Departments of 1 Physiology and Biophysics, 2 Comparative Medicine, and 3 Anesthesiology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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The extent to which
endogenously generated nitric oxide alters Na+ transport
across the mammalian alveolar epithelium in vivo has not been
documented. Herein we measured alveolar fluid clearance and nasal
potential differences in mice lacking the inducible form of nitric
oxide synthase [iNOS; iNOS(/
)] and their corresponding wild-type
controls [iNOS(+/+)]. Alveolar fluid clearance values in iNOS(+/+)
and iNOS(
/
) anesthetized mice with normal oxygenation and acid-base
balance were ~30% of instilled fluid/30 min. In both groups of mice,
fluid absorption was dependent on vectorial Na+ movement.
Amiloride (1.5 mM) decreased alveolar fluid clearance in iNOS(+/+) mice
by 61%, whereas forskolin (50 µM) increased alveolar fluid clearance
by 55% by stimulating amiloride-insensitive pathways. Neither agent
altered alveolar fluid clearance in iNOS(
/
) mice. Hyperoxia
upregulated iNOS expression in iNOS(+/+) mice and decreased their
amiloride-sensitive component of alveolar fluid clearance but had no
effect on the corresponding values in iNOS(
/
) mice. Nasal potential
difference measurements were consistent with alveolar fluid clearance
in that both groups of mice had similar baseline values, which were
amiloride sensitive in the iNOS(+/+) but not in the iNOS(
/
) mice.
These data suggest that nitric oxide produced by iNOS under basal
conditions plays an important role in regulating amiloride-sensitive
Na+ channels in alveolar and airway epithelia.
nitric oxide; osmolality; hyperoxia; nasal potential difference; inducible nitric oxide synthase-deficient mice
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INTRODUCTION |
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IT IS WELL ESTABLISHED that alveolar epithelial cells are capable of transporting Na+ in a vectorial fashion both in vivo and in vitro (for reviews see Refs. 33 and 34). Na ions in the alveolar lining fluid passively diffuse down their electrochemical gradient into alveolar type II (ATII) cells through amiloride-sensitive channels located in their apical membranes (22, 50) and are extruded across the basolateral membranes by various subunits of the ouabain-sensitive Na+-K+-ATPase (8). K ions, which enter the ATII cell in exchange for Na+, exit the cell down their electrochemical gradient, through K+ channels located in the basolateral membrane. It is the active Na+ transport across the alveolar epithelium that creates an osmotic gradient that then drives fluid from the alveolar to the interstitial space. Various studies in humans and animals indicate that active Na+ reabsorption plays an important role in diminishing alveolar fluid and improving oxygenation in both patients with cardiogenic and noncardiogenic edema (35) and in animals with hyperoxic lung injury (38).
Because of their location, alveolar epithelial cells are often exposed
to increased intracellular and extracellular concentrations of reactive
oxygen and nitrogen species. Most mammalian cells have the capacity to
produce nitric oxide (NO) via the oxidative deamination of
L-arginine by either the Ca2+-sensitive (c) or
the Ca2+-insensitive (i) isoform of nitric oxide synthase
(NOS). Pertinent sources of pulmonary NO include activated macrophages
(20) and airway (41) and ATII cells
(40). Consequently, in many pathological conditions, there
may be significant levels of NO present in the alveolar lining fluid
and thus in close proximity to alveolar epithelial cells. The
biological effects of NO depend on its concentration, the biochemical
composition of the target, and the presence of other radicals. NO may
bind to the heme group of soluble guanylate cyclase, resulting in
increased cellular cGMP levels (19); it may react with
superoxide at diffusion-limited rates (6.7 × 109
M1 · s
1) to produce peroxynitrite
(1) and higher oxides of nitrogen, or, in the presence of
an electron acceptor, it may react with thiols to form nitrosothiols
(15). Existing evidence in the literature and our own data
indicate that various redox states of NO modify the activity of both
cation channel proteins and the Na+-K+-ATPase
either by altering signal transduction pathways, by oxidizing or
nitrating ion-transporting proteins directly, or by affecting the
cytoskeleton (5, 14, 17, 21, 26).
However, the extent to which endogenously generated NO alters Na+ transport across the mammalian alveolar epithelium has not been elucidated. This is an important question since, as mentioned above, a number of inflammatory agents are known to upregulate NO production by both inflammatory and alveolar epithelial cells in pathological situations. Furthermore, NO generation by either cNOS or iNOS under baseline conditions may regulate the activity of amiloride-sensitive ion channels in lung epithelial cells. For example, it has been proposed that the lack of iNOS in the airways of mice with cystic fibrosis (25) may contribute to the lack of cAMP-stimulated chloride secretion and the increase in the rate of amiloride-sensitive Na+ transport (7).
Herein we measured levels of amiloride-sensitive alveolar fluid
clearance (AFC) in the lungs of C57BL/6 iNOS-deficient [iNOS(/
)] mice, which have normal phenotype except for decreased ability to clear
pathogens (16), and age- and genetic-matched C57BL/6 wild-type control [iNOS(+/+)] mice by instilling into their lungs isosmotic fluid containing 5% BSA and measuring changes in BSA concentration over a 30-min period. We then repeated these measurements in mice that were exposed to 100% O2 at one atmosphere for
55 h, which upregulated iNOS levels in the inflammatory cells in the lungs of iNOS(+/+) mice. The effects of hyperoxia on the lungs of
these mice were measured by quantitative histopathology. We also
measured potential difference across the nasal airways of iNOS(+/+) and
iNOS(
/
) mice in the presence and absence of amiloride. Because our
data indicate the absence of amiloride-sensitive Na+
transport across both the alveolar epithelium and airways of iNOS(
/
) mice, we measured fluid clearance with a
Na+-free solution to determine if AFC was the result of
active Na+ transport, and we tried to stimulate
amiloride-sensitive fluid clearance by adding forskolin to the
instillate. In contrast to previous studies in which AFC was measured
in situ (12), in isolated perfused lungs
(18), or in ventilated mice with unknown acid-base status
(11), all of our measurements were performed in living,
ventilated, anesthetized mice with adequate oxygenation and acid-base
balance. Results presented here indicate that iNOS(
/
) mice have
normal levels of basal AFC, dependent on vectorial transport of
Na+ moving across alveolar epithelial cells through
amiloride-insensitive pathways that are not stimulated by an increase
in cAMP.
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METHODS |
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Animals.
Pathogen-free C57BL/6NCr [iNOS(+/+)] mice were obtained from
Frederick Cancer Research and Development Center (National Cancer Institute, Frederick, MD). Breeding pairs of
C57BL/6J-NOS2tm1Lau mice [iNOS(/
)] were
obtained from Jackson Laboratory (Bar Harbor, ME). Mice were bred and
maintained in autoclaved microisolator cages (Lab Products, Maywood,
NJ) and were provided with food (Teklad, Madison, WI) and water ad
libitum. All mice used in studies were 8-14 wk old and 20-30
g in weight. Routine surveillance studies in these mice, performed by
one of us (J. R. Lindsey) as previously described
(10), showed that these mice were negative for significant murine pathogens.
Exposure to hyperoxia.
Both iNOS(+/+) and iNOS(/
) mice were exposed to 100%
O2 or room air for 55 h in specially designed
environmental chambers and were housed in a temperature-controlled
environmental facility, as previously described (50). The
cages were flushed with either air or 100% O2 for 30 min,
and then gas flow was maintained at 8 l/min, which resulted in
nondetectable levels of carbon dioxide in the outflow gas. The
O2 concentration in the chamber was monitored continuously
with an O2 analyzer (Ohmeda 5120) and was maintained above
95% in the O2 group. Mice had access to food and water ad libitum throughout the exposure period.
Plasma osmolality.
iNOS(+/+) and iNOS(/
) mice were killed with intraperitoneal
injections of ketamine (8.7 mg/100 g body wt; Phoenix Scientific, St.
Joseph, MO) and xylazine (1.3 mg/100 g body wt; Vedco, St. Joseph, MO).
The brachial artery and vein were severed, 1 ml of blood was collected
in tubes containing 3% EDTA and centrifuged at 9,400 g for
10 min, plasma was collected, and plasma osmolality was measured using
a vapor pressure osmometer (Vapro; Wescor, Logan, UT).
AFC measurement.
iNOS(+/+) and iNOS(/
) mice were anesthetized with an
intraperitoneal injection of diazepam (5 mg/kg body wt; Abbott
Laboratories, Chicago, IL) followed 10 min later by intraperitoneal
ketamine (200 mg/kg; Phoenix Scientific) and were placed on a heating
pad (Braintree, Cambridge, MA). Temperature was monitored continuously with a temperature probe placed under the skin and was maintained at
37°C using a heating pad and lamp. The trachea was exposed and
cannulated with an 18-gauge intravenous catheter trimmed to ~0.5 in.;
the catheter was connected to a mouse respirator (model 687; Harvard
Apparatus, Holliston, MA) via a three-way valve. All mice were
paralyzed with an intraperitoneal injection of pancuronium bromide
(0.04 mg; Gensia Pharmaceuticals, Irvine, CA) and were ventilated with
100% O2 with a tidal volume of 0.2 ml (9-10 ml/kg body wt) and frequency of 160 breaths/min. Preliminary studies showed
that mice instilled with fluid for measurement of AFC (see below) while
ventilated in such a fashion maintained adequate oxygenation and
acid-base balance during the time course of the experiment. Heart rate
and rhythm were monitored continuously via a three-lead
electrocardiogram (Physio-Control, Redmond, WA).
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Measurements of alveolar fluid volume and albumin efflux.
Two critical assumptions in the measurement of AFC are that
1) the instilled protein is not diluted significantly by the
presence of alveolar fluid and 2) there is no significant
flux of albumin across the alveolar epithelium during the experimental
period. To test the first assumption, the alveolar instillate was
withdrawn 30 s after its instillation, and its protein
concentration was measured. The quantity of alveolar fluid leaving the
alveolar space within 30 s from its instillation will be minimal,
so changes in albumin concentration reflect the amount of alveolar
edema present. To test the second assumption, we instilled isosmotic NaCl containing 5% BSA and 1 µCi of 125I-labeled albumin
(specific activity = 1.954 mCi/ml; ICN, Irvine, CA) into the
alveolar spaces of mice from the various experimental groups. All other
procedures were as described above. A blood sample was taken 30 min
postinstillation, and its 125I radioactivity, along with
that of a sample of the instillate, was measured using a gamma counter
(Automatic Gamma System model 1285; Tm Analytic, Elk Grove Village,
IL). The amount of 125I in the mouse blood as a fraction of
125I in the instillate was calculated from the following
equation
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Lung wet-to-dry weight ratio measurement. Uninstilled mice were killed with an intraperitoneal injection of ketamine and xylazine (as above). The chest was opened, and the heart and lungs were removed en bloc. Nonpulmonary tissue was carefully dissected away, and the lungs were blotted, weighed, and placed in an oven at 55°C for 7 days. At that time, the lungs were weighed again, and the wet-to-dry weight ratio was calculated. In a separate group of mice, the lungs were homogenized, and hemoglobin concentration of the homogenate and a blood sample were measured using a total hemoglobin kit (Sigma Diagnostics). The ratio of the blood-free wet-to-dry lung was then calculated as previously described (31). Because of the very small size of the mouse lungs, hemoglobin concentrations in the lung homogenates were very small and barely above background levels. Thus measurements of the bloodless wet-to-dry lung weights were not considered reliable and are not reported here.
Quantitative histopathology.
iNOS(+/+) and iNOS(/
) mice exposed to 100% O2 for
55 h or normoxic conditions were killed by an intraperitoneal
injection of ketamine-xylazine followed by severance of the brachial
blood vessels (as above). The larynx, trachea, and thoracic organs were removed en bloc. The lungs were infused via the trachea to
approximately normal distension with 10% formalin in 70% ethanol and
then fixed in dorsal recumbency for 24 h in this solution. After
fixation, each of the five lobes was trimmed along its airway and
vascular arborization, embedded in paraffin, sectioned at 5 µm, and
stained with hematoxylin and eosin for light microscopy. To confirm
loss of cross striations in pulmonary veins, normally composed of
cardiac muscle (39), some sections were stained by
Mallory's phosphotungstic acid-hematoxylin method. All sections were
coded randomly, and each lobe was scored subjectively
(0-4) for severity of each of the following anatomic
changes: 1) alveolar hemorrhage; 2) alveolar edema; 3) interstitial (peribronchial, peribronchiolar, and
perivascular) edema; 4) interstitial inflammation;
5) pulmonary vein degeneration; and 6)
degeneration and sloughing of bronchiolar epithelium. Scores for each
lesion were weighted according to the percentage that each lobe
contributes to total lung weight in arriving at a total lesion score
for each set of lungs. For each of the six lesions, a "lesion
index" (LI) was calculated by dividing the observed lesion score by
the maximum score possible. Thus the maximum LI possible was 1.0. The
"total lung lesion score" was calculated by adding all of the
lesion indexes for a set of lungs.
Bronchoalveolar lavage.
Mice were killed with an intraperitoneal injection of
ketamine-xylazine, and a trimmed sterile 18-gauge intravenous catheter (Surflo; Terumo Medical, Elkton, MD) was inserted caudally into the
lumen of the exposed trachea. The lungs were then lavaged in situ with
three separate 1-ml washes of sterile saline. The bronchoalveolar
lavage (BAL) fluid was centrifuged, and the cellular pellet was
resuspended in DMEM containing 2% BSA and 2.5 g/l HEPES (pH 7.4).
Total leukocyte count was determined using a hemocytometer. Protein
concentrations of the lavage supernatants were measured using the BCA
protein assay (Pierce). Samples to be used for immunohistochemistry were separated into aliquots in Lab-Tek sterile chamber slides (Nunc,
Naperville, IL) at a concentration of 104 cells/well. Cells
were incubated at 37° for 1 h, washed with PBS, fixed with 4%
parformaldehyde for 10 min, and washed with PBS again. Slides were
stored in 70% ethanol at 20°C until staining.
Immunocytochemistry for iNOS.
Slides containing cells from BAL fluid from iNOS(+/+) and iNOS(/
)
mice were rinsed in PBS and permeabilized with 50 mM lysine-0.1% Triton X-100 for 15 min. Endogenous peroxidase activity was quenched by
treatment with 0.3% hydrogen peroxide in cold methanol for 30 min
followed by three washes with PBS. Nonspecific binding was blocked with
10% goat serum and 10% mouse serum in PBS for 2 h. The primary
antibody, anti-mouse iNOS, NH2-terminus, rabbit polyclonal
IgG (Upstate Biotechnology, Lake Placid, NY), or nonspecific IgG (both
at 0.01 mg/ml in 10% goat serum and 2% BSA in PBS) was applied to the
cells overnight. After being washed in 0.1% Triton in PBS, cells were
incubated with the goat anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody from a DAKO EnVision system
kit (DAKO, Carpintera, CA) for 30 min at 37°C. The cells were then
rinsed in 50 mM Tris · HCl and developed in a filtered solution
of diaminobenzidine from the same kit for 30 s. Slides were
counterstained with methyl green (Fisher Scientific, Fair Lawn, NJ).
For each slide, at least 300 cells were counted and graded as positive
or negative based on the presence or absence of the brown reaction
product in the cell cytoplasm.
Nasal potential difference measurement. Mice were anesthetized by an intraperitoneal injection of a mixture of ketamine (33 mg/100 g body wt; Fort Dodge Laboratories, Fort Dodge, IA), acepromazine (1.2 mg/100 g body wt; Vedco), and xylazine (6 mg/100 g body wt; Vedco). Mice were then placed on their backs, their tail was immersed in 0.9% NaCl, and a catheter was placed in their nasal passages, as previously described (13). The nasal passages were then perfused with lactate Ringer or lactate Ringer with 100 µM amiloride at a rate of 3 µl/min using a perfusion pump. The potential difference across the nares with the tail being the reference was measured with a voltmeter and recorded on a strip chart recorder at a speed of 30 cm/h. Solutions were changed only after the voltage had stabilized.
Statistics. All data were analyzed by ANOVA, using the Bonferroni method for multiple comparisons or Student's t-test when appropriate. All values given are means ± SD, and a P value <0.05 was considered significant.
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RESULTS |
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AFC in normoxic mice.
Arterial blood gases in anesthetized mice 30 min after fluid
instillation are shown in Table
1. Both iNOS(+/+) and iNOS(/
) mice
had normal acid-base balance and arterial PO2
(PaO2) values >80 Torr, consistent with adequate
tissue oxygenation and perfusion. The lower than expected
PaO2 values for this level of inspired O2
are consistent with the presence of a right-to-left shunt resulting from the instillation of fluid in the alveolar space. iNOS(
/
) mice
had significantly higher plasma osmolality compared with iNOS(+/+) mice
[334 ± 5 vs. 321 ± 5 (SD) mosmol/kgH2O;
n = 9; P < 0.001]. We thus adjusted
the instillate osmolality to match the plasma osmolality for each group
of mice.
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Nasal potential difference measurement.
iNOS(+/+) and iNOS(/
) mice had similar baseline nasal potential
difference (NPD) values (
8.3 ± 1.5 vs.
6.2 ± 2.7 mV;
mean ± SD; n = 6; apical membrane compared with
tail). Perfusion of the external nares of the iNOS(+/+) mice with
lactate Ringer solution containing 100 µM amiloride resulted in a
change in NPD of 3.8 ± 1.6 mV, consistent with depolarization of
the apical membrane resulting from the blockade of Na+
entry pathways (Fig. 2A). On
the other hand, no significant change in NPD was observed when the
external nares of iNOS(
/
) mice were perfused with an
amiloride-containing solution (change in NPD = 1 ± 1.4 mV;
Fig. 2B). These findings are consistent with those shown in
Fig. 1 and indicate the lack of amiloride-sensitive Na+
transport across both the alveolar and nasal epithelium of iNOS(
/
) mice.
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Exposure to 100% O2.
None of the iNOS(+/+) or iNOS(/
) mice died during exposure to 100%
O2 for 55 h. Both groups of mice developed sublethal lung injury as shown by flaring of the nostrils and higher lung wet-to-dry weight ratios, total lung lesion scores, and BAL fluid protein concentrations compared with air-breathing controls (Table 2). However, iNOS(
/
) mice developed
less lung injury as shown by a 15% increase of the lung wet-to-dry
weight ratios compared with 33% in the iNOS(+/+) mice and a 160%
increase in total lung lesion score compared with 400% for iNOS(+/+)
mice. These data are consistent with higher endothelial injury in the
iNOS(+/+) mice. Lungs from iNOS(+/+) and iNOS(
/
) mice exposed to
either hyperoxic or normoxic conditions were evaluated by subjective quantification of the following six morphological lesions:
1) alveolar hemorrhage; 2) alveolar edema;
3) interstitial (peribronchial, peribronchiolar, and
perivascular) edema; 4) interstitial inflammation; 5) pulmonary vein degeneration; and 6) sloughing
of bronchiolar epithelium. No alveolar hemorrhage or edema was found in
any of the mice. Mild interstitial edema and inflammation (lymphocytic and neutrophilic) were observed, but differences between groups were
not statistically significant. In both iNOS(+/+) and iNOS(
/
) mice
exposed to hyperoxia, there was hyalinization and patchy to diffuse
loss of cross striations in cardiac muscle, and the severity of this
lesion was significantly greater in iNOS(+/+) mice (LI = 0.51 ± 0.03) than in iNOS(
/
) mice (LI = 0.26 ± 0.13; Table
2). Pulmonary veins were histologically normal in all mice of both
strains exposed to normoxic conditions. In addition, epithelium in the
bronchioles of iNOS(+/+) and iNOS(
/
) mice exposed to hyperoxia had
diffuse, moderate swelling and increased eosinophilia of apical
cytoplasm, with sloughing of nonciliated and ciliated epithelial cells
in their lumens. Sloughed cells were rounded, pyknotic, and frequently
undergoing karyolysis. In some bronchioles, cell losses resulted in
patchy ulcerations. Severity of these changes was significantly greater
in iNOS(
/
) mice (LI = 0.56 ± 0.04) compared with
iNOS(+/+) mice (LI = 0.22 ± 0.2). Both strains of mice had
morphologically normal bronchioles after exposure to normoxic
conditions.
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iNOS immunocytochemistry.
Cells (300) isolated from the BAL fluid of each group of
mice [iNOS(+/+) or iNOS(/
) breathing air or 100% O2]
were counted after immunostaining with a polyclonal anti-mouse iNOS
antibody. In all cases, >95% of these cells were alveolar
macrophages. Approximately 2 and 92% of cells isolated from the BAL
fluid of air- and O2-breathing iNOS(+/+) mice stained
positive for iNOS, respectively. Typical fields are shown in Fig.
3. In contrast, none of the cells from iNOS(
/
) mice stained positive for iNOS.
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AFC in hyperoxic mice.
Exposure of iNOS(+/+) or iNOS(/
) mice to 100% O2 for
55 h did not alter their basal AFC levels (Fig.
4). However, exposure of iNOS(+/+) mice
to hyperoxia decreased their amiloride-sensitive portion of AFC from 61 to 40% of total (Fig. 4). Amiloride (1.5 mM) did not alter AFC of
iNOS(
/
) mice exposed to hyperoxia, similar to what was observed in
normoxia. Hyperoxic exposure did not increase the 30-s AFC value or the
amount of 125I-albumin in the blood (Table 2).
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DISCUSSION |
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The availability of transgenic mice and the considerable interest
in understanding basic mechanisms responsible for the regulation of
fluid transport across the alveolar space in vivo have necessitated the
development of new techniques to measure AFC in mice under physiological conditions. The studies of Garat et al. (12)
and Icard and Saumon (18) were the first to report
successful measurements of amiloride-sensitive alveolar fluid
reabsorption across the lungs of c57 (presumably C57BL/6) mice in situ
and isolated perfused lungs of CD-1 mice, respectively. However, Fukuda
et al. (11) reported recently that lungs of mice in situ
developed a significant amount of interstitial edema, most likely
because of the lack of pulmonary lymph and blood flow, which inhibited
AFC by ~30%. In their studies, Fukuda et al. measured AFC in
anesthetized, ventilated mice but did not report values for arterial
blood gases during the experimental period. We ventilated our mice with
a tidal volume of 9-10 ml/kg body wt, similar to what they
reported. However, to maintain adequate oxygenation and acid-base
balance, we had to ventilate the mice using a much higher respiratory
frequency than Fukuda et al. reported (160 vs. 90 breaths/min).
Furthermore, we carefully adjusted the osmolality of the instillate to
that of the plasma for each strain of mice [321 ± 5 and 334 ± 5 mosmol/kgH2O in the iNOS(+/+) and iNOS(/
) mice,
respectively], since the presence of an osmotic gradient across the
alveolar epithelium may complicate the interpretation of AFC.
Comparison of our results with previous studies is complicated not only
by the fact that our measurements were done under normal physiological
conditions but also by differences in genotype. For example, although
our basal values of AFC in iNOS(+/+) mice are similar to those reported
by Fukuda et al. (11), Ma et al. (30), and
Icard and Saumon (18; ~30% of instilled fluid/30 min corresponding
to ~6 ml · h1 · g dry lung
wt
1), the fraction of AFC blocked by amiloride was very
different (~60% of AFC in our case vs. >90% in the above-mentioned
reports). This was most likely because these investigators used CD-1
mice while we used C57BL/6 mice. Indeed, in a previous study, Garat et
al. (12) reported a 40% inhibition of AFC across the
lungs by amiloride using the in situ model on C57BL/6 mice. These
findings underscore the importance of using properly matched genetic
controls in transgenic mice experiments.
Our studies show that both iNOS(+/+) and iNOS(/
) mice have similar
levels of AFC, which are totally dependent on the presence of active
Na+ reabsorption, as shown by the lack of AFC when
Na+ was replaced by NMDG+Cl
, a
large positive ion with very small permeability across ATII cell
Na+ channels (32). However, AFC in the
iNOS(
/
) mice was totally insensitive to amiloride. This finding was
consistent with the lack of amiloride sensitivity of the potential
difference across the external nares. Amiloride-insensitive fluid
clearance is very important in that it makes up ~50% of fluid
clearance in most animals and in humans (2, 42). This
study shows that these pathways can be increased, which could have
clinical applications in the future.
Our NPD studies in iNOS(/
) mice yielded comparable results to those
performed by Elmer et al. (7), who found that only 25% of
the baseline NPD was eliminated by amiloride compared with our 16%.
They ascribed their finding to the background of the mice.
The lack of amiloride-sensitive fluid clearance in the iNOS(/
) mice
was unexpected and seems paradoxical considering that NO has been shown
to downregulate amiloride-sensitive transport across alveolar
epithelial cells via a cGMP-dependent mechanism (21, 26).
These results imply that there is an optimum level of NO for the
amiloride-sensitive Na+ channel (ENaC) or at least for its
amiloride sensitivity. There are several possibilities that may account
for the lack of amiloride-sensitive fluid clearance in iNOS(
/
)
mice. The ENaC is a heteromultimeric complex of at least three distinct
but homologous subunits,
-,
- and
-ENaC, which were first
cloned from the colon of salt-deprived rats and human lung tissue
(3, 28, 48). Experiments with point mutations suggested
that all three subunits are involved in the channel pore formation
(45). Expression of
-rat (r)ENaC mRNA in adult rat ATII
cells was demonstrated by Northern blot analysis (50), PCR
(9), and in situ hybridization (9, 50).
- and
-rENaC mRNAs were also detected in
large and small airways, but they were less abundant in ATII cells
compared with
-rENaC (9). Our previous biophysical and
biochemical studies in A549 cells, an alveolar epithelial line, showed
that alterations in the stoichiometry of the
-,
-, and
-subunits of ENaC altered both the conductance of Na+
single channels and their response to amiloride. Specifically, incubation of A549 cells with dexamethasone increased expression of
- and
-human (h)ENaC protein levels and decreased the amiloride half-maximal inhibitory concentration (K0.5) for
whole cell currents from 833 ± 69 to 22 ± 5.4 nM (mean ± SE; P < 0.01; see Ref. 27). An
increase in amiloride K0.5 for epithelial
channels of ATII cells in iNOS(
/
) mice may manifest itself as a
lack of response to amiloride.
Admittedly, the concentration of instilled amiloride was quite high
(1.5 mM). However, measurements of the alveolar concentration of
amiloride at the end of the experimental period were considerably and
equally lower (~300 µM) in both types of mice, and its
concentration in the epithelial lining fluid was most likely much lower
because of its binding to albumin and nonuniform distribution in the
alveolar epithelial lining fluid. This finding is consistent with the
results of numerous previous studies showing that, in contrast to its very low inhibition constant (Ki) in vitro
(20-30 nM), much larger concentrations (0.8-1.5 mM) are
needed in the instillate to decrease Na+ transport in vivo
(37). In addition, forskolin, an agent that increases
intracellular cAMP levels, upregulated AFC in the iNOS(+/+) mice by
increasing amiloride-insensitive fluid clearance but failed to increase
AFC in the iNOS(/
) mice. Previous studies in a variety of species
have shown that intratracheal instillation of agents that increase cAMP
upregulate either the amiloride-sensitive (30) or the
amiloride-insensitive fraction of AFC (23). Patch-clamp studies have shown that an increase in cAMP levels of ATII cells results in significant upregulation of both the open time and open
probability of amiloride-sensitive single channels (51). This finding argues against the possibility of inactive channels being
present in the ATII cells of iNOS(
/
) mice.
Another possibility is that Na+ transport across the
alveolar airway epithelium of iNOS(/
) mice occurs through pathways
other than ENaC. These include the Na+-glucose
cotransporter SGLT1 (18), the
Na+-H+ antiport (29), and cyclic
nucleotide-gated cation channels, especially CNG1 (24,
46). SGLT1 has been shown to be present in mouse lung
(18), but transport via this channel seems unlikely because the instillate in our experiments did not contain glucose. CNG1
has been shown to be present in rat lung, to conduct
amiloride-insensitive, cGMP-stimulated Na+ current across
cultured tracheal cells (46), and to mediate a substantial
portion of fluid clearance in sheep (24).
Finally, the fact that neither amiloride nor ouabain decreased AFC
across the alveolar epithelium of iNOS(/
) mice may suggest that
Na+ transport occurred through paracellular pathways. We
believe that this is very unlikely because 1) since the
instillate was isosmotic with the plasma, there was no driving force
for the egress of either Na+ or fluid across the alveolar
epithelium, and 2) ouabain also failed to decrease AFC in
the iNOS(+/+) mice in which 60% of AFC was inhibited by amiloride.
Addition of 0.1-1 mM ouabain to the instillate and to the
perfusion solution of ex vivo animal or human lung preparations
inhibited AFC from 50 to 100% (18, 30, 42-44). The
only study evaluating the effects of ouabain in vivo was performed by
Jayr et al. (23). These authors measured AFC in rats and
reported that addition of 1 mM ouabain to the instillate and
simultaneous intravenous infusion of 0.004 mg of ouabain decreased AFC
from 33 ± 9 to 23 ± 4 (net change in AFC = 30%). As
mentioned, our data indicate that intraperitoneal and intratracheal
instillation of ouabain resulted in progressive atrioventricular block
and death of the mice. Because of the severe hemodynamic
alterations, which most likely effected the measurements of AFC, and
the uncertainty as to whether we achieved a sufficiently high ouabain
concentration on the basolateral membranes of ATII cells, these studies
were not pursued further.
Our previous studies and those of others showed that reactive oxygen
nitrogen intermediates, formed by the reaction of NO with superoxide
and thiols, decrease short-circuit current because of Na+
across cultured monolayers of ATII cells by damaging both apical and
basolateral Na+-transporting pathways (14),
decrease amiloride-sensitive whole cell currents across Xenopus
oocytes injected with -,
-, and
-ENaC (6),
and inhibit both whole and single channel currents across A549
(27), ATII (21), and fetal distal epithelial
(4) cells. In addition, perfusion of the external nasal
passages with an iNOS inhibitor resulted in upregulation of
amiloride-sensitive Na+ transport, suggesting that tonic
release of NO congeners by iNOS downregulated amiloride-sensitive
transport across airway epithelial cells (7).
To gain additional insight into the role of endogenous NO in the
regulation of Na+ transport across the alveolar epithelium
in vivo, we exposed both iNOS(+/+) and iNOS(/
) mice to a sublethal
level of hyperoxia. Exposure to hyperoxia resulted in a significant
upregulation of iNOS in inflammatory cells and damaged the alveolar
epithelium as shown by the large increase in the wet-to-dry weight
ratio and alveolar albumin concentration in both iNOS(+/+) and
iNOS(
/
) mice. However, the degree of lung damage in iNOS(
/
)
mice was lower than in iNOS(+/+) mice as shown by lower increases in
the lung wet-to-dry weight ratio and overall lung index score. We found
no change in basal AFC with 100% O2 for 55 h, but the
amiloride-sensitive portion of fluid clearance decreased from 61% of
total clearance with normoxia to 40% with hyperoxia in iNOS(+/+) mice.
This was a result of upregulation of the amiloride-insensitive
component of AFC by NO. Instillation of DETANONOate, an NO donor, in
the alveolar spaces of rabbits also resulted in a decrease of the amiloride-sensitive portion of the AFC without deceasing its basal value (36). The mechanism responsible for this effect has
not been elucidated.
In these studies, hyperoxia resulted in significant injury to cardiac
muscle in pulmonary veins and to epithelium of bronchioles in both
iNOS(+/+) and iNOS(/
) mice. These changes were clearly degenerative
in character and thus are most likely indicative of toxic effects of
O2. It is not entirely clear whether these changes had any
effect on ion transport, since ATII cells are generally accepted as
mainly responsible for ion transport in the lungs. Although cardiac
muscle does not occur in pulmonary veins of humans, as it does in mice,
and humans have a much different population of cells (far fewer Clara
cells) in their bronchioles than mice, our findings raise the
possibility of toxic effects of hyperoxia on cardiovascular and
bronchiolar tissues of patients.
In summary, our results indicate that iNOS(/
) mice have normal
levels of Na+-dependent fluid transport across their
alveolar and airway epithelia in vivo. However, these mice either lack
amiloride-sensitive pathways or the amiloride IC50 of their
apical transporters is significantly higher than the corresponding
value in their wild-type controls. Thus NO produced by iNOS may be
important in the regulation of amiloride-sensitive Na+
pathways across these epithelia.
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ACKNOWLEDGEMENTS |
---|
We thank Judy Hickman-Davis, Ian Davis, Glenda Davis, Tanta Miles, Mark Philips, and James Fortenberry for technical support and many useful discussions.
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FOOTNOTES |
---|
This study was supported by National Institutes of Health Grants HL-51173, HL-31197, and P30-DK-54781 and by funds from the Veterans Affairs Research Service.
Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St. S., THT 940, Birmingham, AL 35233 (E-mail: Sadis.Matalon{at}ccc.uab.edu).
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. Section 1734 solely to indicate this fact.
Received 31 January 2001; accepted in final form 17 April 2001.
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