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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -adrenergic agonist isoproterenol increased alveolar liquid clearance to ~50% of instilled volume (P < 0.05), whereas the beta 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 beta -adrenergic inhibitor propranolol or the beta 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 beta 1-adrenergic receptors.

amiloride; beta -adrenergic stimulation; adenosine 3',5'-cyclic monophosphate generation; epinephrine; lung

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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 beta -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 beta -adrenergic stimulation with an increase in alveolar liquid clearance and, in that case, which receptor (beta 1 or beta 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 beta -adrenergic stimulation. The third aim was to investigate the fractional inhibition by amiloride on basal and stimulated alveolar liquid clearance to determine whether beta -adrenergic stimulation affects amiloride-sensitive pathways for the removal of excess alveolar liquid.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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), beta -adrenergic agonists (epinephrine, terbutaline, or isoproterenol) or beta -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 weight-1 · h-1; 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-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 10-6 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 10-5 M of the general beta -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 10-4 M of the more specific beta 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 10-6 M epinephrine and 10-4 M of the beta -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 10-6 M epinephrine and 10-4 M of the relatively specific beta 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 10-3 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
ALC = [(V<SUB>I</SUB> − V<SUB>F</SUB>)/V<SUB>I</SUB>] × 100 (1)
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).

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
RITC-Dextran<SUB>extravascular,lung</SUB> = RITC-Dextran<SUB>total,lung</SUB>
− RITC-Dextran<SUB>vascular space,lung</SUB> (2)
where RITC-Dextranextravascular,lung is the RITC-Dextran concentration in the extravascular compartment of the lung, RITC-Dextrantotal,lung is the total RITC-Dextran concentration in the lung, and RITC-Dextranvascular space,lung is the RITC-Dextran 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
V<SUB>Bl</SUB> = 1.039 × [(Q<SUB>H</SUB> × FW<SUB>H</SUB> × Hb<SUB>S</SUB>)/(FW<SUB>S</SUB> × Hb<SUB>Bl</SUB>)] (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 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
FITC-Dextran<SUB>passage</SUB> = (FITC-Dextran<SUB>total,vascular space</SUB>
/FITC-Dextran<SUB>total,instillate</SUB>) × 100 (4)
where FITC-Dextrantotal,instillate is the total amount of FITC-Dextran 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
FITC-Dextran<SUB>total,vascular space</SUB>
= FITC-Dextran<SUB>plasma</SUB> × plasma volume<SUB>total</SUB> (5)
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
plasma volume
= body weight × 0.07 × [(1 − hematocrit)/100] (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 (10-4 M; positive 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 beta -adrenergic receptors by the addition of isoproterenol (10-5 M), terbutaline (10-3, 10-4, or 10-5 M), or epinephrine (10-5 M) to the solution and incubation of the samples for 10 min at 37°C. We used 10-5 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 10-6 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 -70°C until analysis. The cAMP content in each sample was determined with a radioimmunoassay (NEN-DuPont, Boston, MA).

Statistics

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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Table 1.   Alveolar liquid clearance in guinea pigs treated with beta -adrenergic agonists and antagonists over 1 h


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Fig. 1.   Alveolar liquid clearance over 1 h in guinea pigs instilled with 5% albumin (control) and with 5% albumin containing 10-4 M propranolol, 10-6 M epinephrine, or 10-6 M epinephrine + 10-4 M propranolol. Values are means ± 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; dagger  epinephrine + propranolol (by analysis of variance).

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 10-6 M epinephrine and studied them for 1 h. When epinephrine was added to the instillate, the final-to-instilled 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 beta -adrenergic-receptor activation, we added 10-4 M propranolol to the instilled fluid. The increases in the final-to-instilled protein concentration ratio and alveolar liquid clearance seen after epinephrine instillation 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 beta -adrenergic-receptor stimulation, we investigated whether the stimulatory effect was primarily mediated by beta 1-receptor or beta 2-receptor stimulation. We used terbutaline as a relatively specific beta 2-adrenergic agonist and isoproterenol as a general beta -adrenergic agonist. The addition of isoproterenol (10-5 M) to the instillate significantly increased the final-to-instilled protein concentration ratio and the alveolar liquid clearance by the same magnitude as 10-6 M epinephrine (Fig. 2, Table 1). However, the addition of the more specific beta 2-adrenergic agonist terbutaline (10-4 M) did not affect the final-to-instilled protein concentration ratio and hence the alveolar liquid clearance (Fig. 2, Table 1).


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Fig. 2.   Alveolar liquid clearance over 1 h in guinea pigs instilled with 5% albumin (control) and with 5% albumin containing 10-6 M epinephrine, 10-5 M isoproterenol, or 10-4 M terbutaline. Values are means ± 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).

Effect of Atenolol on beta -Adrenergically Stimulated Alveolar Liquid Clearance

To further investigate which beta -receptor subtype could be involved, we used the relatively specific beta 1-adrenergic antagonist atenolol. The addition of atenolol (10-4 M) to the 5% albumin instillate solution containing either epinephrine (10-6 M) or isoproterenol (10-5 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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Fig. 3.   Alveolar liquid clearance over 1 h in guinea pigs instilled with 5% albumin (control) and with 5% albumin containing 10-6 M epinephrine or 10-5 M isoproterenol with (open bars) or without (solid bars) beta 1-adrenergic antagonist atenolol. Values are means ± SD. Addition of 10-4 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; dagger  isoproterenol (by analysis of variance).

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 10-3 M amiloride to the 5% albumin instillate with or without the beta -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 10-6 M epinephrine or 10-5 M isoproterenol, the inhibition was similar compared with the studies in which amiloride was given alone (Fig. 4, Table 2).


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Fig. 4.   Alveolar liquid clearance over 1 h in guinea pigs instilled with 5% albumin (control) and with 5% albumin containing 10-6 M epinephrine or 10-5 M isoproterenol with (open bars) or without (solid bars) sodium-channel inhibitor amiloride. Values are means ± SD. Addition of 10-3 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; dagger  epinephrine; ddager  isoproterenol (by analysis of variance).

                              
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Table 2.   Alveolar liquid clearance in guinea pigs treated with amiloride with or without beta -adrenergic agonists over 1 h

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 10-5 M isoproterenol and 10-5 M epinephrine increased the cAMP content in the lung tissue to a similar level as 10-4 M forskolin, but terbutaline (10-3, 10-4, or 10-5 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 ± 0.98 and 7.90 ± 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).


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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 10-5 M isoproterenol, 10-4 M terbutaline, 10-5 M epinephrine, or 10-4 M forskolin added to incubation medium. Some tissue samples were also incubated with 10-3 or 10-5 M terbutaline. Values are means ± SD; n = 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 10-3 and 10-5 M terbutaline were similar (13.22 ± 2.60 and 14.32 ± 4.06 pmol/mg lung tissue wet weight, respectively) to that with 10-4 M terbutaline. Significant difference (P < 0.05) compared with: * control (37°C); dagger  terbutaline (by analysis of variance).

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 beta -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 FITC-Dextran 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -adrenergic stimulation. The general beta -agonist isoproterenol had similar effects on alveolar liquid clearance as epinephrine, but the more specific beta 2-adrenergic agonist terbutaline had no effect. This finding suggested that the stimulation of alveolar epithelial liquid clearance occurred by the beta 1-adrenergic receptor. Consequently, we tested the specific beta 1-adrenergic antagonist atenolol. Atenolol inhibited the stimulatory effects of epinephrine and isoproterenol. These results strengthen the conclusion that the beta 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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Table 3.   Alveolar liquid clearance in different species at baseline and after stimulation with beta -adrenergic agonists or inhibition with amiloride

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 beta -adrenergic antagonist, as well as atenolol, a beta 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 beta -adrenergic stimulation of alveolar liquid clearance, we investigated whether the guinea pig responded to beta -adrenergic agonists. In several species (3, 4, 15, 27), alveolar liquid clearance increases in response to stimulation by beta -adrenergic agonists (Table 3), whereas in some species (29), there is no response to beta -adrenergic stimulation. The addition of epinephrine or the general beta -adrenergic agonist isoproterenol resulted in significantly increased alveolar liquid clearance rates in the guinea pig studies. This stimulation was totally inhibited when the general beta -adrenergic inhibitor propranolol was added to the instillate. However, when the more specific beta 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 beta 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 beta -adrenergic receptor is stimulated. Because the stimulatory effect of both epinephrine and isoproterenol was completely attenuated by atenolol, a beta 1-receptor inhibitor, the results suggest that alveolar liquid clearance in the guinea pig was stimulated primarily by agonist binding to the beta 1-adrenergic receptor. However, beta 2-adrenergic pathways could not be completely excluded.

Could the observed results be explained by agonist binding to the beta 1-adrenergic receptor? In several studies (8, 12), it has been demonstrated that there are mixed populations of beta -adrenergic receptors in mammalian lungs. In guinea pigs, there are ~20% beta 1-adrenergic and ~80% beta 2-adrenergic receptors (12) in the pulmonary cell membranes. It has also been shown that 21% of the beta -adrenergic receptors on the alveolar type II cell membranes in the guinea pig lung are of the beta 1-subtype (12). Furthermore, guinea pig alveolar type I cells have beta -receptors on their cell membranes (8), but the physiological significance of the beta -receptors on these cells is difficult to evaluate. The exact localization of the beta -adrenergic receptors in the distal lung tissue is not known, but 70% of the total number of beta -adrenergic receptors in the guinea pig lung are in the alveoli (12). Furthermore, there is functional evidence that the beta -adrenergic receptors are localized on both the basolateral and apical sides of the pneumocytes (4, 26). Therefore, there is a high probability that beta -adrenergic receptors may be linked to regulation of alveolar liquid clearance. Our functional data suggest that the beta 1-receptors are the primary receptors regulating alveolar liquid clearance in the guinea pig lung.

Because it has been suggested earlier (16) that different beta -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 beta 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 beta 1-adrenergic receptors in the guinea pig.

Another possible explanation for the increased alveolar liquid clearance after beta 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 beta -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 beta 1-adrenergic-receptor stimulation. Thus the beta 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 beta 2-adrenergic receptor cannot be completely excluded.

    ACKNOWLEDGEMENTS

We thank Dr. Michael A. Matthay for critically reading the manuscript and for all his suggestions.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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