High CO2 levels impair alveolar epithelial function independently of pH.

Abstract

In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO2) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO2 levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO2. The Na,K-ATPase consumes approximately 40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO2 on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.
We found that short-term increases in pCO2 impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO2, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCzeta which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.
Our data suggest that alveolar epithelial cells, through which CO2 is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO2 levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.

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High CO2Levels Impair Alveolar Epithelial Function
Independently of pH
Arturo Briva1,3, Istva ´n Vada ´sz1,4, Emilia Lecuona1, Lynn C. Welch1, Jiwang Chen1, Laura A. Dada1, Humberto E. Trejo1, Vidas Dumasius1, Zaher S.
Azzam1,5, Pavlos M. Myrianthefs1,6, Daniel Batlle2, Yosef Gruenbaum1,7, Jacob I. Sznajder1*
1Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America,
2Division of Nephrology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America, 3Departamento de
Fisiopatologı ´a, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay, 4University of Giessen Lung Center, Justus Liebig University,
Giessen, Germany, 5Ruth & Bruce Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel,
6Intensive Care Unit, Athens University, ‘‘KAT’’ General Hospital, Athens, Greece, 7Department of Genetics, Hebrew University of Jerusalem,
Jerusalem, Israel
Background. In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung
airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange,
but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO2) is a by-product of cellular
energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO2
levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-
neuronal cells sense and respond to CO2. The Na,K-ATPase consumes ,40% of the cellular metabolism to maintain cell
homeostasis. Our study examines the effects of increased pCO2on the epithelial Na,K-ATPase a major contributor to alveolar
fluid reabsorption which is a marker of alveolar epithelial function. Principal Findings. We found that short-term increases in
pCO2impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense
and respond to high levels of CO2, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via
activation of PKCf which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into
intracellular pools. Conclusions. Our data suggest that alveolar epithelial cells, through which CO2is eliminated in mammals,
are highly sensitive to hypercapnia. Elevated CO2levels impair alveolar epithelial function, independently of pH, which is
relevant in patients with lung diseases and altered alveolar gas exchange.
Citation: Briva A, Vada ´sz I, Lecuona E, Welch LC, Chen J, et al (2007) High CO2Levels Impair Alveolar Epithelial Function Independently of pH. PLoS
ONE 2(11): e1238. doi:10.1371/journal.pone.0001238
INTRODUCTION
Pulmonaryedemaoccursinpatientswithcongestiveheartfailureand
acute respiratory distress syndrome and often requires mechanical
ventilation [1,2]. It has been proposed that to prevent ventilator
induced lung injury, patients should be ventilated with low tidal
volumes which may result in hypercapnia [3,4]. Some investigators
have proposed that ‘‘permissive hypercapnia’’ could be beneficial in
patients with lung injury [5,6]. More recent studies have suggested
that hypercapnia may have deleterious effects on the lungs; however,
there has not been an attempt to define whether these effects were
due to high pCO2levels or the associated acidosis [7–10].
Average human respiration generates approximately 450 liters
of carbon dioxide (CO2) per day [11], which, together with CO2
produced from other sources, is removed from the atmosphere by
plants during photosynthesis. The notion of a sensor for CO2has
been proposed in plants and insects. In plants, the stomata of
guard cells close when exposed to high CO2concentrations via
utilization of specific signaling pathways [12] while in Drosophila
a CO2-sensitive receptor has been described in the olfactory
neurons [13]. Recently, it has been reported that mice also can
detect CO2 through the olfactory system involving carbonic
anhydrase [14]. The effects of hypercapnia on excitable cells are
well characterized and include depolarization of glomus cells,
which trigger an increase in alveolar ventilation to maintain
normal CO2levels in the body [15]. In contrast, the effects of CO2
on non-excitable mammalian cells are not well understood. In
vascular smooth muscle cells increased CO2 levels have been
shown to activate mechanisms of cell adaptation, however, they
were thought to be due to the changes in pH occurring during
hypercapnia [16]. A recent report has suggested that renal
epithelial cells respond to changes in CO2concentrations via yet
unidentified mechanisms [17].
Active Na+transport effects edema clearance from the lungs via
apically located sodium channels and basolateral Na,K-ATPase
with water following iso-osmotically the Na+gradient [18–20].
The Na,K-ATPase, a major modulator of cellular homeostasis, is
expressed in all mammalian cells. It consists of a catalytic a-
subunit and a regulatory b-subunit to exchange Na+and K+across
the plasma membrane, consuming ,40% of the energy of the cell
in this process [21]. Inhibition of Na,K-ATPase activity can result
from a decrease in the number of Na,K-ATPase molecules at the
plasma membrane, usually via endocytosis and subsequent
degradation of Na,K-ATPase proteins [22].
We have reported that hypercapnia decreases alveolar fluid
reabsorption (AFR) in rats, however, carbonic anhydrase activity
Academic Editor: Joel Schnur, Naval Research Laboratory, United States of
America
Received August 31, 2007; Accepted November 6, 2007; Published November 28,
2007
Copyright: ? 2007 Briva et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: Supported in part by: HL-85534, T32-HL76139 and DFG/SFB 547.
Competing Interests: The authors have declared that no competing interests
exist.
* To
northwestern.edu
whom correspondence should beaddressed.E-mail: j-sznajder@
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did not have an effect on AFR [23]. Here, we set out to determine
whether the non-excitable alveolar epithelial cell, the site of CO2
elimination in mammals, is affected by elevated CO2levels or the
associated acidosis, focusing on the Na,K-ATPase and the alveolar
epithelial function.
RESULTS
High CO2levels impair alveolar fluid reabsorption
independently of pH
Alveolar fluid reabsorption (AFR), a major function of the lung
epithelium, is contributed by apical Na+channels and basolateral
Na,K-ATPase [24,25]. During lung injury, the alveolar epithelial
Na,K-ATPase function is typically inhibited in association with
impaired AFR [26]. As depicted in Figure 1A, high CO2levels,
independently of extracellular pH, decreased AFR by ,50%,
without affecting the passive fluxes of small or large solutes
indicating that there were no changes in epithelial barrier
permeability (Figure 1B and 1C). Also, the AFR inhibition by
short-term hypercapnia was reversible within one hour of
normalization of CO2levels (Figure 1D). Na,K-ATPase activity
and protein abundance was decreased at the basolateral
membranes isolated from hypercapnia exposed peripheral lung
tissue samples (Figure 2A and 2B).
High CO2levels regulate Na,K-ATPase
independently of pH
We investigated whether cultured alveolar epithelial cells were
sensitive to elevated levels of CO2by examining the effects of CO2
on Na,K-ATPase function. In rat alveolar epithelial type II (ATII)
cells exposed to increasing levels of CO2 (while buffering the
medium to a pH of 7.40) for 30 minutes, the Na,K-ATPase
catalytic activity decreased in a concentration-dependent manner
(Figure 3A) due to the Na,K-ATPase a1-subunit endocytosis from
the plasma membrane into intracellular compartments, as assessed
by cell surface biotinylation (Figure 3B) and live cell imaging in
GFP a1-A549 (Figure 3C). Also, the Na,K-ATPase a1-subunit
endocytosis induced by short-term hypercapnia was reversible
within one hour of normalization of CO2levels (Figure 3D). These
effects were due to high CO2 levels and not to alterations in
extracellular pH (pHe), because neither the Na,K-ATPase activity
Figure1.HighCO2levelsimpairalveolarepithelialfunctioninrats.(A)Isolatedratlungswereperfusedfor1 hwith40or60 mmHgCO2withpHe:7.4or
pHe: 7.2 and alveolar fluid reabsorbtion (AFR) was measured as described in the experimental procedures. Graph represents the mean6SEM, (n=5). (B)
PassivemovementofNa+(closedbars)and3H-Mannitol(openbars)wasmeasuredasdescribedindetailinthesupplementarymethods.Graphrepresents
themean6SEM(n=5).Differencesamonggroupswerenotstatisticallysignificant.(C)Albuminfluxfromthepulmonarycirculationintothealveolarspace
was determined from the fraction of fluorescein isothiocyanate (FITC)-labeled albumin, placed in the perfusate that appeared in the alveolar instillate
during the experimental protocol. Graph represents the mean6SEM, (n=5). Differences among groups were not statistically significant. (D) A three hour
experiment was performed in isolated rat lungs. Lungs were perfused for 1h with 40 mmHg CO2, switched to 60 mmHg CO2for the second hour, and
back to 40 mmHg CO2for the third hour (pHe: 7.4). Alveolar fluid reabsorption (AFR) was measured as described in the experimental procedures. Graph
represents the mean6SEM of 5 independent experiments. Bars in panels A, B and C represent the mean from different groups of animals. Bars in panel D
represent the mean of single samples from the same group of animals. pHe: extracellular pH. *p,0.05; **p,0.01.
doi:10.1371/journal.pone.0001238.g001
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nor the Na,K-ATPase protein abundance at the plasma
membrane were affected by low pHe levels at constant CO2
concentrations (Figure 3A, 3B and 3C). We also observed that the
intracellular pH (pHi) markedly decreased when cells were
incubated at a pHe of 7.20 and normal CO2 concentration
(40 mmHg) (Figure 4). In contrast, incubating cells with increasing
levels of CO2at pHeof 7.40 resulted in a mild and transient
decrease in pHiwhich rapidly returned to baseline levels. These
data suggest that high CO2 levels in alveolar epithelial cells,
independently of pH, promote the endocytosis of Na,K-ATPase
and thus inhibit its activity.
CO2activates PKCf which phosphorylates Na,K-
ATPase a1-subunit at Ser-18
Plasma membrane proteins undergo post-translational modifica-
tions, such as phosphorylation or oxidation which leads them to
endocytose [27]. Previous reports have suggested that upon
stimulation, members of the PKC family translocate from the
cytosol to the membrane compartments and phosphorylate the
plasma membrane Na,K-ATPase leading to its endocytosis.
Specifically, PKCf has been shown to directly phosphorylate the
Na,K-ATPase a1-subunit at Ser-18 [28]. Here, we observed that
exposing cells to high CO2levels translocated both classical (a) and
atypical (f) but not novel (e) PKC isotypes to the plasma
membrane (Figure 5A). To determine whether PKCf was involved
in CO2-induced Na,K-ATPase endocytosis, we incubated the cells
with a selective PKCf inhibitory peptide (1 h, 0.1 mM) and found
that in cells incubated with this inhibitory peptide, but not with
a selective PKCe inhibitory peptide (1 h, 5 mM), CO2-induced
Na,K-ATPase endocytosis was prevented (Figure 5B). To further
determine the role of PKCf, we exposed cells expressing
a dominant negative PKCf, (DN PKCf) to high levels of CO2
and compared them with cells expressing an empty vector. As
shown in Figure 5C, high CO2levels did not cause Na,K-ATPase
endocytosis in cells expressing the DN PKCf . To test whether
these observations have physiological relevance, rats where
infected with adenovirus coding for a DN PKCf and as shown
in Figure 5D, in the rats expressing the DN PKCf the CO2-
induced inhibition of the alveolar fluid reasorption was prevented.
High levels of CO2induced Na,K-ATPase a1-subunit phos-
phorylation as assessed by a ‘‘back phosphorylation’’ assay
(Figure 6A). This phosphorylation was prevented in cells treated
with a PKCf inhibitory peptide suggesting that the Na,K-ATPase
is a target of PKCf (Figure 6A). Furthermore, in A549 cells
expressing a rat Na,K-ATPase a1-subunit where the Ser-18
residue was mutated to alanine (S18A), the high CO2-induced
Na,K-ATPase endocytosis was prevented as assessed by cell
surface biotinylation and live cell imaging with epi-fluorescent
microscopy (Figure 6B and 6C).
DISCUSSION
Increased CO2 levels are observed in patients with impaired
alveolar ventilation such as chronic obstructive pulmonary disease
(COPD) and are predictive of poor prognosis [29]. In patients with
lung injury on mechanical ventilation, acute hypercapnia is
commonly a manifestation of hypoventilation and is associated
with acidosis. Hypercapnic acidosis has been reported to impair
cellular functions such as host inflammatory response and other
deleterious effects including intracranial bleeding, decreased
colonic Na+transport and changes in pulmonary vascular
resistance leading to ventilation/perfusion mismatch [30].
Our study was designed to determine whether short term high
CO2levels or the associated acidosis affect the alveolar epithelial
function, assessed as active Na+transport and thus alveolar fluid
reabsorption. We observed that high CO2levels impaired alveolar
epithelial function which may be of importance for patients with
lung injury, where inability to clear alveolar edema is associated
with increased mortality [31]. To determine whether the impaired
alveolar fluid reabsorption was due to high CO2 levels or to
acidosis we bubbled into the pulmonary circulation high CO2
levels but buffered the solution to maintain a pH of 7.40 and also
conducted experiments with normal CO2levels but acidic pH. As
shown in Figure 1A, high CO2levels either with or without acidosis,
resulted in impaired alveolar fluid reabsorption. These changes were
reversible when normocapnia was restored (Figure 1D). Neither
hypercapnia nor metabolic acidosis caused significant changes in
epithelial permeability to small or large solutes.
We also studied the effects of CO2on the alveolar epithelial cell
Na,K-ATPase, a major contributor to alveolar fluid clearance. We
incubated alveolar epithelial cells with high CO2 levels as
compared to metabolic acidosis and observed that Na,K-ATPase
activity and protein abundance at the plasma membrane de-
creased in high CO2conditions at normal extracellular pH while
lowering the pH with normal CO2 levels had no effect (see
Figure 3). The changes in protein abundance at the plasma
membrane occurred without affecting the total cell pools of the
Na,K-ATPase consistent with the notion that the Na,K-ATPase
were endocytosed from the plasma membrane into intracellular
compartments and not degraded.
These data raise the question of whether these effects are due to
a CO2 specific ‘‘sensor’’ in the alveolar epithelium or due to
changes in intracellular pH. The notion of a sensor for high CO2
levels has been proposed in plants where stomata of Guard cells
close when exposed to high CO2levels [12]. Also, Zhou et al
suggested that in renal cells, CO2levels may be sensed outside the
glomus cells in the carotid bodies [17]. Thus, we measured the
intracellular pH in cells exposed to high CO2levels at normal pH
or normocapnia and low pH by modifying the extracellular
perfusing solution and in both cases we observed a decrease in
intracellular pH which normalized in the high CO2exposed cells
Figure 2. Na,K-ATPase function is impaired in rat lungs exposed to
hypercapnic acidosis. (A) Basolateral membranes (BLM) were purified
from the peripheral lung tissue of rat lungs exposed to 40 mmHg CO2
(pHe: 7.4) or 60 mmHg CO2(pHe: 7.2), and Na,K-ATPase activity was
measured as [c-32P]ATP hydrolysis. Graph represents the mean6SEM,
(n=3). (B) BLM and total membranes were purified from the peripheral
lung tissue of rat lungs treated as (A), and Na,K-ATPase protein
abundance was assessed by Western blot. Graph represents the
mean6SEM, (n=3). Representative blots of Na,K-ATPase a1-subunit at
the BLM and total membrane protein abundance are shown. pHe:
extracellular pH. * p,0.05.
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but remained low in cells exposed to low extracellular pH
(Figure 4). Collectively, these data suggest that high CO2levels
and not acidosis triggered the endocytosis and thus the inhibition
of Na,K-ATPase activity in alveolar epithelial cells.
Previous reports have suggested that inhibition of Na,K-ATPase
activity is related to the phosphorylation status of the Na,K-
ATPase via activation of different PKC isozymes [28,32]. We
found that PKCf and PKCa isozymes were rapidly activated by
high CO2(Figure 5A), however, only PKCf participated in the
signaling pathway leading to Na,K-ATPase endocytosis and
impaired alveolar fluid reabsorption. Furthermore, although we
can not completely exclude the possibility that PKCf may activate
another kinase, our data strongly suggest that PKCf phosphor-
ylated the Na,K-ATPase a1- subunit as phosphorylation was
prevented by a PKCf inhibitory peptide (Figure 6A). It has been
previously reported that Ser-18 in the Na,K-ATPase a1-subunit is
the major site for PKC-mediated phosphorylation [33]. As shown in
Figure 6, in rat GFP-S18A-a1-subunit mutant cells exposed to high
CO2levels the Na,K-ATPase protein abundance at the plasma
membrane was unchanged suggesting that the phosphorylation of
the Ser18 was necessary for the CO2-induced endocytosis.
In summary, we provide evidence that exposing alveolar epithelial
cells to high CO2levelsaffects the function of the alveolar epithelium,
the primary site of CO2elimination in mammals. Exposing cells to
high concentrations of CO2, but not acidosis, resulted in activation of
PKCfwhichdirectlyphosphorylatedtheNa,K-ATPasea1-subunitat
the Ser-18 residue, triggering its endocytosis from the plasma
membrane and causing a decrease in Na,K-ATPase function
independently of pH changes. Notably, these effects were observed
notonlywhencellsandlungswereexposedtoveryhighlevelsofCO2
(,120 mmHg)butalsoattheclinicallyrelevantlevelsinpatientswith
respiratory failure or COPD (pCO2,60–80 mmHg). Thus, we
Figure 3. High CO2levels impair Na, K-ATPase activity independently of pH. (A) ATII cells were exposed to 40, 60, 80 and 120 mmHg CO2with
extracellular pH (pHe): 7.4 or to 40 mmHg CO2with pHe: 7.2 for 30 min, and Na,K-ATPase activity was measured as [c-32P]ATP hydrolysis. Graph
represents the mean6SEM, (n=5). (B) ATII cells were treated as described in (A) and the Na,K-ATPase protein abundance at the plasma membrane
(PM) was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean6SEM, (n=5). Representative blots
of Na,K-ATPase a1-subunit at the PM and total protein abundance are shown. (C) Live cell imaging of GFPa1-A549. Cells were exposed to 40 mmHg
CO2(pHe: 7.4) for 10 min (left panels) and then switched to 120 mmHg CO2(pHe: 7.4) (right, upper panel) or to 40 mmHg CO2(pHe: 7.2) (right, lower
panel) for 30 min. White arrows indicate the plasma membrane Na,K-ATPase. (D) Live cell imaging of GFPa1-A549. Cells were exposed to 40 mmHg
CO2(pHe: 7.4) for 10 min (left panel), switched to 120 mmHg CO2(pHe: 7.4) (middle panel) for 30 min and switched back to 40 mmHg CO2(pHe: 7.4)
for 60 min (left panel). White arrows indicate the plasma membrane Na,K-ATPase * p,0.05, ** p,0.01.
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propose that changes in CO2concentration are sensed not only by
neuronal but also by non-excitable mammalian cells such as the
alveolar epithelial cells. Identification of these sensing mechanisms
and further elucidation of the CO2-mediated signaling pathways will
not only further our understanding of a basic mechanism by which
mammalian cells adapt to hypercapnia, but may also lead to novel
therapeutic approaches in the treatment of lung diseases that are
associated with poor alveolar ventilation.
MATERIALS AND METHODS
Reagents
All cell culture reagents and G418 were from Mediatech Inc
(Herndon, VA). Rat brain protein kinase C (PKC) was purchased
from EMD Biosciences (San Diego, CA). Ouabain was from ICN
Biomedicals Inc. (Aurora, OH).22Na+and [c-32P] ATP were from
GE Healthcare (Piscataway, NJ). Percoll was from Amersham
Bioscience (Uppsala, Sweden). Myristoylated PKCf peptide and
PKCe v1-2 peptide were purchased from Biomol International
(Plymouth Meeting, PA).3H-mannitol was purchased from Perkin
Elmer (Life Sciences, Inc, Boston, MA). All other chemicals were
purchased from Sigma (St. Louis, MO). Na,K-ATPase a1subunit
monoclonal antibody (clone 464.6) was purchased from Upstate
Biotechnology (Lake Placid, NY). Rabbit polyclonal PKCa (C-20),
mouse monoclonal PKCe (E-5), mouse monoclonal PKCf (H-1),
mouse monoclonal GFP (B-2) antibodies, and Protein A/G plus
were purchased from Santa Cruz (Santa Cruz, CA). Rabbit
polyclonal anti-GFP antibody was from Clontech (Palo Alto, CA).
Rabbit polyclonal anti-actin was from Sigma (St. Louis, MO).
Secondary goat anti-mouse-HRP and goat anti-rabbit-HRP were
from Bio-Rad (Hercules, CA).
Animals
Pathogen-free male Sprague-Dawley rats weighing 320–350 g
were used for the isolated lung model and male Sprague Dawley
(200–225 g) were used for alveolar epithelial type II cell isolation
(Harlan, Indianapolis, IN). All animals were provided food and
water ad libitum, were maintained on a 12:12-h light-dark cycle,
and were handled according to National Institutes of Health
guidelines and to Institutional Animal Care and Use Committee
approved experimental protocols.
Isolated-perfused rat lung model
The isolated lungpreparation hasbeen described indetailpreviously
[18]. Briefly, the lungs and heart of anesthetized rats were removed
enbloc.Thepulmonaryarteryandleftatriumwerecatheterizedand
perfused continuously with a solution of 3% bovine serum albumin
(BSA) in buffered physiological salt solution (135.5 mM Na+, 119.1
Cl2, 25 mM HCO32, 4.1 mM K+, 2.8 mM Mg+, 2.5 mM Ca+2,
0.8 mMSO422, 8.3 mMglucose). Trace amounts of FITC-albumin
was also added to the perfusate. The recirculating volume of the
constant pressure perfusion system was 90 ml; arterial and venous
pressures were set at 12 and 0 cm H2O respectively. The vascular
pressures were recorded every 10 seconds with a multichannel
recorder (Cyber Sense Inc. Nicholasville, KY). The lungs were
immersed in a ‘‘pleural’’ bath (100 ml) filled with the same BSA
solution. The entire system was maintained at 37uC in a water bath.
Perfusate pH was maintained at 7.40 by bubbling with a gas mixture
of 95%O2/5%CO2. The lungs were then instilled via the tracheal
cannula in two sequential phases with a total of 5 ml volume of the
BSA solution containing 0.1mg/ml Evans Blue Dye (EBD)-albumin,
0.02 mCi/ml of22Na+and 0.12 mCi/ml of3H-mannitol. Samples
were taken from the instillate, perfusate, and bath solutions after an
equilibration time of 10 minutes from the instillation and again
60 minutes later. To ensure a homogenous sampling of the instillate,
a volume of 2 ml was aspirated and reintroduced into the airspaces
three times before removing each sample. All samples were
centrifuged at 3000 g for 10 minutes. Absorbance analysis of the
supernatant or EBD-albumin was performed at 620 nm in a Hitachi
model U2000 spectrometer (Hitachi, San Jose, CA). Analysis of
FITC-albumin (excitation 487 nm and emission 520 nm) was
performed in a Perkin-Elmer fluorometer (model LS-3B, Perkin-
Elmer, Oakbrook, IL).
The amount of instilled Evans blue dye albumin remains
constant during the experimental protocol, so any change in its
concentration at a given time reflects changes in the airspace
volume. Differences in concentration of Evans blue dye albumin
among samples taken from the instillate at the beginning and after
a determined time reflect the amount of fluid that has been
reabsorbed. The fraction of fluorescein isothiocyanate albumin
that appears in the alveolar space during the experimental
protocol was used to calculate the albumin flux from the
pulmonary circulation into the alveolar space [18,34].
Scintillation counts for22Na+and3H-mannitol were measured
in a Beckman beta counter (model LS 6500, Beckman Instruments
Inc., Fullerton, CA). Levels of pH, pO2and pCO2were monitored
and controlled to the experimentally set parameters by bubbling
more or less CO2and/or adding NaOH or HCl in the pulmonary
circulation perfusate. Samples were quickly processed to avoid
accidental degassing during measurement maneuvers.
The sodium concentration is equal and constant in all the
compartments and since22Na+is instilled only in the airspace, the
disappearance of the radioactive tracer from the airspaces reflects
the total or unidirectional Na+outflux from the airspace (JNa,out)
[18,34]. The passive or bidirectional Na+flux between the
airspace and the other compartments is the difference between
the unidirectional (JNa,out) and active Na+outflux (JNa,,net=
[Na+]J). The passive sodium movement can be calculated by:
JNa,in~½Naz?J(lnCt ? lnC0)=(lnVt ? lnV0)
Where, C0and Ct are the concentrations of22Na+initially and at
Figure 4. Effects of high CO2 and extracellular acidosis on
intracellular pH. Intracelluar pH (pHi) was measured in real time as
the change in fluorescence intensity of ATII cells loaded with BCECF/AM
and exposed approximately 3 min to 40 mmHg, and then for the
indicated time to 60, 80 and 120 mmHg CO2with pHe: 7.4, or 40 mmHg
with pHe: 7.2. pHe: extracellular pH.
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Figure 5. Role of PKCf in CO2-induced Na,K-ATPase a1-subunit endocytosis. (A) ATII cells were exposed for the indicated times to 40 or 120 mmHg
CO2(pHe: 7.4), cytosolic and 1% Triton X-100 soluble fractions were isolated, and translocation of different PKC isoforms was determined by Western
blot with specific antibodies. Representative blots for PKCa, PKCe and PKCf are shown (n=3). (B) ATII cells were incubated with vehicle, 5 mM PKCe
inhibitory peptide or 0.1 mM PKCf inhibitory peptide 1 h prior to being exposed to 40 or 120 mmHg CO2(pHe: 7.4) for 30 min. Na,K-ATPase protein
abundance at the PM was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean6SEM, (n=5).
Representative blots of Na,K-ATPase a1-subunit at the PM and total protein abundance are shown. (C) A549 cells expressing an empty vector or a DN-
PKCf were exposed to 40 or 120 mmHg CO2(pHe: 7.4) for 30 min. The Na,K-ATPase protein abundance at the PM was determined as above. Graph
represents the mean6SEM, (n=5). Representative blots of Na,K-ATPase a1-subunit at the PM and total protein abundance are shown. (D) Isolated rat
lungs from rats infected with Sham-surfactant, with null adenoviral vector (Ad-null), and adenoviral vector with DN PKCf construct (Ad-DN-PKCf)
were perfused for 1 h with 40 mmHg CO2(pHe: 7.4) or with 60 mmHg CO2(pHe: 7.2), and AFR was measured as described in the Methods section.
Graph represents the mean6SEM, (n=5). (E) Lungs from rats infected with Sham, Ad-null and Ad-DN-PKCf were thoroughly rinsed with ice-cold PBS,
tissue was homogenized, and the abundance of PKCf protein abundance was determined by Western blot. Representative Western blots of PKCf and
actin (loading control) are shown. (F) Lung tissues from rats infected with Sham, Ad-null and Ad-DN-PKCf were thoroughly rinsed with ice-cold PBS
and fixed in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was performed as described in the Online Data Supplement. Magnification
x40. *p,0.05, **p,0.01.
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time t respectively, and [Na+] is the constant sodium concentration
in the buffered salt albumin solution. Similarly the mannitol flux
(typically expressing the surface area permeability (PA) is given by:
PA~J(lnMt ? lnM0)=(lnVt ? lnV0)
Basolateral plasma membranes (BLM) isolation
BLM fractions were obtained from tissue collected from the distal
2–3 mm of rat right lungs following serial bronchoalveolar lavage
(PBS 7 ml65) and perfusion of the pulmonary artery (PBS620 ml)
as previously described [35]. Protein fractions enriched for the
BLM domain were obtained generating a 16% percoll gradient
[36].
Determination of Na,K-ATPase activity
Na,K-ATPase activity was measured as previously described
[22,37]. Briefly, Na,K-ATPase activity was calculated from BLM
from tissue or ATII cells exposed to the desired conditions, as the
liberated
activity) and samples assayed in reaction buffer with 2.5 mM
ouabain but devoid of Na+and K+(nonspecific ATPase activity).
Results are expressed as nmol of Pi/mg of protein/hour.
32P difference between the test samples (total ATPase
Total membranes from tissue
Tissue collected from the distal 2–3 mm of rat right lungs as
described above was homogenized in homogenization buffer
(1 mM EDTA, 1 mM EGTA, 10 mM Tris-HCl, pH: 7.5, 1 mg/
ml leupeptin, 100 mg/ml TPCK and 1 mM PMSF), centrifuged at
500 g to discard nuclei and debris, and the supernatant was
centrifuged at 100,000 g, 1 h, 4uC (TL ultracentrifuge, Beckman,
Rotor TLA 100.2). Pellet was considered as the total membrane
fraction.
Western blot analysis
Protein concentration was quantified by Bradford assay [38] (Bio-
Rad, Hercules, CA) and resolved in 10%–15% polyacrylamide
gels (SDS-PAGE). Thereafter, proteins were transferred onto
nitrocellulose membranes (Optitran, Schleider & Schuell, Keene,
NH) using a semi-dry transfer apparatus (Bio-Rad, Hercules, CA).
Incubation with specific antibodies was performed overnight at
4uC. Blots were developed with a chemiluminescence detection kit
(PerkinElmer Life Sciences, Boston, MA) used as recommended by
the manufacturer. The bands were quantified by densitometric
scan (Image J 1.29X, National Institutes of Health)
Alveolar epithelial type II cells isolation and cell
culture
ATII cells were isolated as previously described [39]. Briefly, the
lungs were perfused via the pulmonary artery, lavaged, and
digested with elastase (3 U/ml; Worthington Biochemical, Free-
hold, NJ). ATII cells were purified by differential adherence to
IgG-pretreated dishes, and cell viability was assessed by trypan
blue exclusion (.95%). Cells were resuspended in Dulbecco’s
modified Eagle’s medium containing 10% fetal bovine serum with
2 mM glutamine, 100 U/ml penicillin, 0.25 mg/ml amphotericin
B, and 100 mg/ml streptomycin. Cells were incubated in
a humidified atmosphere of 5% CO2-95% air at 37uC. The day
of isolation and plating is designated cultured day 0. All
experimental conditions were tested in day 2 cells.
Human A549 cells (ATCC CCL 185) expressing the GFP-rat-
Na,K-ATPase-a1-subunit (GFPa1-A549) [28], and GFP-S18A-rat-
Figure 6. PKCf phosphorylates the Na,K-ATPase a1-subunit at Ser 18.
(A) In vitro ‘‘back phosphorylation’’ assay was performed (as described
in the methods section) on the immunoprecipitated Na,K-ATPase a1-
subunit from GFPa1-A549 cells exposed to 40 or 120 mmHg CO2(pHe:
7.4) for 10 min in the presence or absence of PKCf inhibitory peptide
(0.1 mM, 1 h). Upper panel shows a representative autoradiography.
Lower panel depicts a representative Western blot (n=3; p,0.05 when
comparing 40 mmHg vs 120 mmHg). (B) A549 cells expressing the rat
GFPa1-subunit Na,K-ATPase (WT) or the rat GFP-S18A a1-subunit (S18A)
were exposed to 40 or 120 mmHg CO2(pHe: 7.4) for 30 min. The Na,K-
ATPase protein abundance at the plasma membrane (PM) was
determined by biotin-streptavidin pull down and subsequent Western
blot. Graph represents the mean6SEM, (n=5). Representative blots of
Na,K-ATPase a1-subunit at the PM and total protein abundance are
shown. (C) Live cell imaging of A549 cells expressing the rat GFPa1-
subunit Na,K-ATPase (WT) or the rat GFP-S18Aa1-subunit (S18A). Cells
were exposed to 40 mmHg CO2(pHe: 7.4) for 10 min (left panels) and
then switched to 120 mmHg CO2(pHe: 7.4) for 30 min (right panels).
White arrows indicate the plasma membrane Na,K-ATPase. * p,0.05.
doi:10.1371/journal.pone.0001238.g006
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Na,K-ATPase-a1-subunit (40 121) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and
3 mM ouabain to suppress the endogenous Na,K-ATPase a1
subunit. A549 cells expressing an empty vector and DN PKCf
were grown in the presence of G418 [28,41]. Cells were incubated
in a humidified atmosphere of 5% CO2/95% air at 37uC.
For the different experimental conditions, initial solutions were
prepared with DMEM- Ham’s F-12 medium-Tris base (3:1:0.5)
containing 10% fetal bovine serum with 100 U/ml penicillin, and
100 mg/ml streptomycin. The buffer capacity of the media was
modified by changing its initial pH with a Tris base in order to
obtain a pH to 7.4 with the various CO2levels (pCO2of 40, 60, 80
and 120 mmHg). In some additional experiments, modeling
extracellular acidosis, an initial pH of 6.8 was used to result in
a final pH of 7.2 and a pCO2of 40 mmHg. Then media was
placed overnight in a humidified chamber (C-174 Chamber,
Biospherix, Ltd., Redfield, NY) to achieve the desired CO2and
pH before starting the experimental protocols.
Biotinylation of cell surface proteins
Cells were labeled for 20 minutes using 1 mg/ml EZ-link NHS-
SS-biotin (Pierce Chemical Co., Rockford, IL). After labeling, the
cells were rinsed three times with phosphate-buffered saline (PBS)
containing 100 mM glycine to quench unreacted biotin and then
lysed in modified radioimmunoprecipitation buffer (mRIPA;
50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP-40, and 1%
sodiumdeoxycholate, containing
PMSF, 100ug TPCK, 10 mg/ml leupeptin [pH 7.4]). Aliquots
(150 mg of protein) were incubated overnight at 4uC with end-
over-end shaking in the presence of streptavidin beads (Pierce
Chemical Co.). The beads were thoroughly washed and then
resuspended in 30 ml of Laemmli sample buffer solution [42].
Proteins were analyzed by SDS-PAGE and Western blot.
protease inhibitors-1 mM
Live Cell Imaging
Epi-fluorescent microscopy images of GFPa1-A549 cells were
obtained using a Nikon TE2000 (Nikon Instruments Inc, Melville
NY) equipped with an environmental control system chamber
(FCS2 system, Bioptechs Inc, Butler, PA) and a Planapo 60x 1.4
NA objective (Nikon Instruments Inc, Melville, NY). During
imaging, the chamber was perfused with the specific culture media
described above equilibrated to pCO2of 40 or 120 mmHg and
a pHeof 7.4, while in other experiments a pCO2was kept at
40 mmHg and pHewas modified to 7.2. In experiments studying
reversibility, cells were perfused for 10 min with media equili-
brated to pCO2of 40 mm Hg, changed to media equilibrated to
pCO2of 120 mm Hg for 30 min and back to media equilibrated
to pCO2of 40 mm Hg for 1 h. Under all experimental conditions
oxygen was kept at 21%. Images were collected with a Cascade
camera ‘‘TC285’’ EMCCD with on-chip multiplication gain
(Photometrics; Tucson, AZ) driven by MetaMorph Software
(Molecular Devices Corp. Downingtown, PA). For all the
experiments exposure time was 0.5 seconds and to decrease
phototoxic effects 0.25 neutral filter was used.
Intracellular pH measurement
ATII were plated in circular glass cover slips, placed in a chamber
for environmental control system for live cell imaging (FCS2
system, Bioptechs Inc, Butler, PA) and measurements were
obtainedwithamulti-mode
TE2000, Nikon Instruments Inc, Melville NY). Cells were loaded
with 1.5 mM2979-bis-(carboxyethyl)-5,6-carboxyfluorescein
inverted Microscopy(Nikon
(BCECF/AM) (Invitrogen, Carlsbad, CA) for 30 min at 37u C
as described previously [43]. After dye loading, cover slips were
placed in the chamber, maintained at 37u C and continuously
perfused with equilibrated media. BCECF fluorescence in the
chamber was monitored continuously through the desired
excitation wavelength (500 and 440 nm) with an emission
wavelength of 520 nm. For all the experiments the exposure time
was 2 seconds. No neutral filter was used.
Signals were processed by MetaFluor Software (Molecular
Devices Corp. Downingtown, PA).
Cell fractionation
Cells were exposed to 120 mmHg of CO2at 37uC for the desired
times, placed on ice and washed twice with ice-cold PBS. Cells
were scraped in homogenization buffer (1 mM EDTA, 1 mM
EGTA, 10 mM Tris-HCl, pH: 7.5, 1 mg/ml leupeptin, 100 mg/ml
TPCK and 1 mM PMSF) and homogenized by using a Dounce
homogenizer. Homogenates were centrifuged at 500 g to discard
nuclei and debris, and the supernatant was centrifuged at
100,000 g, 1 h, 4uC (TL ultracentrifuge, Beckman, Rotor TLA
100.2). The supernatant was considered the cytosolic fraction. The
pellet containing the crude membrane fraction was resuspended in
homogenization buffer +1% Triton X-100 and centrifuged at
100,000 g, 30 min, 4uC. The supernatant was considered the 1%
Triton X-100 soluble fraction.
Adenoviral infection
Rats were anesthetized with 40 mg/kg Nembutal intraperitoneally
and intubated with a 14-gauge catheter prior to adenoviral
infection. Three experimental groups were studied: Sham-
surfactant (n=5), null adenovirus (n=5), and adenovirus expres-
sing DN PKCf (Cell Biolabs, San Diego, CA) (n=5). A mixture of
adenovirus (46109PFU) in a 50% surfactant (Forest Laboratories
Inc, St. Louis, MO), 50% dialysis buffer vehicle was administered
in four aliquots of 250 ml. Immediately before instillation, a forced
exhalation was achieved by circumferential compression of the
thorax [44]. Compression was relinquished after endotracheal
instillation of 250 ml of virus/vehicle followed by 500 ml of air.
Infected animals were maintained in separate isolator cages for
7 days prior to conducting experimental protocols as described
previously [44].
Hematoxylin and eosin (HE) staining
Lung tissues were rinsed in ice-cold PBS and fixed in 4%
paraformaldehyde overnight. Lungs were embedded in paraffin,
and cut into 4 mm lung tissue sections, which were placed on glass
slides. Slides were deparaffinized in xylene for 5 min (3 times) and
then rehydrated in 100%, 95%, 70% ethanol and PBS.
Hematoxylin and eosin (H&E) staining was performed. Briefly,
slides were stained in hematoxylin for 3 min, rinsed in tap water,
dipped in acid-alcohol 8–12 times, and finally rinsed in tap water.
Next, slides were stained with eosin for 30 s and then dehydrated
with 95% ethanol, 100% ethanol, and xylene. Images were
observed with an Olympus Vanox-s equipped with an Olympus
Japan 138132 objective and were captured using a Nikon Digital
Camera System.
Immunoprecipitation
GFPa1-A549 cells were incubated for 10 min at 5% and 20% CO2
in the presence or absence of the PKCf inhibitory peptide. The
incubation was terminated by placing the cells on ice, aspirating
the media, washing twice with ice-cold PBS and adding
immunoprecipitation buffer (20 mM Tris-HCl, 2 mM EGTA,
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2 mM EDTA, 30 mM Na4P2O7, 30 mM NaF, 1 mM Na3VO4,
1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mg/ml N-tosyl-
L-phenylalanine chloromethyl ketone (TPCK), 10 mg/ml leupep-
tin [pH 7.4]). The cells were then scraped from the plates, frozen
in liquid nitrogen, thawed, sonicated, frozen again, and centri-
fuged for 2 minutes at 14,000 g. After protein determination, SDS
and Triton X-100 were added to each sample to a final
concentration of 0.2% and 1%, respectively. Equal amounts of
protein (700–1000 mg) were then incubated with polyclonal anti-
GFP antibody for 2 hours at 4uC. Protein A/G PLUS-Agarose
was added, and the samples were incubated overnight at 4uC. The
samples were then washed twice with immunoprecipitation buffer
supplemented with 0.2% SDS and 1% Triton X-100 and once
with 20 mM Tris-HCl (pH 7.4).
In vitro phosphorylation
The phosphorylation state of the immunoprecipitated Na,K-
ATPase–GFPa1 subunit was assessed in vitro by the ‘‘back
phosphorylation’’ method [28,45]. The standard reaction mixture
for in vitro phosphorylation of the Na,K-ATPase a1subunit by
purified PKC (150 ng per 150 ml, 30 minutes at 30uC) contained
10 mM MgCl2, 0.25 mM EGTA, 0.4 mM CaCl2,0.32 mg/ml L-
a-phosphatidyl-L-serine,0.03 mg/ml
(DAG), 0.1 mg/ml BSA, and 20 mM Tris-HCl (pH 7.5). The
phosphorylationreactionwas
1,2-dioleoyl-sn-glycerol
started by theadditionof
[c-32P]ATP (final concentration, 100 mM; 1.3 mCi per sample).
The reaction was stopped by placing the tubes on ice and washing
the beads twice with 20 mM Tris-HCl (pH 7.4). Samples were
analyzed by SDS-polyacrylamide gel, transferred to nitrocellulose
membranes and autoradiographed.
Data Analysis
Data are expressed as mean6SEM. Data were compared using
ANOVA adjusted for multiple comparisons with the Dunnet test.
When comparisons were performed between two groups of values
significance was evaluated by non-paired Student’s test and when
comparison were made between repeated measures significance
was evaluated by a paired Students test. A p value ,0.05 was
considered significant.
ACKNOWLEDGMENTS
The authors acknowledge the valuable insights to this manuscript of Dr.
Aaron Ciechanover, Dr.Karen Ridge and Ms. Aileen Kelly.
Author Contributions
Conceived and designed the experiments: JS AB EL. Performed the
experiments: AB IV LW JC VD ZA PM LD EL HT. Analyzed the data:
YG JS AB ZA DB LD EL. Contributed reagents/materials/analysis tools:
DB. Wrote the paper: YG JS AB IV LD EL.
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