Modulation of lung liquid clearance by isoproterenol in rat lungs

F. Saldías, E. Lecuona, E. Friedman, M. L. Barnard, K. M. Ridge, and J. I. Sznajder

Division of Pulmonary and Critical Care Medicine, Michael Reese Hospital, University of Illinois at Chicago, Chicago, Illinois 60616; and Departamento de Enfermedades Respiratorias, Pontificia Universidad Católica de Chile, Santiago, Chile

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

beta -Adrenergic agonists have been reported to increase lung liquid clearance by stimulating active Na+ transport across the alveolar epithelium. We studied mechanisms by which beta -adrenergic isoproterenol (Iso) increases lung liquid clearance in isolated perfused fluid-filled rat lungs. Iso perfused through the pulmonary circulation at concentrations of 10-4 to 10-8 M increased lung liquid clearance compared with that of control lungs (P < 0.01). The increase in lung liquid clearance was inhibited by the beta -antagonist propranolol (10-5 M), the Na+-channel blocker amiloride (10-4 M), and the antagonist of Na-K-ATPase, ouabain (5 × 10-4 M). Colchicine, which inhibits cell microtubular transport of ion-transporting proteins to the plasma membrane, blocked the stimulatory effects of Iso on active Na+ transport, whereas the isomer lumicolchicine, which does not affect cell microtubular transport, did not inhibit Na+ transport. In parallel with these changes, the Na-K-ATPase alpha 1-subunit protein abundance and activity increased in alveolar type II cells stimulated by 10-6 M Iso. Colchicine blocked the stimulatory effect of Iso and the recruitment of Na-K-ATPase alpha 1-protein to the basolateral membrane of alveolar type II cells. Accordingly, Iso increased active Na+ transport and lung liquid clearance by stimulation of beta -adrenergic receptors and probably by upregulation of apical Na+ channels and basolateral Na-K-ATPase mechanisms. Recruitment from intracellular pools and microtubular transport of Na+ pumps to the plasma membrane participate in beta -adrenergic stimulation of lung liquid clearance in rat lungs.

active sodium transport; lung edema clearance; apical sodium channels; sodium-potassium-adenosinetriphosphatase; cytoskeleton

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PULMONARY EDEMA ACCUMULATES as the result of changes in hydrostatic and colloid-osmotic pressure gradients or changes in the vascular or epithelial permeability (11, 19). Over the last decade, lung epithelial Na+ transport has been explored in physiological models of mammalian lungs (1, 10, 15, 29) and alveolar epithelial type II cell monolayers (6, 12, 13, 18, 33, 37). It has been shown that Na+ enters the alveolar epithelial cells through apical Na+ channels and is actively transported out of the cell by basolaterally located Na-K-ATPase (18, 24). Water probably moves along osmotic gradients generated by active Na+ transport out of the alveolus.

Studies in vivo and in isolated perfused fluid-filled lungs have reported that beta -adrenergic agonists enhance active Na+ transport and lung liquid clearance in mammals (3, 7, 15, 30). In cultured alveolar type II (ATII) cell monolayers, the intracellular second messenger cAMP participates in the beta -adrenergic stimulation (6, 13).

Administration of beta -adrenergic agonists terbutaline, epinephrine, and isoproterenol (Iso) has been reported to increase active Na+ transport across the alveolocapillary membrane of rat lungs (7, 15, 30). Recently, it has been reported that incubation of rat ATII cells for 15 min with 1 µM Iso increased 2.5-fold the Na-K-ATPase activity, and this effect was abolished by a cAMP-dependent protein kinase inhibitor (27). Intracellular microtubular transport and cortical cytoskeleton have been shown as well to play a role in signal transduction and recruiting of ion transport-related proteins to the plasma membrane (4, 5, 17, 26). The purpose of this study was to examine the mechanisms by which Iso stimulates lung liquid clearance and focus on the contribution of apical Na+ channels and basolaterally located Na-K-ATPases. Subsequently, we examined whether disruption of the cellular microtubular transport pathway of ion-transporting proteins contributes to Iso-stimulated active Na+ transport.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pathogen-free male Sprague-Dawley rats weighing 280-360 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). A total of 140 rat lungs were studied. All animals were provided food and water ad libitum and were maintained on a 12:12-h light-dark cycle. Iso, amiloride, ouabain, propranolol, colchicine, and beta -lumicolchicine were purchased from Sigma (St. Louis, MO).

Isolated Lungs

The isolated lung preparation was performed as previously described (25, 29, 34). Briefly, rats were anesthetized with 50 mg/kg body wt of pentobarbital sodium. A tracheotomy was performed, and the rats were mechanically ventilated with a tidal volume of 2.5 ml, a peak airway pressure of 8-10 cmH2O, and 100% O2 for 5 min. During the thoracotomy, rats were anticoagulated with 400 U of heparin. The pulmonary artery and left atrium were cannulated, and the pulmonary circulation was flushed of remaining blood by perfusing with a buffered salt albumin solution containing 135.5 mM Na+, 119.1 mM Cl-, 25 mM HCO-3, 4.1 mM K+, 2.8 mM Mg2+, 2.5 mM Ca2+, 0.8 mM SO2-4, 8.3 mM glucose, and 3% bovine albumin, with an osmolality of 300 mosmol/kgH2O. The solution was maintained at pH 7.40 by bubbling a 5% CO2-95% O2 mixture as needed. Two sequential bronchoalveolar lavages were performed with 3 ml of the buffered salt albumin solution containing 0.1 mg/ml of Evans blue dye (EBD; Sigma), 0.02 µCi/ml of 22Na+ (DuPont NEN, Boston, MA), and 0.12 µCi/ml of [3H]mannitol (DuPont NEN). The lungs were then instilled with the volume necessary to leave 5 ml in the alveolar space. Finally, the lungs were immersed in a "pleural bath" reservoir containing 100 ml of the buffered salt albumin solution maintained at 37°C. This allowed us to follow markers that had moved across the pleural membrane or were drained by the lung lymphatics.

Perfusion of the lungs was performed with 90 ml of the same buffered salt albumin solution containing 0.16 mg/ml of fluorescein-tagged albumin (FITC-albumin; Sigma). The perfusate was pumped from a lower reservoir to an upper reservoir by a peristaltic pump and from there flowed through the pulmonary artery and exited via the left atrium. Left atrial and pulmonary arterial pressures were maintained at 0 and 12 cmH2O, respectively, and were recorded via a pressure transducer with a zero reference point at the level of the left atrium. Pulmonary arterial pressure and left atrial pressure were recorded continuously with a multichannel recorder (Gould 3000 oscillograph recorder; Gould, Cleveland, OH).

Samples were drawn from the three reservoirs, air space instillate, pleural bath, and perfusate, at 10 and 70 min after starting the experiment protocol. To ensure homogeneous sampling from the air spaces, 2 ml of instillate were aspirated and reintroduced into the air spaces three times before removing each sample. This has been shown to provide a reproducibly mixed sample in our laboratory (25, 29, 34). All samples were centrifuged at 1,000 g for 15 min. Colorimetric analysis of the supernatant for EBD (absorbance at 620 nm) was performed in a Hitachi model U2000 spectrophotometer (Hitachi, San Jose, CA). Analysis of FITC-albumin (excitation 487 nm and emission 520 nm) was performed in a Perkin-Elmer fluorescence spectrometer (model LS-3B; Perkin-Elmer, Oakbrook, IL). 22Na+ and [3H]mannitol were measured in a beta-counter (Packard Tricarb, Downers Grove, IL). Na+ concentration was measured in an automated microprocessor-controlled analyzer employing the ion-selective electrode technique (Lytening 1; AMDEV, Danvers, MA).

Specific Protocols

Group A. We studied active Na+ transport and lung liquid clearance over 1 h in a control group of rat lungs (n = 10 animals).

Group B. We established the dose-response curve of Iso (10-4 M to 10-10 M) perfused through the pulmonary circulation on lung liquid clearance (n = 6 animals in each group). The doses of Iso selected for these studies were similar to the doses used in other studies of ATII cells in our laboratory (27).

Group C. We determined the effects of Iso given in the instillate (n = 6 animals) and in both instillate and perfusate simultaneously (n = 6 animals) on active Na+ transport and lung liquid clearance.

Group D. We studied the effects of a beta -adrenergic antagonist, propranolol (10-5 M), on lung liquid clearance in a control group (n = 4 animals) and lungs perfused with 10-6 M Iso (n = 6 animals).

Group E. To determine the possible role of Na+ channels and Na-K-ATPase in the stimulatory effects of Iso on liquid clearance, we studied the effect of an Na-K-ATPase antagonist (5 × 10-4 M ouabain) administered in the perfusate alone (n = 6 animals) or with 10-6 M Iso (n = 6 animals), and we evaluated the effect of an Na+-channel blocker (10-4 M amiloride) added to the alveolar instillate alone (n = 5 animals) or with 10-6 M Iso (n = 6 animals).

Group F. To examine the possible role of the intracellular microtubular transport of ion-transporting proteins on the active Na+ transport mediated by Iso, we studied lung liquid clearance in rats treated with colchicine (0.25 mg/100 g body wt injected intraperitoneally ~15 h before the experiments) without beta -agonist stimulation (n = 6 animals) and with 10-6 M Iso added to the perfusate (n = 5 animals). In addition, eight animals were studied after intraperitoneal injection of beta -lumicolchicine (0.25 mg/100 g body wt) ~15 h before experiments with and without beta -adrenergic stimulation. Lumicolchicine is an isomer of colchicine that does not bind tubulin and does not depolymerize microtubules (36). However, it shares other properties of colchicine, such as inhibition of protein synthesis, and it is therefore an appropriate control to demonstrate that the observed effects of colchicine are due to microtubular disruption. The inhibitory effect of colchicine but not lumicolchicine on microtubular transport has been previously reported in bile secretion studies in rats (8).

Group G. To examine the possible association between the active Na+ transport stimulated by Iso and the intracellular microtubular transport, we studied the effect of 10-4 M amiloride added to the alveolar instillate and 5 × 10-4 M ouabain added to the perfusate in colchicine-treated rats without beta -agonist stimulation and with 10-6 M Iso added to the perfusate (n = 6 animals in each group).

Calculations

The alveolar lining fluid volume (VELF) was calculated by instilling 3 ml of fluid [initial volume (V0)] containing a known concentration of albumin tagged by EBD [(EBD)0] into the air space. After brief mixing, a sample was removed, and the EBD concentration at time t [(EBD)t] was determined. The mass of Evans blue-tagged albumin is the same in the instillate [V0(EBD)0] and in the lung after initial mixing [(V0 + VELF)(EBD)t]. Equating the two yields
V<SUB>0</SUB>(EBD)<SUB>0</SUB> = (EBD)<SUB><IT>t</IT></SUB>(V<SUB>0</SUB> + V<SUB>ELF</SUB>) (1)
or
V<SUB>ELF</SUB> = V<SUB>0</SUB>(EBD)<SUB>0</SUB>/(EBD)<SUB><IT>t</IT></SUB> − V<SUB>0</SUB> (2)
Similarly, the alveolar fluid volume at time t is estimated by
V<SUB><IT>t</IT></SUB> = V<SUB>0</SUB>(EBD)<SUB>0</SUB>/(EBD)<SUB><IT>t</IT></SUB> (3)
The movement of Na+ from the alveolar space during a defined period of time is assumed to be accompanied by isotonic water flux and is given by JNa,net = JNa,out - JNa,in, where JNa,net is the net or active Na+ transport, JNa,out is the total or unidirectional Na+ outflux from the rat air space, and JNa,in is the passive bidirectional flux of Na+ between the air space and the other compartments. The volume flux J = JNa,net /[Na+] is the average rate of change in the volume and is given by
<IT>J</IT> = (V<SUB><IT>t</IT></SUB> − V<SUB>0</SUB>)/<IT>t</IT> (4)
where Vt is the volume at time t.

As described (29), the passive movement of 22Na+, JNa,in, is given by
<IT>J</IT><SUB>Na,in</SUB> = [Na<SUP>+</SUP>]<IT>J</IT>(ln C<SUB><IT>t</IT></SUB> − ln C<SUB>0</SUB>)/(ln V<SUB><IT>t</IT></SUB> − ln V<SUB>0</SUB>) (5)
Where Cx is the 22Na+ concentration at time x and [Na+] is the constant Na+ concentration in the buffered salt albumin solution.

Similarly, the volume flux of mannitol [typically expressed as permeability of surface area (PSA)] is given by
<IT>P</IT><SUB>SA</SUB> = <IT>J</IT>(ln <IT>M</IT><SUB><IT>t</IT></SUB> − ln <IT>M</IT><SUB>0</SUB>)/(ln V<SUB><IT>t</IT></SUB> − ln V<SUB>0</SUB>) (6)
where Mx is the [3H]mannitol mass at time x.

Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-albumin that appeared in the alveolar space during the experimental protocol. These calculations were carried out for each sampling period.

ATII Cell Isolation and Western Blot Analysis of Basolateral Membranes

ATII cells were isolated from normal and colchicine-treated rats as previously described (24, 28). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with 10 U/ml of elastase (Worthington Biochemical) for 20 min at 37°C. The tissue was minced and filtered through sterile gauze and 70-µm nylon mesh. The crude cell suspension was purified by differential adherence to IgG-pretreated dishes. ATII cells were homogenized in homogenization buffer, and basolateral membranes (BLMs) were obtained as described by Hammond et al. (14). After several centrifugations to discard the nuclear and mitochondrial pellet, the remaining supernatant was spun at 48,000 g for 30 min. Finally, the BLM fraction was recovered after the membrane pellet was centrifugated in a Percoll gradient (16%) at 48,000 g for 30 min. Na-K-ATPase alpha 1-subunit abundance was determined by Western blot analysis in control and colchicine-treated rats incubated for 15 min with 1 µM Iso. Protein was quantified by Bradford assay, and 2.5 µg of BLM proteins were loaded on each lane of a 10% polyacrylamide gradient gel. Thereafter, they were transferred to nitrocellulose membranes (Optitran, Schleider & Schuell) using a semidry transfer cell (Bio-Rad). Incubation with specific Na-K-ATPase monoclonal alpha 1-antibody (a generous gift from M. Caplan, Yale University, New Haven, CT) at 1:200 dilution was performed overnight at 4°C. Blots were developed with an enhanced chemiluminescence detection kit (Amersham, Arlington Heights, IL) used as recommended by the manufacturer.

Na-K-ATPase Activity

Na-K-ATPase activity was determined in ATII cells isolated from control and colchicine-treated rats as the rate of [32P]ATP hydrolysis in suspended cells in buffer containing (in mM) 5 KCl, 10 MgCl2, 1 EGTA, 50 Tris · HCl, 3 Na2ATP (grade II, Sigma), and [32P]ATP (Amersham) in trace amounts. NaCl was added to a final concentration of 100 mM to examine activity at the concentration at which the Na-K-pump normally operates at maximal transport rate (>70 mM Na+). Cells were transiently permeabilized by thermic shock to make them permeable to [32P]ATP. The reaction was carried out for 15 min at 37°C with and without 1 µM Iso and terminated by placement on ice and addition of TCA-charcoal to absorb nonhydrolyzed [32P]ATP. The ouabain-insensitive ATPase activity was determined in buffer lacking Na+ and K+ but including 1 mM ouabain. Nonspecific ATP hydrolysis was determined in samples in the absence of cells. The 32Pi liberated was quantified by liquid scintillation counting (Packard Tricarb) and expressed as nanomoles of Pi per milligram of protein per minute.

Data Analysis

Data are presented as mean values ± SE; n represents the number of animals in each experimental group. When comparisons were made between two experimental groups, an unpaired Student's t-test was used. When multiple comparisons were made, a one-way analysis of variance was used, followed by a multiple comparison test (Tukey) when the F statistic indicated significance. Results are considered significant at P < 0.05.

    RESULTS
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Materials & Methods
Results
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Lung Liquid Clearance

The lungs of control rats instilled with 5 ml of buffered salt albumin solution cleared ~10% of the instillate in 1 h (0.45 ± 0.03 ml/h), whereas 10-4 to 10-8 M Iso increased lung liquid clearance in a dose-dependent manner up to 149% above that in control lungs (Fig. 1). Lung liquid clearance with 10-9 and 10-10 M Iso was not different from that in control lungs (0.62 ± 0.07 and 0.49 ± 0.05 ml/h, respectively). The effects of 10-6 M Iso added to the instillate (0.82 ± 0.06 ml/h) or the perfusate (0.98 ± 0.05 ml/h) were similar. Iso administered simultaneously by these two routes did not produce an additive effect on lung liquid clearance (0.92 ± 0.06 ml/h). Pulmonary arterial pressure and perfusate flow did not change with the administration of Iso by any route in all experimental groups (data not shown).


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Fig. 1.   Isoproterenol increased lung liquid clearance in isolated perfused rat lungs. Isoproterenol at indicated concentrations was perfused through pulmonary circulation. Bars are means ± SE. CT, control group. * P < 0.01 compared with control lungs.

Propranolol (10-5 M) administered in the instillate and perfusate simultaneously blocked the effect of 10-6 M Iso on active Na+ transport and lung liquid clearance (0.59 ± 0.03 ml/h) but did not affect liquid clearance in control lungs (0.47 ± 0.03 ml/h). Perfusion pressures and flow rates in this group were not different from those in control lungs.

Effects of Colchicine and Lumicolchicine

In colchicine-treated animals, active Na+ transport and lung liquid clearance significantly decreased compared with those in control lungs, and colchicine also partially inhibited the effect of Iso (Fig. 2). However, lung liquid clearance and Na+ transport were not decreased in rat lungs treated with beta -lumicolchicine.


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Fig. 2.   Effects of colchicine (Col; 0.25 mg/100 g body wt) and beta -lumicolchicine (Lumic; 0.25 mg/100 g body wt) on control lungs and lungs perfused with 10-6 M isoproterenol (Iso) through pulmonary circulation. Bars are means ± SE. * P < 0.01 compared with control, Col, and Lumic groups. + P < 0.01 compared with other experimental groups. # P < 0.01 compared with Col and Iso groups.

Air space-instilled amiloride (10-4 M) decreased the active Na+ transport and lung liquid clearance by ~46% compared with those of control lungs. Amiloride (10-4 M) blocked the stimulatory effect of Iso on lung liquid clearance without changing the perfusion flow rates (Fig. 3).


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Fig. 3.   Amiloride (Amil; 10-4 M) instilled into alveolar space inhibited lung liquid clearance in control and Col-treated lungs. Iso did not increase lung liquid clearance in Amil-instilled, Col-treated rat lungs. Bars are means ± SE. * P < 0.001 compared with control group. + P < 0.01 compared with control group.

Ouabain (5 × 10-4 M) perfused through the pulmonary circulation decreased lung liquid clearance and Na+ transport by ~56% compared with control lungs and blocked the stimulatory effect of Iso on lung liquid clearance without changing perfusion flow rates (Fig. 4).


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Fig. 4.   Ouabain (Ouab; 5 × 10-4 M) perfused through pulmonary circulation inhibited lung liquid clearance in control and Col-treated rat lungs. Iso did not increase lung liquid clearance in Ouab-perfused, Col-treated rat lungs. Bars are means ± SE. * P < 0.001 compared with control group. + P < 0.01 compared with Col group. # P < 0.05 compared with Ouab group.

In colchicine-treated rat lungs, amiloride (10-4 M) added to the instillate and ouabain (5 × 10-4 M) added to the perfusate completely blocked basal and Iso-stimulated lung liquid clearance (Figs. 3 and 4).

Effect of Iso on ATII Cells, Na-K-ATPase Activity, and alpha 1-Subunit Protein Expression

As shown in Fig. 5, Na-K-ATPase alpha 1-subunit abundance was determined by Western blot analysis in BLMs of ATII cells exposed to 1 µM Iso in control (Fig. 5A) and colchicine-treated rats (Fig. 5B). The Na-K-ATPase alpha 1-subunit expression significantly increased in BLMs of ATII cells stimulated by Iso, and colchicine inhibited the stimulatory effect of Iso. A representative autoradiogram is shown for the alpha 1-subunit protein expression in control and colchicine-treated rat lungs.


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Fig. 5.   Na-K-ATPase alpha 1-subunit abundance in basolateral membranes of alveolar type II (ATII) cells isolated from control lungs (A) and Col-treated rat lungs (B). Cells were incubated for 15 min with 1 µM Iso. Equal amounts of protein (2.5 µg) were loaded in each lane. Top: representative Western blot of Na-K-ATPase alpha 1-subunit abundance. Bottom: quantitative densitometric scans of 6 experiments. Col blocked increment of Na-K-ATPase alpha 1-subunit expression stimulated by Iso. Bars are means ± SE. * P < 0.01 compared with control group.

In parallel with these changes on lung liquid clearance and protein abundance in ATII cells treated with Iso, we observed a significant increase in the Na-K-ATPase activity in ATII cells isolated from control rats incubated for 15 min with 1 µM Iso (Fig. 6). Colchicine blocked the Na-K-ATPase activity stimulated by Iso.


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Fig. 6.   Na-K-ATPase activity increased in ATII cells isolated from control rats (n = 4) incubated for 15 min with 10-6 Iso. Col blocked stimulatory effect of Iso on Na+-K+ pump activity (n = 4 animals). Bars are means ± SE. * P < 0.01 compared with other experimental groups.

Epithelial Permeability

The movement of protein tracers across the alveolar epithelial barrier was similar to the rates reported in previous studies (3, 15, 29, 34). EBD-bound albumin instilled in the air space was not detected in the perfusate or bath compartments in any of the experimental groups. The movement of FITC-albumin from the pulmonary vascular compartment into the air spaces was not changed in any of the experimental groups. This minimal movement of albumin allows us to accurately assess lung liquid clearance in this model.

As shown in Fig. 7, higher concentrations of Iso (10-4 to 10-6 M) increased epithelial 22Na+ and [3H]mannitol permeability. Propranolol, amiloride, ouabain, and colchicine did not change the passive flux of small solutes (Table 1). The passive movement of 22Na+ and [3H]mannitol across the membrane correlated closely with a slope of ~2.0 for linear regression through the origin (r = 0.77).


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Fig. 7.   Effects of Iso perfused through pulmonary circulation on passive 22Na+ and [3H]mannitol movement. Bars are means ± SE. * P < 0.05 compared with control lungs.

                              
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Table 1.   Passive Na+ and mannitol fluxes in isolated perfused rat lungs

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The isolated perfused liquid-filled lung model is a relatively simple but powerful tool to investigate epithelial transport, but it has limitations that must be considered. The composition of alveolar instillate is arbitrary and may in some way alter the epithelial function. Also, it is not possible to determine which exact cell type is responsible for active transport and whether there is any contribution to transport by the airway epithelium. With consideration of the fact that the rate of active ion transport is low compared with passive bidirectional flux, very subtle variations may not be easily demonstrated (29). However, variables such as temperature, pH, hydrostatic and osmotic pressures, and electrolytes can be tightly controlled in this model (25, 29, 34). Although lungs are not normally filled with liquid, this model is appropriate for the study of lung edema clearance because it reproduces conditions observed during pulmonary edema in which a liquid-liquid interface occurs.

beta -Adrenergic agonists stimulate active Na+ transport and edema clearance in vivo (3), in isolated perfused lungs (7, 30), and in cultured alveolar epithelial type II cells (6, 13). Iso is a nonselective beta -adrenergic agonist with very low affinity for alpha -adrenergic receptors. In agreement with Saumon et al. (30), we have confirmed that Iso increases active Na+ transport and lung liquid clearance in isolated perfused rat lungs by a similar magnitude as shown for terbutaline and epinephrine (3, 7, 15). The effect of Iso on lung liquid clearance was dose dependent (Fig. 1). The magnitude of basal lung liquid clearance was similar to the rate reported previously, ~10% over 1 h in control rat lungs (1, 10, 29, 30). We determined that the beta -adrenergic agonist Iso increased lung liquid clearance via stimulation of beta -adrenergic receptors as well as amiloride-sensitive Na+ channels and alveolar epithelial Na-K-ATPase. These data agree with previous reports showing that beta -adrenergic agonists regulate Na+ channels (37) and Na-K-ATPase activity (27, 33) in ATII cells.

In a similar model, terbutaline exhibited greater effects on active Na+ transport when it was placed in the instillate (7), but in our study, the effects of Iso on active Na+ transport were similar regardless of whether it was added to the instillate, perfusate, or both compartments. Therefore, beta -adrenergic agonists appear equally efficient when placed in the air spaces or the pulmonary circulation, suggesting either that receptors may be present on both sides of the alveolar epithelium or that beta -adrenergic agonists diffuse easily across the alveolocapillary membrane.

In agreement with previous studies in vivo (3, 15) and in isolated lungs (7), baseline lung liquid clearance and Na+ transport were not affected by the beta -antagonist propranolol. However, the experiments with the beta -antagonist propranolol demonstrated that the effects of Iso on lung liquid clearance were mediated via beta -adrenergic receptors. Previous studies have demonstrated that alpha -adrenergic agonists do not increase Na+ transport in alveolar epithelial type II cell monolayers (13).

Evidence for mechanisms by which Iso increased liquid clearance is provided by the experiments with a nonselective Na+-channel blocker (amiloride) and an Na-K-ATPase antagonist (ouabain). Both drugs decreased the Iso-stimulated Na+ transport and lung liquid clearance, suggesting that the stimulatory effect of Iso on edema clearance is linked to Na+ transport via the amiloride-sensitive pathway (Na+ channels) and Na-K-ATPase. In agreement with our findings, beta -adrenergic agonists stimulated the dome formation and short-circuit currents across alveolar epithelial type II cells (13).

The inhibition by amiloride of lung liquid clearance in our study was similar to previous reports (1, 10, 15, 30). The effect of amiloride at these concentrations (10-4 M) probably includes the inhibitory effects on apical Na+ channels, on the Na+-H+ antiport system, and possibly on the basolaterally located Na-K-ATPase (2, 32). However, it does not affect the Na+-glucose cotransport. It has been previously reported that when glucose is replaced by mannitol in the instillate, amiloride almost completely inhibits fluid absorption in isolated perfused lung (30).

Na-K-ATPase and ion channels are associated in epithelial cells with proteins (e.g., actin, ankyrin, and fodrin) that constitute the cortical cytoskeleton, which provides the foundation for spatial distribution of proteins within the plasma membrane (22). Evidence is now emerging for a dynamic regulation of Na+ channels and Na-K-ATPase activity by constituents of the cytoskeleton. In addition to governing cell structure and motility, actin appears to also serve as a signaling molecule required in Na+-channel activation by cAMP-dependent protein kinase (26). Also, there is evidence that Na-K-ATPase exists in intracellular pools and, on specific signaling (i.e., beta -adrenergics), can be rapidly recruited via cell microtubular transport into the plasma membrane (5). Therefore, we studied whether inhibition of microtubular transport to the plasma membrane by colchicine would inhibit the stimulatory effects of Iso on active Na+ transport and edema clearance. Colchicine inhibited lung liquid clearance and Na+ transport in control lungs and rat lungs stimulated by Iso, whereas the isomer beta -lumicolchicine, which shares many colchicine properties with the exception of inhibiting microtubular transport (36), did not inhibit the Iso stimulation of lung liquid clearance. Colchicine decreased basal lung clearance, confirming that the cortical cytoskeleton and intracellular microtubular transport are structurally and functionally linked to Na+ channels and Na-K-ATPase function in polarized epithelial cells (4, 5, 26, 31). The Na-K-ATPase alpha - and beta -subunits are degraded with a half-life of 10-12 h (16), and in our study, colchicine was administered ~15 h before the experiments; therefore, basal lung liquid clearance probably decreased because the microtubular transport pathway was disrupted, inhibiting the movement of Na+-transporting proteins to the plasma membrane (Figs. 2 and 5).

We observed that an Na+-channel blocker (amiloride) and an Na-K-ATPase antagonist (ouabain) completely blocked lung edema clearance stimulated by Iso in colchicine-treated rats (Figs. 2-4). Thus the inhibitory effect of colchicine on the Iso stimulation of lung liquid clearance is probably due to disruption of the vectorial Na+ transport pathway. In agreement with our results, others have reported the modulatory role of the cell microtubular transport and the cytoskeleton on the apical Na+-channel and Na-K-ATPase activity in basal and stimulated conditions (4, 5, 26, 31).

beta -Adrenergic agonists that stimulate Na-K-ATPase activity by increasing cellular cAMP might achieve this effect by interacting with the cell microtubular transport pathway (4, 5, 27). To examine this association, we studied the Na-K-ATPase activity and alpha 1-subunit protein abundance in ATII cells isolated from control and colchicine-treated rat lungs. We observed that Iso increased Na-K-ATPase activity and alpha 1-subunit abundance in BLMs of alveolar epithelial type II cells and that this effect was completely abolished by colchicine (Figs. 5 and 6). We thus reason that cell microtubular transport of Na+ pumps to the cell plasma membrane has a role in active Na+ transport in basal conditions and during stimulation of lung edema clearance by beta -adrenergic agonists.

Na-K-ATPase synthesis occurs in polysomes that become related to the membrane of the rough endoplasmic reticulum, and the alpha beta -subunits are transported through a network of membrane compartments to their functional site in the plasma membrane (5, 23). The Na-K-ATPase upregulation (activity measured by ATP hydrolysis and protein expression in BLMs by Western blot) by Iso occurs in ~15 min in ATII cells (27), and we have demonstrated that Iso increased active Na+ transport in isolated rat lungs within 1 h (Fig. 1). The increase in Na+ transport in our model is due to upregulation of apical Na+ channels and mobilization of Na+ pumps from intracellular pools by translocation to the plasma membrane via cell microtubular transport and possibly other posttranslational events.

In agreement with a previous study (30), we found that the paracellular permeability of the small molecules (22Na+ and [3H]mannitol) was increased by high concentrations of Iso. Changes in the paracellular pathway in epithelia secondary to the action of either catecholamines (21) or cAMP (9) have been reported previously. However, even at higher doses, Iso did not significantly change epithelial permeability to albumin; thus it did not interfere with our ability to estimate the rates of lung liquid clearance.

Restoration of alveolar epithelial permeability and lung edema clearance in patients with acute hypoxemic respiratory failure has been associated with improved clinical outcome (20, 35). In two different models of hyperoxic lung injury, changes in active Na+ transport and lung edema clearance paralleled changes in Na-K-ATPase function in ATII cells (24, 25, 34). Therefore, it is possible that Iso could be utilized as a therapeutic tool to upregulate Na-K-ATPase function and active Na+ transport in patients with pulmonary edema and hypoxemic respiratory failure.

In summary, our data show that Iso increases active Na+ transport and lung liquid clearance in a dose-dependent manner. This stimulation is mediated by the beta -adrenergic pathway in which intracellular microtubular transport of Na+ pumps from inner pools to the cell plasma membrane appears to play an important role.

    ACKNOWLEDGEMENTS

This research was supported, in part, by National Heart, Lung, and Blood Institute Grant HL-48129, American Heart Association Grant 96012890, the Department of Medicine of Michael Reese Hospital, and Pontificia Universidad Católica de Chile.

    FOOTNOTES

Address for reprint requests: J. I. Sznajder, Div. of Pulmonary and Critical Care Medicine, Michael Reese Hospital and Medical Center, 2929 S. Ellis Ave., RC-216, Chicago, IL 60616.

Received 19 May 1997; accepted in final form 4 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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