Regulation of murine airway responsiveness by endothelial nitric oxide synthase

Michel Feletou1, Michel Lonchampt1, Francis Coge1, Jean-Pierre Galizzi1, Claire Bassoullet1, Christelle Merial1, Pascale Robineau1, Jean A. Boutin1, Paul L. Huang2, Paul M. Vanhoutte1, and Emmanuel Canet1

1 Institut de Recherches Servier, 92150 Suresnes, France; and 2 Cardiovascular Research Center and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(-/-)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(-/-) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-L-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine. eNOS(-/-) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-L-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity.

epithelium; immunohistochemistry; methacholine; endothelial nitric oxide synthase knockout mice; N-nitro-L-arginine methyl ester; airway reactivity; reverse transcription-polymerase chain reaction; Western blot


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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THREE ISOFORMS of nitric oxide synthase (NOS), the enzyme that synthesizes nitric oxide (NO) from L-arginine, have been identified, including two constitutive forms, the neuronal (nNOS) and endothelial (eNOS) isoforms, and one inducible (iNOS) isoform (26, 35). Endogenous NO is formed in the lung, its presence has been detected in the exhaled air of humans and in several animal species (14, 19), and it may participate in the inflammatory responses, since exhaled NO is increased in inflammatory airway diseases such as asthma (2). The three NOS isoforms have been identified in the lungs of various species, including humans. The eNOS isoform has been observed in bronchial and large pulmonary blood vessels and in the epithelium; the nNOS isoform has been observed in nonadrenergic noncholinergic nerves and in the epithelium, whereas the iNOS isoform was detected in alveolar macrophages and in the epithelium (1, 33, 34, 37-42). However, the expression and the distribution of these three isoforms are age and species dependent and vary with the experimental conditions (hypoxia, inflammation, etc.; see Refs. 4, 7, 17, 24, 30, 33-35, 37, 41, 42).

NO is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. A paracrine role of NO in bronchial function was suggested by the presence of NOS and soluble guanylate cyclase immunoreactivity, respectively, in the epithelial and smooth muscle cells of the rat bronchus (31). Furthermore, NO is an endogenous neurotransmitter in species such as guinea pigs, horses, and humans (3, 9, 25, 43). Mice subjected to the disruption of the nNOS gene are significantly less responsive to methacholine, indicating that nNOS may promote airway hyperresponsiveness in this species (8). In contrast, NOS inhibition induces airway hyperresponsiveness in the guinea pig (28) and rat (20), suggesting that endogenous NO can also act as a bronchodilator. Interestingly, in rats, a strain-related difference in bronchial responsiveness has been attributed to differences in endogenous NO production in the airways (17). However, the isoform(s) of the NOS involved in this bronchodilatory mechanism and their cellular locations are poorly understood.

The purpose of this work was to determine whether or not eNOS contributes to the regulation of bronchial tone. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone.


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Animals

Specific pathogen-free male Swiss CD-1 mice (Charles River Laboratories, Calco, Italy), C57BL/6 mice, and homozygous mutant mice lacking the gene for eNOS [eNOS(-/-); Iffa Credo, L'Arbresle, France], 8-10 wk of age, were used for the study. The generation of these eNOS-deficient mice has been described elsewhere (16). The mice were maintained under pathogen-free conditions in filter-topped cages in an air-conditioned room at 21 ± 1°C, fed a standard laboratory diet, and given water ad libitum. Mice were killed with an overdose of pentobarbital sodium (ip) or with CO2 inhalation. This study was performed in agreement with the National Research Council Guide for the Care and Use of Laboratory Animals and with the guidelines established by the ethical committee of the Institut de Recherches Servier.

Expression of NOS mRNA

C57BL/6 and eNOS(-/-) mice were killed by CO2 inhalation, and the brains, tracheae, and apical parenchyma of the lungs were rapidly removed and frozen in liquid nitrogen. Experiments were performed on three different batches of tissues, each containing tissue taken from five different mice. The total RNAs were extracted using the RNAqueous-4 PCR protocol (Ambion, Austin, TX), and 2 µg were reverse transcribed with oligo(dT)12-18, in accordance with the first-strand cDNA synthesis protocol from Amersham Pharmacia Biotech (Saint-Quentin, France). PCRs were performed in 100 µl of solution containing 10 mM Tris · HCl, pH 8.3, 1.5 mM MgCl2, 0.2 mM dNTP, 2 µl of single-strand cDNA preparation, 0.3 µM each primer, and 2 units of AmpliTaq gold polymerase (Perkin-Elmer, Courtaboeuf, France). A 30-cycle program at 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min, a hot start at 94°C for 9 min, and a final extension at 72°C for 8 min were performed. The forward and reverse PCR primers were specific to the three NOS isoforms: positions 2,636-2,656 and 3,454-3,470 for eNOS (12), positions 1,819-1,842 and 2,347-2,370 for iNOS (22), and positions 1,781-1,805 and 2,291-2,312 for nNOS (29). The eNOS primers were chosen in the deleted portion of the eNOS gene used to establish the eNOS(-/-) mice (16). The PCR fragments were analyzed by electrophoresis on a 1.5% (wt/vol) agarose gel, transferred to a Hybond-N+ membrane (Amersham), and hybridized with [alpha -32P]dCTP radiolabeled oligonucleotide using terminal transferase (Amersham Pharmacia Biotech). The following probes were used: nucleotides 2,967-2,986 for eNOS (12), nucleotides 2,065-2,086 for iNOS (22), and nucleotides 2,075-2,095 for nNOS (29). One million counts per milliliter were used in 10 ml of hybridation buffer [5× standard saline citrate (SSC), 5× Denhardt's solution, 100 µg/ml tRNA, and 0.1% (wt/vol) SDS] and incubated at 42°C for 16 h. The filter was then washed two times at 50°C for 30 min in 2× SSC and 0.1% (wt/vol) SDS and exposed with Hyperfilm (Amersham). beta -Actin was PCR amplified (540 bp) as described by the manufacturer (Clontech, Palo Alto, CA) and was used as a quantitative control for each cDNA. The radioactivity of the iNOS, eNOS, and nNOS signals was measured with the fluorescence image analyzer FLA-200 (Fuji Photo Film, Tokyo, Japan) and expressed in photostimulated luminescence units. For each sample, the radioactivity was normalized with the corresponding signal obtained for beta -actin. The specificity of PCR products was assessed by Southern blot using selective internal oligonucleotide probes of each NOS isoform. There was no signal when either the mRNA (distilled water control) or RT was omitted from the first-strand cDNA conversion, indicating that the signals observed were not the result of any atmospheric contamination or genomic DNA (data not shown). beta -Actin was used as a quantitative control to confirm that each sample contained a similar amount of cDNA. The amplicon had the expected length as deduced from the sequence of the NOS transcripts (12, 22, 29). These primers led to DNA fragments of 840, 552, and 532 bp for eNOS, iNOS, and nNOS, respectively.

Immunoblotting

The murine tissues were isolated and pooled as described above. Proteins from tracheas, apical parenchyma of the lungs, and brains of both C57BL/6 and eNOS(-/-) mice were resolved by SDS-PAGE (6% wt/vol; see Ref. 23) and electrotransferred to nitrocellulose filters. The blot was incubated for 1 h at 25°C in blocking PBS buffer containing 10% (wt/vol) skimmed milk (Difco) and 0.1% (vol/vol) Tween 20 and then overnight at 4°C with mouse monoclonal anti-eNOS, -iNOS, or -nNOS (0.1 µg/ml; Transduction Laboratories, Lexington, KY) in blocking buffer. Immunoblots were washed three times with washing PBS buffer containing 0.1% (vol/vol) Tween 20 and then incubated for 1 h at 25°C with polyclonal horseradish peroxidase-conjugated goat anti-mouse antibody (Amersham Pharmacia Biotech) diluted at 1:3,500 in PBS buffer containing 1% (wt/vol) BSA and 0.1% (vol/vol) Tween 20. After three washes, peroxidase activity was revealed by enhanced chemliluminescence reagent and exposure to Hyperfilm in accordance to the instructions of the manufacturer (Amersham). Western blot analysis of the SDS-treated protein extracts from positive controls revealed specific immunoreactive polypeptides with a molecular weight of 140, 130, and 155 kDa corresponding to eNOS, iNOS, and nNOS, respectively.

Immunohistochemistry

Mice were killed with an overdose of pentobarbital sodium (ip). Three to five animals of each strain were used, and at least four sections were performed in each organ for each NOS studied. The thorax was opened, and the respiratory tract was fixed by distention with a 4% paraformaldehyde solution in PBS at pH 7.4 (Life Technologies, Eragny, France), injected through the trachea under a pressure of 22 cmH2O. Next, the lungs were removed, fixed for 24 h with the same solution, and embedded in paraffin. Sections (4 µm thick) were collected on silanized slides (DAKO, Carpinteria, CA) and immunostained after deparaffinization in a toluene bath and rehydration in graded alcohol solutions. After light trypsinization (0.1% in PBS for 6 min at room temperature), endogenous biotin-binding activity was suppressed by sequential 20-min incubations, first with 0.1% avidin and then with 0.01% biotin in 0.05 M Tris · HCl, pH 7.4. The sections were incubated for 15 min with 3% H2O2 to block endogenous peroxidases.

To localize NOS isoforms in the murine airway epithelium, immunohistochemistry was performed with previously characterized specific antibodies against each of the NOS isoforms. It was specified by the manufacturers that antibodies against NOS of human origin cross-react with murine NOS. This was confirmed by the Western blot experiments.

nNOS. Slides were incubated for 1 h at room temperature with a rabbit polyclonal anti-human nNOS (1 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). The specificity of the antibody for the nNOS isoform was confirmed by the positive immunoreactivity of neuronal cells of the brain and the lack of immunoreactivity of endothelial cells and macrophages stimulated with lipopolysaccharide (LPS) plus interferon-gamma .

iNOS. Slides were incubated for 1 h at room temperature with a rabbit polyclonal anti-mouse iNOS (1 µg/ml for the lung and 2.5 µg/ml for the trachea; Transduction Laboratories). The specificity of the antibody for the iNOS isoform was demonstrated by the positive immunoreactivity of macrophage cells after stimulation by LPS plus interferon-gamma and by the lack of immunoreactivity in nonstimulated cells, brain neuronal cells, and endothelial cells.

eNOS. The endothelial cells of the aortic sections were stained after a 1-h incubation with eNOS antibodies, whereas in pulmonary tissues with a 1-h incubation, the labeling could be observed under the microscope and detected on the screen of the image analyzer but could not be correctly visualized on the glossy prints. An 18-h incubation period was then performed to provide Fig. 5. Incubations were performed with a rabbit polyclonal anti-human eNOS (1 µg/ml) in 0.05 M Tris and carrier proteins to reduce background (DAKO). Rabbit polyclonal anti-human eNOS (1 µg/ml; Transduction Laboratories and Santa Cruz Biotechnology; batch reference: I148) produced similar results and was tested in parallel. The specificity of the antibody for eNOS was shown by the positive immunoreactivity of endothelial cells in the aorta and the lack of immunoreactivity in neurons and in stimulated macrophages (data not shown).

After being washed in phosphate buffer, sections were incubated successively with biotinylated goat anti-rabbit IgG (10 µg/ml; Sigma) for 60 min and then with streptavidin conjugated to horseradish peroxidase (DAKO) for 10 min (lung) or 20 min (trachea). This was followed by incubation in freshly prepared substrate-chromogen solution of H2O2 (0.03%) and 3-amino-9-ethylcarbazole (DAKO) in 0.1 M acetate buffer, pH 5.2. After intensive rinsing in deionized water, sections were counterstained with Mayer's hematoxylin, air-dried, mounted with glycergel mounting medium (DAKO), examined under a white light microscope (DMLB; Leica, Wetzlar, Germany), and photographed with a video camera (Sony) and an image analyzer (Visiolab 2000; Biocom, Les Ulis, France).

Controls. Antibodies from Transduction Laboratories were raised against 178-, 183-, and 194-amino acid-long peptides (COOH-terminal portion of eNOS, iNOS, and nNOS, respectively). These peptides are not commercially available. Therefore, controls were obtained by replacement of the primary antibody by buffer alone or rabbit IgG (DAKO) at the same concentration that had been used with the various antibodies. In contrast, each antibody from Santa Cruz Biotechnology was raised against a 20-amino acid-long peptide taken from each NH2-terminal portion of the three NOS. These immunogenic peptides are commercially available. Therefore, controls were performed with buffer alone, rabbit IgG (DAKO), and primary antibodies incubated 24 h at 4°C with a 10-fold excess of the corresponding purified immunogenic NOS peptide.

Bronchial Reactivity

Unrestrained conscious mice were placed in a whole body plethysmography chamber (volume 400 ml PLY 32II, version 2.1; Buxco Electronics, Sharon, CT) to analyze the respiratory waveforms (12). After 20-30 min of equilibration, a 20-s aerosol of methacholine was delivered through the aerosolator (Nebulizer 35B; Devilbiss). The airway resistance was expressed as enhanced pause (Penh) = [TE (expiratory time)/40% of Tr (relaxation time) - 1] × Pef (peak expiratory flow)/Pif (peak inspiratory flow) according to the recommendations of the manufacturer. To evaluate the effect of eNOS on airway responsiveness, cumulative dose-response curves to aerosolized methacholine were performed. After each dose of methacholine, a 40-min period of recovery was observed. Mice were subjected to a single dose-response curve of methacholine. Certain mice were treated with either N-nitro-L-arginine methyl ester (30 mg/kg ip), a nonspecific NOS inhibitor, or aminoguanidine (30 mg/kg ip), a specific iNOS inhibitor. Treatments were administered 30 min before evaluation of bronchial reactivity. Experiments were always performed in parallel with contemporary controls. Depending on the protocols, groups included 5-12 mice.

Effectiveness of the Aminoguanidine Treatment

These experiments were performed to assess the effectiveness of the dose and route of administration of aminoguanidine when used in the measurement of airway responsiveness. Male C57BL/6 and eNOS(-/-) mice were subjected to intraperitoneal administration of bacterial LPS (Escherichia coli, serotype 055:B5; 125 mg/kg) or saline solution (10 ml/kg ip). They were treated 5 h later with either aminoguanidine (30 mg/kg ip) or saline (10 ml/kg ip). The mice were then anesthetized with a volatile anesthetic, isoflurane. Under sedation, 1 ml of intracardiac blood was directly withdrawn with a syringe moistened with citrate buffer (3%). The blood was transferred in an EDTA-containing vial maintained at 0°C and then centrifuged at 5,000 rpm (10 min at 4°C). The supernatants were collected and centrifuged (7,000 rpm, 20 min, 4°C) in ultrafiltration tubes (Millipore). The plasmatic concentration of nitrite plus nitrate was assessed using a commercial kit (Cayman, Ann Arbor, MI; Griess method; see Refs. 11 and 15).

The plasmatic levels of nitrite plus nitrate were not significantly different in C57BL/6 and eNOS(-/-) mice. In both strains, LPS (125 mg/kg ip) administration produced a statistically significant increase in nitrite plus nitrate. The aminoguanidine treatment (30 mg/kg ip) produced a statistically significant reduction of the increase in plasmatic nitrite plus nitrate: 30 and 56% reduction in C57BL/6 and eNOS(-/-), respectively (Table 1).

                              
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Table 1.   Plasmatic nitrite plus nitrate in C57BL/6 and eNOS mice with and without LPS administration: effect of aminoguanidine treatment

Statistical Analysis

Data are shown as means ± SE; n is the number of animals that were studied. Statistical analysis was performed either with a Student's unpaired t-test or with a one- or two-way ANOVA followed by Dunnett's post hoc test. P values <0.05 were considered to indicate statistically significant differences.

Reagents

All chemical reagents were obtained from Sigma Chemical (St. Louis, MO), unless otherwise specified.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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nNOS

The mRNA for nNOS was observed in the trachea and brain of both C57BL/6 and eNOS(-/-) mice but not in the lungs (Fig. 1). No signal could be detected in the lung parenchyma, even after 40 cycles of PCR (data not shown). Similarly, the nNOS protein detected by immunoblotting was observed in the trachea and the brain from both C57BL/6 and eNOS(-/-) mice but not in the lungs (Fig. 2). For the three strains of mice [CD-1, C57BL/6, and eNOS(-/-)], positive immunostaining for nNOS was observed inconsistently. When observed, the staining was in the submucosa of the airway epithelium. The staining intensity was stronger in the trachea than in distal airways. Neither epithelial nor endothelial cells were stained (data not shown).


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Fig. 1.   Distribution of endothelial (e) nitric oxide synthase (NOS), inducible NOS (iNOS), and neuronal NOS (nNOS) transcripts in trachea, lung parenchyma, and brain of C57BL/6 and eNOS-deficient [eNOS(-/-)] mice. Total RNA (2 µg) extracted from trachea, lung, and brain of C57BL/6 and eNOS(-/-) mice was amplified by RT-PCR using primers specific to iNOS, eNOS, and nNOS. After 25 cycles of PCR for beta -actin and 30 cycles of PCR for NOS isoforms, the amplified DNAs were analyzed in agarose gel and hybridized with selective internal 32P-labeled oligonucleotide probes. The length of the PCR fragments was estimated by restriction DNA markers (phi X174/HaeIII) and is indicated in base pairs (bp).



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Fig. 2.   Immunoblotting of eNOS, iNOS, and nNOS expressed in trachea, lung, and brain of C57BL/6 and eNOS(-/-) mice. Proteins (100 µg) were separated by SDS-PAGE, transferred to nitrocellulose filters, and incubated with anti-eNOS, -iNOS, or -nNOS antibody. Detection of primary antibody binding was performed with a specific horseradish peroxidase-linked secondary antibody followed by enhanced chemiluminescence-directed exposure to Hyperfilm. Proteins (100 µg) from mouse brain were used as a positive control for eNOS and nNOS. Proteins (10 µg) from RAW 264.7 cells stimulated with interferon-gamma (10 ng/ml) and lipopolysaccharide (LPS; 1 µg/ml) for 12 h were used as a positive control for iNOS. The blots were calibrated with kaleidoscope molecular mass markers (Bio-Rad). The estimated molecular mass of the NOS isoforms is indicated on right. Each blot is representative of three independent experiments.

iNOS

The mRNA for iNOS was observed in the trachea, lungs, and brain of both C57BL/6 and eNOS(-/-) mice (Fig. 1). In contrast, the iNOS protein was not detected by immunoblotting in tissues from either C57BL/6 or eNOS(-/-) mice (Fig. 2). However, the RAW 264.7 cells stimulated with interferon-gamma and LPS were stained, confirming the validity of the antibody. For the three strains of mice [CD-1, C57BL/6, and eNOS(-/-)], the antibody against iNOS specifically revealed a weak immunostaining in the apical cytoplasmic part of the tracheal epithelial cell layer (Fig. 3). Similarly, in the small bronchus (but not in the alveoli) of the wild-type and eNOS(-/-) mice, a staining was also specifically observed in the epithelial cells; although more intense, the labeling was detected only in some of the epithelial cells (Fig. 4). No staining could be observed in pulmonary blood vessels.


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Fig. 3.   Immunolocalization of iNOS in the tracheal epithelium of C57BL/6 (left) and eNOS(-/-) (right) mice. Immunostaining was performed on paraffin sections. Sections were incubated for 1 h with a rabbit polyclonal mouse iNOS antibody (2.5 µg/ml; Transduction Laboratories) and then with biotinylated goat anti-rabbit IgG (10 µg/ml) for 1 h. The staining was performed with streptavidin conjugated to horseradish peroxidase for 20 min. The slides were counterstained with Mayer's hematoxylin (calibration bars = 7.5 µm). A: negative control. In the two strains of mice, there was no labeling when sections were incubated with control IgG serum (2.5 µg/ml). B: immunostaining. For both strains, the labeling (arrows) was observed in the apical part of the epithelial cells. Similar observations were performed with the other antibody from Santa Cruz Biotechnology (data not shown).



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Fig. 4.   Localization of iNOS in bronchial epithelium of C57BL/6 (left) and eNOS(-/-) (right) mice. Immunostaining was performed on paraffin sections. Sections were incubated for 1 h with a rabbit polyclonal mouse iNOS antibody (1 µg/ml; Transduction Laboratories) and then with biotinylated goat anti-rabbit IgG (10 µg/ml) for 1 h. The staining was performed with streptavidin conjugated to horseradish peroxidase for 10 min. The slides were counterstained with Mayer's hematoxylin. A: negative control. In the two strains of mice, there was no labeling when sections were incubated with control IgG serum (1 µg/ml; calibration bars = 15 µm). B: immunostaining. Labeling (arrows) was observed in bronchial epithelial cells of both C57BL/6 and eNOS(-/-) mice. A more intense labeling was consistently observed in eNOS(-/-) compared with C57BL/6 mice (4-5 sections from 4 different mice; calibration bars = 15 µm). C: immunostaining at a higher magnification. Arrows show the immunostaining localized in specific bronchiolar epithelial cells (calibration bar = 3 µm). Similar observations were performed with the other antibody from Santa Cruz Biotechnology (data not shown).

eNOS

The mRNA for eNOS was observed in the trachea, lungs, and brain of C57BL/6 but not in eNOS(-/-) mice (Fig. 1). Similarly, the eNOS protein detected by immunoblotting was observed only in tissues from C57BL/6 mice (Fig. 2). In CD-1 and C57BL/6 mice strains, an intense anti-eNOS labeling was observed in the endothelial cells of the aorta (data not shown). In the lung, the staining occurred especially in the endothelial layer of the large pulmonary blood vessel. However, a diffuse staining of unknown origin could also be observed in the parenchyma. For eNOS(-/-) mice, there was no staining of the aortic endothelial cells (data not shown), but in the lung sections, a diffuse staining was observed in the parenchyma and in the endothelial perinuclear area. However, in contrast to the slides from C57BL/6 mice, the endothelial cytoplasm was not stained (Fig. 5).


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Fig. 5.   Localization of eNOS in sections of murine pulmonary arteries. Left: C57BL/6. Right: eNOS(-/-). Immunostaining was performed on paraffin sections. Sections were incubated for 18 h with a rabbit polyclonal human eNOS antibody (1 µg/ml; Transduction Laboratories) and then with biotinylated goat anti-rabbit IgG (10 µg/ml) for 1 h. The staining was performed with streptavidin-conjugated horseradish peroxidase for 10 min. The slides were counterstained with Mayer's hematoxylin (calibration bars = 8 µm). A: negative control. In the two strains of mice, there was no labeling when sections were incubated with control IgG serum (1 µg/ml). B: immunostaining. In the C57BL/6 mice, a strong continuous labeling of the whole endothelial cytosol was observed along with a diffuse labeling in the parenchyma. In the eNOS(-/-) mice, the diffuse labeling of the parenchyma was still observed together with endothelial perinuclear spots. The endothelial cytosol was unstained. Similar observations were performed with the other antibody from Santa Cruz Biotechnology (data not shown). Note that immunostaining of the aorta showed an intense labeling of the endothelial cells exclusively in tissues from C57BL/6 mice. In contrast to the lung tissues, the incubation time required for the aortas was only 1 h (data not shown).

Airway Responsiveness to Cholinergic Agonists In Vivo

In CD-1 mice, methacholine (30-100 mM) induced a significant dose-dependent increase in Penh. The Penh values in the presence of methacholine were significantly different from basal values at each dose of methacholine tested. N-nitro-L-arginine methyl ester (30 mg/kg ip, a nonspecific NOS inhibitor) did not modify the basal value of Penh but significantly potentiated the increase in Penh produced by methacholine (Fig. 6). N-nitro-L-arginine methyl ester produced both an increase in sensitivity to methacholine and an increase in the response at the highest dose of methacholine tested. In contrast, aminoguanidine (30 mg/kg ip, a specific iNOS inhibitor) did not affect the response to the cholinergic agonist (Fig. 6). In C57BL/6 mice, one of the two parental strains of eNOS(-/-) mice, the basal value of Penh was not significantly different from the value observed in CD-1 mice, and methacholine produced a similar dose-dependent increase in Penh. In this strain of mouse, N-nitro-L-arginine methyl ester did not affect the basal value of Penh either but produced a significant increase in the response to methacholine. The effects of methacholine and N-nitro-L-arginine methyl ester were not significantly different between the two strains of mice (Fig. 7).


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Fig. 6.   Inhaled methacholine (30-100 mM)-induced changes in airway responsiveness in unrestrained conscious CD-1 mice. Effect of treatments with N-nitro-L-arginine methyl ester (L-NAME: 30 mg/kg ip), a nonspecific NOS inhibitor, and aminoguanidine (30 mg/kg ip), a specific iNOS inhibitor. Data are shown as means ± SE (n = 8-12). *Statistically significant difference with control (P < 0.05). Penh, enhanced pause (index of airway resistance).



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Fig. 7.   Inhaled methacholine (100 mM)-induced changes in airway responsiveness in unrestrained conscious C57BL/6 and CD-1 mice. Effect of N-nitro-L-arginine methyl ester treatment (30 mg/kg ip). Data are shown as means ± SE (n = 5). *Statistically significant difference with baseline (P < 0.05). #Statistically significant difference produced by N-nitro-L-arginine methyl ester treatment (P < 0.05). Responses in C57BL/6 mice were not significantly different from those in CD-1 mice.

In eNOS(-/-) mice, the basal value of Penh was similar to that observed in CD-1 or C57BL/6 mice. However, the eNOS(-/-) mice were significantly more sensitive to the bronchoconstricting action of methacholine than the other strains (Fig. 8). In the eNOS(-/-) mice, treatment with N-nitro-L-arginine methyl ester or aminoguanidine did not significantly alter basal values of Penh or the maximum response to methacholine. However, both inhibitors tended to increase the sensitivity to the muscarinic agonist at the intermediate concentration of 30 mM, although the differences observed did not reach statistical significance (Fig. 9).


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Fig. 8.   Inhaled methacholine (10-100 mM)-induced changes in airway responsiveness in unrestrained conscious C57BL/6 and eNOS(-/-) mice. Data are shown as means ± SE (n = 8). *Statistically significant difference between the two strains (P < 0.05).



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Fig. 9.   Inhaled methacholine (10-100 mM)-induced changes in airway responsiveness in unrestrained conscious eNOS(-/-) mice. Effect of N-nitro-L-arginine methyl ester treatment (30 mg/kg ip) and aminoguanidine (30 mg/kg ip). Data are shown as means ± SE (n = 9).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the murine lungs, the three NOS isoforms are expressed. The mRNA and the protein of nNOS are observed in the trachea but could not be detected in the parenchyma. This is in agreement with the immunoreactivity to nNOS that was observed inconsistently and that was confined to the airway submucosa. The immunostaining obtained with nNOS is consistent with labeling of submucosal airway nerves, confirming previous observations of nNOS immunoreactivity in inhibitory nonadrenergic noncholinergic bronchodilator nerves of human airways (38). nNOS has also been detected in epithelial cells of rat and sheep bronchi (34, 40, 42), and in a human respiratory epithelial cell line, nNOS has been identified by RT-PCR (1). However, the present study did not confirm that a specific cell subpopulation of the murine airway epithelium expresses nNOS in a constitutive manner.

The mRNA of iNOS is expressed in the airway and in the lung parenchyma. However, the Western blot did not allow detection of the protein. This is most likely to be explained by a level of expression of the protein that is too low for detection with the antibody tested. Indeed, immunohistochemistry experiments show the presence of iNOS in the tracheal epithelium and, to a lesser extent, in the bronchiolar epithelium. The staining was localized specifically in the airway epithelium, with no immunoreactivity overlap with the immunostaining of other NOS isoforms. The observation that iNOS is expressed in normal murine airway epithelium is in agreement with previous results obtained in normal human, rat, and sheep lungs (1, 21, 32, 34, 39, 40, 42). Since the initial identification of cytokine-induced NOS activity in macrophages of the mouse, iNOS has been shown to be "constitutively" expressed in various tissues such as the lung epithelium of the sheep and the kidney epithelial cells of the rat (34, 36). Whether the expression of iNOS in normal human and rodent airways reflects a truly "constitutive" expression or results from repeated exposure to airborne stimuli remains to be determined. However, resident lung macrophages submitted to the same environmental factors were not immunostained for iNOS, confirming similar results in alveolar macrophages obtained from bronchoalveolar lavage of normal human volunteers. In human respiratory epithelial cells, a rapid loss of iNOS expression is observed ex vivo (13). This suggests that the airway epithelial cells are exposed in vivo to unknown factors that maintain continuous iNOS expression. The anatomical localization of this isoform in close contact with the airspace is likely to play a role in the host defense against environmental agents and pathogens. The expression of iNOS has been also described in sheep airway and vascular smooth muscle cells (34). In the present work, no staining was detected, except in the epithelial cells.

The mRNA and the eNOS protein were observed both in the trachea and in the lung parenchyma of C57BL/6 mice but not, as expected, in the eNOS(-/-) mice (5, 16). In the present study, eNOS was expressed specifically in endothelial cells of bronchial blood vessels and in large pulmonary vessels. In smaller pulmonary blood vessels, the staining was much less consistent. No staining could be observed in the airways. This observation is in agreement with earlier studies in mice (18), sheep (4, 30), and in normoxic rat lungs (24, 41). In contrast, other investigators have reported the expression of eNOS in bronchial epithelial cells of rat and sheep (33, 34, 40, 42). In human bronchial epithelial cells in culture, the expression of eNOS has also been observed (33). However, these results were not confirmed in another study involving human airway specimens (39). Also, the present study did not confirm that a specific cell subpopulation of the murine airway epithelium expresses eNOS in a constitutive manner. Earlier antibodies of eNOS (Santa Cruz Biotechnology) produced an intense labeling of murine epithelial tracheal basal cells. However, Coerts et al. (6) and unpublished observations in our laboratory strongly suggest that this staining, which could not be reproduced with other antibodies, was artifactual. The diffuse labeling observed in the parenchyma and in the perinuclear area of both strains can most likely be attributed to a nonspecific labeling of unknown origin, possibly because of the prolonged incubation period used in the present study (18 h). However, as expected, the endothelial cytosol of the eNOS(-/-) mice was devoid of any staining. It cannot be ruled out that other cell types besides the endothelial cells express the eNOS at a level that remains below detection.

In spontaneously breathing, nonanesthetized CD-1, C57BL/6, and eNOS(-/-) mice, administration of N-nitro-L-arginine methyl ester, a nonspecific inhibitor of NOS, did not affect basal Penh. Interestingly, these basal values of Penh are not significantly different in eNOS(-/-) mice compared with the other strains of mice. This suggests that NO is not a major regulator of basal airway tone. In contrast, NO modulates airway reactivity to bronchoconstricting agents. In CD-1 and C57BL/6 mice, N-nitro-L-arginine methyl ester potentiated the responses to methacholine. This effect of N-nitro-L-arginine methyl ester can be attributed to an inhibition of NOS because this inhibitor was studied at a dose that has been proven to be effective in mice (10). The present study confirms previous results in other species showing that NOS inhibitors produce bronchial hyperresponsiveness both in vivo and in vitro (27, 28).

The origin of the endogenous NO production is unlikely to be the iNOS because the specific inhibitor, aminoguanidine, at a dose that has been proven to be effective in mice (Ref. 10 and the present study), had no effect on the increase in Penh produced by the muscarinic agonist. These results confirm an earlier study showing that, in a similar experimental protocol, unrestrained, conscious iNOS knockout mice had the same airway responsiveness in response to methacholine as the wild-type controls (7). The contribution of nNOS is also most unlikely because mice subjected to disruption of the nNOS gene are significantly less responsive to methacholine, indicating that nNOS promotes airway hyperresponsiveness (8). These results indicate that, in the mouse, the hyperresponsiveness produced by N-nitro-L-arginine methyl ester is likely to be linked to the inhibition of eNOS.

The involvement of eNOS is further supported by the fact that eNOS(-/-) mice are markedly hyperresponsive to inhaled methacholine. Furthermore, in these mice, in contrast to CD-1 and C57BL/6 mice, N-nitro-L-arginine methyl ester does not augment the maximal increase in Penh produced by methacholine. The hyperresponsiveness to inhaled methacholine is likely to be the result of eNOS gene deletion, although an effect on another gene that segregates with the eNOS locus cannot be ruled out. Interestingly, anesthetized mice knocked out for either nNOS, iNOS, or eNOS are not hyperresponsive to the intravenous administration of methacholine (7). Whether the difference observed between these findings and the present study is due to the anesthesia or the route of administration of the bronchoconstrictor (intravenously vs. inhaled) remains to be determined.

However, the nonspecific NOS inhibitor tends to increase the sensitivity of the eNOS(-/-) mice to methacholine, an effect that is mimicked by aminoguanidine. This indicates that the iNOS isoform could be involved in the control of bronchial responsiveness in the eNOS(-/-) mice. Two other unrelated observations in immunohistochemistry and Southern blot, respectively, an apparent increase in labeling of iNOS in bronchial epithelium and an apparent increase in mRNA expression in tracheal, lung, and brain tissues (Southern blots: 0.35 ± 0.04 and 0.47 ± 0.02, ratio of photostimulated luminescence units of iNOS to beta -actin in tracheae from C57BL/6 and eNOS mice, respectively), are suggestive of a role for iNOS in eNOS(-/-) mice airways. Whether this effect can be attributed to a compensatory mechanism of iNOS overexpression, linked to the eNOS gene deletion or to a specificity of this strain of mouse, remains to be more firmly established.

In summary, the present findings demonstrate that the three NOS isoforms are constitutively expressed in murine lung, and their specific anatomical localization suggests a specific function. Furthermore, targeted disruption of the murine eNOS gene induces airway hyperresponsiveness, suggesting that, in contrast to nNOS (8), NO derived from eNOS, possibly from the endothelial cells, plays a role in reducing reactivity of the airways.


    ACKNOWLEDGEMENTS

We acknowledge the contribution of J. Staczek for the measurement of airway resistance, of L. Pennel for immunohistochemistry, and of C. Thomas for statistical analysis. We thank J. M. Polak for helpful critical discussion.


    FOOTNOTES

Address for reprint requests and other correspondence: E. Canet, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France (E-mail: emmanuel.canet{at}fronetgrs.com).

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 17 February 2000; accepted in final form 19 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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