Download Airway surface liquid pH in well-differentiated airway - AJP-Cell

Am J Physiol Cell Physiol
281: C1504–C1511, 2001.
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
Received 23 April 2001; accepted in final form 6 July 2001
(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 ␮l 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
HCO3⫺ directly (2, 11, 20, 24) and/or modify the activities of HCO3⫺ 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
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 941430521 (E-mail: [email protected]; 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.
cystic fibrosis; acidification; trachea; fluorescence microscopy
THE AIRWAY SURFACE LIQUID
C1504
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Jayaraman, Sujatha, Yuanlin Song, and A. S. Verkman. Airway surface liquid pH in well-differentiated airway
epithelial cell cultures and mouse trachea. Am J Physiol Cell
Physiol 281: C1504–C1511, 2001.—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
HCO3⫺/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 (⫺HCO3⫺) and 0.060 pH
units/min (⫹HCO3⫺); the pH recovery rate was reduced by
amiloride and H2DIDS. In anesthetized mice in which the
trachea was surgically exposed for measurement of BCECFdextran 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⫺/HCO3⫺ 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.
AIRWAY SURFACE LIQUID PH
METHODS
Cell culture experiments. Well-differentiated cultures of
bovine tracheal cells were grown on collagen-coated 12-mmdiameter 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 BCECFdextran 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 ␮M), valinomycin (10 ␮M), carbonyl
cyanide m-chlorophenylhydrazone (5 ␮M), and forskolin (10
␮M). 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 HCO3⫺-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 HCO3⫺, the
perfusate consisted of 120 mM NaCl, 25 mM NaHCO3, 3.3
AJP-Cell Physiol • VOL
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 HCO3⫺-free buffer) or
NaH2PO4/Na2HPO4 (for HCO3⫺-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
␮l 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 ␮l 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 ␮g 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 ␮g 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.
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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.
C1505
C1506
AIRWAY SURFACE LIQUID PH
RESULTS
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 (E) and ASL (‚) (left). Also shown is pH measured in the
ASL in the absence (䊐) and presence (■) of CO2/HCO3⫺ (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).
AJP-Cell Physiol • VOL
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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/HCO3⫺ (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/HCO3⫺ (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.
The sensitivity of ASL pH to changes in pH of the
basolateral surface perfusate was measured in the
absence and presence of CO2/HCO3⫺. 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
C1507
AIRWAY SURFACE LIQUID PH
2B shows the kinetics of ASL pH following changes in
perfusate pH in the absence and presence of CO2/
HCO3⫺. 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/HCO3⫺ and
⫺0.027 ⫾ 0.003 (pH 6.0) and 0.025 ⫾ 0.007 (pH 8.0) in
the presence of CO2/HCO3⫺. CO2/HCO3⫺ did not significantly affect the initial rate of pH change.
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/
HCO3⫺. 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/HCO3⫺, as summarized in
Fig. 3C. The approximate twofold increase in the rate
of pH recovery in the presence of HCO3⫺ suggests the
involvement of an HCO3⫺-dependent pathway in ASL
pH equilibration.
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⫹/3HCO3⫺ cotransporter) were added to the
perfusate. Amiloride and omeprazole (dispersed in perAJP-Cell Physiol • VOL
fluorocarbon) 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⫹).
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/HCO3⫺, suggesting that H⫹/OH⫺ transport by Na⫹/H⫹ exchange is the principal mechanism
of ASL pH equilibration in the absence of CO2/HCO3⫺.
Figure 4C summarizes the initial rates of ASL alkalization in response to HCl addition in the absence or
presence of CO2/HCO3⫺. There was significant inhibition of ASL alkalization by amiloride in the absence
but not in the presence of HCO3⫺. 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⫺/HCO3⫺ 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 ␮M) and
glibenclamide (500 ␮M) had no significant effect on
ASL pH, whereas NPPB (200 ␮M), DPC (300 ␮M),
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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/
HCO3⫺ (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
(F) and absence (E) of CO2/HCO3⫺.
C1508
AIRWAY SURFACE LIQUID PH
H2DIDS (100 ␮M), and acetazolamide (100 ␮M) 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
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 ␮M), 4,4⬘-dinitrostilbene-2,2⬘-disulfonic acid (DNDS; 300 ␮M), amiloride (1 mM), omeprazole (300 ␮M), 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 (E) or presence (F) of CO2/HCO3⫺.
C: averaged initial rates (means ⫾ SE) of
ASL pH recovery for measurements done
as in B for 4 sets of cultures.
AJP-Cell Physiol • VOL
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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/
HCO3⫺ (A) and in the presence of CO2/HCO3⫺ (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/HCO3⫺.
C1509
AIRWAY SURFACE LIQUID PH
ASL pH alkalization are summarized in Fig. 5C. The
significant slowing of ASL pH recovery by H2DIDS
suggests the involvement of Cl⫺-HCO3⫺ exchange.
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
HCO3⫺ of 20 ⫾ 3 mM.
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
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.
AJP-Cell Physiol • VOL
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Fig. 5. Role of Cl⫺-dependent transport
in ASL pH regulation. Measurements
were done in the presence of CO2/HCO3⫺.
A: ASL pH (means ⫾ SE, 4–6 cultures)
measured after 2-h incubations with acetazolamide (500 ␮M), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB;
300 ␮M), H2DIDS (100 ␮M), diphenylamine-2-carboxylate (DPC; 500 ␮M),
glibenclamide (500 ␮M), and forskolin
(20 ␮M), 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.
C1510
AIRWAY SURFACE LIQUID PH
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.
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/HCO3⫺, 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 HCO3⫺ in the regulation of ASL
pH. In human and bovine airway cell cultures, Smith
and Welsh (24) reported that short-circuit current was
HCO3⫺ dependent and inhibited by DPC and acetazolamide. In transfected fibroblasts, Poulsen et al. (20)
AJP-Cell Physiol • VOL
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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.
characterized Na⫹-independent, HCO3⫺-dependent pH
regulation in cells expressing wild-type, but not ⌬F508,
CFTR. These studies suggested that CFTR might be
permeable to HCO3⫺. There is limited information on
pH regulation in airway epithelial cells. In cultured
human nasal epithelial cells, intracellular pH in the
absence of HCO3⫺ was shown to involve amiloridesensitive 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 HCO3⫺, pH is regulated by
the amiloride-sensitive Na⫹/H⫹ exchanger, but in the
presence of HCO3⫺, the major pathway involved Na⫹and Cl⫺-independent, HCO3⫺-dependent transport,
possibly the transport of HCO3⫺ by CFTR. They also
reported evidence for an H2DIDS-inhibitable Cl⫺/
HCO3⫺ exchanger. Recently, Choi et al. (4) reported
that CFTR mutants associated with pancreatic insufficiency do not support HCO3⫺ transport, suggesting a
physiological role for CFTR-dependent HCO3⫺ 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⫺/HCO3⫺ 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/HCO3⫺, indicating
that Na⫹/H⫹ exchange is the principal HCO3⫺-independent route for transepithelial H⫹/OH⫺ transport. In
the presence of CO2/HCO3⫺, steady-state ASL pH was
slightly increased (compared with no CO2/HCO3⫺), 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⫺/HCO3⫺ exchange
as the principal route for transepithelial H⫹/OH⫺
transport in the presence of HCO3⫺, 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 HCO3⫺ 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
AIRWAY SURFACE LIQUID PH
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.
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.
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.
3. Boucher RC. Molecular insights into the physiology of the ‘thin
film’ of airway surface liquid. J Physiol 516: 631–638, 1999.
4. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, and
Muallem S. Aberrant CFTR-dependent HCO3⫺ transport in mutations associated with cystic fibrosis. Nature 410: 94–97, 2001.
AJP-Cell Physiol • VOL
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.
6. Erjefält I and Persson CG. On the use of absorbing discs to
sample mucosal surface liquids. Clin Exp Allergy 20: 193–197, 1990.
7. Effros RM, Jacobs ER, Schapira RM, and Biller J. Response
of the lung to aspiration. Am J Med 108, Suppl: 15s–19s, 2000.
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.
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.
10. Higenbottam T. The ionic composition of airway surface liquid
and coughing. Bull Eur Physiopathol Respir 23, Suppl: 25s–27s,
1987.
11. Illek B, Yankaskas JR, and Machen TE. cAMP and genistein
stimulate HCO3⫺ conductance through CFTR in human airway
epithelium. Am J Physiol Lung Cell Mol Physiol 272: L752–
L761, 1997.
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.
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.
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.
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.
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.
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.
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.
19. Pilewski JM and Frizzell RA. Role of CFTR in airway disease.
Physiol Rev 79: S215–S255, 1999.
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.
21. Poulsen JH and Machen TE. HCO3⫺-dependent pH regulation
in tracheal epithelial cells. Pflügers Arch 432: 546–554, 1996.
22. Rao S and Verkman AS. Analysis of organ physiology in
transgenic mice. Am J Physiol Cell Physiol 279: C1–C18, 2000.
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.
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.
25. Wine JJ. The genesis of cystic fibrosis lung disease. J Clin
Invest 103: 309–312, 1999.
26. Wong CH, Matai R, and Morice AH. Cough induced by low
pH. Respir Med 93: 58–61, 1999.
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.
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.
281 • NOVEMBER 2001 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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 acidbase disturbances produced substantial changes in
ASL pH. Metabolic and respiratory acidosis resulted in
decreased ASL pH. Mild metabolic alkalosis created by
HCO3⫺ 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. HCO3⫺ permeabilities), it is difficult to establish more than an empirical relationship among changes
in serum HCO3⫺, 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.
C1511