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. 2000 Jan 1;522(Pt 1):147–157. doi: 10.1111/j.1469-7793.2000.t01-2-00147.xm

The influence of mode of delivery, hormonal status and postnatal O2 environment on epithelial sodium channel (ENaC) expression in perinatal guinea-pig lung

Deborah L Baines *, Hans G Folkesson *, Andreas Norlin *, Colin D Bingle †, Hai Tao Yuan ‡, Richard E Olver *
PMCID: PMC2269744  PMID: 10618159

Abstract

  1. We have studied factors that potentially modulate the expression of mRNA coding for subunits of the amiloride-sensitive sodium channel, αENaC and βENaC, in lungs of vaginally and Caesarean (CS)-delivered late gestation fetal guinea-pigs.

  2. Expression of αENaC and βENaC mRNAs was developmentally regulated in the late gestation fetus, reaching peak levels at term (68 days post conception, PC) and postnatally, respectively. In animals delivered by CS at 65 days PC and term, αENaC mRNA expression was significantly increased by day 1 post partum, reaching levels greater than those normally achieved in vaginally delivered animals at term. In contrast, βENaC mRNA levels remained significantly lower postnatally in animals delivered by CS at 65 days PC compared with those in vaginally and CS-delivered animals at term.

  3. Plasma cortisol and total triiodothyronine (T3) levels increased towards term, were higher 1 day after vaginal delivery but declined towards pre-term levels by day 3. Cortisol levels also increased rapidly in the CS-delivered animals, reaching levels similar to those in vaginally delivered animals at day 1. Plasma T3 levels at days 1 and 3 were significantly lower in animals delivered by CS at 65 days PC.

  4. The increase in αENaC mRNA paralleled the increase in plasma cortisol after delivery, but not T3, and inhibition of cortisol synthesis with 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) after CS delivery suppressed the increase in αENaC mRNA expression.

  5. Concomitant with the increase in αENaC mRNA expression after CS delivery at 65 days PC was an increase in the amiloride-blockable component of lung fluid clearance by day 3 postnatally.

  6. We conclude that in late gestation guinea-pigs delivered by CS there is a significant increase in lung αENaC expression postnatally, which is mediated, in part, by the postnatal rise in cortisol at delivery. This in turn leads to an increase in amiloride-sensitive lung fluid clearance, which is unrelated to labour.

Respiratory outcome in the newborn is linked with fetal maturity and mode of delivery (Cohen & Carson, 1985). Both these factors underlie the ability of the fetus to modulate the ion transport processes required to efficiently reduce lung fluid at birth (Olver, 1981). Removal of fluid from the lung is driven by amiloride-sensitive transepithelial Na+ absorption (Olver et al. 1986; O'Brodovich et al. 1990a), and this component of the lung fluid reabsorption process increases as term approaches. Compared with babies delivered vaginally, those delivered by Caesarean section (CS) without labour have a higher incidence of respiratory distress, altered nasal transepithelial potential differences (PDs) and a lower amiloride-sensitive component of nasal PD, suggesting that labour is critical for the activation of transepithelial Na+ transport (Gowen et al. 1988; Barker et al. 1997).

In both sheep and guinea-pig, the rise in fetally derived adrenaline during labour correlates with an increase in amiloride-sensitive fluid absorption (Olver et al. 1986; Finley et al. 1998) and, in late gestation fetal sheep, exogenous adrenaline can induce fluid absorption (Brown et al. 1983). However, the response to adrenaline is dependent on gestational age and is matured by the affect of corticosteroids and thyroid hormones (Barker et al. 1988, 1990a,b) during fetal development.

The cloned amiloride-sensitive sodium channel subunits α-, β- and γENaC are expressed in lung and alveolar type II pneumocytes (ATII) (McDonald et al. 1994; Voilley et al. 1994; Farman et al. 1997), and the absence of αENaC in transgenic mice has been shown to completely inhibit net fluid absorption at birth (Hummler et al. 1996). The mRNAs coding for α- β- and γENaC are developmentally regulated in the rat and their expression can be modulated by steroid hormones and environmental O2, the levels of which change at birth (Tchepichev et al. 1995; Pitkänen et al. 1996). However, few studies have attempted to investigate the way in which the perinatal changes in these factors are co-ordinated with ENaC mRNA expression in vivo. In addition, little is known about the expression of α- and βENaC mRNA in lung in relation to CS delivery of pre-term animals, a procedure which may impair the ability of these animals to clear lung fluid.

The guinea-pig is an important model for lung development because, unlike rat and mouse lung, alveolarisation begins prenatally, as in human lung (Collins et al. 1986; Pringle, 1986). Preparations of guinea-pig lung have been used in many functional studies of ion transport and fluid clearance (O'Brodovich et al. 1990a; Fyfe et al. 1994; Perks et al. 1997; Woods et al. 1997; Finley et al. 1998; Gambling et al. 1998; Norlin et al. 1998), yet little is known about the expression of ENaC mRNA in these preparations.

In this study we report how the interplay between gestational age, mode of delivery, perinatal hormonal and O2 environments modulate the postnatal expression of ENaC mRNA and lung fluid clearance.

METHODS

All experimental procedures were carried out according to the Home Office Animals (Scientific Procedures) Act, 1986. Fetuses were delivered vaginally at term (68 days) or by Caesarean section (CS) from a total of 40 anaesthetised (halothane, 3% in O2), time-mated guinea-pigs (Dunkin Hartley) at 35, 50, 60, 65 and 68 days post conception (PC). Fetuses delivered by CS at 65 or 68 days PC were resuscitated and fostered onto lactating dams. For oxygen expression studies, 65 day CS-delivered fetuses were revived in air and transferred within 10 min to hypoxia (15% O2) or hyperoxia (95% O2) for periods of 1 to 3 days. In each experiment, lung tissue was prepared from one fetus or neonate within 4 h of vaginal delivery (control) and from the remaining animals after defined time intervals (1, 3, 4, 7, 10 or 60 days postnatal). Litters contained between two and five fetuses and therefore it was not possible to undertake analyses for all time points in every experiment. Data are presented as means ±s.e.m., and values of n reflect the number of animals from independent litters. Dams subjected to CS were humanely killed after delivery. All other animals were humanely killed at the end of the experiment.

Guinea-pigs were treated with metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone; Sigma), a cortisol synthesis inhibitor, by an intraperitoneal injection (50 mg kg−1) after CS delivery and twice daily thereafter for up to 1 day post delivery. Control animals were similarly injected with saline vehicle control.

Lung tissue preparation

Lung tissue was isolated from fetal guinea-lungs by a method previously described by our laboratory (MacGregor et al. 1994; Monaghan et al. 1997), with appropriate modifications for neonatal samples. Fetuses delivered by Caesarean section (see above) and neonates were anaesthetised by intraperitoneal injection with a cocktail of 25% Hypnorm, 25% Hypnovel (Roche, Welwyn Garden City, UK), 50% H2O (8.0 ml kg−1), or an intramuscular injection of 0.65 mg kg−1 Hypnorm (Janssen Pharmaceutical, Oxford, UK). This was followed by an intraperitoneal injection of heparin (1000 units; Leo Laboratories, Buckingham, UK). Tracheal cannulas were inserted for the instillation of solutions into the lungs. Neonatal lungs were firstly instilled with 5–10 ml ice-cold lavage solution (140 mM NaCl, 5 mM KCl, 10 mM Hepes, 20 mM glucose, 2 mM EDTA, pH 7.4). The solution was left for 10 min, removed and followed by a further five washes of similar volume to remove alveolar macrophages. In both fetuses and neonates, the pulmonary circulation was cleared by transcardial perfusion with ice-cold, isotonic saline containing 5 units ml−1 heparin; lung tissue was then further processed for the preparation of RNA.

Preparation of RNA, Northern and slot blots

Lung tissue RNA was prepared by the acid-guanidinium thiocyanate-phenol-chloroform method of Chomczynski & Sacchi (Chomczynski & Sacchi, 1987) using Trizol reagent (Sigma). Total RNA was resuspended in RNase-free H2O and quantified by analysis of optical density (OD) at 260λ by UV spectrophotometry.

RNA samples (5 μg), in a final volume of 30 μl, were denatured in formamide (50%) and formaldehyde (2.2 M), in Mops buffer (0.2 M Mops pH 7.0, 50 mM NaOAc, 5 mM EDTA), for 15 min at 65°C and cooled rapidly on ice. Samples were loaded onto formaldehyde gels (1.1% agarose, 2.2 M formaldehyde in Mops buffer) and electrophoresed in Mops buffer at 1 V cm−1 for 3–4 h. Gels were washed for 20 min in RNase-free water, followed by a 20 min submersion in 0.1 n NaOH and then by 45 min in 20 × sodium chloride-sodium citrate buffer (SSC). RNA was capillary blotted onto Hybond nylon membrane (Amersham Int., Amersham, UK) with 20 × SSC overnight. Membranes were then removed, rinsed in 2 × SSC and the RNA cross-linked to the membrane with UV irradiation (UVP cross-linker).

After establishing that our probes were specific for α- and βENaC mRNAs and that the washing conditions produced discrete bands on Northern blots (Fig. 1), we performed slot blots to analyse larger numbers of RNA samples by image analysis. Slot blots were carried out by resuspending 5 μg denatured RNA in 2 × SSC to a final volume of 100 μl, and then applying the samples to Hybond using Millipore blotting apparatus. Samples were cross-linked as above.

Figure 1. Developmental expression of α- and βENaC mRNA in guinea-pig lung.

Figure 1

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A and B: top, representative Northern blots of total RNA extracted from distal lung tissue (5 μg) prepared from guinea-pigs of the gestational ages (days) shown. An ∼3.7 kb αENaC mRNA (A) and an ∼2.6 kb βENaC mRNA (B) were identified as indicated. The relative migration of 28s (upper line) and 18s (lower line) ribosomal RNA is shown to the right of the blots. Middle, corresponding blots of 18s ribosomal RNA. Bottom, αENaC (A) and βENaC (B) expression levels analysed by electronic autoradiography of slot blots, normalised to 28s (αENaC) and 18s (βENaC) for loading, are illustrated graphically for the gestational ages shown. These data are presented as percentage expression (means ±s.e.m.) relative to levels in vaginally delivered animals at term (day 68), to standardise data between blots. (Unless otherwise stated in the text, graphs represent analysis of n = 4 individuals.) αENaC mRNA reached peak levels at term (68 days PC) whereas βENaC mRNA reached peak levels postnatally. * Significantly different from term expression levels (P < 0.05).

Guinea-pig-specific αENaC and βENaC probes, 761 and 372 bp, respectively, were prepared by RT-PCR from guinea-pig lung RNA using standard protocols and primers designed from rat sequences. The sense and antisense primers, respectively, corresponded to bases 1059–1079 and 1802–1822 of rat αENaC (GenBank accession no. X70497) and 880–900 and 1232–1252 of rat βENaC (GenBank accession no. X77932). An 18s oligonucleotide probe, kindly provided by Dr H. McArdle, Rowett Institute, Aberdeen, UK, was used to correct for any loading variance between samples. A 28s ribosomal RNA probe was used to correct for loading variance in samples used to compile the graph of Fig. 1 and samples obtained from animals exposed to 95% O2.

Probes were labelled with [α-32P]dCTP using a Multiprime Labelling Kit (Amersham Int.). The 18s deoxyoligonucleotide terminal phosphate was replaced with [γ-32P]ATP using a polynucleotide kinase (Promega, UK) catalysed reaction. Unincorporated nucleotides were removed by applying the labelled mixes to a microspin Probe-Quant column (Pharmacia, UK). High specific activity probes (108 c.p.m. μg−1 DNA) were used for hybridisation protocols. Membranes were pre-hybridised with rapid hybridisation buffer (Amersham Int.) for 15–30 min at 65°C. Hybridisation was carried out in the same buffer for 3 h at 65°C with denatured double-stranded αENaC, βENaC or 18s oligonucleotide probes using 5 × 105 c.p.m. ml−1 hybridisation buffer. Blots were washed sequentially in 2 × SSC + 0.1% SDS to 0.1 × SSC + 0.1% SDS for 20 min at 65°C. Membranes were wrapped in plastic film and analysed by electronic autoradiography using a Hewlett Packard InstantImager for 1–12 h or exposed to autoradiographic film at −70°C for 1–12 days. Quantification of RNA extracted from whole lung and applied slot blots was carried out using electronic autoradiography. Four duplicates were made of each slot blot. Northern blots and slot blots were firstly hybridised with either αENaC or βENaC probes and analysed, then stripped in boiling 0.1% SDS and allowed to cool to room temperature. Complete removal of labelled probes from the blots was ascertained by use of electronic autoradiography prior to reprobing for 18s. No blot was probed more than three times. Northern blots were analysed by electronic autoradiography or densitometry of scanned film positive autoradiographs using public domain NIH Image Software.

Samples were corrected for loading by normalising to 18s ribosomal RNA prior to statistical analysis. We found that analysis of our Northern blots and slot blots gave similar results. More samples can be analysed at one time using slot blots thus reducing the variability incumbent in comparing Northern blots. Controls (i.e. day 65/term samples) were run on each blot to allow statistical analysis of controls and test group samples. However, we found that variability in absolute values was still observed between blots, due to differences in hybridisation conditions and specific activity of the probes. Thus, for clarity of the results and figures we found it beneficial to express our data as a percentage relative to normal (vaginal) delivery at term samples.

Plasma hormone assays

One to two millilitres of blood were collected from the aorta of anaesthetised animals (see above) into syringes. Samples were then transferred to 1.5 ml Eppendorf tubes and centrifuged at 7000 r.p.m. for 5 min at 4°C to pellet the blood cells. The plasma was removed and stored in 100 μl aliquots at −70°C until required. Fifty microlitres of plasma (diluted 1:10 for the cortisol assay) were analysed using Milenia (DPC, Los Angeles, USA) cortisol and total triiodothyronine (T3) endpoint enzyme immunoassay kits, using the manufacturer's protocol.

Lung fluid clearance

Guinea-pigs were delivered by CS at 65 days PC (weighing 91 ± 6 g) as described above and taken within 15 min or within 4 h of vaginal delivery at term (weighing 110 ± 2 g) and overdosed with intraperitoneal pentobarbital sodium (80 mg (kg body weight)−1; Apoteksbolaget, Umeå, Sweden). A separate group of guinea-pigs was delivered by CS at 65 days PC, left with foster mothers for 3 days and then overdosed with intraperitoneal pentobarbital sodium and studied for lung fluid clearance. An endotracheal tube (PE-90; Clay Adams, Becton-Dickinson, Parsippany, NJ, USA) was inserted into the trachea through a tracheostomy within 2 min of death. The guinea-pigs were maintained in a decubitus position throughout the experiment and covered with a heating pad to maintain normal body temperature. Continuous positive airway pressure (CPAP) with 5–7 cmH2O was delivered during the 1 h experiment with 100% O2. The airway pressure was measured as described previously (Finley et al. 1998). Fluid instillation was carried out by briefly disconnecting the animals from the CPAP circuit and instilling 10 ml (kg body weight)−1 of a 5% albumin solution (50 mg ml−1 bovine serum albumin (Sigma) in an aqueous solution of 0.9% NaCl), over 10–15 s. The animals were then immediately reconnected to the CPAP circuit for the 1 h experiment. At the end of the experiment, the lungs and heart were removed en bloc. A sample of remaining lung fluid was aspirated by gently advancing a sampling catheter into a wedged position in a distal airway in the lungs. Protein concentrations in the instillates and airspace samples were measured as described previously (Finley et al. 1998). To determine the fractional inhibition of lung fluid clearance by amiloride, an excess of amiloride (10−3 M; Sigma) was added to the 5% albumin instillate solution to ensure an appropriate concentration at the alveolar epithelium, since a fraction is protein bound and another fraction rapidly leaves the airspace due to its low molecular weight.

Clearance of fluid from the distal airspaces of the lungs was measured by the increase in protein concentration of the instilled solution over 1 h. Data are presented as a ratio between final and instilled protein concentration. As previously discussed (Finley et al. 1998), the increase in protein concentration due to removal of water from the airspaces is a direct reflection of lung fluid clearance but does not, however, imply that all fluid is reabsorbed at the alveolar level. Some fluid may be reabsorbed by the distal airways.

As the lung is naturally fluid-filled in utero, we have taken into account that a certain fraction of this fluid would be present in the lungs at the start of the experiment. This fluid is virtually free of protein and will dilute the protein concentration in instillates and thereby influence the calculations of lung fluid clearance. To control for this, we used separate groups of animals at each developmental stage and treatment, into which 1.5-2 ml of the 5% albumin solution was instilled, carefully mixed, and the fluid immediately withdrawn. This fluid was aspirated and reintroduced four times before the final sample was taken. The whole procedure took approximately 1–2 min, and during this time it is unlikely that significant amounts of protein left or entered the airspaces or that significant volumes of fluid were reabsorbed from, or secreted into, the airspaces. As protein would not cross the alveolar epithelial barrier during this short time, any change in protein concentration would represent a dilution by pre-existing fluid. Knowing this, we calculated the pre-existing fluid volume using the relationship:

graphic file with name tjp0522-0147-mu1.jpg

where VL is the pre-existing lung fluid volume (ml), VI is the instilled fluid volume (ml), CI is the protein concentration in instilled fluid (mg ml−1), and CL is the protein concentration in aspirated fluid (mg ml−1). We calculated the pre-existing lung fluid in term vaginally delivered animals to be 1.55 ± 0.86 ml kg−1 (n = 4) and that in day 65 CS-delivered animals to be 4.9 ± 1.64 ml kg−1 (n = 5). These values were significantly different (P < 0.05). The pre-existing fluid volume was used to correct the instilled protein concentrations in our test animals.

All data are presented as means ±s.e.m. The data were analysed with one-way analysis of variance (ANOVA) with Tukey's test as post hoc. Two groups were compared with Student's unpaired t test when appropriate. Differences were considered significant when a value of P < 0.05 was reached.

RESULTS

Developmental expression of α- and βENaC mRNA

RT-PCR of guinea-pig lung using αENaC- and βENaC-specific primers amplified products of 761 and 372 bp, which demonstrated 85 and 87% sequence homology to rat αENaC and βENaC, respectively. The αENaC and βENaC probes recognised mRNAs of approximately 3.7 and 2.6 kb in guinea-pig distal lung, respectively (Fig. 1). This is consistent with observations from several other laboratories where the reported size of αENaC ranges from 3.2 to 3.9 kb and βENaC from 2.2 to 2.4 kb (Canessa et al. 1993, 1994; McDonald et al. 1994; Voilley et al. 1994; Fuller et al. 1995).

Analysis of Northern blots and slot blots indicated that, in the guinea-pig, expression of αENaC and βENaC mRNA was negligible in the lung tissue of the late second trimester fetus (35 days PC) and early third trimester fetus (50 days PC) (data not shown). αENaC mRNA became detectable at 60 days PC and expression increased significantly at 65 days PC (63 ± 8%, P = 0.05, n = 11) to reach maximal levels at term (100 ± 9%). αENaC mRNA levels then fell postnatally towards adult levels (Figs 1A and 2A). βENaC mRNA was detectable from 60 days PC and levels increased significantly from 65 days PC (62 ± 4%, P < 0.05, n = 9) to reach maximal levels at 1 day postnatal (130 ± 21%). Levels did not decline postnatally (P = 0.8, n = 5) in whole lung (Figs 1B and 3A).

Figure 2. Expression levels of αENaC mRNA in the lungs of term vaginally and CS-delivered guinea-pigs.

Figure 2

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RNA (5 μg) was prepared from distal lung tissue of animals at the gestational ages shown, applied to slot blots and hybridised with the guinea-pig αENaC probe. αENaC mRNA expression levels were normalised to 18s RNA for loading and expressed as percentage expression (means ±s.e.m.) relative to levels at term (68 days PC) in vaginally delivered animals, to standardise data between blots. The day of delivery (days PC) and mode of delivery for each group of animals are indicated with arrows. A, animals delivered by CS at 65 days PC (CS 65) compared with animals born vaginally at term (V Term). * Significantly different from 65 days PC (P < 0.05); † significantly different from vaginally delivered animals at term (P < 0.05). B, animals delivered by CS at term (68 days PC; CS Term) compared with animals born vaginally at term (V Term). * Significantly different from vaginally delivered animals at term (P < 0.05).

Figure 3. Expression levels of βENaC mRNA in the lungs of term vaginally and CS-delivered guinea-pigs.

Figure 3

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RNA (5 μg) was prepared from distal lung tissue of animals at the gestational ages shown, applied to slot blots and hybridised with the guinea-pig βENaC probe. βENaC mRNA expression levels were normalised to 18s RNA for loading and expressed as percentage expression (means ±s.e.m.) relative to levels at term (68 days PC) in vaginally delivered animals, to standardise data between blots. The day of delivery (days PC) and mode of delivery for each group of animals are indicated with arrows. A, animals delivered by CS at 65 days PC (CS 65) compared with animals born vaginally at term (V Term). B, animals delivered by CS at term (68 days PC; CS Term) compared with animals born vaginally at term (V Term). * Significantly different from CS at term (P < 0.05).

Effect of Caesarean delivery on expression of αENaC mRNA

Animals delivered by CS at 65 days PC showed significantly increased levels of αENaC mRNA, relative to day of delivery (0 days), after 1 and 3 days in normoxia (133 ± 13%, n = 12, P < 0.05 and 119 ± 13%, n = 10, P < 0.05, respectively). In addition, the level of αENaC mRNA after day 1, but not day 3, was significantly higher (P < 0.05, n = 11) than that in vaginally delivered animals at term (68 days PC; Fig. 2a).

Animals delivered by CS at term also demonstrated a significant increase in αENaC expression (190 ± 18%, n = 4, P < 0.05) at day 1 (69 days PC), compared with term animals delivered vaginally with labour (Fig. 2b).

Compared with levels following CS at 65 days PC, the apparently greater increase in αENaC mRNA following CS delivery at term did not reach significance (P = 0.08). By 71 days PC, αENaC mRNA had declined to similar levels in both test groups. These data indicate that the process of labour is not a prerequisite for an increase in expression of αENaC mRNA at birth.

Effect of Caesarean delivery on expression of βENaC mRNA

In fetuses delivered at 65 days PC by CS, levels of βENaC mRNA remained significantly lower than those in term fetuses delivered vaginally, at both day 1 (61 ± 4%, n = 8, P < 0.05) and day 3 post partum (65 ± 7%, n = 6, P < 0.05) (Fig. 3a). In animals delivered by CS at term (68 days PC), βENaC mRNA expression was similar at term, 1 and 3 days postnatally to that in animals delivered vaginally (104 ± 12%, n = 4, P = 0.35; 117 ± 8%, n = 4, P = 0.6; and 108 ± 44%, n = 3, P = 0.3, respectively) (Fig. 3b). These data indicate that fetal maturity is important in determining expression of βENaC mRNA postnatally.

Effect of environmental oxygen on expression of αENaC mRNA after pre-term CS delivery

Levels of αENaC mRNA were significantly increased at day 1 post partum (P < 0.05, n = 4), reaching similar levels irrespective of whether animals were maintained in 15, 21 or 95% O2 (Fig. 4). However, by day 3, there was a significant difference between animals maintained in hyperoxia (95% O2) and hypoxia (15% O2) (176 ± 10%, n = 4, and 81 ± 21%, n = 4, respectively; P < 0.05).

Figure 4. Expression levels of αENaC mRNA in the lungs of CS-delivered guinea-pigs exposed to different postnatal environmental O2 concentrations.

Figure 4

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αENaC mRNA levels (means ±s.e.m.) in animals delivered by CS 65 days PC and maintained in environmental O2 concentrations of 15, 21 or 95% for up to 3 days postnatally. * Significantly different from 15% O2 (P < 0.05).

Plasma hormone levels

Cortisol

Plasma cortisol levels in the guinea-pig were approximately 10-fold higher than corresponding human levels. This finding was consistent with the high plasma cortisol levels detected in this species by other workers (Keightley & Fuller, 1995; Fenske, 1996). In animals delivered normally at term, plasma cortisol levels increased significantly (P < 0.05, n = 8) during late gestation from 2607 ± 103 nmol l−1 at 65 days PC to reach 3827 ± 147 nmol l−1 at term (68 days PC). In the first few hours after birth, the plasma cortisol concentration increased rapidly (data not shown) and remained significantly higher at day 1 postnatally (5185 ± 115 nmol l−1, P < 0.05) than at day 65 PC. By day 3, plasma cortisol levels had declined and continued to fall towards pre-term levels up to day 7 ex utero (n = 4; Fig. 5a).

Figure 5. Influence of maturity and mode of delivery on plasma cortisol levels in the neonatal guinea-pig.

Figure 5

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Plasma cortisol levels are expressed as standard SI units (nmol l−1) as means ±s.e.m. The day of delivery (days PC) and mode of delivery for each group of animals are indicated with arrows. A, plasma cortisol levels in guinea-pigs delivered vaginally at term (V Term) compared with those in animals delivered by CS at 65 days PC (CS 65). * Significantly different from 65 days PC (P < 0.05). B, animals delivered by CS at term (68 days PC; CS Term) compared with animals delivered vaginally at term (V Term). * Significantly different from 65 days PC (P < 0.05).

Animals delivered by CS at 65 days PC exhibited a significant increase in plasma cortisol concentration at day 1 (P < 0.05, n = 6), which reached levels similar (5743 ± 794 nmol l−1) at 1 day post partum to those of term animals born vaginally. Plasma cortisol then declined towards pre-term levels by day 3 (n = 3; Fig. 5a). Animals delivered by CS at term (68 days PC) displayed a plasma cortisol profile very similar to that of term animals delivered vaginally with labour (Fig. 5B), reaching 5363 ± 628 nmol l−1 (n = 3) at delivery and 5822 ± 266 nmol l−1 (n = 6) at day 1. The maximal level of plasma cortisol achieved by day 1 was similar in both pre-term (65 days PC) and term (68 days PC) CS-delivered animals, even though animals delivered at term (68 days PC) already exhibited significantly higher levels of plasma cortisol at the time of delivery (P < 0.05, n = 3). Animals delivered by CS at 65 days PC into a reduced environmental O2 concentration of 15% had significantly increased plasma cortisol levels by postnatal day 1 (7253 ± 784 nmol l−1, P < 0.05, n = 4), but the levels were not significantly different from those in animals delivered into normoxia (21% O2) at day 1 or 3 (data not shown).

Our data suggest that plasma cortisol is elevated by delivery and that neither the mode of delivery nor moderate hypoxia significantly affects the postnatal levels of cortisol at 1 or 3 days.

T3

In animals delivered vaginally at term, the total plasma T3 concentration increased from 65 days PC (0.76 ± 0.03 nmol l−1, n = 6) to reach significantly higher levels at term (68 days PC) (2.746 ± 0.34 nmol l−1; P < 0.05, n = 4). After birth, the total T3 concentration increased rapidly and remained high at day 1 (3.74 ± 0.48 nmol l−1, n = 3) but fell by day 3 to reach levels similar to those of the pre-term (65 days PC) animal by day 7 (Fig. 6a).

Figure 6. Influence of maturity and mode of delivery on plasma total T3 levels in the neonatal guinea-pig.

Figure 6

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Plasma total T3 levels are expressed as standard SI units (nmol l−1) as means ±s.e.m. The day of delivery (days PC) and mode of delivery for each group of animals are indicated with arrows. A, plasma total T3 levels in guinea-pigs delivered vaginally at term (V Term) compared with those in animals delivered by CS at 65 days PC (CS 65). * Significantly different from 65 days PC (P < 0.05); † significantly lower than V Term (P < 0.05). B, animals delivered by CS at term (68 days PC; CS Term) compared with animals delivered vaginally at term (V Term). * Significantly different from 65 days PC (P < 0.05).

Animals delivered by CS at 65 days PC exhibited a small postnatal increase in the total plasma T3, but levels at day 1 were significantly lower than those in vaginally delivered animals (1.4 ± 0.12 nmol l−1, P < 0.05, n = 6) and did not change by day 3 (Fig. 6a). In contrast, animals delivered by CS at term (68 days PC) exhibited a similar pattern of change in plasma T3 levels to that in vaginally delivered animals. The total T3 concentration increased to 3.5 ± 0.17 nmol l−1 at day 1 but fell to 2.93 ± 0.65 nmol l−1 at day 3 (n = 4; Fig. 6b). At day 1, total T3 levels were significantly lower in animals delivered by CS at 65 days PC than those in animals delivered by CS at term (68 days PC) (P < 0.05, n = 6).

The suppressed levels of plasma T3 in animals delivered by CS at 65 days PC were, at postnatal day 1 and day 3, not significantly altered by maintenance in 15% O2 (1.4 ± 0.1 nmol l−1, n = 5 and 1.0 ± 0.1 nmol l−1, n = 3, respectively).

These data suggest that delivery of pre-term animals at 65 days PC by CS significantly precludes the normal (post vaginal delivery) postnatal rise in plasma total T3 and that moderate hypoxia has no further effect on T3 levels in these animals.

Effect on αENaC expression of inhibition of cortisol synthesis with metyrapone

Animals delivered by CS at 65 days PC and treated with metyrapone up to day 1 had significantly lower plasma cortisol levels than saline control animals (1510 ± 450 and 4990 ± 1030 nmol l−1, respectively; P < 0.05, n = 5). The level of plasma cortisol in the treated animals was not significantly different from levels at delivery at 65 days PC (2270 ± 480 nmol l−1, P = 0.2, n = 4). Similarly, levels of αENaC mRNA were suppressed in animals treated with metyrapone compared with saline-injected controls. Compared with αENaC expression on day 65 PC (100%), levels were 154 ± 12% after saline but only 124 ± 10% after metyrapone treatment, on day 1 (P = 0.05, n = 5; Fig. 7).

Figure 7. Effect of metyrapone on plasma cortisol and αENaC mRNA levels in the pre-term CS-delivered guinea-pig.

Figure 7

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Plasma cortisol levels (nmol l−1; ▪) and percentage expression of αENaC mRNA relative to day 65 PC (□) in guinea-pigs delivered by CS at 65 days PC (CS 65) and treated with saline or metyrapone up to day 1 postnatally (CS 65 + 1). Data are expressed as means ±s.e.m.* Significantly different from CS 65 (P < 0.05).

Lung fluid clearance

The animals delivered by CS at 65 days PC exhibited transient respiratory distress and the lung wet/dry weight ratio was significantly higher in these animals in comparison with animals delivered vaginally at term (7.4 ± 0.3 and 4.9 ± 0.1, respectively; P < 0.05, n = 11), suggesting a higher level of fluid present in the lungs on delivery at 65 days PC.

On the day of delivery (day 0), lung fluid clearance, as determined by the final to instilled protein concentration ratio, was significantly lower in animals delivered by CS at 65 days PC (1.25 ± 0.04) than that in animals born vaginally at term (2.01 ± 0.19; P < 0.05, n = 4). At day 1 postnatally, clearance rates had increased (data not shown) and at day 3 the final to instilled protein concentration ratio was not significantly different from that of the vaginally delivered term animal (2.08 ± 0.24, n = 4; Fig. 8). Addition of 10−3 M amiloride to the instillate did not significantly inhibit lung fluid clearance in the 65 day CS animals at day 0, but amiloride significantly reduced the final to instilled protein concentration ratio to 1.08 ± 0.05 (P < 0.05, n = 4), an inhibition of approximately 93%, at day 3, consistent with the increased expression of αENaC mRNA. The amiloride-sensitive component in the animals delivered by CS at 65 days PC at day 3 was also greater than that seen in animals delivered normally at term. In these vaginally delivered animals, amiloride reduced the final to instilled protein concentration ratio to 1.22 ± 0.07 (P < 0.05, n = 4), an inhibition of approximately 78% (Fig. 8).

Figure 8. Lung fluid clearance and effect of amiloride in the pre-term CS- and vaginally delivered term guinea-pig.

Figure 8

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Lung fluid clearance, shown as final to instilled protein concentration ratio (see Methods), in the presence (□) and absence (control; ▪) of 10−3 M amiloride, for animals delivered by CS at 65 days PC, on the day of delivery (CS 65), and at postnatal day 3 (CS 65 + 3), and for those delivered vaginally at term (V Term). Data are expressed as means ±s.e.m.† Significantly different from CS 65; * significantly different from control (- Amiloride) (P < 0.05).

DISCUSSION

Effect of delivery on steady-state α- and βENaC mRNA levels

Our finding that αENaC and βENaC are developmentally expressed in guinea-pig lung is in agreement with those of several other investigators in other species (Voilley et al. 1994; Tchepichev et al. 1995; Dagenais et al. 1997). The observation that expression of αENaC mRNA was maximal at term and declined postnatally, whereas βENaC was predominantly expressed postnatally, is similar to that described in rat and mouse lung (Tchepichev et al. 1995; Dagenais et al. 1997). However, we have found that fetal maturity and the mode of delivery modulate the normal developmental pattern of α- and βENaC mRNA expression postnatally.

There is an increasing body of evidence that hormones present during the process of labour and/or during fetal development play a role in the regulation of amiloride-sensitive lung fluid absorption (Olver et al. 1986; Barker et al. 1990a,b) and the expression of amiloride-sensitive sodium channel (ENaC) mRNAs (Champigny et al. 1994; Tchepichev et al. 1995; Venkatesh & Katzberg, 1997). We have shown that, in guinea-pigs delivered by CS 3 days pre-term (65 days PC) and at term, steady-state αENaC mRNA levels rapidly increase postnatally. This suggests that labour is not required to increase αENaC mRNA levels in the early postnatal period.

Role of cortisol and T3 in regulating α- and βENaC mRNA levels

Our data show that plasma cortisol level profiles in the late gestation fetal and neonatal guinea-pig closely mimic αENaC mRNA expression. In particular, the rapid rise in plasma cortisol concentration after CS delivery of 65 day PC and 68 day PC animals is paralleled by the rapid rise in αENaC mRNA. Furthermore, inhibition of cortisol synthesis in the CS-delivered animal suppressed αENaC mRNA levels, supporting the hypothesis that the rapid elevation of cortisol post delivery mediates the upregulation of αENaC expression in these animals. Cortisol has been shown to influence the maturation of the response of amiloride-sensitive fluid absorption to adrenaline (Barker et al. 1990a; Wallace et al. 1995), and evidence from several species suggests that steroid hormones can act to increase αENaC mRNA transcription within 8 h (Champigny et al. 1994; Tchepichev et al. 1995; Venkatesh & Katzberg, 1997). Our finding that βENaC expression remained low after CS delivery at 65 days PC but was similar to that in vaginally delivered animals after CS at term, suggests that elevation of ENaC subunit transcription by corticosteroids in vivo may be restricted to the α-subunit. This is consistent with the findings of Tchepichev et al. where treatment of pregnant rats with dexamethasone increased αENaC mRNA but not β- or γENaC mRNA in the fetal lung (Tchepichev et al. 1995). Our finding that maximal plasma cortisol levels were similar in all groups and did not correlate directly to the steady-state level of αENaC mRNA may indicate a threshold effect of the steroid on αENaC transcription. For example, in human lung explants, the induction of increased levels of αENaC mRNA is saturable at 30–100 nM dexamethasone (Venkatesh & Katzberg, 1997). Alternatively, there may be factors present during the process of labour that prevent overexpression of αENaC mRNA.

We have shown that pre-term (65 days PC) CS-delivered animals exhibited significantly lower plasma total T3 levels compared with term CS- or vaginally delivered animals. This phenomenon has been described in human babies delivered pre-term (van Wassenaer et al. 1997) and probably reflects the requirement of specific enzymes induced during labour for conversion of T4 to T3 (Van der Geyten et al. 1997). T3 does have an important role in salt and water transport in the lung (Barker et al. 1990a,b). However, as the pattern of plasma T3 in our studies did not correlate with αENaC mRNA levels, we suggest that T3 does not modulate αENaC mRNA expression in the late gestation guinea-pig lung, a conclusion supported by the finding that T3 does not upregulate αENaC transcription in human fetal lung explants (Venkatesh & Katzberg, 1997). Although the suppressed levels of T3 in CS-delivered pre-term animals paralleled that of βENaC, evidence from pregnant rats treated with thyroid releasing hormone (Tchepichev et al. 1995) and from human explants suggests that βENaC mRNA levels in the perinatal lung are not regulated by T3.

Effect of oxygen on the expression of αENaC

On delivery, the newborn inspires ambient air containing about 21% O2, although the concentration of O2 to which the alveolar epithelium is exposed is significantly lower (approximately 12–14%). Even so, this represents a substantial increase in alveolar PO2 (PA,O2) from about 25 to approximately 100 mmHg. Altered PO2 has been shown by several investigators to modulate fluid transport in the lung (Barker & Gatzy, 1993) and both ENaC expression and activity (Pitkänen et al. 1996; Planes et al. 1997). However, we found that there was no significant difference in the αENaC mRNA levels at postnatal day 1 of animals delivered by CS at 65 days PC, when maintained under hyperoxic (95% O2), normoxic (21%) or hypoxic (15% O2) conditions. In addition, relative hyperoxia had no effect on cortisol or T3 levels. Thus, our data suggest that the rapid upregulation of αENaC within 24 h of CS delivery at 65 days PC is unlikely to be mediated by O2. In support of this hypothesis, we have shown firstly, that term CS-delivered animals also exhibit a rise in αENaC mRNA levels postnatally, whereas term vaginally delivered animals do not – even though both experience a similar increase in PA,O2. Secondly, we find that a range of inspired PO2 values, including extreme hyperoxia, have no differential effect at 24 h (when αENaC transcription is already upregulated) but do at 72 h. Consistent with this delayed response to relative hyperoxia observed in vivo is the observation in rat FDLE (fetal distal lung epithelial) cells that O2-induced increases in α- and βENaC mRNAs are not seen until 48 h (Pitkänen et al. 1996).

αENaC mRNA expression and amiloride-sensitive lung fluid clearance

Our observation that lung fluid clearance (at day 0) is lower in animals delivered by CS at 65 days PC than that in vaginally delivered animals at term is consistent with a greater lung fluid content, as indicated by the increased lung wet/dry weight ratios and transient respiratory distress in these animals. Net fluid transport in the lung is dependent on the relative magnitude of the forces for Cl− secretion and Na+ absorption (Olver et al. 1986), thus Cl− secretion may not be downregulated and/or Na+ absorption upregulated sufficiently to achieve the higher fluid clearance rates of term vaginally delivered animals. Delivery by CS is known to be associated with reduced ion transport (Gowen et al. 1988) and lung function (Olver, 1981) and the negligible amiloride-sensitive component of fluid absorption is consistent with the presence of less αENaC mRNA and consequently less functionally active protein in animals delivered by CS at 65 days PC. The catecholamine terbutaline has been reported to mediate activation of amiloride-sensitive Na+-permeable epithelial channels and to increase their trafficking to the apical membrane (Ito et al. 1997). As there are significantly diminished catecholamine levels following elective CS (Lagercrantz, 1996), a reduced ability to incorporate and activate αENaC protein in the lung apical membrane could also contribute to the reduced absorptive function observed immediately following delivery by CS at 65 days PC.

Our finding that the rate and proportion of amiloride-blockable fluid clearance in the lung increased concomitantly with αENaC, but not βENaC, mRNA expression in the CS-delivered pre-term animals suggests that the increased synthesis of αENaC message contributes to increased function of this channel in the apical membrane. Furthermore, recent evidence from transgenic knockout mice has suggested that the absence of β- or γENaC mRNA in lung only has a small effect on fluid clearance compared with mice lacking αENaC (Hummler et al. 1996; Barker et al. 1998; McDonald et al. 1999). Although we have not studied γENaC in these tissues, its mRNA expression in lung closely follows that of βENaC (Tchepichev et al. 1995).

Interestingly, although the level of fluid clearance was similar, amiloride blocked nearly all the fluid absorption in the 65 day CS-delivered animal by postnatal day 3 but only 78% of that in animals delivered vaginally at term. A significant contribution of an amiloride-insensitive component of sodium transport has been described in several different preparations and species (O'Brodovich et al. 1990a, b; Fyfe et al. 1994; Finley et al. 1998). Thus, the marked reduction of this component in our CS-delivered animals leads us to speculate that, in the absence of labour, the amiloride-sensitive pathway is upregulated in preference to the amiloride-insensitive pathway.

Conclusion

We have shown that late gestation guinea-pigs delivered by CS significantly upregulate αENaC but not βENaC mRNA expression postnatally and that this leads to increased amiloride-sensitive lung fluid clearance in the absence of labour. This upregulation is mediated, at least in part, by the postnatal rise in cortisol concentration at delivery but is not directly related to levels of T3 and PO2, which may, nevertheless, have permissive roles early in the postnatal period. The finding that αENaC mRNA levels increase significantly postnatally in both pre-term and term CS-delivered animals, whereas those in vaginally born animals do not, leads us to speculate that there are alternative pathways for upregulation of amiloride-sensitive fluid absorption in the lung in the absence of labour and that, paradoxically, labour may suppress steady-state αENaC mRNA levels postnatally.

Acknowledgments

We thank Gordon Tennant, Alan Monaghan, Lorraine Gambling, Mark Clunes and Stuart Litchfield for technical assistance. This study was supported by grants from the Wellcome Trust (programme grant no. 039124/Z/4A), the Anonymous Trust, Tenovus Scotland, Swedish Natural Science Research Council, and the Crafoord Foundation.

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