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1 Respiratory Research, The epithelial Na
channel (ENaC) plays a critical role in the active reabsorption of
alveolar fluid at the time of birth or during pulmonary edema. Although
rat (r) ENaC is regulated by glucocorticoids during fetal development,
there are no data regarding the influence of gender hormones on ENaC
expression or function. We report higher levels of mRNAs encoding the
rat epithelial sodium channel; progesterone; 17 THE ACTIVE TRANSPORT of ions by lung epithelium results
in fluid movement across airways and alveolar spaces. Thus the net amount of fluid moving across the epithelium will be influenced by the
relative amounts of epithelial secretion (Cl) and absorption (Na).
Previous work has demonstrated that lung epithelial ion transport is
influenced by several factors, including the stage of lung development
(5, 20, 22, 24, 29). In addition, glucocorticoids augment both the
expression of the rat (r) epithelial Na channel (ENaC) (5, 20, 29) and
the amount of Na transport by distal lung epithelia grown in primary
culture (8). In contrast, there is little information regarding the
influence of gender hormones on lung epithelial ion transport. Zeitlin
et al. (32) have demonstrated that the presence of female gender
hormones within the culture medium alters the amount of tracheal
epithelial short-circuit current
(Isc) that is
inhibitable by the Na transport inhibitor amiloride or by the Cl
secretion inhibitor furosemide. To our knowledge, there have been no
reports regarding the effect of gender hormones on the expression or
function of ENaC in the lung.
We report here that the lungs of postpubertal female rats, relative to
male rats, have higher levels of Animal Preparation and Protocols
Adult rats.
Adult (300-400 g) male and female Wistar rats were purchased from
Charles River (St. Constant, Quebec, Canada) and maintained in a normal
day-night light cycle. We obtained vaginal smears from nonpregnant
mature female rats for three consecutive estrous cycles (4 days in
rats) to determine the stage of the cycle (2). Nonpregnant female rat
lungs were harvested either during the stage of predominant estrogen
(28-30 h, metestrus I stage) or predominant progesterone
secretion. All animals were killed with Euthanyl immediately before
removal of their lungs. To determine the effect of pregnancy on ENaC
mRNA levels, lungs were obtained from pregnant rats on the twentieth
day of gestation (term = 22 days).
Hormonal regulation of female lung epithelial Na channel mRNA
levels and function.
Adult ovariectomized (300-400 g) and immature (80-100 g;
24-25 day postnatal age) female Wistar rats were purchased from
Charles River. Ovariectomy had been performed in the mature female rats 3 wk before study. Both ovariectomized and immature female rats were
used to study the effects of progesterone or estradiol alone. Control
animals were treated with the vehicle only (10% ethanol in saline,
subcutaneously), whereas others received progesterone (1.5 mg/100 g
body wt in 10% ethanol in saline, subcutaneously) or 17 RNA Isolation and Northern Analysis
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
-rENaC subunit or the cystic fibrosis transmembrane conductance
regulator (CFTR) in the lungs of nonpregnant adult female relative to
adult male Wistar rats. Combined, but not separate, administration of
progesterone and 17
-estradiol increased mRNA levels encoding
-rENaC, 
-rENaC, and CFTR within 24 h. We also found a
dose-dependent increase in rENaC functional activity (as assessed by
the amiloride-sensitive short-circuit current across primary monolayer
cultures of alveolar epithelial cells mounted in Ussing chambers) after
a 5-day incubation of cells in medium containing progesterone and
17-estradiol. These findings suggest a gender-dependent influence on
the lung's ability to recover from pulmonary edema and on the degree
of airway fluid hydration in cystic fibrosis.
-estradiol; cystic fibrosis; pulmonary edema
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-rENaC mRNA. These differences likely arise from female gender hormones, since combined, but not separate, intraperitoneal administration of progesterone and
estradiol augments levels of rENaC subunit mRNA in sexually immature
female rats. Moreover, primary cultures of rat lung epithelial cells
from uninjected animals display a dose-dependent enhancement of ENaC
functional activity, as assessed by amiloride-sensitive Isc across
monolayers mounted in Ussing chambers, when incubated for 5 days in
serum-free medium containing the same combination of progesterone and
estradiol.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-estradiol
(2 µg/100 g body wt in 10% ethanol in saline, subcutaneously). Only
immature female rats were used to study the effect of combined
progesterone and 17-estradiol. In this case, immature animals
received a subcutaneous injection of progesterone combined with
17-estradiol. The estradiol dose was fixed at 2 µg/100 g body wt,
and the ratio of progesterone to estradiol was either 250:1, 500:1, or
750:1 (wt/wt). All animals were killed with Euthanyl 8, 24, or 48 h
later (n = 4 in each group).
-, -, and -rENaC
subunits of the Na channel. Blots were then washed (highest stringency:
0.2× SSC, 0.1% SDS, at 42°C for 60 min), and transcripts were visualized using standard autoradiography. Full-length -tubulin cDNA probes were used to assess the integrity of the
mRNA.
View larger version (31K):
[in a new window]
Fig. 1.
-Rat (r) epithelial Na channel (ENaC) mRNA levels of whole lung were
higher in adult female rats during progesterone (prog) and estrogen
(estro) phases of their estrous cycle relative to pregnant (pregn) and
adult male rats. Values are means ± SE.
* P < 0.05 vs. males.
** P < 0.05 vs. pregnant and
males. Inset: all bands from same
representative Northern blot.
View larger version (17K):
[in a new window]
Fig. 2.
-rENaC mRNA levels were higher in adult female rats during
progesterone (prog) and estrogen (estro) phases of their estrous cycle
than in pregnant rats. Prog and adult male levels were intermediate
between pregnancy and estro levels. Except prog vs. male, all
intergroup comparisons were significantly different
(P < 0.05).
Inset: all bands from same
representative Northern blot.
Cystic Fibrosis Transmembrane Conductance Regulator Measurements
Relative quantitation of cystic fibrosis transmembrane conductance regulator mRNA. To determine the effect of gender hormones on steady-state levels of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, quantitative polymerase chain reaction (PCR) was performed on reverse transcripts using the methods of Gilliland et al. (11), as modified by Galea and Feinstein (10), and of Wang et al. (31). The oligodeoxynucleotide primers used were derived from the following rat sequences: antisense primer, 5'-GTAAGGTCTCAGTTAGAATTGAA-3'; sense primer, 5'-CATCTTTGGTGTTTCCTATGATG-3'; and were chosen from highly conserved regions of different exons to avoid the amplification of genomic DNA. These primers were designed to amplify a 482-bp fragment.
An internal standard for CFTR was prepared by removing a region of 270 bp from the 482-bp PCR product as described (28). The resulting 211-bp construct was inserted in pBluescript II KS. A cRNA corresponding to the sense transcript was produced from the Hind III-linearized plasmid using the Riboprobe Gemini System II according to the manufacturer's recommendations. An aliquot of RNA (3 µg) was mixed with 200 ng random hexamers and 1.0 pg control cRNA in a final volume of 10.7 µl, heated to 90°C for 5 min, and quickly chilled on ice. Reverse transcription was performed at 37°C for 1 h in 20 µl of a buffer containing (in mM) 25 tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3, 75 KCl, 3 MgCl2, 10 dithiothreitol, 0.4 each of dATP, dCTP, dGTP, and dTTP, with 1 U/µl ribonuclease inhibitor and 200 U Moloney murine leukemia virus reverse transcriptase. The reaction was stopped on ice for immediate use or stored at
20°C until used. For the PCR amplification, a 3-µl aliquot
of the cDNA reaction products and 25 pmol of each primer were mixed
with buffer to a final volume of 50 µl and final concentration of (in
mM) 20 Tris · HCl, pH 8.4, 50 KCl, 1.5 MgCl2, and 0.2 each of dATP dCTP, dGTP, and dTTP, with 10 µCi
[-32P]dCTP (3,000 Ci/mmol). This mixture was overlaid with 30 µl mineral oil, placed in
a microprocessor-controlled thermocycler (Perkin Elmer or MJ Research),
and heat denaturated at 97°C for 7 min. Taq DNA polymerase (1.35 U/5 µl) was added at the start of a 5-min annealing step at 55°C,
followed by a 1-min extension at 72°C. The reaction was then
submitted to a number of cycles (75 s at 94°C, 1 min at 55°C,
and 1 min at 72°C).
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Isolation of Lung Epithelial Cells
Enriched populations of alveolar type II cells were isolated from the lungs of immature female rats (~125 g) using the methods of Borok et al. (3). Briefly, cells were released from rat lungs by elastase disaggregation. The elastase was then neutralized without serum by 2 mM EDTA, 1% bovine serum albumin (BSA), and 0.1% soy bean trypsin inhibitor in a buffered saline solution containing (in mM) 136 NaCl, 2.2 Na2HPO4, 5.3 KCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 5.6 glucose. The cells were further purified by panning on bacteriological plates coated with immunoglobulin G, then resuspended in minimal completely defined serum-free medium (MDSF). MDSF consisted of Dulbecco's modified Eagle's medium-Ham's F-12 with added BSA (1.25 mg/ml), nonessential amino acids (0.1 mM), glutamine (2 mM), sodium penicillin G (100 U/ml), streptomycin (100 µg/ml), and HEPES sufficient to achieve a final concentration of 25 mM. Cells were then seeded at 106 cells/cm2 on permeant polycarbonate filters (for RNA extraction: Transwell, 24-mm diameter, 0.4-µm pore size; for Ussing chamber studies: Snapwell, 12-mm diameter, 0.4-µm pore size; Costar, Cambridge, MA) in MDSF plus hormones dissolved in dimethyl sulfoxide (DMSO) or in MDSF plus DMSO without hormones (control). Unattached cells were removed on the third day after plating with the provision of new medium, which was again provided on alternate days thereafter. After 5 days, cells were used in Ussing chamber studies or had RNA extracted for Northern blot assessments of relative levels of-rENaC mRNA.
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Ussing Chambers
ENaC functional activity was assessed as the amiloride-sensitive Isc across monolayers of cells in Ussing chambers. The bioelectric properties of these postnatal lung epithelial cells were continuously monitored under open-circuit conditions (23) with a voltage-current clamp amplifier (model VCC600, Physiologic Instrument, San Diego, CA). We maintain open-circuit conditions because Joris and Quinton (15) have presented evidence that sustained short-circuiting leads to a significant underestimation of amiloride-sensitive Na transport. The transmonolayer potential difference was monitored continously, and every 10 s, a bidirectional 1-µA pulse of 0.5-s duration was passed to allow calculation of transepithelial resistance. The Isc was determined periodically by transiently clamping the voltage at zero. The monolayers were bathed by Hanks' balanced salt solution (equilibrated with a gas mixture of 5% CO2-21% O2-74% N2) containing hormones dissolved in DMSO or DMSO alone (final DMSO dilution 1:1,000). After a stable baseline was established, amiloride was added apically (100 µM final concentration).Statistical Analysis
To determine the statistical significance of multiple comparisons, where P > 0.05 for normality of distribution and equality of variance between groups, we used a one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple comparison test. Where P < 0.05 for normality of distribution and equality of variance, we used the nonparametric one-way ANOVA on-ranks test. The tests were performed using SigmaStat for Windows statistical software, version 1.0. A probability (P) value of <0.05 was considered significant.| |
RESULTS |
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Abstract
Introduction
Methods
Results
Discussion
References
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All results are from at least four independent experiments.
Differential Expression of Na Channel Subunits in Male and Female Adult Rats
Northern analyses of whole lung RNA demonstrated higher levels of-rENaC mRNA in females relative to males (Fig. 1).
Nonpregnant females had the highest levels of mRNA encoding -rENaC
(Fig. 1). Also for females, levels of -rENaC mRNA varied depending on whether or not the rat was pregnant and were higher during the
estrogen phase than during the progesterone phase of the estrous cycle
(Fig. 2). -rENaC mRNA levels were not altered by
gender, pregnancy, or the phase of the estrous cycle (data not shown).
Combined, But Not Separate, Administration of Progesterone and
17-Estradiol Increases ENaC mRNA Levels
-rENaC mRNA levels were increased 8 h after a
single dose of combined progesterone and 17-estradiol (Fig.
3). -rENaC mRNA levels were not increased 24 or 48 h
later (data not shown). In contrast, -rENaC mRNA levels were
increased 48 h after combined treatment, with
progesterone-to-17-estradiol ratios of 500:1 or 750:1 (wt/wt) (Fig.
4). Neither progesterone nor 17-estradiol alone
increased the expression of any of the ENaC subunits in either
ovariectomized or immature female rats. However, a decrease in
-rENaC mRNA levels was observed after treatment with either
progesterone or 17-estradiol alone of both ovariectomized (8 h) and
immature (48 h) rats (Fig. 5).
Differential Expression of CFTR in Male and Female Adult Rats
CFTR mRNA levels, as determined by quantitative PCR of reverse transcripts, were lower in adult male rats than in adult females regardless of the stage of the estrous cycle (see changes in Fig. 6). Neither progesterone nor 17-estradiol alone altered the CFTR expression. With 750:1 (wt/wt)
progesterone-to-17-estradiol ratio for 8 h, there was a
statistically significant increase in CFTR mRNA (Fig.
7).
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The observations are summarized in Table 1.
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Combined Administration of Progesterone and
17-Estradiol Increases ENaC Function
-estradiol, a
dose-dependent augmentation in amiloride-sensitive Isc was observed.
Low dose (progesterone 64 nM, 17-estradiol 85 pM; 750:1, wt/wt)
treatment did not change the amiloride-sensitive Isc (2.51 ± 0.18 vs. control 2.39 ± 0.16 µA/cm2;
n = 10, P > 0.05), but after high dose
(progesterone, 2.8 µM; 17-estradiol, 3.7 nM; 750:1, wt/wt)
treatment, the amiloride-sensitive Isc was elevated,
associated with an increase in cellular levels of -rENaC mRNA (Fig.
8).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We report that the lungs of sexually mature female rats, relative to
males, have higher levels of mRNAs encoding -rENaC and CFTR. These
differences likely arise from female gender hormones since combined,
but not separate, administration of progesterone and estradiol augments
levels of mRNA encoding rENaC subunits or CFTR in sexually immature
female rats. The rENaC subunits displayed differential responses to
combined progesterone and estradiol treatment. Whereas -rENaC mRNA
levels increased 8 h after treatment, -rENaC did not respond at the
times evaluated, and -subunit mRNA levels were only increased 24 h
after treatment. These findings are in keeping with previous reports of
differential regulation of rENaC subunits by other steroid hormones. In
the renal inner medullary collecting duct, either glucocorticoids or
mineralocorticoids increase levels of the mRNA encoding the -, but
not the - or -, subunits of rENaC (30). Moreover, the adult rat
colonic epithelium also demonstrates differential regulation of ENaC
subunits by mineralocorticoid (16). We have recently demonstrated
differential regulation of the - vs. - and -subunits of rENaC
both during normal development and in response to glucocorticoids (20). Endogenous levels of -, -, and -ENaC vary between epithelia of
different organs (9, 18). Although the functional consequence of this
is unknown, oocyte expression assays using cRNA coding for -, -,
and -ENaC demonstrate that (in oocytes) -ENaC appears to be
essential for functional channel activity, and the - and -ENaC
greatly enhance amiloride-sensitive Na channel activity (4).
We also report that combined treatment with progesterone and
17-estradiol enhanced rENaC function, as assessed by the
amiloride-sensitive Isc. A single
intraperitoneal injection of progesterone and 17-estradiol did not
alter the amiloride-sensitive
Isc after 5 days
of primary culture in hormone- and serum-free medium. However, in cells
from uninjected animals, a dose-dependent augmentation in
amiloride-sensitive Isc was observed
after 5 days in medium containing progesterone and 17-estradiol.
Taken together, these results suggest that the functional half-life of
ENaC in these cells may be significantly shorter than 5 days.
Although no previous reports are available regarding the effect of gender hormones on the expression of ENaC, steroid hormones can have marked effects on Na transport. Perhaps the best studied is aldosterone, which enhances Na transport in the kidney. However, these results cannot necessarily be extrapolated to the lung, since previous work has demonstrated that aldosterone has no direct effect on distal lung epithelial Na transport (21). Indeed, although it has been shown that aldosterone increases the levels of rENaC mRNA and transmembrane Na transport in fetal distal lung epithelia, it does so through the stimulation of glucocorticoid receptors (5). These latter observations are compatible with work demonstrating that endogenous and exogenous glucocorticoids (5) regulate the expression of the lung epithelial Na channel during fetal development.
Steroid hormones also modulate CFTR, a Cl channel expressed in fetal
distal lung epithelia (17, 27). In our present study, combined
treatment with progesterone and estradiol (750:1, wt/wt) increased CFTR
mRNA levels in lungs of immature female rats. Furthermore, neither
17-estradiol nor progesterone alone altered CFTR mRNA levels,
suggesting complex interactions between the effects of progesterone and
17-estradiol. Compatible with our present observations, Zeitlin et
al. (32) showed that neither estradiol nor progesterone alone affected
the Isc of
cultured rabbit tracheal epithelial cells. Others have demonstrated
that estrogens administered separately can increase CFTR mRNA in a
uterine epithelial cell line (25). However, the effects of estrogen may
be organ or developmentally dependent, since estradiol alone diminishes
CFTR functional activity in fetal rat lung (27).
ENaC represents the rate-limiting step in epithelial Na transport. Its
activity therefore plays a critical role in the kidney's regulation of
total body Na and fluid volume, the absorption of fluid from the
gastrointestinal tract, and the reabsorption of fluid from the lungs'
airways and alveolar spaces (19). The importance of Na transport in
lung disease is emphasized by the recent observation that a transgenic
mouse with an inactivated -mENaC gene dies at birth from retained
fluid within the lungs (13). Perturbation of normal transepithelial
active Na transport is associated with several different diseases. An
excessive activity of the ENaC plays a role in the pathogenesis of
cystic fibrosis airway disease (2, 14) and an inherited form of
systemic hypertension (Liddle's disease) (26). In contrast, inadequate amiloride-sensitive Na channel expression and Na transport is observed
in transient tachypnea of the newborn (12) and may contribute to the
inability of some individuals to recover from pulmonary edema (for
review, see Ref. 19).
Our study has potential implications for the understanding of human disease. In view of an increase in ENaC expression in their lungs, females may have an advantage over males in their ability to clear fetal lung liquid at birth or when they develop pulmonary edema. It has been noted that prematurely born human girls have a lower incidence of the neonatal respiratory distress syndrome (1) than do boys. However, if a similar phenomenon also occurs in the airways, it would be detrimental in cystic fibrosis. Interestingly, female cystic fibrosis patients have a worse prognosis than do male cystic fibrosis patients (7).
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ACKNOWLEDGEMENTS |
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We thank B. Rafii and V. Hannam for excellent technical assistance, Dr. G. Kent for assistance in our assessment of the stage of the estrous cycle, and Z. Borok for helpful advice concerning primary culture of postnatal rat lung alveolar epithelial cells.
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FOOTNOTES |
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This work was supported by Medical Research Council of Canada Group Grant in Lung Development Project 8 (to H. O'Brodovich), a block term grant from the Ontario Thoracic Society, and a grant-in-aid from the Canadian Cystic Fibrosis Foundation (to N. Sweezey).
Address for reprint requests: N. Sweezey, Respiratory Research, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8.
Received 2 August 1996; accepted in final form 20 October 1997.
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Abstract
Introduction
Methods
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Discussion
References
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