INTRODUCTION

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Bronchial tone is regulated by factors originating from multiple sites: nerves, epithelium, and inflammatory cells. One mediator regulating bronchial tone is nitric oxide (NO) (1). This labile mediator is synthesized from L-arginine by the action of NO synthases of which there are three isoforms: endothelial type (eNOS), neuronal type (nNOS), and an inducible type (iNOS), which is expressed under conditions of inflammation. In the airways, eNOS is present in endothelium of blood vessels and epithelial cells of the lung, and the latter may provide an epithelium-derived relaxant tone (2). nNOS is expressed in nerves and is thought to contribute to nonadrenergic noncholinergic (NANC) neurogenic relaxation. However, nNOS has also been identified in epithelial cells (3), and eNOS may be expressed in certain nerves penetrating blood vessels (4). iNOS is found in infiltrating macrophages, epithelium, and possibly in smooth muscle in the lungs of asthmatic patients, and in multiple cell types in animal models of airway inflammation (for review see Reference 1).

In many studies NO synthase inhibitors have been shown to potentiate cholinergic contractile responses, suggesting that endogenously released NO inhibits neural contraction of airway smooth muscle and contributes to neurogenic dilator tone (5). However, there are other studies which suggest that NO synthase inhibitors do not affect cholinergic responses (9, 10). The reasons for these discrepancies are not clear. A major problem in dissecting out the role of NO in modulating neural control of the airways has been the inability to block each isoform of NOS selectively since the currently available drugs are not sufficiently selective for individual isoforms of NOS (11).

In addition to NO, endogenously released prostaglandins (PGs) regulate airway tone. Prostaglandin E₂ (PGE₂) and PGI₂ are released from airway tissue, and epithelial cells lining the airways seem to be the source of these PGs (12). Bronchial smooth muscle contractility is modulated by exogenous (13, 14) and endogenous PGs in various mammalian species including humans (15). Although interactions between NO and PGs have been described, there is little consensus about the effects of such interactions on airways reactivity. Again the inability to study individual isoforms of NOS may have confounded the interpretation of results.

Our hypothesis was that NO derived from either nNOS or eNOS modulates the reactivity of small airways and that the effects of NO are critically dependent upon local prostanoid synthesis. In this study, we have used eNOS- and nNOS-knockout mice to define the role that NO and the prostaglandins may play in the regulation of airway tone. We have studied small bronchi of the type that are likely to be relevant to the control of airway resistance in disease and that may contribute to air-trapping and segmental lung collapse in asthma.

	METHODS

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Reactivity Studies

Male mice weighing 25 to 30 g were killed by dislocation of the neck. The following genotypes were studied: male wild-type (F₁ between male SV129 and female C57BL/6J) and mutant mice lacking either nNOS or eNOS (both on the background of SV129 and C57BL/6J) (16, 17). The lung was removed and placed in Krebs solution of the following composition (mM): NaCl, 118; KCl, 4.69; CaCl₂, 2.5; KH₂PO₄, 1.18; MgSO₄, 1.18; NaHCO₃, 25; glucose, 11; disodium EDTA, 0.027. Third-order branches of the tracheobronchial tree were carefully dissected. Ring segments (2.5 mm in length) were mounted horizontally between two stainless steel wires (40 µm in diameter) in an automated tension myograph (JP Trading, Aarhus, Denmark). Airways were maintained at 37° C in Krebs solution bubbled with 5% CO₂ and 95% O₂ (pH, 7.4). After an equilibration period of 45 min, airways were stretched to an optimal resting tension of 3 mN (determined by preliminary studies to be the tension at which carbachol [CCh, 10 µM = ECmax] produced a maximal response). Each tissue was considered fully equilibrated when the contractile response to repeat applications of CCh (10 µM) was constant.

Electrical Field Stimulation (EFS)

To investigate neuronally mediated contractile responses, tissues were subjected to transmural nerve stimulation. Using the stimulation parameters of 2 to 64 Hz, 20 V, 0.3 ms for 5-s frequency response curves were constructed in each airway until constant. Frequency response curves were then constructed in the absence or presence of drugs as described below:

To confirm that the responses to EFS were neuronal in origin, some airways were pretreated with the Na⁺ channel blocker tetrodotoxin (1 µM, 15 min). To determine whether the EFS responses were due to activation of cholinergic nerves, tissues were pretreated with atropine (1 µM, 30 min).

To investigate the involvement of endogenously released NO in the responses to EFS, frequency response curves were constructed in the absence or presence of the following enzyme inhibitors in the airways of wild-type, eNOS- and nNOS-knockout mice.

1. Modulation of the contractile response to EFS by NO was investigated by construction of frequency-response curves in the absence or presence of the NO synthase inhibitor, N^G-nitro-L-arginine methylester (L-NAME, 300 µM, 30-min pretreatment).

2. To determine whether cyclooxygenase products were involved in modulation of the contractile response to EFS, frequency-response curves were constructed in the absence or presence of the cyclooxygenase inhibitor indomethacin (5 µM, 30-min pretreatment).

3. The possibility that NO and cyclooxygenase products act synergistically, to modulate contractile response to EFS, was investigated by construction of frequency-response curves in airways pretreated with both L-NAME (300 µM) and indomethacin (5 µM).

Exogenous Application of CCh

To determine whether NO and/or cyclooxygenase products modulate excitatory neurogenic responses postjunctionally, concentration-response curves to CCh (10 nM to 100 µM) were constructed in the airways of wild-type mice in the absence (time control) or presence of L-NAME (300 µM), indomethacin (5 µM), or a combination of both inhibitors.

Immunohistochemistry

Lungs were collected and small airways were dissected and removed from four knockout mice, and two wild-type mice. Mice were anaesthetized and ventilated through a tracheal cannula. The left ventricle was cannulated and via this route the animal was perfused with saline (50 ml) followed by 150 ml of a fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The lungs were cut into small blocks and postfixed for a further 4 h in 4% buffered paraformaldehyde at room temperature. The blocks were then rinsed and cryoprotected by immersion overnight at 4° C in 0.1 M PB containing 30% sucrose with continuous stirring. After this time the blocks were frozen and 40-µm-thick sections were cut with a sledge microtome (Leitz). Free-floating sections were processed by the avidin-biotin peroxidase complex (ABC) procedure to visualize immunoreactive sites. All the free-floating sections were incubated for 30 min in PB containing 3% normal goat serum and 0.2% Triton X-100 and then incubated with the primary antibody (antisera to nNOS) 1:2,500 dilution in PB/Triton X-100 overnight at 4° C (18, 19). After several washes in PB, sections were incubated with biotinylated goat antirabbit immunoglobulin for 1 h. To visualize the immunoreactivity, sections were then incubated for 90 min with peroxidase-linked ABC and the activity demonstrated using the nickel-enhancement diaminobenzidine procedure (20).

Control procedures were carried out on sections taken from the airways of two wild-type animals. No immunolabeling was observed when the primary antibody was omitted or replaced with an equivalent concentration of preimmune and normal rabbit serum. The specificity of the nNOS antiserum was demonstrated by incubating the tissue sections with primary antiserum that had been incubated overnight at 4° C with recombinant nNOS (2 µg/ml). This procedure abolished immunostaining in all lung tissue sections.

Drugs

All drugs used were purchased from Sigma (Poole, UK). The nNOS antibody was a gift from Dr. V. Riveros-Moreno of Wellcome Research Laboratories (Beckenham, UK). The normal goat serum was purchased from ICN Biochemicals (Barcelona, Spain) and peroxidase-linked ABC from Vector Laboratories (Burlingame, CA).

Analysis of Data

In all experiments EFS-induced contractile responses are expressed as a percentage of CCh (10 µM)-induced contraction. Data are expressed as mean ± SEM. Data were compared by paired t test and factorial or repeated-measure two-way analysis of variance followed by Fisher's LSD. p < 0.05 was considered significant.

	RESULTS

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The mean diameters of wild-type and eNOS- and nNOS-knockout mice airway used in this study were 403.8 ± 25.5 µm (n = 24), 425.3 ± 20.7 µm (n = 24), and 400.8 ± 23.2 µm (n = 24), respectively, and did not differ significantly from each other. The mean CCh (10 µM)-induced contractions of wild-type and eNOS- and nNOS knockout mice airways were 4.9 ± 0.4 mN (n = 24), 5.6 ± 0.4 mN (n = 24), and 4.7 ± 0.3 mN (n = 24), respectively, and were not significantly different from each other.

Wild-type Controls

EFS (2 to 64 Hz) produced frequency-dependent contraction, as shown in Figure 1. The absolute mean responses to EFS were 0.09 ± 0.2 mN, 0.1 ± 0.02 mN, 0.19 ± 0.03 mN, 0.32 ± 0.05 mN, 0.55 ± 0.08 mN, and 1.02 ± 0.1 for 2, 4, 8, 16, 32, and 64-Hz, respectively, n = 24 in each case. This contraction was abolished by pretreatment with tetrodotoxin (1 µM) or atropine (1 µM) (n = 3 in each case; see Figure 1 for typical responses).

View larger version (7K): [in this window] [in a new window]   Figure 1.   Typical contractile responses to electrical field stimulation of isolated small murine airways (2, 4, 8, 16, 32, 64 Hz) before (Control) and after pretreatment with either tetrodotoxin (A) or atropine (B).

Pretreatment with L-NAME (300 µM, n = 8) had no significant effect on EFS-induced contraction (Figures 2A and 3A), whilst indomethacin (5 µM, n = 8) significantly enhanced the responses by approximately 60 to 170% at each frequency (p < 0.05) (Figures 2B and 4A). Combined pretreatment with L-NAME and indomethacin (n = 8) produced a greater enhancement (approximately 140 to 290%) of the responses to EFS compared with the effect of indomethacin alone (n = 8, p < 0.01) (Figures 2C and 4A). Neither L-NAME nor indomethacin had any effects per se on basal tension of these airways or airways taken from eNOS or nNOS knockouts. Application of the stable prostacyclin analogue iloprost (1 µM, n = 2) had no effect on the basal tension of airways, indicating the absence of elevated tone caused by endogenous mediator release. Basal tension was 2.8 (2.0 to 3.6) and 2.8 (2.1 to 3.5) before and after iloprost.

View larger version (9K): [in this window] [in a new window]   Figure 2.   Typical contractile responses to electrical field stimulation of isolated small murine airways (2, 4, 8, 16, 32, 64 Hz) before (Control) and after pretreatment with (A) L-NAME (300 µM); (B) indomethacin (5 µM); or combined pretreatment with (C ) L-NAME (300 µM) and indomethacin (5 µM).

eNOS Knockouts

Indomethacin significantly enhanced EFS-induced contraction (n = 8, p < 0.01) to a similar degree to that seen with the airways of wild-type controls (Figure 4B), whereas L-NAME alone had no overall effect, although it might have marginally enhanced responses at some frequencies (n = 8) (Figure 3B). As with the wild-type combined pretreatment with indomethacin, L-NAME produced a significantly greater effect than did indomethacin alone (n = 8) (p < 0.01) (Figure 3B).

View larger version (19K): [in this window] [in a new window]   Figure 4.   Contractile responses to EFS (2-64 Hz) of small airways of wild-type mice (A), eNOS-knockout mice (B), and nNOS-knockout mice (C ) before (control; open column) and after (shaded column) pretreatment with indomethacin (5 µM) and combined (hatched column) pretreatment with indomethacin (5 µM) and L-NAME (300 µM). Data are mean ± SEM for n = 8. *p < 0.05 and **p < 0.01 versus the control; # p < 0.05 and ## p < 0.01 compared with the response after pretreatment with indomethacin (5 µM).

View larger version (13K): [in this window] [in a new window]   Figure 3.   Contractile responses to EFS (2-64 Hz) of small airways of wild-type mice (A), eNOS-knockout mice (B), and nNOS-knockout mice (C ) before (control; open column) and after (hatched column) pretreatment with L-NAME (300 µM). Data are shown as mean ± SEM for n = 8. Statistical significance is shown by *p < 0.05 and **p < 0.01 versus the control.

nNOS Knockouts

Pretreatment of small airways of nNOS-knockout mice, with indomethacin, significantly enhanced EFS-induced contraction (n = 8) (p < 0.01) to a similar degree to that achieved in wild type controls (Figure 3C), again L-NAME alone had no effect (Figure 4C). Combined pretreatment with indomethacin and L-NAME (300 µM) did not enhance EFS-induced contraction (n = 8) (p < 0.01) above that achieved with indomethacin alone (Figure 3C).

Postsynaptic Modulation of Cholinergic Neurotransmission

CCh (10 nM to 100 µM) caused concentration-dependent contraction of small airways of wild-type mice. The contractile response to CCh was biphasic. On addition of CCh a rapidly developing contraction was followed by a stable sustained phase. Although the concentration response curve to CCh was unaffected by either indomethacin or L-NAME pretreatment, i.e., no shift of EC₅₀ and no alteration of the maximal response (n = 6 in each case, p = 0.9909 and p = 0.3855, respectively), the contraction became monophasic and stable in the presence of indomethacin. Combined pretreatment with L-NAME (300 µM) and indomethacin (5 µM) did not alter the potency of CCh, although the maximum contraction was increased by approximately 15% (n = 6, p < 0.01) (Figure 5).

View larger version (16K): [in this window] [in a new window]   Figure 5.   Contractile concentration-response curves to carbachol (CCh, 0.01 to 100 µM) of small airways of wild-type mice in the absence (time control) and presence of L-NAME (300 µM), indomethacin (5 µM), or both L-NAME (300 µM) and indomethacin (5 µM). Data are mean ± SEM for n = 6. **p < 0.01 versus the time control.

Immunohistochemistry

Staining of dissected airways from wild-type mice showed an absence of cartilage, confirming the bronchiolar nature of the airways studied. Using a specific polyclonal antibody against nNOS, immunoreactive nerve fibers were found pervading the lung parenchyma of the small airways of wild-type mice. These immunoreactive nerve fibers formed small plexuses that contained few and small vesicles and isolated nerve fibers that exhibited varicose and undulate morphology. Some immunoreactive nerve fibers were found lying parallel to the basal membrane of the bronchiolar epithelium and in some instances penetrating the epithelium and running between the epithelial cells ending close to the lumen of the airway (Figure 6). There was no staining for nNOS in tissues collected from nNOS knockout animals.

View larger version (47K): [in this window] [in a new window]   Figure 6.   Immunostaining of nNOS in small bronchioles of (A) wild-type and (B) nNOS knockout mice. In the airways of wild-type mice nNOS positive staining was found associated with nerve fibres in close proximity to airway smooth muscle and the epithelium. No nNOS staining was found in the airways of nNOS knockout mice.

	DISCUSSION

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The results of this study demonstrate that both NO and PGs modulate neural cholinergic constriction of murine bronchioles. The isoform of NOS responsible for this production of NO in murine small airways is nNOS and its effects are only apparent when prostanoid synthesis is blocked. These results define the enzymatic source of NO involved in the regulation of airway tone in the mouse and indicate that previous contradictory reports may be accounted for by the modulatory effects of prostanoids on endogenous NO.

The segments of airway used in this study were taken from third-order branches of the tracheobronchial tree and had an internal diameter of approximately 400 µm. Histologic examination showed that they could be classified as bronchiolar since they had no cartilage support (21). EFS of the small airways caused reproducible frequency-dependent contraction that was completely blocked by the Na⁺ channel blocker tetrodotoxin and the muscarinic receptor antagonist atropine. Thus, the contractions seen were due to activation of cholinergic nerves.

The contractile response to EFS in the airways of wild-type control mice was enhanced by pretreatment with the cyclooxygenase inhibitor indomethacin, but not by pretreatment with the NOS inhibitor L-NAME. This is similar to some previous reports (9, 10) but it is in marked contrast to several other studies of isolated airways where NOS inhibitors potentiated neural cholinergic contractile responses (5). Search of the literature reveals that the apparent discrepant results with NOS inhibitors, i.e., enhanced cholinergic neurogenic contraction in some studies or no effect in others, may be easily explained. In the "positive" studies indomethacin was present in the organ baths throughout the experiment, and in the "negative studies" it was not (9, 10). Thus, it seems that NOS inhibitors might produce significant contractions when PG synthesis is inhibited, but not when the cyclooxygenase pathway is intact. In the present study we tested this hypothesis directly; combined pretreatment with L-NAME and indomethacin enhanced cholinergic contraction to a significantly greater extent than did indomethacin alone. The simplest explanation of this result is that cyclooxygenase products suppress the synthesis or the effect of NO.

Interaction between the COX and NOS pathways has been described in other systems. In certain inflammatory situations NO has been reported to enhance PG production (22), whereas in other studies PGs inhibited NO production in both healthy and inflammatory states (25, 26). Our studies would be consistent with inhibition of NO production by PGs. The mechanism of this effect is not clear, but Marotta and colleagues (25) found that whereas PGE₂ or PGI₂ inhibited NO production in J774 murine macrophage cells, TXA₂-mimetics or PGF₂ did not. However, in these studies it was suggested that these prostanoid effects were due to inhibition of iNOS expression rather than to inhibition of activity. PGE₂ and PGI₂ are both synthesized in airway epithelium (27), but whether these prostanoids contributed to the effects seen in the present study requires further investigation.

A major problem in dissecting out the role of NO in airways has been the inability to block each isoform of NOS selectively since the currently available drugs block all three isoforms (11), or have uncertain mechanisms of action (30). Often, support for the role of NO in vitro systems is provided by the measurement of NO. Unfortunately the quantities of NO produced by the tissues under investigation are too low to be detected on the currently available assay systems. However, gene knockout mice provide an opportunity to identify biologic roles for individual isoforms. Using the knockouts we identified nNOS as the isoform responsible for the endogenous NO activity in the murine airways. Combined pretreatment with L-NAME and indomethacin significantly enhanced EFS-induced contraction of airways of both wild-type and eNOS-knockout mice above that achieved by indomethacin alone. In contrast in the nNOS knockouts, combined treatment with L-NAME and indomethacin was no more effective than indomethacin alone. These studies clearly identify the nNOS isoform as playing an important role in NO-mediated modulation of neural cholinergic contraction of murine small airways. Indeed support for such a hypothesis is provided by the fact that the immunohistochemical studies clearly demonstrate the presence of nNOS containing nerve fibers in the small airways of wild-type but not nNOS knockout mice. However, the overall magnitude of the effect of indomethacin in the airways of nNOS knockouts was no greater than in those of the wild type. These results may suggest the induction of some other compensatory mechanism in the absence of nNOS during development, but this requires further investigation. The airways of eNOS knockouts responded to EFS no differently from small airways of wild-type mice, indicating that eNOS is not involved in modulating airway tone in mice. Immunostaining for eNOS in the lungs of wild-type mice showed expression of this isoform only in the endothelium lining of blood vessels and not in the airways themselves. This is similar to the situation in humans where constitutive NOS expression has been identified only in endothelial and nerve cell types (31, 32).

The inhibitory effect of NO was not due to an action of NO released after binding of cholinergic neurotransmitter at postjunctional muscarinic receptors since combined pretreatment with L-NAME or indomethacin had no effect on the potency of exogenously applied CCh, although it did cause a small enhancement of the maximum response to CCh. It is possible that the effect of NO is due to a prejunctional action to decrease the release of cholinergic neurotransmitter. However, a previous study has shown that the NOS inhibitor, L-N^G-nitro-arginine, does not change EFS-induced overflow of [³H]acetylcholine from guinea-pig tracheal tissues in the presence of indomethacin (6). An alternative explanation is that NO is released from nerves in small airways and acts directly on the smooth muscle as a functional antagonist to the cholinergic contraction. Studies in which only the luminal side of the airway is bathed may help to address the question of what cell type is important for the effects seen in these studies. Additionally studies investigating the effects of EFS in precontracted airways should yield information regarding the possibility that the modulatory action of NO in this system may be due to functional antagonism.

The PG effect might also be a prejunctional or postjunctional effect. In the absence of indomethacin, CCh induced biphasic contraction, which had an initial transient phase followed by an unstable sustained phase. However, in the presence of indomethacin, CCh-induced contraction was monophasic and stable, indicating that PGs have the capacity to exert a postjunctional effect. The other possibility is that the PGs act at a prejunctional level to inhibit cholinergic neurotransmitter release and, indeed, studies in large canine (33) and human airways (34) support such an hypothesis. Further studies will be required to investigate how these substances regulate neural responses and affect NO responses in the murine airways.

We have studied murine airways to take advantage of genetically modified animals. The application of myography methodology to study murine airways, as described here, should prove useful to take full advantage of knockout technology in understanding airway physiology and pathophysiology. If human small airways behave similarly, it seems that whereas NOS inhibition alone would not cause significant bronchiolar constriction, a combination of NOS inhibition and COX inhibition might increase airway resistance. This could lead to air-trapping and segmental collapse. Our results predict that inhibitors of nNOS are likely to have this effect, whereas selective inhibitors of eNOS and iNOS would not. A further implication of our study is that if functional defects of nNOS are detected in human diseases or in subjects with nNOS polymorphisms, such subjects might show an unduly large bronchoconstrictor response to COX inhibition. This hypothesis might be worth testing in relation to the enigma of aspirin-sensitive asthma.

In conclusion, this study has shown that endogenously produced PGs and NO modulate tone in small airways. The relationship between the inhibitory PGs and NO is cooperative, such that the bioactivity of nNOS located in nerves is inhibited by COX products and only becomes functionally "visible" in the absence of COX activity. In addition, eNOS activity does not appear to affect bronchiolar tone.