J. Appl. Physiol.
88: 416–424, 2000.
Dexamethasone and thyroid hormone pretreatment
upregulate alveolar epithelial fluid clearance in adult rats
HANS G. FOLKESSON,1 ANDREAS NORLIN,1 YIBING WANG,2
PARISA ABEDINPOUR,1 AND MICHAEL A. MATTHAY2
1Department of Animal Physiology, Lund University, S-223 62 Lund, Sweden; and 2Cardiovascular
Research Institute, University of California, San Francisco, California 94143-0130
acute lung injury; alveolar epithelium; amiloride; b-adrenergic agonists; glucocorticoids; pulmonary edema; sodium transport
FLUID CLEARANCE FROM THE DISTAL air spaces of the lung
is driven by active sodium transport across the alveolar
epithelial barrier (4, 7, 16, 26, 30, 31, 34, 36, 44).
Several studies have demonstrated that exogenous
administration of b-adrenergic agonists can stimulate
alveolar fluid clearance (6, 7, 13, 22, 26, 34, 42). Also,
exogenous addition of cAMP can stimulate alveolar
fluid clearance (5, 34). In addition, endogenous release
of catecholamines in pathological conditions increases
alveolar fluid clearance by b-adrenergic receptor stimulation (39, 40) and under normal conditions at birth
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solely to indicate this fact.
416
(18). However, catecholamine-independent pathways
may also increase alveolar fluid clearance. For example, epidermal growth factor and transforming
growth factor-a stimulate alveolar fluid transport (8,
22).
Hormones released from the adrenal glands, such as
cortisol and aldosterone, and the thyroid gland, such as
3,38,5-triiodine-L-thyronine (T3) and L-thyroxine (T4),
have been implicated as important regulators of Na1
channels (2, 37). Aldosterone primarily affects Na1
channels in the kidney (37), whereas cortisol can affect
both kidney and lung Na1 channels (10, 37). Glucocorticoid regulation of alveolar epithelial fluid absorption
has been suggested from several studies in developing
and adult lungs (12, 25, 45, 47). Molecular investigations have suggested that the epithelial Na1 channel
(ENaC) possesses a glucocorticoid regulatory element
(10, 41). Glucocorticoids also promote alveolar epithelial type II cell maturation and differentiation in the
preterm fetus (24). In the adult lung, glucocorticoids
have been recommended by some investigators for the
treatment of pulmonary edema and inflammation in
established clinical lung injury (32). In addition, many
patients with cardiogenic or noncardiogenic pulmonary
edema are treated with glucocorticoids for a variety of
nonpulmonary disorders. Thus the in vivo effects of
glucocorticoids on alveolar epithelial fluid transport are
important.
Therefore, we first tested the hypothesis that alveolar epithelial fluid transport could be upregulated by
glucocorticoids. Adult rats were injected with intramuscular dexamethasone over 48 h before measurement of
in vivo alveolar fluid transport. The dose and administration schedule of dexamethasone were chosen from a
study on glucocorticoid-induced expression of aquaporin-1 water channels in the developing rat lung (27).
The second objective was to determine whether coadministration of dexamethasone and the thyroid hormone T3 would exert an additive effect on stimulating
alveolar fluid clearance. A synergistic effect has been
reported in the fetal near-term sheep lung (2), but no
studies have been done in the adult lung. The third
objective was to determine whether the increase in
alveolar fluid clearance after dexamethasone, T3, and
dexamethasone-plus-T3 pretreatment was secondary to
endogenous catecholamine release or to a proliferative
effect on alveolar epithelial type II cells. We also
investigated whether the sensitivity to b-adrenergic
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Folkesson, Hans G., Andreas Norlin, Yibing Wang,
Parisa Abedinpour, and Michael A. Matthay. Dexamethasone and thyroid hormone pretreatment upregulate alveolar
epithelial fluid clearance in adult rats. J. Appl. Physiol. 88:
416–424, 2000.—The in vivo effect of 48-h glucocorticoid and
thyroid hormone 3,38,5-triiodine-L-thyronine (T3) pretreatment on alveolar epithelial fluid transport was studied in
adult rats. An isosmolar 5% albumin solution was instilled,
and alveolar fluid clearance was studied for 1 h. Compared
with controls, dexamethasone pretreatment increased alveolar fluid clearance by 80%. T3 pretreatment stimulated alveolar fluid clearance by 65%, and dexamethasone and T3 had
additive effects (132%). Propranolol did not inhibit alveolar
fluid clearance in either group, indicating that stimulation
was not secondary to endogenous b-adrenergic stimulation.
With the use of bromodeoxyuridine in vivo labeling, there was
no evidence of cell proliferation. Alveolar fluid clearance was
partially inhibited by amiloride in all groups. Fractional
amiloride inhibition was greater in dexamethasone- and
dexamethasone-plus-T3-pretreated rats than in control animals, but less in T3-pretreated rats. In summary, pretreatment with dexamethasone, T3, or both in combination upregulate in vivo alveolar fluid clearance similarly to short-term
b-adrenergic stimulation. The effects are mediated partly by
increased amiloride-sensitive Na1 transport, because the
stimulated alveolar fluid clearance was more amiloride sensitive than in control rats. These observations may have
clinical relevance because glucocorticoid therapy is commonly
used with acute lung injury.
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
stimulation was altered after dexamethasone pretreatment as well as after the combined pretreatment with
dexamethasone and T3. The fourth objective was to
determine whether the fractional inhibition by amiloride changed after dexamethasone, T3, and dexamethasone-plus-T3 pretreatment.
METHODS
Animals
Preparation of the Instillates
A 5% albumin instillate solution was prepared by dissolving 50 mg/ml of bovine serum albumin (Sigma Chemical, St.
Louis, MO) in an aqueous solution of 0.9% NaCl. For studies
of the potential effects of endogenous epinephrine release on
alveolar fluid clearance after dexamethasone, T3, and dexamethasone-plus-T3 pretreatment, 1024 M of the general b-adrenergic antagonist propranolol (Sigma Chemical) was added
to the 5% albumin instillate solution. To investigate whether
b-adrenergic agonists could stimulate alveolar fluid clearance
after dexamethasone and dexamethasone-plus-T3 retreatment, terbutaline (1024 M; Sigma Chemical) was instilled
with the 5% albumin solution. For the determination of the
fractional inhibition by amiloride on alveolar fluid clearance,
amiloride (1023 M; Sigma Chemical) was instilled with the 5%
albumin solution. We used amiloride at the concentration of
1023 M because ,50% of the amiloride is protein bound and
another significant fraction escapes from the air spaces,
resulting in functional in vivo concentrations closer to 1024 M
(35, 49).
Glucocorticoid and Thyroid Hormone Pretreatment
The rats were injected intramuscularly (im) with 0.35
mg/kg body wt of dexamethasone (water soluble; Sigma
Chemical) dissolved in a 0.9% NaCl solution. The rats were
pretreated on 2 consecutive days ,24 h apart, and also on the
morning of the third day, the same day on which the in vivo
alveolar fluid clearance experiment was done. This dose and
administration protocol were adapted from a study by King
and colleagues (27) on glucocorticoid-induced expression of
aquaporin-1 in developing rat lungs. Control rats were treated
with 0.9% NaCl alone. In some rats, the acute effects from
instillation of dexamethasone simultaneously with the 5%
albumin solution were examined.
In a second set of rats, synergistic effects from pretreatment with dexamethasone and the thyroid hormone T3
(dissolved in concentrated NH4, then diluted 1003 and pH
corrected; Sigma Chemical) were investigated. The rats were
injected with 0.35 mg dexamethasone/kg body wt im and 30
µg T3/rat (im) on 2 consecutive days, ,24 h apart, and also on
the morning of the third day, the day on which the alveolar
fluid clearance experiment was done. This dose and administration protocol of T3 were adapted from a study by Barker
and colleagues (2) on synergistic effects of glucocorticoids and
thyroid hormone on newborn sheep lungs. Rats were also
pretreated with 30 µg T3 im alone/rat on 2 consecutive days,
,24 h apart, and on the morning of the third day, the day on
which the alveolar fluid clearance experiment was done.
General Experimental Protocol
In all rats, after surgery, a 30-min baseline of stable heart
rate and blood pressure was required before fluid instillation.
An instillation tubing (PE-50 catheter; Clay Adams, Becton
Dickinson) was gently passed through the tracheal tube and
into the left lung at the end of the 30-min baseline. Then, 1 ml
of fluid (i.e., 3 ml/kg body wt with or without stimulatory or
inhibitory substances) was instilled over 25 min into the left
lung for the 1-h studies. The 1-h studies began at the start of
instillation of the fluid. The fluid was instilled by injecting
0.04 ml/min by using a 1-ml syringe. After the instillation, the
tubing was withdrawn. The location of the instilled fluid was
clearly visible in the lungs by postmortem examination.
At the end of the experiments, a blood sample was obtained, the abdomen was opened, and the rats were exsanguinated by transecting the abdominal aorta. The lungs were
removed through a median sternotomy. An alveolar fluid
sample (0.1–0.2 ml) was obtained by gently passing the
sampling catheter (PE-50 catheter; Clay Adams, Becton
Dickinson) into a wedged position in the instilled area of the
left lung. We previously reported that fluid aspirated with a
catheter wedged into the distal air spaces is a good reflection
of alveolar fluid protein concentration (6). Protein concentrations in alveolar fluid samples and in plasma samples were
measured by the Lowry method (29) modified for use on
microtiter plates.
Specific Protocols
Group 1: Control (1-h) studies (n 5 9). After the 30-min
baseline period, 3 ml/kg body wt of the 5% albumin solution
were instilled into the left lower lobe. The rats were then
studied for 1 h. After this time, the rats were exsanguinated
and processed as described in General Experimental Protocol.
Group 2: Dexamethasone studies (n 5 13). The rats (n 5 9)
were pretreated for 2 consecutive days with dexamethasone
as described in Glucocorticoid and Thyroid Hormone Pretreatment. Then, on the third day, the animals were anesthetized
and prepared for the alveolar fluid clearance measurements.
After the 30-min baseline period, 3 ml/kg body wt of the 5%
albumin solution were instilled into the left lower lobe. The
rats were studied for 1 h. After this time, the rats were
exsanguinated and processed as described in General Experimental Protocol. In some rats (n 5 4), the acute effects of
dexamethasone were investigated after simultaneous instillation of the 5% albumin solution and the dexamethasone.
Group 3: T3 studies (n 5 4). The rats were pretreated for 2
consecutive days with T3 as described in Glucocorticoid and
Thyroid Hormone Pretreatment. Then, on the third day, the
animals were anesthetized and prepared for the alveolar fluid
clearance measurements. After the 30-min baseline period,
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Surgical preparation and ventilation. Male SpragueDawley rats (n 5 89) weighing 300–350 g (B&K Universal,
Sollentuna, Sweden) were anesthetized with 50 mg/kg body
wt of pentobarbital (Nembutal, Apoteksbolaget, Umeå, Sweden) intraperitoneally (ip). A 2-mm-inner-diameter (PE-240;
Clay Adams, Becton Dickinson, Parsippany, NJ) endotracheal
tube was inserted through a tracheostomy. A PE-50 catheter
(Clay Adams, Becton Dickinson) was inserted into the right
carotid artery to monitor systemic blood pressure and to
obtain blood samples.
Pancuronium bromide (0.3 mg/kg body wt, Pavulon, Organon-Teknika, Boxtel, The Netherlands) was given intraarterially hourly for neuromuscular blockade. The rats were
maintained in the left lateral decubitus position during the
experiments. The rats were ventilated with a constantvolume piston pump (Harvard Apparatus, Dover, MA) with
an inspired oxygen fraction of 1.0 and with peak airway
pressures of 7–9 cmH2O during the baseline period, supplemented with a positive end-expiratory pressure of 4 cmH2O.
The Ethics Review Committee on Animal Experiments at
Lund University approved the experiments.
417
418
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
Hemodynamics and Airway Pressure
Peak airway pressure, systemic blood pressure, and heart
rate were measured by using calibrated pressure transducers
(UFI model 1050BP, BioPac Systems, Goleta, CA) connected
to analog-to-digital converters and amplifiers (MP100 and
DA100, respectively, BioPac Systems) and continuously recorded on an IBM computer with Acknowledge 3.0 software
(BioPac Systems).
Alveolar Fluid Clearance
The increase in alveolar protein concentration over 1 h was
used to measure clearance of fluid from the distal air spaces,
as we have done before (7, 34, 40, 44). Data on alveolar fluid
clearance are shown in two ways. First, alveolar fluid clearance is presented as a ratio between the final aspirated
alveolar fluid protein concentration and the instilled fluid
protein concentration. The ratio of final to instilled protein
concentration provides direct evidence for alveolar fluid clearance, because fluid must be removed for the final alveolar
protein concentration to rise. Because there were no changes
in the epithelial permeability to protein and very little
protein was transported out of the air spaces in any of the
groups (see RESULTS), this method is accurate for measuring
fluid clearance from the distal air spaces of the lungs. The
second method is based on calculating alveolar fluid clearance
(AFC) from the following equation
AFC 5 [(VI 2 VF)/VI] 3 100
(1)
where AFC is alveolar fluid clearance, VI is instilled fluid
volume, and VF is final alveolar fluid volume calculated from
the increase in the concentration of protein in the final
alveolar aspirate from the instilled protein amount.
The term ‘‘alveolar,’’ however, does not imply that all
reabsorption of fluid occurs at the alveolar level; some fluid
reabsorption may occur across distal bronchial epithelium
(1).
Alveolar Type II Cell Proliferation
To investigate whether proliferation of alveolar epithelial
type II cells occurred in response to dexamethasone injections
with or without T3, four additional rats were used. One rat
was used as control and was injected with 0.9% NaCl im three
times during 48 h. The second rat was injected with dexamethasone (0.35 mg/kg body wt) at the same times during 48
h, and the third rat was injected with T3 (30 µg/rat). The
fourth rat was injected with both dexamethasone (0.35 mg/kg
body wt) and T3 (30 µg/rat) together at the same times during
48 h.
Tissue preparation. Forty hours after the first injection
(38), the rats were injected with 100 mg/kg body wt of
bromodeoxyuridine ip (BrdU; Boehringer Mannheim, Mannheim, Germany). Then, 48 h after the first injection, the rats
were killed by an ip overdose of pentobarbital sodium mixed
with heparin for anticoagulation. The abdomen was opened,
and the abdominal aorta was transected to exsanguinate the
animals. The chest was opened through a midline sternotomy.
The lungs were perfused blood free via the pulmonary artery.
When the lungs were free of blood, the perfusate solution was
changed to 4% paraformaldehyde for fixation. The lungs were
then left in the paraformaldehyde for 4 h and then cut into
1-cm3 cubes and transferred into a 30% sucrose solution. The
sucrose-immersed lung tissue was embedded in OCT (Miles,
Elkhart, IN), and the tissue was snap-frozen in liquid nitrogen. Then, 4- to 5-µm sections were cut and mounted on
3-aminopropyl-triethoxysilane-coated slides (Fisher Scientific, Pittsburgh, PA). The slides were stored at 270°C until
stained for BrdU.
Immunohistochemistry. Cell proliferation was determined
by a BrdU assay (38) with a specific antibody. The sections
were incubated in 1% H2O2 (Sigma Chemical) in phosphate-
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3 ml/kg body wt of the 5% albumin solution were instilled into
the left lower lobe. The rats were studied for 1 h. After this
time, the rats were exsanguinated and processed as described
in the General Experimental Protocol.
Group 4: Dexamethasone-plus-T3 studies (n 5 6). The rats
were pretreated for 2 consecutive days with dexamethasone
and T3 as described in Glucocorticoid and Thyroid Hormone
Pretreatment. Then, on the third day, the animals were
anesthetized and prepared for the alveolar fluid clearance
measurements. After the 30-min baseline period, 3 ml/kg
body wt of the 5% albumin solution were instilled into the left
lower lobe. The rats were studied for 1 h. After this time, the
rats were exsanguinated and processed as described in General Experimental Protocol.
Group 5: Effect of propranolol (1024 M) in dexamethasone
(n 5 4)-, T3 (n 5 4)-, dexamethasone-plus-T3-pretreated (n 5
4), and control (n 5 4) rats. The rats were pretreated for 2
consecutive days with saline, dexamethasone, T3, or dexamethasone plus T3 as described in Glucocorticoid and Thyroid Hormone Pretreatment. Then, on the third day, the
animals were anesthetized and prepared for the alveolar fluid
clearance measurements. The general b-adrenergic antagonist propranolol was added to the instillate and instilled into
the saline-, dexamethasone-, T3-, or dexamethasone-plus-T3pretreated rats to determine whether the effects were mediated by an increased release of endogenous epinephrine. After
the 30-min baseline period, 3 ml/kg body wt of the propranololcontaining 5% albumin solution were instilled into the left
lower lobe. The rats were studied for 1 h. After this time, the
rats were exsanguinated and processed as described in General Experimental Protocol.
Group 6: Effect of terbutaline in dexamethasone (n 5 7)-,
dexamethasone-plus-T3-pretreated (n 5 6), and control rats
(n 5 5). The rats were pretreated for 2 consecutive days with
dexamethasone or dexamethasone plus T3 as described in
Glucocorticoid and Thyroid Hormone Pretreatment. Then, on
the third day, the animals were anesthetized and prepared for
the fluid clearance measurements. The b2-adrenergic agonist
terbutaline was added to the instillate to investigate whether
terbutaline had an additional stimulatory effect in dexamethasone-pretreated, dexamethasone-plus-T3-pretreated, and control rats. After the 30-min baseline period, 3 ml/kg body wt of
the 5% albumin solution were instilled into the left lower lobe
and the rats were studied for 1 h. After this time, the rats
were exsanguinated and processed as described in General
Experimental Protocol.
Group 7: Effect of amiloride (1023 M) in dexamethasone
(n 5 4)-, T3 (n 5 4)-, dexamethasone-plus-T3-pretreated (n 5
6), and control rats (n 5 4). The rats were pretreated for two
consecutive days with dexamethasone, T3, or dexamethasone
plus T3 as described in Glucocorticoid and Thyroid Hormone
Pretreatment. Then, on the third day, the animals were
anesthetized and prepared for the fluid clearance measurements. Amiloride, a Na1 channel inhibitor, was added to the
5% albumin instillate to determine the fraction of the fluid
clearance from the distal air spaces that was mediated by
amiloride-sensitive Na1 channels. After the 30-min baseline
period, 3 ml/kg body wt of the amiloride-containing 5%
albumin solution was instilled into the left lower lobe. The
rats were studied for 1 h. After this time, the rats were
exsanguinated and processed as described in General Experimental Protocol.
419
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
Statistics
All the data are summarized and presented as means 6 SD
or means 6 SE when appropriate. The data were analyzed by
one-way ANOVA with Student-Newman-Keuls post hoc test
or Student’s t-test, both paired and unpaired, when appropriate. Statistical significance was set as P , 0.05.
RESULTS
Effect of dexamethasone. There were no changes in
systemic blood pressure and airway pressure after fluid
instillation in the dexamethasone-pretreated rats or in
those that were acutely treated with dexamethasone
compared with control rats or with baseline conditions
in the 1-h studies.
Dexamethasone pretreatment resulted in a significant increase in the ratio of final to instilled protein
concentration over 1 h compared with in control rats
(Fig. 1). There was a similar increase in the calculated
alveolar fluid clearance (Table 1). Dexamethasone alone
increased alveolar fluid clearance by 80% compared
with in control rats. There was no effect on the ratio of
final to instilled protein concentration or alveolar fluid
clearance from the acute dexamethasone exposure compared with control rats (Table 1). Saline injections into
the control rats did not affect alveolar fluid clearance
compared with normal noninjected control rats (data
not shown).
Effect of T3. There were no changes in systemic blood
pressure and airway pressure after fluid instillation in
Fig. 1. Alveolar fluid clearance expressed as ratio of final to instilled
protein concentration in dexamethasone (n 5 9)- and salinepretreated rats (n 5 9). Values are means 6 SD. Dexamethasone
pretreatment continued for 2 days with 1 intramuscular (im) injection of 0.35 mg dexamethasone/kg body wt each day. Alveolar fluid
clearance experiment was done on the 3rd day. Pretreatment with
dexamethasone resulted in an 80% increase in alveolar fluid clearance. * P , 0.05 compared with control rats (ANOVA with Tukey’s
post hoc test).
the T3-pretreated rats compared with control rats or
baseline conditions in the 1-h studies.
T3 pretreatment resulted in a significant increase in
the ratio of final to instilled protein concentration over
1 h compared with control rats, but was not different
from that in dexamethasone-pretreated rats (Fig. 2).
There were similar increases in the calculated alveolar
fluid clearance (Table 1). T3 alone increased alveolar
fluid clearance by 65% compared with in control rats.
Table 1. Effect of dexamethasone and thyroid hormone
pretreatment on alveolar liquid clearance over 1 h
in anesthetized rats
Alveolar Albumin
Concentration, g/dl
Group
Control
Dexamethasone
pretreatment
Dexamethasone
acute treatment
Dexamethasone1T3
T3
Terbutaline
Dexamethasone1
terbutaline
Dexamethasone1
T3 1terbutaline
Propranolol
Dexamethasone1
propranolol
T3 1 propranolol
Dexamethasone1
T3 1 propranolol
Amiloride
Dexamethasone1
amiloride
T3 1 amiloride
Dexamethasone1
T3 1 amiloride
Alveolar Fluid
Clearance, % of
instilled volume
n
Instilled
Final
9
4.63 6 0.48
5.58 6 0.65
17.0 6 2.0
9
4.87 6 0.33
7.04 6 0.45
30.6 6 4.0*
4
6
4
5
4.96 6 0.44
5.78 6 0.25
5.62 6 0.05
4.99 6 0.20
6.22 6 0.77
9.70 6 1.51
7.88 6 0.92
7.21 6 0.50
20.0 6 5.6
39.4 6 8.0*†
28.0 6 8.2*
30.7 6 2.2*
7
5.03 6 0.89
7.47 6 2.10
30.8 6 8.2*
4
4
5.96 6 0.05
4.98 6 0.76
9.77 6 0.93
6.00 6 0.71
38.6 6 5.4*‡
17.3 6 2.2
4
4
4.66 6 0.32
4.65 6 0.29
6.53 6 0.57
6.50 6 0.45
28.6 6 2.5*
28.4 6 3.4*
4
6
4.70 6 0.35
4.71 6 0.15
7.44 6 0.41
5.03 6 0.25
36.9 6 1.6*‡
9.7 6 3.3*
4
6
4.80 6 0.02
5.84 6 0.50
5.68 6 1.13
7.32 6 0.47
13.3 6 1.5†
20.0 6 7.9
4
5.96 6 0.05
7.50 6 0.34
20.4 6 3.6
Values are means 6 SD. n, No. of rats; T3 , 3,38,5-triiodine-Lthyronine. * P , 0.05 vs. control; † P , 0.05 vs. dexamethasone; ‡ P ,
0.05 vs. dexamethasone-terbutaline (ANOVA with Tukey’s post hoc
test).
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buffered NaCl (PBS) with 0.1% Triton X-100 for 5 min and
then washed with PBS. The slides were then incubated with
100 µg/ml Pronase E (Sigma Chemical) in 20 mM Tris · HCl
and 20 mM CaCl2, pH 7.6, for 10–30 min and then rinsed in
PBS. To remove the DNA-binding proteins, the sections were
incubated in ice-cold 0.1 N HCl for 10 min. The DNA was then
denatured by incubating the sections in 2 N HCl for 30 min at
37°C, and then the acid was neutralized with 0.1 M borax
(Sigma Chemical), pH 8.5, for 5–10 min. After washing in
PBS, the sections were incubated in 3% horse serum in PBS
for 30 min at room temperature. Then, the sections were
incubated with an anti-BrdU monoclonal antibody (Dakopatts, Glostrup, Denmark), diluted 1:50 in 3% horse serum
for 1 h at room temperature. After washing of the slides in
PBS, the sections were incubated with a biotinylated goat
anti-mouse IgG Fc (Sigma Chemical) diluted 1:400 in 3%
horse serum in PBS for 1 h at room temperature and washed
in 50 mM Tris-buffered saline (TBS). Then, a streptavidinalkaline phosphate conjugate (Sigma Chemical), diluted 1:20
in TBS, was added to the slides for 30–60 min. The sections
were washed in 0.1 M Tris · HCl (pH 9.5), 0.1 M NaCl, and 50
mM MgCl2. Then, the sections were incubated in freshly
prepared substrate solution [44 µl NBT in 10 ml 0.1 M
Tris · HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2, and 33 µl
BCIP; GIBCO-BRL Life Technologies] at room temperature
for 5 min in the dark. After substrate incubation, the slides
were washed in distilled H2O. After dehydration with alcohol,
the sections were mounted with Canada balsam (Sigma
Chemical).
Cell counting. From each treatment, two random sections
were selected. On each section, five random fields were
selected, and the stained cells were counted on all five fields
in each section and averaged. The cell numbers were then
compared between the different groups.
420
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
Effect of dexamethasone in combination with T3.
There were no changes in systemic blood pressure and
airway pressure after fluid instillation in the dexamethasone-plus-T3-pretreated rats compared with control rats or with baseline conditions in the 1-h studies.
Dexamethasone-plus-T3 pretreatment resulted in a
significant increase in the ratio of final to instilled
protein concentration over 1 h compared with control
rats and a further increase in the above-mentioned
ratio compared with dexamethasone-pretreated animals (Fig. 2). There were similar increases in the
calculated alveolar fluid clearance (Table 1). The combination of dexamethasone and T3 stimulated alveolar
fluid clearance by 132% compared with the 80% stimulation by dexamethasone alone and 65% stimulation by
T3 alone.
Effect of terbutaline and propranolol treatment. The
addition of the b-adrenergic agonist terbutaline to the
instillate of control rats increased the ratio of final to
instilled protein concentration to 1.44 6 0.05 vs. 1.21 6
0.03 for control rats and alveolar fluid clearance was
increased by ,80%, similar to that after dexamethasone pretreatment (Fig. 3). However, addition of terbutaline to dexamethasone-pretreated rats did not result
in a further increase in the ratio of final to instilled
protein concentration (Fig. 3) nor in alveolar fluid
clearance (Table 1) compared with normal rats instilled
with terbutaline. Addition of terbutaline to dexamethasone-plus-T3-pretreated rats also did not result in a
further increase in the ratios of final to instilled protein
concentration (Fig. 3) or in the alveolar fluid clearance
(Table 1) compared with the dexamethasone-plus-T3pretreated rats instilled with the 5% albumin solution.
Propranolol instillation to dexamethasone-pretreated
rats had no effect on alveolar fluid clearance compared
DISCUSSION
Glucocorticoids have been implicated in stimulating
alveolar fluid clearance, especially in the near-term
lung (25, 45, 47), but no studies have investigated
whether glucocorticoids can stimulate alveolar fluid
Fig. 3. Alveolar fluid clearance expressed as the final to instilled
protein concentration ratio in control rats (n 5 9), rats stimulated
with the b2-adrenergic agonist terbutaline (Terb; n 5 5), and dexamethasone (Dex; n 5 9)-, terbutaline-instilled dexamethasone (n 5
7)-, dexamethasone-plus-T3 (n 5 6)-, and terbutaline-instilled dexamethasone-plus-T3-pretreated rats (n 5 4). Values are means 6 SD.
Dexamethasone pretreatment continued for 2 days with 1 im injection of 0.35 mg dexamethasone/kg body wt with or without 30 µg T3
daily. Alveolar fluid clearance experiment was done on the 3rd day.
Instillation of a terbutaline-containing albumin solution to dexamethasone- or dexamethasone-plus-T3-pretreated rats did not further stimulate alveolar fluid clearance compared with corresponding
control rats.
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Fig. 2. Alveolar fluid clearance expressed as ratio of final to instilled
protein concentration in control rats (n 5 9), dexamethasone (n 5 9)-,
3,38,5-triiodine-L-thyronine (T3 ; n 5 4)-, and dexamethasone-plus-T3stimulated rats (n 5 6). Values are means 6 SD. Dexamethasone
pretreatment continued for 2 days with one intramuscular (im)
injection of 0.35 mg dexamethasone/kg body wt with or without 30 µg
T3 daily. Alveolar fluid clearance experiment was done on the 3rd day.
Pretreatment with T3 and dexamethasone resulted in a further
significant stimulation of alveolar fluid clearance to 132% of control
levels. T3 pretreatment alone also stimulated alveolar fluid clearance, but to a lesser extent than dexamethasone pretreatment. * P ,
0.05 compared with control rats. †P , 0.05 compared with dexamethasone- and dexamethasone-T3-pretreated rats (ANOVA with Tukey’s
post hoc test).
with the dexamethasone-pretreated rats (Table 1). Furthermore, instillation of the albumin solution containing propranolol to T3- and dexamethasone-plus-T3treated rats also did not affect alveolar fluid clearance
(Table 1).
Effect of amiloride. To determine whether the increase in alveolar fluid clearance from dexamethasone
pretreatment was dependent on an increase in sodium
uptake by lung epithelial cells, amiloride (1023 M) was
added to the 5% albumin instillate and instilled into
the distal air spaces of the rats. We also determined
whether fractional amiloride inhibition was different
among control, dexamethasone-pretreated, and dexamethasone-plus-T3-pretreated rats. Amiloride inhibited the increase in alveolar fluid clearance from the
distal air spaces in dexamethasone-pretreated rats
(Fig. 4, Table 1). Also, the fractional inhibition by
amiloride was significantly greater in dexamethasonepretreated rats than in control rats (Fig. 5).
Amiloride also inhibited the increase in alveolar fluid
clearance in the dexamethasone-plus-T3-pretreated rats
(Fig. 4, Table 1). The fractional inhibition by amiloride
was significantly greater in dexamethasone-plus-T3pretreated rats than in control rats (Fig. 5). However,
the inhibition by amiloride of T3-stimulated alveolar
fluid clearance did not reach statistical significance
(P 5 0.20) (Fig. 4).
Cell proliferation. Neither of the treatments induced
an alveolar epithelial cell proliferation as assessed by
the BrdU assay (data not shown).
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
clearance in the adult lung. The results of these in vivo
studies in adult rats demonstrated a marked upregulation in alveolar epithelial fluid transport capacity after
48 h of dexamethasone pretreatment. In addition, there
was an independent effect of thyroid hormone pretreat-
Fig. 5. Percent inhibition of alveolar fluid clearance by amiloride
(1023 M) in dexamethasone (n 5 4)-, dexamethasone-plus-T3 (n 5 6)-,
T3 (n 5 4)-, and saline-pretreated rats (n 5 4). Values are means 6
SD. Dexamethasone pretreatment continued for 2 days with 1 im
injection of 0.35 mg dexamethasone/kg body wt with or without 30 µg
T3 daily. The alveolar fluid clearance experiment was done on the 3rd
day. The amiloride-containing albumin solution in the dexamethasonepretreated rats inhibited alveolar fluid clearance more than in
control rats, although amiloride had less effect in T3-pretreated rats.
A higher amiloride sensitivity was also observed after dexamethasoneplus-T3 pretreatment. * P , 0.05 compared with control; † P , 0.05
compared with T3-pretreated rats (ANOVA with Tukey’s post hoc
test).
ment (T3) as well as an additive effect of T3 when given
together with dexamethasone in the mature rat lung.
Amiloride inhibited most (62%) of the dexamethasone- and dexamethasone-plus-T3-stimulated increase
in alveolar fluid clearance. Thus most of the stimulated
increase in alveolar fluid clearance probably occurred
by increasing Na1 uptake through amiloride-sensitive
channels in the alveolar epithelial cells. In normal rats,
amiloride reduced alveolar fluid clearance by ,48%,
similar to the fractional inhibition that has been reported previously by us (23, 26) and other investigators
(4, 16, 30). After dexamethasone pretreatment, this
fraction increased to 62%, suggesting a modest upregulation of the amiloride-sensitive Na1 transport pathways. The amiloride-sensitive fraction was similar in
the rats pretreated with dexamethasone and T3, suggesting that T3 had limited effects on the amiloridesensitive Na1 channels in this model. Also, T3 alone had
a significantly lower amiloride-sensitive fraction (38%)
than dexamethasone-treated (62%) and control animals (48%) (Fig. 5), suggesting that T3 may primarily
upregulate the amiloride-insensitive fraction of alveolar fluid clearance. Also, the time required for the
stimulatory effect by dexamethasone suggests that the
effect occurred by transcriptional and/or translational
mechanisms. Other investigators have reported that
dexamethasone pretreatment increased the mRNA levels for the three subunits of the ENaC (10, 41, 47) as
well as that of the translated protein in the cell
membrane (41). There are also data that dexamethasone pretreatment increases the activity and expression of the basolaterally located Na1-K1-ATPase (3, 25).
Thus it is likely that an upregulated capacity to transport Na1 across the alveolar epithelium is the mechanism responsible for the increased fluid absorption
after dexamethasone pretreatment in the adult rat.
Our data do not precisely determine whether it was
mediated by increases in ENaC expression and function, Na1-K1-ATPase activity and expression, or both.
The data suggest, however, that an increase in ENaC
function may be a significant component because the
fractional inhibition was greater after dexamethasone
and dexamethasone-plus-T3 pretreatment.
One objective of these studies was to test the hypothesis that pretreatment with dexamethasone resulted in
stimulation of clearance of fluid from the distal air
spaces of the lung and to investigate whether the
responsiveness to b-adrenergic stimulation was altered
by the dexamethasone and the dexamethasone-plus-T3
pretreatment. We and other investigators have previously reported that b-stimulatory agents, growth factors, and endogenous catecholamines were associated
with an increase in liquid removal from the alveolar
compartment of the lung (7, 22, 26, 34, 40, 42). It has
also been suggested that ENaC possesses a glucocorticoid regulatory element, potentially making it sensitive
to glucocorticoid regulation (10, 41). We investigated
whether the effects were secondary to endogenous
epinephrine release and compared the effects of dexamethasone pretreatment on alveolar fluid clearance
with the stimulatory effect of b-adrenergic agonists. We
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on June 17, 2017
Fig. 4. Alveolar fluid clearance expressed as final-to-instilled protein
concentration ratio in dexamethasone (n 5 4)-, dexamethasoneplus-T3 (n 5 6)-, T3 (n 5 4)-, and saline-pretreated rats instilled with
amiloride (1023 M)-containing instillate (n 5 4). Values are means 6
SD. Dexamethasone pretreatment continued for 2 days with 1 im
injection of 0.35 mg dexamethasone/kg body wt with or without 30 µg
T3 daily. Alveolar fluid clearance experiment was done on the 3rd day.
Amiloride-containing albumin solution in dexamethasone- and dexamethasone-plus-T3-pretreated rats inhibited alveolar fluid clearance
to a higher degree than in control rats. Inhibition by amiloride in
T3-pretreated rats did not reach statistical significance (P 5 0.20).
* P , 0.05 compared with control rats; † P , 0.05 compared with rats
instilled with 5% albumin solution without amiloride (ANOVA with
Tukey’s post hoc test).
421
422
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
methasone pretreatment in rats (3, 25). It is also
possible that Na1 flux through the amiloride-sensitive
Na1 channels is increased after dexamethasone and
dexamethasone-plus-T3 pretreatment.
The effect from dexamethasone pretreatment was
not secondary to endogenous catecholamine release,
because propranolol did not block the dexamethasone
stimulation of alveolar fluid clearance. We investigated
this possibility because several studies have reported
that endogenous release of epinephrine can regulate
the rate of alveolar fluid clearance (18, 28, 39, 40).
However, the longer-term upregulation by dexamethasone, T3, or dexamethasone-plus-T3 pretreatment of
alveolar fluid clearance in this study was not mediated
by endogenous release of catecholamines. The small
molecular size of propranolol made it likely that adequate concentrations were reached quickly at the
b-adrenergic receptor sites on the basolateral side of
the alveolar epithelial cells. Thus complete blockage
would be expected within the 1-h experimental time.
Another potential concern would be if the per se
injections of dexamethasone, T3, or dexamethasone
plus T3 would cause epinephrine release. To address
this concern, we injected normal rats with 0.9% NaCl at
the same times as they would have received the stimulants. We found no effects from the injections per se,
indicating that no significant epinephrine release occurred. Moreover, a relatively long time period passed
between the injections and the start of the fluid clearance experiments. Also, recent data show that the
stimulatory effect of epinephrine is short lived in vivo
(11).
Could the effect be related to increased cell proliferation after dexamethasone or dexamethasone-plus-T3
pretreatment? Recent experiments have demonstrated
that proliferation of alveolar epithelial type II cells may
be correlated with an enhanced capacity to absorb
alveolar fluid (21, 48). However, we did not observe an
increase in cell proliferation after either of the treatments in this study.
What is the physiological role of glucocorticoids in
the lung? Glucocorticoids have been implicated as
regulators of fetal and neonatal lung maturation (12).
For example, glucocorticoids may be involved together
with b-adrenergic agonists in the regulation of reabsorption of fetal lung fluid from the air spaces at delivery
and onset of breathing, as has been suggested from
several molecular studies (25, 47). Glucocorticoids also
upregulate both alveolar ENaC (45, 47) and Na1-K1ATPase (3, 25) in near-term lungs, suggesting that they
might affect the vectorial Na1 transport that is responsible for maintaining dry alveoli. In adult lung, glucocorticoids have been used in the late phase of acute
respiratory distress syndrome to hasten recovery from
clinical acute lung injury (32).
In summary, dexamethasone pretreatment of rats
increased the rate of alveolar fluid clearance. The
stimulation was further potentiated by pretreatment
with dexamethasone and T3, suggesting that an additive effect by these two hormones can occur in the adult
lung, in contrast to the synergistic effect in the fetal
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on June 17, 2017
also investigated whether addition of b-adrenergic
agonists to dexamethasone- and dexamethasone-plusT3-pretreated rats resulted in an additional stimulation of the fluid clearance. Propranolol did not affect the
dexamethasone-, T3-, or the dexamethasone-plus-T3stimulated alveolar fluid clearance, so it is not likely
that dexamethasone, T3, or dexamethasone plus T3
acted via endogenous epinephrine release. Terbutaline,
the b-adrenergic agonist, and dexamethasone pretreatment were equally potent in stimulating alveolar fluid
clearance. Also, terbutaline had no additional stimulatory effect on alveolar fluid clearance in dexamethasone- or in dexamethasone-plus-T3-pretreated rats.
Could an increased expression of aquaporins in lung
epithelium or endothelium increase alveolar fluid clearance? Aquaporins are present in the lung as observed in
both expression and functional studies (9, 15, 19, 20,
33, 46), and steroid-pretreatment over 48 h increases
aquaporin-1 expression in developing rat lungs (27). It
is not known whether aquaporin-5 expression in alveolar epithelial type I cells (15) is sensitive to glucocorticoid stimulation. Even if that were the case, it is
unlikely that the observed increase in alveolar fluid
clearance in this study could be explained by increased
aquaporin expression. Transepithelial water movement requires an osmotic driving force for water to
cross the barrier created by transepithelial Na1 transport via amiloride-sensitive and -insensitive pathways
and by basolaterally located Na1- K1-ATPase (17).
In several species, a significant fraction of the stimulatory effect from b-adrenergic agonists on alveolar
fluid clearance can also be inhibited by amiloride (7, 26,
34, 40). However, because both basal and stimulated
alveolar fluid clearance by b-adrenergic agonists is
inhibited similarly by amiloride, the data indicate that
b-adrenergic agonists increase alveolar fluid clearance
by stimulation of both amiloride-sensitive and -insensitive pathways. The amiloride-insensitive fraction may
be related, in part, to cation channels that are not
inhibited by amiloride. Recent data (14, 43) suggest the
existence of a rodtype, cyclic, nucleotide-gated cation
channel in the alveolar epithelium that could be involved in fluid movement in the lung. Other pathways
are possible, of course, because no definite proof exists
presently concerning the makeup of the amilorideinsensitive fraction. However, the results from this
study suggest that dexamethasone pretreatment affects the amiloride-sensitive fraction of alveolar fluid
clearance more than it affects the amiloride-insensitive
fraction. In contrast, T3 might stimulate alveolar fluid
clearance by stimulating the amiloride-insensitive fraction of Na1 channels more than the amiloride-sensitive
fraction. These results, together with the time needed
after dexamethasone exposure to reach an effect, indicate also that the amiloride-sensitive Na1 channel is
upregulated after dexamethasone pretreatment. Indeed, amiloride-sensitive ENaC channels possess glucocorticoid regulatory element in their gene sequences
that could allow these channels to be upregulated after
steroid exposure (10, 41). Also, the basolaterally situated Na1-K1-ATPase can be upregulated after dexa-
DEXAMETHASONE ENHANCES FLUID CLEARANCE FROM THE LUNG
lung. Dexamethasone pretreatment affects the amiloride-sensitive fraction of alveolar fluid clearance to a
greater degree than the amiloride-insensitive fraction.
Pharmacological treatment with glucocorticoids and/or
thyroid hormones (T3) may increase the basal transport
capacity of the alveolar epithelium in patients with
pulmonary edema.
This study was supported by grants from the Swedish Natural
Research Science Council, the National Heart, Lung, and Blood
Institute (HL-51854), the Crafoord Foundation for Scientific Research, the Hierta Retzius Foundation for Scientific Research, and
the Magnus Bergwalls Foundation.
Address for reprint requests and other correspondence: H. G.
Folkesson, Dept. of Animal Physiology, Lund Univ., Helgonavägen 3
B, S-223 62 Lund, Sweden (E-mail: [email protected]).
Received 16 March 1999; accepted in final form 29 September 1999.
16.
17.
18.
19.
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