INTRODUCTION

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EXPERIMENTS performed during the last decade demonstrated the importance of active sodium transport in the resorption of alveolar fluid. Vectorial active sodium transport via apical sodium channels and basolateral Na-K-adenosinetriphosphatase (ATPase) in lung epithelium has been studied using a number of experimental models, including cell systems (7, 13), whole animals (4, 17, 20), and isolated perfused lungs (IPLs) (1, 6, 8, 10, 14, 19, 21). Fluid resorption is upregulated by -adrenergic agonists and adenosine 3,5-cyclic monophosphate (cAMP) in each of these models (4, 7, 8, 12, 13, 17, 20, 21).

In the past 10 years, the IPL was used in >200 publications in the Journal of Applied Physiology and American Journal of Physiology (Lung, Cellular, and Molecular Physiology), and many of these studies examined the mechanisms and regulators of lung fluid balance. In some of these studies, perfusate was recirculated during the experiment (1, 8, 10, 14, 19, 21); in others, the perfusate was discarded after a single pass (6, 12). Our laboratory has performed a number of IPL studies by using normal rat lungs. In reviewing the results of these experiments, it appeared that recirculation of the IPL perfusate resulted in a uniform loss of lung weight by the preparation, whereas single pass of the perfusate through the IPL led to variable and much lower rates of weight change. This suggested that there might be a substance accumulating in the perfusate during recirculation that was responsible for stimulating alveolar fluid resorption.

This report describes a series of experiments that confirmed that recirculation of the perfusate accelerates alveolar fluid resorption. Because cyclic nucleotides may be secreted by IPLs (2) and cAMP can stimulate alveolar fluid resorption (4, 7, 8, 12, 13, 17), we assayed the perfusate for cAMP and performed experiments with a cyclic nucleotide agonist, a -adrenergic agonist, a cyclic nucleotide-dependent protein kinase inhibitor, and phosphodiesterase (PDE) to show that cAMP could play a role in accelerating fluid resorption during recirculation of perfusate.

	METHODS

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IPL experiments were performed as previously described (6). Briefly, male Sprague-Dawley rats (Harlan, Madison, WI), 175-225 g, were anesthetized with pentobarbital sodium (80 mg/kg ip). The chest was opened, the pulmonary artery was cannulated, and the lungs were excised and hung on a weight transducer. During recirculation, the perfusate returned to an Erlenmeyer flask by dripping from the left atrium, and the recirculating volume was 50 ml. Perfusion of the pulmonary artery with Ringer solution (37°C, pH 7.4) was initiated via a peristaltic pump at 7 ml/min. The composition of the Ringer solution (in mM) was 137 NaCl, 2.68 KCl, 1.25 MgSO₄, 1.82 CaCl₂, and 5.55 glucose; the Ringer solution also contained 0.5% bovine serum albumin and 1.0% dextran (70 kDa) and was buffered with 12 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid. Pulmonary arterial pressure was monitored continuously via a pressure transducer catheter within the pulmonary arterial cannula. Pulmonary arterial pressure was 5.4 ± 0.3 cmH₂O (n = 31), and this changed by <1 cmH₂O during each experiment. After the preparation stabilized, 2.0 ml of the Ringer solution were instilled into the trachea for assessment of fluid resorption. Pulmonary arterial pressure dropped 10-20% after instillation, because of alveolar recruitment and opening of subtended capillaries, then was constant.

The rate of alveolar fluid resorption from IPLs was taken to be the rate of weight loss of the preparation. In a separate group of experiments (n = 17), alveolar volume change was measured by the change in concentration of fluorescein isothiocyanate (FITC)-labeled dextran (150 kDa) in the instillate, as determined by fluorometry. In these experiments, 3.0 ml of Ringer solution were instilled to facilitate obtaining the final alveolar sample for fluorometry, and the IPL was perfused by single pass (n = 12) or recirculation (n = 5) for 60 min. In this range (2 or 3 ml), the volume of instillate did not alter the effects of recirculation. This is consistent with the findings of Jayr et al. (17), who demonstrated that the volume of alveolar instillate did not affect the percentage of alveolar fluid resorbed in an anesthetized rat. The fluorometrically determined alveolar volume change was compared with the value determined from the weight loss of the same IPL. This is shown graphically in Fig. 1, which demonstrates that weight change correlates well with alveolar volume change.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Alveolar volume change as assessed by change in fluorescein isothiocyanate (FITC)-dextran concentration vs. weight change. A line of identity is shown. Slope of best-fit line is 0.8, r2 = 0.7.

Six sets of experiments were done using this gravimetric technique. In every experiment, whenever a perfusate change was made, the weight loss reached a new steady state within 5-10 min. Each perfusion period was therefore 20-25 min, and we measured the rate of weight loss during the final 15 min.

In the first set of experiments (n = 16), 10 rat lungs were perfused in single-pass fashion followed by recirculation of the perfusate. In four IPLs, recirculation of perfusate was continued for up to 2 h, with persistence of the accelerated rate of weight loss. An additional six rats were perfused by recirculation first, followed by single-pass perfusion. The rates of weight change were calculated for each animal during the single-pass and recirculating periods. Samples of perfusate were obtained at 5-min intervals and frozen at 70°C for subsequent cAMP analysis by enzymatic fluorometric assay, as described previously (22).

In the second set of experiments (n = 2), a period of single-pass perfusion with normal Ringer solution was followed by single-pass perfusion with perfusate that had been recirculated through other IPLs for 60 min and stored frozen at 70°C. This was done to determine the effect of previously recirculated perfusate on the rate of alveolar fluid resorption in a different lung.

In the third set of experiments (n = 4), single-pass perfusion was followed by a baseline period of recirculation of perfusate; then H-8 {N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride}, an inhibitor of cyclic nucleotide-dependent protein kinases (16), was added to the recirculating perfusate at a final concentration of 40 µM.

In the fourth set of experiments (n = 5), an initial period of single-pass perfusion was followed by single-pass perfusion with 100 µM 8-bromoadenosine 3,5-cyclic monophosphate (8-BrcAMP), a specific analog of cAMP that is more membrane permeable than cAMP.

In the fifth set of experiments (n = 5), an initial period of single-pass perfusion was followed by single-pass perfusion with the -adrenergic agonist terbutaline at a concentration of 1 mM. -Adrenergic agonists exert many of their effects via generation of intracellular cAMP and thus should mimic perfusion with 8-BrcAMP.

In the sixth set of experiments (n = 5), an initial period of single-pass perfusion was followed by recirculation, with subsequent addition of PDE to the recirculating perfusate at a concentration of 20 mU/ml. The optimal concentrations for inhibition by H-8 and PDE and stimulation by 8-BrcAMP and terbutaline were determined in a preliminary series of experiments (data not shown).

Statistical analysis of the differences between groups was performed by using a two-tailed paired Student's t-test. Values are means ± SE.

	RESULTS

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In the first set of experiments, recirculation of the perfusate resulted in a faster rate of alveolar fluid resorption (Table 1), regardless of the order in which the perfusion techniques were performed. After a period of single-pass perfusion, recirculation increased weight loss from 0.7 ± 2.0 to 9.0 ± 1.3 mg/min. When recirculation was performed first, the rate of weight loss decreased from 6.3 ± 0.9 to 3.2 ± 0.3 mg/min during single-pass perfusion. Figure 2 shows representative weight vs. time tracings from two experiments: one with single-pass perfusion first and the other with recirculation first.

View larger version (6K): [in this window] [in a new window]   View larger version (7K): [in this window] [in a new window]   Fig. 2.   Representative weight vs. time tracings from isolated perfused lungs (IPLs) of 2 rats. A: single-pass perfusion followed by recirculation; B: recirculation followed by single-pass perfusion. Recirculation resulted in a greater rate of weight loss regardless of order in which perfusion techniques were performed. Pulmonary arterial pressure remained stable in normal range throughout each experiment. Sudden apparent shifts in weight represent investigator adjustment of zero-suppression control.

In the second set of experiments, to assess whether the stimulatory component of the perfusate could be transferred to another lung, a bioassay was performed by single-pass perfusion with thawed aliquots of recirculated perfusate. Figure 3 shows the results of the bioassay. The rate of fluid resorption increased during single-pass perfusion with the thawed aliquots, demonstrating that the activity of the recirculated perfusate was retained, despite freezing.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Weight vs. time tracing from an isolated lung perfused in single-pass fashion with Ringer solution, then with thawed aliquots of recirculated perfusate from 2 other IPLs. Rate of fluid resorption increased when thawed aliquots were perfused. Pulmonary arterial pressure remained stable in normal range. Sudden apparent shifts in weight represent investigator adjustment of zero-suppression control.

Because cyclic nucleotides may be secreted by IPLs (2) and cAMP stimulates alveolar fluid resorption (4, 7, 8, 12, 13, 17), we assayed single-pass and recirculated perfusate for cAMP. Figure 4 shows the results of the cAMP assay performed on the perfusate samples. There was a marked accumulation of cAMP in the perfusate with recirculation, but the levels were consistently low with single-pass perfusion.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Results of enzymatic fluorometric assay for cAMP at 5-min intervals. There is a significant accumulation of cAMP with recirculation of perfusate, in contrast to persistent low levels with single-pass perfusion technique.

To support the hypothesis that increased cAMP was at least partially responsible for the acceleration of fluid resorption caused by recirculation, we performed analog studies using 8BrcAMP, -adrenergic stimulation studies using terbutaline, kinase inhibition studies using H-8, and cyclic nucleotide hydrolysis studies using PDE. A representative tracing is shown in Fig. 5, and Fig. 6 illustrates the effect of PDE on the rate of weight loss during recirculation. Table 2 summarizes the results of these studies.

View larger version (6K): [in this window] [in a new window]   Fig. 5.   Representative weight vs. time tracing from an isolated lung perfused in single-pass fashion, followed by single-pass perfusion with 100 µM 8-bromoadenosine 3,5-cyclic monophosphate (8-BrcAMP). Addition of 8-BrcAMP stimulated weight loss and reproduced effect of recirculation. Pulmonary arterial pressure remained stable in normal range.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of adding phosphodiesterase (PDE) to recirculating perfusate of 5 IPLs. Addition of PDE (20 mU/ml) abolished effect of recirculation.

After the first two sets of experiments comparing single-pass perfusion with recirculation had been performed, a number of IPL preparations showed relatively rapid weight loss (4-5 mg/min) during initial perfusion with the single-pass technique. When H-8 was added to the single-pass perfusate of these IPLs, the rate of fluid resorption was reduced (Fig. 7). Thus we observed, during single-pass perfusion in these normal lungs, heterogeneity of weight loss which appeared to be at least partially mediated by cAMP. Two issues are of note in this regard: 1) even those lungs that lost weight relatively rapidly with single-pass perfusion resorbed fluid more rapidly with recirculation, and 2) a review of all IPL experiments performed in our laboratory in the last several years revealed similar rates of single-pass weight loss in several groups of animals, with about one-half of the groups losing weight rapidly. However, there is no clear-cut trend or pattern to allow prediction of the behavior of a given group of animals. We investigated a number of possible explanations for this heterogeneity, including change in vendor or housing, investigator experience, season of animal purchase, or viral infection, but we have been unable to determine its cause or a temporal pattern to its occurrence.

	DISCUSSION

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Active sodium and fluid resorption is key to the maintenance of a functioning alveolus, as demonstrated by a number of investigators (1, 4, 6-8, 10, 13, 14, 17-21). Many laboratories have used the isolated perfused lung as a model system, using single-pass (6, 12) and recirculation (1, 8, 10, 14, 19, 21) of the perfusate. We sought to determine whether the rate of baseline fluid resorption in this system depends on the method of perfusion of the IPL.

Our first experiments confirmed that recirculation resulted in a consistent and rapid rate of weight loss compared with single-pass perfusion. The rate of fluid resorption seen in our experiments was consistent with that reported in the literature as measured by using IPLs (1, 10, 14, 21) and in vivo (17) and ex vivo (18) preparations (Table 3). Because weight loss reflected predominantly the alveolar volume change, as confirmed by the FITC-dextran experiments, recirculation stimulated fluid resorption from the alveolar space. However, we cannot comment on the rate of interstitial fluid filtration into the alveoli, inasmuch as both methods of assessment measure only net alveolar volume change. Some of the IPLs with single-pass perfusion gained weight, but only very slowly, suggesting that the background rate of fluid filtration into the alveoli is low. Taken together, this suggested that a substance accumulated in the recirculating perfusate that stimulated fluid resorption. Reproduction by passive transfer indicated that this activity was preserved with freezing.

Although the mechanism of the effect of recirculation is not completely determined, we performed a series of initial experiments to look for known stimulators of alveolar sodium transport. Because other investigators reported the secretion of cyclic nucleotides by IPLs (2) and cAMP and -agonists stimulate alveolar fluid resorption (4, 7, 8, 12, 13, 17, 20, 21), we assayed the perfusate for cAMP. There was a marked increase in the concentration of cAMP in the recirculated perfusate. Experiments with the cAMP-specific analog 8-BrcAMP demonstrated that cAMP was capable of the observed effect of recirculation. We cannot explain the quantitative discrepancy between the increase in weight loss induced by recirculation and that by 8-BrcAMP, but the degree of stimulation by 8-BrcAMP in these experiments is similar to that found by other investigators using cAMP analog (12, 21). It is possible that there are other active substances in the recirculating perfusate besides cAMP. In addition, we do not know the site of release of cAMP; if it is assumed that it is acting on the alveolar epithelium, it is difficult to know whether 8-BrcAMP in the perfusate or locally generated cAMP would achieve optimal intracellular concentrations faster. The effect of terbutaline was similar in relative magnitude to that of 8-BrcAMP; this finding is consistent with the known stimulation of intracellular cAMP generation by -adrenergic agonists and their acceleration of alveolar fluid resorption. Finally, the addition of H-8 or PDE prevented stimulation of fluid resorption by recirculation. H-8 inhibits other kinases in addition to cAMP-dependent protein kinase (16), and PDE is not specific for cAMP (3), so we cannot rule out a contribution by guanosine 3,5-cyclic monophosphate (cGMP) or other substances. However, nitric oxide, a known stimulator of cGMP production, reduced active transepithelial sodium transport in alveolar type II cell monolayers (15), suggesting that cGMP has no role in the effect of recirculation; additionally, the effects of 8-BrcAMP are relatively specific for cAMP. Thus the accumulation of cAMP is sufficient to explain the observed findings, but we cannot exclude contributions from other agents, especially given the small quantitative differences between the effects of 8-BrcAMP and recirculation. We did not perform experiments with inhibitors of active sodium transport, since the FITC-dextran data confirmed that weight loss was due to alveolar volume resorption and thus active sodium transport.

View larger version (6K): [in this window] [in a new window]   Fig. 7.   Weight tracing from an IPL that lost weight rapidly with single-pass perfusion. Addition of H-8 to single-pass perfusate reduced rate of weight loss, suggesting that initial rapid rate of weight loss was cAMP or cyclic nucleotide dependent. Pulmonary arterial pressure remained stable in normal range. Sudden apparent shifts in weight represent investigator adjustment of zero-suppression control.

Several questions remain. First, the stimulus for increased production or release of cAMP has not been determined. It is possible that another substance accumulates during recirculation and triggers cAMP release into the perfusate. Second, the source of the cAMP released into the perfusate is not clear. Although type II alveolar epithelial cells likely account for the majority of alveolar fluid resorption and we speculate that they have increased intracellular cAMP with recirculation, it seems unlikely that they are the source of the cAMP. If alveolar type II cells were the source of the cAMP, then recirculation would not increase cAMP levels above that already present within the type II cells and alveolar fluid resorption would not change. We did not measure type II cell cAMP levels, because it takes several hours to isolate the cells, and the isolation may alter the cAMP levels, potentially making the results difficult to interpret.

The mechanisms by which cAMP increases alveolar fluid resorption by the IPL are unknown but likely are mediated by increased activity of Na-K-ATPase and/or transport through sodium channels. Most investigations of the short-term regulation of Na-K-ATPase and sodium channels have used renal tubular epithelia; in this system, cAMP mediates phosphorylation of the -subunit of Na-K-ATPase (5, 11) and insertion of sodium channel-rich membrane vesicles (9). Whether these precise mechanisms are responsible for cAMP effects on alveolar epithelium has not been explored, but terbutaline and cAMP agonists increase Na-K-ATPase activity in alveolar type II cells independent of the intracellular sodium concentration (23).

IPLs perfused using single-pass technique had a variable, but small, rate of fluid resorption. There was some heterogeneity, since a number of animals perfused with single-pass technique lost weight relatively rapidly (4-5 mg/min). These rats still had more rapid weight loss with recirculation, and the rate of single-pass weight loss was reduced by addition of H-8 to the perfusate, suggesting that the elevated basal rate of fluid resorption of these IPLs was cAMP mediated. The reason for the presumably elevated cAMP in these animals is unknown but could be due to stresses such as subclinical viral infections. Thus there is some heterogeneity in the rate of alveolar fluid resorption in normal rat lungs by use of single-pass technique, whereas use of recirculation results in a relatively homogeneous and increased rate of fluid resorption. Heterogeneity of sodium and fluid resorption also has been observed after acute and chronic hyperoxic rat lung injury (6, 24).

In conclusion, the perfusion technique used in the IPL significantly affects the rate of fluid resorption, and investigators using the IPL model should bear in mind that use of the recirculating technique does not measure the true baseline rate of alveolar fluid resorption. The use of recirculation produced a more homogenous and greater rate of fluid resorption, but this appeared to represent a partially stimulated state, probably due at least in part to cAMP. The results of experiments using recirculation of the perfusate should be designed and interpreted accordingly.