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Department of Physiology and Pharmacology, School of Medicine, University of South Dakota, Vermillion, South Dakota 57069
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Zheng, Lu P., Rui Sheng Du, and Barbara E. Goodman.
Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs. J. Appl.
Physiol. 82(1): 240-247, 1997.
We investigated
effects of acute hyperoxia on solute transport from air space to
vascular space in isolated rat lungs. Air spaces were filled with
Krebs-Ringer bicarbonate solution containing fluorescein
isothiocyanate-labeled dextran (FD-20; mol wt 20,000) and either
22Na+
and [14C]sucrose, or
D-[14C]glucose
and
L-[3H]glucose.
Apparent permeability-surface area products for tracers over time (up
to 120 min) were calculated for isolated perfused lungs from control
rats (room air) and rats exposed to >95%
O2 for 48 or 60 h immediately
postexposure. After O2 exposures,
mean fluxes for
[14C]sucrose and FD-20
were significantly higher than in room-air control lungs. However,
amiloride-sensitive Na+ and active
D-glucose fluxes were unchanged
after hyperoxic exposure. Therefore, it is unlikely that decreases in
net solute transport in this lung-injury model contributed to pulmonary
edema resulting from O2 toxicity.
Increased net solute transport shown to help resolve pulmonary edema
after acute hyperoxic exposure must therefore begin during the recovery
period. In summary, our data show increases in passive solute fluxes
but no changes in active solute fluxes immediately after acute
hyperoxic lung injury.
amiloride; phloridzin; oxygen toxicity; pulmonary edema
INTRODUCTION EXPOSURE TO HIGH LEVELS of
O2 is often the treatment of
choice for individuals with severe pulmonary disease, such as cystic fibrosis and acute respiratory distress syndrome, even
though the levels of O2 may be
toxic to the lungs. In laboratory studies, Robinson et al. (18) allowed
baboons to breathe 100% O2 for 2-4 days to achieve acute O2
toxicity. Massive accumulation of edema fluid resulted in distention of
the lymphatic channels and alveolar septal edema. Hyaline membranes
were formed in the lungs, and a variety of inflammatory cells entered
the alveoli. Exudative and proliferative lesions have been found after
hyperoxia in lungs from both humans and baboons. When animals were
continuously exposed to toxic levels of
O2, progressive cellular damage in
many organ systems resulted until the process was stopped by the death
of the animal (3). In hyperoxia, the rate of production of
O2 free radicals is known to be
higher than the rate of degradation of
O2 free radicals (5).
O2 toxicity via free radicals will lead to inactivation of enzymes, perturbation of membrane function by
lipid peroxidation, damage to the cells and genetic material, and
inflammation of the lung. Physiological changes after hyperoxia include
decreases in vital capacity, diffusing capacity, lung compliance, and
tracheal mucus velocity (12). The major pathological features of
O2 toxicity in the lungs are
pulmonary edema, hyaline membrane formation, injury of type I alveolar
epithelial cells, and proliferation of type II alveolar epithelial
cells.
After animals were exposed to pure
O2, inflammatory cells,
predominantly neutrophils, were observed first in alveolar capillaries and then in the interstitium of the lungs (13, 15). With increasing O2 exposure times, the
physiological and pathological changes became severe, leading to
increased solute permeability of the alveolar epithelium and eventually
to pulmonary edema (13). The amount of pure
O2 exposure that induced this
pulmonary O2 toxicity depended on
the animal species and the exposure time (2). For rats, a 3-day
exposure to normobaric 100% O2 is
lethal.
The permeability of the alveolar epithelium to solutes was
significantly increased in hyperoxic rats (4). Holm et al. (11) found
that exposure of rabbits to 100%
O2 for 64 h resulted in greatly
increased transport of the large molecules cyanocobalamin and
cytochrome c, indicating increased
alveolar epithelial permeability. In rabbits that survived hyperoxic
lung injury for 200 h, the permeability values returned to control
levels, indicating the reversibility of or the recovery from the
permeability pulmonary edema. During exposure to high levels of
O2 in hamster lungs, both
endothelial permeabilities to bovine serum albumin and fluorescein isothiocyanate (FITC)-labeled dextran (FD-150; mol wt 150,000) and
epithelial permeabilities to sucrose and bovine serum albumin increased
significantly (21).
In addition to these known alterations in passive alveolar epithelial
permeability, alterations in active transport may be involved in the
severity of alveolar pulmonary edema that occurs in hyperoxia (1, 10,
14, 16, 17, 19, 22). The purpose of this study was to find out whether
altered amiloride-sensitive (active or transcellular)
Na+ transport or
phloridzin-sensitive (active or transcellular)
Na+-D-glucose
cotransport (or both) may contribute to the alveolar pulmonary edema
found after acute hyperoxic exposure (>95% for 60 h) in rats. Our
hypothesis was that active solute transport might be impaired in lungs
from rats acutely exposed to hyperoxia and thus contribute to the
severity of the alveolar pulmonary edema.
METHODS
3 M) was added to the
perfusate after a 60-min control period after the instillation of the
three tracers
(22Na+,
[14C]sucrose, and
FD-20). For the D-glucose
transport experiments, phloridzin (3 × 103 M) was added with the
instillate (containing trace amounts of D-[14C]glucose,
L-[3H]glucose,
and FD-20) before the beginning of the experiment. Phloridzin
experiments were performed on alternate days with similar experiments
with no drug, using the same three tracers. The tracers to be studied
in each group of experiments were carefully chosen to allow
simultaneous measurement of permeability across the blood-gas barrier
through each of the three major routes [transcellular, paracellular, and transcytosis (or large pores)]. The
Na+ and
D-glucose are transported both
transcellularly and paracellularly. The sucrose and
L-glucose are transported
paracellularly. The FD-20 is transported by vesicular transport or
through the few large pores. The air space concentration of FD-20
dropped ~20% during the experiment in the room-air-exposed lungs and
in the O2-exposed lungs.
Therefore, it is not feasible to accurately determine net water
movement out of the air spaces by measuring changes in the
concentration of air space FD-20, as it is with FD-150 and some other
large molecules. Amiloride was a gift from Merck, Sharp, & Dohme
(Rahway, NJ). Radioisotopes were purchased from New England Nuclear
(Boston, MA). All other chemicals and drugs were purchased from Sigma
Chemical (St. Louis, MO).
Isolation and perfusion of rat lungs.
After appropriate exposure periods, rats were anesthetized
(pentobarbital sodium, 60 mg/kg ip) and injected with heparin sodium (1.0 U/g ip). A tracheostomy was performed, and the rats were ventilated with 95% O2-5%
CO2 for 10 min followed by
instillation of 2 ml of Krebs-Ringer bicarbonate solution (KRB) to
flush the gases into the distal air spaces. The tracheostomy was
closed, and the animal was left for 10 min to absorb the gases from the distal air spaces and thereby degas the lungs. Then the pulmonary artery and left atrium were cannulated and the lungs were carefully removed from the chest cavity. Lungs were cleared of blood by perfusing
KRB solution with a constant-flow pump at 10 ml/min through the
pulmonary artery cannula. The KRB was equilibrated with 95%
O2-5%
CO2 to a measured pH of 7.4 at
37°C. The KRB solution consisted of (in mM) 118.5 NaCl, 1.3 CaCl2, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4,
16.6 NaHCO3, 10.0 sucrose, and
0.01% bovine serum albumin.
Isolated lungs were hung in a temperature-controlled Plexiglas chamber
maintained at 37°C by a proportional thermoregulator. Lungs were
suspended from a force transducer (model FT03; Grass, Quincy, MA) for
continuous monitoring of changes in lung weight. Perfusion (pulmonary
artery) pressure was measured continuously with a pressure transducer
(model P23XL; Gould, Quincy, MA). Transducer input was recorded on a
polygraph (model 79D; Grass). Lungs were evaluated for 10 min before
lavage to be sure that preparation weight and perfusion pressure were
constant. The KRB in the reservoir was heated and bubbled with a 95%
O2-5%
CO2 gas mixture to maintain the
temperature of the lungs at 37°C and the pH at 7.4.
Air spaces were gently lavaged three times with 7 ml KRB instillate
containing the tracers. The lavage procedure flushed slowly ~5 ml KRB
in and out of the lungs with no more than 10 cmH2O pressure and then left 3 ml
(1/4 total lung capacity) in the distal air spaces at the end.
Perfusate samples (3 ml) were collected every 5 min from the short left
atrial cannula. The samples were analyzed for radioactivity in a liquid
scintillation spectrometer (model 6800; Beckman Instruments, Irvine,
CA) and for fluorescence with a fluorometer (Farrand, Mt. Vernon, NY).
Samples of the instillate in the air spaces (50 µl) were similarly
analyzed before and after lavage and at the end of the experiment.
The rat acute hyperoxic exposure model is known to cause considerable
variability in the degree of blood-gas barrier damage. Therefore,
strict criteria are necessary for evaluating individual experiments to
be included in the statistical analysis. For these studies, all
experiments (n = 7) with large lung
weight gains (>0.7 g) were eliminated from the statistical analysis
because the lungs were not in a steady state with regard to lung fluid balance. The effects of blood-gas barrier damage due to human technique
during the surgery is difficult to separate from damage caused by the
hyperoxic exposure. To evaluate this, we calculated the percentage of
instilled passively transported tracer (sucrose or
L-glucose) appearing in the
initial left atrial sample (5 min). In room-air-exposed lungs,
experiments from all four groups (n = 7) with high initial leaks (samples 0.7-3.8% initial alveolar concentration) were not included in the statistical analysis (in the 21 included experiments, the range was 0.15-0.37%). In 48-h O2-exposed lungs, experiments
(n = 4) with high initial leaks (0.7-3.9%) were not included in the statistics (in the 23 included experiments, the range was 0.12-0.50%). In 60-h exposed
lungs, experiments (n = 4) with high
initial leaks (1.0-1.7%) were not included in the statistics (in
the 21 included experiments, the range was 0.17-0.84%).
There were four different studies in this investigation. All four
studies included three different groups of rats (room-air controls, and
rats exposed to O2 for 48 and 60 h). Different instilled test solutions were chosen for the different
studies as appropriate. The first two studies were to evaluate the
effects of hyperoxia on tracer fluxes and amiloride-sensitive fluxes
with and without hyperoxia. In study
1, fluxes of the three air space tracers
(22Na+,
[14C]sucrose, and
FD-20) were measured for 120 min in lungs with no drugs added to see
whether there were changes in tracer fluxes over time and to see
whether hyperoxia had a direct effect on the fluxes of the tracers. In
study 2, amiloride-sensitive fluxes were determined by using trace amounts of
22Na+,
[14C]sucrose, and
FD-20 in the instillate before and after adding amiloride to the
perfusate of the same lungs at 60 min. The last two studies were to
evaluate the effects of hyperoxia on
D- and L-glucose fluxes and
phloridzin-sensitive fluxes with and without hyperoxia. In
study 3, fluxes of the three air space
tracers
(D-[14C]glucose,
L-[3H]glucose,
and FD-20) in the lungs with no drugs were measured for 90 min to see
whether there were changes over time and to see the effects of
hyperoxia on tracer fluxes. In study
4,
D-[14C]glucose,
L-[3H]glucose,
and FD-20 were instilled with phloridzin into the air spaces throughout
the experiment. Thus, phloridzin-sensitive
D-glucose fluxes could be
compared with the control (no drug)
D-glucose fluxes in
study 3.
Appearance of tracers in the perfusate (vascular) samples was used as a
measure of the rate of unidirectional movement of tracers across the
alveolar epithelium. Based on an adaptation of Fick's first law of
diffusion (6, 7, 20), apparent permeability-surface area products
(PS) were calculated. Briefly, Fick's first law can be represented as
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Cc) = change in
tracer concentration between the alveolar space
(CA) and the capillary (Cc)
[disintegrations · min1 · cm3
or FU/cm3]. In this
single-pass perfusate system, Cc = 0; thus
CA Cc = CA. By mass balance
considerations, assuming that the disappearance of the tracer from the
air spaces equals the appearance of the tracer in the vascular space,
JS = Cv
, where is the flow rate
during the sample collection time and Cv is the concentration of tracer
in the vascular perfusate sample. Therefore,
PS can be calculated with the equation
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passive
PSD-glucose. Another
way to estimate the active
D-glucose fluxes across the blood-gas barrier is to measure the phloridzin-sensitive component of
total D-glucose fluxes.
However, control
D-glucose fluxes and phloridzin-sensitive
D-glucose fluxes are,
of necessity, measured in experiments in separate lungs. Similarly, we
can use amiloride-sensitive Na+
transport as an estimate of net transcellular sodium transport. Therefore, active or transcellular
PSNa = measured total
PSNa PSNa
remaining after amiloride (amiloride-insensitive).
The statistics comparing differences in results in the same lungs
(i.e., effects of amiloride) were computed by paired Student's t-test. Comparing results from
different lungs for the three groups of rats (i.e., effects of
hyperoxia or phloridzin) used one-way analysis of variance with
appropriate post hoc analysis. Data are reported throughout as means ± SE, with statistical significance being designated at the
P < 0.05 level.
RESULTS
Before the isolated lung experiments, 6.7% (2 of 30) of the rats exposed to >95% O2 for 60 h died, but none of the rats exposed to high O2 for 48 h died. When the chests of the O2-exposed rats were opened during surgery, fluid could be seen around the lungs, implying leakage of fluid into the interstitium and subsequently into the pleural space. Wet-to-dry weight ratios for 48 h hyperoxic lungs (5.9 ± 0.1) and 60 h hyperoxic lungs (6.0 ± 0.1) were significantly higher than those for room-air control lungs (4.6 ± 0.05), implying interstitial and/or alveolar edema in lungs from O2-exposed rats. Perfusion (pulmonary artery) pressures were measured throughout each experiment (see Table 1). There were no significant changes in perfusion pressure during the evaluation period in any experiment or between the room-air control lungs and the lungs exposed to O2 for 48 or 60 h. In all experiments, the left atrial pressure was zero.
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In study 1, the fluxes of 22Na+, [14C]sucrose, and FD-20 in the three groups of rats (room-air control, 48- or 60-h exposure to O2) with no drug added were measured (Table 2). In these experiments, the effects of hyperoxia can be seen, as both PSsucrose and PSFD-20 were significantly higher in the 48- and 60-h O2-exposed lungs than in the room-air control lungs. The results from study 2 investigating amiloride-sensitive fluxes with and without hyperoxia are reported in Table 3. Note that in the experiments in which amiloride was added to the perfusate at 60 min, Na+ fluxes were significantly lower at 75-90 min than at 40-55 min in all three groups of rats. Sucrose and FD-20 fluxes were significantly lower than control values in the presence of amiloride but in many cases in the 48- or 60-h O2-exposed lungs, PSsucrose and PSFD-20 were still significantly higher than the corresponding room-air control values. Thus the pathways available to sucrose and FD-20 in O2-exposed lungs can still lead to increased transbarrier fluxes of these tracers in the presence of amiloride. Note that the change in weight of the lung preparation during the control time period (40-55 min) was different from that during the experimental time period (75-90 min). These changes imply less weight loss and/or slight weight gain after amiloride. This is further support for the measurement of amiloride-sensitive Na+ fluxes as an indicator of net Na+ and water transport.
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Table 4 includes the fluxes of D-[14C]glucose, L-[3H] glucose, and FD-20 in the three groups of rats (room-air control, 48- or 60-h O2-exposed) in experiments with no drug added (study 3) and in experiments on alternate days with phloridzin added (study 4) with the instillate before the beginning of the experiment. The values reported in Table 4 compare the mean fluxes for each of the tracers at 40-55 min between experiments in study 3 (without phloridzin) and in study 4 (with phloridzin). Note that in the experiments with no drugs, there were no significant changes in tracer fluxes in the 48- and 60-h O2-exposed lungs compared with room-air control values.
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As shown in Table 4, when phloridzin was added with the instillate, all D-glucose fluxes in the presence of phloridzin were significantly lower than the D-glucose fluxes in the alternate experiments with no drug. In addition, the PSD-glucose values in the presence of phloridzin in the room-air control lungs and in the 60-h O2-exposed lungs were not significantly different from the corresponding simultaneously measured PSL-glucose values. Thus phloridzin has little effect on the passive (paracellular) pathways available to D-glucose and L-glucose and FD-20.
Total Na+ fluxes were determined as the values before drugs for the amiloride experiments in study 2 (Table 3), with passive (amiloride-insensitive) Na+ fluxes being the values after drugs for the same experiments. Therefore, active (amiloride-sensitive) Na+ fluxes can be calculated for the room-air control lungs and for the 48- and 60-h O2-exposed lungs (Table 5). These active fluxes are not different from each other. Total D-glucose fluxes were determined as the D-glucose fluxes from study 3 (Table 4) with passive D-glucose fluxes being the corresponding simultaneously measured L-glucose fluxes from the same experiments. Therefore, active D-glucose fluxes can be calculated for the room-air control lungs and for the 48- and 60-h O2-exposed lungs. Obviously, because of the large effect of phloridzin on D-glucose fluxes in the O2-exposed lungs, there was still a large component of active D-glucose transport in O2 toxicity.
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DISCUSSION
O2 toxicity can be caused by a long exposure to high concentrations or high pressures of O2. Hyperoxia induces acute lung injury, inflammation, and alveolar pulmonary edema (18). In hyperoxia, the levels of O2 free radicals are higher than normal (5). These high levels of O2 free radicals lead to damage to the epithelia of the lung, causing an increase in the permeability of the epithelia. Type I cells are damaged first, followed by proliferation of type II cells (12). Because alveolar pulmonary edema is a major problem in hyperoxia, the rate of osmotically driven lung fluid absorption could be compromised in hyperoxia. However, the pulmonary edema caused by hyperoxia may result simply from an increase in the passive permeability or "leak" pathway across the pulmonary capillaries and/or epithelia (2).
Our hypothesis was that net solute reabsorption out of the alveolar air spaces was decreased in rats acutely exposed to hyperoxia for 48 or 60 h due to the direct effects of O2 toxicity on solute transport. Therefore, excess fluid would remain in the air spaces, contributing to the severity of the alveolar pulmonary edema produced by the passive leaks. We have utilized an isolated perfused rat lung model with measurements of net unidirectional fluxes of tracer molecules from the distal air spaces into the vascular space to clarify the pathways available for blood-gas barrier transport in room-air control and O2-exposed rat lungs. In our experiments, increased FD-20 and [14C]sucrose fluxes and increased L-[3H]glucose and phloridzin-insensitive D-[14C]glucose fluxes after exposure to high O2 provide evidence for increased passive epithelial permeability in these hyperoxic rat lungs. However, immediately postexposure, the amount of active (amiloride-sensitive) Na+ transport appeared to be unchanged in the 48- and 60-h O2-exposed lungs compared with the room-air control group. The rate of lung weight change (Table 3) after amiloride is significantly different in the 48- and 60-h O2-exposed lungs than in room-air exposed lungs. Thus it appears from the weight change data that there is more active transport to be inhibited in the O2-exposed lungs. However, because of extreme interanimal variability in the rate of lung weight change even in lungs from room-air-exposed rats, we cannot with confidence give major scientific significance to these differences. In focusing on changes in Na+-D-glucose cotransport after hyperoxic exposure, we found that total D-glucose fluxes and active D-glucose fluxes were unchanged in lungs from both 48- and 60-h O2-exposed rats compared with room-air controls. Thus our hypothesis that decreased net solute fluxes measured in lungs immediately postexposure contribute to the severity of the alveolar pulmonary edema was not verified experimentally in rats acutely exposed to >95% O2 for 48 and 60 h.
Amiloride (103 M) is known
to inhibit numerous Na+
transporters but particularly
Na+-selective channels and
Na+/H+
antiporters, which have both been found in the apical membrane (air
space side) of the type II alveolar epithelial cells. In preliminary
results with the use of similar techniques (8) in room-air control rat
lungs, it has previously been shown that addition of amiloride to the
perfusate yields a typical concentration-response inhibition of the
PSNa
from the air spaces to the vascular space for the range of
105 to 2 × 103 M, with maximal
inhibition of 47% at 2 × 103 M, and an inhibitor
constant of 1 × 104
M. In all of our experiments with perfused amiloride, the inhibitory response of the
PSNa
was seen by at least 15 min after the addition of amiloride to the
perfusate reservoir. Based on this evidence and previous studies (7, 9,
20), we have chosen to compare the effects of perfused amiloride on
unidirectional net Na+ fluxes out
of the air spaces for lungs from room-air control, 48- and 60-h
O2-exposed rats.
Because of the lack of unlabeled
D-glucose in the KRB solution,
the fluxes measured in the
D-glucose experiments are those of only the representative radioactively labeled
D-glucose molecules from the
instillate to the perfusate and may represent a small fraction of the
total solute fluxes across the blood-gas barrier. In other words, the
PS values for
D-glucose can provide relevant information about the transbarrier fluxes of
D-glucose by both passive and
active pathways, but the
D-glucose data may not imply similar changes in the transbarrier fluxes of
Na+ and/or water.
Na+ is known to be taken up into
the cells by numerous different transporters, including both
amiloride-sensitive Na+ channels
and
Na+/H+
exchangers and Na+-dependent
cotransporters (Na+-amino acid,
Na+-HCO3,
and Na+-glucose).
In previous studies (7), it was found that perfused phloridzin had no effect on air space to vascular space D-glucose fluxes in rat lungs. In the experiments reported herein, when phloridzin (the Na+-coupled glucose-transport inhibitor) was added with the instillate in lungs from room-air control rats, total D-glucose fluxes from air space to vascular space were decreased by 76% or more. In the phloridzin experiments, total D-[14C]glucose fluxes were significantly higher in the 48-h O2-exposed lungs than in the room-air control lungs and L-glucose fluxes were significantly higher in both 48- and 60-h O2-exposed lungs. The percentages of the total D-glucose fluxes that were due to active transport based on corresponding L-glucose fluxes ranged from 85 to 88% for the room-air controls and 48- and 60-h O2-exposed lungs in study 3. By comparison, the percentage of phloridzin-sensitive D-glucose transport calculated from the mean PSD-glucose values in the experiments with no drugs compared with those in separate lungs in the phloridzin experiments ranged from 76 to 83%.
The permeability of the alveolar epithelium to solutes is known to be significantly increased in hyperoxic animals (11, 21). From the results of our study, increased mean PS values for [14C]sucrose, L-[3H]glucose, and FD-20 show that the passive permeability of the alveolar epithelium was increased after acute exposure of rats to >95% O2 for 48 and 60 h. This increase in alveolar epithelial permeability agrees with PS values for sucrose, which were 4.7 times higher in O2-exposed hamster lungs than in room-air control lungs (21), and with the greatly increased alveolar membrane permeability to cyanocobalamin and cytochrome c in 64-h O2-exposed rabbit lungs (11). However in our 60-h O2-exposed rat lungs, the mean PS value for sucrose was 2.6 times higher and the mean PS value for L-glucose was 2.1 times higher than in room-air control lungs, implying that the blood-gas barrier still remained relatively tight in these hyperoxic rat lungs.
Table 6 is a summary of results from
several laboratories on the effects of hyperoxia on net
Na+ (or water) clearance from the
air spaces. Nici et al. (14) studied adult male rats exposed to >97%
O2 for 60 h followed by recovery
in room air.
Na+-K+-adenosinetriphosphatases
(ATPases) were observed in the type II alveolar epithelial cell
basolateral membranes of these rats by immunocytochemistry. The levels
of total mRNA of the
Na+-K+-ATPase
1- and 
-subunits were
increased three to four times immediately after exposure, and total
lung
Na+-K+-ATPase
protein increased by 24 h after exposure. After the 60-h O2 exposure, there was no visual
evidence of type II cell proliferation. Increased gene expression was
quickly followed by a rise in antigenic Na+-K+-ATPase
membrane protein, which persisted at least 1 wk into the recovery
period. The data suggested that upregulation of
Na+-K+-ATPases
is an early response to pulmonary edema and/or hyperoxic injury
immediately postexposure in rats exposed to >95%
O2 for 60 h.
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In preliminary experiments, Carter et al. (1) also studied lungs from rats acutely exposed in vivo to >95% O2 for 60 h immediately postexposure and during recovery. They found two populations of animals, those with greater epithelial injury (as indicated by an increase in PSsucrose) and those with lesser epithelial injury. As the degree of epithelial injury increased, so did the amiloride- or benzamil-sensitive Na+ transport. In addition, increases in type II alveolar epithelial cell levels of Na+-K+-ATPase mRNA and protein were found after in vitro hyperoxic exposures (1). These data suggested a direct effect of hyperoxia on increasing net Na+ clearance from the air spaces without hormonal or cell-to-cell interactions.
Olivera et al. (17) studied lung liquid clearance and epithelial permeability in isolated rat lungs and Na+-K+-ATPase activity in type II alveolar epithelial cells during acute hyperoxic exposure (100% for 64 h) and at 0, 7, and 14 days of recovery in room air, respectively. Albumin flux into the air spaces was increased immediately postexposure and returned to control values after 7 days of recovery. Na+ and mannitol fluxes recovered to normal only after 14 days recovery. Active Na+ transport and lung liquid clearance were reduced immediately postexposure, increased above control after 7 days recovery, and returned to control after 14 days recovery. Changes in Na+-K+-ATPase paralleled these changes with a decrease in type II cells isolated from rats immediately postexposure and an increase over control in rats recovering for 7 days. These results suggest that Na+-K+-ATPases may be directly involved in the recovery from O2 toxicity and the resolution of alveolar pulmonary edema in this acute lung injury model.
Olivera et al. (16) studied lungs from rats exposed to 85%
O2 for 7 days immediately
postexposure and at 7, 14, and 30 days recovery, respectively. The
model has been called the subacute model for exposure to
O2 at sublethal levels. The
subacute model is known to differ from the acute model that was used in
this laboratory and others (1, 14, 17). Rats exposed to 85% O2 for 5-7 days can tolerate
100% O2 for long periods, whereas rats initially acutely exposed to >95%
O2 can survive for only 60-72
h before death (4). One of the reasons for the differences between the
two models appears to be that only during the 85% O2 exposures do type II alveolar
epithelial cells undergo both hypertrophy and proliferation. Resultant
changes in the rate of production and secretion of surfactant have been
implicated as one of the components of the adaptation to or tolerance
of hyperoxia in the 85%
O2-exposure model. Using the
subacute hyperoxia model, Olivera et al. (16) found that albumin fluxes
in isolated lungs were elevated but returned to normal after a 7-day
recovery. Na+ and mannitol fluxes
were also elevated but required 30 days to return to normal. Active
Na+ transport increased
immediately postexposure and returned to normal after 7 days. In
addition,
Na+-K+-ATPase
activity and protein expression was increased in type II cells after
O2 exposure, and the
1-subunit of
Na+-K+-ATPase
mRNA in type II cells was also increased. In their experiments, there
was an increase of 102% in type II cells. Even so, immunocytochemical analysis suggested increases in
Na+-K+-ATPase
per type II cell. Thus, in the subacute
O2-exposure model, there was
increased active Na+ transport
immediately postexposure associated with both upregulation of
Na+-K+-ATPase
and increased ATPase activity.
Sznajder et al. (19) studied amiloride-sensitive Na+ fluxes and ouabain-sensitive active Na+ transport in isolated perfused lungs from subacutely exposed rats (85% O2 for 7 days). They found increased albumin flux and permeability to small solutes in hyperoxic rat lungs compared with controls. Amiloride inhibited Na+ flux by a greater percentage in rats exposed to hyperoxia than in control rat lungs. Ouabain decreased active Na+ transport by a greater percentage in O2-exposed rats than in control rats. These results suggest that upregulation of both amiloride-sensitive Na+ channels and Na+-K+-ATPases contribute to effective alveolar edema clearance in subacute hyperoxic injury.
Haskell et al. (10) also studied rats exposed to 85% O2 for 7 days. They found higher levels of Na+ channel protein in type II alveolar epithelial cells from O2-exposed rats. Both inward and outward Na+ currents were increased in patch clamps of the cells. Outward currents were equally sensitive to amiloride and ethyl-isopropyl amiloride, implying the presence of L-type or low affinity amiloride-sensitive epithelial Na+ channels. Yue et al. (22) reported increased expression and activity of Na+ channels in type II alveolar epithelial cells of rats exposed to 85% O2 for 7 days followed by 100% O2 for 4 days. Both the number and the open probability of the L-type Na+ channels were increased significantly during exposure to hyperoxia. These studies also implied an increase in transepithelial Na+ transport as a result of exposure to hyperoxia; however, in this case a Na+ entry pathway appeared to be upregulated instead of the Na+ exit pathway. Physiologically, it is known that increases in intracellular Na+ concentration will lead to increases in Na+-K+-ATPase activity.
In summary, passive transport measured by changes in the transbarrier fluxes of sucrose, L-glucose, or FD-20 was significantly increased by exposure to hyperoxia. The present data show that amiloride-sensitive Na+ transport and active D-glucose transport may be neither increased nor decreased immediately postexposure in lungs from rats acutely exposed to >95% O2 for 48 or 60 h. Thus, these data suggest that impairment of active solute transport does not contribute to the lung fluid imbalance immediately postexposure in this acute hyperoxic lung injury model. It is possible that the integrity of the blood-gas barrier has not recovered sufficiently to allow active solute reabsorption to overcome the permeability pulmonary edema. However, addition of amiloride to leaky hyperoxic lungs immediately postexposure does still cause a lung weight loss to change significantly to a lung weight gain. Thus, there is still a significant component of the net Na+ flux that is amiloride-sensitive (transcellular) in the hyperoxic-exposed lungs. It is highly likely that during recovery from hyperoxic pulmonary edema, net solute and water clearance out of the air spaces increase. In this model of acute lung injury, the upregulation of transporters may not yet have led to increased net solute fluxes across the blood-gas barrier immediately postexposure. However, this model should be useful for providing information about transport changes during recovery from hyperoxic injury and/or investigating the signal transduction regulatory mechanisms that may be involved in recovery from hyperoxic alveolar pulmonary edema.
ACKNOWLEDGEMENTS
The authors thank Drs. Douglas Wangensteen, David Ingbar, and Ethan Carter for many helpful discussions and Dr. Peter Reynen, who while a medical student, designed and built the exposure chamber. This study was part of a degree of M.A. in physiology for Lu P. Zheng.
FOOTNOTES
Address for reprint requests: B. E. Goodman, Dept. of Physiology and Pharmacology, School of Medicine, Univ. of South Dakota, Vermillion, SD 57069.
Received 19 January 1995; accepted in final form 28 August 1996.
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Wt, mg/min 
Amiloride-sensitive
22Na
Water clearance