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,
Departments of
*
Anesthesia,
Medicine, and
Surgery, and
Cardiovascular Research Institute, University of California San Francisco, CA 94110; and
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Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenergic agonists. Inhibition of
iNOS restored the normal catecholamine-mediated up-regulation of
alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a
stable analog of cAMP, restored the normal fluid transport capacity of
the alveolar epithelium after prolonged hemorrhagic shock, whereas
direct stimulation of adenyl cyclase by forskolin had no effect.
Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine
attenuated the iNOS-dependent production of NO in the lung and restored
the normal up-regulation of alveolar fluid clearance by catecholamines
after prolonged hemorrhagic shock. Based on in vitro studies with an
alveolar epithelial cell line, A549 cells, the effect of sulfasalazine
appeared to be mediated in part by inhibition of NF-
B activation,
and the protective effect was mediated by the inhibition of IB
protein degradation. In summary, these results provide the first in
vivo evidence that NO, released within the airspaces of the lung
probably secondary to the NF-B-dependent activation of iNOS, is a
major proximal inflammatory mediator that limits the rate of alveolar
epithelial transport after prolonged hemorrhagic shock by directly
impairing the function of membrane proteins involved in the
-adrenergic receptor-cAMP signaling pathway in alveolar
epithelium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenergic activity and release of
oxidant radicals and IL-1 in the airspaces (9, 10, 11),
mostly by neutrophils that accumulate in the lung after the onset of
hemorrhagic shock (9, 12).
NO is one of the important oxidant radicals released during hemorrhagic
shock in lung and liver by the activation of inducible NO synthase
(iNOS).3 NO plays an
essential role in initiating the inflammatory response in these organs
after onset of hemorrhage (13, 14, 15). Moreover, the
inhibition of iNOS by pretreatment with
N-(iminoethyl)-L-lysine
(L-NIL) in hemorrhaged rats was associated with a
significant decrease in the shock-dependent neutrophil infiltration in
the lung and with a significant reduction in the shock-mediated
increase in extravascular lung water (13). However, it is
still unknown whether induced NO could affect the -adrenergic
receptor-cAMP signaling pathway of the alveolar epithelium in vivo and
thus be a proximal mediator responsible for the oxidant-mediated
inhibition of alveolar epithelial fluid transport after prolonged
hemorrhagic shock.
Therefore, the first objective was to determine whether the release of
NO in the airspaces of the lung was responsible for the shock-mediated
failure of the alveolar epithelium to respond to catecholamines.
Hemorrhagic shock was associated with a significant increase in the
production of nitrite secondary to the expression of iNOS in the lung
and a failure of the alveolar epithelium to up-regulate vectorial fluid
transport in response to -adrenergic agonists. Inhibition of iNOS by
two specific iNOS inhibitors restored the normal alveolar fluid
transport capacity after hemorrhagic shock. The second objective was to
determine whether the shock-mediated release of NO within the airspaces
not only affects the function of the -adrenergic receptor, but also
other membrane proteins, such as adenyl cyclase, that are involved in
the -adrenergic receptor-cAMP signaling pathway in alveolar
epithelium. The third objective was to test the hypothesis that the
activation of the transcription factor, NF-B, may be responsible for
the release of NO in the airspaces of the lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung barrier function studies
Surgical preparation and ventilation. Male Sprague Dawley rats weighing 300–350 g were anesthetized with pentobarbital (60 mg/kg i.p.), and anesthesia was maintained with 30 mg/kg of pentobarbital sodium every 2 h. An endotracheal tube (PE 220) was inserted through a tracheotomy. Pancuronium bromide (0.3 mg/kg/h i.v.) was given for neuromuscular blockade. Catheters (PE 50) were inserted into both carotid arteries to monitor systemic arterial pressure, obtain blood samples, and withdraw blood for induction of prolonged hemorrhagic shock. A catheter was also inserted into the jugular vein to monitor central venous pressure. The rats were maintained in the left lateral decubitus position during the experiments and were ventilated with a constant-volume pump (Harvard Apparatus, Holliston, MA) with an inspired oxygen fraction of 1.0, peak airway pressures of 8–12 cm H2O, supplemented with positive end-expiratory pressure of 3 cm H2O. The respiratory rate was adjusted to maintain the PaCO2 between 35 and 40 mm Hg during the baseline period.
Preparation of instillate. A 5% bovine albumin solution was prepared using Ringer’s lactate and adjusted with NaCl to be iso-osmolar with the circulating plasma of the rat, as previously published (8, 9). Anhydrous Evan’s blue dye (0.5 mg) was added to the albumin solution to confirm the location of the instillate at the end of the study, and 1 µCi of 125I-labeled human serum albumin (Frosst Laboratories, Quebec, Canada) was also added to the albumin solution. The 125I-labeled albumin served as the alveolar protein tracer in all experiments. A sample of the instilled solution was saved for total protein measurement, radioactivity counts, and water-to-dry weight ratio measurements so that the dry weight of the protein solution could be subtracted from the final lung water calculation. In some experiments, propranolol, dibutyryl cAMP (dc-AMP), or forskolin was added to the instilled protein solution.
General protocol
In all experiments, after the surgery, heart rate and systemic
blood pressure were allowed to stabilize for 60 min
(Fig. 1
, A–C). The rat was
placed in the left lateral decubitus position to facilitate liquid
deposition into the left lung 300 min after the onset of hemorrhagic
shock. Hemorrhagic shock was induced by withdrawing blood from the
carotid artery to maintain a mean systemic arterial pressure of 30–35
mm Hg for 60 min. This corresponded to the removal of 9–12 ml of
blood. After 1 h of hemorrhagic shock the rats were resuscitated
with intravascular 4% albumin solution in 0.9% NaCl over 30 min to
maintain a central venous pressure <8 mm Hg, as we have done before
(8, 9). The volume of 4% albumin solution administered
was twice the amount of blood withdrawn.
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At the end of the experiment (1 h after the beginning of the alveolar instillation), the abdomen was opened and the rats were exsanguinated by transecting the abdominal aorta. Urine was obtained for radioactivity counts. The lungs were removed through a median sternotomy. An alveolar fluid sample from the distal airspaces (0.1–0.2 ml) was obtained by gently passing the sampling catheter (PE-50 catheter, 0.5 mm internal diameter) into a wedged position in the instilled area of the left lower lobe. After centrifugation, the total protein concentration and the radioactivity of the liquid sampled were measured. The right and left lungs were homogenized separately for water-to-dry weight ratio measurements and radioactivity counts.
Specific protocols
Group 1. Effect of prolonged hemorrhagic shock and fluid resuscitation (n = 23). The rats were hemorrhaged and resuscitated to determine the effect of prolonged hemorrhagic shock and fluid resuscitation on fluid transport across the lung epithelium. Five hours after the onset of hemorrhagic shock, 3 ml/kg of the 5% bovine albumin solution with 1 µCi of 125I-labeled albumin were instilled into the left lung (n = 6). Because there was no up-regulation of alveolar fluid clearance after prolonged hemorrhagic shock, epinephrine was added to the protein solution instilled into the distal airspaces at a concentration of 10-5 M (n = 5). This concentration of intra-alveolar epinephrine has previously been shown to be sufficient to up-regulate alveolar fluid clearance in pilot experiments. The first series of control studies included rats that underwent the same surgical preparation, were studied for the same period of time, but were neither hemorrhaged nor fluid resuscitated (n = 9). The second series of control studies included rats that were not hemorrhaged or fluid resuscitated, but had their distal airspaces instilled with an albumin solution containing 10-5 M epinephrine for the last hour of the experiment (n = 3).
Group 2. Inhibition of iNOS with amino guanidine or L-NIL (n = 23). NO is one of the important oxidant radicals released during hemorrhagic shock and plays an essential role in the initiation of the inflammatory response in the lung (13). Thus, experiments were performed to determine whether the production of NO in the airspaces would affect alveolar fluid clearance after hemorrhagic shock and fluid resuscitation. Rats were pretreated with either amino guanidine (i.v. bolus 180 mg/kg, then an i.v. infusion of 1.04 mg/kg/h) (n = 4) or L-NIL (4 mg/kg i.p. repeated every 2 h) (n = 5), two unrelated iNOS inhibitors. Epinephrine (10-5 M) was added to the solution instilled into the distal airspaces of the lung of hemorrhaged rats. Control studies included rats that were not hemorrhaged or fluid resuscitated, but were given either amino guanidine (i.v. bolus 180 mg/kg, then an i.v. infusion of 1.04 mg/kg/h) (n = 4) or L-NIL (4 mg/kg i.p. repeated every 2 h) (n = 4) and were instilled with a protein solution containing epinephrine (10-5 M). In pilot experiments (n = 6), neither amino guanidine nor L-NIL affected alveolar fluid clearance in control rats.
Group 3. Stimulation with dc-AMP or forskolin
(n = 14).
These experiments were designed to determine whether NO would not only
affect the function of the -adrenergic receptor, but also that of
other membrane proteins, such as adenyl cyclase, involved in the
-adrenergic receptor-cAMP signaling pathway. Thus, forskolin (5
x 10-5 M) (Sigma, St. Louis, MO), an adenyl
cyclase agonist, was added to the protein solution instilled into the
distal airspaces (n = 3). In addition, the instilled
protein solution contained propranolol at a concentration
(10-4 M) that inhibits the -adrenergic
receptors on the alveolar epithelial cells (7), and
aminophylline, a phosphodiesterase inhibitor, to prevent the rapid
degradation of cAMP (10-4 M). Aminophylline was
also given as continuous i.v. infusion at the rate of 1 mg/kg/h that
was started 30 min before onset of hemorrhagic shock. As a positive
control, a second series of hemorrhaged rats had their airspaces
instilled with dc-AMP (10-4 M; Sigma), a stable
analog of cAMP (n = 4). These rats also had their
airspaces instilled with propranolol (10-4 M)
and were given aminophylline (10-4 M) as a
continuous i.v. infusion (1 mg/kg/h).
Control studies included rats that were neither hemorrhaged nor fluid resuscitated, but had their distal airspaces instilled with an albumin solution containing forskolin (5 x 10-4 M) (n = 3) or dc-AMP (10-4 M) (n = 4). In addition, the instilled protein solution contained propranolol (10-4 M) and aminophylline (10-4 M). Aminophylline was also given as continuous i.v. infusion at the rate of 1 mg/kg/h throughout the experiment.
Group 4. Inhibition of NF-B activation with
pyrrolidine dithiocarbamate (PDTC) or sulfasalazine
(n = 21).
Activation of the proinflammatory transcriptional factor, NF-B,
occurs early (15 min) after the onset of septic (16) and
hemorrhagic shock (17). Also, the in vivo inhibition of
NF-B activation prevents endotoxin-dependent iNOS expression in the
lung (18). Thus, experiments were performed to determine
whether inhibition of activation of NF-B in the lung would affect
the release of NO within the airspaces of the lung after prolonged
hemorrhagic shock. Inhibition of NF-B activation was achieved by
adding sulfasalazine (130 mg/kg/day) (n = 4) to the
drinking water for 3 wk or by pretreating rats with PDTC (200 mg/kg
i.p.) (n = 4) 30 min before onset of hemorrhagic shock.
Sulfasalazine has been shown recently to be a potent and specific
inhibitor of NF-B activation by TNF-, LPS, or phorbol myristate
acetate-treated SW620 human colonic epithelial cells (19).
PDTC has been shown to inhibit NF-B activation in a rat model of
septic shock (18). Epinephrine
(10-5 M) was added to the protein solution
instilled into the distal airspaces of the lung of hemorrhaged rats.
Control studies included rats that were not hemorrhaged or fluid
resuscitated, but were pretreated with either sulfasalazine (130
mg/kg/day) (n = 3) to the drinking water for 3 wk or
with PDTC (200 mg/kg i.p.) (n = 4) and were instilled
with a protein solution containing epinephrine
(10-5 M). In pilot experiments
(n = 6), neither sulfasalazine nor PDTC affected
alveolar fluid clearance in control rats.
Measurements
Hemodynamics, pulmonary gas exchange, and protein concentration. Systemic arterial, central venous, and airway pressures were continuously measured. Arterial blood gases were measured at 1-h intervals. Samples from the instilled protein solution, from final distal airspace fluid, and from initial and final blood were collected to measure total protein concentration with an automated analyzer (AA2; Technicon, Tarrytown, NY).
Albumin flux across endothelial and epithelial barriers. Two different methods were used to measure the flux of albumin across the lung endothelial and epithelial barriers, as we have done before (7, 8, 9). The first method measures residual 125I-labeled albumin (the airspace protein tracer) in the lungs as well as accumulation of 125I-labeled albumin in plasma. The second method measures 131I-labeled albumin (the vascular protein tracer) in the extravascular space of the lungs (7, 8, 9).
Alveolar fluid clearance. Changes in the concentration of the nonlabeled bovine albumin and the instilled 125I-labeled albumin over the study period (1 h) were used to measure fluid clearance from the distal airspaces, as we have done before (7, 8, 9). There is a good correlation between the changes in the concentration of instilled nonlabeled bovine albumin and 125I-labeled albumin. Because some reabsorption may have occurred across distal bronchial epithelium, the term alveolar does not imply that all fluid reabsorption occurred at the alveolar level.
Tracer binding measurement. The 125I binding to albumin was measured by adding trichloroacetic acid (20%) to plasma and alveolar fluid samples. None of the fluid samples ever had >1% of unbound iodine present.
Production of nitrite by alveolar macrophages
To determine nitrite production by alveolar macrophages after prolonged shock, a first series of experiments included rats (n = 4) that were hemorrhaged and fluid resuscitated as described in General Protocol. Then, the rats were exsanguinated, and their lungs were inflated to total lung capacity with cold sterile PBS (10 ml) and lavaged twice.
A second series of experiments included rats that were pretreated with L-NIL (4 mg/kg i.p. repeated every 2 h) (n = 4) before they underwent prolonged hemorrhagic shock and fluid resuscitation. At the end of the experiments, the lungs were inflated to total lung capacity with cold sterile PBS (10 ml) and lavaged twice.
A third series of experiments included rats that were pretreated with sulfasalazine (130 mg/kg/day added to the drinking water for 3 wk) (n = 4) before they underwent prolonged hemorrhagic shock and fluid resuscitation as described previously. At the end of the experiments, the lungs were inflated to total lung capacity with cold sterile PBS (10 ml) and lavaged twice.
Control studies included 1) rats (n = 4) that were neither hemorrhaged nor fluid resuscitated and had their airspaces lavaged twice at the end of the studies with cold sterile PBS (10 ml); and 2) rats that were neither hemorrhaged nor fluid resuscitated, but were either pretreated with L-NIL (4 mg/kg i.p. repeated every 2 h) (n = 4) or sulfasalazine (130 mg/kg/day added to the drinking water for 3 wk) (n = 4) and had their airspaces lavaged twice at the end of the studies with cold sterile PBS (10 ml).
In all experiments, the lavage samples were then centrifuged at 800 x g for 10 min at 4°C to remove cells. The cell pellet was resuspended in 1 ml of sterile RBC lysis buffer (Sigma) for 10 min at room temperature. The cells were again recovered by centrifugation at 800 x g for 10 min at 4°C. Then viable cells were counted, and a small aliquot was removed for cytospin and cell differential staining (Diff-Quick; Dade Diagnostics, Aguada, PR). Cell concentration was adjusted by dilution in serum-free RPMI 1640 medium (University of California at San Francisco Cell Culture Facility) to 5 x 105 cells/ml and equal number of cells (1 x 105) were plated on 96-well tissue culture plates (Falcon, Lincoln Park, NJ). Cells were treated with a range of LPS concentrations from 0 to 10 ng/ml (Escherichia coli 055:B5; Sigma). The alveolar macrophages were then incubated at 37°C (5% CO2) for 24 h, and cell supernatants were collected. Nitrite production was quantified in the harvested supernatant using a modified Griess Reagent (Sigma) with measurement of visible light absorption at 540 nm. Cell differential counts were performed manually. Results are expressed as nitrite production (µM) over 24 h x 105 cells.
Western blot measurement for iNOS
Frozen lungs from hemorrhaged and control rats (n = 5 in each group) were thawed in a tissue homogenization buffer (100 mg lung tissue/ml buffer) including 50 mM Tris, pH 7.4, 20 mM NaCl, 10 mM KCl, 0.1 mM DTT, 1 mM EDTA, 1% SDS, and a protease inhibitor mixture. Then, the lung tissue was homogenized with a polytron (3 x 15-s bursts) and the samples were immediately boiled for 5 min at 95–100°C. Samples were sonicated 2 x 15 s at room temperature. Then the samples were centrifuged at 14,000 x g at 4°C for 30 min and the supernatant was quickly frozen at -70°C. The protein concentration was measured using the bicinchoninic acid assay kit with BSA as the standard (Pierce, Rockford, IL). A portion of the lung tissue lysate was then boiled in Laemmli sample buffer for 5 min at 95–100°C before loading 25 µg of total protein per lane on a 10% Tris-glycine SDS-polyacrylamide gel. The electrophoretically separated proteins were subsequently transferred to nitrocellulose membranes, which were blocked with 5% milk protein in PBS for 120 min. To detect iNOS protein, nitrocellulose membranes were immunoblotted using a commercially available 1:500 or 1:1000 dilution of a monoclonal anti-mouse iNOS Ab overnight at 4°C (Transduction Laboratories, Lexington, KY). The blot was then incubated with HRP-labeled goat anti-mouse Ig (dilution 1:2000). Protein bands were visualized using a chemiluminescence method (SuperSignal; Pierce) and quantitated using a digital image analysis system (ChemiImager; Alpha Innotech, San Leandro, CA).
A549 cell experiments
Cell culture.
All experiments involved A549 cells (American Type Culture Collection,
Manassas, VA), a human lung adenocarcinoma cell line representative of
distal respiratory epithelium. These cells have previously been shown
to be a useful model for studying in vitro NF-B regulation in
response to proinflammatory stimuli (20). Cells were
maintained in a room air/5% CO2 incubator at
37°C using DMEM (Life Technologies, Grand Island, NY) containing 10%
FBS and penicillin/streptomycin (Life Technologies).
Transient transfection and luciferase assay.
To measure NF-B regulation in response to proinflammatory stimuli,
A549 cells were transiently transfected with a plasmid in which the
luciferase gene was driven by three tandem NF-B binding motifs
followed by a minimal IFN-
promoter (three binding sites for
NF-B-Luc, a gift of H. Wong, University of Cincinnati, Cincinnati,
OH). This plasmid previously was demonstrated to be a sensitive tool to
specifically evaluate NF-B activation (20).
Cells were transfected in triplicate, in six-well plates, at a density
of 300,000 cells per well by incubation with cationic liposomes
(Fugene; Roche, Indianapolis, IN) for 48 h in DMEM. Then, the
cells were exposed to a mixture of proinflammatory cytokines (TNF-,
IL-1, IFN- ; Boehringer Mannheim, Indianapolis, IN) or its
vehicle at a concentration of 10 ng/ml. A group of cells were
pretreated with sulfasalazine (2 mM) 3 h before exposure to
proinflammatory cytokines. Twenty-four hours later, cellular proteins
were extracted and analyzed for luciferase activity according to the
manufacturer’s instruction (Promega, Madison, WI) using a luminometer.
Luciferase activity was corrected for total cellular protein and
reported as fold-induction over the control cells (cells that were
transfected and treated with cytokine vehicle alone).
EMSA for NF-B and Western blot measurement for
IB.
A549 cells were plated in 100-mm culture dishes at a density of
1,000,000 cells per dish and exposed 24 h later to a mixture of
proinflammatory cytokines (TNF-, IL-1, IFN- ; Boehringer
Mannheim) or its vehicle for 12 min. Then, nuclear protein extraction
was performed on ice with ice-cold reagents. Cells were initially
washed with cold PBS and harvested by scraping. The cells were pelleted
in 1 ml of PBS at 5000 x g at 4°C for 10 min. The
pellets were washed twice with PBS and resuspended in lysis buffer (10
mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 0.4% Nonidet
P-40, and 0.5 mM PMSF). The suspension was incubated for 5 min on ice
and subsequently centrifuged at 5000 x g at 4°C for
10 min. The supernatant (cytoplasmic proteins) was saved for Western
blot analysis, and 1 cell pellet volume of extraction buffer was added
(20 mM HEPES, pH 7.9, 420 mM NaCl, 0.5 mM DTT, 1 mM EDTA, 0.5 mM PMSF).
The suspension was incubated on ice for 15 min and then centrifuged at
16,000 x g at 4°C for 30 min. The supernatant
(nuclear proteins) was collected and kept at -70°C until use. The
protein concentration was determined using the bicinchoninic acid assay
kit with BSA as the standard (Pierce).
EMSA was performed using a NF-B consensus nucleotide probe (5'-ATG
TGA GGG GAC TTT CCC AGG C-3') that was end-labeled with
[-32P]ATP (Amersham Life Science, Arlington
Heights, IL). Nuclear protein (5 µg) was incubated with 100,000 cpm
of 32P-labeled NF-B consensus nucleotide for
20 min in a total volume of 16.5 µl in a binding buffer consisting of
10 mM Tris buffer, pH 7.5, 1 mM MgCl2, 50 mM
NaCl, 0.5 mM DTT, 0.5 mM EDTA, 4% glycerol, and 1 µg of poly(dI-dC)
(Pharmacia, Piscataway, NJ). The specificity of the DNA/protein binding
was determined by competition reactions in which a 100-fold molar
excess of unlabeled NF-B oligonucleotide was added to the reaction.
After incubation, the samples were loaded onto a nondenaturing minigel
(5% Tris-borate-EDTA), and the gel was subjected to electrophoresis at
10 V/cm. Each gel was then dried and was subjected to
autoradiography.
To detect IB protein by Western blotting, samples of cytoplasmic
proteins saved during the extraction of nuclear proteins were boiled in
Laemmli sample buffer for 5 min at 95–100°C before loading 25 µg
of total protein per lane on a 10% Tris-glycine SDS-polyacrylamide
gel. The electrophoretically separated proteins were subsequently
transferred to nitrocellulose membranes, which were blocked with 5%
milk protein in PBS for 120 min. Then, nitrocellulose membranes were
immunoblotted using a 1:2500 dilution of a polyclonal anti-rabbit
IB Ab overnight at 4°C (a gift from W. Greene, Gladstone
Institute, University of California at San Francisco). The blot was
then incubated with HRP-labeled goat anti-rabbit Ig (dilution
1:1500). Protein bands were visualized using a chemiluminescence method
(SuperSignal; Pierce) and quantitated using a digital image analysis
system (ChemiImager; Alpha Innotech).
Cell viability. Cell viability after exposure to different experimental conditions was measured by a trypan blue exclusion test. In pilot experiments, we determined that cell viability was >95% for all experimental conditions.
Statistics
All the data are summarized as mean ± SEM. One-way ANOVA and the Fisher’s exact t test were used to compare experimental with control groups. A p value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To induce hemorrhagic shock, 38 ± 3% of the blood volume
was withdrawn (9.5 ± 0.7 ml of blood) causing systemic arterial
hypotension (34 ± 5 vs 104 ± 4 mm Hg, p <
0.05) and metabolic acidosis (arterial pH: 7.22 ± 0.02 vs
7.42 ± 0.01, p < 0.05) with a calculated base
deficit of the arterial blood of -15.8 ± 2.3 in hemorrhaged rats
vs -0.5 ± 0.4 in controls (p < 0.05) at
the end of the ischemic phase of shock (Fig. 1
).
Hemorrhagic shock was associated with a failure of the alveolar
epithelium to increase vectorial fluid transport in response to
catecholamines, despite the absence of any increase in the alveolar
epithelial permeability to protein. Protein flux
(125I-labeled albumin) from the airspaces to the
plasma was comparable in hemorrhaged and control rats (0.6 ± 0.2
vs 0.4 ± 0.2, NS). The final-to-initial distal airspace unlabeled
protein concentration did not increase in hemorrhaged rats despite
adding epinephrine to the instilled protein solution to maximize the
response of -adrenergic receptors (Table I). In contrast, when epinephrine was
added to the protein solution instilled into the alveolar space of
control nonhemorrhaged rats, the expected increase in airspace
unlabeled protein concentration occurred (Table I). In response to
catecholamines, alveolar fluid clearance significantly increased in
control rats, but not in hemorrhaged rats (Fig. 2). Comparable values were obtained when
alveolar fluid clearance was measured with the
125I-labeled albumin (control rats: 32 ± 2
to 42 ± 2%, p < 0.05; hemorrhaged rats; 33
± 2 to 31 ± 3%, NS). Finally, hemorrhagic shock was associated
with a small increase in lung endothelial permeability to protein as
indicated by the significant increase in extravascular plasma
equivalents of noninstilled lung in hemorrhaged rats compared with
controls (Table II).
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NO has been shown to be one of the important oxidant radicals
released during hemorrhagic shock and plays an essential role in the
initiation of the inflammatory response in the lung (13).
Thus, the second series of experiments was designed to determine
whether the release of NO in the airspaces of the lung secondary to the
expression of iNOS was responsible for the shock-mediated failure of
the alveolar epithelium to respond to catecholamines. Hemorrhagic shock
was also associated with a significant increase in the expression of
iNOS protein in the lung homogenate of hemorrhaged and
fluid-resuscitated rats compared with controls (Fig. 3). There was a significant increase in
the production of nitrite, one of the major end-products of NO, by
alveolar macrophages removed from hemorrhaged rats 6 h after onset
of hemorrhagic shock and cultured ex vivo for 24 h in presence of
increasing concentrations of LPS compared with controls (Fig. 4).
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Inhibition of iNOS in the lung with L-NIL significantly
decreased the production of nitrite by alveolar macrophages removed
from hemorrhaged rats 6 h after onset of hemorrhagic shock
compared with the values measured in hemorrhaged, but not pretreated
rats (Fig. 4). Inhibition of iNOS in the lung also restored the normal
catecholamine-mediated up-regulation of alveolar liquid clearance
(Table I, Fig. 5). Comparable values were
found when alveolar fluid clearance was measured with the
125I-labeled albumin (amino guanidine-pretreated
rats: 43 ± 2%; L-NIL pretreated rats: 42 ±
3%). Finally, inhibition of iNOS in the lung was associated with a
normalization of lung endothelial permeability to protein in
hemorrhaged rats (Table II).
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). Alveolar fluid clearance was significantly increased, as
expected, in control rats pretreated with either amino guanidine
(46 ± 1%) or L-NIL (45 ± 3%) and was
comparable with that measured in control rats (44 ± 3%).
Comparable values were found when alveolar fluid clearance was measured
with the 125I-labeled albumin (data not shown).
In addition, neither amino guanidine nor L-NIL affected the
rate of alveolar fluid clearance in control rats that did not have
their airspaces instilled with epinephrine (data not shown). Airspace instillation of dc-AMP, but not direct stimulation of adenyl cyclase with forskolin, restores normal alveolar epithelial fluid transport after hemorrhagic shock
The third series of experiments was designed to determine whether
NO would not only affect the function of the -adrenergic receptor,
but also that of other membrane proteins, such as adenyl cyclase,
involved in the -adrenergic receptor-cAMP signaling pathway.
Forskolin, an adenyl cyclase agonist, was added to the protein solution
instilled into the distal airspaces of hemorrhaged rats. Hemorrhaged
rats instilled with dc-AMP, a stable analog of cAMP, served as positive
controls. Rats pretreated either with forskolin or dc-AMP underwent a
hemorrhagic shock similar to that of nonpretreated rats.
Direct stimulation of adenyl cyclase by forskolin in hemorrhaged rats
did not restore the normal catecholamine-mediated up-regulation of
alveolar liquid clearance. The final-to-initial distal airspace
unlabeled protein concentration was 1.39 ± 0.04 in hemorrhaged
rats instilled with forskolin vs 1.34 ± 0.02 in hemorrhaged rats
not instilled with forskolin. Also, comparable values were obtained for
alveolar fluid clearance measured either with the nonlabeled bovine
albumin (Fig. 2) or with the 125I-labeled albumin
(34 ± 2 vs 32 ± 2%, p < 0.05). In
contrast, direct stimulation of adenyl cyclase by forskolin
significantly increased vectorial fluid transport across the alveolar
epithelium of control, nonhemorrhaged rats. The final-to-initial distal
airspace unlabeled protein concentration was 1.55 ± 0.02, and
alveolar fluid clearance measured either with the nonlabeled bovine
albumin (Fig. 2) or with the 125I-labeled albumin
(43 ± 2%) was significantly increased. Moreover, there was a
significant increase in the final-to-initial distal airspace unlabeled
protein concentration (1.52 ± 0.02 vs 1.39 ± 0.04) and the
corresponding alveolar fluid clearance in hemorrhaged rats that had
their airspaces instilled with dc-AMP compared with the values measured
in hemorrhaged rats instilled with forskolin (Fig. 2).
Inhibition of NF-B in the lung restores normal alveolar
epithelial fluid transport after hemorrhagic shock
Activation of the proinflammatory transcriptional factor, NF-B,
occurs early (15 min) after the onset of septic (16) and
hemorrhagic shock (17). Also, in vivo inhibition of
NF-B activation prevents endotoxin-dependent iNOS expression in the
lung (18). Thus, the fourth series of experiments was
designed to determine whether inhibition of activation of NF-B in
the lung by sulfasalazine or PDTC would affect the release of NO within
the airspaces of the lung after prolonged hemorrhagic shock and would
restore the physiological catecholamine-mediated up-regulation of
alveolar liquid clearance. Rats pretreated either with sulfasalazine or
PDTC underwent a hemorrhagic shock similar to that of
nonpretreated rats.
Pretreatment with sulfasalazine significantly decreased the production
of nitrite by alveolar macrophages removed from hemorrhaged rats 6
h after onset of hemorrhagic shock (Fig. 4). This decrease in the
production of nitrite in hemorrhaged rats pretreated with inhibitors of
the activation of NF-B was associated with a restoration of the
physiological catecholamine-mediated up-regulation of alveolar liquid
clearance. The final-to-initial distal airspace unlabeled protein
concentration and the corresponding alveolar fluid clearance were
significantly increased in hemorrhaged rats pretreated either with
sulfasalazine or PDTC compared with the values measured in hemorrhaged,
but not pretreated rats (Table I, Fig. 5). Comparable values were found
when alveolar fluid clearance was measured with the
125I-labeled albumin (sulfasalazine-pretreated
rats: 44 ± 2%; PDTC-pretreated rats: 43 ± 3%). Finally,
pretreatment either with sulfasalazine or PDTC was associated with a
normalization of lung endothelial permeability to protein in
hemorrhaged rats (Table II).
Pretreatment of control rats with either sulfasalazine or PDTC did not
affect the ability of the alveolar epithelium to up-regulate vectorial
fluid transport in response to exogenous -adrenergic agonists (Table I). Alveolar fluid clearance was also significantly increased in
control rats pretreated either with sulfasalazine (45 ± 1%) or
PDTC (43 ± 2%) and comparable with that measured in control, but
not pretreated rats (44 ± 3%). Comparable values were found when
alveolar fluid clearance was measured with the
125I-labeled albumin (data not shown). In
addition, the results of preliminary studies indicate that neither
sulfasalazine nor PDTC affected the rate of alveolar fluid clearance in
control rats that did not have their airspaces instilled with
epinephrine (data not shown).
To demonstrate that sulfasalazine inhibits NF-B activation using an
in vitro model relevant to these studies, we conducted several new
experiments using A549 cells, a human alveolar epithelial cell line. In
the first series of experiments, A549 cells were stimulated for 12 min
with cytomix (a mixture of TNF-, IFN-, and IL-1), an in vitro
model representative of the oxidative stress to the alveolar epithelium
observed after hemorrhagic shock. Then, nuclear proteins were extracted
and EMSA performed. Exposure to a mixture of proinflammatory cytokines
for 12 min significantly increased nuclear translocation of NF-B.
Compared with the values measured in cells exposed to cytokine vehicle
only, the intensity of the retarded B oligonucleotide was increased
6-fold in reactions containing nuclear extracts from
cytokine-treated cells (Fig. 6). The
specificity of the EMSA for NF-B was determined by performing
competitive reactions with unlabeled homologous B. Inclusion of a
100-fold excess of the unlabeled homologous B oligomer completely
inhibited binding of the labeled B oligonucleotide (Fig. 6).
Exposure to proinflammatory cytokines also significantly increased
NF-B activation of a -dependent luciferase reporter construct
(3xIgBLuc) (Fig. 7). Finally,
cytokine-mediated increase in the nuclear translocation of NF-B was
associated with a complete degradation of cytoplasmic I-B protein
in A549 cells (Fig. 8).
|
|
|
B. Compared with the values measured in
cells exposed to cytokines, the intensity of the retarded B
oligonucleotide was decreased by 30% in reactions containing nuclear
extracts from sulfasalazine-pretreated cells (Fig. 6). Exposure to
sulfasalazine also completely abolished NF-B activation of a
-dependent luciferase reporter construct (Fig. 7). Finally,
pretreatment with sulfasalazine significantly attenuated the
cytokine-mediated degradation of cytoplasmic I-B protein in A549
cells (Fig. 8). In summary, these experiments indicate that
sulfasalazine decreases cytokine-mediated translocation of NF-B to
the nucleus and activation of NF-B-responsive genes. |
Discussion |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenergic receptor and adenyl cyclase. Third,
shock-mediated release of NO in the airspaces of the lung depends in
part on the activation and nuclear translocation of NF-B. Preservation of the capacity of the alveolar epithelium to actively remove fluid from the airspaces is critical for the survival of patients with acute lung injury (21). Therefore, we developed an experimental animal model to investigate the mechanisms that control alveolar epithelial fluid transport under clinically relevant pathological conditions responsible for the development of acute lung injury in humans, such hemorrhage or sepsis. The release of endogenous catecholamines up-regulated alveolar epithelial fluid transport by cAMP-dependent mechanisms and prevented alveolar flooding after onset of short-term septic or hemorrhagic shock (6, 7, 8). However, this normal up-regulation of alveolar liquid clearance was inhibited by a neutrophil-dependent oxidative injury to the alveolar epithelium after severe hemorrhage (9), although the molecular mechanisms responsible for the oxidative injury to the alveolar epithelium were unknown.
Several lines of evidence indicate that induced NO may contribute to the inflammatory response and subsequent end-organ damage in the lung after severe hemorrhage. First, hemorrhagic shock produced a time-dependent increase in iNOS activity in the lung (14, 15). Second, shock-mediated expression of some proinflammatory mediators, such as IL-6 and G-CSF, are iNOS dependent because their expression was inhibited by pretreatment with L-NIL and was absent in iNOS knockout mice (13). Third, inhibition of iNOS by pretreatment with L-NIL in hemorrhaged rats was associated with a significant decrease in the shock-dependent neutrophil infiltration in the lung and with a significant reduction in the shock-mediated increase in extravascular lung water (13). In addition, selective inhibition of iNOS attenuated lung damage in acute lung injury caused by endotoxin (22, 23, 24). Thus, the first objective of the study was to determine whether the release of NO in the airspaces of the lung was responsible for the decrease in the alveolar epithelial fluid transport after severe hemorrhage. Inhibition of iNOS by two unrelated iNOS inhibitors prevented the hemorrhage-dependent increase in release of nitrite by alveolar macrophages and restored the normal fluid transport capacity across the alveolar epithelium after severe hemorrhagic shock. In addition, inhibition of iNOS caused a normalization of shock-mediated increase in lung endothelial permeability to protein. Thus, these results provide the first in vivo evidence that induction of NO synthesis in the lung is a critical mechanism responsible for the inhibition of the cAMP-dependent up-regulation of alveolar epithelial fluid transport after severe hemorrhage. It is unlikely that the protective effect observed in hemorrhaged and pretreated rats was due to a nonspecific effect of iNOS inhibitors. Amino guanidine can have effects that are unrelated to inhibition of iNOS activity, such as inhibition of diamine oxidase and nonenzymatic glycosylation. However, both iNOS inhibitors are chemically unrelated and both restored a normal alveolar epithelial fluid transport after severe hemorrhage. Moreover, neither amino guanidine nor L-NIL affected alveolar fluid clearance in control rats.
To further explore how NO decreases the capacity of the alveolar
epithelium to remove fluid from the airspaces after prolonged
hemorrhagic shock, the second objective was to determine whether
shock-dependent release of induced NO would also affect the function of
other membrane proteins involved in the -adrenergic receptor-cAMP
signaling pathway in the alveolar epithelium. Airspace instillation of
dc-AMP, but not direct stimulation of adenyl cyclase by forskolin,
up-regulated vectorial fluid transport across the alveolar epithelium
following hemorrhage. In contrast, both dc-AMP and forskolin stimulated
alveolar fluid transport in control nonhemorrhaged rats. Thus, these
results provided good evidence that the shock-mediated release of
induced NO can alter the function of membrane proteins involved in the
-adrenergic receptor-cAMP signaling pathway in the alveolar
epithelium.
There are several mechanisms that could explain why inhibition of
induced NO restores cAMP-dependent alveolar epithelial fluid transport
after prolonged hemorrhagic shock. The first mechanism could involve
NO-mediated inhibition of cation channels on rat alveolar type II cells
by cGMP-dependent and -independent pathways. For example,
S-nitrosoglutathione, a NO donor, suppressed the activity of
nonselective cation channels on the apical surface of rat alveolar type
II cells, an effect mediated by a cGMP-dependent protein kinase
(25). Also, NO released by NO donors directly inhibited
amiloride-sensitive sodium channels and NaKATPase in confluent rat
alveolar type II cell monolayers by a cGMP-independent mechanism
(26). Recently, peroxynitrite, a strong oxidant produced
by the reaction between NO and superoxide, has been reported to
decrease amiloride-sensitive sodium current in oocytes expressing
-rENaC by a mechanism related to the oxidation of critical
amino acid residues in the rENaC protein (27). The second
mechanism could involve an effect of NO on chloride secretion in lung
epithelial cells. NO has been shown to activate noncystic fibrosis
transmembrane conductance regulator (CFTR) chloride channels in human
lung epithelial cells (A549) by a cGMP-dependent mechanism
(28). In addition, the results of a recent study indicate
that catecholamines and agents that increase cAMP can cause a transient
increase in chloride secretion by the alveolar epithelium of rabbits in
vivo (29). Thus, the release of induced NO within the
airspaces of hemorrhaged rats could result in an unopposed chloride
secretion by the alveolar epithelium in the event sodium absorptive
pathways are perturbed by the effect of NO on the apical alveolar
epithelial cation channels. The third mechanism depends on NO-mediated
inhibition of -adrenergic receptor and adenyl cyclase, membrane
proteins involved in the -adrenergic receptor-cAMP signaling pathway
of the alveolar epithelium. In this study, we found that the release of
NO with the airspaces of the lung limited the rate of alveolar fluid
transport by inhibiting the response of the -adrenergic receptor and
adenyl cyclase to their respective agonists. In contrast, airspace
instillation of dc-AMP significantly increased alveolar fluid clearance
in hemorrhaged rats, indicating that the alveolar epithelial apical
cation channels could still respond to cAMP-dependent stimulation. For
several reasons, it is unlikely that NO primarily affected the function
of the alveolar epithelial apical cation channels in our in vivo
experimental model of acute lung injury. First, the basal alveolar
fluid clearance was not affected by the shock-mediated oxidative stress
to the alveolar epithelium. We have previously reported that the
airspace instillation of amiloride, but not propranolol, a
-adrenergic antagonist, further reduced the rate of alveolar fluid
transport in hemorrhaged rats, indicating that NO-mediated oxidative
stress to the alveolar epithelium preferentially affect the
-adrenergic receptor-cAMP pathway after hemorrhage (9).
Second, in this study, there was up-regulation of alveolar liquid
clearance in hemorrhaged rats by bypassing the -adrenergic
receptor-cAMP pathway with the airspace instillation of dc-AMP.
However, a direct effect of NO on the apical cation channels of the
alveolar epithelium might be more relevant in other experimental models
of acute lung injury, such as septic shock, where the activation of
iNOS and release of NO are quantitatively greater than after
hemorrhage.
It has been reported previously that activation of the proinflammatory
transcriptional factor, NF-B, occurred early (15 min) after the
onset of septic (16) and hemorrhagic shock
(17), and that the in vivo inhibition of activation of
NF-B attenuated endotoxin-dependent iNOS expression in the lung
(18). Thus, the third objective of the study was to
determine whether inhibition of activation of NF-B in the lung would
affect the release of induced NO and alveolar fluid clearance after
prolonged hemorrhagic shock. Inhibition of the activation of NF-B
either with PDTC or sulfasalazine significantly attenuated the
iNOS-dependent production of NO in the lung and restored the normal
fluid transport capacity of the alveolar epithelium after prolonged
hemorrhagic shock. To determine whether sulfasalazine would affect the
NF-B pathway in alveolar epithelial cells, we conducted additional
experiments using A549 cells, a human alveolar epithelial cell line.
A549 cells were stimulated for 12 min with cytomix (a mixture of
TNF-, IFN-, and IL-1), an in vitro model representative of the
oxidative stress to the alveolar epithelium observed after hemorrhagic
shock. Pretreatment with 2 mM of sulfasalazine 3 h before exposure
to cytomix 1) significantly decreased the translocation of NF-B to
the nucleus, 2) completely inhibited the cytomix-mediated increase in
luciferase activity in cells transfected with a -dependent
luciferase reporter construct (3xIgBLuc), and 3) significantly
attenuated the cytokine-mediated degradation of IB in the
cytoplasm. Thus, these results clearly indicate that sulfasalazine
attenuates NF-B translocation and activation in alveolar epithelial
cells stimulated by proinflammatory cytokines. These data are also in
accordance with recent studies from other investigators that have
reported that sulfasalazine is a specific inhibitor of NF-B in
colonic epithelial cells stimulated with TNF- (19), an
effect in part dependent on the inhibition of the phosphorylation of
IB by sulfasalazine (30). Interestingly, the effect
of sulfasalazine appeared to be specific for the NFB pathway because
this compound did not block the activation of AP1 by TNF-
(19). Moreover, it was not due to an antioxidant effect
because it was not observed with exposure to sulfasalazine moieties,
5-aminosalicylic acid or sulfapyridine, despite the fact that
sulfasalazine and its moieties have comparable radical scavenging
activities (19). In summary, these in vitro experiments
indicate that sulfasalazine decreases the translocation of NF-B to
the nucleus and the activation of NF-B responsive genes in an
alveolar epithelial cell line.
There are several lines of evidence that the activation of NF-B in
the airspaces of the lung may be an important factor in the development
of the inflammatory response in the lung after fluid resuscitation from
prolonged hemorrhagic shock. First, previous studies have shown that
hemorrhage leads to a rapid in vivo activation in the lung of NF-B
through xanthine oxidase-dependent and -independent mechanisms
(31). Hemorrhage is followed by two phases of NF-B
activation. The first phase, which is xanthine oxidase-independent,
occurs immediately after blood loss. Hemorrhage produces intense
pulmonary vasoconstriction, local release of catecholamines, and
increased generation of reactive oxygen intermediates that can activate
NF-B (17). At later time points, cyclic AMP response
element binding protein (CREB)/NF-B interactions and increased
cytokine expression may contribute to the xanthine oxidase-dependent
activation of NF-B (31). Second, there is experimental
evidence that NF-B activation is required for cytokine induction of
iNOS protein. Indeed, a recent study has identified four NF-B
enhancer elements upstream in the human iNOS promoter that confer
inducibility to TNF- and IL-1 (32). Also, earlier
studies have shown that the iNOS gene is transcriptionally regulated by
NF-B-dependent mechanisms (33, 34), although in some
cells, such as alveolar macrophages, there is also a cAMP-mediated
NF-B-independent pathway for iNOS activation (35).
Moreover, the results of a recent study indicated that low pH, as
observed in hemorrhagic shock, may increase the expression of iNOS
through the activation of NF-B in macrophages (36).
Third, the results presented here are the first in vivo evidence that
the activation of NF-B may contribute to the shock-mediated decrease
in the ability of the alveolar epithelium to actively remove fluid from
the airspaces after prolonged hemorrhagic shock.
For several reasons, it is unlikely that the protective effect observed
in hemorrhaged and pretreated rats was due to a nonspecific effect of
PDTC or sulfasalazine. First, although PDTC has antioxidant effects
unrelated to inhibition of the activation of NF-B that could
contribute to the observed effect in our experimental model
(37), both chemically unrelated NF-B inhibitors, PDTC
and sulfasalazine, restored a normal alveolar epithelial fluid
transport after severe hemorrhage. In addition, in contrast to its
salicylate moiety, 5-aminosalicylic acid, sulfasalazine has recently
been shown in vitro to be a specific inhibitor of the activation of
NF-B by interfering with IB phosphorylation (19, 30). Second, neither PDTC nor sulfasalazine affected alveolar
fluid clearance in control rats.
Clinical implications
What is the clinical importance of the rate of alveolar epithelial fluid transport in patients with acute lung injury? The results of earlier clinical studies indicated that the presence of intact alveolar liquid clearance early after the onset of hydrostatic or high permeability edema was associated with a shorter duration of mechanical ventilation and a better overall outcome (21). However, the rate of alveolar liquid clearance has recently been measured in a large number of patients with acute lung injury and hydrostatic pulmonary edema. Results of these clinical studies indicate that 50–60% of the patients with pulmonary edema have impaired or submaximal alveolar liquid clearance (38, 39). Moreover, impaired or submaximal alveolar liquid clearance was an early predictor of prolonged duration of mechanical ventilation and higher hospital mortality in patients with acute lung injury (38). The mechanisms responsible for this reduction in the rate of alveolar liquid clearance have not been adequately explored, although the results of a recent clinical study revealed up-regulated production of reactive-oxygen-nitrogen intermediates in the alveolar spaces of patients with acute lung injury that was associated with impaired alveolar fluid clearance (40). Thus, this study provides new insights regarding the potential contribution of a NO-dependent oxidant pathway that can inhibit alveolar epithelial fluid transport in vivo following severe hemorrhagic shock, an important cause of acute lung injury in humans.
In summary, these results provide the first in vivo evidence that NO,
released within the airspaces secondary to the NF-B-dependent
activation of iNOS, is a major proximal mediator that can limit the
rate of alveolar epithelial transport after prolonged hemorrhagic shock
by altering the function of membrane proteins involved in the
-adrenergic receptor-cAMP signaling pathway in the alveolar
epithelium. This mechanism may be important to explain the clinical
evidence that some patients with acute lung injury have impaired rates
of alveolar epithelial fluid clearance.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Francois Pittet, Department of Anesthesia and Perioperative Care, Room 3C-38, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, CA 94110.
3 Abbreviations used in this paper iNOS, inducible NO synthase; L-NIL, N-(iminoethyl)-L-lysine; dc-AMP, dibutyryl cAMP; PDTC, pyrrolidine dithiocarbamate.
Received for publication March 13, 2000. Accepted for publication March 6, 2001.
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), hemorrhaged and resuscitated rats
(•), hemorrhaged and resuscitated rats pretreated with
L-NIL (
), and hemorrhaged and resuscitated rats
pretreated with sulfasalazine (
); *, p < 0.05
from control rats.
