SPECIAL COMMUNICATION
Alveolar sodium and liquid transport in mice

Philippe Icard and Georges Saumon

Service de Chirurgie Thoracique et Cardiovasculaire, Centre Hospitalier Universitaire de Caen, 14000 Caen; and Institut National de la Santé et de la Recherche Médicale Unité 82, Faculté Xavier Bichat, 75018 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have developed a simple isolated lung preparation for measurement of liquid and solute fluxes across mouse alveolar epithelium. Liquid instilled into air spaces was absorbed at the rate (Jw) of 3.7 ± 0.32 ml · h-1 · g dry lung wt-1. Jw was significantly depressed by ouabain (P < 0.001) and amiloride (P < 0.001). Omission of glucose from the instillate or addition of the Na+-glucose cotransport inhibitor phloridzin did not affect Jw. However, the low epithelial lining fluid glucose concentration (one-third that of plasma), the larger-than-mannitol permeability of methyl-alpha -D-glucopyranoside, and the presence of Na+-glucose cotransporter SGLT1 mRNA in mouse lung tissue suggest that there is a Na+-glucose cotransporter in the mouse alveolar-airway barrier. Isoproterenol stimulated Jw (6.5 ± 0.45 ml · h-1 · g dry lung wt-1; P < 0.001), and this effect was blocked by amiloride, benzamil, ouabain, and the specific beta 2-adrenergic antagonist ICI-118551 but not by atenolol. Similar stimulation was obtained with terbutaline (6.4 ± 0.46 ml · h-1 · g dry lung wt-1). Na+ unidirectional fluxes out of air spaces varied in agreement with Jw changes. Thus alveolar liquid absorption in mice follows Na+ transport via the amiloride-sensitive pathway, with little contribution from Na+-glucose cotransport, and is stimulated by beta 2-adrenergic agonists.

glucose transport; pulmonary alveoli; pulmonary edema


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALVEOLAR LIQUID IS ABSORBED by an active process that involves the transepithelial transport of solutes by distal respiratory epithelia (23). Studies (28) on isolated lungs have helped to delineate the mechanisms of liquid clearance. Most studies have been performed in rats and showed that Na+ is absorbed across the apical membrane of alveolar epithelial cells by two main transport mechanisms, at least in this species. One is amiloride sensitive and consists of Na+ channels (22); the other is amiloride insensitive and has been identified as Na+-glucose cotransport (28), which is present in airway (20, 31) and alveolar (21) epithelia. Na+ is pumped out from epithelial cells through the basolateral membrane by Na+-K+-ATPase (2).

Liquid absorption can be stimulated by beta -adrenergic agonists and cell-permeable cAMP analogs in rats (28) via stimulation of Na+ transport (6, 27). Studies using isolated hamster (15) and rabbit (9) lungs as well as intact rabbits (33) have shown that the mechanisms of clearance and the response to beta -adrenergic agonists vary from one species to another. For example, hamster air space epithelia do not exhibit Na+-glucose cotransport activity (15), and beta -adrenergic agonists do not increase alveolar liquid clearance in rabbits (9, 33) or hamsters (15). Although mice are now frequently used in physiological studies, there are few experimental works dealing with the mechanisms of alveolar liquid clearance in this species. Reproducible data on the properties of the alveolar epithelial transport system would be useful for studies on pharmaceuticals, genetically modified animals, and the effects of gene therapy. Garat et al. (13) devised a simple in situ lung preparation and found that alveolar liquid absorption was stimulated by beta 1- but not by beta 2-adrenergic agonists in mice (14), in contrast to the situation in rats (4).

The present report describes a simple isolated perfused liquid-filled lung preparation that is suitable for evaluating the fluxes of liquids and solutes across the alveolar-airway epithelial barrier in mice. The properties of the mouse alveolar barrier differ somewhat from those of rats. Alveolar liquid absorption is almost exclusively due to Na+ transport via the amiloride-sensitive pathway. Na+ transport and liquid absorption are stimulated by beta 2-adrenergic agonists. The reasons why these observations differ from those of Garat et al. (13) have been explored.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated lung preparation. This study was done on male CD1 mice (Charles River, St. Aubin-lès-Elbeuf, France) with a mean weight of 30 g. Each mouse was anesthetized by an intraperitoneal injection of phenobarbital and tracheotomized, and the trachea was catheterized with a 20-gauge catheter. The abdomen was opened, and the animal was killed by exanguination. The mouse was placed on a thermostated blanket (Ealing, Les Ulis, France) set at 38°C. The anterior wall of the thorax was removed, and the left ventricle and atrium were opened by a single large incision. A 20-gauge catheter was inserted into the pulmonary artery via the right ventricle and secured with a small bulldog clamp squeezing the ventricle wall. The lungs were perfused with a rotor pump at 1 ml/min. The perfusate was warmed in a glass heat exchanger so that the temperature of the solution at the outflow of the perfusion catheter was 38°C. The perfusion temperature was verified with a small thermistance sensor (YSI, Yellow Springs, OH). The lungs were filled with instillate via the trachea. The heating blanket temperature probe was then inserted under the heart, and the small thermistance sensor was placed in the left ventricle to control the temperature of the lungs and monitor that of the outflowing perfusate. Perfusate was not recirculated. The lungs and heart were covered with a small piece of plastic and by folding the heating blanket; care was taken not to compress the viscera.

After the lungs were cleared of blood by perfusion, the liquid was instilled into the air spaces essentially as described for the rat preparation (see, for example, Ref. 1). As much air as possible was withdrawn from the lungs by gentle suction with a syringe until the airways collapsed. Then, 0.55 ml of solution was instilled into the trachea with a calibrated syringe followed by 0.1 ml of air. The lungs were covered as above until the first 0.06-0.08 ml of instillate sample was recovered 5 min later. The liquid in the lungs was removed with a syringe and thoroughly mixed, a sample was taken, and the liquid was returned to the trachea followed by 0.1 ml of air. The last sample was obtained 15 min later. Sample volumes were determined by weighing.

The solution used for perfusion and air space instillation was Krebs-Ringer bicarbonate supplemented with 0.4% bovine serum albumin. Glucose (1 g/l) was added to the perfusate and instillate as indicated. Ficoll 70 (4%; Pharmacia, Uppsala, Sweden) was added to the perfusate as the oncotic agent. The perfusate was equilibrated with 95% O2-5% CO2 by bubbling. The lungs were removed at the end of the experiment and dried at 80°C for 1 wk. All fluxes were standardized according to the dry lung weight. Isoproterenol, terbutaline, atenolol, amiloride, ouabain, and phloridzin were obtained from Sigma (Saint-Quentin Fallavier, France). Benzamil was obtained from RBI (Natick, MA), and ICI-118551 was obtained from Tocris (Bristol, UK). Osmolarity was measured with a freezing-point osmometer (Advanced Instruments, Needham Heights, MA).

The following tracers were added to the instillate: 0.05 µCi/ml of 125I-labeled albumin (CIS, Gif sur Yvette, France) to follow volume changes, 0.05 µCi/ml of 22Na (Amersham, Les Ulis, France) to measure the Na+ transport rate, and 0.1 µCi/ml of [3H]mannitol (Isotopchim, Ganagobie-Peyrus, France) to assess the passive permeability properties of the alveolar-airway barrier. In some experiments, methyl-alpha -D-[14C]glucopyranoside (alpha -MG), a glucose analog that is specifically taken up by cells by cotransport with Na+, was used to trace glucose uptake. 22Na was omitted in these experiments. 125I and 22Na activities were obtained by gamma counting (Wizard, Wallac, Turku, Finland) and those of 3H and 14C with a scintillation counter (Rack 1214, Wallac) after precipitation of the albumin with 0.6% trichloroacetic acid. Tracer activities were determined with at least 10,000 counts/min. Corrections for spillover were performed with standards.

In situ lung preparation. The in situ lung preparation was the same as that described by Garat et al. (13) with minor modifications. The alveolar instillate consisted in 0.55 ml of either the solution used for isolated lungs or commercial Ringer lactate (Biosedra, Louviers, France) supplemented with 5% bovine serum albumin and containing isoproterenol or terbutaline when appropriate. The volume marker of the instillate was 125I-serum albumin. The alveolar liquid was recovered 7.5-60 min after instillation. A second series of experiments was performed with the instilled solution osmolarity increased by adding NaCl.

Calculations. The rate of liquid absorption (Jw) from the air spaces during a time period (Delta t) was calculated from the changes in 125I-albumin, assuming that albumin does not cross the alveolar-airway barrier, with the equation Jw = V0[(Ct - C0)/(CtDelta t)], where V0 is the volume of alveolar liquid at the beginning of the 15-min period and C0 and Ct are the concentrations of tracer in this liquid at the beginning and end, respectively, of the period. In the isolated lung experiments, the unidirectional fluxes of 22Na, [3H]mannitol and alpha -[14C]MG [expressed as the permeability-surface area product (PA)] were determined from Jw and the changes in these tracer activities (2) with the equation PA = Jw{1 + ln(C0/Ct)/ln [V0/(V0 + JwDelta t)]}.

Bronchoalveolar lavage. Mice were killed by phenobarbital injection. The trachea was rapidly catheterized, the thorax was opened, and three successive bronchoalveolar lavages (BALs) were performed with 0.5 ml of isotonic mannitol. Simultaneously, blood was obtained by cardiac puncture. The glucose and urea concentrations in the retrieved BAL fluid and plasma were determined with commercial kits (Sigma), adjusting the reactant volume to increase the signal-to-noise ratio depending on the lavage medium (30). The epithelial lining fluid (ELF) volume and glucose concentration were calculated with urea as the dilution marker, which was equivalent to using the less-permeable solute Na+ given the short duration of the lavage (30).

RT-PCR. Because the amino acid sequences of Na+-glucose cotransporters are highly conserved, we employed the primers that we designed to amplify rat Na+-glucose cotransporter SGLT1 cDNA (29). Rat and mouse tissues were removed aseptically and immediately frozen in liquid nitrogen. Total RNA was isolated by the guanidinium isothiocyanate-phenol-chloroform method. cDNA was synthesized in a 30-µl reaction mixture containing the RT reaction buffer, 15 nmol of each deoxynucleotide triphosphate, 1.5 nmol of random hexamer primer [pd(N)6; Pharmacia], 450 U of Moloney murine leukemia virus reverse transcriptase (GIBCO BRL, Life Technologies, Gaithersburg, MD), and 10 ng of total RNA. The RT reaction was incubated for 60 min at 42°C and 5 min at 95°C to inactivate the reverse transcriptase. The primers used for PCR were designed to amplify a sequence of the SGLT1 rat cDNA. They were 5'-GCCCATCCCAGACGTACAC-3' (sense) and 5'-GGTCCAGCCCACAGAACAG-3' (antisense). The expected PCR product length was 197 bp. A 50-µl hot-start PCR was done in PCR buffer (20 mM Tris · HCl, pH 8.4, 50 mM KCl, and 1.5 mM MgCl2), 2 µl of RT product, 200 M each deoxynucleotide triphosphate, 10 pmol of each primer, 2 U of Taq DNA polymerase (GIBCO BRL), and cDNA, covered by mineral oil. Temperature cycling was 95, 59, and 72°C for 1 min each. The size of the RT-PCR product was verified by ethidium bromide staining of a 2% agarose electrophoresis gel. RT-PCR products obtained from extracts of rat intestine, kidney, and lung were checked by hybridization with a 20-bp specific probe (5'-CACAGAAGAGCGCATCGACC-3').

Statistical analysis. Results are means ± SE. Paired values were compared with Student's paired t-test. Data were compared by one-way analysis of variance and the Bonferroni multiple comparison test or by the Kruskal-Wallis test when appropriate. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated lungs. Experiments with a large (>12 ml · h-1 · g dry lung wt-1) mannitol PA were discarded (9 of 187) because this might have reflected epithelial lesions. Almost all control values (21 of 23) satisfied the criterion. The basal alveolar liquid absorption rate was 3.7 ± 0.32 ml · h-1 · g dry lung wt-1 (n = 21 experiments). Absorption was almost completely suppressed by apical amiloride (10-3 M; n = 9 experiments) and significantly depressed by ouabain (10-3 or 10-4 M; n = 14 experiments) in the perfusate (Fig. 1). Alveolar liquid absorption was the same as the control value (3.7 ± 0.30 ml · h-1 · g dry lung wt-1; n = 15 experiments; not significant) when initially there was no glucose in the alveolar instillate. However, the glucose concentration in the instillate increased from 0.53 ± 0.086 mmol/l 5 min after instillation to 1.15 ± 0.184 mmol/l at 20 min. Such apical concentrations may have supplied a putative Na+-glucose cotransport with glucose, which would explain why a lack of glucose did not reduce alveolar liquid clearance. We therefore added phloridzin (10-3 M; n = 9 experiments), a specific inhibitor of Na+-glucose cotransport, to the instillate. The lack of glucose and the presence of phloridzin did not affect alveolar liquid absorption. Amiloride reduced alveolar liquid absorption and Na+ efflux to the same extent whether (n = 9 experiments) or not (n = 8 experiments) the instillate contained glucose (Fig. 1). Isoproterenol (5 × 10-6 M; n = 21 experiments) resulted in a nearly twofold increase in alveolar liquid absorption (Fig. 2), which was blocked by amiloride (n = 5 experiments) and 10-4 M benzamil (n = 5 experiments) and depressed by ouabain (n = 6 experiments). The same stimulation of absorption was observed with terbutaline (10-4 M; n = 17 experiments). The beta 2-adrenergic antagonist ICI-118551 (10-4 M; n = 12 experiments) but not the beta 1-antagonist atenolol (10-4 M; n = 16 experiments) inhibited the stimulation produced by isoproterenol (Fig. 2). ICI-118551 did not affect baseline alveolar liquid absorption (n = 6 experiments; Fig. 1).


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Fig. 1.   Alveolar fluid absorption rate in isolated lungs under control (Ctrl) conditions and with amiloride (Amil) in alveolar instillate and ouabain (Ouab) in perfusate. dlw, Dry lung weight. Amil and Ouab significantly decreased absorption. Alveolar liquid absorption was not affected by absence of glucose from instillate (0Gl) or by ICI-118551 (ICI). Amil but not phloridzin (Phz) decreased liquid absorption in absence of glucose. *** P < 0.001 compared with Ctrl.



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Fig. 2.   Alveolar liquid absorption rate in isolated lungs under Ctrl conditions; with isoproterenol (Isop) and Isop plus Amil (+Amil), Isop plus benzamil (+Benz), Isop plus Ouab (+Ouab), Isop plus ICI (+ICI), and Isop plus atenolol (+Ate); and in presence of terbutaline (Terb). Isop significantly increased liquid absorption. Effect of Terb was similar to that of Isop. Amil and Benz almost completely inhibited and Ouab significantly depressed absorption. ICI, which did not affect alveolar liquid absorption under Ctrl conditions, suppressed stimulation produced by Isop. Ate had no significant effect. *** P < 0.001 compared with Isop.

Mannitol PA was 6.0 ± 0.58 ml · h-1 · g dry lung wt-1 under control conditions. Mean values ranged from 4.1 ± 0.59 ml · h-1 · g dry lung wt-1 in experiments with ouabain to 8.1 ± 0.74 ml · h-1 · g dry lung wt-1 in those with isoproterenol plus amiloride (Fig. 3). Mannitol PA values did not differ significantly depending on the experimental condition. Amiloride and ouabain significantly reduced Na+ PA (Fig. 3A). The omission of glucose from the instillate or the addition of phloridzin was without effect. Na+ PA in the presence of isoproterenol was significantly larger than in control conditions. The effect of terbutaline did not reach significance. Amiloride, benzamil, ouabain, and ICI-118551 but not atenolol significantly decreased Na+ PA in the presence of isoproterenol (Fig. 3B). ICI-118551 did not significantly affect Na+ PA under control conditions (Fig. 3A).



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Fig. 3.   A: permeability-surface area product (PA; unidirectional flux) for Na+ and mannitol under Ctrl conditions; in presence of Amil, Ouab, and ICI; and with 0Gl, 0Gl+Amil, and 0Gl+Phz. Amil and Ouab depressed unidirectional Na+ flux. Phz was without effect. Significantly different from Ctrl: * P < 0.05; ** P < 0.01 (both by Kruskal-Wallis test). B: Isop significantly increased Na+ PA. Amil, Benz, Ouab, and ICI but not Ate significantly decreased Na+ PA in presence of Isop. Na+ PA with Terb was between Ctrl and Isop values; however, it did not differ significantly from Ctrl value. Significantly different from Isop: * P < 0.05; ** P < 0.01 (both by Kruskal-Wallis test).

Glucose transport. alpha -MG permeability was significantly larger than mannitol permeability (P < 0.01 by paired t-test; n = 9 experiments). The ratio of alpha -MG to mannitol permeability was 2.3 ± 0.34. Phloridzin significantly decreased this ratio (1.2 ± 0.21; P < 0.05; n = 5 experiments). The results of the BALs performed in five mice are shown in Table 1. The ELF volumes were 4.9-13.3 µl and were slightly larger from lavages 2 and 3 than from the first lavage. The ELF glucose concentration did not significantly differ from one lavage to another. It was about one-third that of plasma. It suggested the presence of a glucose uptake mechanism in the lumen of mouse air spaces. To evidence the presence of Na+-glucose cotransport mRNA in mouse lungs, we performed a RT-PCR with primers for SGLT1. The RT-PCR of the RNA extracted from mouse lung gave an amplification product that corresponded in size to that expected for SGLT1 (Fig. 4).

                              
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Table 1.   ELF volume and glucose concentration determined by BAL



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Fig. 4.   Electrophoresis gel of RT-PCR products obtained with mRNAs extracted from rat kidney cortex (lane 1), rat lung (lane 2), and mouse lung (lane 3). Ladder, 123 bp.

In situ lungs. Instillates containing isoproterenol (n = 6 experiments), no isoproterenol (n = 7 experiments), terbutaline (n = 8 experiments), or Ringer lactate (n = 3 experiments) all gave the same alveolar liquid absorption after 60 min (Fig. 5). The alveolar liquid absorption rate at 15 min was about twice that at 60 min. Terbutaline and isoproterenol had no effect (at least 4 experiments in each situation). A plot of the volume of liquid removed from the air spaces versus time showed saturation kinetics: a large amount of liquid was removed in <10 min followed by an absorption rate that tended toward zero (Fig. 6). We suspected that this nonlinear behavior reflected a combination of different mechanisms of absorption, the faster rate being due to the dissipation of an osmotic gradient. The osmolarity of the Krebs-Ringer bicarbonate was 280 mosM and that of the Ringer lactate was 273 mosM. The plasma osmolarity in four mice used for isolated lung preparations was 319 ± 3.9 mosM. We therefore increased the osmolarity of the Krebs-Ringer bicarbonate to 323 mosM and repeated these experiments. The volume removed from the air spaces then increased linearly with time during the first 20 min, indicating steadiness of alveolar liquid absorption (at least 4 experiments in each situation; Fig. 6). Both isoproterenol (n = 6 experiments) and terbutaline (n = 6 experiments) significantly increased the alveolar liquid absorption rate (Fig. 7).


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Fig. 5.   Alveolar liquid absorption rate of mouse lungs in situ after exchange times of 15 and 60 min. There was no significant difference between absorption rates of Krebs-Ringer Ctrl (C), Ringer lactate (L), Krebs-Ringer+Isop (I), and Krebs-Ringer+Terb (T) experiments for either exchange time.



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Fig. 6.   Volume of conventional (280 mosM) Krebs-Ringer bicarbonate (open circle ) removed from air spaces under Ctrl conditions as a function of time. There was an initial period of rapid liquid removal from air spaces, after which absorption became almost zero. Removal of slightly hypertonic Krebs-Ringer bicarbonate (323 mosM; ) from mouse air spaces increased linearly with time during 1st 20 min.



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Fig. 7.   Alveolar liquid absorption rate of mouse lungs in situ with slightly hypertonic Krebs-Ringer bicarbonate as alveolar instillate. Terb and Isop significantly increased absorption rates (values obtained after 15 and 20 min were identical and thus were pooled) compared with Ctrl value. Absorption rates with Isop and Terb were similar. * Significantly difference from Ctrl, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The simple preparation described in MATERIALS AND METHODS is reasonably easy to set up and use. The perfusate is not recirculated and the lungs and heart are left in the thorax because mouse pulmonary veins are easily twisted by heart displacement during handling. It was difficult to avoid the occasional occurrence of pulmonary edema in preliminary experiments when the viscera were not held together by their natural attachments in the thorax. Even minimal pulmonary edema development precludes measurement of alveolar liquid clearance.

Mechanism of alveolar liquid absorption. Alveolar liquid resorption in mice is the result of active transepithelial Na+ transport as in other species. This is substantiated by the finding that luminal amiloride, a blocker of epithelial Na+ channels, and ouabain, which inhibits Na+-K+-ATPase, significantly decreased alveolar liquid absorption and transepithelial unidirectional Na+ fluxes (i.e., Na+ PA, which sums active and passive fluxes) in the absence of a significant decrease in mannitol PA and thus of passive Na+ transport. Thus vectorial Na+ plays the same key role in adults as during the postnatal period in mice (18).

Alveolar liquid resorption in adult mice is essentially due to Na+ transport via the amiloride-sensitive pathway. There is an indication of Na+-glucose cotransport in the mouse lung, but it does not account for the same substantial part of alveolar liquid absorption as in rats (30). The presence of cotransport in mouse lungs is nevertheless suggested by the low glucose concentration in the ELF as estimated with BAL, which is similar to that observed in rats (30) in which luminal Na+-glucose cotransport activity is well documented (3, 15), by the larger-than-mannitol alpha -MG permeability and by the presence of SGLT1 mRNA in the mouse lung as in rat lung (17, 29). The apparently minor role played by cotransport in mice may be explained by a greater rate of Na+ transport via the amiloride-sensitive pathway, resulting in a much larger liquid absorption than in rats and a proportionally smaller contribution of the glucose-dependent component of Na+ transport.

We found that isoproterenol stimulates alveolar liquid absorption. The lack of effect of atenolol and the inhibition produced by ICI-118551 together with the stimulation observed with terbutaline suggest that beta 2-receptors are involved in the signal transduction. Changes in liquid absorption rates were more easily evidenced than changes in transepithelial Na+ transport despite the fact that stimulation of liquid absorption obviously resulted from an increase in Na+ transport, considering the effect of inhibitors such as amiloride or benzamil. Because Na+ PA sums active and passive fluxes, it is possible that variations of the large (as discussed in Differences between mice and rats) passive epithelial permeability concealed the changes in active fluxes, even in the absence of significant intergroup differences in mannitol PAs.

Differences between in situ and isolated lungs. Garat et al. (13) reported that the beta 2-adrenergic agonists terbutaline and salmeterol did not stimulate alveolar liquid clearance in mice, in contrast to isoproterenol. The stimulation by isoproterenol was abolished by atenolol, suggesting that beta 1-receptors are involved in this signal transduction as in the guinea pig (25). There was thus an important difference between our observations in isolated lungs, which suggested that alveolar liquid absorption was stimulated by beta 2-agonists in mice, and those of Garat et al. (13). We were puzzled because mice are more closely related to rats than to guinea pigs, which belong to a different order (8, 16). We therefore repeated the experiments of Garat et al. (13) and found that the 60-min absorption of Krebs-Ringer solution or a commercial Ringer lactate solution was slower (3 ml · h-1 · g dry lung wt-1) in our hands than the absorption rate of the Ringer lactate in their report (6 ml · h-1 · g dry lung wt-1). We found no effect of isoproterenol or terbutaline on alveolar liquid absorption. The amount of liquid absorbed from mouse air spaces after 1 h is nearly 0.1 ml. Because the lymphatic or pulmonary circulation in these dead animals does not remove this liquid, it should accumulate in the lung vessels and tissue. Because mouse lungs weigh ~0.13 g, this volume far exceeded the fraction of lung weight (30%) or extravascular fluid volume (50%) above which alveolar flooding occurs during pulmonary edema in vivo (34). It is thus possible that the large amount of liquid absorbed saturated the hydration capacity of the lung interstitial spaces. Recirculation of liquid between air spaces and interstitium might have ensued, hindering the accurate determination of the alveolar liquid absorption rate. We therefore shortened the exchange time to limit the amount of liquid absorbed. Absorption was highly nonlinear, displaying saturation kinetics, as might be expected if the above explanation is correct. In a preliminary report, Fukuda et al. (12) also mentioned that absorption is unsteady under similar conditions. The initial transient shown in Fig. 4B suggests the dissipation of a hydrostatic or, more probably, an osmotic gradient (5). There was a large difference between the osmotic pressure of the Krebs-Ringer or Ringer lactate solution and mouse plasma (~13%), which could account for half of the absorbed volume. Increasing the osmolarity of the alveolar instillate to slightly above that of mouse plasma decreased the volume of alveolar liquid absorbed and resulted in a steady absorption rate during the first 20 min (the absorbed volume being at that time approximately equal to the water gain tolerated by the lungs before alveolar flooding occurs during pulmonary edema). These experimental conditions unveiled the stimulation produced by isoproterenol and terbutaline. It is worth mentioning that stimulation of alveolar liquid absorption by terbutaline in a similar isolated mouse lung model had been reported by others (1) while this manuscript was in submission.

Differences between mice and rats. An interesting difference between mice and rats is the greater passive permeability of the mouse alveolar epithelial barrier. Mannitol PA is 1.4 ml · h-1 · g dry lung wt-1 in rats (30), less than one-fourth that in mice. This difference cannot be explained by a greater density of intercellular pathways in mouse alveolar epithelium because the epithelial cell density per unit lung volume does not vary appreciably between species (7). This larger permeability allows small hydrophilic solutes (such as glucose) to more easily cross the epithelial barrier. Thus whereas there was a quasi-steady glucose concentration in alveolar liquid that initially did not contain glucose in rats (30), the glucose concentration increased under the same conditions in mice. However, the lack of effect of lowering the instillate glucose concentration on the clearance rate is probably not due to this increase in apical glucose concentration because phloridzin, at a rather high concentration, did not affect alveolar liquid absorption or unidirectional Na+ flux. These observations suggest a relatively low Na+-glucose cotransport activity in mouse air spaces.

Because amiloride almost completely inhibited alveolar liquid absorption and thus active Na+ transport, the unidirectional Na+ flux was probably close to the passive Na+ flux in the presence of amiloride. The ratio of passive to total Na+ transport rates can thus be estimated from the Na+ PA obtained in the presence of amiloride and under control conditions. This ratio is 2:3 (Fig. 3A), which means that active Na+ transport is only 50% passive transport (probably leakage from the paracellular pathway) and may explain that the differences in Na+ flux were less impressive than those in liquid absorption.

There is general agreement that the alveolar liquid absorption rate is ~2 ml · h-1 · g dry lung wt-1 in the presence of glucose in isolated rat lungs (2, 4, 10, 26, 30). The rate of liquid absorption per unit dry tissue weight obtained with isolated lungs is therefore twice as fast in mice as in rats. Mouse alveoli are smaller than those of rats (24), but there is probably no difference in the alveolar surface area (35) or in the number of pneumocytes (7) per unit lung volume between species. Differences in distal air space size would influence the transport of gases because of the parallel perfusion arrangement and the high rates of uptake (35). They are unlikely to affect alveolar liquid absorption, which is several orders of magnitude slower than, for example, the maximal oxygen uptake by lungs, which is 4 ml · s-1 · kg body wt-1 in mice (35) or ~14 × 103 ml · h-1 · g dry lung wt-1. Neither does alveolar liquid clearance depend on perfusion (19), and it is unlikely that proportionally more lung tissue, giving a comparatively greater exchange surface, is filled with instillate in mice than in rats. It is thus possible that the larger clearance in mice than in rats is due to higher rates of active Na+ transport, an intrinsic property of air space epithelial cells. A higher rate of Na+ transport may result in faster liquid absorption even in the presence of high passive (paracellular) permeability. It has been shown that a 10-fold increase in mannitol PA does not affect alveolar liquid absorption in rat lungs (32), likely because it does not appreciably alter the high Na+ reflection coefficient (11).

Conclusion. The simple isolated lung preparation described in this report may be used to measure alveolar liquid clearance in mice with acceptable reproducibility and to estimate changes in active transepithelial Na+ transport with, however, less accuracy. The rate of liquid absorption is high and is essentially attributable to transepithelial Na+ transport as in other species. Na+ crosses the apical epithelial cell membrane mainly via Na+ channels and is pumped out at the basolateral membrane by Na+-K+-ATPase. Finally, Na+ transport and liquid absorption is efficiently stimulated by beta 2-adrenergic agonists.


    ACKNOWLEDGEMENTS

We thank Paule Loiseau for technical assistance and Anne Bergeron for help with the reverse transcription-polymerase chain reaction technique.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Saumon, INSERM U82, Faculté Xavier Bichat, BP 416, 75870 Paris Cedex 18, France (E-mail: saumon{at}bichat.inserm.fr).

Received 19 March 1999; accepted in final form 21 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1232-L1238
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