Departments of 1 Anesthesiology, 2 Pharmacology and Toxicology, 3 Physiology and Biophysics, and 4 Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35233-6810
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ABSTRACT |
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We assessed whether reactive oxygen-nitrogen
intermediates generated by alveolar macrophages (AMs) oxidized and
nitrated human surfactant protein (SP) A. SP-A was exposed to
lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4)
for 30 min in the presence and absence of 1.2 mM CO2. In
the presence of CO2, lipopolysaccharide-stimulated AMs had
significantly higher nitric oxide synthase (NOS) activity (as
quantified by the conversion of
L-[U-14C]arginine to
L-[U-14C]citrulline) and secreted
threefold higher levels of nitrate plus nitrite in the medium [28 ± 3 vs. 6 ± 1 (SE) nmol · 6.5 h1 · 106
AMs
1]. Western blotting studies of
immunoprecipitated SP-A indicated that CO2 enhanced SP-A
nitration by AMs and decreased carbonyl formation. CO2
(0-1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A
nitration in a dose-dependent fashion. Peroxynitrite decreased the
ability of SP-A to aggregate lipids, and this inhibition was augmented
by 1.2 mM CO2. Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr164 and Tyr166) in the absence of
CO2 and three tyrosines (Tyr164,
Tyr166, and Tyr161) in the presence of 1.2 mM
CO2. These findings indicate that physiological levels of
peroxynitrite, produced by activated AMs, nitrate SP-A and that
CO2 increased nitration, at least partially, by enhancing
enzymatic nitric oxide production.
nitric oxide; peroxynitrite; nitrotyrosine; protein oxidation; alveolar epithelium; acute respiratory distress syndrome; hypercapnia; mass spectrometry
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) A, the most abundant pulmonary
SP, is responsible for the immunomodulatory functions of pulmonary surfactant (33). Previously, Greis et al. (13) have shown that exposure of human SP-A to authentic peroxynitrite
(ONOO), 3-morpholinosydnonimine (SIN-1), or
tetranitromethane in the presence and absence of surfactant lipids
resulted in nitration of two of the eight tyrosines located in its
carbohydrate recognition domain. Nitrated SP-A has a decreased ability
to aggregate lipids and bind mannose in vitro (18, 36) and does not
facilitate the adherence and phagocytosis of Pneumocystis
carinii to rat alveolar macrophages (AMs), a necessary step for the
killing of P. carinii (37).
Exposure of mice or rat alveolar macrophages (AMs) in vivo or in vitro
to diverse stimuli of inflammation such as cytokines [interleukin-1, tumor necrosis factor (TNF)-,
interferon-
], lipopolysaccharides (LPS), various pathogens,
respirable dusts, or oxidant gases causes them to release both nitric
oxide (· NO) via upregulation of inducible nitric oxide
synthase (iNOS) (11) and superoxide
(O
2) via membrane-bound NADPH oxidase
(31). The product of the reaction of · NO with
O
2, which proceeds at a near
diffusion-limited rate constant (6.7 × 109
M
1 · s
1) (23),
is ONOO
. Indeed, LPS-stimulated rat AMs have been
shown to generate ONOO
at a rate of 0.1 nmol · min
1 · 106
cells
1 (20). Due to the relatively higher
concentration of CO2 in plasma (1.2 mM), the majority of
ONOO
generated in biological fluids, such as in the
epithelial lining fluid, will react with CO2 to form an
ONOOCO
2 intermediate that promotes
nitration of phenolics (7, 12, 21, 22). However, the mechanisms
responsible for the nitration of proteins are in dispute. Berlett et
al. (5) reported that in the absence of CO2,
ONOO
is an oxidizing but not a nitrating agent.
Pfeiffer and Mayer (24) have recently shown that
ONOO
does not nitrate free tyrosine in either the
presence or absence of CO2. Furthermore, myeloperoxidase
and eosinoperoxidase may interact with hydrogen peroxide, nitrite, and
halides to produce reactive intermediates capable of nitrating,
chlorinating, and oxidizing a variety of targets including proteins (9,
10). Thus it is unclear whether activated AMs, which lack peroxidase and are present in large quantities in the alveolar epithelial lining
fluid in inflammatory conditions, contribute to the nitration and
oxidation of proteins detected in the edema fluid of patients with
acute lung injury (35).
Herein we report that the reactive species produced by LPS-activated
rat AMs oxidize and nitrate human SP-A. Furthermore, physiological
concentrations of CO2 enhance
ONOO-induced nitration and decrease oxidation. To
investigate the mechanisms involved, we measured the production of
reactive nitrogen species by AMs and the extent of SP-A nitration by
ONOO
in the presence and absence of various levels
of CO2 in vitro and identified the specific tyrosine
residues nitrated by mass spectrometry. Our results indicate that
CO2 enhances SP-A nitration, at least partially, by
enhancing enzymatic · NO production.
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METHODS |
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Materials and animals. Slide-A-Lyzer cassettes
(10-kDa molecular-mass cutoff), trifluoroacetic acid, and
the bicinchoninic acid protein assay reagent kit were obtained from
Pierce Chemical (Rockford, IL). Chymotrypsin (EC 3.4.21.1; from bovine
pancreas), nitrate reductase (EC 1.6.6.2; from Aspergillus
species), and protein G agarose were from Boehringer Mannheim
(Indianapolis, IN). Calmodulin (from bovine brain) was from Calbiochem
(San Diego, CA). Dipalmitoylphosphatidylcholine and egg
phosphatidylglycerol were from Avanti Polar Lipids (Alabaster, AL).
Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate kit
came from Promega (Madison, WI).
L-[U-14C]arginine (11.6 GBq/mmol)
was obtained from NEN Life Science Products (Boston, MA). Enhanced
chemiluminescence Western blotting detection reagents were from
Amersham Pharmacia Biotech (Piscataway, NJ). SIN-1 was a kind gift from
Cassella (Frankfurt, Germany). Horseradish peroxidase-conjugated donkey
anti-mouse and donkey anti-rabbit IgGs were from Accurate Chemical and
Scientific (Westbury, NY). Rabbit polyclonal antibody against human
SP-A and monoclonal anti-nitrotyrosine antibody were generously
provided by Drs. David S. Phelps (Pennsylvania State University,
Hershey, PA) and Joseph S. Beckman (University of Alabama at
Birmingham, Birmingham, AL), respectively. Triton X-100 and EDTA were
from EM Science (a Division of EM Industries, Cherry Hill, NJ). Sodium
nitrite, hydrogen peroxide, and n-butanol were from Fisher
Scientific (Fair Lawn, NJ). Dulbecco's modified Eagle's medium (DMEM)
was from GIBCO BRL (Life Technologies, Grand Island, NY). Alkaline
phosphatase-conjugated monoclonal anti-dinitrophenyl group IgE, HEPES,
3-nitro-L-tyrosine, LPS (serotype 055:B5 from
Escherichia coli), rat IgG, NADPH, sulfanilamide, N-(1-naphthyl)ethylenediamine dihydrochloride, sodium nitrate, sucrose, dithiothreitol, aprotinin, leupeptin, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, 2,4-dinitrophenylhydrozine (DNPH), -mercaptoethanol, guanidine hydrochloride, iodoacetic acid,
ammonium bicarbonate, L-valine, L-citrulline,
FAD, flavin mononucleotide, (6R)-5,6,7,8-tetrahydrobiopterin,
L-arginine, Dowex-50W (200-400 mesh, 8% cross-linked,
Na+ form), EGTA, and S-ethylisothiourea were from
Sigma (St. Louis, MO). ONOO
was synthesized from
sodium nitrite and hydrogen peroxide as described previously (4). Male
Sprague-Dawley rats were obtained from Charles River (Indianapolis, IN).
Purification of human SP-A. Endotoxin-free (< 0.01 endotoxin
unit/ml) SP-A was purified from bronchoalveolar lavage fluid of
patients with alveolar proteinosis by n-butanol extraction as
previously described (16). SP-A was dissolved in 10 mM HEPES, pH 7.4. Protein concentration was determined by the bicinchoninic acid method
and stored in small aliquots at 20°C until used. SP-A purity
was demonstrated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting, and function was
checked by its ability to aggregate lipids as previously described (15,
18, 36).
Exposure of SP-A to LPS-activated rat AMs. AMs were isolated by
pulmonary lavage of rats as previously described (37). AMs were then
suspended in DMEM containing 15 mM HEPES, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 4 µg/ml of gentamicin, and 0.5 µg/ml of
amphotericin B, pH 7.4 (buffer A) and plated on rat IgG-coated,
12-well tissue culture plates at a density of 1 × 106
cells/well at 37°C in room air for 30 min. Unattached cells were then washed away, and LPS (100 ng/ml) was added to buffer A and incubated in room air for an additional 6 h. At this time, human SP-A
was added to the medium to a final concentration of 1 mg/ml for 30 min.
AMs were solubilized with 1% Triton X-100 and immediately frozen to
70°C.
In a second series of experiments designed to investigate the
contribution of intermediates produced by the interaction of CO2 and ONOO in SP-A nitration and
oxidation, AMs were suspended in buffer A supplemented with 25 mM sodium bicarbonate (NaHCO3) and incubated in 5%
CO2-95% air (PCO2
40 Torr, pH = 7.4). HCO
3, PCO2, and pH values in the medium
were measured with a blood gas analyzer at 37°C (model 1306, Instrumentation Laboratory, Lexington, MA).
Measurement of nitrate and nitrite in the medium. The culture
medium was collected after 6 h of activation of AMs with LPS and a
subsequent 30 min of incubation with SP-A. Nitrite
(NO2) alone and total nitrate
(NO
3) plus
NO
2, after the reduction of
NO
3 to
NO
2 with nitrate reductase, were
measured with the Griess reaction. Briefly, 250 µl of each sample
were incubated with an equal volume of 0.2 mM NADPH in 50 mM phosphate
buffer, pH 7.5, at room temperature for 2 h in the presence of 0.2 U/ml
of nitrate reductase, followed by 500 µl of Griess reagent containing
0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine
dihydrochloride for 10 min. Absorbance was measured at 550 nm, and
NO
2 concentration was determined with
NaNO2 and NaNO3 standards.
Measurements of nitric oxide synthase activity. Nitric oxide synthase (NOS) activity in stimulated and control AMs was measured by the conversion of L-[U-14C]arginine to L-[U-14C]- citrulline (27). Briefly, the cells were washed with PBS and suspended in extraction buffer (20 mM HEPES, pH 7.5, containing 320 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, 2 µg/ml of aprotinin, 10 µg/ml of leupeptin, 10 µg/ml of soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride). The cells were then collected by centrifugation, resuspended in a small volume of extraction buffer, and disrupted by sonication twice for 10 s, with 30 s of cooling in ice water in-between use of the Sonifier cell disruptor (model W185, Heat Systems-Ultrasonic, Plainview, NY).
To measure the conversion of L-[U-14C]arginine to L-[U-14C]- citrulline, 18 µl of cell lysate were added to 100 µl of 37°C prewarmed buffer consisting of 50 mM potassium phosphate, pH 7.2, 60 mM L-valine, 1.2 mM L-citrulline, 120 µM NADPH, 1 µM FAD, 1 µM flavin mononucleotide, 10 µM (6R)-5,6,7,8-tetrahydrobiopterin, 100 nM calmodulin, 0.24 mM CaCl2, 1.2 mM MgCl2, and 24 µM L-arginine and L-[U-14C]arginine (2,500 Bq). The reaction mixtures were incubated at 37°C for 10 min while shaking, and the reaction was terminated by the removal of arginine and dilution by the addition of 1.5 ml of cold 1:1 (vol/vol) H2O-Dowex-50W (200-400 mesh, 8% cross-linked, Na+ form) followed by a 20-s vortex. Another 2 ml of water were added and left in the resin to settle for 10 min. An aliquot of 2 ml of supernatant was removed and mixed with 20 ml of scintillation fluid to achieve a homogeneous solution. Radioactivity of the formed L-[U-14C]citrulline was measured with a liquid scintillation counter (LKB 1214 Packbeta liquid scintillation counter). The total activity of NOS was determined from the difference between the L-[U-14C]citrulline produced in the absence and presence of both 2 mM EGTA and 2 mM S-ethylisothiourea (a potent NOS inhibitor). NOS activity is expressed as picomoles of L-citrulline per minute per milligram of protein.
Immunoprecipitation and Western blot detection of SP-A and nitrotyrosine. All samples were precleared by incubation with 25 µl/ml of protein G agarose and spun at 6,000 g for 1 min. The supernatant was collected and incubated with a rabbit polyclonal antibody against human SP-A (1 µg/ml) at 4°C for 2 h. Protein G agarose beads (70 µl/ml) were then added, and the mixtures were incubated for an additional 30 min while rocking. The samples were then spun down at 6,700 g for 2 min, and the supernatant was removed. The beads were washed three times with a lysis buffer (10 mM phosphate buffer containing 200 mM NaCl and 1% Triton X-100, pH 7.4) and resuspended in SDS-PAGE sample buffer. The proteins were separated with 12% SDS-PAGE and transferred to nitrocellulose membranes. SP-A and its extent of nitration were identified by immunoblotting with a rabbit anti-human SP-A antibody and a monoclonal anti-nitrotyrosine antibody (0.2 µg/ml), respectively, followed by horseradish peroxidase-conjugated goat IgG against the appropriate IgG. Immunoreactive protein complexes were detected by enhanced chemiluminescence Western blotting detection reagents. In addition, the extent of SP-A oxidation was detected as carbonyl formation as described in Western blot detection of carbonyl formation in SP-A.
Western blot detection of carbonyl formation in SP-A. After the
addition of 12% SDS (1 volume), samples were derivatized with the
addition of 2 volumes of 20 mM DNPH in 10% (vol/vol) trifluoroacetic acid and incubated at room temperature for 30 min. A negative control
was performed by incubation of a sample with 2 volumes of 10%
trifluoroacetic acid without DNPH. The mixtures were then neutralized
by the addition of ~1.5 volumes of 2 M Tris-30% glycerol-19% -mercaptoethanol and separated with 12% SDS-PAGE. Carbonyls were detected with an alkaline phosphatase-conjugated monoclonal
anti-dinitrophenyl group antibody (IgE) with a nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl-1-phosphate kit.
Effects of CO2 on
ONOO- or SIN-1-induced SP-A
nitration and oxidation. Human SP-A was dissolved in 15 mM HEPES at
pH 7.4. NaHCO3 (12.5, 25, or 37.5 mM) was then added into
the medium, resulting in a steady-state concentration of
CO2 of 0.6, 1.2, and 1.8 mM, respectively, as calculated
with the Henderson-Hasselbach equation [pH = pKa + log([B]/[A]), where pH = 7.4, pKa (acidic dissociation constant) = 6.1, and
[A] and [B] are the
concentrations of conjugated acid (CO2) and base
(HCO
3), respectively]. One milliliter of each sample was placed into 1.5-ml microcentrifuge tubes that were hermetically sealed and incubated at 37°C for at
least 10 min. At that time, ONOO
(0.1, 0.25, or 0.5 mM) or SIN-1 (0.5 mM) was added into each tube, and the mixtures were
incubated at 37°C for 30 min. The extent of SP-A nitration and
oxidation was detected as nitrotyrosine and carbonyl formation,
respectively, by utilizing an enzyme-linked immunosorbent assay (ELISA)
(36) and Western blotting after resolution of the protein on 12%
SDS-PAGE. PCO2 and pH values of the
medium were measured at the end of the experiment with a blood gas
analyzer (model 1306, Instrumentation Laboratory, Lexington, MA).
Lipid aggregation. SP-A lipid aggregation activity was measured by monitoring the aggregation of a mixture of egg dipalmitoylphosphatidylcholine and synthetic phosphatidylglycerol liposomes by SP-A at 400 nm in the presence of 5 mM CaCl2 as previously described (36).
Identification of tyrosine nitration sites on SP-A by mass spectrometry. Two hundred micrograms of normal or nitrated human SP-A were lyophilized and resuspended in 200 µl of argon-purged Tris · HCl buffer containing 6 M guanidine hydrochloride, pH 8.6, as previously described (13). These samples were reduced with dithiothreitol (30-fold molar excess relative to cysteine) at 37°C for 2 h in an argon environment, carboxymethylated with a 5-fold molar excess of freshly prepared iodoacetic acid at room temperature for 2 h in the dark, dialyzed extensively through 10-kDa molecular-mass cutoff Slide-A-Lyzer cassettes at 4°C, lyophilized, and resuspended in 150 µl of 100 mM ammonium bicarbonate. SP-A samples (200 µg) were then digested by 15 µg of chymotrypsin at 37°C for 8 h. Digested SP-A samples were lyophilized and resuspended in 0.1% formic acid solution, and a fraction of the samples (typically 10-20 µl) were then loaded onto a capillary C18 column for liquid chromatography-coupled electrospray ionization tandem mass spectrometry analysis as previously described (13).
Statistical analysis. Statistical analysis among means were determined with one-way analysis of variance and Bonferroni modification of Student's t-test. Results are expressed as means ± SE. Significance was set at P < 0.05 compared with corresponding control values.
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RESULTS |
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SP-A modifications by LPS-activated rat AMs. Incubation of
LPS-stimulated AMs with SP-A for 30 or 60 min in room air resulted in
nitration and oxidation of both of its monomer (35-kDa) and dimer
(58-kDa) bands (Fig. 1). Significantly higher levels of SP-A nitration and decreased levels of carbonyl formation were seen
when SP-A was coincubated with LPS-activated AMs in the presence of 1.2 mM CO2 (Fig. 1). Also, the presence of 1.2 mM
CO2 (24 mM HCO3)
significantly inhibited the degradation of SP-A by LPS-stimulated AMs
(data not shown). This result is consistent with a
CO2-mediated shift from oxidation toward nitration and the
general consensus that oxidized proteins are more susceptible to
protease digestion (6).
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Production of
NO2/NO
3
by AMs: role of CO2. LPS
significantly augmented total · NO production by AMs (Fig.
2). In the presence of 1.2 mM CO2, the
production of total NO
2 plus
NO
3 was threefold higher than in the
absence of CO2. In all cases, the ratio of
NO
2 to
NO
3 remained equal to 1.
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CO2 enhanced total NOS activity by
LPS. Total NOS activity in cultured AMs as measured by
L-[U-14C]citrulline formation from
L-[U-14C]arginine was also
increased by 1.2 mM CO2 (Fig. 3).
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Nitration and oxidation of SP-A by
ONOO: role of
CO2. ONOO
(0-0.5
mM)-mediated SP-A nitration as measured by ELISA was enhanced in a
concentration-dependent fashion by 0-1.2 mM CO2 (Fig.
4). Higher levels of CO2 (1.8 mM) did not further augment ONOO
-induced nitration.
Western blot analysis of ONOO
-treated SP-A showed a
similar enhancement of nitration (Fig. 5)
by CO2 and a decreased level of carbonyl formation (data
not shown). Incubation of SP-A (1 mg/ml) with SIN-1 (0.5 mM) at
37°C for 2 h resulted in a significant degree of nitration that was augmented by ~50% in the presence of 1.2 mM CO2
[0.172 ± 0.009 (SE) vs. 0.255 ± 0.017 mol nitrotyrosine/mol
SP-A, respectively; P < 0.05; n = 4 experiments].
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Enhanced inhibition of SP-A lipid aggregation by
CO2. In the presence of 1.2 mM CO2,
ONOO (0.25 mM) inhibited lipid aggregation by 48.6%
compared with 22.9% with ONOO
alone (Fig.
6). This result is also consistent with the
enhanced nitration in the presence of CO2 (Fig. 4).
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Identification of tyrosine nitration sites on SP-A by mass
spectrometry. Exposure of SP-A to ONOO (0.5 mM)
in room air resulted in nitration of either Tyr164 or
Tyr166 and, to much lesser extent, Tyr220, in
agreement with previous findings by Greis et al. (13). However, when
SP-A was incubated with ONOO
in the presence of 1.2 mM CO2, an additional tyrosine (Tyr161) was
nitrated (Figs. 7 and
8). The sites of SP-A nitration by ONOO
are summarized in Table
1.
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DISCUSSION |
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During lung inflammation, there is a marked upregulation of both
· NO and O2 production by
AMs (3, 14, 25, 26, 31, 33) in close proximity to alveolar epithelial cells and pulmonary SPs. The reaction product of · NO and
O
2 is ONOO
, a
potent oxidant and nitrating agent. Significantly higher nitrotyrosine levels have been measured in the lungs of pediatric patients who died
from acute lung injury (17) and in the lungs of rats exposed to
endotoxin (32) or hyperoxia (15). Pulmonary edema fluid obtained from
the alveolar space of patients with either acute lung injury or
noncardiogenic hydrostatic edema contains very high levels of
NO
3 (>100 µM). Furthermore,
albumin immunoprecipitated from both edema fluid and plasma obtained
from these patients is both nitrated and oxidized (35).
The results of our studies indicate that LPS-stimulated rat AMs produce
reactive oxygen-nitrogen intermediates that nitrate and oxidize human
SP-A. CO2 in concentrations present in the normal alveolar
space and plasma (1.2 mM, which corresponds to a
PCO2 of 40 Torr) diminishes oxidation
and enhances nitration both by increasing the formation of a nitrating
species from ONOO and by increasing · NO
production. Because there are eight tyrosines in the carbohydrate
recognition domain of SP-A, the results of the ELISA studies (Fig. 3)
indicate that in the absence of CO2, exposure of SP-A to
ONOO
(0.5 mM) resulted in nitration of about one of
these eight tyrosines. Sequencing of nitrated peptides indicated that
nitration was equally distributed between Tyr164 and
Tyr166. In the presence of 1.2 mM CO2,
approximately two tyrosines per monomeric SP-A were nitrated. The
results of these studies are in excellent agreement with our mass
spectrometry measurements of chymotryptic fragments of SP-A, indicating
that in the presence of CO2, Tyr161 is also
nitrated. Berlett et al. (5) also reported that CO2 enhances ONOO
nitration of a tyrosine in glutamine
synthetase and, at the same time, diminished methionine oxidation.
However, these authors could not identify nitration in the absence of
CO2 even at much higher concentrations (4 mM) than what we
used in this study.
Previous studies have shown that
ONOOCO2, the species formed from
the interaction of ONOO
and CO2, is a
more efficient nitrating agent than ONOO
itself. Indeed, even small concentrations of
CO2 enhanced
ONOO
-induced nitration of tyrosine (30),
p-hydroxy- phenylacetate (29), and albumin (12) while
decreasing the hydroxylation of phenylalanine (30) and benzoate and the
oxidation of cytochrome c (7). Nitration of SP-A caused by
SIN-1, ONOO
, and tetranitromethane has been shown to
decrease its ability to bind to lipids and mannose (15, 18, 36) and
inhibits its ability to enhance killing of P. carinii by rat
AMs (37).
Results presented herein indicate that in addition to acting as a
nitration-promoting agent, CO2 also enhances the production of · NO by LPS-activated AMs. This effect of CO2
has not been reported previously and is clinically important because
permissive hypercapnia has been proposed as an effective way of
ventilating patients with acute respiratory distress syndrome (1, 2). The exact mechanism of the CO2 effect on · NO
production was not determined. However, it is possible that the
formation of the ONOOCO2 adduct, a
species with a much shorter half-life than ONOO
, may
mitigate the oxidative inactivation of NOS by ONOO
(19, 28).
A recent study (35) has found high levels of
NO2 and
NO
3 (>100 µM) in pulmonary edema fluid from acute lung injury patients and that SP-A was also nitrated, which may contribute to the development and progression of acute lung injury. However, the presence of CO2
has been shown to protect bacteria (Escherichia coli) and
parasites (Trypanosoma cruzi) from killing by
ONOO
(8, 34). Thus although CO2 enhances
ONOO
-induced SP-A nitration, which interferes with
its biophysical and host defense properties, it may decrease injury to
other important components of the alveolar epithelium by limiting
ONOO
oxidation.
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ACKNOWLEDGEMENTS |
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We thank Marion Kirk (University of Alabama at Birmingham Comprehensive Cancer Center Mass Spectrometry Shared Facility, Birmingham, AL) for assistance with the LC-ESMS/MS analysis. This Shared Facility is supported in part by National Cancer Institute Grant P30-CA-13148. We also acknowledge the excellent technical assistance of Carpantato Myles and Glenda Davis and the many helpful comments of Drs. Jacinda B. Sampson, Jason P. Eiserich, and Joseph Beckman.
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FOOTNOTES |
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*
S. Zhu and K. F. Basiouny contributed equally to this
work.
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. §1734 solely to indicate this fact.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-31197 and HL-51173 and Office of Naval Research Grant N00014-97-1-0309.
S. Zhu was partially supported by National Heart, Lung, and Blood Institute Grant HL-07553.
Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., Birmingham, AL 35233-6810 (E-mail: Sadis.Matalon{at}ccc.uab.edu).
Received 1 July 1999; accepted in final form 28 December 1999.
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