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 |
Secretion of HCO
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
-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
-buffered solution, and perfused
continually with an aliquot of similar, lightly buffered solution (LBS)
in which NaCl replaced NaHCO
(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
-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
and Cl
. Isolated distal airways
thus secrete both acid and base equivalents.
airway epithelium; bicarbonate transport; pH
 |
INTRODUCTION |
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
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
, 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
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 |
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
-free solution was similar to KRB, but
HCO
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 ·
(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 (
LBS)
The empirically determined
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
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
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.
LBS containing either bafilomycin A1 (100 nM) or DMA (100 µM) were also determined (Fig. 1,
and
,
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,
) and bafilomycin (10 nM, )
are also shown.
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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 |
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
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
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); , rates induced by stepwise additions of
aliquots of NaOH.
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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).
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).
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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.
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To investigate the mechanisms involved in ACh-induced base equivalent
secretion, we bathed bronchi in HCO
-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
-free solution
containing acetazolamide (Fig.
5A), consistent with previous results showing that ACh stimulates HCO
secretion in
these tissues (18). The effect of ACh on PD was maintained
in HCO
-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 -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).
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Previous studies of these bronchi have shown that ACh stimulates the
submucosal glands to secrete HCO
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
or presence of DMA is consistent with an
earlier study in which we suggested that ACh stimulates secretion of
both Cl
and HCO
(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
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
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 |
Previous studies have shown that HCO
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
] 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
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
-free buffer containing acetazolamide to inhibit
carbonic anhydrase, consistent with the secretion of
HCO
. 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
] 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
.
This HCO
secretion response was also blocked by
inhibiting Na+/H+ exchange with DMA, confirming
earlier studies that the HCO
secretion was almost
entirely dependent on activity of this exchanger (15, 32).
The mechanism for HCO
secretion in these airways is
most consistent with the model proposed by Smith and Welsh
(29) for airway HCO
secretion.
According to this model, HCO
is generated within the
cell from CO2 and H2O, in a reaction catalyzed by carbonic anhydrase. HCO
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
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
secretion, whereas inhibition of
HCO
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
blocker-treated bronchi. The response was abolished, however, when both
Cl
and HCO
secretions were blocked
(18). This interdependence of Cl
and
HCO
secretion makes it difficult to study
HCO
secretion by bioelectrical methods; inhibition
of Cl
secretion to enable HCO
secretion to be measured actually enhances HCO
secretion. The methodology in the current study, however, provides a
means for study of HCO
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
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
secretion.
The model proposed by Smith and Welsh (29) predicts that
both Cl
and HCO
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
secretion and
hyperpolarization, consistent with inhibition of both
HCO
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
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
secretion (29).
In addition to its effects on ACh-evoked HCO
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
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
, 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
] 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
] 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
] 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
-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.
 |
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