INVITED REVIEW
Chloride and potassium channel function in alveolar
epithelial cells
Scott M.
O'Grady and
So Yeong
Lee
Department of Physiology, University of Minnesota, St.
Paul, Minnesota 55108
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ABSTRACT |
Electrolyte transport across the
adult alveolar epithelium plays an important role in maintaining a thin
fluid layer along the apical surface of the alveolus that facilitates
gas exchange across the epithelium. Most of the work published on the
transport properties of alveolar epithelial cells has focused on the
mechanisms and regulation of Na+ transport and, in
particular, the role of amiloride-sensitive Na+ channels in
the apical membrane and the Na+-K+-ATPase
located in the basolateral membrane. Less is known about the identity
and role of Cl
and K+ channels in alveolar
epithelial cells, but studies are revealing important functions for
these channels in regulation of alveolar fluid volume and ionic
composition. The purpose of this review is to examine previous work
published on Cl
and K+ channels in alveolar
epithelial cells and to discuss the conclusions and speculations
regarding their role in alveolar cell transport function.
alveolar fluid clearance; Cl
absorption; K+ secretion; epithelial ion transport
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INTRODUCTION |
THE ALVEOLAR EPITHELIUM is composed of
two major epithelial cell types referred to as type 1 (AI) and type II
(AII) cells (5, 10, 30, 46). The transport and water
permeability characteristics of these cells play a critical role in
regulating the volume and electrolyte composition of alveolar lining
fluid. In the fetal state, alveolar epithelial cells are involved in the secretion of fluid into the developing alveoli and airways (1, 19, 30, 52, 55, 67, 68, 87). Shortly before birth,
fluid secretion is inhibited and a net increase in lung liquid
absorption develops in preparation for breathing air (1, 19, 46,
67, 68, 70). This change in fluid transport occurs as a
consequence of a shift in electrolyte transport properties of the
epithelium from anion secretion to electrogenic Na+
absorption. Moreover, electrogenic Na+ absorption persists
as the basal transport process associated with adult alveolar
epithelial cells (10, 28, 45, 46, 48, 49). Inhibition of
Na+ transport across the apical membrane by amiloride
produces a decrease in fluid absorption from the alveolar space
(47). Most of the in vitro work on Na+
transport across the alveolar epithelium has focused on AII cells, which cover 2-5% of the lung surface area and are known to
produce and secrete surfactant (5, 30). The cellular and
molecular mechanism of Na+ transport and its regulation by
corticosteroids and catecholamimes have been the subject of several
recent review articles and special topic papers (9, 10, 37,
45-49). These articles have focused on the importance of
apically localized, amiloride-sensitive Na+ channels as the
principle pathways for Na+ uptake from the alveolar fluid.
Two Na+ channel phenotypes have been identified with
differing Na+ selectivities and amiloride sensitivities. In
cultures of adult rat AII cells grown in the absence of corticosteroids
or reduced oxygen delivery, the predominant Na+ entry
pathway is a 21-pS nonselective cation (NSC) channel with an
Na+/K+ permeability ratio
(PNa/PK) of 1 (26). In
late-gestation fetal distal lung epithelial cells, a similar NSC
channel was previously identified with single-channel conductance
estimates of 23 and 27 pS (42, 62, 63) and a
PNa/PK ratio of 0.9 (63). When adult rat AII cells were grown in the presence of glucocorticoids and
an apical air interface, the predominant apical conductance was a 6-pS,
highly selective Na+ channel (HSC;
PNa/PK ratio = 80:1) that was blocked by
amiloride [half-maximal inhibition (K0.5) = 37 nM;
(7, 26)]. Similarly, in experiments with A549
human alveolar epithelial cells, it was shown that treatment with
dexamethasone for 24-48 h produced a twofold increase in
amiloride-sensitive whole cell current and decreased the
K0.5 for amiloride from 833 to 22 nM (38). In addition, dexamethasone decreased single-channel conductance from 8.6 to 4.4 pS and increased Na+ selectivity, as reflected by a
shift in reversal potential from 47 to 66 mV. These changes in
biophysical and pharmacological properties of A549 cell Na+
channels were associated with increased expression of mRNA and protein
for both
- and
-epithelial Na+ channel (ENaC)
subunits, but no significant change in
-ENaC expression
(38). It has been previously proposed that different combinations of ENaC subunits can form Na+ channels with
differing biophysical and pharmacological properties (25).
Expression of the
-subunit alone produces NSC channels, whereas
channel proteins consisting of
-,
-, and
-subunits exhibit
biophysical and pharmacological properties consistent with HSC
(26). Thus steroid induction of
- and
-subunit mRNA and protein expression is most likely responsible for the change in
Na+ channel characteristics in A549 cells. However, in
primary rat AII cells, all three ENaC subunits could be detected in the
absence of glucocorticoid treatment. Thus the presence of
-,
-,
and
-subunit proteins within the same cell does not necessarily
result in expression of HSC. This observation would suggest that
steroid-mediated increases in Na+ selectivity and low
single-channel conductance also depend on proper assembly and insertion
of channel subunits into the apical membrane and not solely on the
level of expression of each subunit (26).
It is worth noting that the effects of corticosteroids on alveolar
Na+ transport function are not limited to the apical
membrane. Glucocorticoids have been shown to increase
Na+-K+-ATPase subunit expression and activity
in fetal and adult AII cells (3, 6, 11, 17, 67, 74). In
alveolar cells, the predominant Na+-K+-ATPase
catalytic subunit is
1, which contains binding sites for
Na+, K+, and ATP. The
1-subunit
has also been identified in AII cells and is presumably involved in
pump insertion into the basolateral membrane. In the fetal lung
epithelial cell line FD18, dexamethasone increased steady-state levels
of
1- and
1-subunit mRNA by 3.8- and
2.8-fold, respectively (6). This increase in mRNA was
shown to be the result of increased promoter activity with no change in
RNA stability. In adult rat AII cells, dexamethasone was shown to
increase
1- but not
1-mRNA transcript
levels. Both
1- and
1-protein expression
were increased along with stimulation of Na+-K+-ATPase activity (3). Thus
glucocorticoid receptor stimulation produces transcriptional and
translational regulation of Na+-K+-ATPase
function in AII cells. Moreover, coordinate regulation of apical and
basolateral Na+ transport pathways following glucocorticoid
stimulation enhances the rate of transepithelial Na+
absorption (11).
Until recently, the standard model for electrolyte and fluid transport
across the alveolar epithelium proposed that AII cells were the
principle cell type involved in NaCl transport and that AI cells were
primarily involved in fluid transport and gas exchange (10,
46-48). Recent studies of AI cells in culture, however, have demonstrated amiloride-sensitive Na+ uptake that
exceeds rates of Na+ influx measured in parallel cultures
of AII cells by 2.5-fold (30). In addition,
immunocytochemistry experiments with cultured AI cells revealed the
presence of all three ENaC subunits as well as the
1-
and
1-subunits of the
Na+-K+-ATPase. Western blot analysis of
-ENaC expression indicates that the amount of
-subunit/µg
protein in AI cells exceeds that observed in AII cells by threefold.
These results were further supported by Borok et al. (5),
who used antibodies to specific AI and AII cell marker proteins in situ
and cultured AI cells to distinguish sites of expression for ENaC
and Na+-K+- ATPase subunits. The results
showed immunolocalization of
1- and
1-Na+-K+-ATPase subunits in AI
cells in situ and localization of the
-ENaC subunit in cultured AI
cells. In addition,
1- and
1-Na+-K+-ATPase subunits, as
well as the
-ENaC subunit, were detected by RT-PCR in mRNA samples
obtained from highly purified populations of freshly isolated AI cells.
The results of these recent studies provide evidence in support of a
significant role for AI cells in electrogenic Na+
absorption across the alveolar epithelium. Moreover, the observation that AI cells comprise >95% of the internal surface area of the lung
suggests that most of the Na+ absorption is mediated
primarily by AI cells.
Although considerable interest exists in understanding mechanisms and
regulation of transepithelial Na+ transport and its role in
fluid absorption across the alveolar epithelium, less is known about
Cl
and K+ transport pathways and their
physiological significance in alveolar cells. Chloride secretion by
fetal alveolar epithelial cells is well accepted as important for
establishing the osmotic driving force necessary for fluid secretion
into the lumen of the developing lung (19, 35, 52, 53,
55). The role of Cl
channels and transcellular
Cl
absorption in alveolar fluid clearance in the adult
lung has been controversial, but recent studies in mouse and human
lungs have provided compelling evidence showing that activation of the cystic fibrosis transmembrane conductance regulator Cl
channel (CFTR) is essential for increased fluid clearance following
-adrenergic stimulation (18, 36). The role of specific
K+ channels known to exist in alveolar epithelial cells is
even less well understood. Patch-clamp studies have shown that alveolar epithelial cells express an interesting array of K+ channel
families, including voltage-gated (Kv-type) channels, inwardly
rectifying K+ (Kir) channels, and
Ca2+-activated K+ channels (12, 24, 33,
65, 72, 81). The purpose of this review is to discuss what is
known about Cl
and K+ channels present in
both fetal and adult alveolar epithelial cells and to offer some
speculation on the physiological role of these channels in alveolar
epithelial electrolyte transport.
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CHLORIDE CHANNELS IN ALVEOLAR EPITHELIAL CELLS |
Cl
channels were first identified in cultured
alveolar epithelial cells by single-channel patch-clamp recording
techniques in the mid-1980s. The first Cl
channel
identified in adult rat AII cells was a high-conductance (350-400
pS), voltage-sensitive channel that was located in the apical membrane
(76). The channel was found to be selective for
anions relative to Na+
(PNa/PCl = 0.015) and to have an anion
selectivity of I
> Br
> Cl
> NO
. At that time, the
mechanism of transepithelial Na+ transport across cultured
rat AII cells was not well understood, but the authors did speculate
that this high-conductance Cl
channel might be involved
in transcellular Cl
transport across the alveolar
epithelium. Several years later, a high-conductance (375 pS),
stilbene-sensitive, G protein-regulated Cl
channel was
identified in cell-free patches from freshly isolated fetal guinea pig
AII cells (31). Smaller-conductance Cl
channels (25 pS) were also identified in cell-attached patches. The
authors speculated that these large-conductance, G protein-regulated Cl
channels may play a role in mediating Cl
efflux from cells that are involved in active Cl
secretion.
Chloride secretion and Cl
channel
function in fetal alveolar epithelial cells.
Early studies of fetal alveolar epithelial cell transport function were
carried out on rat alveolar buds that aggregate to form cysts in
submersion culture (34, 53, 54). These cysts accumulate
fluid and exhibited lumen negative transepithelial potentials.
Treatment with the Na+-K+-2Cl
cotransport inhibitor bumetanide produced a 70% decrease in
transepithelial potential (34) and reduced the size and
number of cysts, consistent with inhibition of fluid transport into the
lumen (54). Stimulation with a membrane-permeant analog of
cAMP in combination with the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX) increased the size of the cysts
and the lumen negative transepithelial potential difference from 4.6 to
7.3 mV (53, 54). These effects were also blocked by
bumetanide. Stimulation with the
-adrenergic receptor agonist
isoproterenol or epinephrine also increased the lumen negative
potential difference, consistent with an increase in Cl
secretion in alveolar cells from rat and late-gestation fetal sheep
(53, 83). In more recent studies of rat fetal distal lung
epithelial (FDLE) cells,
-adrenergic receptor stimulation with
isoproterenol was shown to increase a glibenclamide-sensitive Cl
conductance of the apical membrane (9).
Similarly, forskolin treatment of FDLE cells was shown to stimulate
both Cl
and HCO
secretion. The effects
of forskolin were blocked by apical addition of glibenclamide [an inhibitor of CFTR Cl
channels and ATP-sensitive potassium
(KATP) channels] and basolateral treatment with bumetanide
(39). The results of these studies suggest a model (see
Fig. 1A) for alveolar anion
and fluid secretion similar to that proposed for a variety of secretory
epithelia (4, 20). The essential features of this model
include Na+-K+-2Cl
cotransporters, Na+-K+-ATPase enzymes, and
K+ channels located in the basolateral membrane and apical
Cl
channels that are regulated by cAMP. Cl
uptake across the basolateral membrane is mediated by electroneutral Na+-K+-2Cl
cotransport. Recycling
of Na+ and K+ out of the cell depends on
Na+-K+-ATPase activity and K+
channels, respectively. Electrogenic K+ efflux sustains the
electrical driving force for Cl
efflux across the apical
membrane when Cl
channels are activated by increases in
cytosolic cAMP. Although a number of details remain to be elucidated,
this general model of alveolar cell anion secretion appears to account
for the presently available data.

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Fig. 1.
Na+ and Cl transport models for alveolar
epithelial cells. A: anion secretion in early-gestation
fetal alveolar epithelial cells. NSC, nonselective cation channel;
CFTR, cystic fibrosis transmembrance conductance regulator; AC,
adenylyl cyclase. B: transcellular NaCl absorption in adult
rat alveolar epithelial type II (AII) cells. HSC, highly selective
Na+ channel. (See text for discussion.)
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The observations that Cl
and fluid secretion could be
stimulated in fetal alveolar epithelial cells by agents that increased cAMP suggested that CFTR may be involved in mediating Cl
exit across the apical membrane. This hypothesis was initially tested
by in situ hybridization studies with 3H-labeled anti-sense
CFTR probes to localize CFTR mRNA in human fetal lung tissue and
cultured lung explants. CFTR mRNA was detected in first- and
second-trimester lung tissue. AII cells in cultured explants were also
found to contain CFTR mRNA, suggesting a potential role in alveolar
anion and fluid secretion (55). In a subsequent study, a
monoclonal CFTR antibody was shown to label the apical membrane of
epithelial cells in cultured human fetal lung explants. These cells
were shown to exhibit isoproterenol- and forskolin/IBMX-stimulated Cl
secretion that was blocked by apical addition of the
Cl
channel inhibitor diphenylamine-2-carboxylate (DPC)
and by basolateral addition of bumetanide (52). In rat
FDLE cells, CFTR protein was identified by immunocytochemistry, again
consistent with a role in Cl
and HCO
secretion described above (39). Although a direct link
between Cl
channel function and CFTR expression was not
absolutely established by these studies, the in situ hybridization and
immunolocalization results provide compelling evidence for CFTR
expression in fetal alveolar epithelial cells.
CFTR-independent anion secretion has also been shown in fetal mouse
lung following stimulation with kerotinocyte growth factor (KGF).
Treatment with KGF for several hours produced increases in lumen volume
of alveolar buds and disrupted branching morphogenesis (87). This effect was observed in both wild-type and CFTR
knockout mice and was blocked by treatment with bumetanide. In
addition, KGF also inhibited expression of the
-subunit of ENaC,
suggesting a reduction in transepithelial Na+ absorption.
The persistence of anion secretion in the absence of CFTR expression
indicates activation of a second anion conductance in the apical
membrane by KGF. The molecular identity of this anion conductance is
presently unknown.
Cl
channel identification and
function in cultured adult alveolar epithelial cells.
After the original report of high-conductance, voltage-dependent
Cl
channels in adult rat AII cells (31, 76),
there have been differing results and conclusions regarding the
expression of cAMP-activated Cl
channels in these cells.
In a previous study (88), patch-clamp recording techniques
were used to measure single channel and whole cell Cl
currents from AII cells stimulated with 500 µM
8-(4-chlorophenythio)-adenosine 3',5'-cyclic monophosphate
(8-cpt-cAMP), a cell-permeant form of cAMP. Only one of 33 cell-attached patches exhibited Cl
channel activation
following 8-cpt-cAMP stimulation. Moreover, whole cell current
measurements also failed to show increases in Cl
current
after stimulation with either 8-cpt-cAMP or the calcium ionophore
ionomycin. These results lead to the conclusion that only a very small
fraction of adult AII cells expresses cAMP-activated Cl
channels and suggested that Cl
transport occurs
passively across the alveolar epithelium through tight junctions
between the cells. In contrast to the findings of that study, Jiang et
al. (27) demonstrated that stimulation of adult rat
alveolar epithelial cell monolayers with the selective
2-adrenoceptor agonist terbutaline resulted in
activation of Cl
channels located in the apical membrane.
Similarly, forskolin-activated Cl
channels were also
identified in the apical membrane of cultured adult rabbit alveolar
epithelial cells (75). Characterization of
terbutaline-activated Cl
channels in adult rat alveolar
epithelial cells was initially conducted using permeablized monolayers,
where the basolateral membrane was perforated with the pore-forming
antibiotic amphotericin B, thus allowing apical-membrane voltage-clamp
experiments to be performed (27). The terbutaline- or
8-cpt-cAMP activated Cl
channels possessed a near-linear
current-voltage relationship (Fig.
2B) and a reversal potential
that became progressively more positive as the Cl
concentration gradient was decreased across the apical membrane. The
anion selectivity sequence was found to be
SCN
>Br
>Cl
>I
,
and the channel was blocked by known Cl
channel
inhibitors in a concentration-dependent manner (Fig. 3). In a subsequent study, whole cell
perforated patch-clamp experiments revealed that both freshly isolated
alveolar epithelial cells and alveolar cells maintained in monolayer
culture under air-interface conditions expressed terbutaline-activated
Cl
currents that were blocked by
5-nitro-2-(3-phenylpropylamino)benzoate or glibenclamide
(29). In addition, CFTR protein expression was
established by immunocytochemistry, and identification of CFTR mRNA in
monolayer cultures of adult rat alveolar epithelial cells was recently
demonstrated by RT-PCR (40, 61).

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Fig. 2.
A: terbutaline (2 µM)- and amiloride (20 µM)-sensitive current responses obtained from
amphotericin-permeabilized monolayers of adult rat alveolar epithelial
cells voltage clamped at 0 mV (27). Experiments were
performed in Ussing chambers, bathed with KMeSO4
intracellular solution (basolateral) and serum-free DMEM/F-12 media
(apical). B: current-voltage relationship for the
terbutaline- and 8-(4-chlorophenythio)-adenosine 3',5'-cyclic
monophosphate (8-cpt-cAMP)-sensitive conductance in the apical
membrane. Terbutaline (2 µM, n = 12) and 8-cpt-cAMP
(100 µM, n = 5) were applied to the basolateral
bathing solution after pretreatment of monolayers with apical amiloride
(20 µM). [Modified from Jiang et al.
(27).]
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Fig. 3.
Effects of Cl channel blockers on apical
membrane Cl current in adult rat alveolar epithelial cell
monolayers permeabilized basolaterally with amphotericin B
(27). A: pretreatment with 200 µM
glibenclamide (Glib.) nearly abolishes the effects of 2 µM
terbutaline on apical membrane current. B:
concentration-response relationships for
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, n = 6),
Glib. (n = 6), and diphenylamine-2-carboxylate (DPC,
n = 9) inhibition of the terbutaline-sensitive current.
The IC50 values for NPPB, Glib., and DPC were 12, 110, and
640 µM, respectively. [Part B from Jiang et al.
(27).]
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In monolayer cultures of adult rat alveolar cells it has been shown
that Cl
channel activation produces stimulation of
transepithelial Cl
absorption (32, 40, 61).
This flux data support the interpretation that the immediate decrease
in short-circuit current (Isc) produced by
terbutaline stimulation results from Cl
uptake across the
apical membrane through CFTR-like Cl
channels.
Although other Cl
channels have been observed in
patch-clamp studies of cultured alveolar epithelial cells (31,
76), CFTR is the only cAMP-activated Cl
channel
known to be present. Replacement of extracellular Cl
with
a nontransported anion or treatment with Cl
channel
blockers known to inhibit CFTR abolishes this current response
(27, 29). Thus CFTR appears to serve as the major influx
pathway in support of transcellular Cl
absorption in
adult rat alveolar epithelial cells following stimulation by
-adrenergic receptor agonists. The transporter
responsible for Cl
exit across the basolateral
membrane is not certain; however, a likely mechanism would involve
electroneutral cotransport with K+. Such a mechanism would
take advantage of the outwardly directed chemical potential for
K+, and Cl
efflux would not be limited by a
relatively depolarized basolateral membrane. In support of this idea,
recent studies of Cl
transport across the basolateral
membrane indicate that terbutaline stimulates an electroneutral
Cl
efflux mechanism that is dependent on an outwardly
directed K+ concentration gradient (40, 61).
Moreover, RT-PCR experiments revealed that monolayers of adult
rat alveolar epithelial cells contain mRNA for
K+-Cl
cotransporter (KCC) 1, KCC3, and KCC4
isoforms of the KCl cotransporters and that expression of protein for
the KCC1 and KCC4 isoforms was detected by Western blots. Thus these
findings support a model for terbutaline regulation of transcellular
Cl
absorption involving Cl
uptake by CFTR
Cl
channels located in the apical membrane and
Cl
exit across the basolateral membrane mediated by KCl
cotransport (Fig. 1B). In contrast to results obtained
with adult rat alveolar cells, agents that increase cytosolic cAMP in
alveolar cells from adult rabbits produce an increase in
bumetanide-sensitive Isc, consistent with
Cl
secretion (58).
At this time, the distribution of CFTR into AI and AII cells within the
alveolus is not clearly defined. Unlike recent studies describing the
identification and functional characterization of Na+
transport proteins within AI cells (5, 30), there are no data confirming the presence of CFTR or any other Cl
channel proteins in AI cells in the intact lung. As mentioned above,
cultured alveolar epithelial cells have been shown to express CFTR mRNA
and protein, but these cultures most likely contain both AI and AII
cell populations (9, 29, 40, 61). Therefore, further
studies will be needed to establish the localization of Cl
channel proteins in AI cells, and this could have
implications on what additional signaling pathways maybe involved in
regulation of Cl
transport within the alveolus.
Role of CFTR in alveolar fluid clearance in mouse and human lung.
A recent, seminal investigation utilizing in situ perfused lung
experiments with wild-type and
F508 CFTR-expressing mice and ex vivo
human lung perfusion studies demonstrated a direct role for CFTR
Cl
channels in alveolar fluid clearance
(18). In experiments with wild-type mice, instillation of
fluid containing Cl
channel blockers (known to inhibit
CFTR) into the airways and alveolar space completely blocked
isoproterenol-stimulated fluid absorption (Fig.
4A). In addition, mice
expressing the
F508 CFTR mutation failed to exhibit a significant
increase in fluid clearance following isoproterenol stimulation (Fig.
5). Experiments with ex vivo human lungs
also showed that infusion of the CFTR Cl
channel
inhibitor glibenclamide inhibits the effects of terbutaline on fluid
clearance (Fig. 4B). These blocker experiments and the results obtained with
F508 CFTR mice indicate that CFTR activation is critical to stimulation of fluid absorption by adrenergic agonists. To establish that CFTR is involved in Cl
uptake from the
alveolar fluid, 22Na+ and
36Cl
uptake measurements were performed in
the mouse lung at 23°C, a temperature that inhibits alveolar fluid
clearance. It was observed that 22Na+ uptake
from the alveolar fluid was similar to that of
36Cl
under basal conditions, but following
isoproterenol stimulation, a significant increase in
36Cl
uptake was observed with no change in
22Na+ uptake. Moreover, when these experiments
were repeated with
F508 CFTR mice, no increase in
36Cl
removal was detected. These results
indicate that CFTR is involved in 36Cl
uptake
from the alveolar fluid and that isoproterenol stimulation increases
the Cl
conductance of the alveolar epithelium.

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Fig. 4.
Effects of isoproterenol (100 µM) or terbutaline (100 µM) and glibenclamide (100 µM) on fluid clearance in mouse and
human lung (reproduced from Ref. 18). A: fluid
clearance in the in situ perfused lung of wild-type mice at 37°C
expressed as % absorption at 15 min under control conditions
(n = 12), glibenclamide (0.1 mM, n = 6), isoproterenol (0.1 mM, n = 18), and isoproterenol
plus glibenclamide (n = 6). B: fluid
clearance in rewarmed ex vivo human lung at 37°C, expressed as
%absorption at 1 h under control conditions. Data reported as
means ± SE; * P < 0.05 compared with control
(n = 23), glibenclamide (0.1 mM, n = 5), terbutaline (0.1 mM, n = 8), and terbutaline plus
glibenclamide (n = 6). [From Fang et al.
(18).]
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Fig. 5.
Fluid clearance from the distal air spaces of wild-type
(open bars) and F508 CFTR (solid bars) mice (data reproduced from
Ref. 18). Measurements were performed using in situ
perfused lung at 37°C under basal conditions (n = 24 wild type, n = 7 F508) and in the presence of 100 µM isoproterenol (n = 9 wild type, n = 6 F508). Data reported as means ± SE; * P < 0.05 compared with control. [From Fang et al.
(18).]
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Interestingly, the complete inhibition of isoproterenol-stimulated
fluid clearance by Cl
channel blockers in wild-type mice
and the lack of effect of adrenergic stimulation on fluid clearance in
F508 CFTR-expressing mice may indicate that adrenergic stimulation
does not increase apical Na+ conductance in adult mouse
alveolar cells. The same may also be true for perfused human lung,
where blockers of CFTR completely abolished the terbutaline-stimulated
increase in fluid absorption. Alternatively, Na+ and
Cl
uptake across the apical membrane may be tightly
coupled so that inhibition of CFTR results in depolarization that
limits Na+ uptake despite increases in apical membrane
Na+ channel activity.
The issue of whether CFTR is involved in the resolution of alveolar
edema was studied in a hydrostatic volume overload model using
wild-type and
F508 CFTR-expressing mice (18). In this model, volume overload was established by saline infusion resulting in
a
30% increase in wet-to-dry weight ratio of lungs from wild-type and heterozygous
F508 mice. In homozygous
F508 mice, the ratio was 64% with histological evidence for significant alveolar edema. Blockade of endogenous catecholamine effects on the lung epithelium using the
-adrenergic receptor antagonist propranolol produced an
increase in the wet-to-dry weight ratio of lung tissue from wild-type
and heterozygous
F508 CFTR mice to a level comparable with that of
homozygous
F508 mice. These results indicate that CFTR activation is
an important physiological response to fluid accumulation in the distal
airways and alveoli and suggest that patients suffering from cystic
fibrosis (CF) may have a greater susceptibility to pulmonary edema.
Relationship between Cl
channel
activation and Na absorption in adult alveolar cells.
In our initial study of ion transport in monolayer cultures of adult
rat alveolar epithelial cells (27), we suggested that Cl
channel activation by terbutaline plays a role in
stimulating amiloride-sensitive Na+ transport by increasing
the driving force for Na+ entry across the apical membrane.
This explanation was based on the observation that no increase in
amiloride-sensitive Na+ conductance could be detected
following stimulation of monolayers with terbutaline and that
Cl
replacement or voltage clamping the apical membrane to
0 mV abolished the time-dependent increase in
Isc or apical membrane current. This
interpretation has been challenged by results of other investigations that have reported increased apical membrane Na+ channel
activity following adrenergic receptor stimulation (1, 7, 9, 37,
46). The basis for these differing results is most likely
related to differences in culture conditions or perhaps the original
source of alveolar cells, as may be the case for rat FDLE cells grown
under high-O2 conditions as a method to produce the adult
transport phenotype (1, 9, 70). In our experiments,
monolayers were not treated with corticosteroids and were most likely
expressing amiloride-sensitive NSC channels and HSC channels as
suggested by the reversal potential (47 mV) of the amiloride-sensitive
Na+ current. In addition, the cells were grown under
serum-free conditions, and a relatively low concentration of
terbutaline (2 µM) was used to stimulate the cells. One possible
explanation for not observing an increase in Na+
conductance in response to terbutaline is that, in the absence of
corticosteroids, the pool of Na+ channels available for
insertion into the apical membrane may be relatively low compared with
cells cultured in the presence of dexamethasone or aldosterone. Thus
the magnitude of increased Na+ channel activity may have
been too small to detect in voltage-clamp experiments using
amphotericin B-permeablized monolayers. In addition, previous studies
(20) have reported using much higher concentrations of
-receptor agonists (20 µM or more), perhaps leading to greater increases in cytosolic cAMP and subsequently higher levels of Na+ channel expression and activity in the apical membrane.
Another interesting point regarding the cells used in our studies is
that terbutaline stimulation at either 2 or 20 µM does not lead to an
increase in cytosolic calcium concentration (unpublished data). This result was similar to studies by Isohama et al. (23),
where treatment with 10 µM terbutaline had no effect on intracellular Ca2+ concentration, but when added in combination with
phorbol ester, a significant increase in cytosolic Ca2+ was
observed. Thus adrenergic receptor stimulation in combination with
protein kinase C activation was necessary to increase intracellular calcium. This was not the case in alveolar cells grown in the presence
of corticosteroids, where terbutaline alone was sufficient to increase
cytosolic Ca2+ concentration (7). This effect
of terbutaline on intracellular Ca2+ has been shown to be
necessary for NSC channel activation (7, 43, 44) and
presumably explains why activation of these channels was not observed
in our previous studies.
In a recent study (35), the direction of Cl
flux across rat FDLE cell monolayers (grown under conditions where they
exhibited the adult alveolar cell transport phenotype) was shown to be
differentially regulated by adrenergic and purinergic receptor
agonists. These authors observed that
-adrenergic receptor
stimulation with isoproterenol produced an apical-to-basolateral
Cl
flux as measured using Cl
-selective
microelectrodes. Cl
absorption (50-70%) was
inhibited by Cl
channel blockers, indicating that
Cl
channels present in the apical membrane were
responsible for Cl
uptake. In contrast, stimulation with
the P2Y receptor agonist UTP produced an increase in the
basolateral-to-apical Cl
flux. Previous studies on
Na+ transport in rat FDLE cells showed that UTP inhibited
apical Na+ channels, whereas stimulation with isoproterenol
increased apical membrane Na+ conductance (9, 22,
71). Although the mechanisms underlying the actions of
isoproterenol and UTP are not completely understood, it is reasonable
to suggest that the direction of Cl
movement across the
apical membrane depends on the influence of Na+ channel
activation or inhibition on membrane voltage relative to the
Cl
channel reversal potential. Apical membrane
depolarization (by isoproterenol) or hyperpolarization (following UTP)
as a consequence of Na+ channel regulation would contribute
to the driving force and influence the direction of Cl
movement across the membrane. The results of this study would indicate
that Na+ and Cl
movement across the apical
membrane are interdependent and influence each other through changes in
apical membrane potential.
 |
K+ CHANNELS IN ALVEOLAR EPITHELIAL CELLS |
K+ channels in epithelial cells play an essential role
in transepithelial electrolyte and fluid transport. In
Cl
-secreting epithelia for example, basolateral
K+ channels are typically activated in parallel with apical
Cl
channels in response to secretagogue stimulation
(2, 13, 14, 50, 51, 77, 78, 82). K+ efflux
through these channels is essential in preventing significant cell
depolarization, thus helping to sustain the electrical driving force
for electrogenic Cl
exit across the apical membrane
(2, 13, 50, 77, 82). Inhibition of these K+
channels effectively blocks transepithelial Cl
secretion.
K+ channels also play a similar role in
Na+-absorbing epithelia, where K+ exit helps to
offset membrane depolarization produced by electrogenic Na+
influx (8, 85, 86). At this time, an extensive literature on the identity and function of K+ channels in alveolar
epithelial cells is not available. However, studies so far in fetal and
adult alveolar epithelial cells suggest the presence of multiple
K+ channel families and accessory regulatory proteins. The
following section describes what is known about the molecular identity, localization, and proposed function of specific K+ channel
family members found in alveolar epithelial cells.
Kv-type K+ channels.
Two distinct types of K+ channels similar to delayed
rectifier potassium channels have been identified in isolated adult rat alveolar type II cells and human lung adenocarcinoma cell line A549
(12, 33, 65). DeCoursey et al. (12)
identified the potassium channels in adult rat alveolar type II cells
as either normal (n)- or large (l)-type channels, depending on their
voltage dependency and tetraethylammonium (TEA) sensitivity.
Most alveolar type II cells possessed the n-type channel that was
activated at more negative potentials (
30 mV) compared with l-type
channels, which were activated at
10 mV. Peers et al.
(65) also showed two distinct potassium channel currents
in adult AII cells that possessed different voltage dependency and
blocker sensitivities. Low threshold currents were activated at more
depolarized voltages than
40 mV, and these currents were blocked by 2 mM 4-aminopyridine (4-AP). In contrast, high threshold currents were
activated at
20 mV and were 4-AP insensitive. In human A549 cells, a
portion of the K+ current was inhibited by TEA, and the
residual current was blocked by cesium (33). Recently,
more rapidly inactivating potassium channel currents have been
identified in adult rat alveolar epithelial cells (Fig. 6,
A-C). The molecular
identity of Kv channels was determined using RT-PCR, Western blot, and
immunocytochemistry. Eight distinct
-subunits (Kv1.1, Kv1.3, Kv1.4,
Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three
-subunits (Kv
1.1,
Kv
2.1, and Kv
3.1), and two K+ channel-interacting
proteins (KChIP: KChIP2 and KChIP3) were detected in monolayer cultures
of these cells by RT-PCR. Among the
-subunits, Kv1.1, Kv1.3, Kv1.4,
Kv4.2, and Kv4.3 were identified at the protein level by Western blot
analysis and were localized to the apical membrane of adult rat
alveolar epithelial cells by immunocytochemistry (Table
1).

View larger version (12K):
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|
Fig. 6.
Voltage-gated K+ (Kv) channel present in
cultured adult rat alveolar epithelial cells measured using the whole
cell perforated patch-clamp technique (data from Ref. 60).
A: typical Kv currents elicited by step depolarizations from
the holding potential of 60 mV to +10 mV in 5-mV increments.
B: current traces from 2 different cells showing variations
in rates inactivation gating between cells (scale: 100 pA, 200 ms).
C: mean current-voltage (I-V) relationship for Kv
channel currents normalized to whole cell capacitance
(n = 7). It is important to note that this plot shows
the voltage activation curve, and thus the reversal potential does not
provide information on K+ selectivity of the channel.
Previous open channel I-V relationships derived from tail
current analysis gave a mean reversal potential of 80 mV (for the
type n current), indicating a high degree of K+ selectivity
(PK/PNa = 0.02) (12).
D: mean activation and steady-state inactivation curves
normalized to a maximum outward current (I/Imax) at +40 mV
(n = 5). Note that a window current exists between 40
and 10 mV, indicating that Kv channels have a measurable open
probability over this voltage range.
|
|
In considering the role of Kv channels in alveolar cell electrolyte
transport, we find it interesting to note that the K+
concentration in alveolar epithelial lining fluid is greater than
plasma, suggesting K+ secretion (15, 16, 59, 75,
84). The finding that Kv channels are present in the apical
membrane of alveolar epithelial cells suggests the possibility that
these channels are involved in K+ secretion into the
alveolar space. The mean window current, derived from activation and
steady-state inactivation curves obtained from whole cell patch-clamp
experiments, lies between
40 and 10 mV (Fig. 6D). Thus Kv
channels possess significant open probability at voltages within this
range and could contribute to basal potassium secretion, provided that
the apical membrane is sufficiently depolarized. Another possible
function of Kv channels may be related to oxygen sensing. In cultured
alveolar cell monolayers, an electrically silent K+ channel
-subunit, Kv9.3 (79, 80), was detected by RT-PCR (Table
1). Previously, Kv2.1/Kv9.3 heteromeric channels have been shown to be
significantly inhibited by hypoxia in several expression systems,
including COS cells (64) and mouse L cells (21). In addition, chronic hypoxia downregulates mRNA
expression of Kv1.1, Kv4.3, and Kv9.3
-subunits in pulmonary artery
smooth muscle cells (69). Kv
-subunits are also known
to confer O2 sensitivity to Kv4.2 in HEK293 cells
(66). Therefore, Kv
- and
-subunits expressed in rat
alveolar epithelial cells may function as oxygen sensors, perhaps
detecting differences in alveolar ventilation or changes in
O2 diffusion across the apical membrane.
Kir channels.
Alveolar type II cells isolated from fetal guinea pig exhibited Kir
currents that were blocked by 1 mM Ba2+ and regulated by
both protein kinase A and protein phosphatases (PP) including PP-1/2A
and PP2C (56, 57). In this cell preparation, Kir2.1 was
detected by RT-PCR, and we have subsequently identified Kir2.1 mRNA
transcripts in adult rat alveolar cells (Table 1). The localization of
Kir2.1 in either fetal guinea pig AII cells or adult rat alveolar
epithelial cells has not been determined. In a previous study, a Kir
channel (Kir1.1) was identified in the apical membrane of epithelial
cells from the thick ascending limb of Henle's loop (41).
In these cells, K+ uptake into the cell occurs through an
Na+-K+-2Cl
cotransporter and is
recycled back into the tubule lumen through Kir1.1. Thus Kir1.1 plays
an important role in sustaining Na+ and Cl
transport across epithelial cells from this nephron segment. However,
the role of Kir channels in alveolar epithelial cells is unclear. One
possibility could be that Kir2.1 is located in basolateral membrane,
where it may play a role in K+ recycling necessary for
maintaining Na+-K+-ATPase activity. This may be
particularly important under conditions of stimulated Na+
absorption where increased Na+-K+-ATPase
activity may depend on the rate of K+ recycling.
In an earlier study by Sakuma et al. (73), the presence of
ATP-sensitive potassium (KATP) channels in human alveolar
cells was suggested from experiments with
2-(3,4-dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl) pyridine N-oxide (YM934), which increased both potassium
influx into the alveolar space and alveolar fluid clearance in human lung. Addition of glibenclamide, a KATP channel blocker,
inhibited the YM934-stimulated increase in alveolar fluid clearance,
providing additional evidence to suggest a role for KATP
channels. Although KATP channels may be present in human
alveolar epithelial cells, the components of these potassium channels
[Kir6.1, Kir6.2, sulfonylurea receptor (SUR) 1, and SUR2] could not
be detected in primary adult rat alveolar epithelial cells by RT-PCR
(Table 1).
Ca2+-activated
K+ channels.
Two-types of Ca2+-activated K+ channels,
large-conductance (BK) and intermediate-conductance (IK) K+
channels, have been identified in A549 cells. Ridge et al.
(72) observed an ~242-pS K+ channel that was
activated by increases in [Ca2+] and membrane
depolarization. Blockers including Ba2+, TEA, and quinidine
were shown to inhibit this large-conductance K+ channel. BK
channels may play a role in repolarizing cells following depolarization
or calcium entry. However, the physiological role of BK channels in
primary alveolar epithelial cells remains to be resolved. IK channels
were activated by adenosine or nucleoside transport blockers such as
nitrobenzylthioinosine in A549 cells (81). In addition,
clotrimazole, a selective blocker of IK channels, was also shown to
inhibit the channel. In RT-PCR experiments, mRNA transcripts for IK-1
were detected in these cells. However, we were unable to
detect slo1, the principal subunit of BK channels and IK-1 in cultured
adult rat alveolar epithelial cells by RT-PCR. Thus at this time there
is no functional or molecular evidence for expression of these
Ca2+-activated K+ channels in primary rat
alveolar epithelial cells (88).
 |
SUMMARY AND CONCLUSIONS |
Cl
and K+ channels serve multiple
functions in excitable and nonexcitable cells, including prominent
roles in cell signaling, control of cell volume, and regulation of
membrane potential. In alveolar epithelial cells, Cl
and
K+ channels are involved in regulating the volume and ionic
composition of alveolar fluid. Results from previous investigations
with cultured fetal alveolar epithelial cells and more recent work with
adult cells and perfused lung studies has shown that CFTR plays a role in both anion and fluid secretion or NaCl and fluid absorption, depending on stage of development. In the fetal state, anion secretion establishes an osmotic driving force necessary for fluid secretion into
the developing alveoli and airways. In the adult lung, CFTR activation
following adrenergic receptor stimulation appears to be essential for
increasing alveolar fluid clearance. This observation suggests that an
increase in apical membrane anion permeability is necessary for
increased fluid transport across the alveolar epithelium in response to
adrenergic agonists. Whether CFTR modulates the expression or activity
of ENaC in the apical membrane of alveolar epithelial cells is
presently unknown. Evidence that CFTR is important for adrenergic
receptor stimulation of alveolar fluid clearance in the adult human
lung suggests the possibility that patients with CF may be less
responsive to terbutaline treatment under conditions of hydrostatic or
lung injury pulmonary edema.
Investigations over the past 15 yr have shown that alveolar epithelial
cells express a variety of K+ channels, including Kir
channels, Ca2+-activated K+ channels, and Kv
channels. The functional roles of these channels in alveolar epithelial
cell transport function remain to be elucidated, but some ideas have
been suggested. For example, the proposed localization of Kir2.1
channels in the basolateral membrane of fetal guinea pig AII cells
suggests a possible role in K+ recycling, in support of
increases in Na+-K+-ATPase activity. In
contrast, the apical localization of at least some of the conducting
-subunits of Kv channels suggests a role in K+
secretion, or perhaps O2 sensing, given that Kv
-subunits have been previously shown to confer oxygen sensitivity to
certain Kv channels in smooth muscle cells. Clearly, more studies are needed to understand the physiological importance of these channels and
to identify other K+ channel types that are important in
sustaining transepithelial Na+ and Cl
transport across the alveolar epithelium.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
S. M. O'Grady, Univ. of Minnesota, 495 Animal
Science/Veterinary Medicine Bldg., 1988 Fitch Ave., St. Paul MN 55108 (E-mail: ograd001{at}umn.edu).
10.1152/ajplung.00256.2002
 |
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