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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta -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(beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

                              
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Table 1.   Respiratory physiological parameters in nNOS+/+ and nNOS-/- mice

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).

                              
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Table 2.   Total and iNOS activity in lung and brain

    DISCUSSION
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Introduction
Materials & Methods
Results
Discussion
References

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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Abstract
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Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(4):L883-L888
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