1 Institut National de la Santé et de la Recherche Médicale Unité 514, Institut Fédératif de Recherches 53, Centre Hospitalier Universitaire Maison Blanche, 51092 Reims Cedex; 2 Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique Unité Propre de Recherche 411, 06560 Sophia Antipolis; 3 Institut National de la Santé et de la Recherche Médicale Unité 364, Tour Pasteur Faculté de Médecine, 06107 Nice Cedex 02, France; and 4 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The
amiloride-sensitive epithelial Na+
channel (ENaC) is an apical membrane protein complex involved in active
Na+ absorption and in control of
fluid composition in airways. There are no data reporting the
distribution of its pore-forming -,
-, and
-subunits in the
developing human lung. With use of two different rabbit polyclonal
antisera raised against
- and
-ENaC, immunohistochemical
localization of the channel was performed in fetal (10-35 wk) and
in adult human airways. Both subunits were detected after 17 wk of
gestation on the apical domain of bronchial ciliated cells, in
glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of
- and
-subunits was similar in fetal and
adult airways. In large airways, the two subunits were detected in
ciliated cells, in cells lining glandular ducts, and in the serous
gland cells. In the distal bronchioles,
- and
-subunits were
identified in ciliated and Clara cells. Ultrastructural immunogold
labeling confirmed the identification of
- and
-ENaC proteins in
submucosal serous cells and bronchiolar Clara cells. Early expression
of ENaC proteins in human fetal airways suggests that
Na+ absorption might begin
significantly before birth, even if secretion is still dominant.
amiloride; human fetal development; airway epithelium; Clara cell; glandular cell
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CLEARANCE OF SODIUM and water from the mature lung is mediated by a transcellular mechanism, combining diffusion of the luminal Na+ through an apical amiloride-sensitive epithelial Na+ channel (ENaC) and excretion of intracellular Na+ by basolateral Na+-K+-ATPase (1, 26). Water diffuses either through specific water channels or through the paracellular junctions to equilibrate the osmotic pressure (21). This mechanism contributes to the correct hydration of the apical liquid layer in the proximal and distal airways as well as in alveolar tissue.
A membrane transport complex composed of three homologous subunits
called -,
-, and
-ENaC (4, 5, 18, 19, 35, 37) is responsible
for the passive electrodiffusion of the ions through the apical
membrane. The subunits are characterized by a large extracellular
domain located between two transmembrane regions, the
NH2- and COOH-terminal segments
being cytoplasmic (1, 31). The expression of the three subunits is
necessary for maximal functional activity (5). They probably associate into functional heterotetramers that contain two
-ENaC, one
-ENaC, and one
-ENaC (11, 16, 33), i.e., a tetrameric
organization also found for other members of the same gene superfamily
(7).
The three rat ENaC mRNA and protein subunits were detected in many
Na+-absorptive tissues such as the
distal parts of the cortical nephron, the distal colon, and the
reabsorptive ducts of the salivary glands and sweat glands (8, 32). In
the mature rat, - and
-ENaC transcripts were detected in ciliated
cells of nasal and bronchial surface epithelium, in bronchiolar Clara
cells, and in alveolar type II cells (10, 20). Rat
-ENaC transcripts
were also detected in nasal and tracheal gland acini. In humans, all
three mRNAs were identified in the surface airway epithelium, whereas
- and
-transcripts were also found in epithelial cells along
gland ducts and in gland acini (3, 22). In previous reports, we identified rat airway epithelium as an important site of expression of
the three ENaC proteins (32). We also showed that the transcription of
all three ENaC subunits was increased around birth, at a moment when
the respiratory epithelium switches from chloride secretion to
Na+ absorption (23, 24, 36, 37).
However, these initial studies did not address specifically the
distribution and the subcellular localization of the ENaC proteins in
the human airways. To understand how the respiratory epithelium is
modified during lung development, we have now examined the protein
distribution of ENaC in human fetal airway tissues. After having tested
several rabbit polyclonal antibodies against rat or human
-,
-,
and
-ENaC, two of them raised against human
-ENaC and rat
-ENaC reacted with the human lung proteins. They were used for
immunohistochemical localization using optic and electron microscopy in
human fetal and adult lung tissues.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human fetal and adult tissue material.
Ten fetuses ranging from 10 to 36 wk gestational age were obtained
from spontaneous abortions or medical inductions. The age distribution
is shown in Table 1. All fetuses were well
preserved without respiratory abnormality or infection. They were not
associated with either polyhydramnios or oligohydramnios. Adult
respiratory tissue was obtained during postmortem examination from
three patients without hypertension who died from nonpulmonary causes.
The Ethics Committee approved these experiments on human tissues.
Different tissue specimens were collected from different parts of the
fetal and adult airways (tracheae, bronchi, and bronchioles) and
immediately fixed. Samples were fixed in 15% Formalin and embedded in
paraffin, and 3-µm sections were mounted on gelatin-coated slides and
dried overnight at 50°C. Other samples were embedded in optimum
cutting temperature compound (Tissue Tek, Miles, IN), frozen in liquid nitrogen, cut at 20°C, and transferred to gelatin-coated
slides. For electron microscopy, adult tissues were fixed
by immersion for 2 h at room temperature in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.2 (Sigma, St. Louis, MO).
Osmium postfixation was omitted. After fixation, the tissue was washed,
dehydrated through graded alcohol series, and embedded in Epon.
|
Preparation of antibodies.
Several polyclonal antibodies were raised against rat -ENaC, as
described in previous publications (1, 17, 18, 31, 32). The polyclonal
antiserum against
-ENaC was raised against the last 17 cytoplasmic
residues of the human
-subunit (1, 36). The polyclonal antiserum
against
-ENaC was obtained after immunization of a rabbit with an
hapten formed with keyhole limpet hemocyanin and the extracellular
-ENaC peptide Y127GVKI SRKRRI AGS143 (32).
After immunization, antisera were regularly analyzed by ELISA against
pure peptides (Fig. 1,
A and
B). When a specific immune response
was detected, the antisera were characterized by biochemical and
histological techniques. The antibodies used in the present study
correspond to the positive ELISA antisera, which were able either to
immunoprecipitate in vitro translated proteins or to detect the protein
with Western blot or immunohistochemical analysis.
|
Immunohistochemistry.
Control immunohistochemistry of -,
-, and
-ENaC antisera was
done with 6-µm frozen rat lung sections. Other complementary characterization of these antisera has been published elsewhere (1, 17,
18, 32, 36). Some additional characterizations are presented in Fig. 1.
Human airway paraffin sections were deparaffinized with xylene and
successively rehydrated in graded ethanol baths, distilled water, and
0.1 M PBS, pH 7.2, before treatment with 0.4% pepsin in 0.01 N HCl for
10 min at room temperature. Hydrogen peroxide bath was used for 5 min
at room temperature to remove endogenous peroxidase activity. A
blocking reagent (6% goat serum) was added for 5 min. The slides were
then rinsed twice with PBS and pretreated for 10 min with pepsin
(0.04% in 0.01 N HCl). After two rinses, the tissue sections were
incubated for 1 h with primary antibodies diluted to PBS as follows:
anti-
-ENaC, 1:100 and anti-
-ENaC, 1:100. Immunohistochemical
staining was carried out using the streptavidin-biotin LSAB2 technique
(DAKO, Glostrup, Denmark).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of the antibodies.
The antibodies used in the present study have been characterized
already in several previous studies (1, 17, 18, 32, 36). After rabbit
immunization, antisera were regularly analyzed by ELISA against pure
peptides. Figure 1, A and
B, shows the positive signal of immune
sera against pure peptides in ELISA reaction. No signal was detected
with preimmune serum. Specificity of the antibodies was also checked by
histology in rat and human tissues known to express ENaC. Antisera were
positive in rat lung and kidney sections (Fig. 1,
C-F) (23, 40), i.e., two
tissues where an active amiloride-sensitive
Na+ absorption has been described.
A positive signal was also detected in human keratinocytes, in
absorptive cells in the surface epithelium of the colon, and at apical
membrane of sweat glands (data not shown). Initial experiments were
also carried out with an anti--ENaC able to recognize the
NH2-terminal segment of the rat
-ENaC in immunohistochemistry. However, it did not cross-react with
human lung
-ENaC. Therefore, the present study was focused on the
expression during fetal development of the
- and
-ENaC proteins.
Developmental expression of - and
-ENaC subunits in fetal airways.
At 10 wk of gestation, the human fetal trachea and the bronchi are
lined with undifferentiated and polarized columnar epithelial cells and
no glands are developed yet. Between 11 and 16 wk of gestation,
ciliated and secretory cells progressively differentiate in the surface
epithelium along proximal airways. At 13-14 wk, the first glands
grow out from the basal aspect of this epithelium into the lamina
propria. Until 16 wk of gestation, the branching distal airways exhibit
a pseudoglandular pattern and give rise to the future conducting
airways. As shown in Table 1, no
- or
-ENaC immunoreactivity was
detected during the early stages of development (
16 wk).
|
Expression of - and
-ENaC subunits
in adult airways.
In the adult surface epithelium of human trachea and bronchi, the
distribution of both
- and
-ENaC subunits was similar to that
observed in late- gestational fetuses. In paraffin or frozen sections,
the
- and
-ENaC staining was observed in the ciliated cells,
which showed homogeneous apical immunostaining (Fig.
3,
A-C). No
- and
-ENaC
staining was identified in basal cells or in mucous goblet cells. In
the submucosal glands, the
- (Fig.
3D) and
-subunits (Fig.
3E) were detected in the serous cells. Serous cells were identified by an anti-lysozyme antiserum, i.e., a specific marker of these cells (Fig.
3F). In the bronchioles,
- (Fig.
3G) and
-subunits (Fig.
3H) were detected at the apex of the
Clara cells. Clara cells were identified by an anti-CC10 antiserum,
i.e., a specific marker of these cells (Fig.
3I).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results reported in this study demonstrate for the first time an
early expression of - and
-ENaC proteins in the human fetal
airways. Both proteins were detected in the apical membrane of the
ciliated cells and also in bronchiolar Clara cells as well as in serous
cells of the submucosal glands. All these localizations, including
those in the serous cells of the submucosal glands, have been reported
at an RNA level by other investigators using in situ hybridization (3,
10). Taken together with these previous studies, the present work
identifies the different sites of ENaC expression in the airways. In
the human distal lung sections, in which bronchioles were examined,
canalicular and alveolar structures could also be observed. However,
because of a delay between abortion and examination of the fetus, the
distal lumens were not optimally preserved. In contrast, the epithelial
cells lining the bronchi and bronchioles were preserved by the rigid
cartilage and muscle layers, whereas the distal lung was collapsed. In
that zone, flattened epithelial cells were hardly distinguishable from
fibroblasts and endothelial cells. For this reason, we limited our
analysis to the proximal lung and to the distal bronchioles. We
believe that use of alternative animal models, such as rat or mouse,
will be necessary to address the issue of the time-dependent expression of ENaC in alveoli. Our data provide, however, original information regarding the expression of ENaC in the human lung during development.
These results are consistent with the data published by Venkatesh and
Katzberg (34), showing the expression of all three ENaC RNAs as
early as 21 wk of gestation. At 24 wk, they reported mRNA contents
equal to 13, 26, and 32% of the adult values for -,
-, and
-ENaC, respectively. Although the immunohistochemistry is not
quantitative, the level of expression of the two subunits that have
been tested was definitely lower at the end of the second trimester
than during any later periods of gestation (Table 1).
Unfortunately, we were unable to carry out the same study with -ENaC
because our antibodies raised against rat
-ENaC did not react with
the human lung subunit. Previous experiments performed in the rat lung
(32) have shown that
-ENaC is indeed colocalized with
-ENaC and
-ENaC in the airways. Moreover, the expression of the
-subunit
parallels the expression of the
-subunit (10). From this
perspective, the expression patterns of
-ENaC and
-ENaC are
expected to be similar. Because the stoichiometry of each subunit
within a functional complex is a fixed value (7, 11), it is likely that
the less abundant transcripts will be the limiting factor for
expression of the complex. Recent data from Otulakowski et al. (25)
suggest that in human airways the limiting factor corresponds to
-ENaC. Our experiments therefore identify probably the main sites of
expression of the highly
Na+-selective and highly
amiloride-sensitive channel in airways. It remains possible that other
channels, characterized by different biophysical or pharmacological
properties and formed by distinct proteins, could also participate in
lung Na+ homeostasis.
The role conferred to -ENaC is usually more important than the role
conferred to
- or
-ENaC, which is consistent with the fact that
there are two copies of
-ENaC per copy of
- or
-ENaC (11). This is also suggested by knockout of the
-ENaC
gene in the mouse, which is associated with an early death caused in part by defective neonatal lung liquid clearance (13). The situation might be different in humans where inactivation of
-ENaC observed during pseudohypoaldosteronism type I is not associated with major lung defects.
The detection of the ENaC proteins in the lung during early stages of
development does not of course prove a strict parallel to function.
Several disparities between molecular and functional data have been
reported in the literature (1, 26), and the concept of silent
Na+ channels has also been
recognized for years (27). Therefore, it is possible that the channels
are present in the epithelium but are silent. We suspect that the
activation of such a silent pool and hence the activation of lung
liquid clearance might be helpful to clear excess lung liquid during
some pathological situations. The effect of known
Na+ channels stimulators, such as
-adrenergic agonists (1, 2, 21), glucocorticoids (1, 6, 36), oxygen
(28, 29, 38), or growth factors, is certainly worth investigating in pathogen conditions such as premature delivery or respiratory distress syndrome.
In the adult surface epithelium, both - and
-ENaC proteins were
detected by optical microscopy at the apical membrane of ciliated
cells, whereas they were not identified in the mucous goblet cells.
Electron microscopy data also suggest an apical or subapical
localization of the
- and
-ENaC subunits. No signal was found
near the basolateral membrane. Such localization is similar to that of
the cystic fibrosis transmembrane conductance regulator (CFTR) protein,
which is also present in apical vesicles under the apical plasma
membrane of the ciliated cells (30). In the submucosal glands,
- and
-ENaC were specifically expressed in the serous gland cells and were
identified at the level of the secretory granules, which membrane fuses
to the apical membrane during exocytosis. This localization at the
level of secretory cells is consistent with the detection of the
corresponding RNA by in situ hybridization (3, 10). Moreover, a similar
staining was observed with the two different antibodies that recognize two distinct regions of two different ENaC subunits.
- and
-ENaC were also detected by electron microscopy in the lumen of secretory granules (Fig. 4F) and were absent
with control preimmune antisera (Fig.
4D). Although a vesicular
localization of these membrane proteins might appear surprising, it is
not unique and has been reported previously for CFTR (14).
Interestingly, our results suggest that in human airways, ENaC and CFTR proteins could be colocalized, even if their temporal expression throughout development differs (12). Further work will be necessary to quantify the relative levels of expression of the ENaC proteins along the bronchial tree and distal air spaces of the lung and to test whether distal airways can contribute significantly to the distal lung liquid clearance observed at birth or during pathological conditions with alveolar edema.
In conclusion, our work shows that - and
-ENaC proteins are
expressed not only in the apical membrane of the ciliated cells but
also in cells lining glandular ducts, in the serous gland cells, and in
Clara cells. Early expression of ENaC proteins in human fetal airways
suggests that Na+ absorption might
begin significantly before birth, even if secretion is still dominant.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Franck Aguila for the artwork.
![]() |
FOOTNOTES |
---|
This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Association Française de Lutte contre la Mucoviscidose, and National Heart, Lung, and Blood Institute Grant HL-51854.
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 and other correspondence and present address of P. Barbry: IPMC, CNRS, UPR411, 660 Route des Lucioles, 06560 Sophia Antipolis, France (E-mail: barbry{at}ipmc.cnrs.fr).
Received 7 December 1998; accepted in final form 19 July 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barbry, P.,
and
P. Hofman.
Molecular biology of Na+ absorption.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G571-G585,
1997
2.
Bland, R. D.,
and
D. W. Nielson.
Developmental changes in lung epithelial ion transport and liquid movement.
Annu. Rev. Physiol.
54:
373-394,
1992[ISI][Medline].
3.
Burch, L. H.,
C. R. Talbot,
S. M. R. Knowles,
C. M. Canessa,
B. C. Rossier,
and
R. C. Boucher.
Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways.
Am. J. Physiol.
269 (Cell Physiol. 38):
C511-C518,
1995
4.
Canessa, C. M.,
J. D. Horisberger,
and
B. C. Rossier.
Epithelial sodium channel related to proteins involved in neurodegeneration.
Nature
361:
467-470,
1993[ISI][Medline].
5.
Canessa, C. M.,
L. Schild,
G. Buell,
B. Thorens,
I. Gautschi,
J. D. Horisberger,
and
B. C. Rossier.
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
Nature
367:
463-467,
1994[ISI][Medline].
6.
Champigny, G.,
N. Voilley,
E. Lingueglia,
V. Friend,
P. Barbry,
and
M. Lazdunski.
Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones.
EMBO J.
13:
2177-2181,
1994[Abstract].
7.
Coscoy, S.,
E. Lingueglia,
M. Lazdunski,
and
P. Barbry.
The Phe-Met-Arg-Phe-amide-activated sodium channel is a tetramer.
J. Biol. Chem.
273:
8317-8322,
1998
8.
Duc, C.,
N. Farman,
C. M. Canessa,
J. P. Bonvalet,
and
B. C. Rossier.
Cell-specific expression of epithelial sodium channel ,
and
subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.
J. Cell Biol.
127:
1907-1921,
1994[Abstract].
9.
Engelhardt, J. F.,
J. R. Yankaskas,
S. A. Ernst,
Y. P. Yang,
C. R. Marino,
R. C. Boucher,
J. A. Cohn,
and
J. M. Wilson.
Submucosal glands are the predominant site of CFTR expression in the human bronchus.
Nat. Genet.
2:
240-248,
1992[ISI][Medline].
10.
Farman, N.,
C. R. Talbot,
R. Boucher,
M. Fay,
C. Canessa,
B. Rossier,
and
J. P. Bonvallet.
Noncoordinated expression of -,
-, and
-subunit mRNAs of epithelial Na+ channel along rat respiratory tract.
Am. J. Physiol.
272 (Cell Physiol. 41):
C131-C141,
1997
11.
Firsov, D.,
I. Gautschi,
A. M. Merillat,
B. C. Rossier,
and
L. Schild.
The heterotetrameric architecture of the epithelial sodium channel (ENaC).
EMBO J.
17:
344-352,
1998
12.
Gaillard, D.,
S. Ruocco,
A. Lallemand,
W. Dalemans,
J. Hinnrasky,
and
E. Puchelle.
Immunohistochemical localization of cystic fibrosis transmembrane conductance regulator in human fetal airway and digestive mucosa.
Pediatr. Res.
36:
137-143,
1994[Abstract].
13.
Hummler, E.,
P. Barker,
C. Talbot,
Q. Wang,
C. Verdumo,
B. Grubb,
J. Gatzy,
M. Burnier,
J. D. Horisberger,
F. Beermann,
R. C. Boucher,
and
B. C. Rossier.
A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.
Proc. Natl. Acad. Sci. USA
94:
11710-11715,
1997
14.
Jacquot, J.,
E. Puchelle,
J. Hinnrasky,
C. Fuchey,
C. Bettinger,
C. Spilmont,
N. Bonnet,
A. Dieterle,
D. Dreyer,
A. Pavirani,
and
W. Dalemans.
Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands.
Eur. Respir. J.
6:
169-176,
1993[Abstract].
15.
Jeffery, P. K.,
D. Gaillard,
and
S. Moret.
Human airway secretory cells during development and in mature airway epithelium.
Eur. Respir. J.
5:
93-104,
1992[Abstract].
16.
Kosari, F.,
S. Sheng,
J. Li,
D. O. Mak,
J. K. Foskett,
and
T. R. Kleyman.
Subunit stoichiometry of the epithelial sodium channel.
J. Biol. Chem.
273:
13469-13474,
1998
17.
Kretz, O.,
P. Barbry,
R. Bock,
and
B. Lindemann.
Differential expression of RNA and protein of the three pore-forming subunits of the amiloride-sensitive epithelial sodium channel in taste buds of the rat.
J. Histochem. Cytochem.
47:
51-64,
1999
18.
Lingueglia, E.,
S. Renard,
R. Waldmann,
N. Voilley,
G. Champigny,
H. Plass,
M. Lazdunski,
and
P. Barbry.
Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone.
J. Biol. Chem.
269:
13736-13739,
1994
19.
Lingueglia, E.,
N. Voilley,
R. Waldmann,
M. Lazdunski,
and
P. Barbry.
Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins.
FEBS Lett.
318:
95-99,
1993[ISI][Medline].
20.
Matsushita, K.,
P. B. MacCray,
R. D. Sigmund,
M. J. Welsh,
and
J. B. Stokes.
Localization of epithelial sodium channel subunit mRNAs in adult rat lung by in situ hybridization.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L332-L339,
1996
21.
Matthay, M. A.,
H. G. Folkesson,
and
A. S. Verkman.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L487-L503,
1996
22.
McDonald, F. J.,
M. P. Price,
P. M. Snyder,
and
M. J. Welsh.
Cloning and expression of the - and
-subunits of the human epithelial sodium channel.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1157-C1163,
1995
23.
O'Brodovich, H.
Epithelial ion transport in the fetal and perinatal lung.
Am. J. Physiol.
261 (Cell Physiol. 30):
C555-C564,
1991
24.
O'Brodovich, H.,
C. Canessa,
J. Ueda,
B. Rafii,
B. C. Rossier,
and
J. Edelson.
Expression of the epithelial Na+ channel in the developing rat lung.
Am. J. Physiol.
265 (Cell Physiol. 34):
C491-C496,
1993
25.
Otulakowski, G.,
S. Flueckiger-Staub,
L. Ellis,
K. Ramlall,
O. Staub,
D. Smith,
P. Durie,
and
H. O'Brodovich.
Relation between alpha, beta, and gamma human amiloride-sensitive epithelial Na+ channel mRNA levels and nasal epithelial potential difference in healthy men.
Am. J. Respir. Crit. Care Med.
158:
1213-1220,
1998
26.
Palmer, L. G.
Epithelial Na+ channels: function and diversity.
Annu. Rev. Physiol.
54:
51-66,
1992[ISI][Medline].
27.
Palmer, L. G.,
J. H.-Y. Li,
B. Lindemann,
and
I. S. Edelman.
Aldosterone control of the density of sodium channels in the toad urinary bladder.
J. Membr. Biol.
64:
91-102,
1982[ISI][Medline].
28.
Pitkanen, O.,
A. K. Tanswell,
G. Downey,
and
H. O'Brodovich.
Increased PO2 alters the bioelectric properties of fetal distal lung epithelium.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L1060-L1066,
1996
29.
Planes, C.,
B. Escoubet,
M. BlotChabaud,
G. Friedlander,
N. Farman,
and
C. Clerici.
Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells.
Am. J. Respir. Cell Mol. Biol.
17:
508-518,
1997
30.
Puchelle, E.,
D. Gaillard,
D. Ploton,
J. Hinnrasky,
C. Fuchey,
M. C. Boutterin,
J. Jacquot,
D. Dreyer,
A. Pavirani,
and
W. Dalemans.
Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.
Am. J. Respir. Cell Mol. Biol.
7:
485-491,
1992[ISI][Medline].
31.
Renard, S.,
E. Lingueglia,
N. Voilley,
M. Lazdunski,
and
P. Barbry.
Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel.
J. Biol. Chem.
269:
12981-12986,
1994
32.
Renard, S.,
N. Voilley,
F. Bassilana,
M. Lazdunski,
and
P. Barbry.
Localization and regulation by steroids of the alpha, beta and gamma subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney.
Pflügers Arch.
430:
299-307,
1995[ISI][Medline].
33.
Snyder, P. M.,
C. Cheng,
L. S. Prince,
J. C. Rogers,
and
M. J. Welsh.
Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.
J. Biol. Chem.
273:
681-684,
1998
34.
Venkatesh, V. C.,
and
H. D. Katzberg.
Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L227-L233,
1997
35.
Voilley, N.,
F. Bassilana,
C. Mignon,
S. Merscher,
M. G. Mattei,
G. F. Carle,
M. Lazdunski,
and
P. Barbry.
Cloning, chromosomal localization, and physical linkage of the beta and gamma subunits (SCNN1B and SCNN1G) of the human epithelial amiloride-sensitive sodium channel.
Genomics
28:
560-565,
1995[ISI][Medline].
36.
Voilley, N.,
A. Galibert,
F. Bassilana,
S. Renard,
E. Lingueglia,
S. Coscoy,
G. Champigny,
P. Hofman,
M. Lazdunski,
and
P. Barbry.
The amiloride-sensitive Na+ channel: from primary structure to function.
Comp. Biochem. Physiol. A Comp. Physiol.
118:
193-200,
1997[ISI][Medline].
37.
Voilley, N.,
E. Lingueglia,
G. Champigny,
M.-G. Mattéi,
R. Waldmann,
P. Barbry,
and
M. Lazdunski.
The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning.
Proc. Natl. Acad. Sci. USA
91:
247-251,
1994[Abstract].
38.
Yue, G.,
W. J. Russell,
D. J. Benos,
R. M. Jackson,
M. A. Olman,
and
S. Matalon.
Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.
Proc. Natl. Acad. Sci. USA
92:
8418-8422,
1995[Abstract].