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and HCO
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 |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
- or
HCO-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
short-circuit current; amiloride; nystatin; swelling-activated conductance; immunocytochemistry; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
-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
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 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
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
and HCO and HCO and
Na-HCO 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 |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cultured rat FDLE cells demonstrate cAMP-stimulated
Cl and HCO · 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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vs.
HCO secretion across airway cells is driven
by a basolateral bumetanide-sensitive Na+-K+-Cl cotransporter while
HCO secretion
across cultured rat FDLE cells.
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and/or HCO-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, respectively.
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and
HCO and
HCO and HCO
secretion in FDLE cells.
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-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 HCODefining 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 HCOIsc = 4.4 ± 0.4; n = 11). These findings are suggestive
of the presence of CFTR.
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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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, followed
by a sustained increase due mostly to HCO and
HCO-agonists. An increase in HCO
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
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 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.
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ACKNOWLEDGEMENTS |
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We thank G. Davis for valuable assistance in isolating and culturing fetal lung epithelial cells.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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apical) vs. absorptive (apical 
