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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Nitric oxide (NO), a neurotransmitter of inhibitory nonadrenergic noncholinergic (iNANC) nerves in
airways, is a radical with a short half-life, and its function may be modified by airway inflammation.
To test this hypothesis, we examined whether airway allergic inflammation affects iNANC responses
mediated by NO in guinea pigs in vitro. Animals sensitized with ovalbumin (OA) were challenged
with 0.03% OA (OA group) or saline (saline group) by inhalation on 3 consecutive days. On the day
after the final challenge, iNANC responses elicited by electrical field stimulation (2 to 16 Hz) or relaxation responses to 3-morpholinosydnonimine (SIN-1), 10
8 to 104 M, were obtained in the tracheal
strips precontracted by histamine (3 × 106 M) in the presence of atropine and propranolol (both
106 M). The iNANC responses of the OA group were significantly attenuated compared with those
of the saline group (p < 0.05), and the inhibitory effect of a NO synthase (NOS) inhibitor, N
-nitro-
L-arginine methyl ester, on the iNANC responses was abolished in the OA group. SIN-1-induced tracheal smooth muscle relaxation was also significantly affected by antigen exposure (p < 0.05), the
effect of which disappeared in the presence of a NO scavenger, carboxy PTIO (3 × 106 M). The impairment of the iNANC responses after antigen exposure was significantly restored by superoxide
dismutase (1,000 U/ml), especially at lower frequencies. Histochemical demonstration of NADPH-diaphorase-positive nerves representing neural NOS density was not different between the two groups.
These results suggest that allergic airway inflammation impairs neural NO-induced relaxation, presumably by inhibiting the access of neural NO to the airway smooth muscle.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The inhibitory nonadrenergic noncholinergic (iNANC) nerve is the only neural bronchodilator mechanism in human airways (1), where its bronchodilatatory function has been demonstrated in vitro by electrical field stimulation (EFS) (2, 3) as well as in vivo by reflex stimulation (4, 5). The neurotransmitter of this nervous system has been suggested to be vasoactive intestinal peptide (VIP) or other related peptides in several species (6, 7) since these peptides have a potent bronchodilatatory action (3, 8) and the coexistence of these peptides with acetylcholine in airway nerve fibers has been demonstrated histochemically (9). Recently, nitric oxide (NO) has also been recognized as a neurotransmitter of iNANC nerves distributed in various organs (10), including airways (13), by functional studies using inhibitors for NO synthase (NOS). Histochemically, colocalization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity, a marker for NOS in the nervous system (17, 18), and VIP-like immunoreactivity was demonstrated in guinea pig airways (19), in which both VIP and NO have been reported to functionally mediate iNANC relaxation (20). Especially in human airways, NO is thought to mainly mediate iNANC responses (14, 16), and so it is important to elucidate how the neural NO-mediated bronchodilatatory function is regulated by airway inflammatory conditions such as those in asthmatic airways. NO has the characteristics of a radical and is known to be scavenged by superoxide (21, 22). Therefore, it is possible that the neural relaxation mediated by NO is modified in inflamed airways, especially during inflammation involving the release of superoxide. In the present study, in order to elucidate the function of nitric oxidergic bronchodilation in inflamed airways, we examined the effect of antigen-induced airway inflammation on iNANC relaxation in the presence and absence of a NOS inhibitor. In addition, we also examined whether the density of peripheral NOS-containing nerves, as determined by staining for NADPH-diaphorase, is modified by airway allergic inflammation.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Sensitization and Inhalation Challenge
Male Dunkin-Hartley guinea pigs, weighing 200 to 250 g, were sensitized by subcutaneous injection of 0.5 ml of saline containing 10 µg of ovalbumin (OA) and 100 mg of aluminum hydroxide on 2 consecutive days. Three weeks after the sensitization procedure, the animals were divided into two groups and exposed to saline aerosol (saline group) or to 0.03% OA aerosol (OA group) delivered by ultrasonic nebulizer (NE-U12; Omron, Tokyo, Japan) (output, 0.8 ml/min) for 3 min in a Plexiglas® exposure chamber (24.5 × 40.5 × 15.0 cm) under spontaneous breathing. Once severe respiratory distress induced by antigen exposure was observed, the animals were immediately taken out of the chamber and oxygen was administered. This inhalation challenge procedure was repeated for 3 consecutive days for each animal.
Animal Preparation for in vitro Studies
On the day after the final challenge, animals were anesthetized with
an intraperitoneal injection of urethane (2 g/kg) and killed by exsanguination. The trachea and lungs were quickly removed and placed in
Krebs-Henseleit (K-H) solution cooled to 4° C. The composition of
K-H solution was as follows (mM): NaCl, 118; KCl, 5.9; MgSO4, 1.2;
CaCl2, 2.5; NaH2PO4, 1.2; NaHCO3, 25.5; glucose, 5.6. After being
cleaned of the connective tissue, the upper trachea was opened longitudinally by cutting through the cartilage and cut into segments so
that each segment contained three to four cartilage rings since we
have preliminarily confirmed that iNANC relaxation responses are
dominant in the upper rather than in the lower trachea in guinea pigs.
Each segment of the upper trachea was mounted in an organ bath
containing K-H solution maintained at 37° C and gassed with 95% O2
and 5% CO2, giving a pH of 7.4. Strips were connected via silk threads
to force displacement transducers (UL-10GR; Minebea Co., Ltd., Tokyo, Japan) for the measurement of isometric changes in tension, and
the responses were recorded on a polygraph (Rectigraph-8K; San-ei,
Tokyo, Japan). Indomethacin (106 M) was present throughout the
experiment to prevent fading of neural responses caused by endogenous prostaglandin production. The tissues were allowed to equilibrate for 1 h with frequent washing under a resting tension of 1 g,
which was found to be optimal for measuring the changes in tension. A
part of the remaining trachea was used for the histologic confirmation
of antigen-induced airway inflammation, which revealed that all of the
antigen-exposed tissues showed a massive infiltration of inflammatory
cells, including eosinophils, into the airway mucosa and epithelium.
Relaxation Study
After the equilibration period, iNANC relaxation responses elicited
by EFS (50 V at source, 0.5 ms duration, for 20 s) or relaxation responses to exogenously applied 3-morpholinosydnonimine (SIN-1),
108 to 104 M, a NO donor, were obtained in the presence of atropine and propranolol (both 106 M). We chose SIN-1 as a NO donor
because a NO scavenger, carboxy PTIO (cPTIO) (23), significantly
shifted SIN-1-induced concentration-response curves to the right in
the preliminary studies. The concentration-response curves to SIN-1
were also obtained in the presence of cPTIO (3 × 106 M). All of
these relaxation studies were performed in airways precontracted with
histamine (3 × 106 M). To obtain iNANC frequency-response curves,
stimulation frequencies of 2, 4, 8, and 16 Hz were applied in random
order until reproducible responses to the corresponding frequency
were obtained. After obtaining reproducible frequency-response
curves in each tissue, the effect of a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), 104 M, on the iNANC response was also
examined. At the end of the EFS-induced relaxation study, tetrodotoxin (106 M) was applied to confirm the component of neural origin.
In all relaxation studies, the maximal relaxation response of each tissue to papaverine (104 M) was obtained to normalize each relaxation
response. In another set of experiments, the effect of superoxide dismutase (SOD), 1,000 U/ml, on the iNANC response was also examined using tracheal tissues taken from both the saline and the OA
groups, in which the iNANC frequency relaxation response was obtained before and after incubation of each tissue with SOD for 30 min.
NADPH-diaphorase and PGP9.5 Staining
For the histologic study, independent animals from each group were anesthetized with urethane (2 g/kg) on the day after the final challenge. In order to fix the tissues, the thorax was opened and the systemic circulation was first perfused with 60 ml of ice-cold saline and then with 360 ml of 2% paraformaldehyde (PFA) in phosphate buffer (pH, 7.4; 0.1 M) solution. After removal of the trachea and the lungs, the upper trachea was immersed in the same fixative for 12 h at 4° C, and further immersed for 24 h at 4° C in 0.1 M phosphate buffer containing 30% sucrose. The tissues were then sectioned at a thickness of 12 µm with a cryostat, cutting along the longitudinal axis of the airways. All cryosections were mounted on chrome-alum, gelatin-coated glass slides and prepared for the later processing for histochemistry.
NADPH-diaphorase staining was performed by incubating slide-mounted tissue sections with 1 mM 
-NADPH/0.2 mM nitroblue tetrazolium/0.1 M TRIS HCl at pH 8.0/0.2% Triton X-100 for 30 to 60 min at 37° C. The reaction was stopped by rinsing the sections in phosphate-buffered saline (PBS) (pH, 7.2; 0.01 M). In order to normalize
the NADPH-diaphorase-positive nerve length with the total nerve
length around the smooth muscle bundle, the same tissues were then
stained immunohistochemically with a panneuronal marker, PGP9.5
(24). Briefly, after washing in PBS, sections were incubated overnight
with monoclonal antihuman PGP9.5 mouse IgG (1:200 dilution; Biogenesis Ltd, Poole, UK) at 4° C. In order to reduce nonspecific binding of the antibody, the slide-mounted tissues were preincubated with
inactivated normal sheep serum (10%) for 30 min at room temperature. The immunoreactions were visualized by indirect immunoperoxidase methods using horseradish-peroxidase-labeled antimouse Ig
from sheep (1:50 dilution, 2-h incubation at 4° C). The diaminobenzidine reaction was performed in 50 mM TRIS HCl. Endogenous peroxidase activity was reduced by preincubation in 0.03% hydrogen peroxide in methanol for 30 min at room temperature. The tissue sections
were covered with a mixture of glycerol and PBS (2:1, vol/vol), and
examined with an Olympus BHS microscope (Olympus Optical Co.,
Ltd., Tokyo, Japan). The staining for NADPH-diaphorase and PGP9.5
in the tissue sections was photographed with Fuji Neopan F-film (Iso
100; Fuji Photo Film Co., Tokyo, Japan).
Quantitative Analysis of NADPH-diaphorase-positive Nerves
After double staining of the NADPH-diaphorase and PGP9.5, each section was examined with a light microscope in order to quantify the NADPH-diaphorase-positive nerve density. For this purpose, the total length of NADPH-diaphorase-positive nerve fibers around the airway smooth muscle bundle was measured and normalized by the total nerve length visualized with PGP9.5 staining within the corresponding smooth muscle area with image-analyzing software (MacScope; Mitani Co., Fukui, Japan) using an Apple Macintosh computer connected to the microscope. The quantification of NADPH-diaphorase-positive nerves was performed in more than five random images of airway smooth muscle (×400) taken from each section, and it was repeated in three to five sections of the tracheal specimens.
All the studies described here were conducted with the consent of the Ethics Committee for Use of Experimental Animals of the Tohoku University School of Medicine.
Drugs
The following drugs were used: urethane, ovalbumin, tetrodotoxin,
papaverine, N-nitro-L-arginine methyl ester, and 3-morpholinosydnonimine (Sigma Chemical Co., St. Louis, MO), atropine sulphate
(Tanabe Pharmaceutical, Osaka, Japan), propranolol and indomethacin (Sumitomo Chemical Co., Osaka, Japan), aluminum hydroxide,
histamine hydrochloride, paraformaldehyde, nitroblue tetrazolium,
Triton-X, NaN3, hydrogen peroxide, and diaminobenzidine (Wako
Pure Chemical Industries, Osaka, Japan), -NADPH (Oriental Yeast
Co., Ltd., Tokyo, Japan), superoxide dismutase linked to polyethylene
glycol (provided by Takeda Chemical Industries Ltd., Osaka, Japan).
Statistical Analysis
All relaxation responses are expressed as percentages of the maximal
relaxation response to papaverine (104 M) of each tracheal tissue.
Two-way analysis of variance was used to compare the two EFS-induced frequency-response curves before and after L-NAME addition as well as to compare the frequency-response curves obtained
from the control group and those from the OA group. The comparison between the iNANC response before and after SOD addition at the
same frequency of the same tissue was performed by Student's paired
t test. All other data were compared by Student's unpaired t test. For
SIN-1-induced relaxation concentration-response curves, IC50, the
concentration required to produce 50% relaxation of the maximal
plateau response by the agonist was calculated by the interpolation of
each curve. All data are expressed as means ± SEM, and values of
p < 0.05 were considered significant.
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RESULTS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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iNANC Relaxation Response and Effect of L-NAME
In tissues taken from saline-challenged animals, EFS (2, 4, 8, and 16 Hz) produced a frequency-dependent relaxation of airways precontracted by histamine (3 × 106 M) in the presence
of atropine and propranolol, which was significantly reduced
by the addition of L-NAME (104 M, p < 0.05) (Figure 1a),
especially at lower frequencies (2 and 4 Hz). In contrast, the
EFS-induced relaxation response in tissues taken from the OA
group was potently attenuated compared with that of the saline
group in the absence of L-NAME (p < 0.01). In this group,
L-NAME had no significant effect on the EFS-induced frequency-response curve (Figure 1b), indicating that the neural NO-induced component of iNANC relaxation was abolished in
the OA group. The frequency-response curve after L-NAME
treatment in the OA group was still significantly reduced compared with that of the saline group (p < 0.05). The application
of TTX (10 to 6 M) abolished the relaxation responses at all
frequencies examined in all tissues, suggesting that the EFS-induced relaxation in the present study is neural origin.
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Exogenously Applied SIN-1-induced Relaxation
Exogenously applied SIN-1 (108 to 104 M) produced concentration-dependent relaxation of the tracheal tissues from
both groups precontracted by histamine (3 × 106 M) (Figure
2). In the absence of cPTIO (3 × 106 M), the maximal relaxation by SIN-1 was not different between the groups, but IC50
of the SIN-1-induced relaxation curve in the saline group (3.3 ± 0.7 × 107 M) was significantly lower than that in the OA
group (1.2 ± 0.3 × 106 M, p < 0.05) (Figure 2a). This significant difference in the relaxation curve between the groups disappeared in the presence of cPTIO (3 × 106 M) (Figure 2b);
IC50 of the saline group and the OA group was 1.2 ± 0.4 × 106 M and 2.6 ± 1.3 × 106 M, respectively (p > 0.05).
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Effect of SOD on iNANC Response
The EFS-induced iNANC relaxation was not significantly affected by the incubation with SOD (1,000 U/ml) in the tissues
of the saline group, whereas those at 2, 4, and 8 Hz were significantly restored in the OA-challenged tissues (Figure 3).
This reversal effect of SOD in the OA group was not observed
in the presence of L-NAME (104 M) (data not shown).
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p < 0.05; significantly different from the data after SOD of OA group.
Neural NADPH-diaphorase Density in Airway Smooth Muscle
NADPH-diaphorase-positive nerve fibers within PGP9.5-positive nerves were abundantly seen around the smooth muscle layer of the tracheal tissues taken from both the saline and the OA groups (Figure 4). These NADPH-diaphorase-positive nerve fibers were also seen around the vessels in the submucosal layer and beneath the epithelium. The nerve fibers in the smooth muscle layer formed networks surrounding the smooth muscle bundle. Quantitative analysis revealed that the density of the NADPH-diaphorase-positive nerves normalized by PGP9.5 costaining were not significantly different between the saline and the OA groups (p < 0.05), although there was a tendency for NADPH-diaphorase-positive nerves to be less abundant in the OA group (Figure 5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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We have demonstrated that in vitro L-NAME-sensitive iNANC relaxation was markedly impaired by repeated in vivo antigen exposure, and this impairment was significantly restored at lower frequencies by the incubation with SOD only in the absence of L-NAME. We have also shown that the NO scavenger-sensitive part of the exogenously applied SIN-1-induced relaxation was also affected. The density of NADPH-diaphorase-positive nerves, representing NOS-containing nerves, surrounding the airway smooth muscle was not significantly changed by antigen challenge.
In the present study, EFS produced TTX-sensitive iNANC
neural relaxation in airways precontracted by histamine in a
frequency-dependent manner. The neural relaxation of saline-exposed animals was significantly reduced by a NOS inhibitor,
L-NAME (104 M), especially at lower frequencies, which is
comparable with previous studies using guinea pigs (13, 20). In
contrast, the iNANC relaxation of OA-exposed animals was
not significantly affected by L-NAME, indicating that the NO-mediated component of the neural relaxation was markedly
impaired by repeated antigen exposure.
One possible mechanism for this impairment, suggested from the present results, is the scavenging of neural NO during the diffusing process from nerve endings to the effective sites of airway smooth muscle. In the present study, exogenously applied SIN-1-induced relaxation was also significantly affected in the antigen-exposed animals. SIN-1 has been demonstrated to liberate NO (25) and shown to relax tracheal smooth muscle in guinea pigs in vitro (26). Because a NO scavenger, cPTIO, significantly attenuated SIN-1-induced relaxation in the present system, SIN-1-induced tracheal smooth muscle relaxation is assumed to be due to NO release in agreement with the previous report (25). In addition, the difference in IC50 of the SIN-1-induced concentration-response curves between the saline group and the antigen group became insignificant in the presence of cPTIO. Therefore, it is possible that, because neural NO is scavenged in the airways repeatedly exposed to antigen, there is less of it available to relax airway smooth muscle.
A possible candidate substance that scavenges NO is superoxide (21, 22). In the present study, SOD significantly restored the iNANC responses at lower frequencies impaired by antigen exposure only in the absence of L-NAME. This indicates that the NO of neural origin was, at least partially, inactivated by superoxide. Superoxide has been shown to be released from inflammatory cells such as eosinophils (27) and mast cells (28) after antigen exposure. These inflammatory cells were abundantly found in the tissues taken from the antigen-exposed animals. Therefore, NO of neural origin could be scavenged by superoxide during the diffusion. The reversal of iNANC responses by SOD became less apparent at higher frequencies. This seems to be related to the fact that the L-NAME-resistive component of iNANC responses becomes greater at higher frequencies.
There may be another possible mechanism to explain the impairment of the L-NAME-sensitive iNANC relaxation. In order to quantify the density of NOS-containing nerves, we used histochemical staining for NADPH-diaphorase. NADPH-diaphorase is an oxidative enzyme localized to some populations of neurons in the brain (29), and it has been shown to reflect NOS (17, 18), especially in neuronal tissues. The colocalization of NADPH-diaphorase and VIP-like immunoreactivity, which is a candidate neurotransmitter of the L-NAME-resistive component of the iNANC system (20), has already been reported in guinea-pig airways (19). Because there was no significant difference in the NADPH-diaphorase-positive nerve density between the saline and OA groups, the density of NOS-containing nerves appears to have been preserved even in the antigen-exposed animals. Thus, it is unlikely that the impairment of the iNANC response by antigen exposure is due to the decrease in the number of NOS-containing nerves.
The impairment of iNANC relaxation after antigen exposure has been reported, especially concerning the component mediated by VIP. We have previously reported that iNANC relaxation is attenuated by the immediate allergic response in vivo (30), and that this attenuation is abolished by the protease inhibitor for tryptase, an enzyme released from mast cells that degrades VIP (31). Lilly and coworkers (32) also demonstrated that VIP-mediated bronchodilation is reduced after chronic antigen exposure through the degradation of VIP by a tryptic enzyme. In the present study, the iNANC responses after the L-NAME addition was significantly smaller in the antigen-exposed group than in the control group. This suggests that L-NAME-resistive iNANC relaxation, the component presumably mediated by VIP, was also affected by repeated antigen exposure. Thus, airway allergic inflammation affects the neurally originated NO-mediated component, which is thought to be dominant in human airways (14, 16), as well as the VIP-mediated component of the iNANC response. It is possible that the neural NO-mediated airway function does not work properly in the presence of airway allergic inflammation, leading to the exacerbation of asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Kunio Shirato, M.D., Professor and Chairman, First Department of Internal Medicine, Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-71, Japan.
(Received in original form June 10, 1996 and in revised form January 27, 1997).
Acknowledgments: The writers thank Brent Bell for reading the manuscript.
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References |
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METHODS
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DISCUSSION
REFERENCES
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