Female gender hormones regulate mRNA levels and function of the rat lung epithelial Na channel

N. Sweezey1, S. Tchepichev1,2, S. Gagnon1, K. Fertuck1, and H. O'Brodovich1,2

1 Respiratory Research, The Hospital for Sick Children, and 2 Medical Research Council of Canada Group in Lung Development, Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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 17beta -estradiol increased mRNA levels encoding alpha -rENaC, gamma -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 17beta -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.

rat epithelial sodium channel; progesterone; 17beta -estradiol; cystic fibrosis; pulmonary edema

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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
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Abstract
Introduction
Methods
Results
Discussion
References

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 17beta -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 17beta -estradiol. In this case, immature animals received a subcutaneous injection of progesterone combined with 17beta -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).

RNA Isolation and Northern Analysis

Lungs were separated from the heart, great vessels, and large airways and flash frozen in liquid nitrogen for subsequent whole lung RNA isolation (6). Autoclaved diethylpyrocarbonate-treated water was used to dissolve the RNA, which was quantified using spectrophotometric absorbance at 260 nm (A260 = 40 µg RNA). Total RNA (15 µg) was then size fractionated on a 1% agarose gel containing formaldehyde and ethidium bromide using a 1× 3-(N-morpholino)propanesulfonic acid buffer. The RNA was passively transferred to Hybond-N nylon membranes (Amersham, Buckinghamshire, UK), ultraviolet cross-linked, and then baked at 80°C for 2 h. A solution of 50% deionized formamide, 5× Denhardt's solution, 1% sodium dodecyl sulfate (SDS), 5× standard sodium citrate solution (SSC), and 0.2 mg/ml salmon sperm DNA was used for prehybridization for a minimum of 6 h, followed by hybridization in a solution of 50% deionized formamide, 2× Denhardt's solution, 1% SDS, 5× SSC, and 0.2 mg/ml salmon sperm DNA. Hybridization occurred overnight at 42°C with one or more randomly primed (Multiprime DNA kit, Amersham) [32P]CTP-labeled full-length cDNA probes coding for the alpha -, beta -, and gamma -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 alpha -tubulin cDNA probes were used to assess the integrity of the mRNA.


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Fig. 1.   alpha -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.


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Fig. 2.   beta -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 [alpha -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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Fig. 3.   alpha -rENaC mRNA levels increased 8 h after combined progesterone and 17beta -estradiol treatment. * P < 0.05 vs. control. Inset: all bands from same representative Northern blot.


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Fig. 4.   gamma -rENaC mRNA levels increased 24 h after combined progesterone and 17beta -estradiol treatment. * P < 0.05 vs. control. Inset: all bands from same representative Northern blot.

Fifteen percent of the reverse transcription reaction was amplified for 18, 22, 25, 28, and 30 PCR cycles in the presence of radioactive nucleotides. One-fifth of the amplified material was separated on 6% polyacrylamide gel electrophoresis and autoradiographed. Quantitation was performed with a phosphoimage analyzer (Fuji, Stamford, CT) according to the manufacturer's protocol, normalizing the CFTR results for the intensity of the internal control band. The range of exponential amplification was 22-28 cycles for both target and control. Twenty-five cycles were used for the quantitative assay. To ensure that the signals did not come from amplification of contaminating (nontarget) DNA, each RT-PCR was repeated on an identical RNA sample without the reverse transcriptase in the first step of the protocol.

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 alpha -rENaC mRNA.


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Fig. 5.   beta -rENaC mRNA levels were lower after treatment with either progesterone or 17beta -estradiol alone, in ovariectomized rats (8 h) (A) and in immature rats (48 h) (B). * P < 0.05 vs. control. Inset: all bands from same representative Northern blot. Prog, progesterone; Estr, 17beta -estradiol.

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

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 alpha -rENaC mRNA in females relative to males (Fig. 1). Nonpregnant females had the highest levels of mRNA encoding alpha -rENaC (Fig. 1). Also for females, levels of beta -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). gamma -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 17beta -Estradiol Increases ENaC mRNA Levels

Whole lung alpha -rENaC mRNA levels were increased 8 h after a single dose of combined progesterone and 17beta -estradiol (Fig. 3). alpha -rENaC mRNA levels were not increased 24 or 48 h later (data not shown). In contrast, gamma -rENaC mRNA levels were increased 48 h after combined treatment, with progesterone-to-17beta -estradiol ratios of 500:1 or 750:1 (wt/wt) (Fig. 4). Neither progesterone nor 17beta -estradiol alone increased the expression of any of the ENaC subunits in either ovariectomized or immature female rats. However, a decrease in beta -rENaC mRNA levels was observed after treatment with either progesterone or 17beta -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 17beta -estradiol alone altered the CFTR expression. With 750:1 (wt/wt) progesterone-to-17beta -estradiol ratio for 8 h, there was a statistically significant increase in CFTR mRNA (Fig. 7).


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Fig. 6.   Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels were lower in adult males than in progesterone (prog) or estrogen (estro) phases of adult female estrous cycle. * P < 0.05 vs. males. Inset: representative blot of products of polymerase chain reaction amplification of reverse transcripts (RT-PCR) of CFTR mRNA and an internal deletion construct (internal control).


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Fig. 7.   CFTR mRNA levels were increased 8 h after injection with a progesterone-to-estradiol ratio of 750:1 (wt/wt) (P < 0.05 vs. control). Inset: representative RT-PCR analysis.

The observations are summarized in Table 1.

                              
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Table 1.   Effects of estrous cycle and treatment on mRNA

Combined Administration of Progesterone and 17beta -Estradiol Increases ENaC Function

The amiloride-sensitive Isc of cells isolated from treated animals was not significantly different from controls after 4 [2.2 ± 0.4 vs. control 2.02 ± 0.46 (SE) µA/cm2; n = 6] or 5 days in culture [3.18 ± 0.17 vs. control 2.77 ± 0.28 µA/cm2; n = 6]. However, when cells isolated from uninjected animals were incubated for 5 days in a serum-free medium containing progesterone and 17beta -estradiol, a dose-dependent augmentation in amiloride-sensitive Isc was observed. Low dose (progesterone 64 nM, 17beta -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; 17beta -estradiol, 3.7 nM; 750:1, wt/wt) treatment, the amiloride-sensitive Isc was elevated, associated with an increase in cellular levels of alpha -rENaC mRNA (Fig. 8).


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Fig. 8.   Studies of alveolar epithelial cell monolayers. A: incubation of cells in medium containing progesterone (P, 2.8 µM) and 17beta -estradiol (E, 3.7 nM; P/E, 750:1) increased amiloride-sensitive transepithelial electrical potential (Vte). Representative Ussing chamber recording over time is shown. Vertical deflections correspond to brief bidirectional 1-µA pulses passed to allow calculation of transepithelial resistance. P:E, hormone treated. B: amiloride-sensitive short-circuit current (amiloride-sensitive Isc). Values are means ± SE; n = 13 independent experiments. * P = 0.002 vs. control. C: ENaC mRNA levels. Progesterone and 17beta -estradiol (as above) increased cellular levels of ENaC mRNA, whereas 18S RNA levels were unchanged. All bands are from same representative Northern blot.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
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We report that the lungs of sexually mature female rats, relative to males, have higher levels of mRNAs encoding alpha -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 alpha -rENaC mRNA levels increased 8 h after treatment, beta -rENaC did not respond at the times evaluated, and gamma -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 alpha -, but not the beta - or gamma -, 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 alpha - vs. beta - and gamma -subunits of rENaC both during normal development and in response to glucocorticoids (20). Endogenous levels of alpha -, beta -, and gamma -ENaC vary between epithelia of different organs (9, 18). Although the functional consequence of this is unknown, oocyte expression assays using cRNA coding for alpha -, beta -, and gamma -ENaC demonstrate that (in oocytes) alpha -ENaC appears to be essential for functional channel activity, and the beta - and gamma -ENaC greatly enhance amiloride-sensitive Na channel activity (4).

We also report that combined treatment with progesterone and 17beta -estradiol enhanced rENaC function, as assessed by the amiloride-sensitive Isc. A single intraperitoneal injection of progesterone and 17beta -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 17beta -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 17beta -estradiol nor progesterone alone altered CFTR mRNA levels, suggesting complex interactions between the effects of progesterone and 17beta -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 alpha -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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Avery, M. E., and J. Mead. Surface properties in relation to atelectasis and hyaline membrane disease. Am. J. Dis. Child. 97: 517-523, 1959.

2.   Baker, D. E. J. Reproduction and breeding. In: The Laboratory Rat, edited by H. J. Baker, J. R. Lindsey, and S. H. Wesbroth. London: Academic, 1979, p. 154-159.

3.   Borok, Z., A. Hami, S. I. Danto, R. L. Lubman, K. J. Kim, and E. D. Crandall. Effects of EGF on alveolar epithelial junctional permeability and active sodium transport. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L559-L565, 1996[Abstract/Free Full Text].

4.   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[Medline].

5.   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].

6.   Chomcyznski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

7.   Corey, M., and V. Farewell. Determinants of mortality from cystic fibrosis in Canada, 1970-1989. Am. J. Epidemiol. 143: 1007-1017, 1996[Abstract].

8.   Cott, G. R., and A. K. Rao. Hydrocortisone promotes the maturation of Na-dependent ion transport across the fetal pulmonary epithelium. Am. J. Respir. Cell Mol. Biol. 9: 166-171, 1993[Medline].

9.   Duc, C., N. Farman, C. M. Canessa, J.-P. Bonvalet, and B. C. Rossier. Cell-specific expression of epithelial sodium channel alpha , beta  and gamma  subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J. Cell Biol. 127: 1907-1921, 1994[Abstract].

10.   Galea, E., and D. L. Feinstein. Rapid synthesis of DNA deletion constructs for mRNA quantitation: analysis of astrocyte mRNA. PCR Methods Applications 2: 66-69, 1992[Medline].

11.   Gilliland, G., S. Perlin, K. Blanchard, and H. F. Bunn. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87: 2725-2729, 1990[Abstract].

12.   Gowen, C. W., E. E. Lawson, J. Gingras, R. Boucher, J. T. Gatzy, and M. R. Knowles. Electrical potential difference and ion transport across nasal epithelium of term neonates: correlation with mode of delivery, transient tachypnea of the newborn, and respiratory rate. J. Pediatr. 113: 121-127, 1988[Medline].

13.   Hummler, E., P. Barker, and J. Gatzy. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nature Genet. 12: 325-328, 1996[Medline].

14.   Jiang, C., W. E. Finkbeiner, J. H. Widdicombe, P. B. McCray, Jr., and S. S. Miller. Altered fluid transport across airway epithelium in cystic fibrosis. Science 262: 424-427, 1993[Medline].

15.   Joris, L., and P. M. Quinton. Components of electrogenic transport in unstimulated equine tracheal epithelium. Am. J. Physiol. 260 (Lung Cell Mol. Physiol. 4): L510-L515, 1991[Abstract/Free Full Text].

16.   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[Abstract/Free Full Text].

17.   MacLeod, R. J., J. R. Hamilton, H. Kopelman, and N. Sweezey. Developmental differences of cystic fibrosis transmembrane conductance regulator functional expression in isolated rat fetal distal airway cells. Pediatr. Res. 35: 45-49, 1994[Abstract].

18.   McDonald, F. J., P. M. Snyder, P. B. McCray, Jr., and M. J. Welsh. Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel. Am. J. Physiol. 266 (Lung Cell Mol. Physiol. 10): L728-L734, 1994[Abstract/Free Full Text].

19.   O'Brodovich, H. M. The role of active Na+ transport by lung epithelium in the clearance of airspace fluid. New Horizons 3: 240-247, 1995[Medline].

20.   O'Brodovich, H. M., C. M. 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[Abstract/Free Full Text].

21.   O'Brodovich, H. M., B. Rafii, and P. Perlon. Arginine vasopressin and atrial natriuretic peptide do not alter ion transport by cultured fetal distal lung epithelium. Pediatr. Res. 31: 318-322, 1992[Abstract].

22.   Olver, R. E., C. A. Ramsden, L. B. Strang, and D. V. Walters. The role of amiloride-blockade sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J. Physiol. (Lond.) 376: 321-340, 1986[Abstract].

23.   Pitkanen, O., A. K. Tanswell, and H. O'Brodovich. Fetal lung cell-derived matrix alters distal lung epithelial ion transport. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L762-L771, 1995[Abstract/Free Full Text].

24.   Rao, A. K., and G. R. Cott. Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture. Am. J. Physiol. 261 (Lung Cell Mol. Physiol. 5): L178-L187, 1991[Abstract/Free Full Text].

25.   Rochwerger, L., S. Dho, L. Parker, J. K. Foskett, and M. Buchwald. Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line. J. Cell Sci. 107: 2439-2448, 1994[Abstract/Free Full Text].

26.   Shimkets, R. A., D. G. Warnock, C. M. Bositis, C. Nelson-Williams, J. H. Hansson, M. Schambelan, J. R. J. Gill, S. Ulick, R. V. Milora, J. W. Findling, C. M. Canessa, B. C. Rossier, and R. P. Lifton. Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial Na channel. Cell 79: 404-414, 1994.

27.   Sweezey, N., F. Ghibu, and S. Gagnon. Sex hormones regulate cystic fibrosis transmembrane conductance regulator in developing fetal rat lung epithelial cells. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L844-L851, 1997[Abstract/Free Full Text].

28.   Sweezey, N. B., C. Gauthier, S. Gagnon, E. Ferretti, and H. Kopelman. Progesterone and estradiol inhibit CFTR-mediated ion transport by pancreatic epithelial cells. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G747-G754, 1996[Abstract/Free Full Text].

29.   Tchepichev, S., J. Ueda, C. M. Canessa, B. C. Rossier, and H. O'Brodovich. Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am. J. Physiol. 269 (Cell Physiol. 38): C805-C812, 1995[Abstract].

30.   Volk, K. A., R. D. Sigmund, P. M. Snyder, F. J. McDonald, M. J. Welsh, and J. B. Stokes. rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct. J. Clin. Invest. 96: 2748-2757, 1995[Medline].

31.   Wang, A. M., M. V. Doyle, and D. F. Mark. Quantification of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86: 9717-9721, 1989[Abstract].

32.   Zeitlin, P. L., M. Wagner, D. Markakis, G. M. Loughlin, and W. B. Guggino. Steroid hormones: modulators of Na+ absorption and Cl- secretion in cultured tracheal epithelia. Proc. Natl. Acad. Sci. USA 86: 2502-2505, 1989[Abstract].


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