Effects of acute hyperoxic exposure on solute fluxes across

the blood-gas barrier in rat lungs

Lu P. Zheng, Rui Sheng Du and Barbara E. Goodman
J Appl Physiol 82:240-247, 1997. ;

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Effects of acute hyperoxic exposure on solute
fluxes across the blood-gas barrier in rat lungs
LU P. ZHENG, RUI SHENG DU, AND BARBARA E. GOODMAN
Department of Physiology and Pharmacology, School of Medicine,
University of South Dakota, Vermillion, South Dakota 57069
Zheng, Lu P., Rui Sheng Du, and Barbara E. Good-
man. Effects of acute hyperoxic exposure on solute fluxes
across the blood-gas barrier in rat lungs. J. Appl. Physiol.
82(1): 240247, 1997.We investigated effects of acute hyper-
oxia 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 22Na1 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
Na1 and active D-glucose fluxes were unchanged after hyper-
oxic 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
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 24 days to
achieve acute O2 toxicity. Massive accumulation of
edema fluid resulted in distention of the lymphatic
channels and alveolar septal edema. Hyaline mem-
branes 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, progres-
sive 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, perturba-
tion of membrane function by lipid peroxidation, dam-
age 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,
240 0161-7567/97 $5.00 Copyright r 1997 the American Physiological Society
injury of type I alveolar epithelial cells, and prolifera-
tion 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,
Downloaded from http://jap.physiology.org/ at Penn State Univ on February 23, 2013
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 sol-
utes 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 fluores-
cein isothiocyanate (FITC)-labeled dextran (FD-150;
mol wt 150,000) and epithelial permeabilities to su-
crose 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 transcel-
lular) Na1 transport or phloridzin-sensitive (active or
transcellular) Na1-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 hyper-
oxia and thus contribute to the severity of the alveolar
pulmonary edema.
METHODS
Animals and exposures. In the present investigation, iso-
lated perfused lungs from rats were used to study the effects
of acute exposure to hyperoxia on net solute transport. Adult
male Sprague-Dawley rats (200300 g, Sasco, Omaha, NE)
were used. There were three groups of rats for each of the four
studies. Control rats were exposed to room air pumped
through the gassing chamber. The two groups of hyperoxic
rats were exposed to .95% O2 for 48 or 60 h, respectively. O2
 SOLUTE TRANSPORT IN HYPEROXIA
exposure was performed in a custom-made sealed polystyrene
chamber in which animals were housed in individual cages
and given food and water ad libitum. The O2 and CO2
concentrations in the chambers were monitored periodically
(at least three times per day) with an O2 analyzer (model
OM-14, Beckman, Fullerton, CA) and a CO2 analyzer (model
LB-2, Beckman). The O2 concentration in the chamber was
,95%, and the CO2 concentration in the chamber was ,1%.
The chamber gases were recycled through water-absorbent
and CO2-absorbent granules (Chemetron Medical Division,
St. Louis, MO) by an air pump to maintain low CO2 concentra-
tions and normal humidity. Humidity was measured periodi-
cally with a humidity meter in the O2 chamber. O2 was
supplied at a rate of 2.504.00 l/min (depending on the
number of rats in the chamber) throughout the exposure
period.
Materials. The tracers used to investigate blood-gas barrier
function were FD-20 (mol wt 20,000; 0.013 g/ml) with 22NaCl
(0.14 Ci/ml) and [14C]sucrose (0.7 Ci/ml) or L-[3H]glucose (2
Ci/ml) and D-[14C]glucose (0.35 Ci/ml). For the Na1 trans-
port experiments, amiloride (1023 M) was added to the
perfusate after a 60-min control period after the instillation of
the three tracers (22Na1, [14C]sucrose, and FD-20). For the
D-glucose transport experiments, phloridzin (3 3 1023 M) was
added with the instillate (containing trace amounts of
D-[14C]glucose, L-[3H]glucose, and FD-20) before the begin-
ning of the experiment. Phloridzin experiments were per-
formed 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 [transcellu-
lar, paracellular, and transcytosis (or large pores)]. The Na1
and D-glucose are transported both transcellularly and para-
cellularly. The sucrose and L-glucose are transported paracel-
lularly. 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 En-
gland Nuclear (Boston, MA). All other chemicals and drugs
were purchased from Sigma Chemical (St. Louis, MO).
Isolation and perfusion of rat lungs. After appropriate expo-
sure 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 37C. 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 37C by a proportional
thermoregulator. Lungs were suspended from a force trans-
241
ducer (model FT03; Grass, Quincy, MA) for continuous moni-
toring 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 37C 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 (14 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
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scintillation spectrometer (model 6800; Beckman Instru-
ments, Irvine, CA) and for fluorescence with a fluorometer
(Farrand, Mt. Vernon, NY). Samples of the instillate in the air
spaces (50 l) 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 evaluat-
ing individual experiments to be included in the statistical
analysis. For these studies, all experiments (n 5 7) with large
lung weight gains (.0.7 g) were eliminated from the statisti-
cal 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 5 7) with
high initial leaks (samples 0.73.8% initial alveolar concentra-
tion) were not included in the statistical analysis (in the 21
included experiments, the range was 0.150.37%). In 48-h
O2-exposed lungs, experiments (n 5 4) with high initial leaks
(0.73.9%) were not included in the statistics (in the 23
included experiments, the range was 0.120.50%). In 60-h
exposed lungs, experiments (n 5 4) with high initial leaks
(1.01.7%) were not included in the statistics (in the 21
included experiments, the range was 0.170.84%).
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 (22Na1, [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 22Na1, [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 in-
242 SOLUTE TRANSPORT IN HYPEROXIA

stilled with phloridzin into the air spaces throughout the Table 1. Perfusion pressures measured
experiment. Thus, phloridzin-sensitive D-glucose fluxes could in these experiments
be compared with the control (no drug) D-glucose fluxes in
study 3. Room Air 48-h O2 60-h O2
Appearance of tracers in the perfusate (vascular) samples Study n Control Exposure Exposure
was used as a measure of the rate of unidirectional movement Study 1, no drugs 20 6.07 6 0.6 6.07 6 0.9 6.72 6 1.0
of tracers across the alveolar epithelium. Based on an adapta- Study 2, with amiloride 19 3.94 6 0.5 4.06 6 0.8 5.96 6 1.0
tion of Ficks first law of diffusion (6, 7, 20), apparent Study 3, no drugs 12 6.60 6 0.5 6.31 6 1.9 5.67 6 0.6
permeability-surface area products (PS) were calculated. Study 4, with phloridzin 13 5.50 6 1.1 4.95 6 1.0 6.33 6 1.5
Briefly, Ficks first law can be represented as
Values are means 6 SE in Torr. n, No. of animals. There are no
significant differences in any perfusion pressures.
JS 5 PS (CA 2 Cc)

JS is the tracer flux measured in disintegrations per minute

per second or arbitrary fluorescence units (FU)/s. PS 5 zin) used one-way analysis of variance with appropriate post
apparent permeability (active plus passive fluxes) times hoc analysis. Data are reported throughout as means 6 SE,
transfer surface area (cm3/s), and (CA 2 Cc) 5 change in with statistical significance being designated at the P , 0.05

Downloaded from http://jap.physiology.org/ at Penn State Univ on February 23, 2013

tracer concentration between the alveolar space (CA ) and the
capillary (Cc) [disintegrations min21 cm23 or FU/cm3]. In
this single-pass perfusate system, Cc 5 0; thus CA 2 Cc 5 CA.
By mass balance considerations, assuming that the disappear-
ance of the tracer from the air spaces equals the appearance
of the tracer in the vascular space, JS 5 CvQ, where Q 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
Cv(Q)
PS 5
CA
CA might change during the experiment because of either
reabsorption of water from the air spaces or movement of
solute from the alveoli to the perfusate; therefore, CA was
corrected throughout the experiment (6, 7, 20). The use of
Ficks law to calculate PS values assumes that steady-state
fluxes occur. Therefore, the times to analyze the changes in
PS values were chosen to coincide with relatively constant
fluxes of the passively transported tracers [14C]sucrose or
L-[3H]glucose. Thus we chose the control PS values for 22Na1,
[14C]sucrose, and FD-20 to be the average of the steady-state
PS values from 40 to 55 min after lavage, whereas the
experimental PS values after the addition of amiloride to the
perfusate at 60 min were the steady-state PS values from 75
to 90 min. In the experiments in which phloridzin was
present in the instillate throughout the experiment, mean
control fluxes for D-glucose, L-glucose, and FD-20 from study 3
were compared with mean fluxes from study 4 from 40 to 55
min after lavage.
D-Glucose and L-glucose are stereoisomers of each other.
Therefore, we assumed that the PS value for the biologically
inert L-glucose could be used to estimate the passive flux of
D-glucose across the alveolar capillary membrane or passive
PSD-glucose 5 PSL-glucose. Therefore, active PSD-glucose 5 mea-
sured total PSD-glucose 2 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, mea-
sured in experiments in separate lungs. Similarly, we can use
amiloride-sensitive Na1 transport as an estimate of net
transcellular sodium transport. Therefore, active or transcel-
lular PSNa 5 measured total PSNa 2 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
Students t-test. Comparing results from different lungs for
the three groups of rats (i.e., effects of hyperoxia or phlorid-
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 6 0.1) and 60 h hyperoxic lungs
(6.0 6 0.1) were significantly higher than those for
room-air control lungs (4.6 6 0.05), implying intersti-
tial and/or alveolar edema in lungs from O2-exposed
rats. Perfusion (pulmonary artery) pressures were mea-
sured throughout each experiment (see Table 1). There
were no significant changes in perfusion pressure dur-
ing 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.
In study 1, the fluxes of 22Na1, [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, Na1 fluxes were
significantly lower at 7590 min than at 4055 min in
all three groups of rats. Sucrose and FD-20 fluxes were
Table 2. Study 1 (no drugs) with Na1, sucrose,
and FD-20: effects of hyperoxia on tracer fluxes
Condition n PSNa PSsucrose PSFD-20
Room air 7 25.40 6 1.1 2.32 6 0.2 0.22 6 0.04
48-h early 7 33.44 6 1.2* 4.29 6 0.3* 0.47 6 0.10*
60-h early 6 31.07 6 3.5 5.97 6 1.1* 0.95 6 0.32*
Values are means 6 SE in 1025 ml/s; n, no. of animals. PS,
permeability-surface area product; FD-20, fluorescein isothiocyanate-
labeled Dextran 20. * Significantly different from lungs from room
air-control animals by analysis of variance (ANOVA), P , 0.05.
 SOLUTE TRANSPORT IN HYPEROXIA 243

Table 3. Study 2 (amiloride) with Na1, sucrose, 60-h O2-exposed lungs compared with room-air control
and FD-20: amiloride-sensitive fluxes values.
with and without hyperoxia As shown in Table 4, when phloridzin was added with
the instillate, all D-glucose fluxes in the presence of
Condition n PSNa PSsucrose PSFD-20 DWt, mg/min phloridzin were significantly lower than the D-glucose
Room air fluxes in the alternate experiments with no drug. In
Before 5 26.94 6 4.0 3.72 6 0.5 0.59 6 0.16 23.50 6 1.7 addition, the PSD-glucose values in the presence of phlorid-
After 15.88 6 3.3* 2.87 6 0.4* 0.39 6 0.11* 20.40 6 1.6 zin in the room-air control lungs and in the 60-h
48-h O2
Before 6 32.72 6 3.2 7.05 6 1.3 1.26 6 0.41 23.50 6 1.5
O2-exposed lungs were not significantly different from
After 22.10 6 2.6* 6.24 6 1.1* 0.98 6 0.34* 8.47 6 2.7* the corresponding simultaneously measured PSL-glucose
60-h O2 values. Thus phloridzin has little effect on the passive
Before 8 29.53 6 2.3 8.84 6 1.5 1.76 6 0.36 22.06 6 1.7 (paracellular) pathways available to D-glucose and
After 18.07 6 1.5* 7.47 6 1.1* 1.18 6 0.21* 6.26 6 1.7* L-glucose and FD-20.
Values are means 6 SE in 1025 ml/s; n, no. of animals; Dwt, change Total Na1 fluxes were determined as the values
in weight. * Significantly different from same lung by paired t-test; before drugs for the amiloride experiments in study 2
significantly different from lungs from room-air control animals by

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ANOVA, P , 0.05.
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 signifi-
cantly 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 (4055 min)
was different from that during the experimental time
period (7590 min). These changes imply less weight
loss and/or slight weight gain after amiloride. This is
further support for the measurement of amiloride-
sensitive Na1 fluxes as an indicator of net Na1 and
water transport.
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 4055 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
Table 4. Studies 3 and 4 with D-glucose, L-glucose,
and FD-20: effects of hyperoxia on tracer fluxes
and phloridzin-sensitive tracer fluxes with
and without hyperoxia
Condition n PSD-glucose PSL-glucose
Study 3, no drugs
Room air 5 28.66 6 5.6 3.17 6 0.7
O2 , 48-h 5 41.64 6 7.0 5.86 6 1.0
O2 , 60-h 3 44.37 6 3.5 6.68 6 1.5*
Study 4, phloridzin
Room air 4 4.79 6 1.0 4.02 6 0.7
O2 , 48-h 6 9.10 6 1.1* 7.45 6 1.0*
O2 , 60-h 4 8.58 6 1.1* 8.64 6 1.5*
Values are means 6 SE in 1025 ml/s; n, no. of animals. * Signifi-
cantly different from lungs from room-air control animals, by ANOVA;
significantly different from lungs with no drug, by ANOVA, P , 0.05.
(Table 3), with passive (amiloride-insensitive) Na1
fluxes being the values after drugs for the same experi-
ments. Therefore, active (amiloride-sensitive) Na1
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 mea-
sured 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.
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
Table 5. Active and passive fluxes for Na1
and D-glucose
Group n Total Flux Passive Flux Active Flux %Active
PSNa
Room-air exposed 5 26.94 6 4.0 15.88 6 3.3 11.06 6 0.8 42.8
PSFD-20 O2 , 48 h 6 32.72 6 3.2 22.10 6 2.6* 10.62 6 0.9 33.0*
O2 , 60 h 8 29.53 6 2.3 18.07 6 1.5* 11.46 6 0.9 38.9
PSD-glucose
0.67 6 0.29 Room-air exposed 5 28.66 6 5.6 3.17 6 0.7 25.49 6 5.6 87.9
0.97 6 0.15 O2 , 48 h 5 41.64 6 7.0 5.86 6 1.0 35.78 6 6.8 85.9
0.93 6 0.26 O2 , 60 h 3 44.37 6 3.5 6.68 6 1.5* 37.69 6 2.6 85.1
0.59 6 0.08 Values are means 6 SE in 1025 ml/s; n, no. of animals. Data are
1.46 6 0.38 given for room air-exposed and for 48-h or 60-h O2-exposed animals.
1.44 6 0.40 For Na1, passive flux is amiloride-insensitive Na1 flux and active flux
is total Na1 flux 2 amiloride-insensitive Na1 flux from study 2. For
D-glucose, passive flux is L-glucose flux and active flux is total
D-glucose flux 2 L-glucose flux from study 3. * Significantly different
from lungs from room air-control animals, by ANOVA, P , 0.05.
244 SOLUTE TRANSPORT IN HYPEROXIA
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, contribut-
ing 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-sensi-
tive) Na1 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 ex-
posed 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 signifi-
cance to these differences. In focusing on changes in
Na1-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 mea-
sured 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 (1023 M) is known to inhibit numerous
Na1 transporters but particularly Na1-selective chan-
nels and Na1/H1 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 1025 to
2 3 1023 M, with maximal inhibition of 47% at 2 3 1023
M, and an inhibitor constant of 1 3 1024 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 Na1 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 experi-
ments are those of only the representative radioac-
tively 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 Na1 and/or water. Na1 is known to be taken up
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into the cells by numerous different transporters, includ-
ing both amiloride-sensitive Na1 channels and Na1/H1
exchangers and Na1-dependent cotransporters (Na1-
amino acid, Na1-HCO2 1
3 , 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 re-
ported herein, when phloridzin (the Na1-coupled glu-
cose-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 trans-
port 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 sol-
utes is known to be significantly increased in hyperoxic
animals (11, 21). From the results of our study, in-
creased 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 perme-
ability 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 re-
mained relatively tight in these hyperoxic rat lungs.
Table 6 is a summary of results from several laborato-
ries on the effects of hyperoxia on net Na1 (or water)
clearance from the air spaces. Nici et al. (14) studied
 SOLUTE TRANSPORT IN HYPEROXIA
Table 6. Comparison of effects of hyperoxia on active transport in rat lungs
Source Lung Model Hyperoxic Exposure
Carter et al., 1994 Isolated lungs .95% for 60 h
Carter et al., 1994 Type II cells .95% for 48 h
Haskell et al., 1994 Type II cells 85% for 7 days
Nici et al., 1991 Type II cells .97% for 60 h
Olivera et al., 1994 Isolated lungs 85% for 7 days
Olivera et al., 1994 Type II cells 85% for 7 days
Olivera et al., 1995 Isolated lungs 100% for 64 h
Olivera et al., 1995 Type II cells 100% for 64 h
Sznajder et al., 1995 Isolated lungs 85% for 7 days
Yue et al., 1995 Type II cells 85% for 7 days 1
100% for 4 days
Present study Isolated lungs .95% for 60 h
* Preliminary experiments with 2 populations of animals, some with greater epithelial injury and some with lesser epithelial injury.
adult male rats exposed to .97% O2 for 60 h followed by
recovery in room air. Na1-K1-adenosinetriphospha-
tases (ATPases) were observed in the type II alveolar
epithelial cell basolateral membranes of these rats by
immunocytochemistry. The levels of total mRNA of the
Na1-K1-ATPase a1- and b-subunits were increased
three to four times immediately after exposure, and
total lung Na1-K1-ATPase protein increased by 24 h
after exposure. After the 60-h O2 exposure, there was
no visual evidence of type II cell proliferation. In-
creased gene expression was quickly followed by a rise
in antigenic Na1-K1-ATPase membrane protein, which
persisted at least 1 wk into the recovery period. The
data suggested that upregulation of Na1-K1-ATPases
is an early response to pulmonary edema and/or hyper-
oxic injury immediately postexposure in rats exposed to
.95% O2 for 60 h.
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 Na1 transport.
In addition, increases in type II alveolar epithelial cell
levels of Na1-K1-ATPase mRNA and protein were
found after in vitro hyperoxic exposures (1). These data
suggested a direct effect of hyperoxia on increasing net
Na1 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 Na1-K1-
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 immedi-
ately postexposure and returned to control values after
7 days of recovery. Na1 and mannitol fluxes recovered
to normal only after 14 days recovery. Active Na1
transport and lung liquid clearance were reduced imme-
diately postexposure, increased above control after 7
days recovery, and returned to control after 14 days
245
Solute or
Water Clearance Na1 Channels Na1-K1- ATPases
> Amiloride-sensitive 22Na*
> a1-Protein*
> Currents in Na1 patch Upregulation
clamps
> a1 -, b-mRNA
> Active 22Na
> Activity; > a1 -, b1-protein
< Water clearance
< Activity
> Water clearance > %Amiloride-sensitive > %Ouabain-sensitive
> No. and open probability Upregulation
of Na1 channels
No change in 22Na or
D-[14C]glucose
Downloaded from http://jap.physiology.org/ at Penn State Univ on February 23, 2013
recovery. Changes in Na1-K1-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 Na1-K1-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 57
days can tolerate 100% O2 for long periods, whereas
rats initially acutely exposed to .95% O2 can survive
for only 6072 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 produc-
tion 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 ele-
vated but returned to normal after a 7-day recovery.
Na1 and mannitol fluxes were also elevated but re-
quired 30 days to return to normal. Active Na1 trans-
port increased immediately postexposure and returned
to normal after 7 days. In addition, Na1-K1-ATPase
activity and protein expression was increased in type II
cells after O2 exposure, and the a1-subunit of Na1-K1-
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 Na1-K1-ATPase per type II cell.
Thus, in the subacute O2-exposure model, there was
increased active Na1 transport immediately postexpo-
sure associated with both upregulation of Na1-K1-
ATPase and increased ATPase activity.
246 SOLUTE TRANSPORT IN HYPEROXIA
Sznajder et al. (19) studied amiloride-sensitive Na1
fluxes and ouabain-sensitive active Na1 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 Na1
flux by a greater percentage in rats exposed to hyper-
oxia than in control rat lungs. Ouabain decreased
active Na1 transport by a greater percentage in O2-
exposed rats than in control rats. These results suggest
that upregulation of both amiloride-sensitive Na1 chan-
nels and Na1-K1-ATPases contribute to effective alveo-
lar 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 Na1 channel
protein in type II alveolar epithelial cells from O2-
exposed rats. Both inward and outward Na1 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 Na1 chan-
nels. Yue et al. (22) reported increased expression and
activity of Na1 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 Na1 channels were increased
significantly during exposure to hyperoxia. These stud-
ies also implied an increase in transepithelial Na1
transport as a result of exposure to hyperoxia; however,
in this case a Na1 entry pathway appeared to be
upregulated instead of the Na1 exit pathway. Physiologi-
cally, it is known that increases in intracellular Na1
concentration will lead to increases in Na1-K1-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 hyper-
oxia. The present data show that amiloride-sensitive
Na1 transport and active D-glucose transport may be
neither increased nor decreased immediately postexpo-
sure 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 over-
come the permeability pulmonary edema. However,
addition of amiloride to leaky hyperoxic lungs immedi-
ately 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 Na1 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 hyper-
oxic injury and/or investigating the signal transduction
regulatory mechanisms that may be involved in recov-
ery from hyperoxic alveolar pulmonary edema.
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.
This study was supported in part by National Heart, Lung, and
Blood Institute Grant HL-38310.
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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