Departments of 1 Pediatric Respiratory Medicine, 4 Microbiology, 2 Anesthesiology, and 3 Chemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908
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ABSTRACT |
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Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.
S-nitrosothiol; asthma; nitric oxide; gold
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INTRODUCTION |
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S-NITROSOTHIOLS (SNOs) both store and execute nitrogen oxide bioactivities in physiological systems. For example, reactions of S-nitrosohemoglobin deliver both nitric oxide (NO) and low-mass SNOs to dilate precapillary systemic arterioles (20, 29, 49), and S-nitroso-L-cysteine acts independently of homolytic cleavage to NO as a stereoselective neurotransmitter (42). In the airway, S-nitrosoglutathione (GSNO) is an endogenous bronchodilator two log orders more potent than theophylline (14). Concentrations of GSNO and other SNOs tend to be low in the airways of patients with severe asthma (7), although expired NO concentrations are high in this condition (4, 40). We reasoned that the contradiction of bronchodilator SNO deficiency in the hypernitrosopneic (40) asthmatic airway might be partially reconciled if SNO catabolism to NO were accelerated in the asthmatic lung; low SNO levels, high NO levels, and bronchoconstriction could follow. We thus hypothesized that breakdown of GSNO by lung tissue may inhibit its bronchodilator activity.
The ovalbumin-sensitized guinea pig lung was used as a model to test this hypothesis because guinea pigs demonstrate both high expired NO concentrations and bronchoconstriction after ovalbumin sensitization (OS) and because nitric oxide synthases (NOSs) are not upregulated in this model (38). Of note, NOS isoforms themselves may produce SNOs under conditions of high thiol concentrations like those in the lungs (47). Furthermore, pulmonary NO excretion is not uniformly eliminated by high-dose NOS inhibition (21, 38, 56). Here we show that 1) GSNO catabolism is accelerated in the presence of guinea pig lung tissues in general and in the presence of OS lung tissue in particular, 2) soluble proteins responsible for this activity are both NADPH dependent and NADPH independent, and 3) this catabolic activity prevents the guinea pig airway smooth muscle relaxant effect of GSNO. These observations suggest that SNO catabolic pathways may be physiologically relevant in the lung.
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MATERIALS AND METHODS |
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Guinea pig sensitization and tissue harvest. Male 500-g guinea pigs were sensitized to ovalbumin as previously described (38) with certain modifications. Briefly, on day 1, animals were placed in a plastic chamber and exposed for 180 s to 5 ml/kg of aerosolized 1% ovalbumin in 10 mM PBS delivered by a compressed air nebulizer. Control animals were exposed to PBS alone. On days 7 and 14, animals were reexposed. Thirty minutes after the last exposure, animals were euthanized (100 mg/kg of pentobarbital sodium) and exsanguinated through an abdominal incision. The incision was then extended to include a sternotomy and midline neck dissection. The left atrium was opened, the right ventricle was cannulated, and the pulmonary vascular bed was lavaged with 20 ml of Hanks' balanced salt solution (pH 7.4, 4°C). The trachea and lungs were then removed en bloc, cleaned thoroughly, and suspended in proteolysis inhibitor buffer (PIB; 50 mM Tris · HCl, 1 mM EDTA, 100 µg/ml of phenylmethylsulfonyl fluoride, and 10 µg/ml each of soybean trypsin inhibitor, antipain, leupeptin, and pepstatin, pH 7.4, 4°C). The lungs were homogenized (Polytron) in PIB. In selected experiments, tracheal tissue was cut into 2-mm-thick coronal rings for bioassay as previously reported (27, 35); care was taken to leave the lungs intact. Lungs from additional animals were inflated through a catheter in the main stem bronchi with 10% neutral Formalin, cut into 5-µm sections, and stained with hematoxylin and eosin to confirm the presence or absence of inflammation.
Protein purification. Homogenized fractions underwent centrifugation (20,000 g for 15 min at 4°C) and filtration (pore size 20 µm). Initial separation of the supernatant proteins in the PIB was performed across a 1 × 30-cm diethylaminoethyl cellulose (Whatman) column eluted with a 0-250 mM linear gradient of NaCl (pH 7.4). Fractions were screened for biochemical activities (see Biochemical methods) and separated further based on the results of this screening. Certain active fractions were further purified in PIB by NADPH activity binding on a reactive red column after 12 h of dialysis (5 kDa) against 100 volumes of PIB at 4°C (28). The reactive red 120 type 3000 agarose (Sigma) column (NADPH/NADH affinity) was preequilibrated with 10 mM K2HPO4, pH 7.4 (low-salt buffer). The sample was loaded and equilibrated on the column for 7 h and eluted with a 16-h linear gradient to 40% high-salt buffer (2.0 M NaCl and 10 mM K2HPO4, pH 7.4) followed by a 4-h wash with 100% high-salt buffer (flow rate 20 ml/h throughout). Certain active fractions were further loaded onto a cellulose phosphate (Sigma) column (1 × 30 cm) equilibrated in PIB, and elution was performed with a linear gradient of 0-250 mM NaCl in PIB at the flow rate of 2 ml/min.
Biochemical methods.
GSNO was assayed by introducing samples into a reaction chamber (NOA
280, Sievers Instruments, Boulder, CO) containing 1 mM cysteine and 100 µM CuCl in a continuous helium stream (pH 7.0, 50°C) as previously
described (12, 14). This assay is highly specific
and is linear to 5 nM (12). Additionally, GSNO was assayed (pH 7.4, 25°C) by spectrophotometry (335 nm;
absorption coefficient = 586 M1 · cm
1) (13,
16). Xanthine oxidase-mediated GSNO catabolic activity was
measured as GSNO decomposition in PBS (25°C) in 21% oxygen in the presence of 150 µM hypoxanthine (52). Glutathione
peroxidase (GPx) was measured in 50 mM
K2HPO4 buffer (pH 7.0), 1 mM EDTA, 1 mM
NaN3, 0.2 mM NADPH, 1 U/ml of glutathione reductase, 1 mM glutathione, and 0.25 mM H2O2 (total volume of
1 ml); all ingredients except the protein fraction and
H2O2 were combined at the beginning of each
day. Protein (100 µl) was added to 800 µl of the above mixture and
allowed to incubate for 5 min at 25°C before initiation of the
reaction by the addition of 100 µl of H2O2
solution. Absorbance at 340 nm was recorded for 5 min, and the activity
was calculated from the slope of these lines as micromoles of NADPH
oxidation per minute (25, 31, 54). Thioredoxin reductase
was assayed by the addition of 10 µg of active fraction protein to
500 µl of assay mixture containing 100 mM potassium phosphate, 10 mM EDTA, 0.2 mM NADPH, 0.2 mg/ml of BSA, 1% ethanol, and 5 mM
dithio-bis-nitrobenzoic acid, pH 7.0 (25°C). Absorbance at 412 nm was
recorded between the first and second minutes of the reaction, and
activity was calculated as micromoles of 2-nitro-5-thiobenzoate
generated per minute (41). Glutathione was measured with
dithio-bis-nitrobenzoic acid (16) in the presence
[oxidized (GSSG)] or absence [reduced (GSH)] of glutathione
reductase (1 U/ml) and
-NADPH (10 µM); GSSG was taken as the
difference between total glutathione concentration and GSH
concentrations (11). Inhibition of protein fractions with
GSNO breakdown activity was studied by incubation with 100 µM
aurothioglucose (ATG) (25), 100 µM deferoxamine
(53), or 30 µM bathocuproine disulfonate
(18) in PBS (25°C) with active fractions before the
reaction with GSNO. Protein was assayed by the method of Lowry
(16). Kinetic studies were performed by measuring
d[GSNO]/dt in the linear range (10 min) at
uniform protein concentration (and NADPH where applicable) and varying
GSNO concentrations (21°C in PBS). Reagents were purchased from Sigma.
Bioassay. Tracheal rings were preserved in Krebs-Henseleit solution (KHS; in mM: 118 NaCl, 55.4 KCl, 1.10 NaH2PO4, 11.1 glucose, 25.0 NaHCO3, 1.38 MgSO4, and 2.32 CaCl2, pH 7.40, at 4°C) in 95% O2 balanced with CO2 and suspended on force transducers connected to a calibrated chart recorder (Gould Instruments, Latrobe, PA) with 1 g of tension, a previously determined optimum (27). After equilibration and conditioning, rings were rinsed twice, observed to return to baseline tension, and then contracted with 5 µM methacholine (EC50). Rings were then exposed to serially increasing concentrations of GSNO that had been preincubated with active and inactive protein fractions with and without ATG or with KHS alone for 30 min (37°C). Percent relaxation was taken as percent of the initial response to methacholine.
Statistical analysis. Multiple means were compared with the use of ANOVA. Nonparametrically distributed means were compared with the Mann-Whitney rank sum test. Data are presented as means ± SE. P < 0.01 was considered significant.
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RESULTS |
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Guinea pig airway soluble proteins accelerate GSNO breakdown.
Intact lung tissue slices accelerated the rate of GSNO breakdown
sevenfold compared with PBS (0.64 ± 0.04 vs. 0.09 ± 0.01 µM/min; P < 0.001; n = 4 tests
each). OS significantly accelerated breakdown to 0.88 ± 0.05 µM/min (P < 0.01; compared with unsensitized tissue; n = 5 samples each) with matched lung
homogenate protein concentrations from each set of animals
(unsensitized, 6.7 ± 0.37 mg protein vs. sensitized, 6.5 ± 0.34 mg protein). Because there are proposed GSNO catabolic proteins
that require (25, 28, 32, 38) and do not require
(18, 24, 30, 41, 52, 53) NADPH, fractions were screened in
the presence and absence of this cofactor. Two anion exchange protein
fractions from whole lung homogenization of both ovalbumin-sensitized
and control animals accelerated GSNO breakdown. An early (10-30 mM
NaCl) fraction had activity of 6.77 × 1010 ± 0.84 × 10
10
mol · min
1 · mg protein
1 in
the presence of 100 µM NADPH (P < 0.001 compared
with that in the inactive fraction; n = 6 assays; Fig.
1). An additional later (80-110 mM
NaCl) fraction had activity of 9.01 ± 1.20 × 10
11 mol · min
1 · mg
protein
1 (P < 0.01 compared with that in
the inactive fraction; n = 4 assays) in the absence of
NADPH (Fig. 2). All fractions were
screened for catabolic activity in the presence of
hypoxanthine (52); none was identified. Activities were
almost completely eliminated by incubation with 0.25% trypsin in
Hanks' balanced salt solution (60 min at 21°C; Fig. 2) and were
completely eliminated by ultrafiltration (10 kDa) or dialysis (5 kDa).
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Preliminary characterization of the soluble GSNO catabolic activity
of guinea pig lungs.
The NADPH-dependent fraction was further purified by reactive red
chromatography. This fraction did not show activity as thioredoxin or
GPx. Its apparent Michaelis-Menten coefficient
(Km) was ~500 nM (100 µM NADPH). A later
NADPH-independent fractionated protein, likely a separate enzyme,
eluted in the 40 mM fraction during phosphocellulose chromatography. It
had a Km of ~1.3 µM (Fig. 2). Of note, the
NADPH-independent fraction produced GSSG stoichiometrically (59 ± 4.5 µM GSSG from 100 µM GSNO; 45 min at 21°C; n = 3 assays). It was partially reversed (57.4%) after
treatment with ATG but was not inhibited by deferoxamine or
bathocuproine disulfonate (Table 1).
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Inhibition of GSNO-mediated airway smooth muscle relaxation by
airway catabolic proteins.
Consistent with previous reports (27, 35), GSNO caused
dose-dependent relaxation of guinea pig tracheal smooth muscle. The
relaxation was inhibited by preincubation of GSNO with the catabolically active fraction from the guinea pig lung protein homogenate (independent fraction) but not with inactive fractions (25 µg protein/ml each, 30 min at 37°C) in KHS. Relaxation after exposure to 50 µM GSNO (IC50 was not achieved after
incubating with the active fraction) was 56 ± 4.4, 47 ± 2.3, and 24 ± 4.1%, respectively, for GSNO alone
(n = 7 rings), GSNO with inactive fraction
(n = 5), and GSNO with active fraction
(n = 5 rings; P = 0.0002 by ANOVA; Fig.
3). As expected (27), we
also demonstrated a brisk and robust relaxation (73-100%) to
isoproterenol (1 µM) in the presence of the active fraction
(n = 7 rings). Coincubation of GSNO with the active
fraction and with excess (100 µM) gold under the same conditions
partially stabilized GSNO bioactivity (38 ± 4.2% at 50 µM
GSNO; P < 0.05 compared with GSNO and the active
fraction alone; n = 7 rings), consistent with partial
ATG-mediated attenuation of the biochemical activity of the same
fraction (Fig. 3). Of note, relaxation of airway rings from
ovalbumin-sensitized animals by 10 µM GSNO was inhibited relative to
that for control rings (13.7 ± 1.6 vs. 22.6 ± 3.7%
relaxation in ovalbumin-sensitized and control animals;
P < 0.05; n = 7 and 5 rings,
respectively), consistent with previous observations (51)
made in guinea pig whole lung preparations. Blinded analysis of airway
sections confirmed the presence of mucosal thickening and eosinophilic
and lymphocytic airway inflammation in the airways of
ovalbumin-sensitized but not of control guinea pigs (n = 9 sections from 3 animals).
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DISCUSSION |
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SNOs are endogenous relaxants of airway smooth muscle (3, 16, 27, 35, 51). Severe asthma is associated with depletion of GSNO and other SNOs in the airway, suggesting that a SNO deficiency may contribute to airway narrowing in the clinical setting (17). Of note, OS and rechallenge dramatically inhibit the bronchodilation effect of GSNO (51). Here, we have shown in the guinea pig that 1) lung tissue degrades GSNO, 2) this degradation prevents airway smooth muscle relaxation, 3) one of the two soluble catabolic proteins is NADPH dependent, whereas the other is partially inhibited in the presence of ATG, and 4) lung tissue GSNO catabolism is increased after OS. These findings suggest that GSNO breakdown may be relevant to pulmonary pathophysiology.
SNO bioactivities may be distinct from those of NO because they often involve electrophilic reactivity toward organic sulfur, nitrogen, carbon, and transition metal moieties as opposed to free radical reactions (7, 29, 48, 49). In other words, SNOs may act independently of, and in fact be inhibited by (7), degradation to NO. Nitros(yl)ation-mediated bioactivities that may be relevant in the lung include smooth muscle relaxation (3, 15, 16, 27, 34, 35), immune functions (9, 37, 43, 45), neurotransmission (10, 36, 42, 50), modulation of ion channel conductivity and airway hydration (6, 15), inhibition of oxidant stress enzymes (1, 5), and antimicrobial effects (22, 44).
SNOs are formed in the lung by organic and inorganic reactions. NOS, present in the normal airway epithelium, may produce GSNO under conditions like those in the airway (8, 16) in which reduced glutathione concentrations are high (47). Reactions between oxygen, superoxide, and NO to form nitrosating species may proceed at physiologically relevant concentrations (1, 5, 8, 19, 20, 22, 26, 31, 44, 50, 55) and are favored both in the relatively oxidative environment of the airway and in biological membranes. Formation of iron-nitrosyl intermediates has also been proposed to contribute to endogenous SNO formation (53), although these species do not appear to be present in the airway (16). The existence of additional cellular mechanisms for SNO synthesis has been proposed, but these processes are not fully understood (9, 18).
In addition to being directly bioactive, GSNO and other SNOs may act as reservoirs for nitrogen oxides in the airway (16, 17), brain (33), plasma (46), erythrocytes (29, 49), and neutrophils (9). Thus SNO catabolism in these storage pools may serve not only to attenuate nitros(yl)ation-mediated activities but also to 1) upregulate the effects of NO, hydroxylamine, peroxynitrite, and other SNO breakdown products (25, 28, 41, 52) and 2) facilitate transmembrane NOx signaling. In this sense, SNOs may be thought of as capacitors for signals originating from NOS activation. In the airway, as in the brain, GSNO is the principal SNO (16, 33), probably because its formation is favored in transnitrosation equilibria under physiological conditions (39, 46).
Catabolic processes have been previously described for GSNO. These
include inorganic copper- and iron-mediated reactions in vitro
(2, 53) that may have limited relevance in vivo where free
concentrations of these ions are generally extremely low. Our active
soluble protein fractions were >10 kDa in size and were inhibited by
trypsin, which suggested that they were lung proteins. Enzymatic
processes have been proposed to exist in neutrophils, platelets, and
bacteria (9, 18, 22), and catabolic activity has recently
been demonstrated for specific enzymes. Neither of the activities we
identified were likely to have been 1) -glutamyl transpeptidase, which is membrane bound (22);
2) Cu/Zn superoxide dismutase, which should be inhibited by
bathocuproine disulfonate (24); or 3) xanthine
oxidase, which should be augmented by hypoxanthine (52). The gold-inhibitable fraction may be a
seleno protein (25), but it does not have GPx activity.
The NADPH-dependent fraction could be a guinea pig-specific isoform of
NADPH-dependent alcohol dehydrogenase (24). Nitrogen
oxides other than NO may be produced in vitro (data not shown), which
is consistent with this possibility. Work is ongoing to purify those
proteins in quantities sufficient for sequencing and further characterization.
Endogenous SNOs are present in physiologically relevant concentrations. GSNO concentrations as high as 7 µM have been reported in normal brain tissue (22), and airway levels may be on this order in certain settings (16). Of note, the potency of GSNO in relaxing guinea pig airway smooth muscle in our study was slightly lower than that previously reported (27), probably reflecting some degree of inorganic breakdown in physiological (control) buffer incubated at neutral pH for 30 min (16). The mechanism by which SNOs relax airway smooth muscle is unclear and does not exclusively involve liberation of NO with subsequent activation of guanylate cyclase (3, 14). It is therefore possible that, in vivo, GSNO catabolism could activate or inactivate its smooth muscle relaxant effect depending on the product and location. Our data suggest the possibility that inactivation may be more physiologically relevant.
In summary, our findings suggest the presence of novel metabolic
pathways with the potential to affect airway smooth muscle tone.
Specifically, breakdown of the endogenous bronchodilator GSNO by lung
proteins prevents guinea pig airway smooth muscle relaxation in
vitro. We have identified two soluble protein fractions involved in
this process, although other proteins, particularly membrane-associated
-glutamyl transpeptidase, may also be relevant. SNO metabolism may
be involved in several additional aspects of pulmonary biology
including immune, antimicrobial, airway clearance, and neuronal
functions in vivo. Recent evidence that humans with severe asthma have
low airway SNO or GSNO levels suggests that SNO catabolism may be
accelerated in certain patients with asthma. This accelerated
catabolism may in turn contribute to bronchoconstriction and/or to
other elements of asthma pathophysiology such as impaired inflammatory
cell apoptosis. Taken together, these findings suggest that further
efforts to define, localize, and characterize the effects of GSNO
metabolic pathways in the lung may provide a novel direction for
pulmonary research, with the potential for leading to new asthma therapies.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-59337.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. M. Gaston, Univ. of Virginia Health System, Box 386, Charlottesville, VA 22908 (E-mail: bmg3g{at}virginia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 February 2000; accepted in final form 24 April 2000.
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