Department of Internal Medicine, Justus-Liebig-University Giessen, 35385 Giessen, Germany
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ABSTRACT |
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We employed ultrasonic nebulization for homogeneous
alveolar tracer deposition into ventilated perfused rabbit lungs.
22Na and 125I-albumin transit kinetics were
monitored on-line with gamma detectors placed around the lung and the
perfusate reservoir. [3H]mannitol was measured by
repetitive counting of perfusion fluid samples. Volume of the alveolar
epithelial lining fluid was estimated with bronchoalveolar lavage with
sodium-free isosmolar mannitol solutions. Sodium clearance rate was
2.2 ± 0.3%/min. This rate was significantly reduced by
preadministration of ouabain/amiloride and enhanced by pretreatment
with aerosolized terbutaline. The 125I-albumin clearance
rate was
0.40 ± 0.05%/min. The appearance of
[3H]mannitol in the perfusate was not influenced by
ouabain/amiloride or terbutaline but was markedly enhanced by
pretreatment with aerosolized protamine. An epithelial lining fluid
volume of 1.22 ± 0.21 ml was calculated in control lungs. Fluid
absorption rate was 1.23 µl · g lung
weight
1 · min
1, which was blunted
after pretreatment with ouabain/amiloride. We conclude that alveolar
tracer loading by aerosolization is a feasible technique to assess
alveolar epithelial barrier properties in aerated lungs. Data on active
and passive sodium flux, paracellular solute transit, and net fluid
absorption correspond well to those in previous studies in fluid-filled
lungs; however, albumin clearance rates were markedly higher in the
currently investigated aerated lungs.
amiloride; epithelial lining fluid; ion channel; ouabain; protamine; protein clearance
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INTRODUCTION |
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THE MAMMALIAN BLOOD GAS BARRIER is constructed to allow optimum gas exchange while maintaining fluid balance. Under physiological conditions, the alveolar side of the barrier is covered by an extremely thin fluid film, termed epithelial lining fluid (ELF), which allows performance of surface tension regulation and maintenance of basic host defense properties but does virtually not interfere with gas diffusion (32, 33). Flooding of the alveolar space causes life-threatening impairment of gas exchange, making strict regulation of the ELF volume mandatory. This is achieved by the barrier properties of the capillary endothelial and high-resistance alveolar epithelial layers, along with epithelial ion channels and pumps, discovered over the past decade (15, 21).
The vectorial transport of sodium across the epithelial barrier was identified as the main driving force for active fluid absorption from the alveolar compartment (2, 3, 8, 15, 21). Sodium enters the epithelial cells from the apical side, mostly through amiloride-sensitive ion channels, and is pumped out of the cells by a basolaterally located Na-K-ATPase. Basic features of this system have been characterized in epithelial cell culture studies as well as in expression experiments (7, 12, 14, 19). Essential physiological data in this field were provided by models of isolated fluid-filled lungs and studies of intact animals undergoing alveolar instillation with defined solutions. In these experiments, the intercompartmental transit of indicator molecules is monitored to establish the kinetics. For a more detailed characterization of channel and pump features, pharmacological agents may be coapplied with the indicator-containing solution to the alveolar space. Overall fluid absorption is calculated from the concentration of slowly permeable indicator molecules such as albumin enriched in the alveolar instillate on net fluid absorption.
There are, however, drawbacks inherent in this approach of lung fluid filling. This maneuver may per se have an impact on physiological regulations as recently suggested for transepithelial glucose transport (24). The physiological alveolar micromilieu is markedly diluted by the instillate. Ventilation has to be discontinued, which may be disadvantageous in view of the fact that ventilatory stretch represents the most prominent stimulus of epithelial surfactant secretion (34, 35) and has been implicated in variations of active and passive transepithelial solute transport and fluid absorption (30). The influence of ventilator strategies and airborne pharmacological interventions, such as inhaled NO, may not be easily addressed in fluid-filled lung models.
In the present study, a setup for alveolar tracer monitoring and fluid clearance in ventilated perfused lungs was established. For this purpose, aerosol techniques were employed, and nebulization of pharmacological agents was used to characterize active and passive transport mechanisms. In essence, amiloride/ouabain-inhibitable sodium transport and net alveolar fluid clearance were identified, with overall rates comparable to those in fluid-filled lungs. Clearance rates of albumin did, however, range at considerably higher values than those anticipated from these models. Protamine aerosolization enhanced paracellular epithelial permeability as assessed by mannitol passage, without affecting active sodium transport. The currently described technique may thus be employed to characterize active and passive epithelial barrier properties in aerated lungs.
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METHODS |
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Reagents
[3H]mannitol, 22Na, and 125I-albumin were obtained from DuPont de Nemours (Brussels, Belgium). All other biochemicals were obtained from Merck (Darmstadt, Germany). 125I-albumin was dialyzed for 3 h before each experiment in a Slide-A-Lyzer (molecular mass cutoff 10,000 Da; Pierce, Rockford, IL) to remove unbound free 125I and 125I bound to small protein fragments from the albumin fraction. After this procedure, the percentage of the TCA-soluble fraction in the iodinated albumin was >2% throughout.Perfused Rabbit Lungs
The model has been previously described in detail (27). Briefly, rabbits of either sex (mean body weight 2.5 kg; Charles River, Sulzfeld, Germany) were deeply anesthetized with an intravenous injection of ketamine (30-50 mg/kg) and xylazine (6-10 mg/kg). The animals were administered 1,000 U heparin anticoagulant/kg body wt. A tracheotomy was performed, and the animals were ventilated with room air with a Harvard respirator (Cat/Rabbit Ventilator, Hugo Sachs Elektronik, March Hugstetten, Germany). After a midsternal thoracotomy, catheters were placed into the pulmonary artery and the left atrium, and perfusion with Krebs-Henseleit buffer was started. The buffer contained 120 mM NaCl, 4.3 mM KCl, 1.1 mM KH2PO4, 25 mM NaHCO3, 2.4 mM CaCl2, 1.3 mM MgPO4, 240 mg of glucose, and 5 g of hydroxyethylamylopectine as an oncotic agent per 100 ml. To wash out blood, the perfusate was initially not recirculated and the flow was slowly increased to 100 ml/min with a left atrial pressure of 2 mmHg. The lungs were placed freely suspended from a force transducer in a temperature-equilibrated housing chamber at 37°C. They were ventilated with room air supplemented with 4% CO2 to maintain pH of the recirculating buffer between 7.35 and 7.37, inspiratory pressure between 5 and 7 mmHg, a tidal volume of 10 ml/kg body wt, a frequency of 30 breaths/min, and a positive end-expiratory pressure of 1 mmHg. The alternate use of two separate perfusion reservoirs (total circulating volume of 275 ml) allowed for repetitive exchanges of perfusion fluid. Perfusion pressure, ventilation pressure, and the weight of the isolated organ were continuously registered. Lungs selected for the study 1) had a homogeneous white appearance without signs of hemostasis or edema formation, 2) had a pulmonary arterial pressure in the normal range, and 3) were isogravimetric (lung weight gain <0.3 g/h) during an initial steady-state period of at least 30 min. Sterile tubing and perfusion fluids were used throughout. Lung wet weight was calculated as body weight (g) × 0.0024 (27).Aerosol Delivery System
Aerosolization was performed with an ultrasonic nebulizer (DeVilbiss, Somerset, PA). The nebulizer was connected directly to the inspiration loop of the ventilator. Filters were mounted in the outlet of the inspiration and expiration loop to prevent tracer contamination of the environment. The tracer mixture was prepared by adding 5 µCi of 22Na, 5 µCi of 125I-albumin, 27 µCi of [3H]mannitol, or selected compounds to 5 ml of saline. During the 10-min aerosolization period, 1.6 ml of aerosol were generated, and a fraction of ~30% of this 1.6 ml of generated aerosol was deposited inside the lung (as calculated from the radioactivity measurement of lung and perfusate). The amount of total tracer deposition was thus ~0.5 µCi of 22Na, ~0.5 µCi of 125I-albumin, and ~2.7 µCi of [3H]mannitol. To ensure the integrity of 125I-labeled albumin, separate experiments were performed. First, before 125I-albumin was applied to each experiment, the portion withdrawn from the stock solution was repetitively dialyzed in dialysis cassettes (Slide-A-Lyzer, Pierce) with a molecular mass cutoff of 10,000 Da. This "washed" 125I-albumin was then further analyzed for the fraction of unbound 125I by protein precipitation with a 10% TCA solution. After centrifugation and measurement of activity in the sediment and supernatant, the fraction of TCA-soluble 125I (unbound) consistently ranged <0.5%. In further in vitro experiments, the condensate of nebulized 125I-albumin was trapped in a ice-cooled tubing. This condensed aerosol was then analyzed as described above. Again, no disintegration of tracer and albumin was notable. In addition, SDS-PAGE demonstrated integrity of the protein. Thus integrity of the protein and stability of 125I binding to the albumin molecule was demonstrated over the entire route of administration.Measurement of Tracer Exchange
Gamma detectors (Target System Electronic, Solingen, Germany) were connected to an automated high-voltage power supply integrated into a personal computer system with data processing (Fig. 1). Two detectors each were placed directly around the lung and the perfusate reservoir, and tracer movements were continuously registered on-line for 22Na and 125I-albumin. [3H]mannitol kinetics were determined by discontinuous perfusate sampling and end-time bronchoalveolar lavage (BAL); these samples were measured in a
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Calculation of Tracer Kinetics
The mean counts per second spectra for the gamma emitters were registered over 30-s periods. The tracer clearance curve over the lung was referenced to a 100% starting point at the end of nebulization. Corrections were undertaken for the radioactivity contained in the perfusate volume detected by the lung gamma detectors during passage through the lung (intravascular volume and some minor percentage of the tubing, in total ~15 ml). Compared with the radioactivity deposited in the lung, this potential by confounding perfusate activity never surpassed 5.5%, even at the end of experiments. In addition, the decrease rate of lung radioactivity was continuously calculated as percent per minute for 22Na and 125I-albumin. Moreover, the perfusate reservoir radioactivity was continuously monitored. Because the yield of tracer detection differs between the lung tissue and the reservoir (for geometric reasons), the latter was calibrated to match the yield of tracer detection over the lung for both 22Na and 125I-albumin. This allowed direct comparison and display of tracer quantities contained in the lung tissue and in the perfusate reservoir (Fig. 2). In the case of [3H]mannitol, lung-to-perfusate passage was calculated from the tracer appearing in the discontinuous perfusate samples. In addition, mannitol remaining in the alveolar space was quantified by BAL at the end of the experiments.
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BAL
Immediately after the termination of perfusion, the bronchoalveolar space was lavaged with 50 ml of isosmolar mannitol (nonlabeled). The entire volume was bolus injected into the trachea and immediately reaspirated gently; the total lavage time did not exceed 15 s. The recovered lavage fluid (recovery 75-80% in all experiments) was then centrifuged at 300 g for 10 min to separate the cells from the supernatant.Sodium Measurement With an Ion-Selective Electrode
The sodium concentrations in the perfusate and BAL fluid were assessed with a highly sensitive ion-selective electrode (Mettler Toledo). This electrode possesses linearity for sodium in the range from 1 × 10Calculation of Epithelial Lining Layer Volume
Assuming that the concentration of sodium in the epithelial lining layer equals that in the perfusion medium (16) in the absence of sodium in the instillation fluid, the relationship between volume and sodium concentration in the recovered BAL fluid and the ELF can be expressed by (VBAL + VELF) × [Na]BAL(recovered) = VELF × [Na]ELF, where VBAL is the volume of instillate (in this case 50 ml), VELF is the volume of ELF, [Na]BAL(recovered) is the concentration of sodium in the recovered BAL fluid, and [Na]ELF is the concentration of sodium in the ELF (equal to the sodium concentration in the perfusate). Resolution of this equation yields VELF = {VBAL × [Na]BAL(recovered)}/{[Na]ELFExperimental Protocols
Control lungs. After termination of the steady-state period, sham aerosol application of 200 µl of saline was performed followed by aerosol application of tracers 15 min later and on-line monitoring for 120 min without pharmacological intervention. Perfusate samples for the assessment of [3H]mannitol passage were taken after 5, 10, 15, 20, 30, 50, 90, and 120 min. After 120 min, perfusion was stopped and BAL was performed to estimate the ELF volume and tracer content.
Inhibition of active sodium transport.
Ouabain (105 M) was admixed to the perfusate, and
amiloride was applied by ultrasonic nebulization (total volume of 200 µl), resulting in a concentration of 10
4 M in the ELF
(lining layer volume as measured for rabbit lungs under baseline
conditions). The subsequent protocol corresponded to that in control lungs.
-Mimetic stimulation.
Studies were performed in the presence of 10
3 M
terbutaline in the ELF (aerosol technique as given for amiloride).
Influence of alveolar protamine. This basic polycation was administered by nebulization (200-µl volume), resulting in an ELF concentration of 0.69 mg/ml. The subsequent protocol corresponded to that in control lungs.
Combined interventions.
In separate studies, combined application of propranolol or
amiloride/ouabain and terbutaline as well as of amiloride/ouabain and
protamine was performed. Concentrations in these studies corresponded to those given above and 103 M for propranolol.
Conebulization of amiloride with either terbutaline or protamine and
propranolol with terbutaline with a total volume of 200 µl of saline
was performed.
Assessment of lung fluid absorption. In these experiments, performed in the absence of tracer application, the lungs were loaded with a total volume of 1.1 ml of saline with an aerosol technique either after termination of the steady-state phase or at the end of a 1-h perfusion period. Experiments were performed in the absence of drugs or in the presence of amiloride/ouabain (concentrations as given above). Fluid absorption was calculated from differences in the ELF volume as assessed by mannitol lavage.
Statistics
Data are means ± SD except fluid absorption data that are means ± SE. ANOVA was employed for comparison of differences between experimental groups. Tukey's test was used as an a posteriori test for linear contrasts. The significance level for the Tukey's test was set at P = 0.05. ![]() |
RESULTS |
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Control Lungs
The tracer quantities deposited within the 10-min aerosolization period were ~0.5 µCi of 22Na, ~0.5 µCi of 125I-albumin, and ~2.7 µCi of [3H]mannitol. Continuous lung-to-perfusate passage was visualized for both 22Na and 125I-albumin (Fig. 2). As anticipated, the kinetics were markedly different between these two tracers; the initial clearance rates were
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Inhibition of Active Sodium Transport
Coapplication of 10
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-Mimetic Stimulation
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Influence of Alveolar Protamine
Aerosolization of this basic polycation enhanced intercompartmental exchange of both [3H]mannitol and 22Na (Figs. 6 and 9). The initial clearance rate increased to approximatelyCombined Interventions
In experiments with a combined application of terbutaline and amiloride/ouabain or terbutaline and propranolol, the terbutaline-related excess 22Na clearance rate was fully reversed, and levels corresponded to those of control lungs (Correlations and Control Data
In all single experiments, the lung tracer decrease and perfusate tracer appearance corresponded well with each other. For sodium, the correlation of these data under the different interventional regimens is given in Fig. 10. No significant alteration of mean pulmonary arterial pressure or lung weight was noted in any of the experiments. In addition, measurement of the capillary filtration coefficient (1.8 × 10
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Assessment of Lung Fluid Absorption
In lungs perfused for 10 min, an ELF volume of 1.22 ± 0.21 ml was measured. After a perfusion period of 1 h, this increased to 2.48 ± 0.18 (SE) ml. The aerosolization of 1.1 ml of 0.9% saline at the end of the 1-h perfusion period resulted in an ELF volume of 3.55 ± 0.22 (SE) ml (Fig. 11). When aerosolization of 1.1 ml of saline was performed at the onset of the 1-h perfusion period, end-point lavage measured an ELF volume of 3.18 ± 0.21 ml. Thus a reabsorption of 0.37 ml of the excess 1.1-ml volume (35%) within the 1-h period was calculated, which corresponded to a fluid absorption rate of 1.23 µl · g lung wet weight
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DISCUSSION |
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Aerosol Techniques
When generating particles of alveolar-accessible size, as currently done, aerosolization may be a powerful tool to achieve homogeneous alveolar deposition of any solution. Compared with jet aerosolization, the presently employed ultrasonic technique nebulizes fluids at a higher rate, thereby reducing the time necessary for tracer deposition within the lung. When this technique was used for labeled albumin, controls were undertaken to ascertain that neither the protein was fragmented nor the label was dissociated from the protein by the ultrasonic device. The yield of ~30% of total nebulized volume was definitely deposited within the lung as calculated from the tracer measurement of lung and perfusate and, therefore, compares favorably with data in the literature in which deposition fractions ranging from <5% up to 35% are reported (26). Precautions, including the use of appropriate lead shielding and in-line filter systems, were taken to avoid any escape of radiation or tracer into the environment, and regular controls persistently demonstrated the absence of leakage of the entire system. The reproducibility of the aerosol technique is well reflected by the small variation in counts detected after tracer loading between the different lungs. Moreover, when the calculated fluid amount deposited in the lung by the aerosol technique was compared with the increase in the ELF in the subsequently performed mannitol lavage, excellent concordance of the data was noted. The use of highly sensitive gamma detectors, constructed to capture a large percentage of the lung and perfusate reservoir surfaces, allowed highly reproducible data with relatively low absolute tracer quantities to be obtained.Sodium Transit Under Baseline Conditions
Immediately after 22Na loading, lung clearance of this solute was evident, with an "initial" clearance rate (rate determined within 3 min after aerosol loading) ofThe -mimetic agent terbutaline caused an increase in the initial
sodium clearance rate of ~44.4%. Such an effect, although moderate,
is in line with the well-known stimulatory activity of this agent on
the sodium channel-Na-K-ATPase axis in alveolar epithelial cells
(13, 20, 29). However, in preceding experiments in
fluid-filled rabbit lungs, different from all other species (dog,
sheep, rat, and human), no such effect of terbutaline was demonstrated
(28).
When considering this discrepancy, it has to be kept in mind that the ELF terbutaline concentrations currently used are comparably high. Nevertheless, as evident from the controls, the presently observed response to terbutaline may not be ascribed to the nonspecific effects of this high concentration on barrier properties because a reversal of the stimulatory effect was achievable by concomitant application of propranolol. Mannitol clearance from the alveolar space was not changed in the terbutaline-exposed lungs, and the excess sodium clearance rate in response to terbutaline was again blocked by coapplication of ouabain/amiloride.
Fluid Absorption
The currently used technique of ELF measurement was based on a single lavage with an isosmolar sodium-free mannitol solution recovered within a short time frame of <15 s. A previous study (4) has demonstrated that such a short time period avoids significant additional alveolar space entry of sodium during the lavage procedure itself. Moreover, the technique is based on the assumption that the sodium concentration in the alveolar lining layer corresponds to that in the intravascular fluid (16). Indeed, excess fluid loading by aerosolization was fully reflected by an increase in ELF, thus supporting the validity of this technique. Assessed for a 1-h observation period in control lungs, a fluid absorption rate of 2.44 µl · minAlbumin Clearance
For albumin, an initial clearance rate of 0.40%/min was calculated from the data of lung tracer decrease and perfusate tracer accumulation. The overall albumin passage amounted to 18% within 90 min, and a half-life for the alveolar albumin load of 260 min was calculated from the current data. The present transepithelial albumin passage was thus more rapid than previously reported for ventilated lungs (albumin passage after 90 min ~12%; half-life 580 min) (31). The differences to fluid-filled lungs are even bigger. Data for albumin clearance under these conditions range from 0.007%/min in rats (11), 0.02%/min in rabbits (10), and 0.06%/min in sheep (18). The clearance in control lungs ascertained that the markedly higher clearance observed in our aerated lung preparation was not due to albumin tracer dissociation or fragmentation of the protein. The underlying reason for the apparent discrepancy to the albumin clearance data in fluid-filled lungs is presently not known. Theoretically, the following explanations might be operative. First, active transport may contribute to this finding. Other investigators (9) already demonstrated a vesicular transcellular albumin transport in parallel to a pathway in which alveolar macrophages clean the alveolar space from protein by phagocytosis and intercompartmental transport. The much lower absolute protein load of the alveolar compartment in our preparation compared with that in fluid-filled lungs may then be responsible for a relatively higher fraction of iodinated albumin cleared from the alveolar space. Second, an intact periepithelial microenvironment may contribute to effective albumin elimination from the alveolar space by providing local proteolytic activities that may be substantially diluted on fluid filling of the alveolar space. Further studies will be necessary to address this interesting difference in albumin clearance between flooded and nonflooded alveolar space in detail.Paracellular Permeability
Mannitol was used as an indicator for passive small-solute paracellular epithelial permeability because it is a representative for sugarlike molecules without being metabolized by the cells (22). In our model, a typical profile of tracer passage under baseline conditions was obtained that, in its kinetics, corresponded nicely to data known from fluid-filled lungs (6, 25). As mentioned before, influencing the active transport rates for sodium (either by inhibition or activation) did not result in changes in mannitol clearance rates. In contrast, pretreating the alveolar space with the polycation protamine resulted in a markedly increased paracellular permeability as demonstrated by a 2.4-fold augmentation of the initial appearance rate of mannitol in the perfusate. Interestingly, no changes in the physiological variables of the lungs were detectable under these circumstances, including lung perfusion pressures and the capillary filtration coefficient. The changes in epithelial paracellular permeability were the sole response currently observed to the protamine aerosolization. Thus as described before by Saumon and colleagues (23, 25), polycations may be used as elegant tools to selectively modulate this barrier property, which may even be employed for enhancing the systemic delivery of alveolar-deposited agents.In conclusion, we present a new experimental approach that employed coaerosolization of 22Na, 125I-albumin, and [3H]mannitol in ventilated lungs for the assessment of epithelial barrier function under near physiological conditions. Most of our findings are well in line with data on active and passive solute transport that were gained from well-established fluid-filled lung models. We did, however, notice a surprisingly high albumin clearance rate that should stimulate the investigation of possible differences of transepithelial protein transport between fluid-filled and aerated lungs in more detail. The presently described technique, in particular, may be utilized to investigate the impact of those factors, such as airborne noxious substances, ventilator strategies with respect to barotrauma, and nebulized pharmacological agents, on lung epithelial barrier function that may not be easily addressed in fluid-filled lungs.
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ACKNOWLEDGEMENTS |
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This work was supported by the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. Seeger, Dept. of Internal Medicine, Justus-Liebig-Univ. Giessen, Klinikstrasse 36, 35392 Giessen, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 October 1999; accepted in final form 6 December 2000.
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