + secretion may shift to HCO 3 − secretion in distal airways. Distal airways contribute to 85-90% of the total epithelial surface area of conducting airways [10,50]. Moreover, mucus plugging and obstruction of bronchioles are among the earliest events of CF lung disease, suggesting that regulation of epithelial ion transport in distal airways is critical for normal lung physiology [46]. However, the assessment of ion transport in distal airways/bronchioles has been limited because of the small size, complex anatomy and relative inaccessibility of structures [10]. Measurement of transmembrane potential in sheep, porcine, and human bronchioles identified Na + and Cl − conductive pathways [2,6,9]. Aquaporin-mediated transepithelial water permeability was identified in guinea pig bronchioles [18]. Measurement of transepithelial potentials by a capillary-Ussing chamber revealed concurrent fluid secretion and absorption and HCO 3 − secretion in human bronchioles [45]. However, characteristics and cellular mechanisms of HCO 3 − transport in distal airways/bronchioles have not been fully investigated.
In the present study, HCO 3 − transport in surface epithelial cells of native bronchioles was studied by measuring intracellular pH (pH i ) in luminally microperfused freshly dissected mice bronchioles. HCO 3 − transport in bronchioles from a CF mouse model was also studied.

Ethics approval
The study was approved by the Ethical Committee of Nagoya University on Animal Use for Experiment (approval No. M210457-003) and the Recombinant DNA Experiment Safety Committee of Nagoya University (approval No. 20-93).

Isolation of bronchioles from mice lung
A CF mouse model in which the F508del mutation was introduced in the mouse Cftr with the C57BL/6J genetic background (ΔF mouse) [53] was purchased from the Jackson Laboratory (Bar Harbor, ME). ΔF mice and their wild-type littermates were bled in Center for Research of Laboratory Animals and Medical Research Engineering, Nagoya University. Mice (8-10 weeks of age) of either sex were suffocated with CO 2 . The thorax was opened and the ice-cold standard HCO 3 − -buffered solution was gently injected into the trachea to fill the lungs. The lungs were then removed and the segments of conducting bronchioles (the third or fourth branches, 150-180 μm in inner diameter) were micro-dissected using sharpened needles in the ice-cold standard HCO 3 − -buffered solution. − -buffered solution contained N-methyl-d-glucamine (NMDG) in place of NaCl, choline bicarbonate in place of NaHCO 3 , and 10 μM atropine to prevent the possible activation of muscarinic receptors by choline. The Na + -free HEPES-buffered solution contained NMDG-Cl in place of NaCl, and Hepes-acid in place of Na-Hepes. In the HCO 3 − -buffered solution containing 20 mM NH 4 Cl, the concentration of NaCl was reduced to maintain osmolarity. All solutions, except for the 25 mM HCO 3 − -0% CO 2 solution, were adjusted to pH 7.4 at 37 ℃.

Microperfusion of isolated bronchioles
The lumen of the isolated bronchiole segments was microperfused by applying a method to microperfuse isolated pancreatic ducts [22]. One end of bronchiole was cannulated for luminal microperfusion ( Fig. 1a and  b). The concentric pipette arrangement consisted of an outer holding pipette, an inner perfusion pipette, and a silica inner capillary for exchange of solutions. The combination of inner silica capillary and waste line enables rapid exchange of luminal solutions. The lumen was perfused at 20-30 μl/min while the bath was perfused at ~ 3 ml/min and maintained at 37 ℃. The luminal perfusate leaving the other end of the bronchiole was diluted and washed away by the much greater flow of solution through the bath, which prevented the luminal perfusate from gaining access to the basal surface of the bronchiole.

Measurement of intracellular pH of bronchiole surface epithelium
The intracellular pH (pH i ) in the epithelial cells was estimated by microfluorometry using the pH-sensitive fluoroprobe 2' ,7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). After cannulating a bronchiole for luminal microperfusion, the epithelial cells were loaded with BCECF by perfusing the lumen with a solution containing the acetoxymethyl ester BCECF-AM (5 μM) for 10 min. Small regions of the bronchiole surface epithelium (Fig. 1c) were illuminated alternately at 430 and 480 nm and fluorescence was measured at 530 nm (F 430 and F 480 ). Values of pH i were calculated from the F 480 / F 430 ratio after correction for the endogenous tissue fluorescence measured prior to loading with BCECF. Calibration data were obtained by the high K + -nigericin technique [36,48].

Reverse transcriptase-polymerase chain reaction
Messenger RNA expression of several ion transporters and channels was examined in isolated bronchioles and tracheal mucosa by polymerase chain reaction (PCR) ( Table 1). Primers were derived from published sequences with GenBank accession numbers. The PCR protocol was: 96 ℃, 25 s; 60 ℃, 30 s; 72 ℃, 40 s; 35 cycles. Templates for positive controls were complementary DNAs (cDNAs) prepared from lung, kidney, heart, colonic mucosa, brain and stomach mucosa. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers (452 bp) were used for the positive controls.

Statistics
Data are presented as the means ± SD unless otherwise indicated. Tests for statistically significant differences were made with Student's t-test.

Results
Isolated bronchioles from CF mice (ΔF/ΔF mice) were used in experiments shown in Fig. 8. Isolated bronchioles from wild-type mice were used in the other experiments (Figs. 1, 2, 3, 4, 5, 6, 7). basal pH i was 6.94 ± 0.03 (n = 64, mean ± SD). When isolated bronchioles were bilaterally perfused with the standard Hepes-buffered (HCO 3 − -CO 2 -free) solution (pH 7.4), basal pH i was 6.77 ± 0.03 (n = 40). Basal pH i in the presence of HCO 3 − -CO 2 was significantly (p < 0.01) higher compared to that in the absence of HCO 3 − -CO 2 . When NH 4 Cl (20 mM) was applied to the lumen in the presence of HCO 3 − -CO 2 , pH i showed typical time-course changes by NH 4 + pulse [40]. Addition of NH 4 Cl caused quick alkalinization (NH 3 influx) followed by slower decline and removal of NH 4 Cl caused quick acidification (NH 3 efflux) followed by slower recovery to the baseline (Fig. 1d). This suggests that H + /HCO 3 − transport is active in this preparation.

− -CO 2 removal on pH i in microperfused bronchioles
When isolated bronchioles were first bilaterally perfused with the standard HCO 3 − -buffered solution and the luminal perfusate was switched to the standard Hepes-buffered (HCO 3 − -CO 2 -free) solution ( Fig. 2a), pH i quickly increased from 6.94 ± 0.02 to 7.05 ± 0.02 (n = 8) and then gradually decreased towards a value (6.86 ± 0.03) lower than the baseline in 10 min. To distinguish between the separate effects of removal of CO 2 and HCO 3 − from the lumen, a solution was prepared which first contained 25 mM HCO 3 − but which was equilibrated with 100% O 2 (pH: ~ 7.8) and thus was nominally free of CO 2 . When the luminal perfusate was switched to the 25 mM HCO 3 − -0% CO 2 solution, pH i quickly increased to 7.21 ± 0.06 (n = 8) and the alkalinization was sustained (Fig. 2b). Thus, the transient alkalinization and the subsequent recovery (acidification) by removal of luminal HCO 3 − -CO 2 was most likely due to CO 2 diffusion of out of the cell followed by HCO 3 − efflux (Fig. 2a). Most of the HCO 3 − efflux was probably via the apical membrane due to the steep HCO 3 − gradient between the cell and the lumen (HCO 3 − concentration was close to zero). H + influx was not likely involved in the subsequent acidification because Na + -H + exchanger would work for H + extrusion in this condition.

Effects of luminal application of forskolin, CFTR inh -172, H 2 DIDS, and amiloride on apical HCO 3 − efflux in bronchiole epithelial cells
The mechanisms for HCO 3 − efflux across the apical membrane were examined using the protocol of Fig. 2a. After HCO 3 − -CO 2 was removed from the luminal perfusate, forskolin (5 μM), the activator of adenylate cyclase, was applied to the lumen as indicated (Fig. 3a). Stimulation with forskolin transiently accelerated the pH i decline by 97% (p < 0.01) (n = 8, Fig. 3a and f ) of control (without forskolin stimulation: blue line in Fig. 3a). The late phase of pH i decline (at midpoint pH i of 6.95) was also accelerated by 47% (p < 0.05) ( Fig. 3a and g) of control. The data suggest that elevation of intracellular cAMP activates HCO 3 − secretion in a biphasic manner: initial large response followed by sustained activation, in mice bronchiole epithelial cells.
CFTR inh -172 (5 μM) and H 2 DIDS (200 μM) in the lumen inhibited the forskolin-stimulated transient pH i decline by 69% (n = 8, p < 0.01) ( Fig. 3b and f ) and 65% (n = 8, p < 0.01) ( Fig. 3c and f ), respectively. Luminal CFTR inh -172 and H 2 DIDS also slowed down the late phase of pH i decline (at midpoint pH i of 6.95) by 54% (p < 0.01) ( Fig. 3b and g) and 33% (p < 0.05) ( Fig. 3c and g), respectively. The data suggest that both CFTR and H 2 DIDS-sensitive HCO 3 − transporter and/or HCO 3 − -permeable anion channel partly mediate cAMPstimulated HCO 3 − secretion. The forskolin-stimulated transient pH i decline in the presence of both CFTR inh -172 and H 2 DIDS ( Fig. 3d and f ) was significantly (p < 0.05) smaller compared to that in the presence of CFTR inh -172 or H 2 DIDS alone (Fig. 3b, c, and f ). The late-phase of pH i decline in the presence of both CFTR inh -172 and H 2 DIDS ( Fig. 3d and g) was significantly (p < 0.05) slower compared to that in the presence of H 2 DIDS alone ( Fig. 3c  and g).
Luminal application of CFTR inh -172 and H 2 DIDS by themselves induced a transient small dip of pH i (Fig. 3b  and c). The transient pH i dip largely disappeared when CFTR inh -172 and H 2 DIDS were simultaneously applied to the lumen (Fig. 3d). This suggests that CFTR and H 2 DIDS-sensitive HCO 3 − transporter/channel compensate each other for apical HCO 3 − efflux. We speculate on the mechanisms as follows. CFTR inhibition would hyperpolarize the cell, which would induce transient HCO 3 − efflux via a HCO 3 − -permeable anion channel or an electrogenic HCO 3 − transporter (such as 1Cl − -2HCO 3 − exchanger). If H 2 DIDS-sensitive HCO 3 -transport is electrogenic, luminal H 2 DIDS would hyperpolarize the cell, which would induce transient HCO 3 − efflux via CFTR. To examine the role of ENaC in HCO 3 − secretion, a relatively low concentration of amiloride (1 μM) [32] was applied to the lumen. Amiloride (1 μM) in the lumen inhibited the forskolin-stimulated transient pH i decline by 63% (n = 8, p < 0.01) (Fig. 3e and f ), but did not significantly affect the late phase of pH i decline ( Fig. 3e and g).
The data suggest that ENaC is involved in the regulation of HCO 3 − secretion. The transient dip of pH i by luminal amiloride (Fig. 3e)

likely indicates apical HCO 3
− efflux which is accelerated by membrane hyperpolarization.
When isolated bronchioles were bilaterally perfused with the standard HCO 3 − -buffered solution, application of CFTR inh -172 (5 μM) to the lumen caused a transient increase of pH i by 0.022 ± 0.003 unit (n = 8, data not shown). The pH i increase was not observed in the absence of HCO 3 − -CO 2 and enhanced by 77% (p < 0.05) by stimulation with forskolin (5 μM) (data not shown). The data suggest that CFTR is involved in HCO 3 − secretion in a physiological condition.

Effects of luminal Cl − removal on pH i in bronchiole epithelial cells
To examine the activity of Cl − -HCO 3 − exchange in the apical membrane, effects of luminal Cl − removal on pH i were examined. When isolated bronchioles were bilaterally perfused with the standard Hepes-buffered HCO 3 − -CO 2 -free solution, removal of luminal Cl − (by replacement with gluconate) caused a slight decline of pH i (Fig. 4a and e). In contrast, when isolated bronchioles were bilaterally perfused with the standard HCO 3 − -buffered solution, luminal Cl − removal caused a reversible increase of pH i by 0.14 ± 0.03 unit (n = 8) over ~ 4 min period (Fig. 4b and e), most likely due to influx of luminal HCO 3 − in exchange for intracellular Cl − . When the activity of apical Cl − -HCO 3 − exchange is shown as the initial rate of pH i increase upon luminal Cl − removal, the activity is not affected by forskolin (5 μM) in the lumen (Fig. 4c and e) and largely (p < 0.01) inhibited by H 2 DIDS (200 μM) in the lumen (Fig. 4d and e). The data suggest that H 2 DIDS-sensitive Cl − -HCO 3 − exchanger is localized in the apical membrane. lumen caused a recovery (Fig. 5a). When the activity of luminal Na + -dependent H + extrusion is shown as the initial pH i increase (ΔpH for 1 min) upon restoration of luminal Na + , the activity was completely (p < 0.01) inhibited by amiloride (100 μM) in the lumen (Fig. 5b and f ). The data suggest that NHE is localized in the apical membrane. The activity of luminal Na + -dependent H + extrusion in the presence of HCO 3 − -CO 2 ( Fig. 5c and f ) was significantly (p < 0.05) greater compared to that in the absence of HCO 3 − -CO 2 ( Fig. 5a and f ), partially inhibited by amiloride (100 μM) in the lumen (Fig. 5d and f ), and completely inhibited by a combination of amiloride (100 μM) and H 2 DIDS (200 μM) in the lumen (Fig. 5e  and f ). The data suggest that NBC is localized in the apical membrane.

Effects of luminal amiloride on pH i in bronchiole epithelial cells
While lower concentrations of amiloride inhibit ENaC with IC50 of 1 μM [32], higher concentrations of amiloride (0.5-1 mM) also inhibit apical NHE in human bronchial epithelium [49]. Figure 6 shows the effects of various concentrations of luminal amiloride (1, 10, and 100 μM) on basal pH i (Fig. 6). To examine the relative contribution of ENaC and apical NHE in H + /HCO 3 − transport in a physiological condition, we examined concentration-dependent effects of luminal amiloride rather than a more specific inhibitor of NHE such as ethylisopropyl amiloride (EIPA).
In the absence of HCO 3 − -CO 2 , luminal application of amiloride caused concentration-dependent decline of pH i (Fig. 6a, c, e, g).
In the presence of HCO 3 − -CO 2 , luminal application of 1 μM amiloride caused an increase in pH i by 0.03 ± 0.01 (n = 8, Fig. 6b and g). Luminal 100 μM amiloride caused a transient increase followed by a continuous decline in pH i by 0.06 ± 0.01 (n = 8) in 5 min ( Fig. 6f and g). Luminal 10 μM amiloride ( Fig. 5d and g) caused an intermediate pattern of pH i changes of those by 1 μM and 100 μM amiloride.
Thus, the effects of lower concentration of apical amiloride on basal pH i were dependent on the presence of HCO 3 − -CO 2 , which suggests that ENaC is involved in the regulation of HCO 3 − transport. The data also indicate that apical NHE is involved in the regulation of basal pH i .

Basal pH i and apical HCO 3 − efflux in bronchiole epithelial cells from CF mice
Basal pH i in the presence of HCO 3 − -CO 2 in isolated bronchioles from ΔF/ΔF mice (6.97 ± 0.02, n = 6) was slightly but significantly (p < 0.05) higher compared to bronchioles from wild-type mice (6.94 ± 0.02, n = 8, blue line) (Fig. 8a and d). Initial increase of pH i (ΔpH) by removal of luminal HCO 3 − -CO 2 was also significantly (p < 0.01) greater in ΔF/ΔF bronchioles compared to wild-type bronchioles (Fig. 8a and e). The data suggest that basal HCO 3 − secretion is impaired in CF bronchioles. The rate of pH i decline at midpoint pH i of 6.95 was significantly (p < 0.05) slower in CF bronchioles compared to wild-type bronchioles ( Fig. 8a and g).
Stimulation with luminal forskolin (5 μM) transiently accelerated pH i decline (apical HCO 3 − efflux) in ΔF/ ΔF bronchioles (Fig. 8b and f ) and the acceleration was comparable to wild-type bronchioles (blue line). Forskolin failed to accelerate the late phase of pH i decline (at midpoint pH i of 6.95) in ΔF/ΔF bronchioles ( Fig. 8b and  g). The data suggest that cAMP stimulation transiently activated HCO 3 − secretion in CF bronchioles probably  [16,21]. Application of ACh (10 μM) to the lumen transiently accelerated pH i decline (apical HCO 3 − efflux) in wild-type bronchioles (blue line in Fig. 8c) and the acceleration was greater than forskolin (Fig. 8f, p < 0.01). The ACh-induced transient acceleration of pH i decline was reduced by 45% (p < 0.01) in ΔF/ΔF bronchioles ( Fig. 8c and f ). Luminal ACh did not affect the late phase of pH i decline in both wild-type and ΔF/ ΔF bronchioles ( Fig. 8c and g). The data indicate that ACh stimulation transiently activated HCO 3 − secretion in wild-type bronchioles and that the ACh-induced , and brain (Br) and reverse transcribed. PCR was performed using each cDNA as template and with gene-specific primers (Table 1). GAPDH was used as a reference. enhancement of HCO 3 − secretion was substantially reduced in CF bronchioles. This suggests that CFTR partly mediates ACh-induced HCO 3 − secretion in addition to CaCC in mice bronchiole epithelial cells.

Discussion
In the present study, HCO 3 − transport in surface epithelial cells of native bronchioles was studied by measuring pH i in luminally microperfused freshly dissected mice -CO 2 in wild-type and ΔF/ΔF bronchioles. # p < 0.01. f Early-phase pH i decline (ΔpH for 1 min) just after stimulation with forskolin or acetylcholine. # p < 0.01 compared to wild-type. *p < 0.01, **p < 0.05 compared with control (without stimulation). & p < 0.01. g Late-phase pH i decline (ΔpH/min at midpoint pH i of 6.95) under stimulation with forskolin or ACh (red dashed lines in a-c). *p < 0.05 compared with control (without stimulation). # p < 0.05 compared to wild-type bronchioles. HCO 3 − transport in bronchioles from CF mice was also studied. Although some connective tissue was attached to the outside of bronchioles (Fig. 1), surface epithelial cells were successfully loaded with BCECF from the lumen and pH i was measured as long as 30 min. The present study focused on HCO 3 − /H + transport across the apical membrane, since rapid exchange of luminal solutions was achieved in our preparation [22].

Intracellular pH in surface epithelial cells of mice bronchioles
Human and rodent bronchioles are lined with columnar to cuboidal epithelium which is composed of ciliated and nonciliated (Clara) cells [35]. In the present study, basal pH i of surface epithelial cells in isolated mice bronchioles was ~ 6.94 in bilateral (bath and lumen) presence of 25 mM HCO 3 − and 5% CO 2 . The value is similar to the basal pH i of cultured human nasal epithelial cells (~ 6.94) in the same experimental condition [37]. The relatively low basal pH i likely resulted from higher pCO 2 in the lumen compared to the physiological in vivo situation where the luminal side of the epithelial layer is exposed to air.

Ion transporters and channels localized in the apical membrane of bronchiole epithelial cells
In the present study, functional studies suggested that CFTR and H 2 DIDS-sensitive HCO 3 − transporter and/or HCO 3 − -permeable anion channel mediate cAMP-stimulated HCO 3 − secretion and ENaC, H 2 DIDS-sensitive Cl − -HCO 3 − exchangers, NHE, and NBC are involved in HCO 3 − /H + transport across the apical membrane of surface epithelial cells of mice bronchioles (Fig. 9). This is supported by mRNA expression of Cftr, ENaC subunits, and Slc4, Slc9, and Slc26 families of transporters (Fig. 7).
The activity of H 2 DIDS-sensitive Cl − -HCO 3 − exchanger was detected in the apical membrane (Fig. 4) and probably mediated part of cAMP-stimulated HCO 3 − secretion (Fig. 3c). The candidate molecules are Slc4a2 (Ae2), Slc4a3 (Ae3), Slc26a4 (Pendrin), Slc26a6, and Slc26a9 of which mRNA expression was detected in isolated bronchioles (Fig. 7). In human bronchial epithelia, SLC26A4 (Pendrin) colocalized with CFTR in the apical membrane of ciliated surface cells and mediated most of HCO 3 − secretion when pretreated with IL-4 [24]. SLC26A9 is prominently expressed in brain and on apical membrane of airway epithelial cells and gastric mucosa [3,31]. A missense variant of SLC26A9 found in a patient of diffuse bronchiectasis failed to activate CFTR in a heterologous expression system [7]. While Slc26a4 (Pendrin) is H 2 DIDS-insensitive, Slc26a9 is sensitive to H 2 DIDS. Thus, Slc26a9 is likely the major apical Cl − -HCO 3 − exchanger in mice bronchioles.
Apical H + secretion via H + /K + ATPase and vacuolar H + -ATPase was reported in airways [52]. Our present study identified the activities of NHE and NBC in the apical membrane of mice bronchioles (Figs. 5, 6) which may contribute to the regulation of intracellular and ASL pH.

Mechanisms and regulation of HCO 3 − secretion in bronchiole epithelial cells
Surface airway epithelial cells as well as serous cells of the submucosal glands secrete Cl − and HCO 3 − in response to agents increasing intracellular cAMP (VIP, noradrenaline, etc.) and/or Ca 2+ (ACh, histamine, etc.) [41]. It is generally accepted that cAMP-mediated secretion involves CFTR and Ca 2+ -mediated secretion involves CaCC encoded by TMEM16A/ANO1 [15]. Cyclic AMP-and Ca 2+ -mediated agonists independently and additively increased HCO 3 − secretion in human bronchioles [45]. However, it has been noted that muscarinic responses of fluid secretion are reduced in submucosal glands from patients with cystic fibrosis [42] and a recent study demonstrated a crosstalk of CFTR and TMEM16A in CFBE cells [29].
We assume that continuous decline of pH i following alkaline load (Figs. 2, 3, 8) demonstrates time course of HCO 3 − secretion into the lumen which is perfused with the HCO 3 − -free solution. Forskolin biphasically stimulated HCO 3 − secretion: transiently accelerated HCO 3 − secretion just after application and increased the rate of steady-state HCO 3 − secretion (Fig. 3a). ACh transiently accelerated HCO 3 − secretion, but did not increase the steady-state HCO 3 − secretion (Fig. 8c). The data indicate that both cAMP-mediated and Ca 2+ -mediated pathways are involved in HCO 3 − secretion in mice bronchiole epithelial cells.
A relatively low concentration of amiloride in the lumen inhibited transient phase of forskolin-stimulated HCO 3 − secretion (Fig. 3e). The data suggest that ENaC is involved in the regulation of HCO 3 − transport, which is consistent with amiloride (1 μM)-induced pH i increase in the presence of HCO 3 − -CO 2 (Fig. 6). The cellular mechanisms for the involvement of ENaC in HCO 3 − secretion are not clear.

HCO 3 − secretion in CF bronchiole epithelial cells
ASL pH was more acidic in trachea of CF pigs under basal and methacholine-stimulated conditions [38]. Lower pH of ASL was also observed in nasal epithelium of CF patients [34,52], while the other study did not find differences in ASL pH of bronchus between CF patients and control [43]. Combination of forskolin and 3-isobutyl-1-methylxanthine alkalinized ASL of cultured bronchial epithelium of normal subjects but acidified CF ASL [12].
In the present study, HCO 3 − secretion was studied in bronchioles isolated from a CF mouse model in which the F508del mutation (most frequent pathogenic variant of CFTR) was introduced (ΔF mouse) (Fig. 8). Although CF mice do not display severe lung disease as observed in humans, an impaired ability to stretch/expand the peripheral lung compartment and increased distances between gas exchange surfaces which are early pulmonary phenotype of human CF were found in young (8-16 weeks old) ΔF/ΔF mice [14]. Our present study demonstrated higher level of basal pH i in the presence of HCO 3 − -CO 2 and larger increase of pH i by removal of luminal HCO 3 − -CO 2 in CF bronchioles (Fig. 8), which indicate that basal HCO 3 − secretion is reduced in CF distal airways.
The effects of forskolin and ACh on HCO 3 − secretion in CF bronchioles (Fig. 8) were unexpected. While forskolin stimulation transiently accelerated HCO 3 − secretion in CF bronchioles (comparable to wild-type bronchioles, Fig. 8b), ACh-induced acceleration of HCO 3 − secretion was substantially reduced in CF bronchioles (Fig. 8c). The data are consistent with the presence of a crosstalk of cAMP-and Ca 2+ -mediated pathways of HCO 3 − secretion. The data also suggest that a cAMP-activated HCO 3 − -permeable anion channel or HCO 3 − transporter was upregulated in CF bronchioles.
The present study has some limitations. (1) The intracellular buffering capacity is not measured and the rate of H + /HCO 3 − flux is not inferred from changes in pH i . (2) Information of membrane potential is not available and the electrochemical potential gradient for HCO 3 − across the apical membrane is not accurately predicted.
(3) RT-PCR of isolated bronchioles does not identify