SPECIAL TOPIC
Alveolar Epithelial Ion and Fluid Transport
cAMP
regulation of Cl
and HCO
secretion
across rat fetal distal lung epithelial cells
Ahmed
Lazrak1,
Ulrich
Thome2,
Carpantanto
Myles1,
Janice
Ware2,
Lan
Chen1,
Charles J.
Venglarik3, and
Sadis
Matalon1,2,3,4
Departments of 1 Anesthesiology,
2 Pediatrics, 3 Environmental Health
Sciences, and 4 Physiology and Biophysics,
University of Alabama at Birmingham, Birmingham, Alabama 35233
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ABSTRACT |
We
isolated and cultured fetal distal lung epithelial (FDLE) cells from
17- to 19-day rat fetuses and assayed for anion secretion in
Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (Isc) and transepithelial resistances
were 7.9 ± 0.5 µA/cm2 and 1,018 ± 73
· cm2, respectively (means ± SE;
n = 12). Apical amiloride (10 µM) inhibited basal
Isc by ~50%. Subsequent addition of forskolin (10 µM) increased Isc from 3.9 ± 0.63 µA/cm2 to 7.51 ± 0.2 µA/cm2
(n = 12). Basolateral bumetanide (100 µM) decreased
forskolin-stimulated Isc from 7.51 ± 0.2 µA/cm2 to 5.62 ± 0.53, whereas basolateral
4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of
HCO
secretion, blocked the remaining
Isc. Forskolin addition evoked currents of
similar fractional magnitudes in symmetrical Cl
- or
HCO
-free solutions; however, no response was seen
using HCO
- and Cl
-free solutions. The
forskolin-stimulated Isc was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced Isc across monolayers having
nystatin-permeablized basolateral membranes. Immunolocalization
studies were consistent with the expression of cystic fibrosis
transmembrane conductance regulator (CFTR) protein in FDLE cells. In
aggregate, these findings indicate the presence of cAMP-activated
Cl
and HCO
secretion across rat FDLE cells mediated via CFTR.
short-circuit current; amiloride; nystatin; swelling-activated
conductance; immunocytochemistry; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
ADULT ALVEOLAR type II
cells actively absorb Na+, with this process playing an
important role in limiting the extent of alveolar edema following
injury to the alveolar epithelium (15, 37). In contrast,
the fetal alveolar epithelium in utero actively secretes Cl
into the developing alveolar space, driving the fluid
secretion necessary for fetal lung growth (16, 22). Thus
compared with fetal plasma, the fetal lung liquid contains a high
concentration of Cl
and almost no protein
(22). Agents that increase intracellular cAMP levels
increase Cl
secretion across fetal lung cultures ex vivo
(2), human fetal alveolar epithelial cell monolayers in
vitro (16), and promote secretion of fetal lung liquid in
fetal sheep in vivo (5).
However, the possible contribution of other ions, such as bicarbonate
(HCO
), to fetal transport, has not been previously
elucidated.
-Adrenergic stimulation of isolated adult Clara or
immortalized Calu-3 human airway cells was shown to induce electrogenic
transepithelial secretion of both Cl
and
HCO
(12, 34). The influx of
HCO
into the fetal fluid may be especially important
because it may increase its pH which in turn may affect a number of
enzymatic functions, production of pulmonary surfactant, and even the
rate of production of fetal fluid.
Herein, we isolated fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses, cultured them on permeable supports until they
formed resistive monolayers (36-48 h), mounted them in Ussing
chambers, and measured short-circuit currents
(Isc) before and after addition of forskolin, a
substance known to increase intracellular cAMP levels. We then
characterized 1) the contributions of Cl
and
HCO
to baseline and forskolin-stimulated Isc across intact monolayers and 2)
the apical membrane Cl
conductances following
permeabilization of the basolateral membranes using the pore-forming
antibiotic nystatin. Our results indicate the presence of significant
basal and cAMP-activated anion currents across rat FDLE cells and show
that both Cl
and HCO
contribute to
these currents by their movement through cystic fibrosis transmembrane
conductance regulator (CFTR)-like channels.
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MATERIALS AND METHODS |
FDLE cell isolation.
The isolation procedure has been described previously
(23). In brief, lungs of 17- to 19-day gestation fetal
rats (term = 22 days) were digested in a solution containing
0.125% trypsin and 0.4 mg/ml DNase in Eagle's minimum essential
medium (MEM) for 10 min. Digestion was stopped by the addition of MEM
containing 10% fetal bovine serum (FBS). Cells were collected by
centrifugation and resuspended in 15 ml of MEM containing 0.1%
collagenase and DNase. This solution was incubated for 15 min at
37°C. The collagenase activity was neutralized by the addition of 15 ml of MEM containing 10% FBS. The cells were plated twice for 1.5 h to remove contaminating fibroblasts. The supernatant contained
epithelial cells with >95% purity. Cells were counted and seeded on
permeable Transwell culture inserts (Corning, NY) with 0.33 cm2 surface area and 0.4-µm pore size. They were then
seeded at a density of 5 × 104 cells per filter,
cultured in DMEM with 10% FBS and 1% penicillin/streptomycin (apical
and basolateral vol: 500 and 1,000 µl, respectively), and exposed to
21% O2-5% CO2 mixture in a humidified
incubator for 36-48 h. The transepithelial resistance
(Rt) was monitored after ~36 h in culture
using an epithelial voltohmmeter equipped with chopstick-style
electrodes (World Precision Instruments, Sarasota, FL).
Transepithelial transport studies.
All experiments were conducted upon achievement of monolayer confluence
within 36-48 h after isolation of FDLE cells. Confluent monolayers
with Rt
1 k
· cm2 were
mounted in modified Ussing chambers connected to a transepithelial voltage clamp (Physiological Instruments, San Diego, CA) that allowed
continuous measurement of the Isc
(10). Changes in Rt were monitored
by imposing a 5-s voltage pulse (2 or 4 mV) across the monolayer every
minute. Rt was calculated using Ohm's Law. The
composition of the apical and basolateral bathing solutions was (in
mM): 145 Na+, 5 K+, 125 Cl
, 1.2 Ca2+, 1.2 Mg2+, 25 HCO
, 3.3 H2PO
, 0.8 HPO
, and
10 glucose (basolateral) or 10 mannitol (apical), pH 7.4. All solutions
were gassed with 95% O2-5% CO2 using an
airlift and warmed to 37°C.
Isc was allowed to stabilize before beginning
each experiment (5-10 min). We then added 10 µM amiloride to the
apical side of the monolayers to block Na+ absorption.
Forskolin (10 µM) was added to both sides of the monolayer to
evaluate possible effects of cAMP on transepithelial anion transport.
To determine the dependence of Isc on
Cl
and HCO
, NaCl or NaHCO3
were replaced with equimolar amounts of sodium gluconate or
Na+-HEPES, respectively. In a third set of ion substitution
experiments, both Cl
and HCO
were
replaced with sodium gluconate and Na+-HEPES. In
experiments conducted in the absence of HCO
, solutions in Ussing chambers were gassed with 100% O2
instead of 95% O2 and 5% CO2.
Bumetanide (100 µM) and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS;
5 mM), both instilled in the basolateral compartment, were used to
inhibit the contribution of the
Na+-K+-2Cl
and
Na-HCO
cotransporters to the generation of anion
currents (8). To avoid the generation of osmotic
gradients, equal amounts of Na+ sulfate were added into the
apical compartment. In an additional set of experiments, the
Cl
channel blockers glibenclamide (200 µM) or DIDS (200 µM) were added into the apical compartments, and changes in
Isc were recorded.
To evaluate the apical membrane Cl
conductance,
monolayers were mounted in Ussing chambers under short-circuit
conditions in the presence of either a basolateral to apical (125:5 mM)
or an apical to basolateral (5:125 mM) Cl
gradient, and
the pore-forming antibiotic nystatin (200 µM) was added into the
basolateral compartment. Under these conditions, the basolateral
membrane is eliminated as a barrier to the flow of monovalent ions, and
Isc provides a direct measure of the apical membrane Cl
conductance. Spontaneously activating
Isc were tested for sensitivity to increased
extracellular osmolality, achieved by adding 10 or 30 mM sucrose into
both compartments. Forskolin-stimulated Isc were
tested for sensitivity to glibenclamide (3-300 µM), added to the
apical compartments of the Ussing chambers.
CFTR immunolocalization.
FDLE cells were grown on transparent cyclopore filters (Falcon).
Monolayers were fixed in methanol at
20°C for 15 min followed by
postfixation using formaldehyde (3%) in PBS for 20 min. Nonspecific protein binding was blocked with 1% (wt/vol) bovine serum albumin. Samples were treated with either a polyclonal antibody raised against
the nucleotide binding domain-1 region of CFTR (a kind gift
from Dr. D. Bedwell, Univ. of Alabama at Birmingham) or a monoclonal
antibody against the COOH terminus of CFTR (Genzyme). The antibody
raised against the NBD-1 region has been previously characterized and
found to be specific for CFTR (4). Texas red X-labeled
anti-mouse IgG and Oregon green-labeled anti-rabbit IgG (Molecular
Probes) were used as secondary antibodies. Samples were counterstained
with the nuclear dye bisbenzimide. In some cases, filters were cut and
folded cell-side out during mounting to enable cross-sectional views
using the technique developed by Tousson et al. (33). CFTR
immunolocalization was assessed using a Lietz Orthoplan inverted
epifluorescence microscope equipped with a step motor, filter
wheel assembly (Ludl Electronics Products, Hawthorn, NY), and 83,000 filter set (Chroma Technology, Brattleboro, VT). Images were captured
with a SenSys-cooled charge-coupled device high resolution digital
camera (Photometrics, Tucson, AZ). Partial deconvolution of images was
performed using IP Lab Spectrum software (Scanalytics, Fairfax, VA).
Statistical analysis.
Results are expressed as means ± SE. Statistical significance
among means was determined by Student's t-test (2 samples)
or ANOVA followed by the Bonferroni modification of the
t-test, corrected for multiple comparisons.
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RESULTS |
Cultured rat FDLE cells demonstrate cAMP-stimulated
Cl
and HCO
secretion.
After 36-48 h in culture, monolayers of rat FDLE cells were
mounted in Ussing chambers and bathed on both sides with standard Ringer solutions. The monolayers generated a basal
Isc of 7.9 ± 0.54 (means ± SE;
n = 12) µA/cm2 and had an
Rt of 1,018 ± 73
· cm2 (means ± SE; n = 12). A representative trace illustrating the effects of amiloride and
forskolin on Isc across rat FDLE monolayers is
shown in Fig. 1. Approximately
50% of the basal Isc was inhibited after
addition of apical amiloride (10 µM). This result is
consistent with the presence of electrogenic Na+ absorption
via an amiloride-sensitive epithelial Na+ channel, most
likely ENaC. On average, amiloride reduced the Isc from 7.9 ± 0.54 to 3.9 ± 0.63 µA/cm2 (n = 12; P < 0.01). Subsequent addition of forskolin (10 µM) to both sides of the
monolayer significantly increased Isc on average
from 3.9 ± 0.63 to 7.51 ± 0.2 µA/cm2
(n = 12), presumably by stimulating anion secretion.

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Fig. 1.
Representative trace showing the short-circuit current
(Isc) response across fetal distal lung
epithelial (FDLE) cells bathed in the presence of Ringer solution. Rat
FDLE cells were isolated, grown on filters, and mounted in Ussing
chambers as described in MATERIALS AND METHODS. Amiloride
(Amil; 10 µM) was added to apical bathing solution of the monolayers
at the time indicated to abolish the Isc due to
active Na+ absorption. Forskolin (Forsk; 10 µM) was then
added to both sides of the monolayer to increase intracellular cAMP.
Bumetanide (Bumet; 100 µM) and 4,4'-dinitrostilbene-2,2'-disulfonate
(DNDS; 5 mM) were added to the basolateral compartment. These
experiments were repeated 6 times each with similar results.
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Next we investigated the possible contribution of Cl
vs.
HCO
secretion to the basal and forskolin-stimulated Isc across FDLE cells using inhibitors.
Specifically, Cl
secretion across airway cells is driven
by a basolateral bumetanide-sensitive Na+-K+-Cl
cotransporter while
HCO
secretion is mediated by a basolateral
Na-HCO
cotransporter that is inhibited by DNDS but
not bumetanide (8). As shown in Fig. 1, addition of
basolateral bumetanide (100 µM) decreased a component of the
forskolin-stimulated Isc. On average Isc was reduced from 7.51 ± 0.2 µA/cm2 to 5.62 ± 0.53 (n = 6).
Figure 2 further demonstrates the
presence of significant forskolin-stimulated Isc
after pretreatment of monolayers with bumetanide. The
bumetanide-insensitive current was reduced to 3.1 ± 0.25 (n = 12) upon addition of DNDS (5 mM) to the
basolateral bathing solution (Figs. 1 and 2). Although this value was
lower than the amiloride-insensitive component of
Isc, the difference was not statistically
significant. Together, these data provide pharmacological evidence for
cAMP-stimulated HCO
and Cl
secretion
across cultured rat FDLE cells.

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Fig. 2.
Representative trace showing the
Isc response across FDLE cells bathed in the
presence of Ringer solution. Amiloride (10 µM) was added to apical
bathing solution of the monolayers at the time indicated to abolish the
Isc due to active Na+ absorption.
Bumetanide (100 µM) was then added to the basolateral compartment
before addition of forskolin (10 µM). Once a stable
Isc was achieved, DNDS (5 mM) was added to the
basolateral compartment. These experiments were repeated 6 times, each
with similar results.
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In subsequent studies, we tested the anion dependency of
Isc across rat FDLE cells. Specifically, we
replaced bath Cl
and/or HCO
with large
organic anions (e.g., gluconate and HEPES). We found that the basal
Isc was not affected by replacement with a
HCO
-free solution (Fig.
3), whereas it was reduced by
Cl
-free solutions (Fig. 4).
However, in both cases, forskolin elicited a current, which on average
was lower than that observed with normal Ringer (Figs. 3 and 4). As
shown in Fig. 4, in HCO
-free solutions, pretreatment
with bumetanide before forskolin abolished the forskolin-induced
increase in Isc. Figure 4 also shows that addition of bumetanide after forskolin totally decreased the
forskolin-induced Isc. Together, Figs. 3 and 4
show that rat FDLE cells possess two components of cAMP-stimulated
current that depend on both HCO
and
Cl
, respectively.

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Fig. 3.
Representative trace showing the
Isc response across FDLE cells bathed in the
presence of Cl -free solutions (NaCl was replaced with an
equimolar amount of sodium gluconate). Amiloride (10 µM) was added to
apical bathing solution of the monolayers at the time indicated to
abolish the Isc due to active Na+
absorption. Once a stable baseline was achieved, forskolin (10 µM)
was added to both compartments. Subsequent addition of the
loop-diuretic bumetanide (100 µM) did not alter the
Isc. These experiments were repeated 4 times,
each with similar results.
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Fig. 4.
Representative traces showing the
Isc responses across FDLE cells bathed in the
presence of HCO -free solutions. NaHCO3
was replaced with an equimolar amount of sodium-HEPES. Top:
bumetanide (100 µM) was added to the basolateral compartment before
addition of forskolin (10 µM). As expected, forskolin did not
increase Isc. Bottom: addition of
forskolin increased Isc to a peak value, which
then returned to a stable baseline. The difference between the peak and
plateau may be due to transient release of HCO from
the cells. Addition of bumetanide totally inhibited the
Isc. These experiments were repeated 4 times,
each with similar results.
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Figure 5 shows that forskolin had little
effect on Isc when both Cl
and
HCO
were replaced with gluconate and HEPES,
respectively. The transient increase in Isc was
likely due to secretion of residual intracellular Cl
and
HCO
. These data indicate that the forskolin response
depends on both Cl
and HCO
. Figure
6 summarizes and compares results from
the pharmacological and ion substitution experiments demonstrating the
presence of cAMP-stimulated HCO
and Cl
secretion in FDLE cells.

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Fig. 5.
Representative trace showing the
Isc response across FDLE cells bathed in the
presence of both Cl - and HCO -free
media. Addition of forskolin (10 µM) resulted in only a transient
increase of Isc, most likely due to the movement
of intracellular Cl and HCO or to any
residual Cl and HCO left in the
chambers. The Isc returned to its baseline value
within 5 min. These experiments were repeated 5 times, each with
similar results.
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Fig. 6.
Mean values (± 1 SE) are shown for the baseline
Isc, response to amiloride (10 µM), forskolin
(10 µM), bumetanide (100 µM), and DNDS (5 mM). All values of
Isc following addition of amiloride were
significantly different from their corresponding baselines.
* Significantly different (P < 0.05) compared with
the corresponding amiloride values; #significantly
different (P < 0.05) compared with the corresponding
forskolin values; +significantly different
(P < 0.05) compared with the corresponding bumetanide
values.
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To test the possibility that HCO
entered FDLE cells
as CO2, which was then converted to HCO
by the action of carbonic anhydrase, we added 100 µM acetazolamide (a
carbonic anhydrase inhibitor) to both the apical and basolateral compartments of FDLE monolayers bathed in Cl
-free
solutions after the addition of 10 µM forskolin. Acetazolamide did
not alter Isc (mean
Isc =
0.04 µA/cm2;
n = 9). These data suggest that HCO
does not enter the cells as CO2.
Defining the apical membrane Cl
conductance
functionally.
Initially, we compared the effects of the Cl
channel
blockers glibenclamide (200 µM both sides) and DIDS (200 µM apical)
on intact monolayers to identify the apical membrane ion channel(s) mediating anion secretion across FDLE cells. Monolayers were bathed in
symmetrical normal Ringer solutions and pretreated with a
Cl
channel blocker. Glibenclamide markedly attenuated the
Isc response to forskolin
(
Isc) from 3.8 ± 0.6 (n = 12) to 0.8 ± 0.1 µA/cm2 (n = 11;
means ± SE; P < 0.01). This value was not
different from the response seen in the absence of both
Cl
and HCO
(Fig. 6). In contrast,
apical DIDS had no significant effect on forskolin-induced
Isc (
Isc = 4.4 ± 0.4; n = 11). These findings are suggestive
of the presence of CFTR.
We next evaluated the apical plasma membrane Cl
conductances by permeabilizing the basolateral membrane with nystatin
(200 µg/ml) in the presence of transepithelial Cl
gradients. Initially, we measured the Isc
arising from a 125 mM basolateral to a 5 mM apical Cl
"secretory" gradient. The representative current tracing in Fig. 7A shows that basolateral
nystatin addition produced a small initial decrease in
Isc, followed by a large spontaneous increase in
Isc. On average, the Isc
increased by 24.5 ± 0.9 µA/cm2 (means ± SE;
n = 10). The subsequent addition of forskolin increased the Isc further, by 29.6 ± 2.3 µA/cm2 (means ± SE; n = 10).
Eliminating the transepithelial Cl
gradient by increasing
Cl
concentration in the apical bath to 125 mM caused the
Isc to drop rapidly (Fig. 7A).

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Fig. 7.
Evaluation of the apical membrane Cl
conductance following nystatin treatment of the basolateral membranes
in the presence of a 125:5 basolateral-to-apical Cl
gradient (i.e., secretory). A: representative current trace.
Nystatin (200 µM) was added to the basolateral solution at the time
indicated to increase the anion permeability of the basolateral
membrane. Subsequently, forskolin (10 µM) was added to both
compartments. Finally, we eliminated the Cl gradient by
adding 120 mM Cl to the apical bath. These experiments
were repeated 10 times with similar results. B: comparison
of the nystatin-induced Isc
( Isc-nystatin) in the absence and presence of
sucrose, added into both the apical and basolateral compartments, just
before the addition of forskolin. Data represent means ± SE;
number of measurements were control: n = 5; 10 mM
sucrose: n = 5; 30 mM: n = 7;
* P < 0.05 compared with 0 mM.
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Previous studies have shown that addition of polyene antibiotics such
as nystatin or amphotericin B to bathing solutions containing Cl
can cause cells to swell and thus activate plasma
membrane conductances (9). Therefore, we added sucrose to
both bath solutions to determine whether the nystatin-induced current
across FDLE cells was swelling mediated. These data are summarized in
Fig. 7B. These results show that 10 mM sucrose reduced the
nystatin-activated current by >50%, whereas 30 mM sucrose abolished
it. We were unable to increase Cl
conductance in intact
monolayers either by fourfold decreases in bath osmolality or by
isoosmotic urea (data not shown).
When the Cl
gradient was directed in the absorptive
direction (i.e., 125 mM apical to 5 mM basolateral), we observed a
small increase in Isc (3.7 ± 0.2 µA/cm2) following nystatin treatment and no
swelling-activated conductance, which was the expected result since the
basolateral solution contained the impermeant anion gluconate
(9). A representative current tracing is shown in Fig.
8A. The subsequent addition of
forskolin to the bathing solutions significantly increased the
Isc without altering the conductance of the
monolayers (Fig. 8). A typical response of the forskolin-induced
Isc to glibenclamide is shown in Fig.
9, while the dose-response relationship
to glibenclamide is shown in Fig. 10.
Glibenclamide inhibited the forskolin-induced Isc with an IC50 of ~25 µM.
Maximal inhibition (~85%) was seen at nearly 100 µM. Apical DIDS
(200 µM) did not alter the forskolin-induced Isc in permeabilized monolayers (data not
shown). Glibenclamide has been shown to block CFTR with an inhibition
constant of ~30 µM (29). However, it can also block
outwardly rectifying chloride channels (26). Fortunately,
disulfonic stilbenes can be used to distinguish between these two
possibilities. Extracellular DIDS blocks outwardly rectifying chloride
channels (35). DIDS can also block CFTR but only from the
cytosolic side (13). Thus the inhibition constant for
glibenclamide observed in this study (25 µM) and lack of effect of
apical DIDS provide functional evidence that a cAMP-activated
glibenclamide-sensitive channel, like CFTR, mediates anion secretion
across the apical membranes of FDLE cells.

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Fig. 8.
A: representative current trace from an FDLE
monolayer bathed with an "absorptive" Cl gradient
(i.e., 5 mM basolateral to 125 mM apical). All other conditions are as
in Fig. 7. Notice the lack of increase in Isc
following the addition of nystatin into the basolateral compartment.
B: comparison of the magnitudes of the nystatin- and
forskolin-induced Isc in the presence of
secretory (basolateral apical) vs. absorptive (apical basolateral) Cl gradients. Data are means ± SE;
n = 10. * P < 0.001 from the
corresponding value obtained with the opposite gradient.
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Fig. 9.
Representative trace showing a dose-response inhibition
of the forskolin-induced Isc by glibenclamide
(3-300 µM) added at the times shown by the arrows in the apical
compartment. The monolayer was bathed with an absorptive
Cl gradient (i.e., 5 mM basolateral to 125 mM apical).
Changes in conductance (mS) of the monolayer following addition of
apical amiloride (10 µM), basolateral nystatin (200 µM), and
forskolin (10 µM) into both compartments are also shown. These
experiments were repeated 5 times each with similar results.
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Fig. 10.
Dose-response relations for glibenclamide inhibition of
the forskolin-induced Isc across permeabilized
monolayers in the presence of an absorptive Cl gradient
(i.e., 5 mM basolateral to 125 mM apical) as described in Fig. 9. Data
are means ± SE (n = 9). The line represents the
best fit using regression. IC50 for the forskolin-induced
current was 26 µM.
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Immunocytochemical localization of CFTR in rat FDLE cells.
Having obtained functional evidence for a CFTR-like channel in rat FDLE
cells, we then used antibodies raised against CFTR for immunostaining.
Staining consistent with CFTR was observed in rat FDLE cells using two
different anti-CFTR antibodies (Figs. 11 and 12). Figure 11 compares
cross-sectional views of rat FDLE that were stained with or without a
monoclonal antibody raised against the COOH terminus of CFTR. In this
case, the secondary antibody was Texas red X-labeled goat anti-mouse
IgG. Although there was some slight background staining associated with
both protocols, the overall pattern of staining was consistent with the
presence of significant levels of CFTR protein in FDLE cells. Figure
12 shows a typical en face view of a
rat FDLE monolayer stained with a polyclonal antibody raised against
the first nucleotide binding fold of CFTR. A companion monolayer was
treated with rabbit IgG to serve as a control. Oregon green-labeled
goat anti-rabbit IgG was used as a secondary antibody, and the nuclei
were stained with bisbenzimide.

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Fig. 11.
Photomicrographs of FDLE cells
treated with a monoclonal anti-cystic fibrosis transmembrane
conductance regulator (CFTR) antibody raised against the first
nucleotide binding fold of CFTR (top) and
nonantibody-treated control cells (bottom). Both monolayers
were exposed to Texas red X-labeled secondary antibodies and
counterstained with the nuclear dye bisbenzimide. The cross-sectional
view was obtained by folding the filters and viewing the edge using
confocal microscopy as previously described
(33).
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Fig. 12.
Photomicrographs of FDLE cells treated with a monoclonal anti-CFTR
antibody raised against the COOH terminus of CFTR. The secondary
antibody was Texas red X-labeled goat anti-mouse IgG. The staining is
consistent with the presence of significant levels of CFTR in FDLE
cells.
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DISCUSSION |
The main conclusions of these studies are 1) baseline
Isc across intact 19-day FDLE monolayers is
mediated partly by Na+ absorption and partly by an
unidentified pathway; 2) incubation of amiloride-treated
FDLE monolayers with forskolin, an agent that increases cAMP levels,
evokes a sustained increase in anion secretion, consisting of a
combination of Cl
and HCO
current;
3) in the presence of a secretory Cl
gradient,
permeabilization of the basolateral membrane with nystatin activates a
"swelling" conductance; 4) in the presence of an
absorptive Cl
gradient, permeabilization of the
basolateral membrane with nystatin reveals a cAMP-stimulated
glibenclamide-sensitive apical membrane anion conductance similar to
CFTR; and 5) immunostaining provides further evidence for
the expression of CFTR-like channels in rat FDLE cells.
Surprisingly, in a previous study, FDLE cells from 18- to 21-day
gestation rat fetuses cultured on porous supports in the presence of
serum and mounted in Ussing chambers exhibited
Isc that were almost completely inhibited by
amiloride. Inhibitors of
Na+-K+-2Cl
cotransporter,
Na+-glucose cotransporters, or Cl
channels
had no significant effect on Isc (24,
28). On the basis of these findings, it was concluded that
near-term FDLE cells actively transport Na+ through
amiloride-sensitive pathways, similar to adult alveolar type II cells,
but have little or no Cl
secretion. The presence of
Na+ absorption and the lack of Cl
secretion
were attributed to the fact that FDLE cells are cultured in room air
(21%), which promotes expression of the various subunits of the ENaC
protein and downregulates Cl
transporters in these cells
(27). Thus differences in gestational age (17-19 days
in our studies vs. 18-20 days in the previously mentioned studies)
as well as differences in oxygenation due to the depth of the
air-liquid interface may account for these differences. However,
testing these possibilities is beyond the scope of the present study.
In other studies, significant levels of Cl
secretion
occur across rat and human FDLE cells cultured in serum-free media
(1, 3, 16). Because the fetal lung secretes
Cl
in utero (6), one would expect that FDLE
cells would also be capable of Cl
secretion as observed
herein. It is interesting to note that agents increasing intracellular
cAMP have also been shown to activate Cl
channels in the
apical membranes of adult alveolar type II cells (11, 20).
Our results are consistent with the recently proposed model for anion
secretion across Calu-3, a human airway serous cell line (8,
12). Calu-3 cells also have a basal Isc
due to HCO
secretion (30) and a
forskolin response consisting of a transient increase in
Isc due, in part, to Cl
, followed
by a sustained increase due mostly to HCO
secretion
(8). Secretion of both Cl
and
HCO
has been reported in a variety of secretory
epithelia such as duodenum, pancreas (21), bile duct
(36), and Clara cells (34). Marunaka et al.
(14) also identified the presence of
HCO
current following stimulation of rat FDLE cells
with
-agonists. An increase in HCO
flux may
increase the pH of the fetal fluid, which may have important
consequences both in the volume of secreted fluid and other homeostatic
functions of the alveolar epithelium, such as surfactant secretion and reabsorption.
The fact that DNDS inhibited the bumetanide-insensitive fraction of the
forskolin-induced Isc and acetazolamide
pretreatment of FDLE cells, bathed in Cl
-free solutions,
had no effect on Isc suggests that
HCO
entered the cells via the
Na+-HCO
cotransporter (8,
12). However, DNDS has been shown to have several other
nonspecific effects that may influence the entry of
HCO
, including blocking K+ channels.
Quantifying the precise effect of basolateral DNDS on rat FDLE cells
and testing several alternative possibilities is beyond the scope of
the present study.
We have identified two distinct anion conductive pathways in the apical
membranes of FDLE cells: a cAMP-activated, DIDS-insensitive conductance, likely CFTR; and a cAMP-independent, DIDS-sensitive, swelling-activated conductance. Immunocytochemical studies also indicate the presence of CFTR at the apical membranes of FDLE cells in
agreement with our functional data and earlier studies (16).
Previous studies suggest that HCO
ions may be
secreted across the apical membranes through CFTR. For example,
incubation of normal but not cystic fibrosis human airway epithelial
cells with forskolin led to HCO
secretion,
consistent with the notion that HCO
was secreted
through CFTR (31). Also, Poulsen et al. (25) demonstrated the existence of 10-pS Cl
channels in
forskolin-treated fibroblasts transfected with wild-type CFTR but not
F508-CFTR. Our observations are consistent with this precept (see below).
The swelling conductance was activated by the pore-forming antibiotic
nystatin since the drug-induced pores are permeable mostly to small
univalent cations and less to anions (selectivity ratio ~7:1). Hence,
addition of nystatin to NaCl- or KCl-containing solutions is expected
to cause cell swelling that can be prevented by addition of impermeant
anions such as gluconate or sulfate into the basolateral compartment
(9). This explains why we observed a significant increase
in Isc across FDLE cells when the
Cl
gradient was oriented from the basolateral to the
apical side of monolayers and not when the gradient was oriented in the
opposite direction. The ability of 30 mM sucrose to abolish the
nystatin activation lends further credence to our hypothesis that this is a swelling-activated conductance, although we cannot exclude the
contribution of Ca2+-activated Cl
conductances and the voltage-sensitive Cl
channels. This
is consistent with the observation of both mRNA and protein expression
of CIC-2, a voltage- and volume-activated Cl
channel (32), in 19-day fetal rat lung, with levels
decreasing significantly after birth (19), in agreement
with our functional measurements.
A number of previous studies have reported anatomically normal lungs in
newborn and older human infants with cystic fibrosis, although they
lacked functional CFTR (7, 17). Thus the
swelling-activated conductance identified in the FDLE cells may be
activated by another unidentified mechanism and play an important role
in fluid secretion in utero under conditions in which normal CFTR may
be lacking.
 |
ACKNOWLEDGEMENTS |
We thank G. Davis for valuable assistance in isolating and
culturing fetal lung epithelial cells.
 |
FOOTNOTES |
This work was supported in part by National Institutes of Health Grants
HL-31197, HL-51173, and P30-DK-54781.
Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St. S., THT 940, Birmingham, AL 35249 (E-mail:
Sadis.Matalon{at}ccc.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.
10.1152/ajplung.00370.2001
Received 19 September 2001; accepted in final form 27 November 2001.
 |
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