INTRODUCTION

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REDUCED PULMONARY EDEMA and increased clearance of lung liquid in patients with hypoxemic respiratory failure have been associated with decreased length of stay of patients in the intensive care department and improved survival (12, 16, 19, 35). Pulmonary edema clearance is an active metabolic process dependent mostly on active Na⁺ transport across the lung epithelium (3, 7, 9, 28, 30). Previous studies have shown that adrenergic- and dopaminergic-agonist drugs increased active Na⁺ transport and edema clearance (2, 7, 14, 29, 30, 36). Among the mechanisms responsible for increasing Na⁺ transport across the alveolar epithelium, Na⁺-K⁺-ATPase appears to play a major role (31). In previous studies during the proliferative phase of hyperoxic lung injury, which is characterized by proliferation of alveolar type II cells (AT2), we observed increased active Na⁺ transport in association with upregulation of Na⁺-K⁺-ATPase in AT2 cells (20, 21, 34).

Other stimuli such as growth factors have been shown to promote lung cell growth and proliferation of alveolar epithelial cells (13). Specifically, epidermal growth factor (EGF) has been demonstrated to promote certain aspects of proliferation and differentiation of epithelial cells in fetal lungs (24). Recent studies showed that adult rat AT2 cells in culture transcribe the EGF gene, translate a corresponding EGF polypeptide, and transduce the EGF signal (25). Also, in alveolar epithelial cell monolayers, exogenous EGF increased short-circuit currents and increased Na⁺-K⁺-ATPase function (4).

We therefore postulated that if, in the rat alveoli, EGF upregulates Na⁺-K⁺-ATPase, this could result in an increase in active Na⁺ transport across the lung epithelium with a consequent increase in lung liquid clearance. To test this hypothesis we utilized a novel apparatus to deliver 1- to 3-µm aerosols containing EGF to the respiratory tract of spontaneously breathing rats. Forty-eight hours post-EGF delivery, active Na⁺ transport and lung liquid clearance were measured from the air spaces in an isolated perfused lung model. We found that active Na⁺ transport and lung liquid clearance were increased in the lungs of rats administered EGF compared with rats in which saline was delivered and with control rats, which had no intervention. Additionally, rats administered EGF to their respiratory tract had increased Na⁺-K⁺-ATPase activity in AT2 cells harvested after the isolated lung experiments.

	MATERIALS AND METHODS

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Pathogen-free male Sprague-Dawley rats (330 ± 15 g) were purchased from Charles River Laboratories (Raleigh, NC). A total of 16 rats was studied. The animals were randomized into three groups. In six rats an aerosol containing 20 µg of EGF in 1 ml of saline was delivered to the respiratory tract. In five rats, saline alone was aerosolized and delivered to the respiratory tract. Five rats not receiving aerosols were used as the control. The groups were age and weight matched.

Delivery of aerosols to the respiratory tract. Aerosols were delivered to the respiratory tract of two groups of the rats by using a nose-only aerosol delivery system (Fig. 1) (39). Each rat was lightly sedated with 0.08 g/kg of intramuscular ketamine and placed within a bodysuit (Alice Chatham King, Los Angeles, CA) suspended from two rails within a plethysmograph. The head was immobilized by using a neck restraint system as shown in Fig. 1. The sling support system was advanced such that the rat's nose fit snugly through a small hole in a latex diaphragm stretched over a nose cone. The rat was able to spontaneously breathe air flowing through the zero-dead-space aerosol delivery port. The cover was placed on the plethysmograph. Pressure in the plethysmograph was monitored with a Validyne MP-45 pressure transducer connected to its carrier amplifier (Validyne CD12, Validyne, Northridge, CA). The pressure tracing was monitored on a chart recorder. Aerosols of EGF were generated from 20 µg of EGF in 1 ml saline by a Aerotech II nebulizer (Cadema) operated at 9 l/min. An aerosol of either EGF (Bachem, Torrance, CA) or saline was delivered to the respiratory tract for 20 min.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Schematic depiction of body plethysmograph with nose-only aerosol delivery system. System was constructed from stainless steel and aluminum. Cross-sectional inset shows design that enables aerosol to flow past rat's nose. Air or aerosol flows toward rat's nose and escapes through 8 circumferential ports in delivery tube. A valve (not shown) is used to deliver either air or aerosol to rat. Thus a continuous flow of air or aerosol past the nose allowed rats to breathe spontaneously. Aerosol was generated by using an Aerotech II nebulizer.

Isolated lungs. The isolated lung preparation was performed by using a modification of a previously described method (20, 21, 28, 34). Briefly, rats were anesthetized with 30 mg/kg body weight of pentobarbital sodium and anticoagulated with 1,000 U heparin administered intraperitoneally. A tracheotomy was performed, and the rats were mechanically ventilated with a tidal volume of 2.5 ml to a peak airway pressure of 8-10 cmH₂O and 100% O₂ for 5 min. After exsanguination, the heart and lungs were removed en bloc. The pulmonary artery and the left atrium were cannulated with triple-lumen catheters. The pulmonary circulation was flushed of the remaining blood by perfusing with 30 ml buffered salt albumin (BSA) solution containing (in mM) 135.5 Na⁺, 119.1 Cl, 25 HCO₃, 4.1 K⁺, 2.8 Mg²⁺, 2.5 Ca²⁺, 0.8 SO₄, and 8.3 glucose, as well as 30 g/l bovine albumin, pH 7.45 and osmolality of ~300 mosm/kgH₂O. The lung air spaces were flushed twice with 5 ml of BSA solution containing 0.1 mg/ml Evans blue dye (EBD)-tagged albumin (Sigma Chemical, St. Louis, MO), 0.02 µCi/ml ²²Na⁺ (Amersham, Chicago, IL), and 0.12 µCi/ml [³H]mannitol (DuPont-NEN, Boston, MA), after which the same solution was instilled such that lungs retained 7 ml. Finally, the lungs were immersed in a covered "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.

Calculations for isolated perfused rat model. The derivation of all equations involved in the mathematical model of edema clearance has been previously described in detail (28). Concentration of EBD-albumin was used to estimate air space volume. Because virtually all EBD-albumin remains in the airspace, we may calculate instillate volume (V) at a given time t as

(1)

where V₀ is the initial volume instilled and [EBD]₀ and [EBD]_t are the concentrations of EBD-albumin at times 0 and t, respectively. The removal of Na⁺ from the alveolar space during a define period of time is probably accompanied by isotonic water flux and is given by

(2)

where J_Na,net is the net or active Na⁺ transport, J_Na,out is the total or unidirectional Na⁺ outflux, and J_Na,in is the back flux of Na⁺ into the alveolar fluid by passive bidirectional movement. Because Na⁺ concentration remains constant in all compartments, the net Na⁺ flux (which we refer to as active Na⁺ transport) from the air space is

(3)

where [Na⁺] is Na⁺ concentration. The unidirectional fluxes of Na⁺ from the alveolar space, a result of active transport and passive movement, were calculated as

(4)

Similarly, the (unidirectional) volume flux of [³H]mannitol (typically expressed as permeability of surface area, PA) was calculated as

(5)

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. For reasons of comparison, fluxes are reported as volume fluxes (volume/time) by using the appropriate solute concentrations.

Cell isolation. AT2 cells were isolated from the isolated perfused lungs after completion of the experimental protocol by using previously described methods (8, 11, 26). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase (30 U/ml, Worthington Biochemicals) for 20 min at 37°C. The tissue was minced and filtered through sterile gauze and 150- and 15-µm nylon mesh. The AT2 cells were purified by differential adherence to IgG-pretreated dishes. Cells were suspended in DMEM (Irvine Scientific) containing 10% fetal bovine serum with 2 mM L-glutamine, 40 µg/ml gentamicin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Identification of AT2 cells was based on the presence of lamellar inclusions. Lamellar bodies were stained with Papanicolaou stain, tannic acid, or alkaline phosphatase. Viability of the AT2 cells was >92%, as determined by exclusion of trypan blue stain. The number of AT2 cells and viability were similar to cell isolations from rats in which no physiological experiments were conducted.

Membrane preparation for Na⁺-K⁺-ATPase activity. Briefly, alveolar epithelial cells were washed three times with PBS and then scraped with a rubber policeman in 1 ml of homogenization buffer (5 mM histamine-imidazole; 2 mM EDTA; 1 mM EGTA; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml leupeptin; and 60 µg/ml soybean trypsin inhibitor) and rinsed with an additional 1 ml of the same buffer. Alveolar epithelial cells were resuspended in 1 ml of homogenization buffer and disrupted in a Teflon homogenizer for 20 strokes. The homogenate was centrifuged (~5 min, 1,000 g, 4°C) to remove unbroken cells and debris. The supernatant was then centrifuged at 60,000 g at 4°C for 1 h. The high-speed supernatant was designated the cytosolic fraction. This pellet was resuspended in 500 µl homogenization buffer and designated the membrane fraction. Total protein concentrations of cytosol and membrane fractions were determined by Bio-Rad protein assay with BSA as standard.

Na⁺-K⁺-ATPase activity. Na⁺-K⁺-ATPase activity was determined under maximal rate conditions in membrane fractions of isolated AT2 cells after completion of the isolated lung experimental protocol as previously described (11, 26). Briefly, the total activity reaction mixture contained (in mM) 130 NaCl, 20 KCl, 3 ATP, 3 MgCl₂, and 30 imidazole in a final volume of 500 µl; the ouabain-sensitive reaction mixture contained the same as the total activity reaction mixture with 3 mM ouabain. Na⁺-K⁺-ATPase activity was determined by preincubating the microsomal fraction at 37°C in buffer for 30 min. The results were corrected for spontaneous hydrolysis of ATP. The reaction was stopped with 1% trichloroacetic acid, and the precipitated proteins were pelleted. P_i was determined by the method that utilized a molybdate-H₂SO₄ solution with Fiske reducing agent (11). The specific activity of the enzyme is expressed as micromoles P_i per milligram protein per hour.

Data analysis. 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 and Duncan's means comparison test were used. Results were considered significant when P < 0.05.

	RESULTS

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Unidirectional Na⁺ flux out of the air space, which accounts for both the active and the passive Na⁺ movement across the lung epithelium, increased in rat lungs to which aerosolized EGF was administered compared with rats in which either aerosolized saline was administered or no treatment was given (Fig. 2), (P < 0.05). The increase in unidirectional Na⁺ flux was due to ~40% increase in active Na⁺ transport in rats aerosolized with EGF (39 ± 2 nM/s) compared with saline-aerosolized (29 ± 3 nM/s) and control (27 ± 1 nM/s) rats. This change in active Na⁺ transport resulted in an increase in lung liquid clearance (estimated by the concentration of EBD) in rat lungs to which aerosolized EGF was administered compared with both of the other groups, control rats and those to which aerosolized saline was delivered (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effects of epidermal growth factor (EGF) on unidirectional Na+ flux (y-axis). Unidirectional Na+ flux increased significantly in lungs from rats aerosolized with EGF compared with saline-aerosolized and control rats. Values are means ± SE. Bars, SE. * Significantly different from other groups (P < 0.01).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of EGF on lung liquid clearance measured by concentration of Evans blue dye (EBD) in air space liquid. Lung liquid clearance increased in rats aerosolized with EGF compared with control and saline-aerosolized rats. Values are means ± SE. Bars, SE. * P < 0.05.

Passive Na⁺ movement across the lung epithelium, measured by the difference between unidirectional Na⁺ flux and active Na⁺ transport, and the passive movement of small solutes, assessed by [³H]mannitol fractional loss, were not different in the three groups studied (Fig. 4). EBD-bound albumin was not detected in the rat perfusate or bath compartments in any of the four experimental groups. Also, the minimal movement of FITC-albumin from the perfusate into the air spaces was similar in all three groups (data not shown). The difference between the EBD-albumin and FITC-albumin measurements probably represents a higher sensitivity of FITC detection, which moves from a large (90-ml) space into a much smaller compartment (7 ml), whereas EBD-albumin is moving from the 7-ml compartment to a 13-fold-larger compartment, falling below the sensitivity of detection. To the extent that albumin movement is equal in both directions, this may indicate a minute undetected loss of EBD-albumin from the air space, resulting in a minimal underestimation of the calculated liquid clearance. This movement represented flux of FITC-albumin from the pulmonary circulation into the air spaces of 0.01% of FITC-albumin mass.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of EGF on passive solute movement. Both passive Na+ outflux from air space and fractional mannitol loss did not change in all experimental groups. Values are means ± SE. Bars, SE.

Our model is a closed system and allows measurement of the mass balance of the tracers at each time point during the experiment. Greater than 94% of ²²Na⁺ and [³H]mannitol was recovered in the three measured compartments (perfusate, air space, and pleural bath) for all experimental groups. Perfusate flow, measured at 10 and 70 min after initiation of the experiment, was not significantly different from control rats (14 ± 3 ml/min) in rats aerosolized with EGF (16 ± 4 ml/min) or with saline (15 ± 6 ml/min). We found that the concentration of Na⁺ in all compartments in all experimental conditions was constant (~140 mM), suggesting a "steady-state" isotonicity among the three compartments.

Na⁺-K⁺-ATPase activity in isolated AT2 cells measured after completion of the physiological experimental protocol increased by more than twofold in rats with EGF aerosols delivered compared with rats with saline aerosols delivered and control rats (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effects of EGF on Na+-K+-ATPase hydrolytic activity in alveolar epithelial type II cells (AT2; µmol Pi · mg protein1 · h1; y-axis) from rats aerosolized with EGF compared with rats aerosolized with saline and control rats. Values are means ± SE. Bars, SE. * Significantly different from control group (P < 0.01).

	DISCUSSION

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Proliferation of AT2 cells in rats exposed to oxygen has been shown to be associated with increased Na⁺-K⁺-ATPase activity in rat AT2 cells and with increased Na⁺ transport and increased lung liquid clearance (20, 21, 34). These studies suggested that the increased Na⁺-K⁺-ATPase activity was due, in part, to a increased number of Na⁺-K⁺-ATPases per cell, as reported in other tissues when increased Na⁺ pumping was needed to cope with threatening extracellular ionic imbalance (5). Growth factors have been shown to stimulate the proliferation of the alveolar epithelium either directly or indirectly by affecting fibroblasts and other interstitial cells (15, 27) and thus could have a role in mounting defense mechanisms against lung injury. For example, the hepatocyte growth factor has been shown to stimulate rat AT2 cell proliferation (22), and the keratinocyte growth factor has been reported to be protective against hyperoxic lung injury, possibly by inducing cellular repair and increasing active Na⁺ transport (17, 23, 37). We reasoned that EGF, a growth factor shown to have functional receptors in AT2 cells and increase short-circuit current in alveolar epithelial cell monolayers without apparent AT2 cell proliferation (4, 25), could have a role against lung edema by upregulating alveolar epithelial Na⁺-K⁺-ATPase and increasing lung liquid clearance in vivo. Additionally, even though EGF is a weak mitogen and 48 h probably are not enough for alveolar epithelial hyperplasia, the fact that there were no changes in permeability for ²²Na+, [³H]mannitol, and albumin is consistent with the absence of proliferation. In a previous study, where there is proliferation of alveolar epithelial cells, we reported that a significant increase in alveolar epithelial permeability for ²²Na+, [³H]mannitol, and albumin was observed (21).

EGF is a peptide growth factor that activates gene transcription via mitogen-activated kinases in epithelial cells (18, 32). In the lungs EGF has been reported to play a role in branching morphogenesis in mice and to contribute to fetal lamb and rabbit lung maturation (6, 33). EGF receptors have been localized to several fetal organs, among them the lungs. Raaberg et al. (25) have estimated ~50 EGF receptors per AT2 cell and postulated that EGF mediates novel and yet undescribed mechanisms of regulation of AT2 cell function. Recently described effects of EGF in AT2 cell include effects on tight junction permeability, increasing short-circuit current in AT2 cell monolayers, as well as upregulation of Na⁺-K⁺-ATPase via transcriptional and/or translational mechanisms (4). Specifically, EGF added to the basolateral surface of alveolar epithelial monolayers had a severalfold increase in transepithelial resistance and short-circuit current (equivalent to ion transport) (4). These effects were abolished by ouabain and partially inhibited by the addition of amiloride as well as the more specific Na⁺ channel inhibitors such as benzamil and ethylisopropyl amiloride. These effects were also prevented by incubation with tyrphostin RG-50864, a reversible specific inhibitor of the EGF receptor tyrosine kinase.

Our study extends these previous observations and supports the notion that EGF mediates the upregulation of Na⁺-K⁺-ATPase in AT2 cells in vivo, resulting in an important physiological effect by increasing active Na⁺ transport and lung liquid clearance when delivered by aerosol to the rat alveolar surface. We observed that when EGF aerosols were delivered to rat lungs they had increased unidirectional Na⁺ flux out of the air spaces due to the increased active Na⁺ transport across the lung epithelium. The EGF-mediated increase in active Na⁺ transport and lung liquid clearance was not associated with changes in epithelial permeability for either small or large solutes (see Figs. 2-4). The increased Na⁺ transport resulted in ~40% increase in lung liquid clearance 48 h post-EGF aerosol delivery compared with that in lungs from rats exposed to saline aerosols and from control rats. This was associated with upregulation of Na⁺-K⁺-ATPase in AT2 cells harvested at the end of the isolated lung protocol (see Fig. 5). These findings support the hypothesis that in vivo delivery of EGF aerosols increases active Na⁺ transport and lung edema clearance, possibly by upregulating Na⁺-K⁺-ATPases located in AT2 cells (4).

Our study was not designed to specifically determine whether the increased Na⁺-K⁺-ATPase in alveolar epithelial cells is the result of recruitment of the Na⁺ pumps from intracellular pools and inserting them into the plasma membrane or de novo transcription and translation of new proteins. However, Na⁺-K⁺-ATPase activity was measured under maximal rate conditions, which suggests that there were more Na⁺-K⁺-ATPases expressed in EGF-treated AT2 cells. Additionally, in a model of lung hyperplasia that occurs by exposing rats to 85% O₂ for 7 days where the number of AT2 cells increased, we reported an increase in Na⁺-K⁺-ATPase activity per cell (20). However, in a pneumonectomy model of hyperplasia rats that is also associated with increased AT2 cell numbers, there was no increase of Na⁺-K⁺-ATPase activity per cell. Thus we are confident in reporting that EGF had an effect on increasing Na⁺-K⁺-ATPase per cell and that the increase in pump activity is not a reflection of more AT2 cells.

Additionally, we conducted a few isolated rat lung experiments 12 and 24 h postaerosolization of the same doses of EGF (W. Olivera, unpublished observations) without changes in active Na⁺ transport and lung liquid clearance. These studies suggest that EGF does not contribute to the short-term regulation of the lung Na⁺-K⁺-ATPase (recruitment and/or translocation) but rather that long-term regulation (transcription and/or translation) is necessary to upregulate the Na⁺-K⁺-ATPase in the alveolar epithelium as reported (4, 18, 32).

A limitation of our and other similar studies is the lack of information on the contribution of alveolar type I cells to the Na⁺ transport mechanisms. It is has been reported that alveolar type I cells contain Na⁺-K⁺-ATPases (26) but, because of the difficulties in isolating these cells, we did not explore their role in EGF-induced changes of Na⁺ transport in this study.

The zero-dead-space nose-only exposure system facilitated the deposition of aerosols containing EGF in the lungs of lightly sedated rats. In 20 min of exposure to aerosols, a quantity of EGF (32 ng) was deposited in the lungs, which caused a significant physiological effect 48 h later. An estimation of the EGF deposited in the nonciliated airways was made by measuring the percentage of an insoluble particle that is retained after 24 h when all particles deposited in the ciliated airways are assumed to have been removed (1). Such quantitation is important to compare the doses between this and future studies of agents with therapeutic potential for upregulating the Na⁺-K⁺-ATPase and enhancing alveolar edema clearance.

Increased lung edema clearance is associated with improved survival in mechanically ventilated patients with hypoxemic respiratory failure (12, 16, 19). Our study demonstrates that the increase in active Na⁺ transport and lung edema clearance is associated with increased alveolar epithelial Na⁺-K⁺-ATPase activity in rat lungs with in vivo delivery of EGF aerosols. Our results suggest that the upregulation of alveolar epithelial Na⁺-K⁺-ATPases can protect against lung injury by clearing edema. Conceivably, the alveolar epithelium may be targeted with aerosols delivering therapeutic agents as a possible strategy for the treatment of patients with pulmonary edema (10).