Alveolar liquid clearance in the anesthetized
ventilated guinea pig
ANDREAS NORLIN, NEELU FINLEY, PARISA ABEDINPOUR, AND HANS G. FOLKESSON
Department of Animal Physiology, Lund University, S-223 62 Lund, Sweden
amiloride; b-adrenergic stimulation; adenosine 38,58-cyclic
monophosphate generation; epinephrine; lung
THE GENERAL PARADIGM for transepithelial liquid movement in the lung is that active salt transport drives
osmotic water transport. Results from several studies
(22, 24) have demonstrated that hydrostatic or oncotic
pressure changes across the alveolocapillary barrier
cannot account for clearance of excess alveolar liquid.
Instead, clearance of excess alveolar liquid from the
distal air spaces of the lungs is driven by an active ion
transport, primarily sodium transport, across the alveolar epithelium (2, 4, 7) and partly across the distal
airway epithelium (1). Inhibitors of sodium uptake and
transport have inhibited alveolar epithelial liquid clearance (for a review, see Ref. 23), confirming that active
sodium transport across the alveolar epithelium is the
principal mechanism that accounts for clearance of
excess alveolar liquid from the air spaces.
Alveolar liquid clearance has been studied by several
experimental techniques and models such as isolated
alveolar epithelial type II cells (21), isolated perfused
lungs (2, 7), and intact animal models (3, 4, 13, 15, 22,
24, 29). Exogenous administration of b-adrenergic agonists stimulates alveolar liquid clearance under normal
conditions in several animal species (3, 4, 7, 13, 15) and
under selected pathological conditions, e.g., hyperoxic
lung injury (14, 20). Also, endogenous release of catecholamines under some pathological conditions (26) has
been reported to increase alveolar liquid clearance by
b-adrenergic-receptor stimulation. Non-catecholaminedependent pathways may also regulate alveolar liquid
clearance (5, 13).
Guinea pigs have been used in studies on pathological conditions such as oxygen-induced lung injury (18)
and in asthma research (17). Some in vitro studies on
lung liquid secretion in fetal guinea pigs (19) and on
liquid absorption in newborn guinea pigs (25) have
been done, but the functional and regulatory mechanisms of alveolar liquid clearance in the adult guinea
pig lung have not been elucidated. Also, the guinea pig
type I and II pneumocytes appear similar to the human
pneumocytes (10), and the guinea pig may thus be a
useful model for the human lung. Therefore, we decided
to study alveolar liquid clearance in the guinea pig,
with the first aim being to develop an in vivo technique
to measure and determine the basal alveolar liquid
clearance. The second aim was to determine, by functional studies, whether the guinea pig responds to
b-adrenergic stimulation with an increase in alveolar
liquid clearance and, in that case, which receptor (b1 or
b2 ) mediates this response. As a part of this aim, we
wanted to investigate whether changes in intracellular
adenosine 38,58-cyclic monophosphate (cAMP) levels
were involved in mediating the stimulated liquid clearance after b-adrenergic stimulation. The third aim was
to investigate the fractional inhibition by amiloride on
basal and stimulated alveolar liquid clearance to determine whether b-adrenergic stimulation affects amiloride-sensitive pathways for the removal of excess alveolar liquid.
MATERIALS AND METHODS
Animals
Sixty-eight male guinea pigs of the Dunkin-Hartley strain
(Sahlins Försöksdjursfarm, Malmö, Sweden) weighing 450–
750 g were used in the study. The animals were kept in a
12:12-h night-day rhythm and were fed with standard guinea
pig chow (SDS, Witham, Essex, UK) and had water ad libitum.
The protocol for these studies was approved by the Ethical
Review Committee on Animal Experiments at Lund University (Sweden).
Preparation of Instillates
A 5% albumin solution was prepared by dissolving 50
mg/ml of bovine serum albumin (mol wt 67,000; Sigma, St.
Louis, MO) in 0.9% NaCl. Fluorescein isothiocyanate (FITC)conjugated Dextran 70 (FITC-Dextran; mol wt 70,000; 50
µg/ml; Sigma) was then added to the 5% albumin solution as
1040-0605/98 $5.00 Copyright r 1998 the American Physiological Society
L235
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Norlin, Andreas, Neelu Finley, Parisa Abedinpour,
and Hans G. Folkesson. Alveolar liquid clearance in the
anesthetized ventilated guinea pig. Am. J. Physiol. 274 (Lung
Cell. Mol. Physiol. 18): L235–L243, 1998.—Alveolar liquid
clearance was examined in ventilated, anesthetized guinea
pigs. An isosmolar 5% albumin solution was instilled into the
lungs. Alveolar liquid clearance was studied over 1 h and was
measured from the increase in alveolar protein concentration
as water was reabsorbed. Basal alveolar liquid clearance was
38% of instilled volume. The high basal alveolar liquid
clearance was not secondary to endogenous catecholamine
release. Compared with control animals, epinephrine and the
general b-adrenergic agonist isoproterenol increased alveolar
liquid clearance to ,50% of instilled volume (P , 0.05),
whereas the b2-adrenergic agonist terbutaline was without
effect. The stimulation of alveolar liquid clearance by epinephrine or isoproterenol was completely inhibited by the addition
of the general b-adrenergic inhibitor propranolol or the
b1-adrenergic inhibitor atenolol. Alveolar liquid clearance
was inhibited by the sodium-channel inhibitor amiloride by
30–40% in control animals and in animals treated with
epinephrine or isoproterenol. Isoproterenol and epinephrine,
but not terbutaline, increased adenosine 38,58-cyclic monophosphate in in vitro incubated lung tissue. The results suggest
that alveolar liquid clearance in guinea pigs is mediated
partly through amiloride-sensitive sodium channels and that
alveolar liquid clearance can be increased by stimulation of
primarily b1-adrenergic receptors.
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ALVEOLAR LIQUID CLEARANCE
an alveolar protein permeability tracer. The FITC-Dextran
was filtered through a PD-10 dextran column (PharmaciaUpjohn, Uppsala, Sweden) before it was added to the 5%
albumin solution to separate free unbound FITC molecules
from the FITC-conjugated Dextran 70. A sample of the
instillate was saved for total protein measurement and
fluorescence analysis.
In some studies (see Specific Experimental Protocols),
b-adrenergic agonists (epinephrine, terbutaline, or isoproterenol) or b-adrenergic antagonists (propranolol or atenolol)
were added to the instillate solution. The sodium-channel
inhibitor amiloride was added to the instillate solution in
some studies (see Specific Experimental Protocols).
Surgical Procedures and Ventilation
General Experimental Protocol
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.
A 30-min baseline period with stable blood pressure and
heart rate was required before fluid instillation into the
lungs. Ten minutes into the baseline period, a solution
containing 2.5 mg/ml of rhodamine B isothiocyanate (RITC)conjugated Dextran 70 (RITC-Dextran; Sigma) was injected
intra-arterially (2 ml/kg body weight were injected, giving
,0.07 mg RITC-Dextran/ml blood). RITC-Dextran was run
through a PD-10 column in the same way as FITC-Dextran to
separate free unbound RITC from the injected RITCconjugated Dextran 70 before the intra-arterial injection.
Blood samples (1 ml) were obtained 10 and 20 min after the
RITC-Dextran injection.
After the baseline period, the animal was temporarily
disconnected from the ventilator, and soft instillation tubing
(Silastic, Dow Corning, Midland, MI) was gently passed
through the endotracheal tube into the distal air spaces of the
lungs. The instillate (6 or 9 ml/kg body weight) was delivered
over 10–15 s, and the animal was then immediately reconnected to the ventilator.
After 58 min, 10 ml of blood were withdrawn, and at 60
min, the animal was given 30 mg of pentobarbital sodium
intra-arterially. The lower abdomen was opened, the animals
were exsanguinated by transecting the abdominal aorta, and
Specific Experimental Protocols
Group 1: Control studies. After the baseline period, the
guinea pigs (n 5 7) were instilled with 6 ml/kg body weight of
the 5% albumin instillate into the lungs. In some studies, the
guinea pigs (n 5 4) were instilled with 9 ml/kg body weight of
the instillation solution to control for surface area effects on
alveolar liquid clearance. The animals were studied for 1 h
and then were exsanguinated and processed as described in
General Experimental Protocol.
Group 2: Epinephrine studies. After the baseline period,
the guinea pigs (n 5 6) were instilled with 6 ml/kg body
weight of the 5% albumin instillate containing 1026 M
epinephrine (NM Pharma, Stockholm, Sweden) into the
lungs. The animals were studied for 1 h and then were
exsanguinated and processed as described in General Experimental Protocol.
Group 3: Isoproterenol studies. After the baseline period,
the guinea pigs (n 5 6) were instilled with 6 ml/kg body
weight of the 5% albumin instillate containing 1025 M of the
general b-adrenergic agonist isoproterenol (Sigma) into the
lungs. The animals were studied for 1 h and then were
exsanguinated and processed as described in General Experimental Protocol.
Group 4: Terbutaline studies. After the baseline period, the
guinea pigs (n 5 7) were instilled with 6 ml/kg body weight of
the 5% albumin instillate containing 1024 M of the more
specific b2-adrenergic agonist terbutaline (Sigma) into the
lungs. The animals were studied for 1 h and then were
exsanguinated and processed as described in General Experimental Protocol.
Group 5: Propranolol studies. After the baseline period, the
guinea pigs (n 5 4) were instilled with 6 ml/kg body weight of
the 5% albumin instillate containing 1026 M epinephrine and
1024 M of the b-antagonist propranolol (Sigma) into the
lungs. In some animals (n 5 4), the instillate was the 5%
albumin solution containing 1024 M propranolol alone. The
animals were studied for 1 h and then were exsanguinated
and processed as described in General Experimental Protocol.
Group 6: Atenolol studies. After the baseline period, the
guinea pigs (n 5 6) were instilled with 6 ml/kg body weight of
the 5% albumin instillate containing 1026 M epinephrine and
1024 M of the relatively specific b1-adrenergic antagonist
atenolol (Sigma) into the lungs. In one group of animals (n 5
4), the instillate was the 5% albumin solution containing 1025
M isoproterenol and 1024 M atenolol, and in another group
(n 5 4), the instillate was the 5% albumin solution with 1024
M atenolol alone. The animals were studied for 1 h and then
were exsanguinated and processed as described in General
Experimental Protocol.
Group 7: Amiloride studies. After the baseline period, the
guinea pigs (n 5 5) were instilled with 6 ml/kg body weight of
the 5% albumin instillate containing 1023 M of the sodiumchannel inhibitor amiloride (Sigma) into the lungs. Amiloride
was used at 1023 M because ,50% is bound to the protein in
the instillate, and because of its relatively low molecular
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The guinea pigs were anesthetized by an intraperitoneal
injection of pentobarbital sodium (40 mg/kg body weight;
Apoteksbolaget, Umeå, Sweden). A 2.0-mm-ID endotracheal
tube (PE-240, Clay Adams, Becton Dickinson, Sparks, MD)
was inserted through a tracheostomy after the animals were
anesthetized. A 0.58-mm-ID catheter (PE-50, Clay Adams,
Becton Dickinson) was then inserted in the left carotid artery
to monitor systemic blood pressure, administer drugs, and
obtain blood samples. Pancuronium bromide (0.3 mg · kg body
weight21 · h21; Pavulon, Organon Teknika, Boxtel, The Netherlands) was administered through the arterial catheter for
neuromuscular blockade. The animals were ventilated with a
constant-volume piston pump (Harvard Apparatus, Nantucket, MA) with an inspired oxygen fraction of 1.0 and tidal
volume set to reach a peak airway pressure of 10–12 cmH2O
during the baseline period.
Peak airway pressure, blood pressure, and heart rate were
measured with calibrated pressure transducers (UFI model
1050BP, BioPac Systems, Goleta, CA) connected to analog-todigital converters and amplifiers (MP100 and DA100, respectively, BioPac Systems) and continuously recorded on an IBM
computer with Acknowledge 3.0 software (BioPac Systems).
then the lungs and heart were carefully removed en bloc from
the thorax through a midline sternotomy. A PE-50 catheter
(Clay Adams, Becton Dickinson) was gently passed to a
wedged position in the instilled lung, and a sample of the
remaining alveolar liquid was aspirated. The right and left
lungs were then homogenized separately for fluorescence
measurements. Parts of the lung homogenates were centrifuged (15,000 g for 20 min), and the supernatants were
collected. Blood samples were centrifuged (3,500 g for 5 min),
and the plasma was collected. Hematocrit was measured on
the last blood sample.
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ALVEOLAR LIQUID CLEARANCE
weight, amiloride leaves the air spaces rapidly (25, 30). The
actual concentration of amiloride in the air spaces was,
therefore, probably close to 1024 M. In two other groups,
amiloride was added to the 5% albumin instillate solution
simultaneously with either 1026 M epinephrine (n 5 4) or
1025 M isoproterenol (n 5 4). The animals were studied for 1 h
and then were exsanguinated and processed as described in
General Experimental Protocol.
Hemodynamic Parameters and Airway Pressure
VBl 5 1.039 3 [ (QH 3 FWH 3 HbS)/(FWS 3 HbBl) ]
Systolic and diastolic systemic blood pressures, heart rate,
and peak airway pressure were measured at the start of the
experiments, after 10 and 20 min into the baseline period,
immediately after instillation, 30 min after instillation, and
at the end of the experiment.
Alveolar Liquid Clearance
(1)
(3)
where QH is the weight of the lung homogenate, FWH is the
fraction of water in the lung, HbS and FWS are the hemoglobin concentration and the fraction of water, respectively, in
the supernatant obtained after centrifugation of the lung
homogenate, and HbBl is the hemoglobin concentration in the
last blood sample. The fraction of water in the lung was
obtained by gravimetric measurements of the lung, as done
before (4, 13, 24, 29). The density of blood was set to 1.039
g/ml.
To estimate the epithelial-to-endothelial passage of FITCDextran, fluorescence was measured in the instillate and in
the blood plasma before and after the experiment. The same
assumption as for the passage of RITC-Dextran was made for
the passage of FITC-Dextran across the alveolar epithelium;
i.e., it had the same passage as albumin across the epithelium
because of their similar molecular weights. The passage of
FITC-Dextran (FITC-Dextranpassage ) from the alveolar spaces
to the blood was calculated by Eq. 4
FITC-Dextranpassage 5 (FITC-Dextrantotal,vascular space
/FITC-Dextrantotal,instillate) 3 100
(4)
where FITC-Dextrantotal,instillate is the total amount of FITCDextran in the instilled fluid and FITC-Dextrantotal,vascular space
is the total amount of FITC-Dextran in the vascular compartment as calculated by Eq. 5
where VI is the instilled volume and VF is the final alveolar
volume (calculated from the protein concentrations in the
instilled and final alveolar liquids). The term alveolar does
not, however, imply that all reabsorption of liquid occurs at
the alveolar level; i.e., some liquid reabsorption may occur
across the distal bronchial epithelium because it can also
transport sodium (1).
where FITC-Dextranplasma is the FITC-Dextran concentration
in plasma and plasma volumetotal is the total plasma volume
(in ml) as estimated by Eq. 6
Endothelial and Epithelial Permeability to Protein
plasma volume
To estimate the clearance of the vascular tracer RITCDextran into the extravascular compartments of the lungs
(interstitium and air spaces), the total extravascular RITCDextran accumulation in the alveolar liquid and in the lung
homogenate was measured spectrophotofluorometrically (CytoFluor 2300, Millipore, Bedford, MA). The passage of RITCDextran molecules across the endothelial-epithelial barrier
was considered to be equal to that of albumin because they
have similar molecular weights (70,000 vs. 67,000). The
calculation of the endothelial protein passage was done using
the RITC-Dextran concentrations in the different compartments and applying them in Eq. 2
RITC-Dextranextravascular,lung 5 RITC-Dextrantotal,lung
2 RITC-Dextranvascular space,lung
(2)
where RITC-Dextran extravascular,lung is the RITC-Dextran concentration in the extravascular compartment of the lung,
RITC-Dextrantotal,lung is the total RITC-Dextran concentra-
FITC-Dextrantotal,vascular space
5 FITC-Dextranplasma 3 plasma volumetotal
5 body weight 3 0.07 3 [ (1 2 hematocrit)/100]
(5)
(6)
where body weight is in grams.
cAMP Generation
Lungs from three guinea pigs were perfused blood free with
0.9% NaCl via the pulmonary artery and were used for the
determination of intracellular cAMP under basal and stimulated conditions. Duplicate samples of blood-free distal lung
tissue (25–30 mg) were incubated in 0.25 ml of 5 mM
tris(hydroxymethyl)aminomethane (Merck, Darmstadt, Germany) in 0.9% NaCl (pH 7.4), 1 mM 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor; Sigma), 0.1 mM ascorbic acid (Merck), and 0.1 mM HCl (Merck), as done earlier (9).
Basal cAMP content was determined after incubation at 4°C
for 10 min, and basal production of cAMP was studied after a
10-min incubation at 37°C. Stimulation of cAMP generation
was studied after the addition of forskolin (1024 M; positive
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The increase in alveolar concentration of the instilled
protein over 1 h was used to measure the clearance of liquid
from the distal air spaces (across the alveolar epithelium and
distal airway epithelium) as done before (3, 4, 13, 15, 22, 26,
29). Data on alveolar liquid clearance are shown in two ways.
First, alveolar liquid clearance is presented as a ratio of the
final aspirated alveolar fluid protein concentration to the
instilled fluid protein concentration. The final-to-instilled
protein concentration ratio provides direct evidence for alveolar liquid clearance because liquid must be transported from
the air spaces for the final alveolar protein concentration to
rise. Because there were no changes in epithelial and endothelial permeability to protein (i.e., very little protein left the air
spaces in any of the groups; see RESULTS ), this method is
accurate for measuring liquid clearance from the distal air
spaces of the lungs. The second method is based on calculating alveolar liquid clearance (ALC; expressed as percentage
of instilled volume) using Eq. 1
ALC 5 [(VI 2 VF)/VI] 3 100
tion in the lung, and RITC-Dextranvascular space,lung is the RITCDextran concentration in the vascular compartment in the
lung.
To calculate RITC-Dextranvascular space,lung, the RITC-Dextran measurement in the last plasma sample was multiplied
by the blood volume in the lungs corrected for hematocrit. The
blood volume (VBl ) in the lungs at the end of the experiment
was calculated from Eq. 3
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ALVEOLAR LIQUID CLEARANCE
Statistics
All data are presented as means 6 SD. The data were
analyzed with one-way analysis of variance with Tukey’s test
post hoc. Differences were considered significant when a P
value of ,0.05 was reached.
RESULTS
Alveolar Liquid Clearance Under Basal Conditions
Anesthetized ventilated guinea pigs were instilled
with either 6 or 9 ml/kg body weight of the 5% bovine
serum albumin solution in 0.9% NaCl. After 1 h, a
sample of the instilled solution was aspirated from the
distal air spaces. The increase in total protein concentration during the 1-h period was used as a measurement of the liquid that had been cleared from the distal
air spaces of the lungs. The results are presented as the
final-to-instilled protein concentration ratio (Table 1)
and the alveolar liquid clearance (Fig. 1). Basal alveolar liquid clearance was high, and 38% of the instilled
liquid was removed over 1 h. The increase in alveolar
protein concentration was similar whether 9 or 6 ml/kg
body weight were instilled (the final-to-instilled protein
concentration ratios were 1.59 6 0.20 and 1.63 6 0.12,
respectively).
Table 1. Alveolar liquid clearance in guinea
pigs treated with b-adrenergic agonists and
antagonists over 1 h
Alveolar Protein
Concentration, g/dl
Condition
Control
Control 1 propranolol
(1024 M)
Control 1 atenolol (1024
M)
Epinephrine (1026 M)
Epinephrine (1026 M)
1 propranolol
(1024 M)
Epinephrine (1026 M)
1 atenolol (1024 M)
Isoproterenol (1025 M)
Isoproterenol (1025 M)
1 atenolol (1024 M)
Terbutaline (1024 M)
n
Instilled
Final
Final-toInstilled Protein
Concentration
Ratio
7 4.68 6 0.86 7.62 6 1.42
1.63 6 0.12
4 5.21 6 0.41 8.49 6 1.53
1.63 6 0.23
4 4.68 6 0.27 7.67 6 0.72
6 4.89 6 0.33 9.68 6 1.29
1.64 6 0.18
1.98 6 0.24*
4 4.81 6 0.39 7.51 6 0.49
1.57 6 0.20†
6 4.78 6 0.22 7.83 6 0.67
6 4.58 6 0.50 9.17 6 1.04
1.64 6 0.19†
2.01 6 0.22*
4 4.82 6 0.14 7.39 6 0.69
7 4.83 6 0.40 7.88 6 0.62
1.53 6 0.12‡
1.64 6 0.12
Values are means 6 SD; n, no. of animals. Significant difference
(P , 0.05) compared with: * control; † epinephrine; ‡ isoproterenol (by
analysis of variance).
tion were completely inhibited (Fig. 1, Table 1). The
addition of propranolol to the control instillate did not
affect basal alveolar liquid clearance (Fig. 1, Table 1).
Effect of Isoproterenol and Terbutaline on Alveolar
Liquid Clearance
When it had been established that epinephrine acted
via b-adrenergic-receptor stimulation, we investigated
whether the stimulatory effect was primarily mediated
by b1-receptor or b2-receptor stimulation. We used
terbutaline as a relatively specific b2-adrenergic ago-
Effect of Epinephrine on Alveolar Liquid Clearance
To determine whether the guinea pig responded to
epinephrine stimulation with an increase in alveolar
liquid clearance, we instilled anesthetized and ventilated guinea pigs with the 5% albumin solution containing 1026 M epinephrine and studied them for 1 h. When
epinephrine was added to the instillate, the final-toinstilled protein concentration ratio and hence the
corresponding alveolar liquid clearance was significantly increased compared with control levels (Fig. 1,
Table 1). To investigate whether this stimulation was
mediated by b-adrenergic-receptor activation, we added
1024 M propranolol to the instilled fluid. The increases
in the final-to-instilled protein concentration ratio and
alveolar liquid clearance seen after epinephrine instilla-
Fig. 1. Alveolar liquid clearance over 1 h in guinea pigs instilled with
5% albumin (control) and with 5% albumin containing 1024 M
propranolol, 1026 M epinephrine, or 1026 M epinephrine 1 1024 M
propranolol. Values are means 6 SD. Treatment with epinephrine
stimulated alveolar liquid clearance by 30% compared with control
treatment. Stimulatory effect with epinephrine was completely inhibited by propranolol. Propranolol by itself did not affect basal alveolar
liquid clearance. Significant difference (P , 0.05) compared with:
* control; † epinephrine 1 propranolol (by analysis of variance).
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control; Sigma) in 2.5% dimethyl sulfoxide (DMSO; Sigma) to
the solution and incubation for 10 min at 37°C. DMSO (2.5%)
alone was added to control for the possible effects of DMSO
(vehicle for forskolin). Intracellular cAMP was measured
after stimulation of b-adrenergic receptors by the addition of
isoproterenol (1025 M), terbutaline (1023, 1024, or 1025 M), or
epinephrine (1025 M) to the solution and incubation of the
samples for 10 min at 37°C. We used 1025 M epinephrine
because the endothelium of the lungs is a major site for
epinephrine metabolism (6). Therefore, the effective epinephrine dose was probably close to 1026 M. All reactions were
stopped with 0.25 ml of 10% trichloroacetic acid (Sigma). The
samples were then homogenized and centrifuged (4,000 revolutions/min for 15 min at 4°C). The supernatants were
extracted with ether (5:1) three consecutive times to remove
the trichloroacetic acid. The remaining ether was evaporated
in a 70°C water bath for 30 min. The samples were stored at
270°C until analysis. The cAMP content in each sample was
determined with a radioimmunoassay (NEN-DuPont, Boston, MA).
ALVEOLAR LIQUID CLEARANCE
L239
nist and isoproterenol as a general b-adrenergic agonist. The addition of isoproterenol (1025 M) to the
instillate significantly increased the final-to-instilled
protein concentration ratio and the alveolar liquid
clearance by the same magnitude as 1026 M epinephrine (Fig. 2, Table 1). However, the addition of the more
specific b2-adrenergic agonist terbutaline (1024 M) did
not affect the final-to-instilled protein concentration
ratio and hence the alveolar liquid clearance (Fig. 2,
Table 1).
Effect of Atenolol on b-Adrenergically Stimulated
Alveolar Liquid Clearance
Effect of Amiloride on Basal and Stimulated Alveolar
Liquid Clearance
To investigate the contribution to alveolar liquid
clearance by amiloride-sensitive pathways under basal
and stimulated conditions, we added 1023 M amiloride
to the 5% albumin instillate with or without the
b-adrenergic agonists. When amiloride alone was added
to the instillate, the final-to-instilled protein concentration ratio and the corresponding alveolar liquid clearance were significantly decreased by 40 and 32%,
respectively, compared with the control levels (Fig. 4,
Table 2). When amiloride was administered to animals
stimulated with either 1026 M epinephrine or 1025 M
isoproterenol, the inhibition was similar compared
Fig. 2. Alveolar liquid clearance over 1 h in guinea pigs instilled with
5% albumin (control) and with 5% albumin containing 1026 M
epinephrine, 1025 M isoproterenol, or 1024 M terbutaline. Values are
means 6 SD. Treatment with isoproterenol resulted in an increase in
alveolar liquid clearance similar to that after epinephrine treatment.
Addition of terbutaline had, however, no stimulatory effect. * Significant difference compared with control, P , 0.05 (by analysis of
variance).
Fig. 3. Alveolar liquid clearance over 1 h in guinea pigs instilled with
5% albumin (control) and with 5% albumin containing 1026 M
epinephrine or 1025 M isoproterenol with (open bars) or without
(solid bars) b1-adrenergic antagonist atenolol. Values are means 6
SD. Addition of 1024 M atenolol to instillate completely inhibited
stimulatory effect of both epinephrine and isoproterenol. Atenolol did
not affect alveolar liquid clearance under basal conditions. Significant difference (P , 0.05) compared with: * epinephrine; † isoproterenol (by analysis of variance).
with the studies in which amiloride was given alone
(Fig. 4, Table 2).
Generation of cAMP
Distal lung tissue from three different guinea pigs
was incubated at 37°C for 10 min to determine the
generation of intracellular cAMP under basal conditions and when stimulated with epinephrine, isoproterenol, or terbutaline. Stimulation of cAMP with forskolin was used as a positive control. Stimulation with
Fig. 4. Alveolar liquid clearance over 1 h in guinea pigs instilled with
5% albumin (control) and with 5% albumin containing 1026 M
epinephrine or 1025 M isoproterenol with (open bars) or without
(solid bars) sodium-channel inhibitor amiloride. Values are means 6
SD. Addition of 1023 M amiloride to instillate inhibited alveolar
liquid clearance by 30–40% in all groups, indicating that both basal
and stimulated alveolar liquid clearances were mediated partly
through amiloride-sensitive pathways. Significant difference (P ,
0.05) compared with: * control; † epinephrine; ‡ isoproterenol (by
analysis of variance).
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To further investigate which b-receptor subtype could
be involved, we used the relatively specific b1-adrenergic antagonist atenolol. The addition of atenolol (1024
M) to the 5% albumin instillate solution containing
either epinephrine (1026 M) or isoproterenol (1025 M)
completely inhibited the stimulatory effects of epinephrine and isoproterenol (Fig. 3, Table 1). There was no
effect of atenolol alone on basal alveolar liquid clearance (Fig. 3, Table 1).
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ALVEOLAR LIQUID CLEARANCE
Table 2. Alveolar liquid clearance in guinea pigs
treated with amiloride with or without b-adrenergic
agonists over 1 h
Alveolar Protein
Concentration, g/dl
Condition
Instilled
Final
7 4.68 6 0.86 7.62 6 1.42
1.63 6 0.12
5 4.96 6 0.01 6.85 6 1.04
6 4.89 6 0.33 9.68 6 1.29
1.38 6 0.21*
1.98 6 0.24*
4 4.51 6 0.06 7.24 6 0.53
6 4.58 6 0.50 9.17 6 1.04
1.61 6 0.13†
2.01 6 0.22*
4 4.96 6 0.25 7.72 6 0.76
1.56 6 0.10‡
Values are means 6 SD; n, no. of animals. Significant difference
(P , 0.05) compared with: * control; † epinephrine; ‡ isoproterenol (by
analysis of variance).
1025 M isoproterenol and 1025 M epinephrine increased
the cAMP content in the lung tissue to a similar level as
1024 M forskolin, but terbutaline (1023, 1024, or 1025 M)
increased the cAMP level to a much lower degree that
was not significantly different from the control level
(Fig. 5). The cAMP content in tissue samples incubated
at 4°C was similar to control tissues incubated at 37°C
(5.47 6 0.98 and 7.90 6 1.41 pmol/mg tissue wet
weight, respectively). Also, 2.5% DMSO (the vehicle for
forskolin) had no effect on the generation of cAMP (data
not shown).
Mean arterial pressure, heart rate, and peak airway
pressure before and after instillation were similar in all
groups. Also, the different treatments did not dramatically affect these parameters except that the
b-adrenergic antagonists slightly decreased heart rate
and blood pressure. There were no differences in the
low endothelial permeability, measured as extravascular plasma equivalents as described in MATERIALS AND
METHODS, among the different experimental groups.
Moreover, there were no measurable amounts of FITCDextran in the last blood sample from any of the
groups, and hence there were no detectable increases in
the epithelial-endothelial barrier permeability in any
of the groups.
DISCUSSION
There were three major findings in this study. First,
the guinea pig has a comparatively high basal clearance of excess alveolar liquid; in fact, it is among the
highest basal alveolar liquid clearances found in any
species (Table 3). This high basal alveolar liquid clearance cannot be explained by circulating endogenous
catecholamines or the release of endogenous catecholamines because neither propranolol nor atenolol decreased the basal alveolar liquid clearance. Second, the
addition of exogenous epinephrine significantly increased alveolar liquid clearance via b-adrenergic stimulation. The general b-agonist isoproterenol had similar
effects on alveolar liquid clearance as epinephrine, but
the more specific b2-adrenergic agonist terbutaline had
no effect. This finding suggested that the stimulation of
alveolar epithelial liquid clearance occurred by the
b1-adrenergic receptor. Consequently, we tested the
specific b1-adrenergic antagonist atenolol. Atenolol inhibited the stimulatory effects of epinephrine and isoproterenol. These results strengthen the conclusion
that the b1-adrenergic receptor subtype is the primary
receptor stimulating alveolar liquid clearance in the
guinea pig. Third, we also investigated the mechanism
responsible for alveolar liquid clearance by adding the
Table 3. Alveolar liquid clearance in different species
at baseline and after stimulation with b-adrenergic
agonists or inhibition with amiloride
Fig. 5. cAMP content in distal lung tissue incubated for 10 min at
4°C under control conditions and in distal lung tissue incubated for
10 min at 37°C under control conditions and with 1025 M isoproterenol, 1024 M terbutaline, 1025 M epinephrine, or 1024 M forskolin
added to incubation medium. Some tissue samples were also incubated with 1023 or 1025 M terbutaline. Values are means 6 SD; n 5 6
animals for all treatments. Both epinephrine and isoproterenol
significantly increased intracellular cAMP levels similar to that of
positive control forskolin. cAMP contents after incubation with 1023
and 1025 M terbutaline were similar (13.22 6 2.60 and 14.32 6 4.06
pmol/mg lung tissue wet weight, respectively) to that with 1024 M
terbutaline. Significant difference (P , 0.05) compared with: * control
(37°C); † terbutaline (by analysis of variance).
Species
Baseline
b-Adrenergic
Stimulation
Amiloride
Sensitivity
Ref.
No.
Guinea pig
Rat
Rabbit*
Sheep†
Dog†
Human†‡
38
33
36
8
3
3
31
45
0
56
150
72
32
39
81
42
NA
40
This study
15
29
4, 22
3
27
All values, except for guinea pig, are adapted from other studies.
Baseline (control condition) is expressed as percent alveolar liquid
clearance over 1 h. b-Adrenergic stimulation is expressed as percent
increase compared with baseline. Amiloride sensitivity is expressed
as percent decrease compared with baseline. * Values corrected for
25-min instillation time. † Studies done originally over 4 h and values
extrapolated to 1 h. ‡ Resected lung lobe model. NA, not available.
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Control
Control 1 amiloride
(1023 M)
Epinephrine (1026 M)
Epinephrine (1026 M)
1 amiloride
(1023 M)
Isoproterenol (1025 M)
Isoproterenol (1025 M)
1 amiloride
(1023 M)
n
Final-toInstilled Protein
Concentration
Ratio
Hemodynamic Parameters and Epithelial-Endothelial
Permeability
ALVEOLAR LIQUID CLEARANCE
fore, the volume of instilled fluid and thereby surface
area is not an important variable for the observed
interspecies variations in alveolar liquid clearance. The
reasons for the observed species differences in alveolar
liquid clearance are unknown but could be related to
variations in the number or basal activity of sodium
channels and/or Na1-K1-adenosinetriphosphatase molecules in the alveolar epithelium.
Clearance of liquid across the alveolar epithelium
has been studied in several animal species as well as in
the human lung (3, 4, 7, 15, 24, 27, 29). Alveolar liquid
clearance over 1 h in the guinea pigs in our study was
38% of the instilled volume under normal basal conditions (Fig. 1). In comparison with other animal species
(3, 4, 13, 15, 27, 29), alveolar liquid clearance in the
guinea pig appeared to be higher (Table 3). The comparatively high basal alveolar liquid clearance in the
guinea pig could have been related to release of or high
circulating levels of endogenous catecholamines such
as epinephrine. Endogenous epinephrine regulates alveolar liquid clearance under some pathological conditions (26). To test whether endogenous epinephrine
could be responsible for the high basal alveolar liquid
clearance, we added propranolol, a general b-adrenergic antagonist, as well as atenolol, a b1-adrenergic
antagonist, to the instilled fluid. The basal alveolar
liquid clearance was not affected by propranolol or
atenolol, proving that the high basal alveolar liquid
clearance was not secondary to released or circulating
endogenous epinephrine.
Because there is considerable interspecies variability
in the response to b-adrenergic stimulation of alveolar
liquid clearance, we investigated whether the guinea
pig responded to b-adrenergic agonists. In several
species (3, 4, 15, 27), alveolar liquid clearance increases
in response to stimulation by b-adrenergic agonists
(Table 3), whereas in some species (29), there is no
response to b-adrenergic stimulation. The addition of
epinephrine or the general b-adrenergic agonist isoproterenol resulted in significantly increased alveolar liquid clearance rates in the guinea pig studies. This
stimulation was totally inhibited when the general
b-adrenergic inhibitor propranolol was added to the
instillate. However, when the more specific b2-adrenergic agonist terbutaline was added to the instillate, no
stimulatory effect was observed. One reason for the
failure of terbutaline to stimulate alveolar liquid clearance may have been that we used too low a dose (1024
M). However, this is unlikely because the dose used in
our study was in the high range of the terbutaline doses
used in other studies (15, 24, 29). Also, there is evidence
that terbutaline, when binding to the b2-adrenergic
receptor subtype in the guinea pig distal lung, has a 14
times lower functional effect as measured by a much
lower increase in intracellular cAMP compared with
isoproterenol (16). Another possible explanation for the
inability of terbutaline to stimulate alveolar liquid
clearance in the guinea pig could be explained by which
b-adrenergic receptor is stimulated. Because the stimulatory effect of both epinephrine and isoproterenol was
completely attenuated by atenolol, a b1-receptor inhibi-
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sodium-channel inhibitor amiloride to the instillate of
both control and stimulated (epinephrine or isoproterenol) animals. In control animals as well as in animals
stimulated with epinephrine or isoproterenol, amiloride decreased alveolar liquid clearance similarly by
30–40%. This finding indicates that stimulation of
alveolar liquid clearance in the guinea pig depends on
both amiloride-sensitive and amiloride-insensitive pathways because there were no differences in the fractional
inhibition by amiloride among the groups.
The first aim of this study was to develop and adapt
the in vivo method for the measurement of alveolar
liquid clearance and to determine the basal alveolar
liquid clearance in the guinea pig. The method we
developed differed slightly from the methods used in
other animal species of comparable size (rats and
rabbits) (13, 15, 29). Therefore, one possible reason for
the variability in basal alveolar liquid clearance among
species could be related to differences in the methodology and the experimental conditions. These include
differences in the duration of instillation and differences in instilled volume, i.e., the surface area exposed
to the instilled fluid. We instilled the fluid over a
comparatively short time [10–15 s vs. 20–30 min in
other studies (13, 26)], and we also instilled a larger
volume of liquid per kilogram of body weight than in
prior studies (13, 24, 26, 29). There were two reasons
for this change in protocol. First, because of the observed high basal alveolar liquid clearance, we needed
to instill a larger volume than in previous investigations to be able to collect a sample of alveolar fluid after
1 h. Second, because of the anatomic properties of the
guinea pig, instillation over a 20- to 30-min period was
impossible because the animals frequently developed a
pneumothorax when that method was used. Therefore,
we decided to instill the fluid over a shorter time with
the animal briefly disconnected from the ventilator.
One way to compensate for the different instillation
times is to disregard the time of instillation and only
compare the experimental time after instillation. This
would be allowed if the alveolar liquid clearance was
linear with time over the study period. Matthay et al.
(22) reported that the alveolar liquid clearance in sheep
increased linearly with time over the first hours after
liquid instillation. Therefore, we corrected earlier studies (3, 4, 24, 29) for the time of instillation, and results
from these studies were considered to have been done
during a shorter time. The other factor of possible
importance could be that the higher instilled volume of
fluid resulted in a greater surface area for sodium and
water uptake. We evaluated this issue in two ways.
First, to investigate whether there were any differences
in alveolar liquid clearance due to different instilled
volumes, we instilled one group of control guinea pigs
with 9 ml/kg body weight and compared it with the 6
ml/kg body weight group. There were no differences in
alveolar liquid clearance between the two groups. Second, earlier work from other laboratories (15, 24, 29)
demonstrated that alveolar liquid clearance was the
same when volumes ranging from 2 to 6 ml/kg body
weight were instilled in sheep, rats, and rabbits. There-
L241
L242
ALVEOLAR LIQUID CLEARANCE
rats (5, 13). Even though these hormones and growth
factors might be released by b-adrenergic substances,
most of the hormones require de novo protein synthesis, and, therefore, the observed effects over 1 h in these
studies were likely from direct effects.
In other animal species, alveolar liquid clearance
depends in part on amiloride-sensitive pathways (2, 4,
13, 15, 26, 27, 29). In most other animal species studied,
intra-alveolar amiloride inhibits alveolar liquid clearance to a similar extent to what was seen in this study
on guinea pigs (Table 3). Both basal and stimulated
alveolar liquid clearance by epinephrine or isoproterenol were inhibited similarly by amiloride. This
indicates that epinephrine and isoproterenol increase
alveolar liquid clearance by stimulation of both amiloride-sensitive and amiloride-insensitive pathways. One
possible explanation for the fractional inhibition by
amiloride may be the existence of not yet identified
cation channels that are not inhibited by amiloride. In
fact, recent data (11, 28) suggest the existence of a
rod-type cyclic nucleotide-gated cation channel in the
alveolar epithelium that may be involved in fluid
movement in the lung.
In summary, guinea pigs have a high basal alveolar
liquid clearance compared with other animal species.
Alveolar liquid clearance is mediated through both
amiloride-sensitive and amiloride-insensitive sodium
channels. Epinephrine upregulates alveolar liquid clearance in guinea pigs primarily through b1-adrenergicreceptor stimulation. Thus the b1-adrenergic receptor
may be the key receptor that is responsible for stimulating vectorial sodium and liquid transport in the guinea
pig lung, although limited involvement of the b2adrenergic receptor cannot be completely excluded.
We thank Dr. Michael A. Matthay for critically reading the
manuscript and for all his suggestions.
This study was supported by grants from the Swedish Natural
Science Research Council, the Crafoord Foundation, the Åke Wiberg’s
Foundation, the Magnus Bergwall Foundation, The Hierta-Retzius
Foundation, and the Royal Physiographic Society in Lund, Sweden.
Address for reprint requests: H. G. Folkesson, Dept. of Animal
Physiology, Lund Univ., Helgonavägen 3 B, S-223 62 Lund, Sweden.
Received 17 March 1997; accepted in final form 14 November 1997.
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