Department of Animal Physiology, Lund University, S-223 62 Lund, Sweden
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ABSTRACT |
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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 -adrenergic agonist
isoproterenol increased alveolar liquid clearance to ~50% of
instilled volume (P < 0.05), whereas
the
2-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
-adrenergic inhibitor propranolol or the
1-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
3',5'-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
1-adrenergic
receptors.
amiloride; -adrenergic stimulation; adenosine
3',5'-cyclic monophosphate generation; epinephrine; lung
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INTRODUCTION |
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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 -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
-adrenergic-receptor stimulation. Non-catecholamine-dependent
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 -adrenergic stimulation with an increase in alveolar
liquid clearance and, in that case, which receptor (
1 or
2) mediates this response. As
a part of this aim, we wanted to investigate whether changes in
intracellular adenosine 3',5'-cyclic monophosphate (cAMP)
levels were involved in mediating the stimulated liquid clearance after
-adrenergic stimulation. The third aim was to investigate the
fractional inhibition by amiloride on basal and stimulated alveolar
liquid clearance to determine whether
-adrenergic stimulation
affects amiloride-sensitive pathways for the removal of excess alveolar
liquid.
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MATERIALS AND METHODS |
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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 an alveolar protein permeability tracer. The FITC-Dextran was filtered through a PD-10 dextran column (Pharmacia-Upjohn, 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), -adrenergic agonists
(epinephrine, terbutaline, or isoproterenol) or
-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
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 weightPeak airway pressure, blood pressure, and heart rate were measured with 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).
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 RITC-conjugated 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 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.
Specific Experimental Protocols
Group 1: Control studies. After the baseline period, the guinea pigs (n = 7) were instilled with 6 ml/kg body weight of the 5% albumin instillate into the lungs. In some studies, the guinea pigs (n = 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 = 6) were instilled with 6 ml/kg
body weight of the 5% albumin instillate containing
106 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 = 6) were instilled with 6 ml/kg
body weight of the 5% albumin instillate containing
105 M of the general
-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 = 7) were instilled with 6 ml/kg
body weight of the 5% albumin instillate containing
104 M of the more specific
2-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 = 4) were instilled with 6 ml/kg
body weight of the 5% albumin instillate containing
106 M epinephrine and
10
4 M of the
-antagonist
propranolol (Sigma) into the lungs. In some animals
(n = 4), the instillate was the 5%
albumin solution containing
10
4 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 = 6) were instilled with 6 ml/kg body weight of the 5% albumin
instillate containing 106 M
epinephrine and 10
4 M of
the relatively specific
1-adrenergic antagonist
atenolol (Sigma) into the lungs. In one group of animals
(n = 4), the instillate was the 5%
albumin solution containing
10
5 M isoproterenol and
10
4 M atenolol, and in
another group (n = 4), the
instillate was the 5% albumin solution with
10
4 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) were instilled with 6 ml/kg body weight of the 5% albumin
instillate containing 103 M
of the sodium-channel inhibitor amiloride (Sigma) into the lungs.
Amiloride was used at 10
3 M
because ~50% is bound to the protein in the instillate, and because
of its relatively low molecular weight, amiloride leaves the air spaces
rapidly (25, 30). The actual concentration of amiloride in the air
spaces was, therefore, probably close to
10
4 M. In two other groups,
amiloride was added to the 5% albumin instillate solution
simultaneously with either
10
6 M epinephrine
(n = 4) or
10
5 M isoproterenol
(n = 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
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
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
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(1) |
Endothelial and Epithelial Permeability to Protein
To estimate the clearance of the vascular tracer RITC-Dextran into the extravascular compartments of the lungs (interstitium and air spaces), the total extravascular RITC-Dextran accumulation in the alveolar liquid and in the lung homogenate was measured spectrophotofluorometrically (CytoFluor 2300, Millipore, Bedford, MA). The passage of RITC-Dextran 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
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(2) |
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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(3) |
To estimate the epithelial-to-endothelial passage of FITC-Dextran, 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
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(4) |
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(5) |
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(6) |
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 (10Statistics
All data are presented as means ± 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 |
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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 ± 0.20 and 1.63 ± 0.12, respectively).
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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 10Effect of Isoproterenol and Terbutaline on Alveolar Liquid Clearance
When it had been established that epinephrine acted via
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Effect of Atenolol on -Adrenergically Stimulated
Alveolar Liquid Clearance
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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 10
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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 10
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Hemodynamic Parameters and Epithelial-Endothelial Permeability
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 ![]() |
DISCUSSION |
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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 -adrenergic stimulation. The
general
-agonist isoproterenol had similar effects on alveolar
liquid clearance as epinephrine, but the more specific
2-adrenergic agonist
terbutaline had no effect. This finding suggested that the stimulation
of alveolar epithelial liquid clearance occurred by the
1-adrenergic receptor.
Consequently, we tested the specific
1-adrenergic antagonist
atenolol. Atenolol inhibited the stimulatory effects of epinephrine and
isoproterenol. These results strengthen the conclusion that the
1-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 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.
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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. Therefore, 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 Na+-K+-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 -adrenergic antagonist, as well as
atenolol, a
1-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 -adrenergic stimulation of alveolar liquid clearance, we
investigated whether the guinea pig responded to
-adrenergic agonists. In several species (3, 4, 15, 27), alveolar liquid clearance
increases in response to stimulation by
-adrenergic agonists (Table
3), whereas in some species (29), there is no response to
-adrenergic stimulation. The addition of epinephrine or the general
-adrenergic agonist isoproterenol resulted in significantly
increased alveolar liquid clearance rates in the guinea pig studies.
This stimulation was totally inhibited when the general
-adrenergic
inhibitor propranolol was added to the instillate. However, when the
more specific
2-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
(10
4 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
2-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
-adrenergic receptor is stimulated.
Because the stimulatory effect of both epinephrine and isoproterenol
was completely attenuated by atenolol, a
1-receptor inhibitor, the results suggest that alveolar liquid clearance in the guinea pig was
stimulated primarily by agonist binding to the
1-adrenergic receptor. However,
2-adrenergic pathways could not
be completely excluded.
Could the observed results be explained by agonist binding to the
1-adrenergic receptor? In
several studies (8, 12), it has been demonstrated that there are mixed
populations of
-adrenergic receptors in mammalian lungs. In guinea
pigs, there are ~20%
1-adrenergic and ~80%
2-adrenergic receptors (12) in
the pulmonary cell membranes. It has also been shown that 21% of the
-adrenergic receptors on the alveolar type II cell membranes in the
guinea pig lung are of the
1-subtype (12). Furthermore,
guinea pig alveolar type I cells have
-receptors on their cell
membranes (8), but the physiological significance of the
-receptors on these cells is difficult to evaluate. The exact localization of the
-adrenergic receptors in the distal lung tissue is not known, but 70% of the total number of
-adrenergic receptors in the
guinea pig lung are in the alveoli (12). Furthermore, there is
functional evidence that the
-adrenergic receptors are localized on
both the basolateral and apical sides of the pneumocytes (4, 26).
Therefore, there is a high probability that
-adrenergic receptors
may be linked to regulation of alveolar liquid clearance. Our
functional data suggest that the
1-receptors are the primary receptors regulating alveolar liquid clearance in the guinea pig lung.
Because it has been suggested earlier (16) that different
-adrenergic agonists differ in their capacity to stimulate
intracellular cAMP, we investigated the ability of isoproterenol,
terbutaline, and epinephrine to stimulate cAMP generation in distal
lung tissue. Isoproterenol and epinephrine both increased the cAMP
content to a level comparable to the level that occurred after
stimulation with forskolin. Terbutaline, however, increased the cAMP
content only slightly and not significantly compared with the control level. To verify the lack of response after terbutaline treatment, we
studied two additional concentrations of terbutaline
(10
3 and
10
5 M), both of which
resulted in levels of cAMP similar to that with
10
4 M terbutaline
treatment. The results are consistent with the work of Johansson (16),
which demonstrated that terbutaline is less efficient compared with
isoproterenol for increasing intracellular cAMP in the distal guinea
pig lung. An alternative explanation is that terbutaline predominantly
binds to
2-adrenergic
receptors, and perhaps these receptors are not coupled to generation of
intracellular cAMP in the guinea pig lung. Both of these explanations
are consistent with the interpretation that increased alveolar liquid
clearance is mediated through
1-adrenergic receptors in the
guinea pig.
Another possible explanation for the increased alveolar liquid
clearance after 1-adrenergic
stimulation is that the administered drugs released other hormones or
cytokines that upregulated clearance. It is known that cortisol and
aldosterone decrease alveolar fluid production in near-term guinea pigs
(19) and that several other endogenous substances such as growth
factors can increase the alveolar liquid clearance in rats (5, 13).
Even though these hormones and growth factors might be released by
-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
1-adrenergic-receptor
stimulation. Thus the
1-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
2-adrenergic receptor cannot be completely excluded.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael A. Matthay for critically reading the manuscript and for all his suggestions.
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FOOTNOTES |
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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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