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
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ABSTRACT |
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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 · h1 · 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-
-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
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
2-adrenergic agonists.
glucose transport; pulmonary alveoli; pulmonary edema
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INTRODUCTION |
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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 -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
-adrenergic agonists vary from one species to another.
For example, hamster air space epithelia do not exhibit
Na+-glucose cotransport activity
(15), and
-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
1- but not by
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
2-adrenergic agonists. The
reasons why these observations differ from those of Garat et al. (13)
have been explored.
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MATERIALS AND METHODS |
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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--D-[14C]glucopyranoside
(
-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
(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)/(Ct
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
-[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 + Jw
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.
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RESULTS |
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Isolated lungs. Experiments with a
large (>12
ml · h1 · 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
2-adrenergic antagonist
ICI-118551 (10
4 M;
n = 12 experiments) but not the
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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Mannitol PA was 6.0 ± 0.58 ml · h1 · 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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Glucose transport. -MG permeability
was significantly larger than mannitol permeability
(P < 0.01 by paired
t-test;
n = 9 experiments). The ratio of
-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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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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DISCUSSION |
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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 -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
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
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
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
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 · h1 · 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 · h1 · 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 2-adrenergic agonists.
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ACKNOWLEDGEMENTS |
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We thank Paule Loiseau for technical assistance and Anne Bergeron for help with the reverse transcription-polymerase chain reaction technique.
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FOOTNOTES |
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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.
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