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
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ABSTRACT |
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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
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INTRODUCTION |
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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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MATERIALS AND METHODS |
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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(Expression of NOS mRNA
C57BL/6 and eNOS(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(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-.
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- 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)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(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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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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nNOS
The mRNA for nNOS was observed in the trachea and brain of both C57BL/6 and eNOS(
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iNOS
The mRNA for iNOS was observed in the trachea, lungs, and brain of both C57BL/6 and eNOS(
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eNOS
The mRNA for eNOS was observed in the trachea, lungs, and brain of C57BL/6 but not in eNOS(
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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(
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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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DISCUSSION |
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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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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