Adrenergic stimulation of Na+
transport across alveolar epithelial cells involves activation of
apical Cl
channels
Xinpo
Jiang,
David H.
Ingbar, and
Scott M.
O'Grady
Departments of Physiology and Medicine, University of Minnesota,
Minneapolis, Minnesota 55455
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ABSTRACT |
Alveolar epithelial cells were isolated from adult
Sprague-Dawley rats and grown to confluence on membrane filters. Most
of the basal short-circuit current
(Isc; 60%) was
inhibited by amiloride (IC50 0.96 µM) or benzamil (IC50 0.5 µM).
Basolateral addition of terbutaline (2 µM) produced a rapid decrease
in Isc, followed by a slow recovery back to its initial amplitude. When
Cl
was replaced with
methanesulfonic acid, the basal
Isc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of
28 mV. Increasing Cl
concentration in the
basolateral solution shifted the reversal potential to more depolarized
voltages. These results were consistent with the existence of a
terbutaline-activated Cl
conductance in the apical
membrane. Terbutaline did not increase the amiloride-sensitive
Na+ conductance. We conclude that
-adrenergic
stimulation of adult alveolar epithelial cells results in an increase
in apical Cl
permeability and that
amiloride-sensitive Na+ channels are not directly affected
by this stimulation.
chloride absorption; cystic fibrosis transmembrane conductance
regulator; sodium channel; glibenclamide; amiloride; alveolar type II
cells; ion transport
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INTRODUCTION |
CHLORIDE SECRETION across the airway epithelium
provides the driving force for fluid secretion into the airways of the
fetal lung and is essential for normal lung development. Lung fluid secretion rate decreases significantly a few days before birth (4). At
the same time, increases in Na+
and fluid absorption occur across the pulmonary epithelium (37). Before
and shortly after birth, airway and alveolar fluid is removed, allowing
for efficient gas exchange. Active
Na+ transport across the alveolar
epithelium is thought to play a major role in this transition (8, 37,
39, 40), and there is evidence to suggest that fluid
clearance is enhanced by
-adrenergic receptor agonists in both fetal
(10, 22, 37, 41, 54) and adult lung (6, 23, 48). These changes in
transepithelial transport correlate with an increase in plasma
epinephrine concentration and an increase in
-adrenergic receptor
expression in pulmonary epithelial tissue late in gestation (10, 15,
55, 56). In addition, levels of epithelial
Na+ channel (ENaC) mRNA and of
Na+-K+-ATPase
mRNA, protein, and activity increased late in gestation and remained
high in adult lung (28, 38, 53). These results support the idea that
stimulation of fluid absorption by
-adrenergic receptor agonists
results from an increase in net
Na+ absorption (47). This
hypothesis is supported by experiments using isolated perfused rat
lungs (17, 26), fetal lung epithelium (34, 36, 51), and isolated rat
alveolar type II cells (14, 49, 58) and by in vivo
experiments (7, 24, 29). In isolated perfused rat lung (21, 29, 30) and
in vivo studies (24, 29), fluid absorption was
considerably reduced by Na+
channel blockers. Channels with high and low affinity for amiloride coexist in the apical membrane of fetal rat lung epithelial cells (32),
with different cation selectivity [permeability ratio (PNa/PK) = 0.9 (42),
PNa/PK > 10 (52, 53)]. In adult rat, high-affinity amiloride-sensitive
Na+ channels have been identified
in cultured alveolar type II cells (14, 16, 46). In addition,
low-affinity amiloride-sensitive Na+ channels are present in
freshly isolated (33) and cultured (57, 58) adult rat alveolar type II cells.
In contrast to the results of experiments using fetal airway and distal
lung epithelial cells (3, 5, 30, 50), adult alveolar type II cells
absorb Cl
in response to
-adrenergic stimulation. This conclusion was based on isotopic flux
measurements using monolayers of adult rat alveolar type II cells (31).
The specific mechanisms involved in
-adrenergic stimulation of
Cl
absorption are presently
unknown. The objective of this study was to determine the effect of
-adrenergic receptor stimulation and 8-(4-chlorophenylthio)adenosine
3',5'-cyclic monophosphate (CPT-cAMP) on
Cl
and
Na+ transport properties of
cultured primary adult alveolar epithelial cells.
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MATERIALS AND METHODS |
Materials.
Male Sprague-Dawley rats weighing 150-174 g were purchased from
Harlan (Indianapolis, IN). Elastase was purchased from Worthington Biochemical (Freehold, NJ). Rat IgG, DNase I, nonessential amino acids,
BSA, L-glutamine, HEPES, trypsin
inhibitor, terbutaline, and glibenclamide were obtained from Sigma
Chemical (St. Louis, MO). DMEM-Ham's F-12 nutrient mixture in a 1:1
ratio (DMEM/F-12) and penicillin-streptomycin were purchased from GIBCO
BRL (Grand Island, NY). Nitex mesh (120 and 40 µm) was purchased from
Tetko (Elmsford, NY). Tissue culture-treated Transwell polycarbonate filters were obtained from Corning Costar (Cambridge, MA). PBS was
obtained from Celox Laboratories (Oakdale, MN). Amiloride was obtained
from Merck Sharp & Dohme Research Laboratories (West Point, PA).
Benzamil,
5-(N-ethyl-N-isopropyl)-amiloride
(EIPA), N-phenylanthranilic
acid [also called diphenylamine-2-carboxylic acid (DPC)],
5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and CPT-cAMP were
purchased from RBI (Natick, MA). All chemical reagents used for
electron microscopy were obtained from Electron Microscopy Sciences
(Fort Washington, PA). All other chemicals were purchased from Sigma Chemical.
Cell preparation and culture.
Alveolar epithelial cells were isolated from adult rat lungs using a
modification of the protocol described by Borok et al. (9). Rats were
anesthetized with an intraperitoneal injection of pentobarbital. Lungs
were perfused with solution
2 (in mM: 140 NaCl, 5 KCl, 2.5 NaH2PO4,
1.3 MgSO4, 2.0 CaCl2, 6 glucose, and 10 HEPES).
After removal, the lungs were repeatedly lavaged with
solution
1 (in mM: 140 NaCl, 5 KCl, 2.5 NaH2PO4,
6 glucose, and 10 HEPES) and solution
2 to eliminate macrophages. The lungs then were filled with elastase-containing solution (2.7 U/ml in solution
2) and were incubated at 37°C
for 30 min in a shaker bath. Elastase was neutralized by stop solution
[2 mM EDTA, 1% BSA, 0.1% soybean trypsin inhibitor, and 0.15/ml
DNase I in a buffered saline solution containing (in mM) 136 NaCl, 2.2 Na2HPO4, 5.3 KCl, 10 HEPES, and 5.6 glucose]. Finely minced tissues were filtered through 120- and 40-µm Nitex mesh. Cells were further purified by panning on IgG-coated petri dishes to remove remnant macrophages and suspended directly in serum-free DMEM/F-12 medium supplemented with 1.25 mg/ml BSA, 0.1% nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml streptomycin. The cells were then seeded onto Transwell membrane filters (4.52 cm2, 0.4-µm pore
size) at a density of 1.5 × 106
cells/cm2 to prepare confluent
monolayers. The medium was changed every other day. The resistance of
the monolayers was measured using an epithelial voltohmmeter (WPI, New
Haven, CT). High-resistance monolayers formed on
day 4 or 5. All the measurements were
performed on day
5, 6,
or 7 following isolation.
Ussing chamber measurements.
High-resistance monolayers on Transwell inserts were mounted in Ussing
chambers and bathed with identical solutions [either serum-free
DMEM/F-12 medium or Cl
-free
Ringer solution containing (in mM) 120 sodium methanesulfonate, 10 potassium methanesulfonate, 30 mannitol, 3 calcium gluconate, 0.7 MgSO4, 20 NaHCO3, and 0.3 NaH2PO4,
at 37°C, bubbled with 95% O2-5%
CO2] on each side to
eliminate any chemical driving force across the monolayer. Monolayer
potential difference (luminal side as reference), short-circuit current
(Isc), and
resistance were measured with voltage-clamp circuitry from JWT
Engineering (Overland Park, KS). Workbench data acquisition software
(Kent Scientific) was used to record the data. To measure apical
membrane conductance, amphotericin B (10 µM) was added to the serosal
solution to eliminate the basolateral membrane as a barrier to ion
movement. Potassium methanesulfonate Ringer solution (in mM: 120 potassium methanesulfonate, 30 mannitol, 3 calcium gluconate, 0.7 MgSO4, 20 KHCO3, 0.3 KH2PO4,
and 10 NaCl) was used as the serosal solution and either serum-free
DMEM/F-12 medium or sodium methanesulfonate Ringer solution (in mM: 120 sodium methanesulfonate, 30 mannitol, 3 calcium gluconate, 0.7 MgSO4, 20 NaHCO3, and 0.3 NaH2PO4)
was used to bathe the apical surface of the monolayer.
Cl
concentration
([Cl
]) in the
basolateral solution was manipulated by replacing potassium methanesulfonate with equimolar KCl to observe changes in reversal potentials
(Erev). For the
halide permeability experiments, the basolateral solution contained (in
mM) 70 potassium methanesulfonate, 30 mannitol, 3 calcium gluconate,
0.7 MgSO4, 20 KHCO3, 0.3 KH2PO4, and 50 KCl. The apical solution was similar to the basolateral solution, except that 50 mM KCl was replaced with 50 mM KSCN, KCl, KBr,
or KI. A World Precision Instrument (Sarasota, FL) epithelial voltage
clamp was used in combination with a Dagan LM-12 analog-to-digital interface (Dagan, Minneapolis, MN) and pCLAMP software (Axon
Instruments) to run the voltage-clamp protocol and record the resulting
currents. Current-voltage
(I-V)
relationships of apical membranes were obtained by imposing a voltage
step command protocol (see Figs. 4, 11, and 13) with a holding
potential of 0 mV. The currents before and after addition of compound
were subtracted to obtain the compound-sensitive components of the
current. The halide permeability ratios
PX/PCl (where X is SCN
,
Br
, or
I
) were calculated from
reverse potential
(Erev)
measurements using the Goldman-Hodgkin-Katz equation
Erev = (RT/zF) · ln[(PX · [X
])/(PCl · [Cl
])],
where R is the gas constant,
T is absolute temperature,
z is valence, and
F is Faraday's constant.
Permeabilized epithelial preparations have been used by several
investigators to examine the conductance properties of both apical and
basolateral membranes (25, 43). The best results with this technique
have been obtained using high-resistance epithelia in which a
significant fraction of the total transepithelial current flows through
the transcellular pathway. An implicit assumption of this technique is
that paracellular permeability remains relatively constant following
agonist stimulation or treatment with specific blockers of membrane
transport proteins. Significant changes in paracellular permeability
will produce errors in estimating both Erev and
conductance for specific ion channels. In this study, the
Erev values
obtained for the amiloride-sensitive and terbutaline-activated current responses are similar to data previously published for amiloride Na+ channels and for the cystic fibrosis
transmembrane conductance regulator (CFTR) (1, 25, 43).
Electron microscopy.
Alveolar epithelial cells were grown to confluence on the Transwell
membrane filters and mounted in Ussing chambers to measure ion
transport activities on day
7. After the experiments, the monolayers were fixed with 2% glutaraldehyde in 0.1 M sodium
cacodylate for 30 min at room temperature. The monolayers then were
washed four times with 0.1 M sodium cacodylate (pH 7.4) and postfixed with 1% OsO4 in cacodylate for 1 h at room temperature. After three quick rinses with 0.1 M sodium
cacodylate, the membranes were removed from the inserts with a scalpel
and cut into strips ~2 × 0.5 mm. Strips were dehydrated with
ethanol and embedded in epon. Thin sections were stained with uranyl
acetate and lead citrate and examined using a Jeol CX100 electron microscope.
Statistics.
All data are presented as means ± SE, and
n is the number of monolayers studied.
The IC50 values for amiloride,
benzamil, EIPA, NPPB, glibenclamide, and DPC were determined by using a four-parameter logistic function to fit the data. The
IC50 of each compound was derived
from the equation used to fit the concentration-response relationship.
Differences between means were analyzed by using either a paired or
unpaired t-test as appropriate.
 |
RESULTS |
Cell characterization and basal bioelectric properties.
Isolated alveolar type II cells undergo a number of phenotypic changes
with time in culture, including decreased ability to secrete surfactant
lipid (20), gradual loss of lamellar bodies (19), and expression of
type I cell surface epitopes (18). These changes are due to their
gradual transformation to alveolar type I cells (13, 18). Previous
studies have shown that cultured alveolar epithelial cells can be
successfully maintained under completely defined serum-free conditions
(9). Cultured alveolar epithelial cells maintained some alveolar type
II cell morphological characteristics for at least 7 days following
isolation under the culture conditions used in these studies. The cells
exhibited an uneven cuboidal shape and had microvilli associated with
the apical membrane (Fig. 1), but lamellar
bodies could not be clearly distinguished. Alveolar epithelial cells
reached confluence on Transwell membrane filters within 5 days after
isolation and exhibited mean maximum transepithelial resistance of
3,898 ± 194
· cm2 on
days
6 and
7 (Fig.
2). The mean
Isc was 1.96 ± 0.15 µA/cm2
(n = 38), and the mean
potential difference was 1.33 ± 0.18 mV (n = 38, luminal side as reference).

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Fig. 1.
Morphology of alveolar epithelial cells cultured on Transwell membrane
filters 7 days following isolation. Cells maintained some type II cell
characteristics, exhibiting an uneven cuboidal shape and containing
microvilli. Scale bar, 1 µm.
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Fig. 2.
Time course of transepithelial resistance development of alveolar
epithelial monolayers (n = 12). Mean
maximum transepithelial resistance was 3,898 ± 194 · cm2.
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Effects of
Na+ channel
blockers on Isc.
Figure 3 shows the effects of
Na+ channel blockers on the basal
Isc of intact
alveolar epithelial monolayers mounted in Ussing chambers and bathed
with the same serum-free DMEM/F-12 medium on both apical and
basolateral sides. In Fig. 3A,
addition of amiloride (20 µM) to the apical solution immediately
inhibited 59.95 ± 2.60% of the basal
Isc
(n = 10), after which the current remained relatively constant. The concentration-response relationships for amiloride and related analogs are shown in Fig.
3B. The rank order of potency for
inhibition by these compounds was benzamil
amiloride > EIPA, with
mean IC50 values of 0.50, 0.96, and 260 µM, respectively. To further characterize the properties of
the amiloride-sensitive conductance, the basolateral membrane barrier was eliminated by treatment with amphotericin B. After
permeabilization, an initial determination of the apical membrane
I-V
relationship was made, followed by addition of 20 µM amiloride to the
apical solution. The
I-V
relationship for the amiloride-sensitive current is reported in Fig.
4. The
Erev for the
current was 46.46 ± 3.44 mV, indicating a high selectivity for
Na+ over
K+ (12.5:1). Treatment of
monolayers with terbutaline (2 µM) produced no significant change in
either the slope or the
Erev (49.58 ± 4.29 mV) of the amiloride-sensitive
I-V
relationship, indicating that terbutaline does not increase the
amiloride-sensitive Na+
conductance of the apical membrane (Fig. 4).

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Fig. 3.
Effects of Na+ channel blockers on
short-circuit current
(Isc).
Monolayers were mounted in Ussing chambers and bathed on both apical
and basolateral sides with same serum-free DMEM/F-12 medium.
A: representative
Isc tracing
showing apical addition of amiloride (20 µM;
n = 10). Initial mean potential
difference and conductance values were 1.53 ± 0.40 mV and 10.13 ± 2.22 mS (n = 10; luminal side as
reference), respectively. B: apical
addition of Na+ channel blockers
decreases Isc in
a dose-dependent fashion, with rank order of potency of benzamil amiloride > 5-(N-ethyl-N-isopropyl)-amiloride
(EIPA). IC50 values were 0.50, 0.96, and 260 µM for benzamil (n = 4), amiloride (n = 5), and EIPA
(n = 5), respectively. Initial mean
potential difference and conductance values for benzamil, amiloride,
and EIPA were 1.03 ± 0.41 mV and 10.75 ± 3.08 mS
(n = 4), 1.05 ± 0.26 mV
and 11.15 ± 2.45 mS (n = 4), and
0.90 ± 0.14 mV and 10.43 ± 2.39 mS
(n = 4), respectively.
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Fig. 4.
Current-voltage
(I-V)
relationship for amiloride-sensitive conductance in apical membrane.
Experiments were performed with amphotericin-permeabilized monolayers
mounted in Ussing chambers and bathed with potassium methanesulfonate
Ringer solution on basolateral side and serum-free DMEM/F-12 medium on
apical side. A: representative tracing
of amiloride-sensitive current in absence of terbutaline recorded in
5-mV step increments from 60 to +80 mV.
B: mean reversal potentials
(Erev) for
amiloride-sensitive current in absence ( ) or presence ( ) of
terbutaline were 46.47 ± 3.44 (n = 9) and 49.58 ± 4.29 mV (n = 6),
respectively. Amiloride (20 µM) was applied to apical bathing
solution, and terbutaline (2 µM) was applied to basolateral bathing
solution. Initial mean potential difference and conductance values were
2.80 ± 0.87 mV and 4.25 ± 0.544 mS
(n = 9) for control and 1.50 ± 0.27 mV and 5.20 ± 0.72 mS (n = 6)
for terbutaline, respectively.
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Effects of terbutaline and CPT-cAMP on
Isc.
Addition of the
-adrenergic agonist terbutaline (2 µM) to the
basolateral bath caused a rapid decrease in
Isc, followed by a slow rebound toward the initial basal current (Fig.
5A). The mean peak current drop stimulated by terbutaline was 3.2 ± 0.42 µA (n = 7). Subsequent addition
of amiloride (20 µM) to the apical bathing solution blocked
62.5 ± 1.8% (n = 5) of the
remaining current. Pretreatment with 20 µM amiloride significantly
decreased the amplitude of the terbutaline-sensitive current response
(from 3.2 ± 0.42 µA to 1.06 ± 0.10 µA,
n = 5, P < 0.01) and completely eliminated
the recovery of the
Isc to its
initial level (Fig. 5B). Addition of
100 µM CPT-cAMP to basolateral bath produced an
Isc response that
was identical to that of terbutaline (Fig. 6). The peak inward current produced by
CPT-cAMP was 1.88 ± 0.26 µA
(n = 5). Addition of 20 µM amiloride
to the apical solution following CPT-cAMP produced a response similar
to that shown in Fig. 5A. When
serum-free DMEM/F-12 medium was replaced with
Cl
-free Ringer solution,
the response to either terbutaline or CPT-cAMP was totally inhibited,
indicating that the
Isc decrease was
Cl
dependent (Fig.
7). Subsequent addition of 20 µM
amiloride to the apical solution under
Cl
-free conditions produced
a response similar to that observed with serum-free DMEM/F-12 medium.
Note, however, that the amiloride-insensitive Isc under
Cl
-free conditions was
significantly reduced compared with the amiloride-insensitive Isc observed in
serum-free DMEM/F-12 medium (Fig.
5A). The amiloride-sensitive Isc was not
significantly different between cultures maintained in serum-free
DMEM/F-12, normal Ringer solution, and
Cl
-free Ringer solution
(6.18 ± 0.50, 5.22 ± 0.81, and 5.85 ± 0.98 µA,
respectively; Fig.
8A). The
amiloride-insensitive
Isc was not
significantly different between normal Ringer solution (1.33 ± 0.46 µA) and Cl
-free Ringer
solution (0.57 ± 0.22 µA). However, the magnitude of
amiloride-insensitive
Isc was
significantly different between serum-free DMEM/F-12 medium (4.24 ± 0.56 µA) and normal Ringer solution (1.33 ± 0.46 µA)
or Cl
-free Ringer solution
(0.57 ± 0.22 µA). When nonessential amino acids were added to
cultures bathed in Cl
-free
Ringer solution, the amiloride-insensitive residual
Isc increased to
a level similar to that observed in serum-free DMEM/F-12 medium (3.26 ± 0.84 µA, Fig. 8B),
suggesting that amino acid-coupled Na+ transport was responsible for
a large portion of the amiloride-insensitive Isc. A similar
response was observed when nonessential amino acids were added to
cultures bathed in normal Ringer solution. To determine whether a
portion of the basal
Isc was due to
Cl
secretion, the loop
diuretics bumetanide and furosemide were added to the basolateral
solution in an attempt to block
Cl
entry into the cells. No
response was observed following treatment with up to 200 µM
bumetanide and 200 µM furosemide, indicating the absence of loop
diuretic-sensitive Cl
secretion as a component of the basal
Isc.

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Fig. 5.
Representative
Isc tracings
showing effects of basolateral administration of terbutaline (2 µM)
on Isc with or
without pretreatment with apical amiloride (20 µM). Monolayer filters
were mounted in Ussing chambers and bathed on both apical and
basolateral sides with same serum-free DMEM/F-12 medium.
A: addition of terbutaline produced a
rapid decrease in
Isc, followed by
a slow increase back to basal
Isc. Further
addition of amiloride blocked most of remaining
Isc
(n = 5). Initial mean potential
difference and conductance values were 1.69 ± 0.38 mV and 7.76 ± 1.42 mS (n = 5), respectively.
B: in presence of amiloride, addition
of terbutaline produces a rapid sustained decrease in
Isc without
secondary recovery phase (n = 5).
Initial mean potential difference and conductance values were 1.92 ± 0.82 mV and 7.74 ± 2.42 mS
(n = 5), respectively.
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Fig. 6.
Representative
Isc tracing
showing effects of basolateral administration of 8-(4-chlorophenylthio)
(CPT)-cAMP (8 cpt-cAMP; 100 µM) on
Isc. Monolayer
filters were mounted in Ussing chambers and bathed on both apical and
basolateral sides with same serum-free DMEM/F-12 medium. Addition of
CPT-cAMP produces a rapid decrease in
Isc, followed by
a slow increase back to basal
Isc. Further
addition of amiloride blocked most of remaining
Isc
(n = 5). Initial mean potential
difference and conductance values were 1.28 ± 0.52 mV and 13.45 ± 6.17 mS (n = 5), respectively.
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Fig. 7.
Representative
Isc tracings
showing effects of basolateral administration of 2 µM terbutaline
(A) or 100 µM CPT-cAMP
(B) on
Isc under
Cl -free conditions.
Monolayers were mounted in Ussing chambers and bathed with same
Cl -free Ringer solution on
both apical and basolateral sides. No response was produced by either
compound, whereas addition of amiloride (20 µM) to apical solution
produced a sustained decrease in
Isc
(n = 4 for each compound). Initial
mean potential difference and conductance values were 1.9 ± 0.89 mV
and 5.83 ± 2.56 mS (n = 4) for
terbutaline and 1.05 ± 0.56 mV and 8.34 ± 3.05 mS
(n = 4) for CPT-cAMP, respectively.
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Fig. 8.
A: amiloride-sensitive and
amiloride-insensitive
Isc when
monolayers were mounted in Ussing chambers and bathed on both apical
and basolateral sides with same serum-free DMEM/F-12 medium
(n = 10), normal Ringer
(n = 9), or
Cl -free solution
(n = 7). Amiloride-sensitive
Isc was not
significantly different between groups (6.18 ± 0.53, 5.22 ± 0.81, and 5.85 ± 0.98 µA, respectively). Magnitude of
amiloride-insensitive
Isc was
significantly different between serum-free DMEM/F-12 medium (4.24 ± 0.56 µA) and normal Ringer (1.33 ± 0.46 µA) or
Cl -free solution (0.57 ± 0.22 µA) but not significantly different between normal Ringer
and Cl -free Ringer
solution. Initial mean potential difference and conductance values for
serum-free medium, normal Ringer, and
Cl -free solution were 1.53 ± 0.40 mV and 10.13 ± 2.22 mS
(n = 10), 1.04 ± 0.28 mV and 8.33 ± 1.30 mS (n = 9), and 1.44 ± 0.46 mV and 8.06 ± 2.73 mS (n = 7), respectively. B: representative
Isc tracing
showing effects of nonessential amino acids on
Isc. Nonessential
amino acids (1.4 mM at each arrow) were added to both apical and
basolateral solutions (n = 5).
Monolayers were mounted in Ussing chambers and bathed with same
Cl -free Ringer solution on
both apical and basolateral sides. Initial mean potential difference
and conductance values were 1.10 ± 0.41 mV and 8.70 ± 2.86 mS
(n = 5), respectively.
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Properties of terbutaline- or CPT-cAMP-activated
Cl
current.
To further define the properties of the terbutaline- or
CPT-cAMP-activated current, amphotericin B was added to the basolateral solution to eliminate the basolateral membrane as a resistive barrier.
The effects of amiloride and terbutaline on current flow across the
apical membrane are shown in Figs. 9 and
10, respectively. The amplitude of the
current activated by apical addition of 2 µM terbutaline (14.06 ± 4.26 µA) was not significantly different from the terbutaline
response of monolayers pretreated with 20 µM amiloride (15.29 ± 3.38 µA). The magnitude of the current blocked by amiloride (6.63 ± 1.23 µA) also was not significantly different after
pretreatment of the monolayers with terbutaline (7.63 ± 0.92 µA).
Replacement of Cl
with
methanesulfonate in both apical and basolateral solutions completely
inhibited the terbutaline-sensitive current response but had no effect
on the amiloride-sensitive current (Fig. 7). The
I-V
relationships for the terbutaline- and CPT-cAMP-sensitive difference
currents are shown in Fig. 11. Figure
11A shows a representative tracing
of the terbutaline-sensitive current obtained by subtracting the basal
current recorded at each voltage step from the current recorded after
addition of terbutaline (2 µM) to the basolateral solution. The
terbutaline- and CPT-cAMP-sensitive currents exhibited nearly linear
I-V
relationships, with mean
Erev of
28.42 ± 2.68 (n = 12) and
26.32 ± 2.50 mV (n = 5),
respectively (Fig. 11B). Increasing
[Cl
] in the
basolateral solution from 10 to 35 mM shifted the
Erev of the
terbutaline-sensitive current to more depolarized voltages (from
28.42 ± 2.68 to
12.51 ± 1.90 mV; Fig.
12), indicating that the
terbutaline-sensitive current was carried by
Cl
. To characterize the
anion permeability properties of the terbutaline-sensitive conductance,
a series of biionic experiments was performed to determine the halide
permeability sequence (Fig. 13). The mean Erev of the
terbutaline-sensitive
I-V
relations for SCN
,
Br
,
Cl
, and
I
were
13.99 ± 2.67,
9.20 ± 1.15, 0.20 ± 0.22, and 3.04 ± 0.76 mV,
respectively. The halide permeability sequence was
SCN
> Br
> Cl
> I
(relative permeabilities
for SCN
,
Br
,
Cl
, and
I
were 1.76, 1.45, 1, and
0.89, respectively).

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Fig. 9.
Representative
Isc tracings
showing effects of basolateral addition of 2 µM terbutaline followed
by apical addition of 20 µM amiloride
(A) or apical addition of 20 µM
amiloride followed by basolateral addition of 2 µM terbutaline
(B). Experiments were performed with
amphotericin-permeabilized monolayers mounted in Ussing chambers and
bathed with potassium methanesulfonate Ringer solution on basolateral
side and serum-free DMEM/F-12 medium on apical side. Voltage across
monolayers was clamped at 0 mV. Initial mean potential difference and
conductance values were 1.08 ± 0.42 mV and 6.67 ± 1.62 mS (A;
n = 8) and 1.11 ± 0.42 mV and 5.46 ± 1.23 mS (B;
n = 8).
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Fig. 10.
Terbutaline (Terb)- and amiloride (Amil)-sensitive current responses
obtained from amphotericin-permeabilized monolayers voltage clamped at
0 mV. Pretreatment with amiloride (n = 8) did not significantly affect magnitude of terbutaline-sensitive
current (15.29 ± 3.38 µA with pretreatment vs. 14.06 ± 4.26 µA without pretreatment). In addition, pretreatment with
terbutaline (n = 8) did not
significantly affect amiloride-sensitive current (7.63 ± 0.92 µA
with pretreatment vs. 6.63 ± 1.23 µA without pretreatment).
Initial mean potential difference and conductance values were same as
in Fig. 9.
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Fig. 11.
I-V
relationship for terbutaline- and CPT-cAMP-sensitive conductance
in apical membrane. Experiments were performed with
amphotericin-permeabilized filters mounted in Ussing chambers and
bathed with potassium methanesulfonate Ringer solution on basolateral
side and serum-free DMEM/F-12 medium on apical side. A:
representative tracing of terbutaline-sensitive current recorded in
response to 10-mV voltage step increments from 70 to +80 mV.
B: I-V plot for terbutaline- and
CPT-cAMP-sensitive currents with mean Erev of
28.42 ± 2.68 (n = 12) and 26.32 ± 2.50 mV (n = 5), respectively. Terbutaline (2 µM) and CPT-cAMP
(100 µM) were applied to basolateral bathing solution after
pretreatment of monolayers with apical amiloride (20 µM). Initial
mean potential difference and conductance values were 1.71 ± 0.39 mV and 3.67 ± 1.28 mS (n = 12) for terbutaline
and 1.76 ± 0.60 mV and 6.63 ± 1.79 mS (n = 5) for
CPT-cAMP, respectively.
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Fig. 12.
Increasing basolateral Cl concentration
([Cl ]) produced a shift in
terbutaline-sensitive Erev. Experiments were
performed with amphotericin-permeabilized filters mounted in Ussing
chambers and bathed with potassium methanesulfonate Ringer solution on
basolateral side and serum-free DMEM/F-12 medium on apical side.
Changes in [Cl ] were achieved by
replacing potassium methanesulfonate with equimolar KCl. Terbutaline (2 µM) was applied to basolateral bathing solution after pretreatment of
monolayers with apical amiloride (20 µM). Erev
were plotted against logarithm of basolateral
[Cl ]. Linear regression analysis was
used to fit data (R = 0.983, n = 5-12). Initial mean potential difference and conductance values
for 10, 20, and 35 mM Cl solutions were 1.71 ± 0.39 mV and 3.67 ± 1.28 mS (n = 12), 2.28 ± 0.81 mV
and 4.56 ± 0.973 mS (n = 5), and 2.50 ± 0.65 mV and
3.25 ± 0.22 mS (n = 5), respectively.
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Fig. 13.
Halide permeability of terbutaline-sensitive current. Experiments were
performed with amphotericin-permeabilized filters mounted in Ussing
chambers and bathed with modified potassium methanesulfonate Ringer
solution on basolateral side and modified potassium methanesulfonate
Ringer solution with 50 mM KCl replaced with 50 mM KSCN, KCl, KBr, or
KI on apical side. Terbutaline (2 µM) was added to basolateral
solution. Mean
Erev for
SCN ,
Br ,
Cl , and
I were 13.99 ± 2.67, 9.20 ± 1.15, 0.20 ± 0.22, and 3.04 ± 0.76 mV, respectively. Initial mean potential difference and
conductance values for 50 mM KSCN, KCl, KBr, and KI were 0.80 ± 0.34 mV and 11.41 ± 5.78 mS, 0.24 ± 0.31 mV and 4.59 ± 1.10 mS, 0.05 ± 0.44 mV and 10.87 ± 4.22 mS, and
0.14 ± 0.26 mV and 10.55 ± 3.42 mS, respectively
(n = 5 for each).
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Blocker pharmacology.
NPPB, glibenclamide, and DPC were used to block the
terbutaline-sensitive current in amphotericin-permeabilized monolayers. Apical addition of NPPB, glibenclamide, or DPC following pretreatment with amiloride and terbutaline revealed that these compounds could inhibit the terbutaline-activated current in a concentration-dependent manner but that they had no effect on the amiloride-sensitive current.
Figure
14A
shows a representative trace of the effect of glibenclamide on
terbutaline-activated current in amphotericin-permeabilized monolayers.
The IC50 values for NPPB,
glibenclamide, and DPC were 12, 110, and 640 µM, respectively (Fig.
14B).

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Fig. 14.
Effects of Cl channel
blockers on terbutaline-sensitive current. Experiments were performed
with amphotericin-permeabilized filters mounted in Ussing chambers and
bathed with potassium methanesulfonate Ringer solution on basolateral
side and serum-free DMEM/F-12 medium on apical side. Monolayer voltage
was clamped at 0 mV. 5-Nitro-2-(3-phenylpropylamino) benzoic acid
(NPPB), glibenclamide, and
N-phenylanthranilic acid (DPC) were
added to apical solution. A:
representative tracing of effect of apical glibenclamide (200 µM at
each arrow) on terbutaline-activated current.
B: concentration-response relationship
for NPPB, glibenclamide, and DPC inhibition of terbutaline-sensitive
current. IC50 values for NPPB,
glibenclamide, and DPC were 12 (n = 6), 110 (n = 6), and 640 µM
(n = 9), respectively. Initial mean
potential difference and conductance values for NPPB, glibenclamide,
and DPC were 1.53 ± 0.40 mV and 9.29 ± 2.10 mS
(n = 6), 1.31 ± 0.40 mV and 10.70 ± 3.11 mS (n = 6), and 1.49 ± 0.57 mV and 12.07 ± 1.91 mS (n = 9), respectively.
|
|
 |
DISCUSSION |
Cultured alveolar epithelial cells used in this study were grown on
large Transwell membrane filters (4.5 cm2), where they form confluent
monolayers with a high transepithelial resistance. The basal electrical
properties of these monolayers agreed with those reported in earlier
studies using similar cell isolation procedures (2, 5, 14, 35). The
cells exhibited apical microvilli and an uneven cuboidal shape similar
to that of native alveolar epithelial cells. In addition, a significant portion of the basal
Isc was blocked
by addition of amiloride to the apical solution, consistent with
previous results using cultured adult rat alveolar epithelial cells (9,
14, 31).
In our initial experiments, we examined the effects of amiloride
analogs on basal
Isc and observed
that the rank order of potency was benzamil > amiloride > EIPA. The
IC50 values and rank order of
potency for these inhibitors were similar to results reported in
previous studies (9) and indicated the presence of high-affinity,
amiloride-sensitive Na+ channels
active under basal conditions. To determine the
Na+/K+
permeability ratio of the apical
Na+ conductance, we examined the
amiloride-sensitive
I-V
relationship using amphotericin-permeabilized monolayers. The
Erev of the
amiloride-sensitive current was +46 mV. The estimated
PNa/PK
ratio calculated from the constant field equation was 12.5:1, a value
similar to that of the cloned rat 

-ENaC
(PNa/PK = 10:1) expressed in Xenopus oocytes
(12). Thus alveolar epithelial cells in culture for a period of 6 days
or more express a Na+ conductance
that exhibits a relatively high selectivity for
Na+. However, in freshly isolated
adult rat alveolar epithelial cells, low-affinity amiloride-sensitive
Na+ channels were previously
observed (33). These channels had relatively low selectivity for
Na+ over
K+ and were effectively blocked by
EIPA over a concentration range similar to that of amiloride (33).
These differences indicate that culture conditions may significantly
affect the expression of Na+
channels with different selectivity and pharmacological properties.
Previous studies with adult rat alveolar epithelial cells in culture
demonstrated that stimulation with terbutaline increased net
Na+ and
Cl
absorption (31). These
flux experiments were performed under short-circuit conditions,
indicating that a transcellular pathway for
Cl
absorption was activated
by
-adrenergic stimulation. In the present study, basolateral
addition of terbutaline produced an immediate decrease in current,
followed by a slow recovery back to the initial
Isc. This effect
was also observed when monolayers were treated with CPT-cAMP, a
membrane-permeant, nonmetabolizable analog of cAMP. The results
obtained from experiments with terbutaline were similar to those
previously reported by Cheek et al. (14). In the present study,
replacement of Cl
in both
apical and basolateral solutions with methanesulfonate eliminated the
effects of terbutaline and CPT-cAMP, suggesting that the decrease in
Isc was dependent
on extracellular
[Cl
]. The
amiloride-insensitive
Isc in serum-free
DMEM/F-12 medium was presumably due to
Na+-amino acid cotransport.
Na+-amino acid cotransport in
alveolar epithelial cells was previously reported by Brown et al. (11).
When nonessential amino acids were added into normal Ringer solution or
Cl
-free Ringer solution,
the nearly zero
Isc was increased
to a level similar to that observed in serum-free DMEM/F-12 medium. Active Cl
secretion
dependent on bumetanide-sensitive
Na+-K+-2Cl
cotransport was not detected in our studies. The
I-V
relationships for the terbutaline- and CPT-cAMP-activated conductances
had similar Erev,
suggesting Cl
as the
current-carrying ion. Changing the basolateral
[Cl
] from 10 to
20 and then to 35 mM shifted the
Erev toward 0 mV, thus confirming the presence of a terbutaline- and cAMP-dependent Cl
conductance in the
apical membrane.
To determine the selectivity properties of the terbutaline- and
cAMP-dependent Cl
conductance, we performed a series of biionic anion substitution experiments using SCN
,
Br
, and
I
as replacement anions and
measured the shift in
Erev for each replacement anion and determined permeability ratios relative to
Cl
. The order of
selectivity was SCN
> Br
> Cl
> I
for these experiments. In
addition, a linear
I-V
relationship was observed under asymmetrical
Cl
conditions. The anion
selectivity properties of the terbutaline-sensitive anion conductance
observed in this study, along with the linear character of the
I-V
relationship in asymmetrical
Cl
-containing solutions,
were similar to those of CFTR (1). Moreover, apical addition of NPPB,
glibenclamide, or DPC produced inhibition of the terbutaline-activated
Cl
current in a
concentration-dependent manner. The
IC50 values for these compounds
were consistent with inhibition of CFTR (27). These experiments
indicate that terbutaline, presumably acting through cAMP, activates
Cl
channels in the apical
membrane with functional and pharmacological properties similar to
those of CFTR.
To investigate the possibility that terbutaline directly increases
Na+ absorption by increasing
apical membrane Na+ permeability,
we measured the effects of terbutaline and CPT-cAMP on
Isc when
Cl
was replaced with
methanesulfonate. Under these conditions, neither terbutaline nor
CPT-cAMP produced any change in
Isc. In addition, the amiloride-sensitive
Isc after
terbutaline in Cl
-free
solution was not significantly different from the amiloride-sensitive Isc measured in
Cl
-containing solution
without terbutaline. Moreover, no change in amiloride-sensitive current
was observed in amphotericin-permeabilized monolayers voltage clamped
at 0 mV following treatment with terbutaline. Finally, no significant
change in the slope or
Erev of the
amiloride-sensitive I-V
relation was detected after treatment with terbutaline. These results
indicate that the amiloride-sensitive
Na+ conductance in these cells is
not directly activated by terbutaline.
The data presented in this study suggest the following model to explain
the mechanism of
-adrenergic stimulation on transepithelial Na+ and
Cl
transport in alveolar
epithelial cells (Fig. 15). Terbutaline binds to
-adrenergic receptors located in the basolateral membrane that are coupled to adenylyl cyclase, resulting in an increase in
intracellular cAMP. We propose that cAMP, presumably acting through
protein kinase A, activates a CFTR-like
Cl
channel in the apical
membrane and that Cl
enters
the cell. Cl
influx is
consistent with the initial decrease in
Isc and occurs as
a result of a substantially depolarized apical membrane due to the
constitutive influx of Na+ through
amiloride-sensitive Na+ channels.
The time-dependent recovery of the
Isc after
terbutaline is due to an increase in
Na+ absorption. This increase,
however, results from an increase in driving force for
Na+ uptake as a consequence of
membrane hyperpolarization produced by
Cl
influx. This would
explain the observation that
Isc recovery is
completely blocked by pretreatment with amiloride. It would also
explain how terbutaline could increase net
Na+ absorption under conditions in
which no change in apical Na+
conductance could be observed. The
Na+ that enters the cell across
the apical membrane is transported across the basolateral membrane by
the
Na+-K+-ATPase.
Cl
, entering across the
apical membrane, is transported across the basolateral membrane by
unknown transport pathways. One possibility is KCl cotransport, since
it could couple Cl
efflux
to an outwardly directed K+
concentration gradient and has been shown to mediate electroneutral Cl
efflux across the
basolateral membrane of
Cl
-absorbing epithelia
(45). Another possibility is a basolateral Cl
channel, as proposed for
sweat duct epithelial cells (44). For this to be feasible, however, the
basolateral Cl
conductance
must have an Erev
that is more depolarized than the basolateral membrane potential.

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Fig. 15.
Cell model showing proposed mechanisms for
Cl and
Na+ transport across adult rat
alveolar epithelial cells. PKA, protein kinase A.
|
|
In conclusion, we propose that the net effect of
-adrenergic
stimulation is to produce a tight coupling between
Na+ and
Cl
influx across the apical
membrane that results in an increase in net NaCl absorption. This
increase in salt absorption sets up the osmotic driving forces required
for movement of fluid across the epithelium. We believe that the
mechanism for transcellular Cl
transport outlined above
provides an explanation for the previously reported increase in net
Cl
absorption stimulated by
-adrenergic agonists (31). Whether this mechanism reflects the
actions of
-adrenergic agonists in vivo is presently
unknown. The transport properties of cultured epithelial cells may not
reflect those of alveolar type II cells in vivo.
However, the results of this study do suggest some interesting experiments that should provide some insight into the physiological role of CFTR in alveolar fluid absorption.
 |
ACKNOWLEDGEMENTS |
We thank Pat Jung of the Acute Lung Injury Strategic Center of
Research Morphology Core for help with electron microscopy of alveolar
epithelial cells, Dr. Richard Lubman for help with the cell isolation
protocol, Rob Bair for help with cell isolation, and Dr. Doug
Wangensteen for helpful comments and discussions.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-50152.
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. §1734 solely to indicate this fact.
Address for reprint requests: S. M. O'Grady, Dept. of Physiology,
University of Minnesota, 495 Animal Science/Veterinary Medicine Bldg.,
1988 Fitch Ave., St. Paul, MN 55108.
Received 27 January 1998; accepted in final form 20 August 1998.
 |
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