INTRODUCTION

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Pulmonary edema accumulates as the result of changes in hydrostatic or colloid-osmotic pressure gradients in the pulmonary circulation and/or increased permeability of the alveolocapillary barrier (1). Intermittent positive-pressure mechanical ventilation with high tidal volume (HVT) causes lung injury and pulmonary edema that mimics abnormalities observed in the acute respiratory distress syndrome (2). It has been reported that HVT ventilation increases microvascular filtration coefficient in isolated lungs (5) and produces a high permeability pulmonary edema in intact animals (6). It was also demonstrated that tidal volume excursion, and not peak airway pressure, is responsible for the pulmonary abnormalities associated with mechanical ventilation, which was accordingly named "volutrauma" rather than barotrauma (2, 7). Pulmonary edema formation in ventilator-associated lung injury (VALI) has been attributed to capillary stress failure, stretch pore phenomenon, depletion, and/or inactivation of surfactant constituents and release of proteolytic enzymes and proinflammatory mediators in the lung such as metalloproteinases and cytokines (8).

Intermittent positive-pressure ventilation with high tidal volume and high inflation pressures (as much as 45 cm H₂O) produces lung injury and pulmonary edema in rats (6, 7, 13). Recently, it has been reported that VALI decreases active Na⁺ transport, and lung edema clearance in association with downregulation of Na,K-ATPase activity in alveolar epithelial type II cells isolated from rats exposed to HVT ventilation for 40 min (14).

It has been shown that -adrenergic agonists increase active Na⁺ transport and lung edema clearance in animal models and human lungs (15). The -adrenergic agents stimulate lung edema clearance by upregulating apical Na⁺ channels and basolateral Na,K-ATPase in rat alveolar epithelium (19, 20). Also, it has been shown that -adrenergic stimulation increases lung edema clearance in hyperoxic-injured rat lungs (21, 22).

This study was designed to determine whether -adrenergic agonists could improve the lung's ability to clear edema in rats exposed to VALI. We have demonstrated that both terbutaline (TERB) instilled into air spaces and isoproterenol (ISO) perfused through the pulmonary circulation accelerate lung edema clearance in rats ventilated with HVT. Experiments in rats treated with colchicine suggest that the stimulatory effects of -adrenergic agonists on active sodium transport are probably mediated by recruitment of ion-transporting proteins from intracellular pools to the plasma membrane in rat alveolar epithelium.

	METHODS

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Pathogen-free, male, Sprague-Dawley rats weighing 280 to 320 g were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). A total of 120 rat lungs were studied. All animals were provided food and water ad libitum and maintained on a 12 h:12 h light-dark cycle. Isoproterenol, terbutaline, amiloride, ouabain, choline chloride, colchicine, and -lumicolchicine were purchased from Sigma Chemical Co. (St. Louis, MO).

Mechanical Ventilation

Rats were anesthetized by intraperitoneal injection of 50 mg/kg body weight pentobarbital, tracheotomized, and mechanically ventilated in a rodent ventilator (Model 683; Harvard Apparatus, South Natick, MA). Adult rats were ventilated for 40 min with HVT of 40 ml/kg to peak airway pressures of 35 cm H₂O and a respiratory rate of 40 breath/min and compared with control nonventilated rats. It has been previously reported that this experimental protocol causes lung injury and pulmonary edema and decreases active Na⁺ transport and lung edema clearance in rats (14).

Specific Protocols

Group A. Control group (n = 10 rats) instilled with 5 ml buffered salt albumin solution (BSA) into the air spaces.

Group B. To examine -adrenergic agonists effects on alveolar epithelial Na⁺ transport, 10⁵ M TERB was instilled into the air spaces or 10⁶ M ISO was perfused through the pulmonary circulation (six in each group).

Group C. Lung edema clearance in 10 rats exposed to HVT ventilation for 40 min.

Group D. HVT ventilated rat lungs instilled with 10⁵ M TERB into air spaces or perfused with 10⁶ M ISO through the pulmonary circulation (six in each group).

Group E. To examine the alveolar epithelial Na⁺ transport pathway in rats exposed to HVT ventilation, we studied the effect of a Na⁺ channel blocker (amiloride 10⁴ M) instilled into rat air spaces and a Na,K-ATPase antagonist (ouabain 5 × 10⁴ M) perfused through the pulmonary circulation in rat lungs perfused with 10⁶ ISO (six in each group).

Group F. To determine whether there was significant edemagenises in HVT ventilated rat lungs as compared with control lungs, the lungs were instilled with a modified BSA solution. The BSA solution was modified by substituting sodium chloride with choline chloride (130.5 mM final concentration) (six in each group).

Group G. We examined the contributory role of intracellular microtubular transport system on lung edema clearance modulation by -adrenergic agonists in VALI-exposed rats. We studied lung edema clearance in rats treated with colchicine (0.25 mg/100 g BW injected intraperitoneally ~ 15 h before the isolated-perfused rat lung experiments) alone or associated to 10⁶ M ISO perfused through the pulmonary circulation. Finally, we studied the effects of -lumicolchicine (0.25 mg/100 g BW injected intraperitoneally ~ 15 h before experiments) alone or associated with -adrenergic stimulation (six in each group). -Lumicolchicine is an isomer of colchicine that does not affect cell microtubular transport but it shares other properties of colchicine such as inhibition of protein synthesis (23). Therefore, it is considered 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 cell microtubular transport has been previously reported on bile secretion and hepatic ultrastructure studies and alveolar epithelial Na⁺ transport modulation by -adrenergic agonists in rats (18, 24).

Isolated Lungs

The isolated lung preparation was performed as previously described (14, 18). Briefly, rats were anesthetized with 50 mg/kg body weight of pentobarbital, tracheotomized, and mechanically ventilated with a tidal volume of 2.5 ml, peak airway pressure of 8 to 10 cm H₂O, and 100% oxygen for 5 min. The chest was opened via a median sternotomy, after which 400 U heparin sodium was injected into the right ventricle. After exsanguination, the heart and lungs were removed en bloc. The pulmonary artery and left atrium were catheterized, and the pulmonary circulation was flushed of remaining blood by perfusing with BSA solution containing in 135.5 mM Na⁺, 119.1 mM Cl, 25 mM HCO₃, 4.1 mM K⁺, 2.8 mM Mg⁺², 2.5 mM Ca⁺², 0.8 mM SO₄², 8.3 mM glucose, 3% bovine albumin, and osmolality of 300 mosm/kg H₂O. The solution was maintained at pH 7.40 by bubbling a mixture of 5% CO₂ and 95% O₂ as needed. Two sequential bronchoalveolar lavages (BAL) were performed with 3 ml of BSA solution containing 0.1 mg/ml Evans blue dye (EBD; Sigma), 0.02 µCi/ml ²²Na⁺ (Dupont, NEN, Boston, MA), and 0.12 µCi/ml ³H-mannitol (Dupont, NEN). The volume of the epithelial lining fluid (ELF) was estimated by the dilution of EBD in the first BAL. 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 BSA 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 BSA solution containing 0.16 mg/ml 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. Pulmonary artery and left atria pressures were maintained at 12 and 0 cm H₂O and recorded via a pressure transducer with a zero reference point at the level of the left atrium. Pulmonary artery and left atrium pressures were recorded continuously with a multichannel recorder (Gould 3000 Oscillograph Recorder; Gould Inc., Cleveland, OH). Pulmonary circulation pressures and flow rates were measured periodically during the experiments.

Samples were drawn from the three reservoirs: air-space instillate, "pleural bath," and perfusate at 10 and 70 min after starting the experimental 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 and in previous work (14, 18). 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 Inst., San Jose, CA). Analysis of FITC-albumin (excitation 487 nm and emmission 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 betacounter (Packard Tricarb; Packard Instrument Co., Downers Grove, IL).

Calculations

The alveolar lining fluid volume (VELF) was calculated by instilling 3 ml of fluid (V₀) containing a known concentration of albumin (EBD)₀, tagged by Evans blue dye into the air space. After brief mixing, a sample was removed and the Evans blue dye concentration at time t (EBD)_t was estimated. The amount of Evans blue dye is the same in the instillate [V₀(EBD)₀] and in the lung after initial mixing [(V₀ + VELF)(EBD)_t]. Equating the two yields:

(1)

or

(2)

Similarly, the alveolar fluid volume at time t is estimated by:

(3)

The movement of sodium for the alveolar space during a defined period of time is assumed to be accompanied by isotonic water flux and is given by: J_Na,net = J_Na,out  J_Na,in, where J_Na,net is the net or active Na⁺ transport, J_Na,out is the total or unidirectional Na⁺ out flux from the rat air spaces, and J_Na,in is the passive bidirectional flux of Na⁺ between the air space and the other compartments. The volume flux J = J_Na,net/ [Na⁺] is the average rate of change in the volume and is given by:

(4)

As described by Rutschman and colleagues (25), the passive movement of ²²Na⁺, J_Na,in, is given by :

(5)

where C(x) is the ²²Na⁺ concentration at time x and [Na⁺] is the constant Na⁺ concentration in the BSA solution.

Similarly, the volume flux of mannitol (typically expressed as PA, permeability-surface area product) is given by:

(6)

where M(x) 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 appears in the alveolar space during the experimental protocol. These calculations were carried out for each sampling period.

ATII Cell Isolation, Total RNA Isolation, and RT-PCR Analysis

ATII cells were isolated from adult rat lungs as previously described (14, 18). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase 30 U/ml (Worthington Biochemical Corp., Freehold, NJ) 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 immunoglobulin G-pretreated dishes, and cell viability was assessed by trypan blue exclusion (> 95%).

Total cellular RNA was extracted from ATII cells isolated from control rats and rats exposed to HVT for 40 min that were incubated with 1 µM ISO for 60 min, using RNeasy total RNA kit (Qiagen Inc., Santa Clarita, CA), as described by the manufacturer, based on the method described by Chomczynski and Sacchi (26). RNA was quantified by measurement of absorbance at 260 nm. The reverse transcriptase (RT) reaction was performed using the Superscript Preamplification System (GIBCO-BRL, Gaithersburg, MD) following the manufacturer's instructions. Briefly, 1 µg of total RNA was converted into cDNA, after denaturing at 70° C for 15 min, by incubation with a buffer containing oligo-dT primers, the reverse transcriptase (RT) enzyme, and dNTPs mix for 50 min at 42° C. The RT enzyme was then inactivated by incubation at 70° C for 15 min and the RNA removed by incubation with RNAse H for 20 min at 37° C. The resultant cDNAs were amplified by PCR using a Perkin Elmer 4800 Thermal Cycler (Perkin Elmer-Cetus, Norwalk, CT). Specific primers for the Na,K-ATPase ₁- and ₁-subunits and rat glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (Clontech Laboratories, Palo Alto, CA) were used (Table 1). The concentration of MgC1₂ was 1.5 mM for each primer pair. For ₁ and ₁ isoforms, amplification was performed as follows: 94° C × 2 min (initial denaturalization), 25 cycles of 94° C × 1 min, 53° C × 1 min 30 s, and 72 ° C 2 min, followed by a final extension at 72° C × 7 min. For the G3PDH, amplification was performed as follows: 94° C × 2 min (initial denaturalization), 21 cycles of 94° C × 45 s, 60° C × 45 s, and 72° C × 2 min, followed by a final extension at 72° C × 7 min. The data were obtained during the exponential phase of the PCR reaction. The amplified bands were analyzed by agarose gel electrophoresis and quantified by densitometric scan (Eagle Eye II; Stratagene, La Jolla, CA) and normalized against the internal control, glyceraldehyde-3 phosphate dehydrogenase.

Data Analysis

Data are presented as mean values ± SEM. When comparisons were made between two experimental groups Student's unpaired 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 were considered significant at p < 0.05.

	RESULTS

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Epithelial Permeability

The epithelial lining fluid (ELF) volume, estimated by the dilution of EBD in the first BAL, did not change significantly in rats ventilated with HVT for 40 min (Table 2). However, alveolar epithelial permeability to small solutes (²²Na⁺ and ³H-mannitol) increased in rats ventilated with HVT for 40 min as compared with control nonventilated rats. As shown in Table 2, and as previously reported, isoproterenol and terbutaline mildly increased the passive flux of small solutes in control nonventilated rats (16, 18).

The movement of albumin across the alveolar epithelial barrier was small, similar to the previously reported rates in normal rat lungs (14, 16, 18, 22, 25). Evans Blue dye-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 circulation into air space was minimal and not affected by HVT ventilation or -adrenergic agonists (Table 2).

Lung Edema Clearance

Terbutaline instilled into the air spaces and isoproterenol perfused through the pulmonary circulation increased lung edema clearance ~ 60 to 100% over basal levels in control nonventilated rats (from 0.50 ± 0.02 ml/h to 0.81 ± 0.04 ml/h and 0.99 ± 0.05 ml/h, respectively) (Figure 1). In ventilator- associated lung injury, active Na⁺ transport and lung edema clearance decreased by ~ 50% when compared with control rats (p < 0.001). TERB and ISO restored the lung's ability to clear edema in rats exposed to HVT ventilation for 40 min (from 0.25 ± 0.03 ml/h to 0.64 ± 0.02 ml/h and 0.88 ± 0.09 ml/h, respectively) (Figure 1). To determine that the decrease in lung edema clearance was due to changes in the active Na⁺ transport and not to edema accumulation, choline chloride was iso-osmotically substituted for sodium chloride in the BSA. As shown in Figure 2, in the lungs ventilated with HVT and instilled with choline chloride, there was no fluid reabsorption, as Na⁺ is needed to drive active Na⁺ transport and clearance. Additionally, there was no fluid accumulation, confirming that at the low hydrostatic pressures across the pulmonary circulation utilized in our model there was no edema formation.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Terbutaline and isoproterenol increases lung edema clearance in rats ventilated with high tidal volume (HVT) for 40 min and in control nonventilated rats. ***p < 0.001 as compared with control rats, and &p < 0.001 as compared with HVT ventilated rats. CT = control group; TERB = 105 M terbutaline instilled into air space, ISO = 10 6 M isoproterenol perfused through the pulmonary circulation.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Choline chloride was substituted for sodium chloride in the BSA solution. There was no fluid reabsorption or edema formation in isolated lungs from either rats ventilated with high tidal volume (HVT) for 40 min or control nonventilated lungs (CT). Bars represent means ± SEM.

The Na⁺ channel blocker amiloride and the Na,K-ATPase antagonist ouabain inhibited the stimulatory effect of ISO in HVT ventilated rats (Figure 3). Pulmonary circulation flow rates were not affected by -adrenergic agonists, amiloride, or ouabain treatment in any experimental group (Table 3).

View larger version (11K): [in this window] [in a new window]   Figure 3.   Amiloride instilled into air space and ouabain perfused through the pulmonary circulation inhibited lung edema clearance stimulation by isoproterenol in rats ventilated with high tidal volume (HVT) for 40 min. Bars represent means ± SEM; ***p < 0.001 and *p < 0.05 as compared with ventilator-injured rat lungs. CT = control; ISO = 106 M isoproterenol; OUA = 5 × 104 M ouabain; AMIL = 104 M amiloride.

We examined the contributory role of the cellular microtubular transport system on -adrenergic modulation of alveolar epithelial Na⁺ transport and lung edema clearance in rats ventilated with HVT. Disruption of intracellular microtubular transport by colchicine prevented isoproterenol stimulation of lung edema clearance in HVT ventilated rats, whereas the inactive isomer -lumicolchicine did not affect active Na⁺ transport stimulated by ISO (Figure 4). Colchicine and -lumicolchicine did not change lung permeability to small and large solutes in any experimental group (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 4.   Effect of colchicine and -lumicolchicine on lung edema clearance in ventilator-injured rat lungs. Colchicine inhibited lung edema clearance stimulation by ISO in rats ventilated with HVT. Bars represent means ± SEM; &p < 0.001 as compared with CT, COL, ISO+COL, and LUMIC ventilator-injured rat lungs. CT = control; ISO = 106 M isoproterenol; COL = 0.25 mg/100 g BW colchicine; LUMIC = 0.25 mg/100 g BW -lumicolchicine.

We also studied whether the Na,K-ATPase ₁ and ₁ mRNAs were affected by ISO in ATII cells isolated from control and ventilator-injured rat lungs. The ₁ and ₁ mRNA levels, evaluated by RT-PCR, did not change in ATII cells incubated with 1 µM ISO for 60 min after isolation from either control or HVT ventilated rats (Figure 5).

View larger version (12K): [in this window] [in a new window]   Figure 5.   Effect of high tidal volume (HVT) ventilation and isoproterenol on alveolar epithelial Na,K-ATPase 1 and 1 subunits mRNA expression. Total RNA was isolated from ATII cells from control rats and rats ventilated with HVT for 40 min. (A) Representative cDNA amplification using RT-PCR and specific primers for 1 and 1 Na,K-ATPase isoforms. (B) Quantitative densitometric scans of four different experiments. Densitometric values were normalized to the G3PDH internal control. cDNA expression in control rats was arbitrarily defined as 1. Data are presented as mean ± SEM.

	DISCUSSION

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Mechanical ventilation is frequently used to support the ventilatory function of patients with acute hypoxemic respiratory failure and pulmonary edema. However, it has been reported that mechanical ventilation with high inflation pressures and high tidal volumes can cause capillary stress fracture of the alveolocapillary barrier, depletion or inactivation of surfactant components, and release of proinflammatory mediators, which causes leakage of fluid, protein, and blood constituents into the lung interstitium and alveolar spaces (2, 8). Therefore, artificial ventilatory support may potentially worsen the pulmonary function by causing alveolar overdistension, especially in patients with acute respiratory distress syndrome and preexisting injured lungs (2).

Positive-pressure ventilation of rats with high tidal volumes for short periods of time produces lung injury and high permeability pulmonary edema (6, 7, 13). In this model, the increased tidal volume excursions, and not peak airway pressures, increase pulmonary microvascular filtration and extravascular lung water (7). It has been reported that adult rats ventilated with high tidal volume (40 ml/kg and to peak airway pressures of 35 cm H₂O) for 40 min causes lung injury and decreases alveolar fluid reabsorption, in association with downregulation of alveolar epithelial Na,K-ATPase function (14). -adrenergic agonists increase active Na⁺ transport and lung edema clearance in healthy and in injured rat lungs by upregulating the apical Na⁺ channels and basolateral Na,K-ATPase function in rat alveolar epithelium (15, 20).

In the present study, we examined whether the -adrenergic agonists terbutaline instilled into air spaces and isoproterenol perfused through the pulmonary circulation could improve active Na⁺ transport and lung edema clearance in rats ventilated with HVT. Mechanical ventilation of rats with HVT for 40 min decreased active Na⁺ transport and lung edema clearance and increased somewhat lung permeability to small solutes, Na⁺ and mannitol. However, the epithelial lining fluid volume and the movement of albumin from the pulmonary circulation into alveolar space were of similar magnitude than in control nonventilated rats. Without significant changes in lung permeability for albumin, the isolated perfused rat lung model is considered an accurate method to assess lung edema clearance, as we have previously reported in normal, hyperoxia-injured, and ventilator-injured rat lungs (14, 18, 25, 27, 28). Also, in this model of ventilator-associated lung injury there was no edema accumulation as outlined in Table I. The data show that the -adrenergic agonists, terbutaline and isoproterenol, restore the lung's ability to clear edema in rats exposed to VALI. The stimulatory effects of TERB and ISO were significantly higher in ventilator-injured rat lungs (increasing ~ 150% and ~ 250% above basal levels, respectively) than in healthy rat lungs where TERB and ISO increased lung edema clearance ~ 62% and ~ 98% over basal levels, respectively. Thus, we reason that the alveolar epithelial damage observed in VALI is probably not of such severity as to damage the epithelium function irreversibly and preclude rat alveolar epithelium from responding to -adrenergic stimulation. Rather it appears that VALI downregulates alveolar epithelial Na,K-ATPase, which can be upregulated by -adrenergic stimulation (14, 18). Recently, it has been reported that -adrenergic agonists also increase lung edema clearance in rats exposed to moderate and to severe hyperoxic lung injury (21, 22). As previously reported, the effect of -adrenergic agonists was of similar magnitude when they were instilled into alveolar space or added to the pulmonary circulation, suggesting that -adrenergic receptors are homogeneously distributed in rat alveolar epithelium or that the small size of -adrenergic agents allow them to diffuse across alveolocapillary barrier (15).

It has also been suggested that isoproterenol could attenuate high permeability pulmonary edema and reduce pulmonary vascular permeability by stabilization of the endothelial cell cytoskeleton (29, 30). Parker and Ivey (29) have reported that isoproterenol significantly attenuates increases in high vascular pressure-induced capillary filtration coefficient (K_fc). These effects are probably mediated by higher intracellular cAMP levels and by inhibiting the active ATP and Ca⁺²- dependent contraction of cytoskeleton myofibrils in endothelial and epithelial cells (29). However, ISO and TERB did not significantly affect alveolocapillary barrier permeability in VALI-exposed rats (see Table 1). Therefore, -adrenergic effects in HVT-ventilated rats were probably not mediated by pulmonary vascular endothelium modulation. In fact -adrenergic agonists have been shown to increase alveolor epithelial permeability to small solutes in normal rat lungs (16, 18).

To examine the active Na⁺ transport pathway in rat alveolar epithelium, we studied the effects of the Na⁺ channel blocker amiloride and the Na,K-ATPase antagonist ouabain in rats exposed to VALI. Both amiloride and ouabain inhibited the stimulatory effects of ISO on lung edema clearance in rats ventilated with HVT, suggesting that -adrenergic effects could be mediated by upregulation of apical Na⁺ channels and basolateral Na,K-ATPase function in rat alveolar epithelium, as previously reported in cultured ATII cells isolated from healthy rats (18).

The sodium pump plays a critical role translocating sodium molecules against an electrochemical gradient in the alveolar epithelium (14, 18, 20, 27). The Na,K-ATPase may be regulated at different levels, including transcription, translation, protein degradation rate, recruitment from intracellular pools to the plasma membrane, recycling from the plasma membrane, and structural changes of Na⁺ pumps in the plasma membrane (32, 33). It has been shown that -adrenergic agonists after short-term exposure (15 to 60 min) increases lung edema clearance in rat lungs by recruitment of Na⁺ pumps from intracellular pools to basolateral membranes of alveolar type II cells (18, 33). Therefore, we studied whether lung edema clearance modulation by -adrenergic agonists in rats exposed to VALI could also be mediated by recruitment and translocation of ion transporting proteins to the plasma membrane in rat alveolar epithelium by pretreating rats with colchicine and its isomer -lumicolchicine. Disruption of intracellular microtubular transport by colchicine inhibited the effects of -adrenergic agonists on active Na⁺ transport and lung edema clearance in rats ventilated with HVT, whereas the isomer -lumicolchicine did not affect the stimulatory effects of -adrenergic (Figure 4). Accordingly, we speculate that -adrenergic agonists stimulate lung edema clearance by promoting the recruitment of Na⁺ pump proteins to plasma membrane in alveolor epithelium in rats exposed to VALI. However, it is also possible that other pathways involved in lung edema clearance regulation such as Na⁺ channels, Na⁺-glucose cotransporter, Na⁺, H⁺ exchanger and water channels, which were out of the scope of the present study, could be involved in -adrenergic regulation of alveolar epithelial Na⁺ transport in ventilator-injured lungs (19, 34, 35).

Finally, alveolar epithelial Na,K-ATPase ₁ and ₁ mRNA steady state levels were not affected by HVT ventilation and -adrenergic short-term exposure (60 min). Thus, our data suggest that the changes in alveolar epithelial Na⁺ transport and lung edema clearance produced during VALI and -adrenergic stimulation were not due to transcriptional modifications of Na,K-ATPase. These data differ somewhat from a recent report where the Na,K-ATPase ₁ mRNA expression was increased in alveolar epithelial cells exposed to terbutaline for 48 h (35).

In conclusion, we have shown that -adrenergic agonists improve alveolar fluid reabsorption in ventilator-associated lung injury. Improvement of lung edema clearance by -adrenergic stimulation is probably mediated by recruitment and translocation of ion-transporting proteins from intracellular pools to the cell plasma membrane in alveolar epithelium. The outcomes of patients with acute lung injury and pulmonary edema are related to the ability of the lung to clear edema. Conceivably, -adrenergic agonists could be utilized as a therapeutic strategy to improve functional recovery of patients with acute hypoxemic respiratory failure and pulmonary edema.