Airway surface liquid pH in well-differentiated
airway epithelial cell cultures and mouse trachea
Sujatha
Jayaraman,
Yuanlin
Song, and
A. S.
Verkman
Departments of Medicine and Physiology, Cardiovascular Research
Institute, University of California, San Francisco, California
94143-0521
 |
ABSTRACT |
Airway surface liquid (ASL) pH has
been proposed to be important in the pathophysiology of cystic
fibrosis, asthma, and cough. Ratio image analysis was used to measure
pH in the ASL after staining with the fluorescent pH indicator
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at an
air-liquid interface was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of HCO
/CO2. Steady-state ASL pH changed in parallel to changes in bath pH and was
acidified by Na+ or Cl
replacement but was
not affected by the inhibitors amiloride, glibenclamide, or
4,4'-dinitrostilbene-2,2'-disulfonic acid. In response to sudden
acidification or alkalization of the ASL by ~0.4 pH units by
HCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pH
units/min (
HCO
) and 0.060 pH units/min
(+HCO
); the pH recovery rate was reduced by
amiloride and H2DIDS. In anesthetized mice in which the
trachea was surgically exposed for measurement of BCECF-dextran
fluorescence through the translucent tracheal wall, ASL pH was
7.14 ± 0.01. ASL pH was sensitive to changes in blood pH created
by metabolic (HCl or NaHCO3 infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, and
H+/OH
transport between the ASL and
basolateral fluid involves amiloride-sensitive Na+/H+ exchange and stilbene-sensitive
Cl
/HCO
exchange. The rapid response of
ASL pH to changes in systemic acid-base status may contribute to airway
hypersensitivity in asthma and other airway diseases.
cystic fibrosis; acidification; trachea; fluorescence microscopy
 |
INTRODUCTION |
THE AIRWAY SURFACE
LIQUID (ASL) is the thin layer of liquid at the air-facing
epithelial surface in the upper and lower airways. The regulation of
ASL volume, ionic composition, and pH is believed to be important in
normal airway physiology and in the pathophysiology of genetic and
acquired diseases of the airways such as cystic fibrosis and asthma
(3, 10, 15, 19). Abnormalities of the ASL may induce
bronchoconstriction and the cough reflex and interfere with epithelial
cell ionic homeostasis and airway defense mechanisms such as
antimicrobial activity and bacterial clearance (10, 17, 23,
26). The determination of ASL composition has posed a
considerable challenge because of its small volume (2-3 µl
of ASL per square centimeter of mucosal surface). Invasive sample
methods utilizing filter paper and microcapillary tubes have yielded a
wide range of ionic concentrations (1, 5, 9, 13, 14, 23,
28) and have been criticized because of potential perturbation
of the airway surface and sampling of intracellular and interstitial
fluids by capillary suction (3, 6, 19, 25). We recently
developed a minimally invasive in situ approach to measure ASL volume,
ionic composition, and osmolality (12). The ASL is stained
with ion-sensitive fluorescent indicators and viewed with a microscope
equipped with z-scanning confocal optics and ratio image detection. The
ASL in airway cell cultures and in the in vivo mouse trachea was
approximately isotonic and not dependent on cystic fibrosis
transmembrane conductance regulator (CFTR) Cl
channels.
Initial measurements of ASL pH in living anesthetized mice showed a pH
~7 (12).
On the basis of evidence that CFTR may transport HCO
directly (2, 11, 20, 24) and/or modify the activities of
HCO
exchangers (4), it has been
proposed that ASL pH may be abnormal in cystic fibrosis. There is
evidence that ASL acidity stimulates neurogenic reflexes in asthma
(26), and it has been postulated that lung damage in
cystic fibrosis occurs because the airways are unable to effectively
neutralize acid in repeated occurrences of subclinical gastric acid
aspiration (7). The ASL is a unique compartment in terms
of its low volume, exposure to an air interface, and contact with a
large surface area of beating cilia. The ASL in vivo is exposed to air
with time-varying CO2, O2, and moisture content
during inspiration and expiration. Theoretically, the determinants of
ASL pH include the composition of air at the tracheal mucosal surface
and of blood at the serosal surface, the activities of membrane
transporters at the epithelial cell apical and basolateral membranes,
and the permeability properties of the paracellular pathway; there may
also be neuroendocrine regulatory mechanisms. Given the limited
information about the transporting properties of the airway epithelium
and the complexity of the system, it is difficult to predict a priori
the value and principal determinants of ASL pH and whether ASL pH is
tightly regulated.
The purpose of this study was to define the principal determinants of
ASL pH. Measurements were done on well-differentiated primary cultures
of bovine airway epithelial cells grown at an air-liquid interface and
in the in vivo mouse trachea. The bovine airway cell culture model was
chosen as a well-established system for which there exists a
considerable body of data on ion transport mechanisms, including an
analysis of intracellular pH regulation (21). The
polarized cell culture system permitted measurements of ASL pH in
response to transporter agonists/inhibitors, ion substitution, and
imposed pH gradients. The mouse trachea was studied as an in vivo model
that permitted the testing of transporter agonists/inhibitors as well
as clinically important systemic acid-base disturbances. The data
reported here establish empirically the major determinants of ASL pH
and the transporting systems involved in transepithelial pH
equilibration. An important unanticipated finding was the lack of a
strict regulatory mechanism maintaining absolute ASL pH.
 |
METHODS |
Cell culture experiments.
Well-differentiated cultures of bovine tracheal cells were grown on
collagen-coated 12-mm-diameter Costar snapwell inserts with
polycarbonate semipermeable membranes at an air-liquid interface at
37°C in a 5% CO2-95% air atmosphere (27).
Culture medium was changed every 2-4 days. Cultures were generally
used 25-30 days after plating, at which time the electrical
resistance was >300
cm2, and the transepithelial
potential difference was >20 mV. Cell inserts were mounted (cells
facing upward) in a stainless steel perfusion chamber in which the
undersurface of the insert was perfused as described previously
(12). The perfusate bathed the cell basolateral surface.
The cell mucosal surface containing the ASL faced upward. The chamber
was maintained at 37°C using a PDMI-2 microincubator (Harvard
Apparatus) positioned on the stage of an upright epifluorescence
microscope and enclosed in a 100% humidified air-5% CO2
tent maintained at 37°C. For pH measurements, the ASL was stained
with the dual-excitation wavelength pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
conjugated to dextran (40 kDa, Molecular Probes), dispersed in a low
boiling point perfluorocarbon (Fluorinert FC-72, boiling point 56°C,
3M Company).
Measurement protocols.
The pH sensitivity of BCECF-dextran in the ASL was calibrated by
incubating the cell culture inserts with high-K+ perfusates
(120 mM KCl, 20 mM NaCl, 1 mM CaCl2, 1 mM
MgSO4, and 20 mM HEPES) containing nigericin (10 µM),
valinomycin (10 µM), carbonyl cyanide
m-chlorophenylhydrazone (5 µM), and forskolin (10 µM). Perfusates were titrated to specified pH (6.5-8.0) and equilibrated with cells for 4 h to set ASL pH, a time at which pH
equilibration was found to be complete. For experiments involving HCO
-free conditions, the perfusate consisted of
HEPES buffer (124 mM NaCl, 5.8 mM KCl, 10 mM glucose, 1 mM
CaCl2, 1 mM MgCl2, and 20 mM HEPES, pH 7.4),
and the apical cell surface was exposed to a 100% humidified air
atmosphere. For experiments in the presence of HCO
, the perfusate consisted of 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM
K2HPO4, 1.2 mM MgCl2, 1.2 mM
CaCl2, and 10 mM glucose, pH 7.4, and the apical surface
was exposed to a humidified 5% CO2-95% air atmosphere. In
some experiments, perfusate pH was adjusted to 6.0-8.0 by
titration with HCl/NaOH (for HCO
-free buffer) or
NaH2PO4/Na2HPO4 (for
HCO
-containing buffer). For ion substitution
experiments, perfusate Na+ was replaced by
choline+ or Cl
by gluconate
. In
experiments involving recovery from acute ASL acidification or
alkalization, 20-50 µl of a perfluorocarbon suspension of
HCl/NaOH (prepared by brief sonication) was added onto the ASL to
change pH by 0.4-0.5 pH units. In some experiments, transport
inhibitors were added to the perfusate or to both the perfusate and the
ASL as described in RESULTS.
Measurements in mouse trachea in vivo.
Mice (25-35 g body wt) were anesthetized with ketamine (60 mg/kg
body wt) and xylazine (8 mg/kg) 15 min after pretreatment with atropine
(1 mg/kg intraperitoneal) to prevent secretions as discussed previously
(12). A midline incision was made in the neck to expose
the trachea for measurement of fluorescence through the translucent
tracheal wall. Unless otherwise indicated, the ASL was stained by
instillation of 5 µl of the BCECF-dextran suspension in
perfluorocarbon using a microcatheter passed through a feeding needle
that was introduced via the mouth. The mouse was positioned on the
microscope stage for fluorescence measurements as described below.
Arterial blood (0.2-0.3 ml) was sampled through a PE-10 catheter
inserted into the carotid artery, and blood pH and
PCO2 were measured using a blood gas analyzer
(Ciba Corning Diagnostic). After completion of the
measurements, mice were euthanized by an overdose of pentobarbital (150 mg/kg). Animal protocols were approved by the University of California
San Francisco Committee on Animal Research.
In some experiments, amiloride (10 mg/kg of 1 mM solution) was injected
intraperitoneally 30 min before anesthesia, and 2.7 µg of amiloride
(dispersed in perfluorocarbon) was instilled into trachea together with
BCECF-dextran. ASL pH was measured 10 min after the perfluorocarbon
instillation. Mice were treated with glibenclamide by intraperitoneal
injection of 0.3 ml of a 1 mM glibenclamide solution and intratracheal
instillation of 4.9 µg of glibenclamide in perfluorocarbon. To create
acute metabolic acidosis or alkalosis, HCl (0.5 meq H+) or
NaHCO3 (0.3 meq) were injected intraperitoneally. ASL pH was measured after 20 min. To create respiratory acidosis or alkalosis, the upper trachea of anesthetized, paralyzed (pancuronium, 1 mg/kg intraperitoneal) mice was cannulated with PE-90 tubing, and mice were
mechanically ventilated with room air (tidal volume 8 ml/kg, respiratory rate 90 respirations/min) using a mouse constant
volume ventilator (Harvard Apparatus). Acute hyperventilation was
produced by increasing the respiratory rate to 140 respirations/min for 10 min before ASL pH measurements and arterial blood gas analysis. Acute hypoventilation/hypercarbia was produced by decreasing
respiratory rate to 90 respirations/min and addition of 5%
CO2 (95% O2, to prevent hypoxia) to the
inspired gas.
Fluorescence microscopy.
The chamber containing the cultured cells or the mouse was positioned
on the stage of a Leitz upright fluorescence microscope with a
Technical Instruments coaxial-confocal attachment. Fluorescence was
detected using a Nikon ×50 extra-long working distance air objective
(numerical aperture 0.55, working distance 8 mm) for ratiometric
measurement of pH at 440- and 490-nm excitation wavelengths and a
535-nm emission wavelength. Background fluorescence (unstained cells or
trachea) was <1% of total fluorescence.
 |
RESULTS |
Cell culture experiments.
The ASL of tracheal cells cultured at an air-liquid interface was
stained with the pH indicator BCECF-dextran by addition of microliter
quantities of a low boiling point perfluorocarbon containing the
dispersed indicator. The perfluorocarbon evaporated within a few
seconds, permitting the BCECF microparticles to dissolve rapidly in the
ASL. The cells on the porous support were mounted in a 37°C perfusion
chamber in which the basolateral surface was perfused, and the
apical surface was exposed to a 100% humidified atmosphere.
Figure 1A shows an in
situ calibration of the ratio of BCECF:fluorescence at 490- and
440-nm excitation wavelengths (F490/F440). ASL
pH was set using perfusates containing high K+ and
ionophores. The pKa of BCECF-dextran in the ASL was 7.05, not different from that in saline. Figure 1A also shows the
averaged results from a series of measurements done in cells in the
absence and presence of CO2/HCO
(in both
perfusate- and atmosphere-bathing apical surface); the individual data
(right) represent results obtained from different cultures
with standard errors determined from measurements done at
4-8 different locations. Averaged ASL pH was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of
CO2/HCO
(P < 0.01), lower than the perfusate pH of 7.40. Figure 1B shows images
of the ASL surface recorded at excitation wavelengths of 490 nm
(left) and 440 nm (middle) together with a
computed ratio image (right) showing excellent pH uniformity
throughout the ASL.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 1.
Airway surface liquid (ASL) pH measurement in bovine
tracheal epithelial cells using
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-dextran. A: calibration of the dependence of
BCECF-dextran fluorescence excitation ratio
(F490/F440) as a function of pH in solution
( ) and ASL ( ) (left). Also
shown is pH measured in the ASL in the absence ( ) and
presence ( ) of CO2/HCO
(SE, n = 10-14 cultures). Right: pH
measured in individual cultures. Each point represents the mean ± SE of 4-8 measurements in different locations in each culture.
B: ratio image analysis of ASL pH. Images are shown at
490-nm (left) and 440-nm (middle) excitation
wavelengths along with pseudocolored ratio image
(right).
|
|
The sensitivity of ASL pH to changes in pH of the basolateral surface
perfusate was measured in the absence and presence of CO2/HCO
. Figure
2A shows the ASL pH at 2 h after changing perfusate pH from 7.4 to 6.0 or 8.0. ASL pH changes
paralleled changes in perfusate pH. The pH changes were reversible, and
cells remained viable after incubation in the acidic and alkaline media
as shown by transepithelial resistance (400-800
cm2) and trypan blue dye exclusion. Figure 2B
shows the kinetics of ASL pH following changes in perfusate pH in the
absence and presence of CO2/HCO
. The
initial rates of pH change (in pH units/min) were
0.030 ± 0.003 (perfusate pH 6.0) and 0.023 ± 0.005 (pH 8.0) in the absence of
CO2/HCO
and
0.027 ± 0.003 (pH
6.0) and 0.025 ± 0.007 (pH 8.0) in the presence of
CO2/HCO
.
CO2/HCO
did not significantly affect the
initial rate of pH change.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Dependence of ASL pH on perfusate pH in tracheal cell cultures.
A: ASL pH measured after incubating cultures for 2 h in
perfusate at pH 6.0 and 8.0 in the absence (open bars) and presence
(solid bars) of CO2/HCO (means ± SE, n = 4-6 cultures). B: time course
of ASL pH (means ± SE, n = 4 cultures) in
response to changes in perfusate pH to 8.0 (left) and 6.0 (right) in the presence ( ) and absence
( ) of CO2/HCO .
|
|
The time course of ASL pH recovery was measured in response to sudden
acidification or alkalization of the ASL by 0.3-0.5 pH units by
addition of perfluorocarbon containing dispersed HCl or NaOH. Perfusate
pH was maintained at 7.4. Measurements were done in the absence (Fig.
3A) and presence (Fig.
3B) of CO2/HCO
. The average
ASL buffer capacity, computed from the decreased pH produced by
addition of known molar quantities of H+, was 14 ± 3 mM H+/pH unit, much less than that of cytoplasm (generally
>50 mM H+/pH unit) because of the relatively low protein
content in ASL. ASL pH returned to its initial values over 5-10
min, with substantially more rapid pH equilibration in the presence of
CO2/HCO
, as summarized in Fig.
3C. The approximate twofold increase in the rate of pH
recovery in the presence of HCO
suggests the
involvement of an HCO
-dependent pathway in ASL pH
equilibration.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
ASL pH regulation in response to sudden ASL acidification or
alkalinization. Time course of ASL pH after alkalinization by direct
addition of NaOH (top) or acidification by HCl
(bottom) in the absence of
CO2/HCO (A) and in the
presence of CO2/HCO (B).
C: averaged initial rates (means ± SE) of ASL pH
recovery (shown as positive values) for measurements done as in
A and B for 4 sets of cultures.
*P < 0.02 comparing ± CO2/HCO .
|
|
The contribution of Na+-dependent transport to ASL pH
regulation was studied using inhibitors and Na+
substitution. The inhibitors (1 mM each) amiloride (epithelial Na+ channel and Na+/H+ exchanger),
furosemide (Na+-K+-2Cl
cotransporter), omeprazole (K+/H+ exchanger),
or 4,4'-dinitrostilbene-2,2'-disulfonic acid
(Na+/3HCO
cotransporter) were added to the perfusate. Amiloride and omeprazole (dispersed in perfluorocarbon) were also added to the ASL. Figure
4A shows ASL pH measured at 2 h after inhibitor addition. There was no significant effect of
these compounds on steady-state ASL pH. Also shown is the acidified ASL
following incubation of cells for 2 h with Na+-free
medium (choline+ replacing Na+).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Role of Na+-dependent transport in ASL pH regulation.
A: ASL pH (means ± SE, 4-6 cultures) measured
after 2-h incubations with furosemide (200 µM),
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; 300 µM), amiloride
(1 mM), omeprazole (300 µM), or after replacement of Na+
by choline+ (Na+-free buffer).
*P < 0.02 compared with control. B: time
course of ASL pH recovery after acidification of ASL by HCl in the
presence of amiloride and absence ( ) or presence
( ) of CO2/HCO .
C: averaged initial rates (means ± SE) of ASL pH
recovery for measurements done as in B for 4 sets of
cultures.
|
|
Kinetic studies were done to identify functionally the Na+
transporter(s) involved in restoring ASL pH in response to a
transepithelial pH gradient. Figure 4B shows the time course
of ASL pH in response to rapid ASL acidification by HCl addition.
Amiloride caused a remarkable inhibition of ASL alkalization in the
absence of CO2/HCO
, suggesting that
H+/OH
transport by
Na+/H+ exchange is the principal mechanism of
ASL pH equilibration in the absence of
CO2/HCO
. Figure 4C summarizes
the initial rates of ASL alkalization in response to HCl addition in
the absence or presence of CO2/HCO
. There was significant inhibition of ASL alkalization by amiloride in
the absence but not in the presence of HCO
. Alkalization was also blocked by perfusate Na+ substitution.
Similar experiments were done to investigate the role of
Cl
transporters in the regulation of ASL pH. The
inhibitors included H2DIDS
(Cl
/HCO
exchanger), glibenclamide,
diphenylamine-2-carboxylate (DPC),
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (Cl
channel, CFTR), and acetazolamide (carbonic anhydrase), and the CFTR
agonist forskolin was tested. All compounds were added to the
perfusate, and the Cl
transport inhibitors (dispersed in
perfluorocarbon) were also added onto the ASL. Figure
5A shows that after a 2-h
incubation, forskolin (20 µM) and glibenclamide (500 µM) had no
significant effect on ASL pH, whereas NPPB (200 µM), DPC (300 µM),
H2DIDS (100 µM), and acetazolamide (100 µM) mildly
acidified the ASL. Also shown is the acidified ASL following incubation
of cells for 2 h with Cl
-free perfusate
(gluconate
replacing Cl
). Kinetic studies
were done in which the time course of ASL alkalization was measured in
response to addition of HCl to the ASL. Figure 5B shows the
slowing of ASL alkalization in the presence of NPPB and
H2DIDS. The averaged rates of ASL pH alkalization are
summarized in Fig. 5C. The significant slowing of ASL pH
recovery by H2DIDS suggests the involvement of
Cl
-HCO
exchange.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Role of Cl -dependent transport in ASL pH regulation.
Measurements were done in the presence of
CO2/HCO . A: ASL pH
(means ± SE, 4-6 cultures) measured after 2-h incubations
with acetazolamide (500 µM), 5-nitro-2-(3-phenylpropylamino)benzoic
acid (NPPB; 300 µM), H2DIDS (100 µM),
diphenylamine-2-carboxylate (DPC; 500 µM), glibenclamide (500 µM),
and forskolin (20 µM), or after replacement of Cl by
gluconate (Cl -free buffer). B:
time course of ASL pH recovery after ASL acidification by HCl in the
presence of H2DIDS or NPPB (no inhibitor control from Fig.
3B shown for comparison). Control (no inhibitor) data (from
Fig. 2B) is shown for comparison. C: averaged
initial rates (means ± SE) of ASL pH recovery for measurements
done as in B for 4 sets of cultures. *P < 0.02 compared with control.
|
|
In vivo mouse trachea experiments.
ASL pH was measured in mouse trachea after staining the tracheal lumen
with BCECF-dextran. The perfluorocarbon suspension of BCECF-dextran was
introduced into the trachea using a small feeding needle that was
passed through the mouth and then promptly withdrawn to permit
spontaneous breathing. Fluorescence was detected through the
translucent tracheal wall after surgical exposure of the trachea by a
midline neck incision. Figure 6 shows
images of the trachea at excitation wavelengths of 440 nm
(left) and 490 nm (middle) and the computed ratio
image (right), showing a quite uniform pH distribution. The
average ASL pH in anesthetized, spontaneously breathing mice was
7.14 ± 0.01 (SE, n = 4 mice). Arterial blood gas
analysis in these mice (room air) showed a PO2
of 107 ± 23 mmHg, a PCO2 of 52 ± 9, pH of 7.20 ± 0.04, and a computed HCO
of
20 ± 3 mM.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
ASL pH in mouse trachea. After surgical exposure of the
trachea in anesthetized mice, the tracheal mucosa was stained with
BCECF-dextran and imaged as described in METHODS.
Excitation ratio imaging of pH in mouse trachea. Fluorescence
micrographs of BCECF-dextran stained ASL in mouse trachea (emission 535 nm) measured at 440-nm (left) and 490-nm (middle)
excitation wavelengths and pseudocolored ratio image
(right). See text for averaged pH values. Arrow points to
unstained blood vessel.
|
|
ASL pH in mouse trachea was measured in response to the transport
inhibitors amiloride and glibenclamide after introduction both
systemically, by intraperitoneal injection, and directly onto the
tracheal mucosa by instillation in a perfluorocarbon suspension. Figure
7 summarizes ASL pH along with arterial
blood pH (A) and PCO2
(B). There was no significant effect of the inhibitors on
ASL or blood pH. The effects of acute metabolic and respiratory acid-base disturbances were studied. As described in
METHODS, metabolic acidosis and alkalosis were produced by
intraperitoneal injection of HCl or NaHCO3; respiratory
alkalosis and acidosis were produced in paralyzed, ventilated mice by
hyperventilation or hypoventilation (with 5% CO2). Figure
7 shows that the maneuvers produced the predicted changes in blood pH
and PCO2. Significant ASL acidification was
produced by either HCl addition or hypoventilation/CO2 breathing. Although blood pH was increased comparably by
NaHCO3 addition or hyperventilation/CO2, ASL pH
was increased significantly only by acute respiratory alkalosis.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7.
Sensitivity of ASL pH to systemic acid-base status. Acute
metabolic and respiratory acid-base disturbances were produced in
anesthetized mice as described in METHODS. Averaged ASL pH
is shown with arterial blood pH (A) and
PCO2 (B; means ± SE,
n = 4-7 mice). *P < 0.02 compared
with control mice.
|
|
 |
DISCUSSION |
The purpose of this study was to characterize ASL pH using an
established airway cell culture model and the in vivo mouse trachea.
The complementary cell culture and in vivo systems were chosen to be
able to test specific transporter agonists and inhibitors, perform ion
substitution maneuvers, and examine the role of
CO2/HCO
, as well as to study the
integrated physiological response of ASL pH to in vivo acid-base
disturbances. The ratioable pH indicator BCECF-dextran was ideal for
these measurements because of its excellent sensitivity to pH changes
near pH 7.0 (7% change in fluorescence ratio for 0.1 unit pH change),
permitting single measurements of ASL pH to better than 0.05 pH unit
accuracy. We found that steady-state ASL pH is principally determined
by serosal fluid/blood pH. Although specific Na+ and
Cl
transporters were found to be involved in the
transient response to imposed pH gradients across the airway
epithelium, ASL pH was not tightly regulated and thus subject to
potentially large variations in response to changes in systemic
acid-base status.
Several lines of evidence have suggested a potentially important role
for HCO
in the regulation of ASL pH. In human and
bovine airway cell cultures, Smith and Welsh (24) reported
that short-circuit current was HCO
dependent and
inhibited by DPC and acetazolamide. In transfected fibroblasts, Poulsen
et al. (20) characterized Na+-independent,
HCO
-dependent pH regulation in cells expressing
wild-type, but not
F508, CFTR. These studies suggested that
CFTR might be permeable to HCO
. There is limited
information on pH regulation in airway epithelial cells. In cultured
human nasal epithelial cells, intracellular pH in the absence of
HCO
was shown to involve amiloride-sensitive
Na+/H+ exchange (18). Poulsen and
Machen (21) carried out a more extensive study of
intracellular pH regulation in bovine tracheal epithelial cells. They
found that in the absence of HCO
, pH is regulated by
the amiloride-sensitive Na+/H+ exchanger, but
in the presence of HCO
, the major pathway involved
Na+- and Cl
-independent,
HCO
-dependent transport, possibly the transport of
HCO
by CFTR. They also reported evidence for an
H2DIDS-inhibitable Cl
/HCO
exchanger. Recently, Choi et al. (4) reported that CFTR
mutants associated with pancreatic insufficiency do not support
HCO
transport, suggesting a physiological role for
CFTR-dependent HCO
transport.
The cell culture studies here showed that although steady-state ASL pH
is determined mainly by perfusate pH, transient responses to sudden
changes in ASL or perfusate pH involved Na+ and
Cl
transporters. Ion substitution and inhibitor studies
suggested the involvement of amiloride-sensitive
Na+/H+ exchange and
H2DIDS-sensitive Cl
/HCO
exchange. The recovery of ASL pH in response to sudden acidification of
the ASL was strongly inhibited by amiloride or Na+
replacement in the absence of CO2/HCO
, indicating that Na+/H+ exchange is the
principal HCO
-independent route for transepithelial
H+/OH
transport. In the presence of
CO2/HCO
, steady-state ASL pH was
slightly increased (compared with no
CO2/HCO
), and the recovery from changes
in ASL pH was accelerated approximately twofold in an
H2DIDS-inhibitable manner. Steady-state ASL pH was mildly
decreased by Cl
replacement and by various
Cl
transport inhibitors. Activation of CFTR by forskolin
did not affect ASL pH. Our results are best explained by
Cl
/HCO
exchange as the principal route for transepithelial H+/OH
transport in the
presence of HCO
, although a contribution of CFTR
cannot be easily assessed because of the imperfect specificity of the
Cl
transport inhibitors and the complexity of the system.
Assessment of the role of CFTR in HCO
will require comparative measurements in CFTR-expressing vs. cystic fibrosis cells
as well as single cell/membrane experiments.
ASL pH in the in vivo mouse trachea was measured using a minimally
invasive procedure in which the trachea was exposed by a skin incision
in the neck, and the ASL was stained with BCECF-dextran using a blunt
feeding needle introduced via the mouth. Measurements were made without
direct contact with the tracheal mucosa or invasion of the tracheal
wall. We showed previously that measured ASL depth, salt content, and
pH remained stable over time (12). Additionally, [Na+] and pH were not different as measured by direct dye
addition through a tracheal window or by the less invasive procedure
used here of dye addition through a feeding needle and measurement through the intact tracheal wall.
ASL pH in mouse trachea was 7.14 when blood pH was 7.2. The mild
systemic acidosis in control mice is probably related to the
anesthesia, which was maintained at a minimal level using ketamine/xylazine. Mice are quite susceptible to hypoventilation during
anesthesia (22), which probably accounts for the slightly lower ASL pH (6.9-7.0) in our preliminary measurements in mice anesthetized using pentobarbital (12). As summarized in
Fig. 7, acute systemic acid-base disturbances produced substantial changes in ASL pH. Metabolic and respiratory acidosis resulted in
decreased ASL pH. Mild metabolic alkalosis created by
HCO
administration did not change ASL pH, whereas
respiratory alkalosis (hyperventilation) producing comparable
elevation in blood pH resulted in increased ASL pH. The principal
conclusion from the mouse experiments is that ASL pH changes rapidly in
response to systemic acid-base status. Because of the complex
determinants of ASL pH in the in vivo model (e.g., changing
PCO2 during inspiration/expiration, differential CO2 vs. HCO
permeabilities), it is difficult to establish more than an
empirical relationship among changes in serum HCO
,
arterial blood PCO2, and ASL pH.
The pH of ASL has been proposed to be important in the physiology of
the cough reflex and airway reactivity. Wong et al. (26) reported that lowering airway pH by citric acid instillation into the
human trachea induced cough, possibly by stimulation of
stretch/irritant receptors (16). Our data in mouse trachea
indicate that ASL pH is affected by serum pH and
PCO2 and that ASL pH can change rapidly in
response to changes in systemic acid-base status. These findings
suggest that substantial changes in ASL pH are produced in clinically
relevant situations such as lactic acidosis and acute changes in
ventilation. Fine et al. (8) reported evidence of
increased airway reactivity and bronchoconstriction after inhalation of
buffered HCl or H2SO4. In severe asthma
associated with CO2 retention, acute ASL acidosis might be
an important exacerbating factor that causes further
bronchoconstriction and impairment of ventilation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
HL-60288, HL-59198, DK-35124, and DK-43840 and National Cystic Fibrosis
Foundation Research and Development Grant R613.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, Univ. of California, San Francisco, San Francisco, CA
94143-0521 (E-mail: verkman{at}itsa.ucsf.edu;
http://www.ucsf.edu/verklab).
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.
Received 23 April 2001; accepted in final form 6 July 2001.
 |
REFERENCES |
1.
Baconnais, S,
Tirouvanziam R,
Zahm JM,
Bentzmann S,
Peault B,
Balossier G,
and
Puchelle E.
Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts.
Am J Respir Cell Mol Biol
20:
605-611,
1999[Abstract/Free Full Text].
2.
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].
3.
Boucher, RC.
Molecular insights into the physiology of the `thin film' of airway surface liquid.
J Physiol
516:
631-638,
1999[Abstract/Free Full Text].
4.
Choi, JY,
Muallem D,
Kiselyov K,
Lee MG,
Thomas PJ,
and
Muallem S.
Aberrant CFTR-dependent HCO
transport in mutations associated with cystic fibrosis.
Nature
410:
94-97,
2001[ISI][Medline].
5.
Cowley, EA,
Govindaraju K,
Guilbault C,
Radzioch D,
and
Eidelman DH.
Airway surface liquid composition in mice.
Am J Physiol Lung Cell Mol Physiol
278:
L1213-L1220,
2000[Abstract/Free Full Text].
6.
Erjefält, I,
and
Persson CG.
On the use of absorbing discs to sample mucosal surface liquids.
Clin Exp Allergy
20:
193-197,
1990[ISI][Medline].
7.
Effros, RM,
Jacobs ER,
Schapira RM,
and
Biller J.
Response of the lung to aspiration.
Am J Med
108, Suppl:
15s-19s,
2000[Medline].
8.
Fine, JM,
Gordon T,
Thompson JE,
and
Sheppard D.
The role of titratable acidity in acid aerosol-induced bronchoconstriction.
Am Rev Respir Dis
135:
826-830,
1987[ISI][Medline].
9.
Govindaraju, K,
Cowley EA,
Eidelman DH,
and
Lloyd DK.
Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection.
Anal Chem
69:
2793-2797,
1997[ISI][Medline].
10.
Higenbottam, T.
The ionic composition of airway surface liquid and coughing.
Bull Eur Physiopathol Respir
23, Suppl:
25s-27s,
1987[Medline].
11.
Illek, B,
Yankaskas JR,
and
Machen TE.
cAMP and genistein stimulate HCO
conductance through CFTR in human airway epithelium.
Am J Physiol Lung Cell Mol Physiol
272:
L752-L761,
1997[Abstract/Free Full Text].
12.
Jayaraman, S,
Song Y,
Vetrivel L,
Shankar L,
and
Verkman AS.
Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH.
J Clin Invest
107:
317-324,
2001[Abstract/Free Full Text].
13.
Joris, L,
Dab I,
and
Quinton PM.
Elemental composition of human airway surface fluid in healthy and diseased airways.
Am Rev Respir Dis
148:
1633-1637,
1993[ISI][Medline].
14.
Knowles, MR,
Robinson JM,
Wood RE,
Pue CA,
Mentz WM,
Wager GC,
Gatzy JT,
and
Boucher RC.
Ion composition of airway surface liquid of patients with cystic fibrosis compared with normal and disease-control subjects.
J Clin Invest
100:
2588-2595,
1997[Abstract/Free Full Text].
15.
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].
16.
Lowry, RH,
Wood AM,
and
Higenbottam TW.
Effects of pH and osmolality on aerosol-induced cough in normal volunteers.
Clin Sci (Lond)
74:
373-378,
1997[ISI][Medline].
17.
Matsui, H,
Grubb BR,
Tarran R,
Randell SH,
Gatzy JT,
Davis CW,
and
Boucher RC.
Evidence for periciliary liquid layer depletion, not abnormal ion composition in the pathogenesis of cystic fibrosis airway disease.
Cell
95:
1005-1015,
1998[ISI][Medline].
18.
Paradiso, AM.
Identification of Na+-H+ exchange in human normal and cystic fibrotic ciliated airway epithelium.
Am J Physiol Lung Cell Mol Physiol
262:
L757-L764,
1992[Abstract/Free Full Text].
19.
Pilewski, JM,
and
Frizzell RA.
Role of CFTR in airway disease.
Physiol Rev
79:
S215-S255,
1999[Medline].
20.
Poulsen, JH,
Fischer H,
Illek B,
and
Machen TE.
Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci USA
91:
5340-5344,
1994[Abstract].
21.
Poulsen, JH,
and
Machen TE.
HCO
-dependent pH regulation in tracheal epithelial cells.
Pflügers Arch
432:
546-554,
1996[ISI][Medline].
22.
Rao, S,
and
Verkman AS.
Analysis of organ physiology in transgenic mice.
Am J Physiol Cell Physiol
279:
C1-C18,
2000[Abstract/Free Full Text].
23.
Smith, JJ,
Travis SM,
Greenberg EP,
and
Welsh MJ.
Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.
Cell
85:
229-236,
1996[ISI][Medline].
24.
Smith, JJ,
and
Welsh MJ.
cAMP stimulated bicarbonate secretion across normal, but not cystic fibrosis, airway epithelia.
J Clin Invest
89:
1148-1153,
1992[ISI][Medline].
25.
Wine, JJ.
The genesis of cystic fibrosis lung disease.
J Clin Invest
103:
309-312,
1999[Free Full Text].
26.
Wong, CH,
Matai R,
and
Morice AH.
Cough induced by low pH.
Respir Med
93:
58-61,
1999[ISI][Medline].
27.
Wu, DX,
Lee CY,
Uyekubo SN,
Choi HK,
Bastacky SJ,
and
Widdicombe JH.
Regulation of the depth of surface liquid in bovine trachea.
Am J Physiol Lung Cell Mol Physiol
274:
L388-L395,
1998[Abstract/Free Full Text].
28.
Zabner, J,
Smith JJ,
Karp PH,
Widdicombe JH,
and
Welsh MJ.
Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro.
Mol Cell
2:
397-403,
1998[ISI][Medline].
Am J Physiol Cell Physiol 281(5):C1504-C1511
0363-6143/01 $5.00
Copyright © 2001 the American Physiological Society