Effect of airway acidosis and alkalosis on airway vascular smooth muscle responsiveness to albuterol

Abstract

Background:
In vitro and animal experiments have shown that the transport and signaling of β2-adrenergic agonists are pH-sensitive. Inhaled albuterol, a hydrophilic β2-adrenergic agonist, is widely used for the treatment of obstructive airway diseases. Acute exacerbations of obstructive airway diseases can be associated with changes in ventilation leading to either respiratory acidosis or alkalosis thereby affecting albuterol responsiveness in the airway. The purpose of this study was to determine if airway pH has an effect on albuterol-induced vasodilation in the airway.

Methods:
Ten healthy volunteers performed the following respiratory maneuvers: quiet breathing, hypocapnic hyperventilation, hypercapnic hyperventilation, and eucapnic hyperventilation (to dissociate the effect of pH from the effect of ventilation). During these breathing maneuvers, exhaled breath condensate (EBC) pH and airway blood flow response to inhaled albuterol (ΔQ̇aw) were assessed.

Results:
Mean ± SE EBC pH (units) and ΔQ̇aw (μl.min(-1).mL(-1)) were 6.4 ± 0.1 and 16.8 ± 1.9 during quiet breathing, 6.3 ± 0.1 and 14.5 ± 2.4 during eucapnic hyperventilation, 6.6 ± 0.2 and -0.2 ± 1.8 during hypocapnic hyperventilation (p = 0.02 and <0.01 vs. quiet breathing), and 5.9 ± 0.1 and 2.0 ± 1.5 during hypercapnic hyperventilation (p = 0.02 and <0.02 vs quiet breathing).

Conclusions:
Albuterol responsiveness in the airway as assessed by ΔQ̇aw is pH sensitive. The breathing maneuver associated with decreased and increased EBC pH both resulted in a decreased responsiveness independent of the level of ventilation. These findings suggest an attenuated response to hydrophilic β2-adrenergic agonists during airway disease exacerbations associated with changes in pH.

Trial registration:
Registered at clinicaltrials.gov: NCT01216748 .

Full text

R E S E A R C H A R T I C L E Open Access
Effect of airway acidosis and alkalosis on airway
vascular smooth muscle responsiveness to
albuterol
Jose E Cancado
1†
, Eliana S Mendes
1*†
, Johana Arana
1
, Gabor Horvath
2
, Maria E Monzon
1
, Matthias Salathe
1†
and Adam Wanner
1†
Abstract
Background: In vitro and animal experiments have shown that the transport and signaling of β
2
-adrenergic
agonists are pH-sensitive. Inhaled albuterol, a hydrophilic β
2
-adrenergic agonist, is widely used for the treatment of
obstructive airway diseases. Acute exacerbations of obstructive airway diseases can be associated with changes in
ventilation leading to either respiratory acidosis or alkalosis thereby affecting albuterol responsiveness in the airway.
The purpose of this study was to determine if airway pH has an effect on albuterol-induced vasodilation in the
airway.
Methods: Ten healthy volunteers performed the following respiratory maneuvers: quiet breathing, hypocapnic
hyperventilation, hypercapnic hyperventilation, and eucapnic hyperventilation (to dissociate the effect of pH from
the effect of ventilation). During these breathing maneuvers, exhaled breath condensate (EBC) pH and airway blood
flow response to inhaled albuterol (ΔQ

aw
) were assessed.
Results: Mean ± SE EBC pH (units) and ΔQ

aw
(μl.min
-1
.mL
-1
) were 6.4 ± 0.1 and 16.8 ± 1.9 during quiet breathing,
6.3 ± 0.1 and 14.5 ± 2.4 during eucapnic hyperventilation, 6.6 ± 0.2 and -0.2 ± 1.8 during hypocapnic hyperventilation
(p = 0.02 and <0.01 vs. quiet breathing), and 5.9 ± 0.1 and 2.0 ± 1.5 during hypercapnic hyperventilation (p = 0.02
and <0.02 vs quiet breathing).
Conclusions: Albuterol responsiveness in the airway as assessed by ΔQ

aw
is pH sensitive. The breathing maneuver
associated with decreased and increased EBC pH both resulted in a decreased responsiveness independent of the
level of ventilation. These findings suggest an attenuated response to hydrophilic β
2
-adrenergic agonists during
airway disease exacerbations associated with changes in pH.
Trial registration: Registered at clinicaltrials.gov: NCT01216748.
Keywords: Airway surface liquid pH, Airway blood flow, Respiratory alkalosis, Respiratory acidosis, Albuterol
Background
In vitro and animal experiments have shown that trans-
port of and signaling by β
2
-adrenergic agonist are pH-
sensitive. At acidic pH, the transport of β
2
-adrenergic
agonists across the airway epithelium is decreased [1], β
2
-
adrenergic receptor function is impaired [2,3], endothelial
function is diminished [4-6], and systemic vascular
smooth muscle tone is increased [7]. Conversely, epithelial
β
2
-adrenergic agonist transport is increased at alkaline pH
[1]. The effects of alkalosis on β
2
-adrenergic receptor
function, endothelial function and systemic vascular
smooth muscle tone are less clear, with studies showing
minimal or no changes in β
2
-adrenergic signaling [5], but
an increase in vascular smooth muscle tone [7].
Inhaled albuterol, a hydrophilic β
2
-adrenergic agonist,
is widely used for the treatment of obstructive airway
disease. Acute exacerbations of obstructive airway dis-
eases can be associated with changes in ventilation lead-
ing to either respiratory acidosis or alkalosis. The
* Correspondence: emendes@med.miami.edu
†
Equal contributors
1
Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University
of Miami School of Medicine, Miami, FL 33136, USA
Full list of author information is available at the end of the article
© 2015 Cancado et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Cancado et al. BMC Pharmacology and Toxicology (2015) 16:9
DOI 10.1186/s40360-015-0008-y

resulting changes in airway pH could have an effect on
albuterol responsiveness. We therefore sought to test the
hypothesis that the magnitude of vasodilation in the
airway caused by inhaled albuterol could be altered by
changes in airway pH. To investigate this possibility, we
determined the effect of airway surface pH on airway
blood flow (Q

aw
) responsiveness to inhaled albuterol in
healthy subjects by manipulating airway pH through
ventilatory maneuvers. Healthy subjects were chosen be-
cause the required respiratory maneuvers would be diffi-
cult to impose on patients with airflow obstruction. Q
aw
was chosen as a “biomarker”of albuterol responsiveness
because airflow responses would only be marginally
sensitive to albuterol in healthy subjects.
Methods
Subjects
Ten healthy lifetime non-smokers participated in the
study. The exclusion criteria were as follows: 1) a phys-
ician diagnosis of cardiovascular or pulmonary disease; 2)
the use of cardiovascular or airway medication; 3)abody
mass index >30; and 4) a forced expiratory volume in
1 second (FEV
1
) < 80% of predicted and FEV
1
-to-forced
vital capacity ratio < 0.7. All subjects had been free of an
acute respiratory infection for at least 4 weeks before be-
ginning the study, and no subject had an acute respiratory
infection during the study. The study was approved by the
Western Institutional Review Board and by the Human
Subjects Research Office at the University of Miami. A
signed informed consent was obtained from the subjects.
The study is registered at clinicaltrials.gov: NCT01216748.
Measurements
Airway blood flow (Q

aw)
A previously validated soluble inert gas uptake method
was used to measure Q

aw
[8,9]. The subjects first inhaled
room air to total lung capacity. After exhaling 500 mL,
they rapidly re-inhaled the same volume of a pre-mixed
gas consisting of 10% dimethylether (DME), balance ni-
trogen. After a predetermined breathhold time, the
subjects then exhaled through a critical flow orifice to
standardize the expiratory flow. During the entire man-
euver, the instantaneous concentrations of DME and ni-
trogen were measured at the airway opening with a mass
spectrometer (Perkin-Elmer; Pomona, CA). The maneu-
ver was performed with two breathhold times each of 5
and 15 sec in random order. The DME concentration
(F
DME
) at the end of phase 1 of the nitrogen wash-in
curve (defining a virtual anatomical dead space, V
D
)was
obtained. The difference in F
DME
between the two
breathhold times (ΔF
DME
) multiplied by V
D
was used to
calculate DME uptake (V

DME
) over the intervening
10 sec. From V

DME
, the mean DME concentration be-
tween the two breathholds (F
mDME
) and the solubility
coefficient for DME in blood and tissue (α), was calculated
using the Fick principle (Q

aw
=V

DME
/(α•F
mDME
). Q

aw
was
normalized for V
D
;therefore,V
D
cancels out and wasn’t
measured. Q

aw
was expressed as μl.min
-1
.mL
-1
,whereμl.
min
-1
reflects blood flow and mL reflects the virtual ana-
tomical deadspace. At each Q

aw
determination, data from
two 5 sec and two 15 sec breathholds were analyzed.
AQ

aw
determination took less than 5 min.
Blood pressure and arterial oxygen saturation (SaO
2
)
by pulse-oximetry were monitored at each measurement
point. Mean systemic arterial pressure (perfusion pres-
sure for airway blood flow) was calculated as diastolic
pressure plus 1/3 pulse pressure.
Spirometry
For spirometry (Forced Expired Volume in one sec-
ond/FEV1, Forced Vital Capacity/FVC, FEV
1
/FVC), a
Koko spirometer was used (Ferraris Respiratory, Louis-
ville, CO). The tracing with the highest FVC of three
forced vital capacity maneuvers was analyzed. Pre-
dicted normal values were taken from Crapo et al [10].
The values were expressed in absolute values and per-
cent of predicted.
Exhaled Breath Condensate (EBC) pH was obtained as
recommended by an American Thoracic Society/Euro-
pean Respiratory Society task force [11]. The EBC samples
were collected with the condenser temperatures close to
0°C. We determined EBC pH immediately following sam-
ple collection without argon purging [12], using a Thermo
Orion 3 Star pH Meter and Micro pH Electrode (Thermo
Scientific Orion Inc., Carlsbad, CA). During the different
breathing maneuvers, EBC samples were collected by
directing the subject’s exhaled breath into a pre-cooled
(-10°C) tube for 5 min, using the disposable R-tubes® from
Respiratory Research System (Charlottesville, VA). Over
this period of time, approximately 0.5-1 mL of condensate
was collected. For further standardization, the subjects
were not allowed to drink or eat for at least one hour be-
fore the EBC samples were collected [13,14].
Ventilation
Compressed air was lead through a calibrated airflow
regulator (Dakota Instruments, Orangeburg, NY) and an
anesthesia bag to a one-way valve at the mouthpiece. Dur-
ing the ventilatory maneuvers, the airflow was adjusted to
keep the anesthesia bag from collapsing or overinflating
until a steady state was reached [15]. The airflow was read
at that point and expressed as l.min
-1
. The system had a
deadspace of 100 mL between the mouthpiece and the
valve separating inspiration from expiration. Subjects wore
a nose clip for all measurements.
Cancado et al. BMC Pharmacology and Toxicology (2015) 16:9 Page 2 of 7

Respiratory maneuvers
Different respiratory maneuvers were used to change
airway pH as reflected by EBC pH.
The same measurements were made in all subjects dur-
ing quiet breathing, hypercapnic hyperventilation, hypo-
capnic hyperventilation and eucapnic hyperventilation. To
induce hypercapnic hyperventilation, we employed a modi-
fication of a previously described procedure [15]. While
monitoring S
a
O
2
using pulse oximetry and end-tidal CO
2
by mass-spectrometry (Perkin-Elmer, Pomona, CA) on a
breath by breath basis, CO
2
was bled into the inspired air
to achieve an end-tidal pCO
2
of at least 55 mmHg,
expected to result in a decrease in systemic pH of about
0.1 pH units. For hypocapnic hyperventilation, the subjects
were instructed to breathe fast and deep until their end-
tidal pCO
2
fell to 30 mmHg, corresponding to a systemic
pH increase of about 0.1 pH units. For eucapnic hyperven-
tilation, the subjects were instructed to increase their venti-
lation to the highest level of ventilation recorded in the
previous two hyperventilation maneuvers, while CO
2
was
bled into the inspired air to maintain end-tidal pCO
2
at
40 mmHg. This maneuver was used to separate the effect
of ventilation from the effect of pH on albuterol respon-
siveness. The same mouthpiece set-up was used for the
measurement of Q

aw
, EBC pH, and ventilation.
Protocol
The subjects were instructed to abstain from ingesting
alcoholic beverages the night before each study day and
not to ingest caffeinated drinks for at least 12 hours be-
fore the study. The subjects were also instructed not to
use phosphodiesterase type 5 inhibitors for 12 hours be-
fore coming to the laboratory.
There were 6 visit days. On day 1, informed consent
was obtained and the subjects underwent a physical
examination to ensure good general health. In females,
a urine pregnancy test was performed to rule out
current pregnancy. Then, spirometry was performed to
ensure normal lung function. For technical reasons,
EBC pH, Q

aw
responses to albuterol and the level of
ventilation could not be assessed simultaneously
during the breathing maneuvers. Therefore, these pa-
rameters were measured during different breathing
maneuvers on different days in random order (quiet
breathing, hypercapnic hyperventilation, hypocapnic
hyperventilation and eucapnic hyperventilation
.
Exhaled breath condensate collection
For each respiratory maneuver, the subjects breathed at
the respective ventilatory level for 2 minutes followed by
a 5 minutes EBC collection while maintaining the same
breathing pattern.
Determination of ventilation
This was done during the different respiratory maneu-
vers as described for the EBC collection. Ventilation was
measured during the 5 min steady state period.
Q

aw
response to albuterol
This was done during the four breathing protocols as
described above. During the 5 min steady state breathing
period, Q

aw
was first measured with a short break in the
breathing maneuver. After resuming the designated breath-
ing maneuver, the subjects inhaled albuterol (180 μg) deliv-
ered by a metered dose inhaler using a holding chamber
during a brief interruption of the breathing maneuver. The
subjects then continued to perform the prescribed respira-
tory maneuver for another 5 min. Q

aw
was again measured
15 min after drug administration during quiet breathing.
Albuterol responsiveness was expressed as the difference
between pre-and post albuterol Q

aw
(ΔQ

aw
).
Statistical analysis
Values are presented as mean ± standard error (SE). Dif-
ferences between the groups were analyzed by a non-
parametric Kruskal-Wallis ANOVA test followed, when
significant, by the Mann-Whitney Utest for compari-
sons between groups. Values were expressed as mean ±
SE and a p value less than 0.05 was accepted as a statis-
tically significant difference. All statistics were analyzed
with SPSS software (Statistical Product and Services So-
lutions, version 18.0; SPSS Inc., Chicago, IL).
Results
The demographics and baseline characteristics of study
participants are shown in Table 1, consistent with good
cardiovascular and respiratory health. All subjects com-
pleted the protocol.
Table 1 Demographics and baseline characteristics of
study participants (visit 1)
Subjects
N10
Mean age (range), yr 37 ± 8 (24-53)
Sex (M/F) 3 / 7
Heart rate, beats/min 65 ± 12
Systolic BP, mmHg 106 ± 5
Diastolic BP, mmHg 68 ± 4
SAT O
2
99 ± 1
FEV
1
, liters 3.26 ± 0.61
FEV
1
, %predicted 104 ± 1
Values are mean ± SE.
N =number of subjects; M, male; F, female; BP, blood pressure; SAT O
2
, arterial
oxygen saturation measured by pulse oximetry; FEV
1
, forced expiratory volume
in 1 second.
Cancado et al. BMC Pharmacology and Toxicology (2015) 16:9 Page 3 of 7

Ventilation and EBC pH
The levels of ventilation at the time of albuterol admin-
istration during the four respiratory maneuvers are
shown in Table 2. Hypercapnia and hypocapnia changed
EBC pH, while eucapnic hyperventilation had no effect
on EBC pH. Thus, it was possible to unlink the level of
ventilation from the changes in EBC pH, which presum-
ably is a reflection of airway surface liquid pH.
Airway blood flow response to albuterol
Mean systemic blood pressure and oxygen saturation were
not different at the Q

aw
measurement points (baseline,
pre-albuterol and post albuterol). The lack of changes in
mean systemic blood pressure obviated the need to ex-
press the airway blood flow responses as airway blood flow
conductance. Vasodilator responses therefore were re-
ported as ΔQ

aw
.
Baseline mean Q

aw
values were similar before the four
breathing maneuvers and remained unchanged during
the subsequent breathing maneuvers as reflected by the
pre-albuterol values (Table 3). All subjects had similar
albuterol response to the different breathing maneuvers
.
Albuterol increased mean Q

aw
significantly, by 46.2 and
33.8% 15 min post drug inhalation during quiet breath-
ing and eucapnic hyperventilation, respectively (Table 3,
Figure 1). In contrast, albuterol had no effect on mean
Q

aw
during hypercapnic hyperventilation (4.9%) or hypo-
capnic hyperventilation (-1.3%) maneuvers, which were
associated with a decrease or increase in EBC pH.
Discussion
The purpose of this study was to determine if respiratory
acidosis and alkalosis have an effect on the physiological
response to inhaled albuterol in airway tissue and if the
effect is related to the ventilation-associated changes in
airway pH as reflected by exhaled breath condensate (EBC)
pH. In order to demonstrate the role of pH in the observed
changes in albuterol responsiveness associated with re-
spiratory acidosis and alkalosis, it was necessary to unlink
the changes in EBC pH from the changes in ventilation.
This was done by comparing quiet breathing with eucapnic
hyperventilation, where ventilation changes while pH is
kept constant. Albuterol responsiveness was the same dur-
ing the two maneuvers, suggesting that hyperventilation
per se did not alter albuterol responsiveness. Likewise, the
preserved albuterol responsiveness during eucapnic hyper-
ventilation ruled out the possibility that cooling and drying
oftheairwaycouldhavebeenthecauseoftheblunted
albuterol responsiveness during respiratory alkalosis and
acidosis. Eucapnic hyperventilation was investigated last in
ordertobeabletoreproducethehighestlevelofventila-
tion achieved in any of the other maneuvers. We therefore
are confident that ventilation per se had no effect on albu-
terol responsiveness. The level of ventilation during quiet
breathing was higher than one would have expected in
healthy subjects at rest (mean 14.4 L
.
min
-1
). It has previ-
ously been reported that wearing a nose clip and breathing
through a mouthpiece increases tidal volume and minute
ventilation [16]. In addition, the breathing setup we used
for our study included a 100 mL deadspace, another stimu-
lus for increasing tidal volume and respiratory rate.
In our study, the intended target of albuterol was
airway vascular smooth muscle contained in the airway
wall. Since the different respiratory maneuvers by them-
selves had no effect on Q

aw
we were able to assess the
effect of respiratory acidosis and alkalosis on albuterol
responsiveness. In some systemic vascular beds, hyper-
capnic acidosis causes relaxation and hypocapnic alkal-
osis causes constriction, resulting in corresponding
blood flow changes [5]. The airway circulation appears
not to be subject to this regulation at least in the range
of pCO
2
changes seen in the present study in which
changes in pH had no effect on Q

aw
; however, they
affected albuterol responsiveness. We allowed 5 min for
albuterol absorption during the four breathing maneu-
vers, and measured Q

aw
15 min after drug inhalation.
This was done because in previous studies we found that
the maximum response typically occurs after 15 min
while a vasodilator response to inhaled albuterol is
already seen after 5 min [17].
We found that both airway alkalosis and acidosis
attenuated albuterol responsiveness. The pH-sensitivity
of albuterol responsiveness could have been related to a
combination of several factors, including absorption
and transport of albuterol from the airway surface to
theairwayvascularsmoothmuscle,β
2
-adrenergic re-
ceptor function, vascular endothelial function or vascu-
lar smooth muscle responsiveness.Invitroand animal
experiments suggest that all of these functions can be
pH-dependent.
Acidosis
The majority of the currently used β
2
-adrenergic bron-
chodilators, including albuterol, cannot freely diffuse
across the epithelial cell membrane because they are
hydrophilic and carry a transient or permanent positive
Table 2 Ventilation and exhaled breath condensate pH
during respiratory maneuvers
Challenges V
(L
.
min
-1
) EBC pH (units)
Quiet breathing 14.4 ± 4.2 6.39 ± 0.14
Eucapnic hyperventilation 35.5 ± 3.4* 6.31 ± 0.08
Hypocapnic hyperventilation 35.2 ± 3.3* 6.59 ± 0.15**
Hypercapnic hyperventilation 24.4 ± 2.9* 5.88 ± 0.14**
V
, ventilation.
EBC, exhaled breath condensate.
*p < 0.05 vs. quiet breathing.
**p <0.02 vs. quiet breathing.
Cancado et al. BMC Pharmacology and Toxicology (2015) 16:9 Page 4 of 7

charge at physiological pH. Thus, the epithelium of the
airway becomes a barrier to these agents, requiring cel-
lular or paracellular transport across the epithelial lining
of the airway to reach their intended target tissues in-
cluding airway vascular smooth muscle. We have previ-
ously demonstrated the existence of an organic cation
transport machinery in the human airway epithelium
and showed that this process is largely mediated by the
organic cation/carnitine transporter OCTN2, which is
likely involved in the delivery of inhaled hydrophilic
cationic bronchodilators to the airway tissue [1]. We
showed that cationic drug uptake is pH dependent, with
about 3-fold lower rates at an acidic pH (5.7) than alka-
line pH (8.2). This mechanism could have been fully or
Table 3 Effects of respiratory maneuvers on airway blood flow (Q

aw
)
Pre-maneuver Q

aw
Q

aw
during steady state Q

aw
15 min post albuterol
Quiet breathing Mean ± SE 38.1 ± 1.4 37.2 ± 1.5 53.7 ± 2.1*
Median 37.7 35.7 54.3
25% quartile 33.8 34.1 46.9
75%quartile 42.1 41.9 58.7
Eucapnic hyperventilation Mean ± SE 41.8 ± 4.3 42.6 ± 4.3 56.4 ± 4.0*
Median 40.7 42.7 49.7
25% quartile 34.8 34.8 46.7
75% quartile 48.8 48.8 61.7
Hypocapnic hyperventilation Mean ± SE 42.9 ± 2.8 45.2 ± 3.4 45.1 ± 2.9
Median 40.1 42.2 43.7
25% quartile 37.2 35.0 38.8
75% quartile 49.1 49.7 54.1
Hypercapnic hyperventilation Mean ± SE 42.1 ± 2.4 44.8 ± 3.8 46.7 ± 4.0
Median 37.5 44.7 43.7
25% quartile 35.5 34.1 38.4
75% quartile 46.2 57.1 52.2
Q

aw
is expressed in μl.min
-1
.mL
-1
. * p < 0.05 vs. steady state.
Figure 1 Relative albuterol-induced changes in airway blood flow (ΔQ

aw
) during four breathing maneuvers. Values are mean ± SE. *p <
0.01 and ** p <0.02 vs quiet breathing and eucapnic hyperventilation.
Cancado et al. BMC Pharmacology and Toxicology (2015) 16:9 Page 5 of 7

partially responsible for the blunted albuterol respon-
siveness during respiratory acidosis associated with a de-
creased airway surface liquid pH. We have also shown
that albuterol crosses the airway epithelium via the para-
cellular route [18]. The paracellular pathway can also
mediate pH-dependent permeability to pH-dependent
changes in negative charges.
It has also been reported that acidosis can cause rapid
desensitization and uncoupling of β
2
-adrenergic recep-
tors [2], possibly leading to albuterol unresponsiveness
as seen in the present investigation.
Albuterol-induced vasodilation is endothelium-dependent,
involving endothelial relaxant factors including nitric oxide
[19]. Although the observations on the effects of intracellu-
lar and extracellular acidosis and pCO
2
on endothelial func-
tion have not been consistent, the majority of studies have
shown that acidosis can impair endothelial function [4-6]. It
is likely that airway surface liquid pH is a reflection of extra-
cellular pH, but changes in both extracellular and intracellu-
lar pH have been implicated in the effect of acidosis on
endothelial function. Thus, endothelial dysfunction could
have had a role in the blunted albuterol responsiveness in
our study. Finally, airway vascular smooth muscle function
could be directly affected by acidosis. In particular, acidosis
can lead to smooth muscle cell hyperpolarization, which in
turn could attenuate albuterol-induced vasodilation [5].
Alkalosis
Respiratory alkalosis also attenuated albuterol-induced
vasodilation in our study. In vitro, alkalosis increases the
transport of organic cations such as albuterol across the
airway epithelium via the transcellular and paracellular
routes [18]. Alkalosis may also increase β
2
-adrenergic re-
ceptor ligand binding [3]. Finally, alkalosis has been shown
to cause endothelium-dependent vasodilation without
altering endothelial nitric oxide synthase function [5]. All
of these actions would be expected to potentiate inhaled
albuterol-induced vasodilation. The mechanistic explan-
ation for our observation that respiratory alkalosis has the
same attenuating effect on albuterol responsiveness as
acidosis remains unclear at this time.
Conclusions
Patients with airway disease are likely to have highly vari-
able airway surface liquid pH and adrenergic airway
smooth muscle responsiveness [20-23]. Therefore, we de-
cided to investigate the pH dependence of β
2
-adrenergic
responsiveness as a marker of albuterol responsiveness in
healthy subjects with normal β
2
-adrenergic smooth muscle
responsiveness in whom the airway surface liquid pH can
be artificially manipulated. From a clinical perspective,
airway smooth muscle would have been a more meaningful
airway wall target to assess responsiveness to inhaled albu-
terol. However, healthy subjects do not have an increased
airway smooth muscle tone and responses to albuterol
would have been too small to study the effects of respira-
tory acidosis and alkalosis. We chose not to include
patients with asthma or COPD in the investigation because
they have a blunted airway blood flow response to albute-
rol due to endothelial dysfunction [18], and because meas-
uring airflow responses by pulmonary function testing
would have been technically difficult under the experimen-
tal conditions of the study.
Our in vivo observation showed that both respiratory
acidosis and alkalosis blunt albuterol responsiveness in
the airway wall, although it is not known whether the
effect is driven by intracellular or extracellular pH or
pCO
2
and which of the above-mentioned mechanisms
may be involved. In this study we found that albuterol
responsiveness as assessed by Q

aw
in the airway is
blunted by acidosis and alkalosis, using Q

aw
as a bio-
assay. It remains to be shown whether the clinical bene-
fits of inhaled albuterol, i.e., bronchodilation may be less
than expected during acute respiratory acidosis and
alkalosis, which can be associated with exacerbations of
asthma and COPD.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
AW: Conception and design, analysis and interpretation of data, drafting of
the manuscript, critical revision of the manuscript for important intellectual
content, supervision and final approval of the version to be published. MS:
Conception and design and manuscript editing. ESM: Acquisition of data,
statistical analysis, and interpretation of data. JEC: Acquisition and analysis of
data, GH: Conception and design of the study. MEM: Sample analysis and
interpretation. JA: Acquisition of data. All authors read and approved the
final manuscript.
Authors’information
Matthias Salathe and Adam Wanner are senior authors contributed equally
to this paper.
Funding
The study was supported by NIH 1R01HL060644.
Author details
1
Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University
of Miami School of Medicine, Miami, FL 33136, USA.
2
Department of
Pulmonology, Semmelweis University School of Medicine, Budapest,
Hungary.
Received: 15 October 2013 Accepted: 16 March 2015
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