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. 1999 Aug 15;519(Pt 1):261–272. doi: 10.1111/j.1469-7793.1999.0261o.x

Role of endogenous cortisol in basal liquid clearance from distal air spaces in adult guinea-pigs

Andreas Norlin *, Deborah L Baines *, Hans G Folkesson *
PMCID: PMC2269482  PMID: 10432356

Abstract

  1. We investigated the role of endogenous cortisol in the modulation of distal air space liquid clearance in adult guinea-pigs. Cortisol synthesis was inhibited with the 11-β-hydroxylase inhibitor metyrapone (0–7 days pretreatment). After cortisol synthesis inhibition, distal air space liquid clearance was measured by the increase in concentration of an instilled 5 % albumin solution after 1 h.

  2. Two days of metyrapone pretreatment resulted in a 46 ± 19 % decrease in plasma cortisol levels compared with control, which was paralleled by a 60 ± 13 % decrease in distal air space liquid clearance. The Na+ channel inhibitor amiloride inhibited 40 ± 22 % of distal air space liquid clearance in control animals but did not inhibit distal air space liquid clearance in the metyrapone-pretreated group. Co-injection of dexamethasone prevented the inhibition by metyrapone and the amiloride sensitivity of distal air space liquid clearance was greater than in control animals. After 7 days of metyrapone pretreatment, plasma cortisol levels and distal air space liquid clearance were not significantly different from normal, but amiloride sensitivity was greater than in control animals (91 ± 37 %).

  3. Pretreatment with emetine, a protein synthesis inhibitor, reduced distal air space liquid clearance in control animals and in dexamethasone-co-injected animals, but failed to inhibit distal air space liquid clearance after metyrapone pretreatment. Expression of the epithelial sodium channel α-subunit (αENaC) mRNA in lung tissue was decreased after 2 days of metyrapone pretreatment and after 7 days pretreatment or after co-injection with dexamethasone, αENaC mRNA expression was restored towards control levels.

  4. Thus, endogenous cortisol is important for maintaining normal liquid balance in the adult guinea-pig lung and a critical regulatory pathway is by modulation of ENaC expression and/or function.

Uptake and transport of Na+ across the distal air space epithelium provides the osmotic driving force for clearance of excess liquid from the distal air spaces of the lungs (Basset et al. 1987; Berthiaume et al. 1987; Matalon et al. 1996; Matthay et al. 1996). The significance of vectorial Na+ transport for clearance of excess liquid and maintenance of liquid balance in the adult lung has been demonstrated in vivo (Berthiaume et al. 1987; Jayr et al. 1994; Folkesson et al. 1996; Norlin et al. 1998). In various animal species, Na+ transport across the distal air space epithelium can be stimulated by β-adrenergic agonists, such as adrenaline or terbutaline (Berthiaume et al. 1987; Jayr et al. 1994; Folkesson et al. 1996; Norlin et al. 1998). Also, endogenous release of catecholamines during pathological conditions (Pittet et al. 1994; Modelska et al. 1997) and at birth (Finley et al. 1998) stimulates distal air space liquid clearance. The effect of β-adrenergic agonists is mediated via increases in intracellular cyclic adenosine monophosphate (cAMP) (Berthiaume, 1991; Finley et al. 1998; Norlin et al. 1998). Other endogenous, non-catecholaminergic substances have been demonstrated to stimulate distal air space liquid clearance and/or Na+ transport. For example, transforming growth factor-α increases distal air space liquid clearance in rats (Folkesson et al. 1996). Acute and/or long-term administration of epidermal growth factor increases Na+ uptake by rat alveolar type II cells (Borok et al. 1996; Kemp et al. 1998) and keratinocyte growth factor increases Na+ transport across alveolar epithelial cell monolayers (Borok et al. 1998) and in vivo (Wang et al. 1998).

Glucocorticoid hormones have been suggested as regulators of alveolar epithelial Na+ uptake and transport in both adult and fetal lungs (Renard et al. 1995; Tchepichev et al. 1995; Barquin et al. 1997; Ingbar et al. 1997). In adult rats, steroid hormones have been demonstrated to regulate mRNA expression for the α-, β- and γ-subunits of the amiloride-sensitive epithelial Na+ channel (ENaC) (Renard et al. 1995; Tchepichev et al. 1995). Dexamethasone has been shown to stimulate Na+,K+-ATPase expression in fetal and adult rat lungs (Barquin et al. 1997; Ingbar et al. 1997), and dexamethasone pretreatment of adult rats has been shown to increase distal air space epithelial liquid transport (Folkesson et al. 1999).

In this study, we hypothesized that endogenous cortisol regulates distal air space liquid clearance in adult animals by regulation of ENaC in epithelial Na+ transport. The first aim was to investigate the effects of a reduction in plasma cortisol levels over 2–7 days on distal air space liquid clearance in adult guinea-pigs. The second aim was to determine if the amiloride-sensitive fraction of distal air space liquid clearance was altered by the reduction of plasma cortisol levels, and whether this effect was correlated with mRNA expression levels for the α-subunit of the amiloride-sensitive ENaC (αENaC). The third aim was to investigate whether the synthesis of the ENaC protein was altered after reduction in plasma cortisol levels.

METHODS

Animals

Eighty-nine male Dunkin-Hartley guinea-pigs (AB Sahlins Försöksdjursfarm, Malmö, Sweden), weighing 559 ± 100 g, were used in the study. The animals were kept at a 12:12 h night-day rhythm and were fed with standard guinea-pig chow (SDS, Witham, Essex, UK), with water ad libitum.

The Ethical Review Committee on Animal Experiments at Lund University had approved the protocol for these studies.

Preparation of instillates

A 5 % albumin solution was prepared by dissolving 50 mg ml−1 bovine serum albumin (Sigma) in 0.9 % NaCl (Pharmacia-UpJohn, Uppsala, Sweden). An instillate sample was saved for total protein measurement. In some studies (see ‘Specific experimental protocols’), the Na+ channel inhibitor amiloride (10−3 M; Sigma), the β-adrenergic antagonist propranolol (10−4 M; Sigma), or the β-adrenergic agonist isoprenaline (10−5 M; Sigma) were added to the instillate.

Pretreatment with glucocorticoid synthesis inhibitor

The guinea-pigs were pretreated with the 11-β-hydroxylase inhibitor metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone; Sigma) over two, four or seven consecutive days. Metyrapone (62.5 mg ml−1) was dissolved in 24 % ethanol in 0.9 % NaCl and was injected subcutaneously (s.c.) in the morning and in the evening (25 mg kg−1 at each time to reach a total daily dose of 50 mg kg−1). In the morning of the day of experiment, the animals received one-half the daily dose of metyrapone and were prepared for the distal air space liquid clearance experiment. The dose of metyrapone selected was adopted from its higher ranges of clinical dosage. Two animals were given a higher dose of metyrapone (100 mg kg−1) to investigate if the effect was maximal or sub-maximal. Since there were no differences in the results between these guinea-pigs and the guinea-pigs that received 50 mg kg−1 of metyrapone, the lower dose was used and all animals were combined into one group. A separate group of animals (vehicle controls) were injected s.c. for 2 days with 24 % ethanol (vehicle for metyrapone).

Pretreatment with dexamethasone

Metyrapone-pretreated guinea-pigs were co-injected with the glucocorticoid dexamethasone (Sigma), for two consecutive days (0.01 or 0.35 mg kg−1 day−1). Dexamethasone was injected s.c. into the back, simultaneously with the metyrapone injection on the morning of each day and on the morning of the distal air space liquid clearance experiment. The lower dose of dexamethasone was selected to give a plasma steroid concentration equivalent to normal cortisol levels (see Results below). To calculate the dose we assumed a total body volume of 500–700 ml and, compared with cortisol, a 7-fold higher glucocorticoid receptor affinity for dexamethasone, as shown for human fetal lung (Baxter & Rousseau, 1979). The protocol for the dexamethasone pretreatment was adopted and modified from a study on steroid-induced expression of aquaporin 1 in fetal rat lung by King and co-workers (King et al. 1996).

Pretreatment with emetine

Guinea-pigs pretreated with metyrapone (50 mg kg−1 day−1) for 2 days with or without dexamethasone (0.35 mg kg−1 day−1) and control animals were given the protein synthesis inhibitor emetine (2.0 mg kg−1; Sigma) intratracheally 15 h before the distal air space liquid clearance experiment. Emetine was dissolved in 0.9 % NaCl and instilled in a volume of 1.0 ml kg−1 into the lungs as previously described in the rat (Folkesson et al. 1990). The animals were briefly anaesthetized with diethylether and mounted in their upper incisors on a slanting board. A blunt and slightly angled injection needle was passed into the distal trachea and the emetine solution was injected quickly into the lungs. After instillation, the guinea-pigs were kept in an upright position for 1 min to allow for maximal distribution of the instilled liquid in the distal lung. The dose of emetine was determined empirically and was one-fifth the near-lethal dose described for injections into rat brain (Lopez-Mascaraque & Price, 1997).

Surgical procedures and ventilation

We have previously described the surgical procedure and instillation technique for distal air space liquid clearance studies in guinea-pigs (Norlin et al. 1998). Guinea-pigs were anaesthetized by intraperitoneal pentobarbital sodium (40 mg kg−1; Apoteksbolaget, Umeå, Sweden). A 2.0 mm i.d. endotracheal tube (PE-240, Clay Adams, Becton Dickinson & Co., Sparks, MD, USA) was inserted through a tracheostomy after the anaesthesia. A 0.58 mm i.d. catheter (PE-50, Clay Adams, Becton Dickinson & Co.) was inserted in the left carotid artery to monitor systemic blood pressure, administer drugs, and to obtain blood samples. Pancuronium bromide (0.3 mg kg−1 h−1; Pavulon, Organon Teknika, Boxtel, The Netherlands) was administered through the arterial catheter for neuromuscular blockade. We determined the depth of anaesthesia and muscle relaxation from pupil dilation, heart rate, blood pressure, and breathing reflexes. Supplemental anaesthesia of 20 mg kg−1 pentobarbital sodium was given hourly or when needed. The animals were ventilated with a constant-volume piston pump (Harvard Apparatus, Nantucket, MA, USA) with an inspired oxygen fraction of 1.0 and tidal volumes set to drive peak airway pressures to 10–12 cmH2O and with a positive end expiratory pressure of 3 cmH2O during the baseline period.

Peak airway pressures, blood pressures and heart rates were measured with calibrated pressure transducers (UFI Model 1050BP, BioPac Systems, Goleta, CA, USA) connected to analog-to-digital converters and amplifiers (MP100 and DA100, respectively, BioPac Systems) and continuously recorded on an IBM computer with Acknowledge version 3.2.4 software (BioPac Systems).

General experimental protocol

The distal air space liquid clearance experiment was always done 1–2 h after the last injection of the pretreatment. Immediately after surgery, the animals were placed in the left decubitus position on a slanting board. A heating pad covered the animals during the experiments to control and maintain normal body temperature. After 30 min of stable blood pressure and heart rate, the animal was temporarily disconnected from the ventilator and a soft instillation tubing (Silastic, Dow Corning) was passed through the endotracheal tube to rest just above the bronchial carina. The instillate (6 ml kg−1) was delivered to both lungs over 10–15 s and the animal was immediately reconnected to the ventilator.

After 58 min, 5 ml of blood was withdrawn, and an overdose of pentobarbital sodium was administered. At 60 min the lower abdomen was opened and the animals were exsanguinated by transection of the renal artery. The lungs and heart were carefully removed en bloc from the thorax through a mid-line sternotomy. A PE-50 catheter (Clay Adams, Becton Dickinson & Co.) was passed to a wedged position in the lung and a sample of the remaining distal air space liquid was aspirated. Lungs from each experimental group were immediately frozen in liquid nitrogen and stored at −70°C until mRNA analysis. Blood samples were centrifuged (3500 g, 5 min) and plasma was collected and stored at −70°C until further analysis. Haematocrit and total plasma protein were measured in the last blood sample.

Specific experimental protocols

Control studies

After the baseline period, the guinea-pigs (n = 8) were instilled with the 5 % albumin solution into both lungs. After 1 h, the animals were exsanguinated and processed as described in ‘General experimental protocol’.

Cortisol inhibition studies

Guinea-pigs were pretreated with metyrapone (50 mg kg−1 day−1) for two consecutive days (n = 6), four consecutive days (n = 4), or seven consecutive days (n = 6). Another group (n = 4) of guinea-pigs pretreated with ethanol alone (vehicle) was also studied. Distal air space liquid clearance was measured in all animals as described in ‘General experimental protocol’. To investigate if the decrease in circulating cortisol affected endogenous adrenalin release or the efficiency of the β-adrenergic signalling pathway in the lungs, guinea-pigs pretreated for 2 days with metyrapone were instilled with the 5 % albumin instillate with 10−4 M of the β-adrenergic antagonist propranolol (n = 3) or 10−5 M of the β-adrenergic agonist isoprenaline (n = 3).

Dexamethasone pretreatment studies

Metyrapone-pretreated guinea-pigs were co-injected for two consecutive days with dexamethasone (0.35 mg kg−1, n = 5 or 0.01 mg kg−1 day−1, n = 6). Distal air space liquid clearance was measured in all animals as described in ‘General experimental protocol’.

Amiloride studies

The guinea-pigs were pretreated with metyrapone (50 mg kg−1 day−1) for two consecutive days (n = 8) or seven consecutive days (n = 6). Also, two separate groups of guinea-pigs that were given metyrapone for 2 days were co-injected with dexamethasone, 0.35 mg kg−1 day−1 (n = 6) or 0.01 mg kg−1 day−1 (n = 4). Untreated guinea-pigs (n = 8) were also studied. Amiloride (10−3 M) was added to the 5 % albumin instillate and distal air space liquid clearance was measured in all animals as described in ‘General experimental protocol’.

Emetine studies

The guinea-pigs were pretreated with metyrapone (50 mg kg−1 day−1) for two consecutive days (n = 4) or with metyrapone (50 mg kg−1 day−1) and dexamethasone (0.35 mg kg−1 day−1) in parallel for two consecutive days (n = 4). Untreated guinea-pigs (n = 4) were also studied. Fifteen hours before the distal air space liquid clearance experiment, the animals were intratracheally instilled with 2.0 mg kg−1 of the protein synthesis inhibitor emetine in 1 ml kg−1 0.9 % NaCl. Distal air space liquid clearance was measured in all animals as described in ‘General experimental protocol’.

Haemodynamic parameters and airway pressure

Systolic and diastolic systemic blood pressures, heart rates and peak airway pressures were measured at the start of experiments, 10 min before instillation, and then immediately after instillation, at 30 min after instillation, and at the end of the experiment.

Distal air space liquid clearance

Distal air space liquid clearance was measured as previously described (Berthiaume et al. 1987; Jayr et al. 1994; Folkesson et al. 1996; Finley et al. 1998; Norlin et al. 1998). We present distal air space liquid clearance as the final-to-instilled protein concentration ratio, i.e. the ratio between the protein concentration of the final aspirated distal air space liquid and the protein concentration of the instilled liquid. An increase in protein concentration in the distal air space liquid over the experimental time period (1 h) is direct evidence for liquid leaving the air spaces. Because there were no changes in epithelial and endothelial permeability to protein, this method is accurate for measuring liquid clearance from the distal air spaces of the lungs. Some liquid reabsorption may occur across the distal bronchial epithelium because it also absorbs sodium (Ballard et al. 1992).

Plasma cortisol

Cortisol levels in plasma samples from each animal were measured with a commercially available enzyme-linked immunosorbent assay (ELISA; Milenia Cortisol, Diagnostic Products Corporation, Los Angeles, CA, USA). Intra-assay variability was < 6.9 % and inter-assay variability < 8.0 %.

Preparation and analysis of mRNA

Whole lung tissue RNA was isolated using the acid guanidium thiocyanate-phenol-chloroform method (Chomczynski & Sacchi, 1987) with TRIzol Reagent (Sigma). The RNA pellet was suspended in diethyl pyrocarbonate-treated (RNase-free) water and quantified by analysis of optical density at 260 nm (1 unit of optical absorbance at 260 nm (A260 nm) = 40 μg).

Slot blots

RNA samples (5 μg) were denatured in a buffer containing 6 × standard sodium citrate buffer (SSC) and 7.4 % formaldehyde as described by Kacimi et al. (1995). The samples were loaded onto a Hybond-N nylon membrane (Amersham) by a vacuum manifold (Millipore Corp.). The nylon membrane was rinsed in 2 × SSC and the RNA was cross-linked to the nylon membrane by UV irradiation (1200 J cm−2; UVP Cross-Linker, Hoeffer Scientific Instruments, San Francisco, CA, USA).

Northern blots

RNA (10 μg) was denatured with 50 % formamide and 6.5 % formaldehyde in Mops buffer (0.1 M 3-(morpholino)-propanesulfonic acid, pH 7.0, 40 mM sodium acetate, 5 mM EDTA, pH 8.0). RNA was loaded onto a 1.1 % agarose gel containing 33 % formaldehyde in Mops buffer (pH 7.0) and electrophoretically separated for 3–4 h. Gels were washed for 20 min in RNase-free water followed by 20 min submersion in 0.1 n NaOH followed by 45 min in 20 × SSC. The RNA was capillary blotted to a Hybond-N+ nylon membrane (Amersham) overnight with 20 × SSC. The nylon membrane was rinsed in 2 × SSC and the RNA was cross-linked to the membrane, either by alkalization of the membrane for 20 min in 50 mM NaOH or by UV cross-linking (700 J cm−2).

Hybridization and analysis

A guinea-pig-specific αENaC (α-cENaC) probe of 761 bp was prepared by reverse transcription PCR from guinea-pig lung RNA using primers designed from rat sequences. The sense primer corresponds to bases 1059–1079 and antisense corresponds to bases 1800–1820 of αENaC. The amplified sequence is 85 % homologous to the rat sequence for this region. The probe hybridizes to a mRNA of 3.7 kb in the guinea-pig lung (authors' unpublished observations; Genbank access no. AF071230). The probe was labelled with 32P-dCTP by a multiprime labelling kit (Amersham). Loading variances between the RNA samples were assayed by an 18S ribosomal RNA oligonucleotide, kindly provided by Dr H. McArdle (The Rowett Institute, Aberdeen, UK). The 18S probe was labelled by replacing the terminal phosphate with [γ-32P]-ATP, with a polynucleotide kinase (Promega UK, Southampton, UK) catalysed reaction. Unbound nucleotides were removed with a microspin Probe-quart column (Pharmacia Biotech Ltd, St Albans, UK). High specific activity probes (108 counts min−1 (μg DNA)−1) were used for the hybridization protocols. Both Northern and slot blot membranes were prehybridized in Rapid Hyb (Amersham) hybridization buffer for 15 min at 65°C, prior to hybridization with αENaC or 18 s probes for 3 h at 65°C. The membranes were then washed sequentially at 65°C for 20 min in three different concentrations of SSC solutions (2 × SSC, 1 × SSC and 0.1 × SSC) + 0.1 % SDS. The blots were wrapped in plastic film and transcripts were visualized by autoradiography. The mRNA was quantified either by image analysis of 32P disintegrations in the specific area of the hybridized product by a phospho-imager (Hewlett-Packard Co.) or by densitometry analysis of scanned film-positive autoradiographs with the public domain NIH image software.

Statistics

All data are presented as means ± standard deviations. Data were analysed with one-way analysis of variance (ANOVA) with Tukey's post hoc test or with Student's t test when appropriate. Differences were considered significant when P ≤ 0.05.

RESULTS

Cortisol inhibition studies

Two days of metyrapone pretreatment decreased distal air space liquid clearance significantly (60 ± 13 %) compared with untreated controls (Fig. 1A). The decrease in distal air space liquid clearance was accompanied by a 46 ± 19 % decrease in plasma cortisol levels in metyrapone-pretreated animals compared with control (Fig. 1B). Four days of metyrapone pretreatment did not further alter distal air space liquid clearance (Fig. 1A) and the low plasma cortisol levels persisted (Fig. 1B). After 7 days of metyrapone pretreatment, distal air space liquid clearance had returned to control levels (Fig. 1A) and plasma cortisol levels were similar to control levels (Fig. 1B). Pretreatment for 2 days with the vehicle for metyrapone (24 % ethanol) did not affect the distal air space liquid clearance (final-to-instilled protein concentration ratios were 1.58 ± 0.18 (n = 4) and 1.60 ± 0.12 (n = 8) with and without pretreatment).

Figure 1. Effects of metyrapone pretreatment on distal air space liquid clearance and plasma cortisol levels.

Figure 1

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A, distal lung liquid albumin composition after 1 h in guinea-pigs instilled with the 5 % albumin solution after 0–7 days pretreatment with the 11-β-hydroxylase inhibitor metyrapone (50 mg kg−1 day−1), expressed as the final-to-instilled protein concentration ratio. Distal air space liquid clearance was significantly (P < 0.05) decreased after 2 days (n = 6) of metyrapone pretreatment compared with control (n = 8). Distal air space liquid clearance remained low up to 4 days (n = 4), but was similar to control after 7 days (n = 6) of metyrapone pretreatment. * P < 0.05 compared with control, † P < 0.05 compared with 7 days metyrapone pretreatment; ANOVA with Tukey's post hoc test. B, plasma cortisol levels in guinea-pigs pretreated with metyrapone for 0–7 days displayed as a box plot. Cortisol levels correlated well with the decreased distal air space liquid clearance. After 2 days of metyrapone pretreatment (n = 7), plasma cortisol level was 46 ± 19 % of normal levels (n = 6). This level was maintained after 4 days (n = 4) of metyrapone pretreatment, but after 7 days (n = 8), when distal air space liquid clearance was back to normal, there was a clear tendency towards an increase in plasma cortisol levels. The lower and upper limits in the boxes represent the 25th and 75th percentiles of the data range, respectively, and horizontal lines inside boxes are median values. Vertical lines below and above the boxes represent the 10th and 90th percentiles, respectively. All observations outside these limits are represented as filled circles. * P < 0.05 compared with control; ANOVA with Tukey's post hoc test.

Does depletion of endogenous cortisol affect release of or circulating plasma levels of endogenous catecholamines? We added 10−4 M propranolol to the instillate of guinea-pigs pretreated for 2 days with metyrapone to block the action of catecholamines on β-adrenergic receptors. Intraluminal administration of propranolol did not alter distal air space liquid clearance in metyrapone pretreated animals (final-to-instilled protein concentration ratio was 1.21 ± 0.09 (n = 3) after propranolol instillation as compared with 1.22 ± 0.10 (n = 6) after metyrapone pretreatment). A decrease in circulating cortisol may affect synthesis or function of many different proteins in the cell. Therefore, we added 10−5 M of the β-adrenergic agonist isoprenaline to the instilled albumin solution to investigate if the β-adrenergic receptors, a secondary signalling system, or components of the signal pathway were affected. Pretreatment with metyrapone for two consecutive days did not affect the sensitivity of distal air space liquid clearance to stimulation by intra-alveolar isoprenaline. After cortisol depletion, distal air space liquid clearance measured as final-to-instilled protein concentration ratio was increased by 159 % by isoprenaline. The final-to-instilled protein concentration ratio was increased to 1.57 ± 0.34 (n = 3) when isoprenaline was added to the instillate compared with 1.22 ± 0.10 (n = 6) in animals pretreated for 2 days with metyrapone.

We then determined if replacement of endogenous cortisol with an exogenous glucocorticoid (dexamethasone) would restore distal air space liquid clearance to normal levels in metyrapone-pretreated animals. Distal air space liquid clearance was restored towards baseline levels in the guinea-pigs pretreated with metyrapone that received either 0.35 or 0.01 mg kg−1 day−1 of dexamethasone for 2 days (Fig. 2A). Pretreatment with metyrapone and dexamethasone (0.35 mg kg−1 day−1) decreased plasma cortisol levels by 70 % compared with levels in animals pretreated with metyrapone alone (310 ± 446 nmol l−1 (n = 9) compared with 2218 ± 905 nmol l−1 (n = 6)).

Figure 2. Effects of metyrapone and dexamethasone pretreatment on distal air space liquid clearance and αENaC mRNA expression.

Figure 2

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A, distal lung liquid albumin composition after 1 h in guinea-pigs instilled with the 5 % albumin solution after 2 days pretreatment with the 11-β-hydroxylase inhibitor metyrapone (50 mg kg−1 day−1) alone or combined with dexamethasone (0.35 or 0.01 mg kg−1 day−1) expressed as the final-to-instilled protein concentration ratio. Distal air space liquid clearance was reduced by 60 ± 13 % after 2 days of metyrapone pretreatment (n = 6) compared with control (n = 8). This reduction was abolished when endogenous cortisol was replaced with dexamethasone (0.35 mg kg−1 day−1, n = 5). Also, a lower dose of dexamethasone (0.01 mg kg−1 day−1, n = 6) clearly tended to restore the decreased distal air space liquid clearance after 2 days of cortisol depletion. Values are means ± standard deviations. * P < 0.05 compared with control, † P < 0.05 compared with metyrapone + dexamethasone (0.35 mg kg−1 day−1); Student's t test. B, typical Northern blot of total RNA (10 μg) extracted from whole lung after pretreatment with metyrapone with and without dexamethasone. Upper blot, the αENaC probe detected a ≈3.7 kb mRNA in all samples. Expression levels of αENaC were decreased after metyrapone pretreatment for 2 days compared with control. Also, after combined metyrapone and dexamethasone pretreatment, mRNA expression was not different from control. Lower blot, same samples after hybridization with an 18S oligonucleotide probe. Relative expression of αENaC mRNA (control = 100 %) was calculated from image analysis data of slot blots corrected for loading variance by comparison to 18S ribosomal RNA levels.

Amiloride studies

The Na+ channel inhibitor amiloride (10−3 M) was added to the instilled liquid to investigate the effect of metyrapone pretreatment on liquid transport driven by Na+ flow through amiloride-sensitive channels. In control guinea-pigs, amiloride significantly inhibited distal air space liquid clearance by 40 ± 22 % (Fig. 3A). Amiloride did not affect the attenuated distal air space liquid clearance after 2 days of metyrapone treatment (Fig. 3A). After 7 days metyrapone pretreatment, when distal air space liquid clearance had returned to normal levels, the amiloride-sensitive fraction of distal air space liquid clearance was greater (91 ± 37 %) than the fraction for control animals (Fig. 3A). The amiloride-sensitive fraction of clearance by guinea-pigs pretreated with both metyrapone and dexamethasone was also elevated compared with control animals (Fig. 4). The amiloride-sensitive fraction of distal air space liquid clearance was 68 ± 18 %, with 0.01 mg kg−1 day−1 dexamethasone and 79 ± 45 % with 0.35 mg kg−1 day−1 dexamethasone.

Figure 3. Effects of metyrapone pretreatment and amiloride on distal air space liquid clearance and αENaC mRNA expression.

Figure 3

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A, distal lung liquid albumin composition after 1 h in guinea-pigs instilled with the 5 % albumin solution in untreated guinea-pigs or after 2 or 7 days pretreatment with the 11-β-hydroxylase inhibitor metyrapone (50 mg kg−1 day−1) under control conditions (▪) or with 10−3 M amiloride (□) added to the instillate and expressed as the final-to-instilled protein concentration ratio. Amiloride inhibited 40 ± 22 % of distal air space liquid clearance under normal (untreated) conditions (n = 8 in both groups). Amiloride sensitivity was completely abolished after 2 days metyrapone pretreatment (control n = 6; amiloride, n = 8). After 7 days, in contrast, amiloride sensitivity was restored to an even higher level than in control animals (n = 6 in both groups). Values are means ± standard deviation. * P < 0.05 compared with control group without amiloride, † P < 0.05 compared with 7 day metyrapone-pretreated group without amiloride; Student's t test. B, a typical Northern blot of total RNA (10 μg) extracted from whole lung after metyrapone and dexamethasone pretreatment. Upper blot, the αENaC probe detected a ≈3.7 kb mRNA in all samples. Expression levels of αENaC were decreased after metyrapone pretreatment for 2 days compared with control conditions. After 7 days of metyrapone pretreatment, αENaC mRNA expression was not different from control. Lower blot, the same samples after hybridization with an 18S oligonucleotide probe. Relative expression of αENaC mRNA (control = 100 %) was calculated from image analysis data from slot blots corrected for loading variance by comparison to 18S ribosomal RNA levels.

Figure 4. Effects of metyrapone and dexamethasone pretreatment and amiloride on distal air space liquid clearance.

Figure 4

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Distal lung liquid albumin composition after 1 h in guinea-pigs instilled with the 5 % albumin solution after 2 days of pretreatment with the 11-β-hydroxylase inhibitor metyrapone (50 mg kg−1 day−1) or 2 days of pretreatment with metyrapone and 0.01 or 0.35 mg kg−1 day−1 of dexamethasone under normal conditions (▪) or with 10−3 M amiloride (□) added to the instillate. Distal air space liquid clearance is expressed as the final-to-instilled protein concentration ratio. Amiloride (n = 6) did not inhibit distal air space liquid clearance in animals pretreated with metyrapone alone (n = 8). In animals that received metyrapone + dexamethasone (0.01 mg kg−1 day−1, n = 6) pretreatment, amiloride (n = 4) inhibited 68 ± 18 % of the distal air space liquid clearance. When the higher dose of dexamethasone (0.35 mg kg−1 day−1, n = 5) was co-injected with metyrapone, 79 ± 45 % of distal air space liquid clearance was inhibited by amiloride (n = 6). Values are means ± standard deviation. * P < 0.05 compared with low-dose dexamethasone group without amiloride, † P < 0.05 compared with high-dose dexamethasone group without amiloride; Student's t test.

Emetine studies

Emetine inhibited protein synthesis in control animals and animals pretreated with metyrapone alone, or with metyrapone and dexamethasone (0.35 mg kg−1 day−1). Distal air space liquid clearance was inhibited by 70 ± 15 % in control animals after inhibition of protein synthesis (Fig. 5). Similarly, emetine inhibited 67 ± 39 % of distal air space liquid clearance in animals pretreated with metyrapone and dexamethasone for 2 days. Emetine did not significantly affect distal air space liquid clearance in guinea-pigs pretreated with metyrapone alone.

Figure 5. Effects of emetine instillation on distal air space liquid clearance after metyrapone and dexamethasone pretreatment.

Figure 5

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Distal lung liquid albumin composition after 1 h in guinea-pigs instilled with the 5 % albumin solution after 2 days of pretreatment with the 11-β-hydroxylase inhibitor metyrapone (50 mg kg−1 day−1) or 2 days of combined pretreatment with metyrapone and dexamethasone (0.35 mg kg−1 day−1) under normal conditions (▪) or with emetine (□; 2.0 mg kg−1) instillation 15 h before the distal air space liquid clearance experiment. Under control conditions, distal air space liquid clearance was inhibited by 70 ± 15 % after emetine instillation (control, n = 8; emetine, n = 4). Emetine instillation did not affect the distal air space liquid clearance in the metyrapone-pretreated animals (control, n = 6; emetine, n = 4). However, when the endogenous cortisol was replaced with dexamethasone (0.35 mg kg−1 day−1), emetine instillation again inhibited distal air space liquid clearance by a fraction similar to control conditions (control, n = 5; emetine, n = 4). Values are means ± standard deviation. * P < 0.05 compared with control group without emetine, † P < 0.05 compared with metyrapone + dexamethasone without emetine; Student's t test.

αENaC expression studies

We collected lungs from each group and analysed mRNA levels for αENaC. Two days of metyrapone pretreatment reduced mRNA levels for αENaC (Fig. 3B); after 7 days metyrapone pretreatment, αENaC mRNA levels were not significantly different from those before metyrapone pretreatment (Fig. 3B). When dexamethasone was administered simultaneously with metyrapone, αENaC mRNA levels were greater than the levels in untreated control animals (Fig. 2B).

Haemodynamic parameters and blood parameters

Heart rate and blood pressure were measured in all animals during the distal air space liquid clearance measurements. There were no significant changes in either heart rates or blood pressures that could result in the observed changes (heart rate varied by < 10 % and blood pressure by < 15 %). Neither were there any changes due to treatments between baseline periods and experimental periods (none of the parameters varied by > 10 % over the experimental period). Haemodynamic parameters, haematocrit (< 5 % variation between and within groups), and plasma protein concentration (< 10 % variation between and within groups) were always within the normal variation in all the experimental groups.

DISCUSSION

This study demonstrates that endogenous cortisol is important for maintaining basal distal air space liquid clearance in adult guinea-pigs. Steroid hormones have been shown to regulate αENaC mRNA synthesis in the colon (Renard et al. 1995) and kidney (Volk et al. 1995). We hypothesized that endogenous cortisol synthesis inhibition would decrease Na+ uptake and distal air space liquid clearance. We found that after inhibition of cortisol synthesis with the 11-β-hydroxylase inhibitor metyrapone, the fractional inhibition of distal air space liquid clearance by the Na+ channel inhibitor amiloride was negligible. This suggested that the decrease in distal air space liquid clearance after cortisol inhibition was indeed related to a decrease in amiloride-sensitive Na+ uptake pathways across the distal air space epithelium.

In this study, adult guinea-pigs were pretreated with metyrapone for two, four, or seven consecutive days. When guinea-pigs were pretreated with metyrapone for 2–4 days, distal air space liquid clearance was decreased. After 7 days of pretreatment, distal air space liquid clearance had returned to a normal level. These changes in distal air space liquid clearance correlated well with changes in plasma cortisol levels. The results suggest that changes in plasma cortisol may regulate and control basal distal air space liquid clearance. The return of distal air space liquid clearance to a normal level over 7 days is likely to be related to the partly restored plasma levels of endogenous cortisol after prolonged treatment with metyrapone. Evidence suggests that this effect may be mediated by a feedback mechanism where adrenocorticotropin (ACTH) plays a key role in increasing cortisol synthesis when plasma cortisol levels are suppressed (Axelrod & Reisine, 1984). However, distal air space liquid clearance, but not plasma cortisol concentration, returned to normal levels after 7 days of metyrapone pretreatment. This may indicate that there could be additional mechanisms for the regulation of distal air space liquid clearance. Lung cells may develop alternative regulatory pathways of distal air space liquid clearance to compensate for low cortisol plasma levels, e.g. other hormone receptors could be up-regulated. Alternatively, the lungs of metyrapone-pretreated guinea-pigs became more sensitive to endogenous cortisol.

Since removal of endogenous cortisol decreased the rate of distal air space liquid clearance, we investigated whether supplying cortisol-depleted animals with exogenous steroid (dexamethasone) would restore distal air space liquid clearance to normal levels. When dexamethasone was administered concomitantly with metyrapone for 2 days, the inhibition of distal air space liquid clearance by metyrapone alone was reversed. A relatively high dose of dexamethasone (0.35 mg kg−1 day−1) restored distal air space liquid clearance to near-normal levels, but a dose equivalent to normal plasma cortisol levels (0.01 mg kg−1 day−1) was as efficient. This regulation of distal air space liquid clearance appears thus to be quantal, i.e. an all-or-none effect of dexamethasone, which is an atypical response to a steroid hormone; however, it is hard to determine from two doses only where on the dose-response curve the applied dexamethasone concentrations were. Liley and co-workers (Liley et al. 1988) demonstrated that surfactant protein A (SP-A) production by human fetal lung cells in vitro was regulated in a dose-dependent fashion by corticosteroids. It should be noted, however, that the dose-response relationship may be quite different in the in vivo situation, which is more complex than the in vitro cell system. Dexamethasone was selected instead of cortisol because it is more stable, has a longer half-life, and is a more specific glucocorticoid agonist than cortisol (Baxter & Rousseau, 1979), which also binds to the mineralocorticoid receptor and could act via this pathway. However, there is little evidence suggesting that mineralocorticoids can affect distal air space liquid clearance (Renard et al. 1995), implying that glucocorticoid receptor binding by cortisol is the major pathway for the regulation of distal air space liquid clearance.

What are the mechanisms by which cortisol exerts its effects on distal air space liquid clearance? Amiloride is an inhibitor of Na+ channels, among them ENaC, which has been shown to be an important pathway for Na+ uptake from distal air spaces and driving fluid absorption (Hummler et al. 1996; Matalon et al. 1996; Matthay et al. 1996). In this study, amiloride inhibited distal air space liquid clearance by ∼40 % in control guinea-pigs, a result similar to that reported in our previous study in guinea-pigs (Norlin et al. 1998). However, in animals pretreated with metyrapone for 2 days, there was no amiloride-induced inhibition. This finding suggests that the amiloride-sensitive channels were the pathways primarily affected by metyrapone pretreatment. We used 10−3 M amiloride since a large fraction of amiloride becomes protein bound and another fraction diffuses from the air spaces and therefore the effective amiloride concentration in the distal air spaces was probably significantly lower (O'Brodovich et al. 1990; Yue & Matalon, 1997). The amiloride-sensitive pathways that may have been affected by this amiloride concentration are the ENaC and the Na+-H+ exchanger. However, a vectorial transepithelial transport of Na+ is required to clear liquid from the distal air spaces, and the Na+-H+ exchanger is mainly believed to be associated with cell volume regulation (Shaw et al. 1990; O'Brodovich et al. 1991). Moreover, inhibition of the sodium channel, but not of the Na+-H+ exchanger or the Na+-glucose symport, slows liquid clearance from the newborn guinea-pig lung (Shaw et al. 1990; O'Brodovich et al. 1991). Consequently, it is unlikely that these pathways mediated the observed effects. Although we cannot exclude an effect on these systems, the main effect is probably on the amiloride-sensitive ENaC.

After the replacement of endogenous cortisol with exogenous dexamethasone, the amiloride-sensitive fraction of distal air space liquid clearance increased. Also, after 7 days of metyrapone pretreatment, when distal air space liquid clearance was restored to normal levels, amiloride sensitivity was again increased. This result suggests that the amiloride-sensitive pathways were increased compared with normal animals when steroid levels were replaced after endogenous plasma cortisol depletion. There are two possible explanations for this change in amiloride sensitivity. The first is that amiloride-sensitive pathways have been altered in their sensitivity to amiloride. It has been reported that amiloride binds to and inhibits ENaC during its open state (for review, see Garty & Palmer, 1997) and that binding affinity is dependent on subunit composition of the channel (Fyfe & Canessa, 1998). Therefore, if increased circulating steroids increase Po for ENaC or if the channel subunit composition is altered, amiloride could potentially inhibit the channel more efficiently and fractional inhibition of distal air space liquid clearance by amiloride would consequently increase. A second explanation is that there was an increase in the number of amiloride-sensitive channels (i.e. ENaC) in the membrane. We found that αENaC mRNA expression was reduced in animals pretreated with metyrapone, but in animals given both metyrapone and dexamethasone, as well as those given metyrapone for 7 days, αENaC mRNA levels were similar to those in normal animals. These data suggest that αENaC mRNA transcription contributes, at least in part, to the number of functional channels in the membrane and the amiloride-sensitive proportion of distal air space liquid clearance. Others have also shown that steroids increase ENaC mRNA levels, which results in a functional increase in amiloride-sensitive short-circuit current (Isc) across cell monolayers and single channel Po (Champigny et al. 1994; Venkatesh & Katzberg, 1997). mRNA expression for the αENaC was decreased in animals pretreated with metyrapone over 2 days. The decrease in αENaC mRNA expression in 2 day metyrapone-pretreated animals was 20 % compared with control, but the loss of amiloride sensitivity in these animals was 100 %. Developmental changes in expression of αENaC mRNA are similar in whole lung preparations and preparations from isolated alveolar epithelial type II cells (Finley et al. 1998). The apparent discrepancy between the decrease in αENaC mRNA expression and the fractional amiloride-induced inhibition of distal air space liquid clearance may be explained in various ways. First, total content of mRNA may be a mix of newly synthesized mRNA and degradation products of original mRNA. Second, the main action from cortisol may be by regulating de novo protein synthesis as has been shown in other systems (Verdi & Campagnoni, 1990; Ogata et al. 1995). In our study, emetine reduced distal air space liquid clearance in control animals and in animals that had been concomitantly pretreated with metyrapone and dexamethasone, but had no inhibitory effects in metyrapone-pretreated animals. An increased translation and cell membrane insertion of ENaC may mask an increased expression of mRNA for ENaC, because the mRNA produced will be consumed at a much higher rate than in the normal animal. Similarly, the observation that although αENaC mRNA was not significantly reduced in the metyrapone-treated animals after 2 days but both distal air space liquid clearance and amiloride sensitivity were affected suggests that cortisol may also be required for efficient processing of αENaC protein. It is also possible that the protein turnover is faster than the mRNA turnover or that cortisol is affecting synthesis or insertion of protein into cell membranes. In fact, in Madin-Darby canine kidney epithelial cells, ENaC has been found to have a relatively high turnover rate of about 2 h (Staub et al. 1997). Thus, if cortisol affects multiple steps of ENaC synthesis or degradation, the observed decrease in distal air space liquid clearance may be rapid, and could potentially be achieved without a total inhibition of mRNA synthesis and expression.

This study also indicates that there may be residual liquid absorption from the lungs of control animals through amiloride-insensitive pathways. Little is known about the ion channels that may be involved in these processes but a candidate has recently been cloned that transports sodium but is amiloride insensitive and is expressed in the lungs (Ding et al. 1997; Schwiebert et al. 1997). Even though we detected differences in the proportion of amiloride-sensitive to amiloride-insensitive distal air space liquid absorption between control animals, metyrapone-treated animals, and animals treated with metyrapone and dexamethasone, we cannot rule out the possibility that endogenous cortisol may also have effects on the amiloride-insensitive pathways for fluid absorption.

Cortisol may regulate secretion of catecholamines from the adrenal glands (Axelrod & Reisine, 1984). In the hedgehog, cortisol depletion may result in a subsequent excessive output of catecholamines (Brown et al. 1982a,b; Critchley et al. 1982) from the adrenal medulla and peripheral nervous system. The excessive release of endogenous catecholamines, primarily adrenaline, would be likely to increase distal air space liquid clearance via β-adrenergic stimulation (Berthiaume et al. 1987; Jayr et al. 1994; Pittet et al. 1994; Folkesson et al. 1996; Matthay et al. 1996; Finley et al. 1998; Norlin et al. 1998). However, we found that intra-alveolar propranolol did not affect distal air space liquid clearance in those animals, suggesting that no stimulation via endogenous release of catecholamines occurred. The failure of propranolol to decrease distal air space liquid clearance could, however, be due to the fact that propranolol is given into the distal air spaces, since circulating catecholamines act on basolateral adrenergic receptors. This is not likely to be the case since this administration route has been demonstrated to inhibit endogenous β-adrenergic stimulation from catecholamines in several experimental models (Jayr et al. 1994; Pittet et al. 1994; Modelska et al. 1997; Finley et al. 1998; Norlin et al. 1998). Another possible effect of cortisol depletion is alteration of cellular mechanisms other than vectorial Na+ transport mechanisms. To investigate this, we added the β-adrenergic agonist isoprenaline to the instillate of one metyrapone-pretreated group of guinea-pigs. Isoprenaline stimulated distal air space liquid clearance to normal levels. Accordingly, it is unlikely that the depletion of cortisol affected the β-adrenergic receptor and signalling system, although the effects on other cell systems have not been excluded.

We conclude that endogenous basal plasma levels of cortisol are important for maintaining normal lung liquid balance and distal air space liquid clearance in adult guinea-pigs. Cortisol modulates the amiloride-sensitive Na+ transport pathways in the lung, since amiloride sensitivity disappeared in cortisol-depleted animals along with a simlutaneous decrease in the expression of αENaC mRNA. Our study shows that cortisol exerts its effect on distal air space liquid clearance through regulation of de novo synthesis of Na+ channel proteins (ENaCs).

Acknowledgments

This work was supported by grants from the Swedish Natural Science Research Council, the Crafoord Foundation, The Wellcome Trust Programme (039124), the Royal Physiographic Society in Lund, the Magnus Bergwall Foundation, the Tenovus Tayside, the Anonymous Trust, and the Hierta Retzius Foundation.

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