Contribution of type I NOS to expired gas NO and bronchial
responsiveness in mice
George T.
De Sanctis1,
Sanjay
Mehta2,
Lester
Kobzik3,
Chandri
Yandava1,
Aiping
Jiao1,
Paul L.
Huang4, and
Jeffrey M.
Drazen1
1 Pulmonary and Critical Care
Division, Department of Medicine and
3 Department of Pathology,
Brigham and Women's Hospital;
4 Cardiac Unit and Cardiovascular
Research Center, Massachusetts General Hospital, Harvard Medical
School, Boston, Massachusetts 02115; and
2 Respiratory Division, Department
of Medicine, London Health Sciences Centre-Victoria Campus, University
of Western Ontario, London, Ontario, Canada N6A 4G5
 |
ABSTRACT |
Nitric oxide (NO)
can be measured in the expired gas of humans and animals, but the
source of expired NO (FENO) and
the functional contribution of the various known isoforms of NO
synthase (NOS) to the NO measured in the expired air is not known.
FENO was measured in
the expired air of mice during mechanical ventilation via a tracheal
cannula. FENO was significantly
higher in wild-type B6SV129J +/+ mice than in mice with a targeted
deletion of type I (neural) NOS (nNOS,
/
) (6.3 ± 0.9 vs. 3.9 ± 0.4 parts/billion, P = 0.0345, for +/+ and
/
mice, respectively), indicating
that ~40% of the NO in expired air in B6SV129 mice is derived from
nNOS. Airway responsiveness to methacholine (MCh), assessed by the log
of the effective dose of MCh for a doubling of pulmonary resistance
from baseline
(ED200RL),
was significantly lower in the
/
nNOS mice than in the
wild-type mice
(logED200RL,
2.24 ± 0.07 vs. 2.51 ± 0.06 µg/kg, respectively;
P = 0.003). These findings indicate that nNOS
significantly contributes to baseline
FENO and promotes airway
hyperresponsiveness in the mouse.
asthma; bronchoconstriction; neuronal nitric oxide synthase; knockout; nitric oxide
 |
INTRODUCTION |
NITRIC OXIDE (NO) is a modulator of airway function; in
cat and guinea pig tracheal smooth muscle, modulation of the enzymatic capacity to produce NO is associated with diminished contractile responses (1, 4, 15, 19, 24) to a variety of stimuli. In these
experiments, NO was thought to contribute in an inhibitory fashion to
airway tone because inhibition of the endogenous synthesis of NO, using
competitive inhibitors of nitric oxide synthase (NOS), was associated
with enhanced airway responses. In contrast, patients with asthma,
compared with nonasthmatic individuals, have elevated levels of NO in
their exhaled air (16, 17, 21, 22). Furthermore, it is known that, in
subjects with asthma, treatment with steroids is associated with an
improvement in pulmonary function and a decrement in the amount of NO
in the exhaled air (18, 21, 28). These data from patients with asthma
indicate that NO can contribute in a procontractile manner to the
airway narrowing.
The NO that appears in the exhaled air derives from endogenous
production by one of three known NOSs, namely type I or neural NOS
(nNOS), type II or immune NOS (iNOS), and type III or endothelial NOS
(eNOS) (12, 25). Although it has been speculated that types I and III
NOS are the source of NO under basal, i.e., not inflamed, conditions,
appropriately selective enzyme inhibitors are not available that can be
used to establish this point unequivocally. To ascertain the effects of
selective type I NOS deficiency on the amount of NO in the exhaled air
and to ascertain the effects of this deficiency on basal airway tone
and the response to bronchoconstrictor agonists, we measured exhaled NO
and pulmonary mechanical responses to methacholine (MCh) in wild-type
+/+ B6SV129J mice and B6SV129 mice harboring a homozygous targeted
disruption of the nNOS gene (14). Our data indicate that nNOS
contributes significantly to the NO in the exhaled air and that NO
derived from nNOS functions in a procontractile role in the mouse.
 |
MATERIALS AND METHODS |
Animals.
Male and female specific pathogen-free (SPF) wild-type B6SV129J nNOS
+/+ mice were purchased from Jackson Laboratory (Bar Harbor, ME) and
were acclimatized for 7-10 days after arrival. Male and female SPF
nNOS
/
mice (14) were bred in a barrier facility. All
animals were housed in isolation cages under SPF conditions; blood from
sentinel animals was routinely tested to ensure their SPF status. All
animals were studied at 7-8 wk of age.
Immunohistochemistry.
At the termination of the experiment, animals were killed by
exsanguination under surgical anesthesia, and lung and ileal (small
bowel) tissue samples were collected, inflated with optimum cutting
temperature (OCT) compound, and snap frozen.
Immunostaining was performed on OCT-treated, snap-frozen, 2%
paraformaldehyde-fixed (5 min) cryostat tissue sections. A standard
avidin-biotin-based immunoperoxidase protocol was used (13) with
diaminobenzidine as the chromogen. Immunostaining was performed using a
rabbit anti-NOS I antibody (Transduction Laboratories, Lexington, KY) or control normal rabbit immunoglobulin G (Sigma Chemical, St. Louis,
MO).
Genotype analysis by polymerase chain reaction.
The polymerase chain reaction (PCR) amplification was carried out as
previously described (14, 27) with minor modifications. Briefly, the
primers used for the detection of nNOS were as follows: B1 primer,
5'-CCT TAG AGA GTA AGG AAG GGG GCG GG-3'; and B2 primer, 5'-GGG CCG ATC ATT GAC GGC GAG AAT GAT G-3' (14). The PCR
mixture (25 µl) contained 500 ng of genomic DNA, 10 pmol of each
primer, PCR buffer (Boehringer Mannheim), 2.5 mM
MgCl2, 200 µM each dCTP, dATP,
and dTTP, 50 µM dGTP, 150 µM 7-deaza-dGTP, 5% dimethyl sulfoxide, and 1.5 units of Taq polymerase. The
PCR conditions were as follows: 6 min at 94°C followed by 15 s at
94°C, 23 s at 60°C, and 30 s at 60°C. Chain elongation was
continued after the last cycle for 5 min. Twenty microliters of PCR
product were electrophoresed on a 1.5% agarose gel with the products
visualized using ethidium bromide under ultraviolet light. The absence
of the 404-bp product confirmed the disruption of the nNOS gene in the
/
mice, whereas the band was observed in wild-type +/+
mice.
Measurement of expired NO levels.
The animals were ventilated with inspired air that was essentially NO
free [<1 part/billion (ppb)] and that had an
O2 fraction of 0.21. For the
measurement of expired NO levels
(FENO), mixed expired gas samples
were collected in a Mylar bag over 5 min from the expiratory port of
the ventilator; all samples were analyzed within 30 min of collection
as previously described (23). The expired gas sample was vigorously
mixed manually for 5-10 s before determination of the NO
concentration by chemiluminescence (42S NO analyzer; Thermo
Environmental Instruments, Franklin, MA). The NO analyzer was
calibrated daily against a reference gas of known NO concentration
(Matheson Scientific Gas, Houston, TX).
Changes in FENO during airway
contractile responses.
A baseline mixed expired gas sample was collected before each dose of
MCh. To assess changes in FENO
levels during bronchoconstriction after MCh administration, collection
of mixed expired gas was started 90 s before administration of a dose
and continued until 210 s after administration of the dose, for a total
collection time of 300 s. The FENO
level after each dose of MCh was thus referenced to the baseline level
immediately preceding the dose. In pilot studies, the 33 and 100 µg/kg doses of MCh had no significant effect on either pulmonary
resistance (RL)
or FENO levels (data not shown);
thus mixed expired gas was collected only before and after the 330, 1,000, and 3,300 µg/kg doses of MCh.
Measurement of airway responsiveness.
Airway responsiveness was measured as previously described (8). Each
mouse was anesthetized with an intraperitoneal injection of
pentobarbital sodium (70-80 mg/kg; Anthony Products, Arcadia, CA).
When an acceptable stage of surgical anesthesia was reached, the metal
portion of a 19-gauge tubing adapter was inserted into the trachea and
was secured in place. An internal jugular vein was cannulated with a
saline-filled Silastic catheter (0.06 cm outer diameter, 6-8 cm
length, <0.005 ml volume) attached to a 0.1-ml Hamilton microsyringe
(Hamilton, Reno, NV) and was used to administer MCh
(acetyl-
-methylcholine chloride; Sigma Chemical). A thoracotomy was
performed so that pleural pressure would equal body surface pressure.
The tracheostomy tube was passed through a hole in the plethysmograph
chamber and was connected via three ports of a four-way connector to a
rodent ventilator (model no. 683; Harvard Apparatus, Division of Ealing
Scientific, Natick, MA). The ventilator was set to provide 150 breaths/min with tidal volumes of 5-6 µl/g and a positive
end-expiratory pressure of 3-4
cmH2O. Mice were placed in a
sealed constant-mass plethysmograph consisting of a 1-liter bottle
insulated from the ambient environment by 0.5-in. foam padding and
containing copper mesh to maintain isothermal conditions. The pressure
difference between the plethysmograph chamber and the 1-liter reference
chamber was detected with a transducer (Celesco model LCVR, 0-2
cmH2O, Canoga Park, CA). Changes in lung volume were determined from measured changes in plethysmograph pressure. An electrical signal proportional to flow was obtained by
electrical differentiation of the volume signal. The delay between the
volume and flow signals was <0.5 ms. Transpulmonary pressure was
measured as the pressure difference between the pressure at the airway
opening, measured from the fourth port of the four-way connector, and
the pressure in the plethysmograph itself. The plethysmograph system
has been shown to be without significant amplitude distortion or phase
shift up to 30 Hz.
RL was determined
with the use of signals derived from transpulmonary pressure and lung
volume (8). Dose-response curves to MCh were obtained by administering
sequentially increasing doses of MCh (33 to 3,300 µg/kg) in a 20- to
35-µl volume. The volume of fluid injected with each dose produced no
measurable physiological effects. The peak response to each dose was
obtained from 15 serial measurements after injection of MCh. Each
measurement was calculated via a cross-correlation technique from a
number of breaths recorded during the measurement interval. Because the pulmonary response to MCh peaks later and dissipates more slowly with
increasing doses, the interval for each of the measurements was
increased from 4 s (10 breaths) to 10 s (25 breaths) as the MCh dose
was increased. Furthermore, enough time was allowed to elapse between
MCh doses such that predose measurements of
RL returned to
within 10% of the value obtained before the preceding dose of MCh. A
large breath (3 times tidal volume) was administered to standardize
volume history before each dose of MCh. Pulmonary responses were
recorded on a dedicated microcomputer. Each animal's dose-response
curve was log transformed and then was subjected to regression analysis
to calculate the dose required for a twofold increase in
RL
(ED200RL).
In vitro
L-arginine-to-L-citrulline
conversion (maximal NOS activity) assay.
The total and Ca2+-independent
fraction of total NOS activity were assessed as previously described by
Mehta et al. (23). Mouse lung parenchyma and brain parenchyma were
homogenized in five volumes of ice-cold buffer [50 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, pH
7.5] containing (in mM) 1 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 1.25 CaCl2, and 0.2 phenylmethylsulfonyl fluoride, as well as (in µg/ml) 10 pepstatin, 10 antipain, 10 leupeptin, 10 chymostatin, and 10 soybean trypsin inhibitor (all from Sigma). After centrifugation at 9,000 g for 15 min at 4°C, the pellet
was washed twice with an equal volume of buffer containing 1 M KCl, and
the supernatants were pooled. Maximal in vitro NOS activity was
measured by the conversion of L-[3H]arginine
to
L-[3H]citrulline.
Fifty-microliter samples were incubated in 50 mM Tris · HCl (pH 7.5) containing 1.25 mM
CaCl2, 1 mM EGTA, 1 µM flavin
mononucleotide, 1 µM FAD, 1 µM tetra-hydrabiopterin, 1 mM NADPH,
and 20 µM
L-[2,3,4,5-3H]arginine
(0.3 Ci/mmol) in a final reaction volume of 100 µl. The reaction was
carried out at 37°C for 45 min and was stopped by adding 900 µl
of 20 mM sodium-acetate (pH 5.4) containing 1 mM
L-citrulline, 2 mM EGTA, and 2 mM EDTA. Reaction mixes were applied to cation exchange resin (AG
50W-X8, Na+ form; Bio-Rad,
Hercules, CA) columns and were eluted two times with distilled water,
the eluents were pooled, and radioactivity was determined by liquid
scintillation spectrophotometry.
All lung and brain homogenate samples were incubated as above in the
presence of 100 µM
NG-monomethyl-L-arginine
(L-NMMA) to assess background
non-NOS-dependent conversion of
L-[3H]arginine
to
L-[3H]citrulline.
Samples were also incubated in the presence of excess 3 mM EGTA to
assess the Ca2+-independent
fraction of total NOS activity. The sample protein concentration was
determined by the biuret reaction (Micro BCA; Pierce, Rockford, IL)
with bovine serum albumin as the standard. Total and
Ca2+-independent
non-L-NMMA-inhibitable in vitro
maximal pulmonary NOS activities are expressed as picomoles
L-citrulline produced per
milligram protein per minute.
Statistical analysis.
Computations were performed with the JMP 3.15 (SAS Institute, Cary, NC)
statistical package. Differences between mean values were assessed by a
Wilcoxon/Kruskal-Wallis test for nonparametric data and a
Tukey-Kramer honestly significant differcence test for parametric
data. Results are expressed as means ± SE and, unless
otherwise stated, were considered statistically significant at
P < 0.05.
 |
RESULTS |
Immunohistochemistry.
Immunoperoxidase staining for nNOS in peripheral airways and proximal
gut confirmed the presence of nNOS in the wild-type +/+ animals.
Staining of similar sections of airways and gut demonstrated an absence
of staining in the nNOS-deficient group (Fig.
1). Immunohistochemistry and reverse
transcriptase (RT)-PCR revealed no alterations in types II and III NOS
in the type I knockout mice (Huang, unpublished observations).

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Fig. 1.
Representative histological sections from the mice in this study.
Immunodetection of neural nitric oxide synthase (nNOS):
immunoperoxidase staining of cryostat sections of airways and gut
confirms the presence of nNOS antigen in wild-type +/+ mice
(A, airway;
B, intestinal wall) and its absence in
nNOS knockout mice (C, airway;
D, intestinal wall). Absence of staining in nNOS
/ colon and by control rabbit immunoglobulin G not shown.
Original magnification, ×200. Arrows in A
and C indicate nNOS positive nerves.
|
|
Genotype confirmation by PCR.
PCR amplification reactions were carried out in nNOS
/
and wild-type +/+ animals to detect nNOS. The absence or presence of
the 404-bp product was confirmed in the appropriate groups of nNOS
/
and wild-type +/+ mice, respectively (Fig.
2).

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Fig. 2.
Genotype analysis by polymerase chain reaction. nNOS is absent in the
nNOS knockout (KO) mice (absence of 404-bp product) and present in the
wild-type controls.
|
|
FENO during MCh-induced
bronchoconstriction.
Mean values ± SE for FENO are
presented in Table 1. Both wild-type and
nNOS-deficient mice had measurable
FENO levels. In wild-type +/+ mice
(n = 12), the mean basal
FENO was 6.3 ± 0.9 ppb. In
contrast, the FENO in the nNOS
/
mice (n = 18) was
significantly lower (3.9 ± 0.4 ppb) than that in the wild-type +/+
controls (P = 0.034). Therefore, nNOS
accounts for ~40% of the total
FENO when comparing the
FENO levels of the nNOS +/+ and
/
groups. Analysis of expired gas collected before and
after the 330, 1,000, and 3,300 µg/kg doses of MCh revealed no
significant increase in FENO in
either the nNOS +/+ or the nNOS
/
group. The fractional change in FENO during
bronchoconstriction (from baseline, taken before delivery of the MCh
dose) in the nNOS +/+ and
/
groups for the 330, 1,000, and 3,300 µg/kg doses of MCh are as follows: 0.99 ± 0.095 vs.
1.17 ± 0.095, 0.98 ± 0.096 vs. 1.16 ± 0.096, and 1.29 ± 0.10 vs. 1.32 ± 0.10, respectively.
Airway resistance and responsiveness in wild-type
+/+ and
nNOS-deficient
/
mice.
There were no significant differences in baseline
RL between the
wild-type and nNOS
/
groups (1.83 ± 0.04 vs. 1.84 ± 0.04 cmH2O · ml
1 · s).
In nNOS
/
mice, the
logED200RL
was 2.51 ± 0.06 (n = 17) and, in
the wild-type +/+ mice, was 2.24 ± 0.07 µg/kg
(n = 12); the nNOS group was 1.9-fold
less responsive (calculated from the difference of the log values) than
the wild-type group (P = 0.003; Fig.
3).

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Fig. 3.
Bronchial responsiveness to methacholine in wild-type nNOS +/+ and nNOS
knockout / mice. nNOS +/+ mice were significantly more
responsive to methacholine challenge than the nNOS /
mice.
ED200RL,
effective dose of methacholine for a doubling of pulmonary resistance
from baseline. Values are means ± SE. The log
ED200RL is significantly
(P = 0.003) higher in nNOS / mice compared with nNOS
+/+ mice.
|
|
In vitro
L-arginine-to-L-citrulline
conversion (maximal NOS activity) assay.
Mean values ± SE for total and
Ca2+-independent NOS activity are
presented in Table 2. Both wild-type +/+
and nNOS-deficient
/
mice had measurable total and
Ca2+-independent NOS activity in
lung and brain. In the lung there was no significant difference between
the +/+ and
/
mice with respect to either total NOS or
iNOS activity. In the brain, there was no significant difference
between the +/+ and
/
mice with respect to iNOS activity.
However, there was a significant difference between the +/+ and the
/
mice in the mean total NOS activity (expressed as pmol
L-citrulline · mg
protein
1 · min
1
in brain). In the nNOS
/
mice
(n = 3), there was significantly (P = 0.003) lower total activity (0.93 ± 0.29 pmol
L-citrulline · mg
protein
1 · min
1)
compared with the wild-type +/+ (n = 3) controls (14.3 ± 2.1 pmol
L-citrulline · mg
protein
1 · min
1).
 |
DISCUSSION |
NO derived from nNOS contributes significantly to the NO in the mixed
expired gas of B6SV129 mice. Moreover, there was a small, i.e.,
1.9-fold, but significant decrease in airway responsiveness in the nNOS
/
mice from that in wild-type mice, indicating that nNOS-derived NO contributes in a procontractile manner to the airway
response to MCh. These results indicate that nNOS contributes ~40%
of the NO measured in mixed expired air and that the NO so produced
acts in a procontractile fashion to enhance MCh responsiveness in the
mouse.
NO is formed enzymatically by the stoichiometric conversion of
L-arginine to
L-citrulline. Endogenously
produced pulmonary NO has been shown to be a modulator of airway
function in health and disease. NO levels have been measured in the
expired gas of animals and humans and are reported to be increased in
asthmatics (16, 17, 21, 22) and in animal models of allergic
inflammation (23). Three distinct isoforms of NOS, each encoded by
separate genes, have been identified, and all catalyze the formation of NO. Of the three isoforms (11), the relative contribution of nNOS to
both FENO levels and airway
responsiveness is unknown. Even though nNOS is thought to be a
"low-NO-output" form of the enzyme, our data indicate that it
contributes a substantial fraction of the NO appearing in exhaled air.
Our data do not allow us to ascertain if this is a direct effect of
nNOS gene disruption on the cells contributing to the
FENO per se or an indirect effect modifying conditions such as air or blood flow in such a way as to
diminish the amount of NO recovered from the lung.
Type I NOS, also called nNOS, is an isoform found in high
concentrations in certain neuronal cells, both in the peripheral nervous system and in the central nervous system (5, 7, 20).
nNOS-containing nerve fibers have been localized in airway smooth
muscle, around submucosal glands, around blood vessels, and in airway
intrinsic ganglia (10, 20), but the role of nNOS in baseline airway
tone or in modifying the response to bronchoconstrictor stimuli had not
been elucidated. Our data clearly demonstrate that, in the mouse, nNOS
contributes in a procontractile fashion to the airway response to MCh.
Of interest, a recent analysis for linkage between the asthma phenotype
and markers on human chromosome 12 has identified a region on the
chromosome near the known location of human type I NOS (2).
The mechanism whereby mice deficient in type I NOS exhibit decreased
MCh responsiveness cannot be ascertained from the data acquired in this
study. There are, however, a number of possible mechanisms worthy of
consideration in this regard. First, although NO has been shown in
intact guinea pigs and isolated human airways to be a mediator of
nonadrenergic noncholinergic (NANC) neural bronchodilation, this has
been a prorelaxant rather than a procontractile mechanism (3, 6, 9, 24,
26). It is tempting to speculate that such NANC neurotransmission may
be procontractile in the mouse. It is also possible that other
mechanisms involving the integrative cardiopulmonary response of nNOS
knockout, such as changes in reflex homeostasis, result in the
diminished MCh responsiveness we observed. Irrespective of mechanism,
the diminished MCh response observed clearly indicates that NO derived
from neural sources augments the contractile response to a standard
bronchoprovocative stimulus.
In summary, the measurement of decreased
FENO levels in nNOS
/
knockout mice vs. wild-type nNOS +/+ mice demonstrates,
for the first time, that nNOS contributes significantly to the total FENO. There were no alterations in
total or Ca2+-independent (iNOS)
activity in the lungs of the nNOS knockout mouse vs. the wild-type
controls. In addition, whereas there were no differences in iNOS
activity in the brain between wild-type and nNOS mice, there were
significant differences in brain total NOS activity between the groups.
These differences can be attributed to the targeted deletion of the
nNOS gene in the latter group. Similar findings have been demonstrated
by immunohistochemistry and RT-PCR (Huang, unpublished observations).
We presume that the remaining FENO
is derived from eNOS because the animals were raised in viral
antigen-free conditions, and thus, in the absence of immune activation,
the remaining NO in the expired gas is derived from eNOS. Whereas the
decrease in FENO is not associated
with any changes in baseline lung resistance, the nNOS
/
knockout mice are significantly less responsive to MCh challenge than
their wild-type counterparts. Because the targeted deletion of the type I NOS gene resulted in only a 1.9-fold difference in airway
responsiveness between the wild-type and knockout mice, nNOS most
probably plays a secondary role in modulating this phenotype in the
mouse. It remains to be investigated whether there are strain-related
differences in the functional importance of nNOS-derived NO and what
role eNOS may play in modulating cholinergic responses in the mouse. Our findings implicate the involvement of nNOS-derived NO, perhaps in a
NANC excitatory system, in airway narrowing in the mouse.
 |
ACKNOWLEDGEMENTS |
G. T. De Sanctis was supported by National Heart, Lung, and Blood
Institute Grant HL-36110 and the American Lung Association. S. Mehta is
the recipient of fellowships from the Medical Research Council of
Canada, the Fonds de Recherches en Santé du Québec, and the
Canadian Lung Association. P. Huang is an established investigator of
the American Heart Association and was funded by National Institute of
Neurological Disorders and Stroke Grant NS-33335.
 |
FOOTNOTES |
Address for reprint requests: J. M. Drazen, Pulmonary Division, Brigham
and Women's Hospital, 75 Francis St., Boston, MA 02115.
Received 4 February 1997; accepted in final form 9 July 1997.
 |
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