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Am J Physiol Lung Cell Mol Physiol 284: L855-L862, 2003. First published January 10, 2003; doi:10.1152/ajplung.00348.2002
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Vol. 284, Issue 5, L855-L862, May 2003

Secretion of acid and base equivalents by intact distal airways

S. K. Inglis, S. M. Wilson, and R. E. Olver

Tayside Institute of Child Health, University of Dundee, Dundee DD1 9SY, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 ± 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when pHLBS was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 µM) but was inhibited by 100 nM bafilomycin A1 (by 76 ± 13%), suggesting involvement of vacuolar-H+ ATPase. Addition of ACh (10 µM) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 µM), an inhibitor of Cl- secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 µM) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-. Isolated distal airways thus secrete both acid and base equivalents.

airway epithelium; bicarbonate transport; pH


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE DEPTH AND COMPOSITION of the airway surface liquid (ASL), which lines the airway epithelium, must be strictly controlled to allow the processes that keep the airways clean and free from infection to function normally. For instance, variations in the pHASL can affect ciliary beat frequency (5, 26), mucus rheology (10, 23, 31), airway smooth muscle tone (28), and the integrity of the epithelium itself (11). However, the mechanisms by which pHASL is controlled are not well understood. It is certainly clear that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is secreted by airway submucosal glands as an essential component of gland liquid secretion (4, 15, 16, 21, 22), but, despite this secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the ASL is relatively acidic (6, 8, 19, 20, 25). It is therefore possible that the ASL is acidified, either by surface epithelium or by proximal regions of the gland ducts.

That abnormal ASL pH may be important in disease is suggested by findings that expired breath condensate is markedly more acidic than normal in patients with inflammatory diseases such as asthma, bronchiectasis, and chronic obstructive pulmonary disease (13, 24). This acidosis in asthmatic patients can be normalized following anti-inflammatory therapy (13, 24). Although acidic breath condensate thus seems to be a characteristic of some inflammatory lung diseases, these studies should be interpreted with caution since the mechanisms that underlie breath condensate are not fully understood (12) and a correlation between breath condensate pH and pHASL has not been proven. A preliminary study suggests that nasal ASL is also acidic in cystic fibrosis (CF) (7), and this accords with the finding that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is impaired in CF airway epithelium (29). The possible role of acidic ASL in the pathophysiology of these inflammatory lung diseases is yet to be determined.

The aim of the current study was to determine the mechanisms by which airway epithelia can modulate the pHlumen. Importantly, intact distal bronchi isolated from porcine lungs were used since these airways possess both glandular and surface epithelia, both of which potentially contribute to regulation of pHlumen.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Solution composition and drugs. Modified Krebs-Ringer bicarbonate (KRB) solution contained (in mM) 112 NaCl, 4.7 KCl, 2.5 CaCl2, 2.4 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.6 D-glucose. Solution pH was maintained at 7.4 by continuous gassing with 5% CO2 in O2. Lightly buffered solution (LBS) contained (in mM) 137 NaCl, 4.7 KCl, 2.5 CaCl2, 2.4 MgSO4, 1.2 KH2PO4, and 11.6 D-glucose. Sufficient NaOH was added to bring pH to approximately pH 7. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution was similar to KRB, but HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was replaced with equimolar HEPES, pH was adjusted to 7.4 with HCl, and the solution was gassed with 100% O2. All drugs were added from freshly prepared stock solutions in DMSO, such that the final concentration of DMSO was no more than 0.001%, except ACh, which was added from a freshly prepared aqueous stock. All drugs were obtained from Sigma Chemical.

Isolation of distal bronchi. Cotswold pigs (15 kg) were obtained from a local supplier, sedated with inhaled halothane, and killed with an intravenous overdose of pentobarbital sodium in accordance with UK and institutional regulations. Apical and middle lobes of the right and left lungs were excised and placed in KRB solution. Distal bronchi were carefully dissected free from the surrounding parenchyma, and side branches were tightly ligated with suture.

Measurement of bioelectrical properties. Bronchial bioelectrical properties were measured as described previously (18). Isolated bronchi {length 1.82 ± 0.04 cm, outside diameter 0.35 ± 0.01 cm, average luminal surface area 1.36 cm2 [where surface area = length · external diameter · 0.682 · pi  (32), n = 86]} were placed in a bath of KRB and slowly warmed from room temperature to 37°C. Bronchi were then tied onto two polyethylene cannulas. The tip of each cannula was precoated with partially cured Sylgard-184 to establish an electrical seal between the airway epithelium and cannula. Once cannulated, the lumina of the bronchi were perfused continuously with KRB solution. Perfusion was driven by a peristaltic pump (Masterflex L/S).

To measure transepithelial potential difference (PD), we placed a glass microelectrode filled with 3.6% agar-3 M KCl within the lumen of the cannula supporting the proximal end of the bronchus and coupled to a high impedance electrometer (WPI model 705). The reference bridge (polyethylene tubing filled with 3.6% agar-3 M KCl) was placed in the KRB bathing solution and connected to the electrometer via a calomel electrode. Electrometer voltage output was continuously recorded both on a chart recorder and directly to computer disk using a Powerlab computer interface (AD Instruments, Hastings, UK).

Measurement of luminal pH. Once PD had stabilized (20-30 min), the luminal perfusion was switched from KRB solution to LBS (pH ~7). Initially, the LBS perfusate was allowed to run to waste for ~3 min to remove any residual KRB from the airway. Thereafter the LBS perfusate was returned to the reservoir of LBS (total volume 10 ml), which was continually stirred with a magnetic stirrer and gassed with 100% O2 to displace dissolved CO2. This 10-ml aliquot of LBS was thus continually recirculated (3 ml/min) through the airway lumen, and its pH was continuously recorded to computer disk using a pH electrode (Thermo Russell, Auchtermuchty, Fife, UK) placed in the reservoir and connected to a pH Pod and Powerlab interface (AD Instruments). The relatively rapid perfusion rate was chosen to ensure rapid mixing of perfusing LBS with the bulk of the aliquot. In initial experiments, pH was recorded with a conventional pH meter (model RL150, Thermo Russell) and noted manually every 30 s.

Determination of LBS buffering capacity and calculation of rates of acid/alkali secretion. Measured aliquots of NaOH (1 M) were added to a known volume of LBS, and the evoked changes in pH were used to calculate the buffering capacity (beta LBS)
&bgr;<SUB>LBS</SUB> = <FR><NU>change in [H<SUP>+</SUP>] (mM)</NU><DE>change in pH</DE></FR>
The empirically determined beta LBS varied with pHLBS (Fig. 1), and this relationship was used to convert changes in pHLBS recorded during an experiment into the rate at which acid or base equivalents was secreted into the luminal LBS. Specifically, we calculated the mean rates of acid (see Fig. 3C) secretion by multiplying the mean rate of change of pHLBS during the initial 5 min of circulation of LBS by the beta LBS at the midpoint of that 5-min period. We calculated the rates of base secretion (Figs. 3D and 5A) in a similar fashion using the mean rate of change of pHLBS 10 min before and 10 min following ACh addition. We constructed the time course showing the rate of base equivalent secretion in response to ACh (Fig. 4B) by multiplying the rate of change in pHLBS during consecutive 3-min time periods by the beta LBS at the midpoint of the pHLBS range measured during that time period. This was divided by three to give the base equivalent secretion per min. beta LBS containing either bafilomycin A1 (100 nM) or DMA (100 µM) were also determined (Fig. 1, open circle  and down-triangle, respectively). To ensure that perfusion of the LBS through the airway lumen had no effect on its buffering capacity, we added known volumes of 1 M NaOH to LBS circulating through cannulated bronchi. We determined buffering capacities of the circulating LBS from the evoked changes in pH using the relationship described above, and their dependence on pHLBS is shown in Fig. 1.


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  		Fig. 1.   Effect of pH on buffering capacity. Aliquots of NaOH were added to a measured volume of lightly buffered solution (LBS), and the evoked change in pH was recorded. Buffering capacity was calculated as described in METHODS. Data points were obtained from 7 different batches of LBS over a period of 6 mo and are fitted to a Gaussian curve. Buffering capacities of LBS circulating through cannulated bronchi () were also calculated from experiments in which aliquots of NaOH were added to circulating LBS, and the resultant, immediate alkalinization was recorded. Buffering capacities of LBS containing dimethylamiloride (DMA, 100 µM, down-triangle) and bafilomycin (10 nM, open circle ) are also shown.

Statistics. Experiments followed a strictly paired protocol so that each test bronchus was paired to a control tissue isolated from the same lung. Paired Student's t-test was therefore used to compare responses of control and test bronchi. Data were judged to be significantly different if P < 0.05. Data are reported as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Resting PD was -4.44 ± 0.20 mV (n = 68). This is in good agreement with previous electrometric studies of these tissues (14, 18).

Effect of the unstimulated bronchus on pHlumen. Normally, the pHLBS was adjusted to approximately pH 7 with NaOH before the experiment. When this LBS (mean pH 6.99 ± 0.05, n = 50) was circulated through the bronchial lumen, it was acidified (by 0.065 ± 0.010 pH units) to reach an average pH of 6.93 ± 0.04 in ~10 min (n = 50) (Fig. 2A). The mean initial rate of secretion of acid equivalents, the product of the rate of fall in pHlumen during the first 5 min of acidification, and the beta LBS (see METHODS), was 1.68 ± 0.14 µmol/h (n = 50) at initial pHLBS of 6.99 ± 0.05. This initial acidification was accompanied by a significant depolarization of transepithelial PD (0.47 ± 0.11 mV, n = 22). The pHLBS then remained relatively stable but did have a tendency to drift upward, presumably as a result of the driving force for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> movement into the lumen. To ensure that the changes in pHLBS originated from the cannulated bronchi, we circulated LBS through the perfusion system in the absence of an airway; a short piece of polyethylene tubing was pulled onto the cannulas to complete the perfusion circuit. Circulation of LBS in the absence of a bronchus had no effect on pHLBS (Fig. 2B). In three separate experiments the mean pHLBS for 5 min before circulation through the system (6.993 ± 0.028) was not different from the mean pHLBS for 5 min after the onset of circulation (6.991 ± 0.029).


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  		Fig. 2.   Effect of starting pHLBS on the magnitude of acidification that occurs when LBS circulates through the airway lumen. A: representative experiments showing change in pHLBS occurring once LBS circulates through airway lumen. B: effect on pHLBS of circulating LBS through the system when airway is not present (note different axes scaling). C: data are the results of similar experiments to those shown in A and show the effect of initial pHLBS on the total fall in pHLBS on circulation through the airway lumen. , Initial rates of acid secretion that occur when LBS is circulated through the airway lumen (see A); triangle , rates induced by stepwise additions of aliquots of NaOH.

To test whether the magnitude of acidification was dependent on starting pHLBS, we varied the initial pHLBS by altering the quantity of NaOH added (Fig. 2). As initial pHLBS increased, the magnitude of acidification also increased (Fig. 2, A and C). However, despite this greater acidification, the pHLBS did not fall to as low a level as in experiments with approximately neutral starting pHLBS (Fig. 2A).

To investigate further the effect of pHLBS on acidification, in a separate series of experiments we added an aliquot of NaOH to the circulating LBS to increase pHLBS, and the evoked change in pHLBS was noted. Once pH had reached its new, stable level, another aliquot of NaOH was added to further increase pHLBS, and the evoked change in pHLBS was again noted. Up to twelve consecutive aliquots of NaOH were added. Once again, a higher pHLBS evoked a greater fall in pH (Fig. 2C).

Under resting conditions, the bronchi thus secrete acid equivalents into the lumen. To investigate the mechanism underlying this acidification, we added DMA (100 µM), an Na+/H+ exchanger inhibitor, to the circulating LBS. Figure 3 shows that this had no effect on either the magnitude of acidification or the rate of acid equivalent secretion. However, inclusion of bafilomycin A1 (100 nM), an inhibitor of vacuolar (v)-H+ ATPase, in the LBS significantly inhibited both the magnitude and rate of acidification (Fig. 3, A-C). To investigate the reversibility of the inhibitory effect, we first perfused bronchi with LBS containing bafilomycin A1 and recorded the acidification. The bronchi were then perfused with KRB for 20 min to wash out the bafilomycin A1 before being perfused with a second aliquot of LBS that did not contain bafilomycin A1. The second acidification in the absence of bafilomycin (0.065 ± 0.009 pH units) was significantly greater than the acidification in the presence of bafilomycin (0.031 ± 0.004 pH units, n = 4, P < 0.05), demonstrating that the effect of bafilomycin was reversible (Fig. 3D). In paired, control bronchi, the acidification levels of two consecutive aliquots of LBS that did not contain bafilomycin A1, separated by a 20-min perfusion with KRB, were not significantly different (acidification of first aliquot 0.057 ± 0.005 pH units, second aliquot 0.059 ± 0.015 pH units, P > 0.05). Bafilomycin A1 also significantly inhibited the depolarization of transepithelial PD associated with the initial acidification (control depolarization 0.46 ± 0.13 mV, depolarization after bafilomycin A1 treatment 0.27 ± 0.10 mV, n = 5, P < 0.05). beta LBS was unaffected by the presence of either DMA or bafilomycin A1 (Fig. 1).


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  		Fig. 3.   Effect of luminal DMA (100 µM) and bafilomycin A1 (100 nM) on the initial acidification of luminal LBS. A: representative experiments showing change in pH with time. B: mean (± SE) fall in pH following circulation of LBS through the lumen. Experiments were carried out on paired bronchi isolated from the same animal. One control bronchus was untreated; the other was treated with either DMA (n = 5) or bafilomycin (n = 5). C: mean (± SE) rate of secretion of acid equivalents during initial 5 min of LBS perfusion calculated using appropriate buffering capacities (see Fig. 1 and METHODS). D: typical experiment showing bafilomycin (Baf) reversibility. LBS containing bafilomycin was circulated through the bronchial lumen, and subsequent acidification was recorded. Krebs-Ringer bicarbonate (KRB) containing no bafilomycin was then washed through the lumen for 20 min. Finally, a second aliquot of LBS not containing bafilomycin was circulated through the lumen, and the resultant acidification was recorded. E: mean (± SE) rate of change of luminal pH in 10 min before (open bars) and 10 min following (solid bars) ACh (10 µM) addition in paired bronchi either untreated (control) or treated with bafilomycin (n = 4). * Significant difference from control (paired Student's t-test).

Effect of ACh on pHlumen. Once pHLBS had stabilized, the gland secretagogue ACh (10 µM) was added to the bathing solution. This evoked an alkalinization of luminal LBS (rate of increase in pHlumen from 0.0009 ± 0.0002 to 0.0026 ± 0.0002 pH units/min, n = 37, P < 0.05, Fig. 4A) and increased the rate of secretion of base equivalents into the luminal solution (from a mean of 0.20 ± 0.06 µmol/h for the 10 min before ACh addition to a peak of 1.16 ± 0.11 µmol/h and mean for 10 min after ACh addition of 0.61 ± 0.08 µmol/h, n = 12, P < 0.05, Fig. 4B). This was accompanied by a hyperpolarization of transepithelial PD (1.64 ± 0.11 mV, n = 37, Fig. 4C).


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  		Fig. 4.   Effect of ACh on luminal pH and transepithelial potential difference (PD). A: representative experiment showing change in pHlumen in response to basolateral ACh (10 µM). B: data from experiment in A converted to rate of base secretion as described in METHODS. C: effect of ACh on transepithelial PD measured concurrently with the pHlumen data shown in A.

To investigate the mechanisms involved in ACh-induced base equivalent secretion, we bathed bronchi in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free (HEPES-buffered) solution containing acetazolamide (1 mM) for ~1.5 h before the experiment. When LBS was subsequently circulated through the lumen, it initially acidified. However, the alkalinization response to ACh was abolished in the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution containing acetazolamide (Fig. 5A), consistent with previous results showing that ACh stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in these tissues (18). The effect of ACh on PD was maintained in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution and was not significantly different from the effect on control bronchi (Fig. 5B).


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  		Fig. 5.   Effect of various drugs on response to ACh. A: mean rate of change of luminal pH in 10 min before (open bars) and 10 min following (solid bars) ACh (10 µM) addition. Experiments were carried out on paired bronchi isolated from the same animal. One control bronchus was treated solely with ACh; the other was pretreated with either HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution plus acetazolamide (ace, 1 mM, n = 4), DMA (100 µM, n = 8), bumetanide (Bumet, 100 µM, n = 6), DMA + Bumet (n = 7), or 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 300 µM, n = 7). B: mean hyperpolarization evoked by ACh under each of the conditions detailed in A. Data are means ± SE. * Significant difference from pre-ACh; #Significant difference from control (Student's paired t-test, P < 0.05).

Previous studies of these bronchi have shown that ACh stimulates the submucosal glands to secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and that this is blocked by DMA (15, 32). We therefore investigated the effect of DMA on ACh-evoked alkalinization. Pretreatment with DMA (100 µM) had no effect on the initial fall in pHlumen (control 0.051 ± 0.010, DMA treated 0.055 ± 0.012 pH units, n = 3) but blocked the ACh-induced alkalinization (Fig. 5A). The hyperpolarization was maintained in the presence of DMA.

The maintenance of the hyperpolarization response in either the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or presence of DMA is consistent with an earlier study in which we suggested that ACh stimulates secretion of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (18). We therefore investigated the effect of bumetanide (100 µM), an inhibitor of Na+/K+/2Cl- cotransport and hence of Cl- secretion in these tissues. Pretreatment with bumetanide significantly increased the rate of alkalinization both before addition of ACh and in the presence of ACh (n = 6, P < 0.05, Fig. 5). The hyperpolarization evoked by ACh was unaffected by bumetanide (Fig. 5B). Inhibiting Cl- secretion thus significantly increases the rate of base equivalent secretion.

To inhibit both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, we pretreated bronchi with both DMA and bumetanide. This had no effect on the initial acidification (fall in pHlumen in control bronchi 0.053 ± 0.007, in DMA plus bumetanide-treated bronchi 0.042 ± 0.003 pH units, n = 7, P < 0.05) but blocked both the alkalinization and hyperpolarization responses to ACh (Fig. 5, A and B). To further investigate the effect of inhibiting Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, we pretreated bronchi with the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 300 µM), previously shown to block the liquid secretion response to ACh in these tissues (4). NPPB pretreatment significantly increased the initial acidification of LBS (fall in pHlumen in control bronchi 0.044 ± 0.005, in NPPB-treated bronchi 0.072 ± 0.012 pH units, n = 7, P < 0.05). The subsequent pH and bioelectric responses to ACh were almost completely abolished (Fig. 5, A and B).

Bafilomycin A1, which inhibits the initial acidification of luminal LBS (Fig. 3), had no effect on the subsequent response to ACh (increase in the rate of alkalinization in controls 0.0015 ± 0.0002; after bafilomycin A1 treatment 0.0018 ± 0.0004 pH units/min, increase in PD in controls 1.99 ± 0.32; after bafilomycin A1 2.37 ± 0.68 mV, n = 4, Fig. 3E).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, together with Cl- secretion, drives liquid secretion from submucosal glands (4, 15, 16, 21). One may therefore predict that the pH of ASL is likely to be relatively alkaline, particularly during periods of glandular secretion. For example, using a value for [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] of 27.6 mM measured in glandular secretions from distal bronchi (32) and normal PCO2 in the distal airways of 40 mmHg, we would predict pH of 7.47. However, numerous measurements of ASL pH both in vitro, for example in native ferret trachea (pH 6.85; Ref. 24) and human primary airway cell cultures (pH 6.44 and 6.81; Refs. 6 and 19, respectively), and in vivo on human nasal epithelium (8, 19) have shown that ASL is acidic relative to plasma. The aim of the present study, therefore, was to determine whether freshly isolated distal bronchi, which contain both submucosal glandular and surface epithelia, secrete both acid and alkali equivalents to control luminal pH.

Secretion of acid equivalents. LBS acidified when it was circulated through the lumen of an isolated, cannulated bronchus, indicating that the airway was secreting acid equivalents into the lumen. The magnitude of this acidification was dependent on the starting pH; a higher starting pHLBS evoked a greater acidification. However, this greater acidification was not sufficient to lower pHLBS to the same level as that reached following acidification of LBS with lower initial pH. The resultant, minimum stable pH reached following acidification was therefore not constant but varied with starting pH.

Acidification of luminal pH has been described in studies of both native (2) and cultured (9, 30) airway epithelium. In native sheep trachea, this acid secretion was reduced by 5-(N-ethyl-N-isopropyl)amiloride, implying a role for Na+/H+ exchange (2). However, the lack of effect of DMA in our current study excludes a significant role for this transporter in acidifying the lumen in distal bronchi. Indeed, the acidification in our study seems to be mediated almost entirely by bafilomycin A1-sensitive v-H+ ATPase. Further studies by Acevedo and Newton (1) indicate that v-H+ ATPase might play a modest role in the luminal acidification in native trachea. Interestingly, this transporter does not appear to be functional in the ciliated cells isolated from the surface epithelium (27), suggesting that v-H+ ATPase is expressed in a subset of nonciliated cells either in the surface epithelium or perhaps in the proximal collecting or ciliated ducts of the submucosal glands.

In contrast, in a study of cultured airway surface epithelia, Fischer et al. (9) found no evidence for involvement of either v-H+ ATPase or Na+/H+ exchange in luminal acidification. Instead, it seemed to be mediated by a zinc-sensitive H+ conductance. As there is normally no driving force for efflux of acid equivalents from cells, they suggest that there is a source of acid equivalents close to the apical membrane that drives proton secretion through this conductance. However, there is no evidence at present that such an acidic region exists within airway epithelial cells. The reason for this apparent difference in the mechanism for acidification is not clear but may result from the different cell types used in the two studies. Fischer et al. (9) used human airway epithelial cells both in primary culture and a cell line. It is possible that these cultures did not contain the cell types responsible for the bafilomycin-sensitive acidification measured in our study. Alternatively, the mechanism for acidification may be species specific. Indeed, Fischer et al. refer in their discussion to unpublished studies showing bafilomycin-sensitive acidification in bovine trachea. Our study is thus consistent with bafilomycin A1-sensitive v-H+ ATPase being primarily involved in acid secretion in distal airways.

Secretion of base equivalents. Addition of ACh to the bathing solution evoked both an alkalinization of pHlumen and a hyperpolarization of transepithelial PD, consistent with secretion of base equivalents. We previously used bioelectric techniques (18) and videomicroscopy (17) to show that ACh evoked both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion across isolated, perfused distal bronchi and stimulated liquid and particulate secretion from the submucosal glands.

The alkalinization response to ACh was blocked in bronchi bathed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free buffer containing acetazolamide to inhibit carbonic anhydrase, consistent with the secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The average rate of ACh-evoked base equivalent secretion was 0.28 µmol · cm-2 · h-1. This is in excellent agreement with the rate of 0.38 µmol · cm-2 · h-1 calculated from measurements of the [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in liquid secreted in response to ACh in distal bronchi (32). Together, these results strongly suggest that the alkalinization response evoked by ACh results from secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

This HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion response was also blocked by inhibiting Na+/H+ exchange with DMA, confirming earlier studies that the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion was almost entirely dependent on activity of this exchanger (15, 32). The mechanism for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in these airways is most consistent with the model proposed by Smith and Welsh (29) for airway HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. According to this model, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is generated within the cell from CO2 and H2O, in a reaction catalyzed by carbonic anhydrase. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> leaves the cell through an apical anion conductance, likely to be the cystic fibrosis transmembrane conductance regulator (CFTR) (4), whereas the H+ also generated in this reaction leaves the cell via Na+/H+ exchange. Inhibition of this exchanger therefore effectively blocks HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. As an extension to this model, Smith and Welsh proposed that inhibition of Cl- secretion by bumetanide, which inhibits basolateral Na+/K+/2Cl-, would enhance the rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, whereas inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion would enhance Cl- secretion (29). Our bioelectric study was consistent with this model, since ACh increased ion transport by a similar magnitude in control, bumetanide-pretreated and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> blocker-treated bronchi. The response was abolished, however, when both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretions were blocked (18). This interdependence of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion makes it difficult to study HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion by bioelectrical methods; inhibition of Cl- secretion to enable HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion to be measured actually enhances HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. The methodology in the current study, however, provides a means for study of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion while Cl- secretion is intact. In addition we were able to demonstrate that inhibition of Cl- secretion with bumetanide did indeed increase the rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion both at rest and following ACh as predicted. The hyperpolarization response to ACh was maintained under these conditions, presumably as a result of this increased rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion.

The model proposed by Smith and Welsh (29) predicts that both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit the cell through an apical anion conductance, and previous studies have shown that inhibitors of CFTR certainly block ACh-evoked liquid secretion in distal bronchi (4). The current study shows that NPPB blocks ACh-evoked HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and hyperpolarization, consistent with inhibition of both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- secretion. The combination of DMA and bumetanide similarly blocks both the alkalinization and hyperpolarization responses to ACh. This is in contrast to their independent inhibitory effects on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- secretion, respectively, that do not block ACh-evoked hyperpolarization. Our results are thus consistent with the Smith and Welsh model for airway epithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (29).

In addition to its effects on ACh-evoked HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, NPPB also significantly enhanced the initial acidification. The most likely explanation for this is that although the net initial change in pHlumen is an acidification, a low rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion occurs even in the absence of ACh; once this is blocked by NPPB, a larger initial acidification is revealed.

Secretion of acid and alkali equivalents by the airways. Our findings that ACh stimulates alkalinization of the airway lumen are in contrast to a recent study that measured secretion of individual porcine submucosal glands by monitoring the appearance of droplets of fluid at the points on the tracheal surface where submucosal gland ducts open onto the surface (22). Although this study confirmed that cholinergically induced gland liquid secretion is driven by secretion of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, it found that the secretions were slightly acidic relative to the bath and that the pH of the secretions were the same under both basal and carbachol-stimulated conditions. This is surprising given that we clearly measure luminal alkalinization in response to ACh and that the [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] of secretions collected from the lumen of bronchi exposed to ACh are ~27 mM (32). The authors postulated that anions and liquid are secreted in the glandular acini and are then acidified as they pass through the proximal gland ducts before they reach the airway surface (22). The finding that [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in ACh-evoked airway liquid is higher when secreted volumes are large than in small secreted volumes (3) is consistent with this hypothesis that gland liquid is acidified, either within the proximal gland ducts or by surface epithelium, since secretion of acid equivalents or absorption of base equivalents would have more effect on [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in small volumes. Our study certainly confirms that airways are capable of secreting both acid and base equivalents, and it is possible that acidification of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-rich glandular secretions occurs within proximal regions of the gland duct. However, we clearly detect net alkalinization of airway luminal fluid in response to ACh. Our results are thus more consistent with the hypothesis that acidification occurs on the airway surface and that the secreted fluid monitored by Joo et al. (21) is exposed to sufficient surface epithelium to enable significant acidification to take place. Because the bronchi in our studies were continually perfused, the luminal fluid may not have been in contact with the surface epithelium long enough for acidification to take place. Clearly, the balance between secretion of acid and alkali equivalents is extremely important to enable regulation of pHASL. Further studies are needed to determine the mechanisms underlying this regulation and the potential role for abnormal pHASL in pathophysiology of inflammatory lung diseases.


    ACKNOWLEDGEMENTS

The authors thank Dr. Steve Ballard for helpful comments and Maree Constable for excellent technical help.


    FOOTNOTES

This work was supported by a Wellcome Trust Research Career Development Fellowship (S. K. Inglis).

Address for reprint requests and other correspondence: S. K. Inglis, Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, Univ. of Dundee, Dundee DD1 9SY, UK (E-mail: s.k.inglis{at}dundee.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 10, 2003;10.1152/ajplung.00348.2002

Received 18 October 2002; accepted in final form 26 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Acevedo, M, and Newton IP. A proton pump contributes to acid secretion by sheep tracheal epithelium (Abstract). J Physiol 482: 27P, 1995.

2.   Acevedo, M, and Steele LW. Na+-H+ exchanger in isolated epithelial tracheal cells from sheep. Involvement in tracheal proton secretion. Exp Physiol 78: 383-394, 1993[Abstract].

3.   Ballard, ST, and Trout L. Bicarbonate concentration in ACh-induced airway liquid is dependent upon the volume but not rate of secretion (Abstract). FASEB J 16: A819, 2001.

4.   Ballard, ST, Trout L, Bebok Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 277: L694-L699, 1999[Abstract/Free Full Text].

5.   Clary-Meinesz, C, Mouroux J, Cosson J, Huitorel P, and Blaive B. Influence of pH on ciliary beat frequency in human bronchi and bronchioles. Eur Respir J 11: 330-333, 1998[Abstract].

6.   Coakley, RD, Paradiso AM, Grubb BR, Gatzy JT, Chadburn JL, and Boucher RC. Abnormal airway surface liquid pH regulation in cultured CF bronchial epithelium. Pediatr Pulmonol Suppl 20: 194, 2000.

7.   Davies, MG, Davies JC, Geddes DM, and Alton EW. Nasal epithelial pH is lower in patients with cystic fibrosis. Pediatr Pulmonol Suppl 20: 194, 2000.

8.   England, RJA, Homer JJ, Knight LC, and Ell SR. Nasal pH measurement: a reliable and repeatable parameter. Clin Otolaryngol 24: 67-68, 1999[ISI][Medline].

9.   Fischer, H, Widdicombe JH, and Illek B. Acid secretion and proton conductance in human airway epithelium. Am J Physiol Cell Physiol 282: C736-C743, 2002[Abstract/Free Full Text].

10.   Holma, B. Influence of buffer capacity and pH-dependent rheological properties of respiratory mucus on health effects due to acidic pollution. Sci Total Environ 41: 101-123, 1985[Medline].

11.   Holma, B, Lindegren M, and Anderson JM. pH effects on ciliomotility and morphology of respiratory mucosa. Arch Environ Health 32: 216-226, 1977[ISI][Medline].

12.   Hunt, J. Exhaled breath condensate: an evolving tool for noninvasive evaluation of lung disease. J Allergy Clin Immunol 110: 28-34, 2002[ISI][Medline].

13.   Hunt, JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TAE, and Gaston B. Endogenous airway acidification. Implications for asthma pathophysiology. Am J Respir Crit Care Med 161: 694-699, 2000[Abstract/Free Full Text].

14.   Inglis, SK, Collett A, McAlroy HL, Wilson SM, and Olver RE. Effect of luminal nucleotides on Cl- secretion and Na+ absorption in distal bronchi. Pflügers Arch 438: 621-627, 1999[ISI][Medline].

15.   Inglis, SK, Corboz MR, and Ballard ST. Effect of anion secretion inhibitors on mucin content of airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 274: L762-L766, 1998[Abstract/Free Full Text].

16.   Inglis, SK, Corboz MR, Taylor AE, and Ballard ST. Effect of anion transport inhibition on mucus secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 272: L372-L377, 1997[Abstract/Free Full Text].

17.   Inglis, SK, Corboz MR, Taylor AE, and Ballard ST. In situ visualisation of bronchial submucosal glands and their secretory response to acetylcholine. Am J Physiol Lung Cell Mol Physiol 272: L203-L210, 1997[Abstract/Free Full Text].

18.   Inglis, SK, Corboz MR, Taylor AE, and Ballard ST. Regulation of ion transport across porcine distal bronchi. Am J Physiol Lung Cell Mol Physiol 270: L289-L297, 1996[Abstract/Free Full Text].

19.   Ireson, NJ, Tait JS, MacGregor GA, and Baker EH. Comparison of nasal pH values in black and white individuals with normal and high blood pressure. Clin Sci (Lond) 100: 327-333, 2001[Medline].

20.   Jayaraman, S, Song Y, and Verkman AS. Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea. Am J Physiol Cell Physiol 281: C1508-C1511, 2001.

21.   Joo, N, Wu JV, Krouse ME, Saenz Y, and Wine JJ. Optical method for quantifying rates of mucus secretion from single submucosal glands. Am J Physiol Lung Cell Mol Physiol 281: L458-L468, 2001[Abstract/Free Full Text].

22.   Joo, NS, Saenz Y, Krouse ME, and Wine JJ. Mucus secretion from single submucosal glands of pig: stimulation by carbachol and vasoactive intestinal peptide. J Biol Chem 277: 28167-28175, 2002[Abstract/Free Full Text].

23.   King, M. Viscoelastic properties of airway mucus. Fed Proc 39: 3080-3085, 1980[ISI][Medline].

24.   Kostikas, K, Paptheodorou G, Ganas K, Psathakis K, Panagou P, and Loukides S. pH in expired breath condensate of patients with inflammatory airway diseases. Am J Respir Crit Care Med 165: 1364-1370, 2002[Abstract/Free Full Text].

25.   Kyle, H, Ward JP, and Widdicombe JG. Control of pH of airway surface liquid of the ferret trachea in vitro. J Appl Physiol 68: 135-140, 1990[Abstract/Free Full Text].

26.   Luk, CK, and Dulfano MJ. Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants. Clin Sci (Lond) 64: 449-451, 1983[Medline].

27.   Maddox, ADM, and Acevedo M. In the absence of bicarbonate and CO2, Na+-H+ antiport mediates all pH recovery from acidification in ciliated cells isolated from sheep trachea (Abstract). J Physiol 489: 103P, 1995.

28.   Ricciardolo, FLM, Rado V, Fabbri LM, Sterk PJ, Di Maria GU, and Geppeti P. Bronchoconstriction induced by citric acid inhalation in guinea pigs: role of tachykinins, bradykinin, and nitric oxide. Am J Respir Crit Care Med 159: 557-562, 1999[Abstract/Free Full Text].

29.   Smith, JJ, and Welsh MJ. cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J Clin Invest 89: 1148-1153, 1992[ISI][Medline].

30.   Smith, JJ, and Welsh MJ. Fluid and electrolyte transport by cultured human airway epithelia. J Clin Invest 91: 1590-1597, 1993[ISI][Medline].

31.   Tam, PY, and Verdugo P. Control of mucus hydration as a Donnan equilibrium process. Nature 292: 340-342, 1981[Medline].

32.   Trout, L, Gatzy JT, and Ballard ST. Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. Am J Physiol Lung Cell Mol Physiol 275: L1095-L1099, 1998[Abstract/Free Full Text].

Am J Physiol Lung Cell Mol Physiol 284(5):L855-L862
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