1 Department of Surgery and
Medical Research Council of Canada Group in Mechanisms of Organ
Injury, Distal lung epithelial cells
(DLECs) play an active role in fluid clearance from the
alveolus by virtue of their ability to actively transport
Na+ from the alveolus to the
interstitial space. The present study evaluated the ability of
activated macrophages to modulate the bioelectric properties of DLECs.
Low numbers of lipopolysaccharide (LPS)-treated macrophages were able
to significantly reduce amiloride-sensitive short-circuit current
(Isc) without
affecting total
Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the
Isc. The effect
was reversed by the addition of
N-monomethyl-L-arginine
to the medium, implying a role for nitric oxide. We hypothesized that
macrophages exerted their effect by expressing inducible nitric oxide
synthase (iNOS) in DLECs. The products of LPS-treated macrophages
increased the levels of iNOS protein and mRNA transcripts in DLECs as
well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in
macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute
respiratory distress syndrome and suggest a novel mechanism whereby
nitric oxide might alter cell function by altering its ion-transporting
phenotype.
distal lung epithelium; macrophages; lung injury
ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is
characterized by the presence of hypoxemia, reduced lung compliance,
and increased permeability pulmonary edema that results in diffuse
alveolar infiltrates on chest radiographs. This pathological process
develops as a result of the disruption of the alveolar-capillary
membrane with leakage of protein-rich fluid exudate and migration of
inflammatory cells (neutrophils and macrophages) into the air space
(reviewed in Ref. 40). Under normal circumstances, the distal lung
epithelial cells (DLECs) that line the terminal airways and alveoli
provide a tight physical barrier to the movement of interstitial fluid into the alveolar space. Injury to this layer may result in alveolar edema that contributes to the clinical characteristics of ARDS.
Recent studies have demonstrated that the DLECs also play an active
role in fluid clearance from the alveolus by virtue of their ability to
actively transport Na+ from the
alveolus to the interstitial space. Vectorial transport of
Na+ (with
Cl Our laboratory has previously investigated the ability of alveolar
macrophages (M Materials and Solutions
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and water following)
across monolayers of these cells is mediated by the presence of
Na+ channels on their apical
surface that permit Na+ entry down
their electrochemical gradient into the cell and
Na+-K+-adenosinetriphosphatase
(ATPase) on the basolateral surface of the cell that extrudes
Na+ (3, 30). Both in vitro and in
vivo experiments suggest a physiological role for this transport
mechanism. Studies performed under conditions where the bioelectric
properties may be measured show that monolayers grown on porous
supports exhibit unidirectional apical-to-basolateral transport of
Na+ and are able to establish a
significant potential difference (apical negative) across the
monolayer. These processes are inhibited by the
Na+-channel blockers amiloride and
benzamil as well as by the
Na+-K+-ATPase
inhibitor ouabain and are stimulated by
-adrenergic agonists and
membrane-permeant analogs of adenosine 3',5'-cyclic
monophosphate (3, 27-30). The existence of this process in vivo is
supported by several lines of evidence. First, tracer studies in
isolated perfused rat lung preparations using
22Na+
have clearly demonstrated active
Na+ reabsorption from the alveolar
spaces (9). Second, physiological studies in animals have shown a role
for an amiloride-sensitive process in fluid clearance from the alveolus
(1, 2, 20). Finally, there is evidence supporting the existence of a
comparable system in humans. Sakuma et al. (38) have reported
amiloride- and ouabain-inhibitable alveolar fluid clearance in resected
human lung at rates comparable to those observed in animal experiments. Considered together with the in vitro data, these observations suggest
that this fluid-resorptive mechanism may play an active role in the
resolution phase of ARDS. In this regard, a study by Matthay and
Wiener-Kronish (26) correlated the clinical resolution of ARDS with the
ability of patients to concentrate alveolar fluid protein, an indirect
measure of Na+ transport-mediated
fluid clearance from the alveolar space.
s) to modulate DLEC
Na+-transport activity as a model
whereby activated alveolar M
s might contribute to the development
and persistence of ARDS in the septic patient. These studies
demonstrated that lipopolysaccharide (LPS)-treated alveolar M
s were
able to reduce total and amiloride-sensitive short-circuit current
(Isc) in
primary cultures of DLECs (7). The observation that this inhibitory
effect was dependent on
L-arginine metabolism suggested
a role for nitric oxide (NO) as a key mediator of this effect. Because
DLECs incubated with the supernatants of LPS-treated M
s exhibited
alterations in
Isc similar to
those seen in the coculture system, we hypothesized that NO derived from DLECs might, at least in part, be responsible for the effect. The
present studies demonstrate that the soluble products of LPS-treated M
s decrease the amiloride-sensitive
Na+ transport of the DLECs without
affecting total
Isc. These
changes correlated with an increase in the expression of inducible NO synthase (iNOS) at both the mRNA and protein levels in DLECs. However,
treatment of DLECs with the NO donor
S-nitroso-N-acetylpenicillamine (SNAP) alone did not reproduce the effect, suggesting that NO production is necessary but not sufficient to account for the observed
alterations in amiloride-sensitive
Isc in DLECs
exposed to M
products. These data therefore suggest a mechanism
whereby NO might contribute to altered cell function by altering its
ion-transporting phenotype.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Alveolar M Isolation
Epithelial Cell Isolation and Culture
DLECs were harvested from late-gestation fetal rats and grown in primary culture according to methods previously described (28). In brief, the lungs were excised from timed-gestation 20- or 21-day (term = 22 days) Wistar rat fetuses and minced into 1-mm3 pieces. The lung fragments were incubated at 37°C with 0.125% trypsin and 0.002% deoxyribonuclease, and the dissociated cells were then passed through a Nitex 100 (B. and S. H. Thompson, Scarborough, Canada) mesh filter. The cells were then incubated with 0.1% collagenase and purified with differential adhesion techniques. These cells are >99% pure epithelial cells and consist of mature and precursor type II epithelial cells or distal airway cells, and hence we refer to these cells as DLECs (30). Previous experiments have demonstrated that epithelial cells cultured in this manner transport Na+ via amiloride-sensitive and -insensitive mechanisms (28). These cells possess amiloride-sensitive whole cell Na+ currents but no detectable ClThe harvested epithelial cells were immediately seeded (1 × 106 cells/cm2) onto Transwell tissue culture-treated porous polycarbonate filters (total surface area 4.7 cm2; Costar, Cambridge, MA). All cells were grown in MEM with 10% fetal bovine serum and penicillin-streptomycin at 37°C in a humidified 95% air-5% CO2 environment. Nonadherent epithelial cells were removed 24 h after they were seeded. Epithelial monolayers were subsequently studied 3 or 4 days after they were seeded. Cells used in patch-clamp experiments were seeded (5 × 105 cells/cm2) onto translucent porous Nunc filter inserts (Whatman Scientific) and were confluent when studied 4 days later.
Epithelial Cell Monolayer Bioelectric Properties
The bioelectric properties of epithelial cell monolayers were studied by placing the filters in Ussing chambers (MRA International, Clearwater, FL) that contained warmed HBSS and 22.4 mM NaHCO3 that was circulated with an air lift of a 95% air-5% CO2 gas mixture (7, 30). Measurements of the bioelectric properties of the monolayer were made with KCl agar-calomel half-cells and silver-silver chloride electrode-saline agar bridges that were connected to a high-impedance millivoltmeter that could function as a voltage-current clamp with automatic fluid-resistance compensation (VCC 600 Physiologic Instruments, San Diego, CA). The transepithelial monolayer potential difference (PD) was recorded continuously under open-circuit conditions with a linear-chart recorder (Linear Recorder 585, Baxter, Toronto, Canada). Every 10 s, a 0.5-s duration 1-µA pulse of current was delivered across the monolayer so that the measured change in PD enabled the calculation of resistance (R) using Ohm's law. Transepithelial R is a sensitive measurement of epithelial monolayer permeability to ions and in large part reflects the barrier function of epithelial tight junctions. Every 15 min, the transepithelial PD was temporarily clamped to 0 mV so that Isc could be recorded. Isc represents the net movement of positive charge from the apical to the basolateral side of the epithelial membrane, with the basolateral side of the monolayer having a positive PD relative to the apical side of the monolayer.Four groups of epithelial cell monolayers were studied to determine the
effect of alveolar Ms and/or LPS on monolayer permeability (R) and ion transport
(Isc). The
apical side of confluent epithelial monolayers was exposed to control
medium, LPS alone (10 µg/ml), M
s alone (1-6 × 105
cells/cm2), or both LPS (10 µg/ml) and M
s (1-6 × 105
cells/cm2) for varying times. In
some studies, the NO donor SNAP (0.1 mM) was added to the monolayers
every 4 h for four doses before evaluation of the bioelectric
properties. Monolayers were then rinsed with MEM and placed in Ussing
chambers containing freshly prepared HBSS. After baseline bioelectric
properties were determined, the Na+-transport blocker amiloride
(0.1 mM apically), the
Na+-K+-2Cl
cotransport inhibitor bumetanide (0.1 mM basally), and the NSC inhibitor flufenamic acid (0.45 mM apically) were added sequentially to
the monolayer, thus enabling the calculation of amiloride-sensitive current and nonselective current as a percentage of the total current
in all experimental groups. The amiloride-sensitive
Isc likely
reflects blockade of Na+ channels
because our laboratory has previously shown that amiloride blocks 12- and 25-pS Na+-permeant
channels (31, 42) and whole cell cation conductances (43) in the apical
membrane of these epithelial cells. This amiloride dose was chosen
based on previous dose-response studies performed on similar cells
(28). Furthermore, dimethylamiloride, an amiloride analog with high
potency for the
Na+/H+
antiport, does not affect in vivo lung water clearance (28), DLEC
Isc (30) or whole
cell Na+ currents (43).
In some studies, alveolar Ms were incubated in coculture but were
physically separated from the DLEC monolayer. To accomplish this,
alveolar M
s (2 × 106)
were added to the bottom of the Ussing chambers and allowed to adhere
for 2 h before insertion of the filters coated with the epithelial cell
monolayers into the Ussing apparatus. This coculture setup was
incubated for a further 16-24 h in the presence or absence of LPS,
at which time the bioelectric properties were investigated.
iNOS Expression in Alveolar Ms and DLECs
Northern blot analysis. Total RNA was
extracted with the method of Chomczynski and Sacchi (4). Briefly,
cells were washed with cold
Ca2+- and
Mg2+-free HBSS and lysed with
guanidium thiocyanate. The cell lysate was then recovered from the dish
with a sterile cell scraper and transferred to Eppendorf tubes.
After RNA extraction and spectrophotometric quantitation, 10 µg of total RNA were electrophoresed in a 1.2% agarose gel
containing formaldehyde, blot transferred to a nylon membrane, and
then ultraviolet cross-linked. Membranes were hybridized with the
32P random-labeled cDNA probe for
the murine M iNOS (kindly provided by Dr. Dennis Stuehr, Cleveland
Clinic, OH; see Ref. 10), washed at room temperature in 1× sodium
chloride-sodium phosphate-EDTA and 0.1% sodium
dodecyl sulfate for 30 min, and exposed overnight to Kodak film at
70°C. Comparable RNA loading between lanes was assessed by probing with a cDNA probe for rat
-tubulin.
Western blot analysis. Alveolar Ms
and DLECs were recovered separately for Western blot analysis by lysing
cells in the inner and outer wells, respectively, with boiling Laemmli
buffer (22). Cell lysates (50 µg for DLECs and 5 µg for M
s) were
separated with 8% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and subsequently transferred to nitrocellulose with the
Bio-Rad Mini Trans-Blot system. The blot was blocked with 5% nonfat
milk (Bio-Rad) in tris(hydroxymethyl)aminomethane-buffered
saline for 30 min at room temperature and then exposed for
1 h while being shaken at room temperature to a 1:2,500 dilution of a
rabbit polyclonal antibody generated against a synthetic peptide
derived from the COOH-terminal sequence of the mouse M
iNOS protein
(kindly provided by Dr. Carl Nathan, Cornell University Medical
College, New York, NY). The blot was then washed three times with
antibody buffer solution and incubated with a 1:25,000 dilution of goat
anti-rabbit immunoglobulin G conjugated to horseradish peroxidase. The
blots were washed, dried, and quantitated with an enhanced
chemiluminescence detection system (ECL, Amersham).
Nitrite Analysis
The nitrite content of the cell-free supernatants was measured by reacting samples (100 µl) with the Griess reagent (100 µl) for 10 min at 37°C according to established methods (12). Absorbance at 540 nm was then measured, and nitrite concentrations were calculated from a linear standard curve generated between 0 and 128 mM sodium nitrite.Detection of Nitrotyrosine Residues
Cells were cultured on glass coverslips in six-well tissue culture dishes. Coverslips were then washed three times with ice-cold PBS and fixed in 100% methanol (Statistics
The results are means ± SE of n experiments unless otherwise indicated. The statistical significance of the differences between the means of multiple groups or the means of an individual group at multiple time points was determined by one-way analysis of variance followed by Newman-Keuls multiple intergroup comparisons. Student's unpaired two-tailed t-test was used to assess significance between two groups. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Effect of M Number on the Bioelectric Properties of
the Epithelium
|
Because our previous studies had demonstrated that LPS-stimulated Ms
induced a reduction in amiloride-sensitive
Isc several hours
before having an effect on total
Isc, we
postulated that the lower numbers of M
s might exert a selective
effect on amiloride-sensitive Isc. To test this
possibility, amiloride was added after the measurements had stabilized
to determine the amiloride-sensitive component of the
Isc. O'Brodovich
and colleagues (28, 30) and others (3, 34) have previously demonstrated
that amiloride decreases Na+
transport without affecting total transepithelial
R. Although total
Isc did not
differ among groups, DLEC monolayers incubated with LPS-treated M
s
(2 × 106/filter)
demonstrated a marked reduction in the amiloride-sensitive component of
the Isc compared
with all other groups (Fig.
2A, Table
1). A time course of this effect
demonstrated that some degree of inhibition occurred by 4 h and then
was progressive over the next several hours (Fig.
2B). By 16 h, DLECs exposed to
LPS-treated M
s exhibited essentially no measureable
amiloride-sensitive Isc. This effect
was present whether M
s were in direct contact with the DLECs
(contact) or were physically separated from the DLEC monolayer. To
investigate the source of the amiloride-insensitive Isc, monolayers
were treated with flufenamic acid (0.45 mM) and bumetanide (0.1 mM). As
shown in Table 1, DLECs exposed to LPS-treated M
s, either in direct
contact or physically separated, exhibited a significant increase in
the flufenamic acid-sensitive component. Bumetanide-sensitive
Isc remained low
(<1%) in all groups. Considered together, these data show that
soluble products derived from LPS-treated M
s are able to induce
significant qualitative and quantitative alterations in DLEC ion
transport. Specifically, they are able to virtually obliterate the
amiloride-sensitive
Isc and increase the amount of flufenamic acid-sensitive
Isc while having
no effect on bumetanide-sensitive
Isc, total
Isc, or monolayer
R.
|
|
Induction of DLEC iNOS Expression by Stimulated
Ms
|
|
The time course of iNOS protein expression is illustrated in Fig.
5. No detectable iNOS protein
was seen in control DLECs over the 10-h study period. By contrast,
epithelial cells cultured in the presence of Ms and LPS demonstrated
a small amount of iNOS protein by 4 h, which increased to maximum
values between 6 and 10 h. At 6 h, M
s recovered from the
same study expressed a large amount of iNOS protein. The induction of
iNOS protein activity was reflected in increased nitrite release by the
Griess reaction. To detect nitrite produced by DLECs, two approaches were taken. First, we generated M
supernatants by treating cells with LPS or vehicle overnight and then added this cocktail to DLECs.
The cocktail was washed away after 6 h, the DLECs were thoroughly
washed, and then the DLECs were further incubated in fresh medium for
16 h. Nitrite levels in the supernatant were then measured. The nitrite
level increased from undetectable in studies in which the cocktails
were derived from unstimulated M
s to 14.6 ± 3.9 µM
(n = 6 experiments) in studies in
which the cocktails were from LPS-treated M
s. Second, M
s were
cocultured physically separated from DLECs but bathing in the same
medium in the presence or absence of LPS for 6 h. The cells were then washed, and the production of nitrite was detected in DLECs cultured separately in fresh medium for 16 h. Exposure of DLECs to
LPS-stimulated M
s increased nitrite release to 71.6 ± 4.2 µM
(n = 6 experiments). By contrast,
LPS-treated M
s (3 × 106) released 204.6 ± 3.0 µM (n = 3 experiments). LPS-treated
DLECs in the absence of M
s repeatedly released small concentrations of nitrites (<5 µM). These data thus support the iNOS gene and protein expression studies, demonstrating that iNOS activity is less in
DLECs than in M
s.
|
Role of NO as a Mediator of Altered Bioelectric Properties in DLECs
After iNOS expression in LPS-stimulated DLECs was demonstrated, studies were performed to discern the role of NO as the effector molecule of the observed changes in amiloride-sensitive Isc. A previous study (7) showed that L-NMMA was able to prevent MA recent study (17) has implicated peroxynitrite as playing an
important role in NO-mediated effects on target cells including DLECs.
To discern the generation of peroxynitrite in DLECs when cultured with
LPS-stimulated Ms, cocultures were blotted with anti-nitrotyrosine
antibodies and studied by confocal microscopy. As shown in Fig.
6, nitrotyrosine residues were detected in
M
s in LPS-treated M
-DLEC cocultures but not in DLECs. Specificity was confirmed by the lack of fluorescence when the primary antibody was
omitted (Fig. 6C) or competed with
peptide (Fig. 6D).
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DISCUSSION |
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Resolution of pulmonary edema after acute lung injury is dependent on
the successful treatment of the primary process responsible for fluid
leakage into the air space coupled with efficient fluid reabsorption
from the alveolar space. Fluid transport out of the alveolar space is
largely mediated by epithelial cell vectorial transport of
Na+ from the apical to the
basolateral surface (reviewed in Ref. 27). The clinical importance of
this epithelial Na+ transport
mechanism is illustrated by data showing that survival from either high
pressure (congestive heart failure) or high permeability (ARDS)
correlated with the ability of the lungs to concentrate its air space
fluid (26), presumably via the active absorption of
Na+ and
Cl, with water following.
Such a speculation was supported by a recent report (38) that
demonstrated that the distal lung regions of the human lung also absorb
fluid by active Na+ transport. In
the present study, we have utilized an in vitro model to investigate
cellular interactions that might contribute to reduced
Na+ transport by DLECs, leading to
impaired recovery from pulmonary edema. We originally chose to study
M
s because of data showing their potential role in the pathogenesis
of lung injury during the early stages before neutrophil influx. In
addition, their ability to release a diverse range of inflammatory
mediator molecules coupled with their close apposition to DLECs in vivo
suggested the possibility that they might be capable of modulating DLEC function. Using a M
-DLEC coculture system, we demonstrated that M
products released in response to LPS are able to induce iNOS gene and
protein expression in DLECs and stimulate iNOS activity in these cells.
The NO generated in this system is necessary but not sufficient to
cause the observed reduction in the amiloride-sensitive Isc and the
increase in the flufenamic acid-sensitive component of total
Isc in DLECs.
This occurred without affecting total
Isc or the
R of the monolayer. These observations
suggest a mechanism whereby NO produced locally in the lung might
interact with other proinflammatory molecules to exert effects that
impair Na+ transport during acute
lung inflammation.
Increased iNOS expression with enhanced local production of NO in the
lung has been demonstrated after systemic LPS administration in rats
(21, 25, 41, 46). Several possible cellular sources for NO generation
in this setting have been suggested, including both interstitial and
alveolar Ms, DLECs, bronchial epithelial cells, fibroblasts, and
pulmonary vascular smooth muscle cells. The present study confirms the
ability of M
s and DLECs to express iNOS in response to appropriate
stimuli. Importantly, they provide a physiological model whereby cells
might interact to augment NO production. Specifically, although LPS
alone was unable to induce iNOS in DLECs, coculture with M
s in the
presence of LPS resulted in iNOS expression. This occurred when M
s
and DLECs were physically separated to allow evaluation of the cell
source of iNOS. These data suggest that M
s products released in
response to LPS treatment induced iNOS expression in the DLECs. This is supported by the temporal delay in iNOS expression by the DLECs, presumably the time required for LPS to stimulate the synthesis and
release of M
products into the medium. The nature of these products
was not evaluated in the present study. However, other investigators
have defined various cytokines that either alone or in combination
induce DLEC iNOS expression. These include interleukin-1
, tumor
necrosis factor-
, and interferon-
(13, 33, 35, 36). A recent
study by Gutierrez et al. (13) reported that LPS alone is capable of
stimulating a modest amount of NO release from DLECs. However, this
effect was observed at a later time point (>24 h) than that evaluated
in the present study. In relative terms, M
s appear to be much more
potent generators of NO than DLECs in the in vitro setting. In the
context of the in vivo setting, the close apposition of M
s to DLECs
in vivo would readily permit M
-derived NO as well as endogenous
production by DLECs to contribute to NO-mediated events occurring in
the DLECs.
Although NO appears to be necessary for the reduction in
amiloride-sensitive
Isc in DLEC
monolayers, the data suggest that it must act in concert with other
cellular products to exert its effects. One possible candidate is
peroxynitrite. Peroxynitrite is generated through a chemical reaction
of NO with superoxide anion (18). In separate studies, this molecular
species was shown to be secreted by activated Ms into the epithelial
cell lining fluid (19) and also to inhibit amiloride-sensitive
22Na+
uptake in alveolar type II cells (17). The half-life of this molecule
is <1 s, making it unlikely that M
-derived peroxynitrite is the
active species when the cells are physically separated in the in vitro
separate-culture model. However, this does not preclude an effect in
vivo where the cells are closely apposed. In this regard, the presence
of nitrotyrosine residues has been reported in histological sections of
lung tissue with ARDS due to sepsis (14). Localization along the
blood-gas barrier is consistent with an effect of peroxynitrite on
DLECs (14). In the present study, we were unable to detect
nitrotyrosine residues in DLECs as evidence of their exposure to
peroxynitrite. Although this suggests that peroxynitrite was not
involved, we cannot totally rule out the possibility of an inadequate
sensitivity of the immunohistochemistry in the DLECs. Alternatively,
S-nitrosothiols produced by the
interaction of NO with thiols may mediate the effect of NO on DLECs.
S-nitrosoglutathione, the predominant
form found in alveolar lining fluid, has a prolonged half-life (~3 h)
and bioactivity that includes bronchodilation, inhibition of
receptor-ligand interaction, and inhibition of enzyme function (11).
Importantly, S-nitrosoglutathione
levels were found to be markedly increased in the lavage fluid of
patients with pneumonia, suggesting a possible role for this product in vivo (11).
The mechanism whereby NO and its cofactor might exert their effects on
DLECs requires further study. Many of the effects of NO and its
by-products are known to be mediated via a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway. There
are well-characterized NSC channels in epithelial cells of the rod retina (8) and inner medulla collecting duct of the kidney (24) that
are respectively upregulated and downregulated by cGMP. Studies from
our group demonstrated that neither atrial natriuretic peptide (the
second messenger of which is cGMP) nor 8-bromo-cGMP (a
membrane-permeant analog of cGMP) altered the bioelectric properties of
DLECs within 30 min of exposure (29). However, our present and previous
work has demonstrated that the LPS-treated M effect requires several
hours of incubation. This longer time-dependent effect may indicate
additional mechanisms, possibly related to cGMP generation. Another
possible explanation relates to the recent report by Rotin et al. (37)
indicating that
cytoskeleton-Na+-channel
interactions determine the apical localization of the
-subunit of
the epithelial cell Na+ channel in
DLECs. Coupled with a previous study by Compeau et al. (7)
demonstrating that LPS-treated M
s induced cytoskeletal changes in
DLECs, these data suggest an effect related to altered traffic to or
retention of Na+ channels at the
apical membrane. In this regard,
S-nitrosoglutathione has been shown to
stimulate ADP ribosylation of F-actin in neutrophils (5).
Finally, NO or its metabolites may modulate both transcriptional and
posttranscriptional events in the cell (16, 44), suggesting a possible
effect on expression of the Na+
channel itself or of its regulatory proteins.
The Ussing chamber studies demonstrated that the ion-transport
characteristics of the DLECs were markedly changed as a result of
exposure to LPS-stimulated Ms; there was a virtual disappearance of
amiloride-sensitive
Isc with a
concomitant increase in its flufenamic acid sensitivity. Normally,
~70% of the
Isc of the DLEC
monolayer is amiloride and benzamil sensitive (28-30). It has been
previously demonstrated that the amount of amiloride-sensitive Isc is increased
when DLECs are cultured in serum-free medium (29), whereas it is
decreased when DLECs are cultured from more immature fetal rat lungs
(34) or when DLECs are grown on an immature fetal lung cell-derived
matrix (32). Exposure of rats to sublethal hyperoxia has also been
shown to upregulate non-amiloride-inhibitable Isc (15, 47). The
ionic nature of the amiloride-insensitive Isc is not
completely understood. The weight of evidence suggests that the
amiloride-insensitive
Isc is
Na+ transport. Previous studies
have demonstrated that the
Isc of the DLECs
is entirely dependent on the presence of
Na+ in the bathing medium (28),
that amiloride-insensitive
22Na+
transport is present in tracheal epithelium (23), and that flufenamic
acid, which inhibits NSC channels in colonic epithelium (39), markedly
decreased the Isc
in LPS-M
-exposed DLEC monolayers. In addition, as previously
described (28, 34), the classic inhibitor of
Cl
secretion, bumetanide,
had no influence on DLEC
Isc. However, Pitkanen et al. (32) have noted that
Cl
depletion has a modest
effect on the DLEC
Isc, and
flufenamic acid has also been reported to block
Ca2+-activated
Cl
channels in
Xenopus laevis oocytes (45).
Therefore, one cannot rule out the possibility that some
Cl
secretion is present in
the DLECs but that the basolateral entry pathway for
Cl
is via an
Na+-dependent
bumetanide-insensitive transporter.
In summary, the present study demonstrates that cell-cell interactions in the lung during inflammation may impair fluid resorption and thus resolution of pulmonary edema. The central role for NO generation in this process suggests alternative approaches to the prevention and treatment of lung injury.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the contributions of Jeffrey R. Weidner and Richard A. Mumford (Merck Research Laboratory, Rathway, NJ), who were responsible for the preparation of the synthetic peptide used to develop the anti-inducible nitric oxide synthase rabbit serum. Anti-nitrotyrosine antibodies were kindly provided by Dr. H. Ischiropoulos of the University of Pennsylvania (Philadelphia).
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FOOTNOTES |
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This work was supported by the Medical Research Council (MRC) of Canada Group in the Mechanisms of Organ Injury, the MRC Group in Lung Development, and the Physicians' Services Incorporated Foundation.
H. O'Brodovich is a career scientist of the Heart and Stroke Foundation of Ontario (Canada).
Address for reprint requests: O. D. Rotstein, Toronto Hospital, 200 Elizabeth St. EN9-232, Toronto, Ontario, Canada M5G 2C4.
Received 21 February 1996; accepted in final form 3 December 1997.
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