cAMP-induced changes of apical membrane potentials of confluent H441 monolayers
Ahmed Lazrak1 and
Sadis Matalon1,2
Departments of 1Anesthesiology and
2Physiology and Biophysics, Schools of Medicine and
Dentistry, University of Alabama at Birmingham, Birmingham, Alabama
35294
Submitted 2 December 2002
; accepted in final form 11 April 2003
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ABSTRACT
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We recorded apical membrane potentials (Va) of H441
cells [a human lung cell line exhibiting both epithelial Na+ (ENaC)
and CFTR-type channels] grown as confluent monolayers, using the
microelectrode technique in current-clamp mode before, during, and after
perfusion of the apical membranes with 10 µM forskolin. When perfused with
normal Ringer solution, the cells had a Va of -43 ±
10 mV (means ± SD; n = 31). Perfusion with forskolin resulted
in sustained depolarization by 25.0 ± 3.5 mV (means ± SD;
n = 23) and increased the number, open time, and the open probability
of a 4.2-pS ENaC. In contrast to a previous report (Jiang J, Song C, Koller
BH, Matthay MA, and Verkman AS. Am J Physiol Cell Physiol 275:
C1610C1620, 1998), no transient hyperpolarization was observed. The
forskolin-induced depolarization of Va was almost totally
prevented by pretreatment of monolayers with 10 µM amiloride or by
substitution of Na+ ions in the bath solution with
N-methyl-D-glucamine. These findings indicate that cAMP
stimulation of Na+ influx across H441 confluent monolayers results
from activation of an amiloride-sensitive apical Na+ conductance
and not from Va hyperpolarization due to Cl-
influx through CFTR-type channels.
forskolin; epithelial sodium channel; cystic fibrosis trans-membrane conductance regulator; current clamp; patch clamp; single channel currents; amiloride; glibenclamide
THE AMILORIDE-SENSITIVE epithelial sodium
(Na+) channel (ENaC) plays a fundamental role in the regulation of
fluid movement across epithelial cells. In the lungs, the transepithelial
transport of Na+ ions through ENaC, or ENaC-type channels,
generates the osmotic driving force for water movement from the alveolar to
the interstitial space, which reduces the amount of fluid in the alveolar
space under both physiological and a number of pathological conditions
(23,
34).
The existence of amiloride-sensitive Na+ absorption across the
alveolar epithelium in vivo has been demonstrated in rats, rabbits, hamsters,
mice, guinea pigs, sheep, and humans
(6,
9,
24,
26,
27,
30). Furthermore, alveolar
type II (ATII) cells isolated from the lungs of these species grown to
confluence on filters and mounted in Ussing chambers generated a spontaneous
potential difference and short-circuit current (Isc) that
was partly inhibited by amiloride with an IC50 of
0.85 µM
(2,
7). On the basis of these
findings, it has been proposed that Na+ ions diffuse passively
across the apical membranes of ATII cells through these channels, down an
electrochemical gradient maintained by an Na+-K+-ATPase
pump.
Direct evidence for the existence of an ion channel in the apical membranes
of ATII cells was derived from electrophysiological measurements performed on
isolated ATII cells. In these cells, three different types of channels were
identified: Ca2+-activated cation channels
(5); Ca2+
independent, moderately selective cation channels with unitary conductances
between 20 and 25 pS (10,
35,
36); and highly selective (4
pS) ENaC-type Na+ channels
(11,
22). The basic biophysical
properties of these channels depend on the culture conditions: ATII cells
grown on air-liquid interface or in the presence of steroids (such as
aldosterone) expressed channels with 6.6 pS unitary conductance and with very
high selectivity for Na+ over K+
(PNa/PK >80) where P is
permeability. These channels are inhibited by submicromolar concentrations of
amiloride (K0.5 = 37 nM)
(11); however, if cells are
cultured in the absence of steroids they express either nonselective or poorly
selective (PNa/PK = 7) 25-pS channels
with an amiloride IC50 of
1 µM (reviewed in Ref.
22). Currently, there is
significant controversy as to what type of channel is expressed in alveolar
epithelial cells in vivo: the higher-than-expected K+ concentration
in the alveolar epithelial lining fluid of anesthetized rabbits and the
reduction of these values following application of amiloride
(28) are consistent with the
presence of non-selective or poorly selective amiloride-sensitive channels at
the apical side of alveolar epithelial cells. However, it is likely that both
types of channels are present at the alveolar epithelium and that their
relative expression is regulated by a number of hormones and environmental
factors. Furthermore, recent studies indicate that alveolar type I cells,
which form >97% of the alveolar epithelium, also have amiloride-sensitive
Na+ channels, the biophysical properties of which have not been
studied at present (14).
There is also considerable evidence that agents that increase intracellular
cAMP upregulate Na+ transport in a number of species, including
humans, in vivo and ex vivo, and across isolated ATII cells (reviewed in Refs.
23,
25). Patch-clamp measurements
have shown that
-agonists and permeable analogs of cAMP increase the
number and/or the open probability (Po) of the active
Na+ channels in ATII cells, depending on the type of channels
expressed (3,
36). On the other hand, Jiang
et al. (13) and O'Grady et al.
(29) have proposed that the
cAMP-induced increase of Na+ transport is merely due to an increase
of the driving force across the apical membranes, secondary to an activation
of CFTR-type Cl- conductance. Thus the mechanism by which an
increase in cAMP increases Na+ absorption across lung epithelial
cells containing both ENaC and CFTR-type channels is still in dispute.
Herein we cultured H441 cells on transparent membranes until they formed
confluent monolayers and measured the apical membrane potential
(Va) and Na+ single channel activity before,
during, and after increasing the cytoplasmic cAMP concentration by perfusing
these cells with forskolin, an adenyl cyclase activator. H441 cells are
derived from human Clara cells found in the bronchiolar epithelium, which
normally lacks mucous cells and produces a mucous-poor, watery proteinaceous
secretion. H441 cells express both ENaC (this study) and CFTR channels
(16) in the absence of hormone
supplementation. Furthermore, in contrast to ATII cells, they exhibit stable
recording of Va when impaled with low-resistance
microelectrodes. Our data clearly demonstrate that agents that increase cAMP
activate an apical Na+ conductance, by directly increasing the
activity and the Po of an ENaC-type channel, and
depolarize Va.
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MATERIALS AND METHODS
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Cell culture. H441 cells were obtained from ATCC and were grown in
RPMI 1640 medium supplemented with 2 mM L-glutamine, 1.5 g/l sodium
bicarbonate, 4.5 g/l glucose, 10 mM HEPES (pH 7.4), and 10% fetal bovine
serum. Cells were seeded in 75-cm2 flasks and incubated in a
humidified atmosphere of 5% CO2-95% O2 at 37°C until
they reached confluence and then were passaged weekly. For consistency, only
cells between passages 82 and 97 were used for the present
studies. The cells were subcultured on Millipore membranes (12 mm in diameter,
Millicell-CM; Millipore, Bedford, MA), and the medium was replaced every other
day. Transepithelial resistance (Rt) was measured daily
with an Epithelial Voltohmmeter equipped with chopstick-style electrodes
(World Precision Instruments, Sarasota, FL). All measurements were conducted
on H441 cells monolayers between the 4th and 6th days after the initial
seeding, the time at which they typically formed confluent monolayers. Filters
containing H441 cells were rinsed with normal Ringer solution (NRS) and
transferred to the recording chamber mounted on the stage of an inverted
microscope (Olympus). Both the apical and basolateral sides of the monolayers
were perfused continuously with solutions of the same ionic composition at
room temperature (2022°C), using two gravity-driven perfusion
systems.
All H441 cells were initially perfused with NRS containing (in mM) 143
NaCl, 5.4 KCl, 1.8 CaCl2, 2 MgCl2, 10 glucose, and 10
HEPES (pH 7.4). The osmolality of this solution, measured with a Vapor
Pressure Osmometer (Vapro Wescor, Logan, UT), was 300 ± 5
mosmol/kgH2O. In some experiments, cells were perfused with
solutions in which NaCl was replaced with equimolar concentrations of
Na-gluconate or N-methyl-D-glucamine (NMDG)-Cl. The
osmolality of all solutions was adjusted to 300 mosmol/kgH2O with
mannitol, and the pH was adjusted to 7.4 with 10 mM HEPES and 1 N NaOH. Once
stable recordings were obtained, amiloride (10 µM), forskolin (10 µM),
or glibenclamide (100 µM) was added into the solutions perfusing the apical
side of the monolayers.
Patch-clamp measurements. The cell-attached mode of the
patch-clamp technique (8) was
used to detect the discrete activity of the amiloride-sensitive Na+
channels on the apical membranes of H441 confluent cell monolayers. The
pipettes were made from LG16-type capillary glass (Dagan, Minneapolis, MN)
with a two-stage vertical puller (PIP5; HEKA, Pfalz, Germany). They were
back-filled with a solution of the following ionic composition (in mM): 145
Na+-gluconate, 1.8 CaCl2, 2 MgCl2, 5.5
mannitol, and 10 HEPES, pH 7.4. Pipette resistance, when filled with this
solution, was
15 M
. The offset potential was corrected with an
amplifier (Axopatch 200; Axon Instruments, Foster City, CA) just before the
giga-seal formation. Before recording channel activity, we perfused the cells
with a solution containing (in mM): 134 K+-gluconate, 10 KCl, 5
MgCl2, 10 HEPES, and 5.5 glucose, pH 7.4, which depolarized
Va to a mean value of -3 mV (n = 7). The patch
potential was then calculated from the following equation:
(Vpatch = Va -
Vpipette), where Va = -3 mV and
Vpipette is the applied potential. The data were sampled
at 25 kHz and filtered at 12 kHz. During analysis, a 300-Hz
low-pass digital filter was used. The amplitude and Po of
the channels were calculated from all event histograms, constructed from at
least 10 min of recordings, as previously described
(18,
19). Recordings were either
continuous or appended to each other to satisfy this condition.
Current-voltage (I-V) relationships were constructed from
steady-state currents measured at 300 ms from the start of voltage pulses,
using Clampfit Program (Axon Instruments) and Origin (Microcal Software,
Northampton, MA). The conductance was measured as the slope conductance of
I-V relationships.
Measurements of Va. Microelectrodes were made
from 0.5-mm inner diameter glass capillaries (World Precision Instruments)
using the P87 micropipette puller (Narishige, Tokyo, Japan) and were filled
with 300 mM KCl solution at pH 7.4 (10 mM HEPES). When the pipette was filled
with this solution, its tip had a resistance ranging from 125 to 175 M
.
The cell membrane resistance of single cells (i.e., cells not part of
confluent monolayers) was 415 ± 47 M
(means ± SD;
n = 12). The ground electrode, an Ag-AgCl pellet, against which the
membrane potential was measured, was connected to the bath via an agar bridge
(2% agar in 150 mM NaCl solution). All measurements were performed in current
clamp mode using an IE-251A amplifier (Warner Instruments, Hamden, CT). The
data were stored onto the hard drive of a computer equipped with
digital/analog and analog/digital converter (Digidata 200) and analyzed using
the PClamp software (Axon Instruments, Union City, CA).
Chemicals. All chemicals were purchased from Sigma-Aldrich (St.
Louis, MO). Amiloride was dissolved in water; forskolin was dissolved in
ethanol (ethanol's maximal concentration in the bathing solution was
<0.1%). Glibenclamide was dissolved in DMSO.
Statistics. All data were analyzed by ANOVA, using the Bonferroni
method for multiple comparisons or Student's t-test when appropriate.
All values given are means ± SD and a P value of < 0.05 was
considered significant.
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RESULTS
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All measurements were performed on H441 confluent cell monolayers
[Rt = 1.9 ± 0.36 K
·cm (means ±
SD; n = 15)] unless stated otherwise. When cells were perfused with
NRS, Va ranged from -30 to -50 mV (means ± SD = -43
± 10 mV; n = 31; see Table
1). Only cells with stable Va for at least 3
min were used for further measurements. When the apical side of the cells was
perfused with NRS containing 10 µM amiloride, Va
hyperpolarized by
17 mV and recovered to its initial value when amiloride
was washed out (Fig. 1). This
finding suggests the presence of a basal Na+ influx across the
apical membranes of H441 monolayers through amiloride-sensitive pathways.

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Fig. 1. Effects of amiloride on apical membrane potential (Va).
Typical record of Va of an H441 cell monolayer during
perfusion of the apical side with normal Ringer solution (NRS), followed by
NRS containing 10 µM amiloride (Amil). In both cases the basolateral
compartment was perfused with NRS alone. Note the hyperpolarization of the
apical membrane potential during the perfusion with amiloride and its
spontaneous recovery to the control value when amiloride was washed out. This
recording is a typical result that was reproduced 11 times.
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Single channel recordings using the cell-attached mode showed the presence
of an Na+ channel with 4.2 pS unitary conductance and long-lasting
open states, consistent with the biophysical properties of ENaC
(Fig. 2). In a number of
experiments (n = 5), the upper part of the pipette (5 µl for a
total volume of 10 µl) was filled with a solution containing 4 µM
amiloride (for a final concentration of 2 µM). As shown in
Fig. 2C,2 µM
amiloride induced the complete cessation of channel activity in the patch.
Recordings performed on H441 cell groups forming incomplete monolayers show
the presence of two Na+ conductances (4.2 and 20 pS;
Fig. 3). Although we did not
perform single Cl- channel measurements, H441 cells have been shown
to express a CFTR-type chloride channel
(16).

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Fig. 2. Single channel currents in a H441 cell confluent monolayer. Single channel
currents recorded in a cell-attached patch mode from the apical membrane of an
H441 cell that was part of a confluent monolayer, at a holding potential of
-100 mV (Vholding = Vapical -
Ppipette). The pipette was filled with 145 mM
Na+; the cell was perfused with a solution containing 145 mM
K+, which depolarized the membrane potential to about -3 mV (see
MATERIALS AND METHODS). The recording (A) and amplitude
distribution histogram (B), constructed by appending single channel
events of 3 different recordings (10 min total), point to the presence of a
channel characterized by a unitary conductance of 4.2 ± 0.43 pS (mean
± SD; n = 1,010 events for a 10-min recording). The single
channel activity was totally blocked by 2 µM amiloride in the pipette
solution (C). Results from typical experiments reproduced at least 13
times under both experimental conditions. C, closed.
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Fig. 3. Single channel currents in a H441 cell, which was not part of a confluent
monolayer. Single channel currents recorded from a cell-attached patch of an
H441 cell, at a holding potential of -100 mV (Vholding =
Vapical - Ppipette). The pipette was
filled with 145 mM Na+; the cell was perfused with a solution
containing 145 mM K+, which depolarized the cell to 0 mV. As can be
seen, in contrast to the record shown in
Fig. 2A, 2 distinct
conductances were found at 20 and 4.2 pS. This was a typical recording that
was reproduced >8 times.
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In subsequent experiments, we evaluated the effects of cAMP on H441
Va. As shown in Fig.
4 and Table 1,
perfusion of the apical sides of monolayers with NRS containing forskolin (10
µM) resulted in a sustained and fully reversible depolarization of
Va (
Va = 25.0 ± 3.5 mV;
mean ± SD; n = 23). Furthermore, when amiloride (10 µM) was
added into the solution at the plateau of the forskolin response,
Va rapidly hyperpolarized and returned to a baseline that
was less negative than when perfused with NRS alone
(Fig. 4). The forskolin-induced
Va depolarization was attenuated significantly when
amiloride was added in the apical perfusion solution
(Fig. 5) or when we replaced
Na+ in the apical and basolateral baths with equimolar
concentrations of NMDG (Fig.
6). In the latter case, addition of glibenclamide into the
perfusion medium totally abolished the forskolin-induced depolarization. Thus
the resulting depolarization was most likely due to Cl- secretion
through cAMP-stimulated CFTR channels. However, significant depolarization was
observed when cells were reperfused with NRS containing forskolin
(Fig. 7). On the other hand,
the amplitude of the forskolin-induced depolarization was not affected when
Cl- in the perfusion solution was mostly replaced with equimolar
concentrations of gluconate (Fig.
8). In this case, the depolarization was preceded by a transient
hyperpolarization most likely due to the efflux of K+ ions. Because
previous studies have shown that agents that increase cAMP may also increase
intracellular Ca2+ in lung epithelial cells
(21), this K+
efflux may have occurred through Ca2+-activated
K+ channels.

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Fig. 4. Effects of forskolin (Forsk) on Va. Initially, both the apical
and basolateral compartments of the cells shown in A and B
were perfused with NRS. Once stable, a recording of Va was
obtained (-35 and -45 mV, respectively), the apical compartments were perfused
with NRS containing 10 µM forskolin, which resulted in a significant and
sustained depolarization. Va returned to its control value when the
apical solution was switched to NRS alone (A) or when amiloride (10
µM) was added into the perfusate (B). In both cases, the membrane
depolarization during forskolin perfusion is consistent with the activation of
a Na+ conductance in these cells. Results are of typical
experiments, which were reproduced at least 23 and 11 times, respectively.
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Fig. 5. Amiloride prevents the forskolin-induced depolarization of H441
Va. Initially, both the apical and basolateral
compartments of this H441 monolayer were perfused with NRS. After
establishment of a stable Va (around -30 mV), the apical
compartment was perfused with NRS containing 10 µM amiloride, which caused
an immediate hyperpolarization of Va to about -57 mV.
Subsequent perfusion with NRS containing both forskolin and amiloride (10
µM each) resulted in very modest depolarization of Va
compared with the observed depolarization when cells were perfused with
forskolin alone (Fig. 4).
Result of a typical experiment that was reproduced at least 6 times.
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Fig. 6. Sodium ions are necessary for the forskolin-induced depolarization in H441
cells. Both the apical and basolateral compartments of an H441 cell were
perfused with solutions in which Na+ ion was substituted with
equimolar amounts of N-methyl-D-glucamine
(NMDG+, an impermeant cation). When a new stable
Va value was attained, the apical compartment was perfused
with a solution containing NMDG-Cl and 10 µM forskolin. This resulted in a
very modest Va depolarization, which was completely
reversed when glibenclamide (100 µM) was added into the solution perfusing
the apical compartment. Result of a typical experiment that was reproduced at
least 5 times.
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Fig. 7. Sodium ions are necessary for the forskolin-induced depolarization in H441
cells monolayers. After perfusion with NMDG-Cl, a monolayer was perfused with
NRS containing 10 µM forskolin. It resulted in a significant and sustained
depolarization of Va. Result of a typical experiment that
was reproduced successfully at least 4 other times.
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Fig. 8. Chloride ions are not necessary for the forskolin-induced
Va depolarization. Both the apical and basolateral
compartments of an H441 cell monolayer were perfused with solutions in which
Cl- ion was replaced with equimolar concentrations of gluconate
(gluc). Once a stable Va value (-44 mV) was obtained, the
apical compartment was perfused with a Na-gluconate solution containing 10
µM forskolin. This resulted in a transient hyperpolarization, followed by a
sustained depolarization. The amplitude of Va
depolarization was similar to that seen when monolayers were perfused with
NaCl containing 10 µM forskolin (Fig.
4 and Table 1).
Result of a typical experiment that was reproduced 7 times.
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As an aggregate, our findings show that the forskolin-induced
depolarization in H441 monolayers results from the entry of Na+
ions through newly activated amiloride-sensitive Na+ or cation
channels. Indeed, as shown in Fig. 9,
B and C, perfusion of H441 cells with forskolin
increased the number of active channels in the patches from two to three and
their Po from 0.23 ± 0.03 to 0.55 ± 0.05
(mean ± SD; n = 7; P < 0.05) without affecting the
unitary conductance. Because the number of channels in the patches was not
determined, it is possible that forskolin activated existing quiescent
channels. In any case, our results differ from those of Chen et al.
(3), who report that
stimulation of ATII cells with terbutaline increased the number but not the
Po of 4-pS Na+-selective channels.

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Fig. 9. Effects of forskolin on single channel activity of H441 cells. Single
channel currents recorded in cell-attached mode from 2 H441 monolayers at a
holding potential of -100 mV (Vholding =
Vapical - Ppipette). The pipettes were
filled with 145 mM Na+, and the cell monolayers were perfused with
a solution containing 145 mM K+. A: the monolayer was
perfused with NRS; B: the monolayer was perfused with NRS containing
10 µM forskolin. C and D: an expanded view of B
and the associated current amplitude distribution histogram. The histogram was
constructed from a 10-min recording. As can be seen, forskolin induced an
increase of both the number of the active channels in the patches and their
open probability without affecting their unitary conductance (4.2 pS). Result
of a typical experiment that was successfully repeated 13 times.
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DISCUSSION
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Our results clearly demonstrate that perfusion of confluent monolayers of
H441 cells with forskolin, an agent known to increase intracellular cAMP
levels in epithelial cells, resulted in significant depolarization of their
apical membrane potential; this effect was decreased either by adding
amiloride into the perfusate or by replacing Na+ ion with equimolar
concentrations of NMDG+, a large impermeant positive ion.
Furthermore, perfusion with forskolin increased both the number of active
channels and the Po of a 4-pS conductance of an ENaC-type
channel. Previously, Jiang et al.
(13) proposed that exposure of
confluent monolayers of ATII cells to either
-adrenergic agents or
cell-permeable analogs of cAMP stimulates an apical CFTR-type Cl-
conductance, resulting in the hyperpolarization of the ATII cell
Va. These authors speculate that the observed increase in
the Na+ component of the Isc across these
monolayers following
-adrenergic stimulation was the result of an
increased driving force for Na+ ions across the apical membranes
instead of a direct effect on Na+ conductance. It should be
stressed that Jiang et al.
(13) provide no direct
evidence for the development of membrane hyperpolarization following increases
of intracellular cAMP levels. Our direct measurement of Va
in H441 cells shows no evidence of transient Va
hyperpolarization during perfusion of the cell monolayers with NRS containing
forskolin. Instead, our measurements of Va and the
biophysical properties of Na+ single channels are strong evidence
that perfusion of H441 cells with forskolin increased the total Na+
conductance, in agreement with both measurements of
Isc and Na+ single channel activity in
ATII cells (3,
17,
19,
35,
36). Our findings are also in
agreement with theoretical analysis using the Nernst equation. It shows that
the driving force for Na+ ions across the apical membrane
(Va - ENa) where
ENa is the sodium reversal potential of H441 or ATII cells
is about -100 mV, and thus, as suggested
(33), a significant degree of
hyperpolarization, unlikely to be achieved by the influx of Cl-
ions, would be needed to explain the large increase in Na+ current
following an increase in intracellular cAMP. On the contrary, as shown in
Fig. 5, perfusion of H441 cells
with forskolin in the presence of amiloride-depolarized
Va, consistent with Cl- secretion and not
absorption.
Admittedly, our measurements were conducted in a different cell type than
those of Jiang et al. (13)
(H441 vs. cultured ATII cells). The reason for choosing this system was the
biophysical properties of Na+ channels in ATII cells vary according
to the culture conditions. For example, ATII and A549 cells cultured in the
presence of steroids express 4-pS ENaC-type channels
(11,
20); however, in the absence
of steroids, they express mainly nonselective 20-pS cation channels
(11,
35). We were concerned that
steroids may alter the response of these cells to agents that increase cAMP
and thus opted to use H441 cells, which express both ENaC and CFTR-type
channels without being treated with steroids. It should be kept in mind that
the biophysical properties of Na+ ion channels of alveolar
epithelial cells in vivo have not been determined: as mentioned above, ATII
cells express a variety of cation channels
(23), and recent data are
consistent with the presence of amiloride-sensitive Na+ influx
across isolated alveolar type I cells
(14). In other systems,
Uyekubo et al. (32) show that
forskolin tripled fluid absorption across open-circuited primary cultures of
bovine tracheal epithelial cells and that the effect was inhibited by CFTR
blockers. Microelectrode studies suggest that the magnitude of the absorptive
response to forskolin in bovine cells depends on the size of an inwardly
directed electrochemical driving force for Cl- movement across the
apical membrane. However, this effect was not seen in human tracheal cells,
perhaps due to the maximum stimulation of CFTR under control conditions.
There is considerable evidence that increases in cAMP and cAMP-dependent
protein kinase A (PKA) increase both the Po and the number
of channels in alveolar epithelial cells. Addition of terbutaline or PKA into
the bath solution of ATII cells patched in the cell-attached and inside-out
mode, respectively, doubles the Po of a 27-pS nonselective
Na+ channel without affecting its single channel conductance
(36). Similar results were
obtained following the addition of PKA plus ATP to the presumed cytoplasmic
side of planar bilayers containing a putative immunopurified ATII
Na+ channel protein
(31). Berdiev et al.
(1) showed that PKA
phosphorylates both the 135-kDa and the 70-kDa polypeptides of the
immunopurified ATII Na+ channel protein. Finally, perfusion of A549
cells with forskolin significantly increased the whole-cell
amiloride-sensitive Na+ current and the NPo of
an 8.6-pS Na+ channel in cell-attached patches
(20) where N refers
to the number of channels. These data support the hypothesis that
phosphorylation of the Na+ channel complex (or of cytoskeletal
proteins interacting with this complex) is involved in cAMP activation.
On the other hand, there is also significant evidence showing that cAMP may
promote insertion of new channel protein from a cytoplasmic pool to the apical
membranes. Chen et al. (3)
report that exposure of ATII cells to agents that increase cAMP upregulated
Po of the 2025-pS nonselective cation channel while
it increased the numbers but not the Po of a 4-pS ENaC
channel. These results are consistent with insertion of new ENaC channels in
the apical membrane via cAMP-dependent processes. These observations are also
consistent with the findings of Kleyman et al.
(15), who report that exposure
of A6 cells to increasing intracellular cAMP doubled the amount of ENaC
protein in the apical membrane of A6 cells. However, as mentioned previously,
our data indicate significant increases in the Po of a
4-pS channel in H441 cells. In any event, these data provide strong evidence
that an increase in intracellular cAMP activates existing Na+
channel in the apical membranes by a variety of mechanisms.
Movement of Na+ ions from the alveolar to the interstitial space
necessitates the simultaneous movement of an anion (such as Cl-) to
preserve the electro-neutrality. A number of in vivo studies suggest the
movement of Cl- ion occurs via transcellular vs. para-cellular
pathways. Nielsen et al. (27)
found that the substitution of Cl- ions with methanesulfonate leads
to the total block of the basal Na+-dependent fluid clearance
across rabbit lungs. Under these conditions, forskolin induced Cl-
secretion instead of Na+ absorption. However, addition of
methanesulfonate into the alveolar space may have depolarized the apical
membrane (due to the efflux of Cl- ions), decreasing the driving
force for Na+. Fang et al.
(4) show that perfusion of
mouse lungs in situ with solutions containing 50% of the normal
[Cl-] resulted in a significant inhibition of Na+
dependent alveolar fluid clearance. Interestingly,
F508 mice, lacking
surface expression of CFTR, had normal levels of AFC, indicating that
transcellular movement of Cl- ions may occur via a variety of
Cl- channels. On the other hand, stimulation of clearance by
isoproterenol upregulated AFC and Cl- absorption in wild-type mice
but not in
F508. On the basis of these findings, the authors propose
that functional CFTR is necessary for the cAMP-induced stimulation of
Na+ transport in vivo. However, recently published findings
(12) show that
-adrenergic stimulation increased Cl- transport across the
alveolar epithelium in 50% of CFTR(-/-) mice, indicating the involvement of
complex mechanisms in fluid clearance. In any event, these studies show that
transcellular Cl- movement through CFTR and other types of
Cl- channels plays an important role in the vectorial transport of
Na+ ion in vivo; however, they provide absolutely no evidence of
membrane hyperpolarization as the critical factor as suggested by Jiang et al.
(13).
 |
ACKNOWLEDGMENTS
|
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The authors thank T. Myles and S. Muturi for technical support and Dr. Ian
Davis for helpful comments and suggestions.
DISCLOSURES
This work was supported in part by National Institutes of Health Grants
HL-31197, HL-51173, and P30 DK-54781.
 |
FOOTNOTES
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Address for reprint requests and other correspondence: S. Matalon, Dept. of
Anesthesiology, Univ. of Alabama at Birmingham, 901 19th St. S., BMR2 Rm. 224,
Birmingham, AL 35205-3703 (E-mail:
Sadis{at}uab.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
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