Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

Sha Zhu1,*, Khaled F. Basiouny1,*, John P. Crow1,2, and Sadis Matalon1,3,4

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 h-1 · 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha , interferon-gamma ], 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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), beta -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 congruent  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 (NO-2) 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% beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 HCO-3) 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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Fig. 1.   CO2 enhanced nitration (A) and suppressed oxidation (B) of surfactant protein (SP) A by lipopolysaccharide (LPS)-activated alveolar macrophages (AMs). SP-A was added to AMs activated with LPS (100 ng/ml) for 6 h and coincubated for an additional 30 or 60 min in absence (-) and presence (+) of 1.2 mM CO2. SP-A was immunoprecipitated with a polyclonal rabbit anti-human SP-A antibody, and protein nitration was detected with Western blotting with a polyclonal anti-nitrotyrosine antibody. SP-A oxidation as assessed by carbonyl formation was detected by Western blotting with a monoclonal anti-dinitrophenyl group antibody after derivatization of protein with 2,4-dinitrophenylhydrozine (DNPH). Nos. on left, molecular mass.

Production of NO-2/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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Fig. 2.   Enhancement of AM nitrite (NO-2) plus nitrate (NO-3) production by LPS and CO2. AM activation and SP-A exposure were performed as in Fig. 1. NO-2 plus NO-3 was measured in medium at that time with Griess reagent. Values are means ± SE; n = 6 experiments. * P < 0.01 compared with AMs cultured in absence of 1.2 mM CO2. # P < 0.01 compared with AMs cultured in presence of 1.2 mM CO2 but in absence of LPS.

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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Fig. 3.   CO2 enhanced nitric oxide synthase (NOS) activation by LPS. AM activation and SP-A exposure were performed as in Fig. 1. NOS activity was measured as conversion of L-[U-14C]arginine to L-[U-14C]citrulline. Values are means ± SE; n = 4 experiments. * P < 0.01 compared with AMs cultured in absence of 1.2 mM CO2. # P < 0.01 compared with AMs cultured in presence of 1.2 mM CO2 but in absence of LPS.

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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Fig. 4.   Enhancement of peroxynitrite (ONOO-)-mediated SP-A nitration by CO2. SP-A (0.1 mg/ml in 15 mM HEPES buffer, pH 7.4) was exposed to indicated concentrations of ONOO- in absence (0 mM) and presence of CO2 (0.6, 1.2, and 1.8 mM). Corresponding PCO2 values in medium, measured with a blood gas analyzer, were 0, 20, 40, and 59 Torr, respectively (means of 2 measurements). Nitrotyrosine was quantified by quantitative ELISA with nitrated bovine serum albumin as a standard as described in METHODS. Values are means ± SE; n >=  4 experiments. All values obtained in presence of CO2 are significantly different from corresponding values in absence of CO2.



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Fig. 5.   Western blotting analysis of ONOO--nitrated SP-A in absence and presence of CO2. SP-A was exposed to ONOO- as in Fig. 4. SP-A protein was analyzed by Western blotting as described in METHODS. Blotting was repeated 3 times with identical results. Nos. on left, molecular mass.

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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Fig. 6.   Enhanced inhibition of SP-A lipid aggregation by CO2. SP-A was exposed to ONOO- as in Fig. 4. Lipid aggregation activity of SP-A was measured as aggregation of dipalmitoylphosphatidylcholine-egg phosphatidylglycerol liposomes at 400 nm in presence of 10 mM CaCl2. Values are means ± SE; n = 4 experiments. * P < 0.01 compared with untreated SP-A control value. # P < 0.01 compared with SP-A treated with 0.25 mM ONOO- in absence of 1.2 mM CO2.

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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Fig. 7.   Liquid chromatography-coupled electrospray ionization tandem mass spectrometry (LC-ESMS/MS) detection of chymotryptic fragment Val158-Lys-Lys-Tyr161 (158VKKY161) of SP-A treated with ONOO- in absence (A) and presence (B) of 1.2 mM CO2. SP-A was digested with chymotrypsin and analyzed by LC-ESMS/MS as described in METHODS. An ion with a mass-to-charge ratio (m/z) = 537 representing 158VKKY161 was detected. Another ion with m/z = 582, corresponding to nitrated form of this, was identified in ONOO--treated SP-A in presence of CO2. Rel Int, relative intensity.



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Fig. 8.   Tandem mass spectrometry analysis of nitrated form of chymotryptic fragment 158VKKY161. Singly charged ions of nitrated peptide (m/z = 582) were focused into a collision cell to produce ion fragments and analyzed in the 3rd quadrupole of mass spectrometer as described in METHODS. A collision-induced dissociation spectrum of m/z = 582 with predicted Y ion series is shown.


                              
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Table 1.   Identification of sites of tyrosine nitration in SP-A exposed to ONOO-


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During lung inflammation, there is a marked upregulation of both · NO and O-2 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 ONOOCO-2, 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 ONOOCO-2 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 NO-2 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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