INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There is increasing evidence that endogenously produced nitric oxide (NO) may have both beneficial and detrimental effects in asthma (1). These differential properties have been attributed to a dual physiologic and pathologic role of NO, depending on the enzyme responsible for its generation. NO is a highly reactive radical formed from the semi-essential amino acid L-arginine by the action of three isoforms of the enzyme NO synthase (NOS) (2). The constitutive isoforms (cNOS) are expressed in neurones (nNOS, NOS 1) and endothelial cells (eNOS, NOS 3) of the airway (3) and are involved in the physiologic regulation of the airway. The third isoform is induced following exposure to pro-inflammatory cytokines (iNOS, NOS 2) and is expressed in epithelial cells and inflammatory cells of the airway (4), generating 1,000-fold higher amounts of NO than cNOS and may be responsible for the pathologic effects of NO in asthma. In keeping with this, biopsy samples from patients with asthma show increased iNOS expression in epithelial cells (5), and raised levels of NO are found in exhaled air of patients with asthma (6), suggesting inflammation, which is a key feature of asthma, is responsible for the increased expression of this enzyme. Furthermore, exhaled NO levels rise further during asthma exacerbations (7) and are lowered after treatment with corticosteroids (6).

The mechanisms by which NO may be deleterious in asthma are poorly understood, but there is evidence to suggest that excess NO generation may enhance the inflammatory processes underlying asthma (8) as well as producing epithelial cell shedding (13), a characteristic feature of asthma. In contrast, there is also considerable evidence that NO may be bronchoprotective in asthma (1) and have mast cell-stabilizing properties (14, 15). NO generated from nNOS is a neurotransmitter released by inhibitory nonadrenergic, noncholinergic (iNANC) nerves (16) and counteracts cholinergic bronchoconstriction (17). Inhalation of high concentrations of NO has a small bronchodilating effect in patients with asthma (18), and inhibition of its production with NOS inhibitors increases airway responsiveness in experimental animals (19, 20) and patients with asthma (21).

Allergen challenge is a useful clinical model to study the mechanisms underlying asthma, and especially inflammation, since inhaled allergens are likely to be an important mechanism contributing to persistent asthmatic inflammation. The response to inhaled allergen is variable, but in approximately 50% of sensitized adults it is characterized by a bi-phasic response (22); the initial early asthmatic response (EAR), reflecting predominantly airway smooth muscle contraction, is maximal between 10-30 min and resolves within 2 h after allergen inhalation. This is followed by the late asthmatic response (LAR) 3-8 h later, which reflects both airway smooth muscle contraction and inflammation, and may continue for 24 h or longer (23). While development of the EAR is a useful model to study mast cell function, since smooth contraction is produced by the action of spasmogenic mediators released from mast cells, development of the LAR is useful to study inflammatory mechanisms because it is characterized by an influx of inflammatory cells, particularly eosinophils, and is associated with a gradual rise in exhaled NO levels 8-21 h after allergen challenge (24). It is not known whether this increase in NO contributes to the inflammatory process underlying the LAR or whether it is simply a marker of asthmatic inflammation. Furthermore, it is possible that endogenous NO may exert both beneficial and deleterious effects throughout the response to inhaled allergen. First, it may be bronchoprotective during the EAR as a result of mast cell stabilization, in addition to counteracting bronchoconstriction through release from iNANC neurones. Second, it may enhance the inflammatory processes underlying the LAR and exaggerate airway responses during this time.

To investigate this, we examined the effect of nebulized N^G-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, on exhaled NO levels and airway responses to inhaled allergen throughout the EAR and LAR, in a group of patients with mild asthma. We hypothesized that if endogenous NO exerted mast cell-stabilizing and bronchoprotective properties, as well as pro-inflammatory properties when generated in excess, inhibition of its production might increase airway responses during the EAR but decrease airway responses during the LAR.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Twenty-two male patients took part in the study. Female patients were not studied to avoid menstrual cycle effects on exhaled NO levels. All patients were nonsmokers, had mild asthma with an FEV₁ greater than 70% of predicted, and demonstrated a positive skin test of 6 mm or more in response to common airborne allergens (Dermatophagoides pteronyssinus, mixed grass pollen, or cat fur). None had an exacerbation of asthma or a respiratory tract infection in the preceding 6 wk and, apart from house dust mite, there was no current exposure to allergens to which the subjects were sensitized. All patients were steroid-naive and took no regular medications for their asthma, apart from treating occasional symptoms with intermittent, short-acting ₂-agonists. Written informed consent was obtained from each patient and the study was approved by the Ethics Committee of the Royal Brompton Hospital.

Study Protocol

Each patient attended the unit on three occasions. The first visit was for a screening allergen challenge to determine the response to inhaled allergen. Patients were then separated into two groups, depending on whether they demonstrated both an early and late asthmatic response (dual responders) or an isolated early response (single responders). At least 3 but not more than 5 wk later, patients attended the unit in the morning for the first of the two study visits, which were separated by 3-5 wk and conducted in the same manner. After baseline lung function, blood pressure, heart rate, and exhaled NO measurements, patients inhaled nebulized L-NAME (170 mg in 4-ml 0.9% saline) or placebo (4-ml 0.9% saline) in a double-blind, randomized, crossover manner. Twenty minutes later exhaled NO levels, blood pressure, and pulse rate were measured, immediately followed by allergen challenge. Dual responders remained in the unit overnight and, in order to maintain reduced exhaled NO levels throughout the early and late phase response, inhaled two further doses of nebulized L-NAME 170 mg or placebo 3.5 and 7 h after the first dose. In these patients blood pressure, pulse, and exhaled NO levels were measured at 30 min and then at hourly intervals for 10 h after allergen challenge. The following morning a further exhaled NO level measurement was made 21 h postchallenge. Single responders underwent the same experimental protocol but with the last measurement made 3 h after allergen challenge. After the final measurement, all patients received nebulized salbutamol 2.5 mg and budesonide 1 mg prior to discharge.

Intradermal Skin Prick Tests

Stock solutions of allergen extract (10,000 U/ml) for Dermatophagoides pteronyssinus, mixed grass pollen, and cat fur (Soluprick; ALK, Horsholm, Denmark) as well as a positive and negative control were applied intradermally to the volar aspect of the forearm. The wheal and flare response was measured 15 min later and the extract producing the largest wheal was used for allergen challenge.

Allergen Challenge

At the screening visit fresh dilutions of freeze-dried allergen extract (Aquagen SQ; ALK, Horsholm, Denmark) were made up with 0.9% saline from a stock solution of 100,000 U/ml to give final concentrations of 200, 500, 1,000, 2,500, 5,000, 12,500, 25,000, and 50,000 U/ml. Each solution was administered from a hand-held nebulizer attached to a breath-activated dosimeter (Mefar, Brescia, Italy) with a delivery time of 1 s per breath. The nebulizer delivers particles with an aerodynamic mass median diameter of 3.5-4.0 µm at an output of 9 µl per breath. Pulmonary function was assessed by measurement of FEV₁ with a dry wedge spirometer (Vitalograph, Buckingham, UK).

A standard protocol was followed for each allergen challenge. After baseline and postsaline FEV₁ measurements, starting with an allergen concentration of 200 U/ml, serially increasing concentrations of allergen were inhaled. FEV₁ was measured 5 and 10 min after each allergen concentration and the challenge terminated when a greater than 15% fall in FEV₁ from the postsaline value was observed within 10 min of inhalation, representing an adequate early-phase response. After the final concentration of allergen, FEV₁ measurements were taken at 5, 10, 20, 30, 45, and 60 min and thereafter in duplicate at 30-min intervals up to 10 h. A late asthmatic response was defined as a fall in FEV₁ of greater than 15% from the postsaline value on at least three occasions, between 4 and 10 h after allergen exposure. For subsequent allergen challenges during the second two visits, the cumulative dose of allergen producing the initial greater than 15% fall in FEV₁ was administered as a bolus over five breaths after saline control.

All bronchodilators were withheld for at least 8 h before allergen challenge and not permitted until 3 h after challenge for single responders and 10 h for dual responders.

Exhaled Nitric Oxide Measurements

Exhaled NO was measured using a chemiluminescence analyzer (LR 2000; Logan Research, Rochester, UK) sensitive to NO from 1 to 1,000 parts per billion (ppb, by volume). Subjects wore a noseclip and exhaled slowly from total lung capacity over 20-30 s against resistance provided by a mouthpiece and a wide-bore teflon tube connected to the analyzer. NO was sampled continuously at 250 ml/min from a side arm attached to the mouthpiece. A display unit provided visual guidance for the subject to maintain pressure and exhalation flow rate within the range 3 (0.4) mm Hg and 6 (0.09) L/min, respectively. Results of the exhalation analysis were displayed graphically on a plot of NO and CO₂ concentrations, pressure, and flow against time. Three successive recordings of end-exhaled NO levels were made and the mean value used in the analysis. The analyzer was calibrated daily using NO-free certified compressed air to set absolute zero and then a certified concentration of NO in nitrogen of 90 ppb and 500 ppb (BOC Special Gases; Surrey Research Park, Guildford, UK) and certified 5% CO₂ (BOC). Ambient air NO level was recorded and the absolute zero adjusted prior to all measurements. All NO measurements were performed blind by an independent observer who took no further part in the study.

Nebulized L-NAME

Solutions of L-NAME were made up fresh on each study day. L-NAME and placebo were aerosolized by a jet nebulizer (Model CR60; Medic-Aid, Sussex, UK) and inhaled through a mouthpiece with a noseclip worn throughout inhalation. Each solution was inhaled by tidal breathing over 12 min.

Blood Pressure and Heart Rate

Systolic and diastolic blood pressure and heart rate were measured by an Accitorr 1A monitor (Datascope Corp., Montvale, NJ).

Analysis of Data

All results were expressed as means ± standard error of the mean (SEM) unless otherwise stated. Airway responses to inhaled allergen were expressed as percent change from the postsaline value. The effect of saline and L-NAME on airway responses to inhaled allergen were determined by comparing the maximum percent fall in FEV₁ from baseline and the area under the percent fall in FEV₁ time curve (AUC) during the early and late response. AUC was calculated using the trapezoidal method and determined for the early (AUC_{0-2 h}) and late (AUC_{4-10 h}) asthmatic responses. Differences between these variables after each challenge were evaluated by paired t tests. In addition, between-challenge comparisons were made for airway responses at each time point after L-NAME and saline using a paired t test with the Bonferonni correction for multiple comparisons. Exhaled NO levels, pulse, and blood pressure were compared with repeated measures analysis of variance (ANOVA), and each measurement compared with a Bonferonni t test. Statistical significance was taken as p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Of the 22 patients who underwent screening allergen challenge, 10 were dual responders and 12 single responders. Characteristics of these groups are given in Table 1. Inhalation of three sequential doses of nebulized L-NAME was well tolerated, and there were no clinically significant adverse effects or alterations in haematological or biochemical parameters.

Single Responders

Exhaled NO levels. Baseline exhaled NO levels did not significantly differ prior to L-NAME or saline inhalation (Table 2). Nebulized L-NAME reduced exhaled NO levels by 77 ± 5% (28 ± 8 ppb to 7 ± 3 ppb, p < 0.01) 20 min after inhalation. This reduction was maintained throughout the early phase response (Table 2, Figure 1). Saline inhalation did not significantly alter exhaled NO levels prior to allergen challenge (23 ± 6 ppb to 23 ± 7 ppb, NS) and there was no significant change in levels during the 3 h after challenge (Table 2, Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Change in exhaled NO levels before and after allergen challenge following inhalation of L-NAME (closed diamonds) and saline (closed circles) in single responders demonstrating an early-phase response. BL = baseline level; Post = level 20 min post L-NAME/ saline nebulization. Data are mean ± SEM. *p Value < 0.01 versus baseline.

Airway responses to inhaled allergen. There were no significant differences between the airway responses to inhaled allergen after nebulized saline or L-NAME at any of the time points measured (Figure 2). The mean maximum fall in FEV₁ after L-NAME and saline was 21.2 ± 2.9% and 23.8 ± 3.0%, respectively (NS, Table 3). The mean AUC _{0-2 h} after L-NAME and saline was 20.2 ± 3.9 and 24.9 ± 4.4 % FEV₁/h, respectively, and did not significantly differ (Table 3).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Airway responses to allergen challenge following inhalation of nebulized L-NAME (closed diamonds) and saline (closed circles) in single responders demonstrating an early-phase response. FEV1 is expressed as percent change from the post-saline value. Data are mean ± SEM.

Blood pressure and heart rate. No significant alterations in blood pressure or heart rate were observed (Table 4).

Dual Responders

Exhaled NO levels. Baseline exhaled NO levels did not significantly differ prior to L-NAME or saline inhalation (Table 2). Nebulized L-NAME reduced exhaled NO levels by 71 ± 7% (17 ± 3 ppb to 5 ± ppb, p < 0.01) 20 min after inhalation. The two further doses of L-NAME maintained this reduction throughout the early and late phase responses (Table 2, Figure 3). Twenty-one hours after allergen challenge, and approximately 14.5 h after the last dose of L-NAME, exhaled NO levels had risen to 19 ± 4 ppb, which was not significantly different from baseline pre-challenge levels (Table 2, Figure 3). Saline inhalation did not significantly alter exhaled NO levels prior to allergen challenge (17 ± 3 ppb to 16 ± ppb, NS), but there was a gradual increase in levels from 4 h (19 ± 26%). However, this increase only reached significance 21 h after challenge when levels were 88 ± 31% (17 ± 3 ppb to 30 ± 5 ppb, p < 0.05) above baseline pre-challenge levels (Table 2, Figure 3).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Change in exhaled NO levels before and after allergen challenge following inhalation of L-NAME (closed diamonds) and saline (closed circles) in dual responders demonstrating an early- and late-phase response. Vertical arrows represent times at which each dose of L-NAME or saline was administered relative to allergen challenge. BL = baseline level; Post = NO level 20 min after first L-NAME/saline nebulization. Data are mean ± SEM. *p Value < 0.01 versus baseline. **p Value < 0.05 versus baseline.

Airway responses to inhaled allergen. There were no significant differences between the airway responses to inhaled allergen after nebulized saline or L-NAME at any of the time points measured (Figure 4). The mean maximum fall in FEV₁ did not significantly differ after L-NAME and saline during either the early (0-2 h) asthmatic response (31.2 ± 4.5% and 31.8 ± 5.8%, respectively) or late (4-10 h) asthmatic response (27.4 ± 3.9% and 30.6 ± 4.5%, respectively (Table 3). Mean area under the percent change in FEV₁ time curves did not significantly differ: The AUC_{0-2 h} after L-NAME and saline was 37.6 ± 8.4 and 36.7 ± 8.4 % FEV₁/h, respectively (NS, Table 3), and 106.2 ± 18.9 and 117.1 ± 22.4 % FEV₁/h, respectively for the AUC_{4-10 h} (NS, Table 3).

View larger version (13K): [in this window] [in a new window]   Figure 4.   Airway responses to allergen challenge following inhalation of nebulized L-NAME (closed diamonds) and saline (closed circles) in dual responders demonstrating an early- and late-phase response. FEV1 is expressed as percent change from the post-saline value. Data are mean ± SEM.

Blood pressure and heart rate. Diastolic blood pressure increased at a single time point compared with baseline 10 h after allergen challenge and 3.5 h after the last dose of L-NAME (61 ± 2 versus 70 ± 4 mm Hg, p < 0.05). Apart from this, no other significant alterations in blood pressure or heart rate were observed (Table 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This is the first study to examine the role of endogenous NO during allergen-induced bronchoconstriction in patients with asthma. Despite significant reductions in exhaled NO levels prior to challenge and during the early and late asthmatic responses, nebulized L-NAME did not alter allergen-induced bronchoconstriction in either single or dual responders. This suggests that in patients with asthma, NO generated endogenously in the airway does not protect against or contribute to the processes underlying the airway responses to inhaled allergen.

In both groups of patients exposure to inhaled allergen produced significant reductions in FEV₁ consistent with early and late asthmatic responses. In addition, exhaled NO levels increased from baseline during the late asthmatic response, reaching significance 21 h after allergen challenge in those patients demonstrating a dual response during saline treatment, and is likely to reflect the induction of iNOS during this time. This may appear at odds with the findings of our previous study (24), in which we demonstrated a significant elevation of exhaled NO levels 8 h after challenge. In the current study exhaled NO levels increased from baseline by approximately 19% at 4 h to 28% at 10 h postchallenge. While this increase was lower, the rising trend in exhaled NO levels was in keeping with our previous study, and it is likely the smaller number of patients studied accounts for the lack of statistical significance attached to this increase. Nebulized L-NAME reduced exhaled NO levels by approximately 75% in both groups of patients, and additional doses administered at 3.5-h intervals maintained this reduction through to 10 h postchallenge. Twenty-one hours after allergen challenge, however, and at least 14 h after the last dose of L-NAME, exhaled NO levels returned only to baseline, suggesting a residual effect of L-NAME even at 21 h.

We were surprised that L-NAME did not increase bronchoconstriction during the early asthmatic response, given recent evidence that nebulized NOS inhibitors increase airway responsiveness to bradykinin, methacholine, histamine, and adenosine monophosphate (AMP) in patients with asthma (21, 25). The mechanisms by which endogenous NO may be bronchoprotective to these agents is not clearly understood, but it is likely that NO generated from iNANC neurones may be important, since this counteracts cholinergic bronchoconstriction (17). There is no basal release of NO from these neurones, which must be stimulated to exert a bronchoprotective effect (17). It is possible that while bradykinin may release NO from a variety of neural and non-neural cell types directly, AMP and histamine exert additional reflex vagal bronchoconstriction through stimulation of airway sensory nerves (26, 27), which may also activate iNANC pathways, evoking NO release. Inhaled allergen has no immediate effect on airway sensory nerves, and there is no evidence that it induces the release of basely expressed NO in patients with asthma. In addition, recent evidence in experimental animals suggests that iNANC NO-mediated effects are impaired in the presence of airway inflammation, possibly as a result of scavenging of neural NO by superoxide released from inflammatory cells (28). It is therefore possible that following allergen inhalation, the bronchoprotective mechanisms exerted by iNANC neurones are not only impaired because of underlying airway inflammation, but also not initiated, because allergen does not stimulate the release of NO from these neurones either directly or indirectly.

Airway mast cells play a pivotal role in the orchestration of the early and late response to inhaled allergen, and there is considerable evidence that endogenous NO regulates the reactivity of mast cells in experimental animals (14, 29). Immunoglobin E (IgE)-mediated mast cell degranulation results in the release of a wide variety of spasmogenic mediators in addition to a number of pro-inflammatory cytokines (30). Thus, exposure to inhaled allergen has the capacity to evoke all the key features of asthma, including bronchoconstriction and airway inflammation. NO is produced by mast cells constitutively (31). NOS inhibitors have been demonstrated not only to increase histamine release from activated mast cells in vitro (32) but also to produce all the features of mast cell-induced inflammation in vivo (33), suggesting that endogenous NO may protect against the effects of inhaled allergen. We have previously demonstrated that the mast cell-regulating properties of NO may not be functionally important in vivo, since L-NAME did not increase airway responsiveness to another mast cell stimulus, AMP, to any greater extent than histamine in patients with mild asthma (25). The observations of the current study support this, as we would have anticipated a more vigorous bronchoconstrictor response during the early phase after L-NAME if endogenous NO stabilized airway mast cells.

We did not observe any effect of NO inhibitor on the late-phase response to allergen. There is considerable evidence to suggest that NO generated from iNOS is able to enhance the inflammatory processes underlying asthma. NO is chemotactic for eosinophils and in experimental animals chronic treatment with L-NAME inhibits eosinophil migration in vivo and ex vivo (11), and this property appears to represent a direct effect of NO on the eosinophil itself. As well as having local effects such as increasing vascular permeability and edema formation (9), NO also increases the production of prostaglandins through an action on cyclo-oxygenase enzyme (34), which may further contribute to the inflammatory process. In addition, NO suppresses the T helper 1 (Th1) subset of T lymphocytes, which in turn enhances the activity and stimulation of Th2 lymphocytes (10). Th2 lymphocytes are responsible for interleukin 4 (IL-4) and interleukin 5 (IL-5) production, which are pivotal in the orchestration of asthmatic inflammation, including IgE expression and recruitment of eosinophils into the airway. These pro-inflammatory properties of NO lead us to predict that inhibition of endogenous NO might attenuate the late asthmatic response. Increased expression of iNOS is likely to account for the elevated levels of exhaled NO observed during this time and, although the rise was small in our study, it started at a time corresponding to the initial fall in FEV₁ during the late-phase response. Despite significant reductions in exhaled NO during the late phase after L-NAME, bronchoconstriction during this time was not attenuated, suggesting elevated NO and late-phase bronchoconstriction are not causally related. It therefore seems likely that the mechanisms through which iNOS-derived NO enhances airway inflammation do not take immediate effect and are not responsible for the processes initiating the late-phase response. A more probable explanation is that increased iNOS expression and elevated NO levels occur as a consequence of late-phase inflammation and do not contribute to the inflammatory processes between 4 and 10 h postchallenge. Whether very high levels of NO generated 21 h after challenge contribute to airway inflammation at this time remains to be determined. It could be argued that NO derived from nNOS might exert a bronchoprotective effect during the late response and counteract any deleterious effect of NO generated from iNOS. This seems unlikely as, in addition to the impaired iNANC NO-mediated responses in inflamed airways (28), high levels of NO produced by iNOS downregulate cNOS in the airways.

It is difficult to compare the results of this study to animal models of allergen challenge. In experimental animals inhaled allergen challenge produces a rapid increase in exhaled NO associated with acute bronchoconstriction, and this rise returns to baseline within 20 min despite continuing bronchoconstriction (35). This acute increase in NO in experimental animals is also observed after challenge with other spasmogens, such as histamine and leukotriene C₄ (36), a phenomenon that is not observed in humans (37). Furthermore, in rats allergen challenge results in increased expression of iNOS in lung tissue (38), while in the guinea pig model repeated administration of allergen is not associated with a continued and sustained rise in exhaled NO levels or increased expression of iNOS (39). It seems unlikely that in experimental animals rapid increases in local NO production reflect release from basely expressed NOS through a direct action of the spasmogen. This acutely released NO is then able to exert a bronchoprotective effect, possibly through a direct action on smooth muscle. In support of this, NOS inhibitors increase the acute bronchoconstrictor responses to inhaled allergen in ovalbumin-sensitized guinea pigs (36, 39). As we have observed in our study, in patients with asthma the early response to inhaled allergen is not associated with a rise in exhaled NO, suggesting that there is no basal NO release during acute bronchoconstriction. This may further explain who NO inhibition did not increase bronchoconstriction during the early asthmatic response.

We have previously demonstrated that in patients with asthma nebulized L-NAME 170 mg reduced exhaled NO levels to the same extent as L-NAME 54 mg, but only the higher dose increased airway responsiveness to AMP and histamine (25, 40). There are two speculative interpretations of this effect: Either L-NAME is able to exert an additional unknown effect on the mechanisms of airway responsiveness separate from NO inhibition, or the higher dose of L-NAME is able to penetrate deeper into the airway parenchyma and inhibit NO not responsible for that measured in exhaled air. In the current study we have continued to use the higher dose of L-NAME but failed to show any effect on airway responses to inhaled allergen, suggesting L-NAME is not exerting any additional effect in this model. It is conceivable that since inhaled allergen is a very potent mast cell stimulus and induces release of multiple mediators, the degree of NO inhibition was insufficient to attenuate these responses. Thus, it is possible that, in order to observe any effect of L-NAME on allergen challenge responses, even higher doses will need to be administered. In this study three repeated doses of L-NAME produced a consistent reduction in exhaled NO levels, was safe, well tolerated, and without clinically significant alterations in pulse, blood pressure, hematological, or biochemical parameters. It is not known whether higher doses of L-NAME will be as well tolerated, and this will need to be evaluated in future studies. The small but statistically significant rise in diastolic blood pressure after the third dose of nebulized L-NAME may indicate this is the maximum time period over which this drug can be given without producing clinically important increases in blood pressure. In animal studies effects of NO inhibition on inflammatory parameters have been determined with significantly higher doses of NOS inhibitors used in this study, administered over longer periods and in some cases by the intravenous route. There are both ethical and technical issues related to the administration of NOS inhibitors in human volunteers by this route and over prolonged periods.

In conclusion, this study suggests that endogenous NO does not appear to regulate the responses to inhaled allergen in patients with asthma. In the setting of allergen exposure, a major factor contributing to the asthmatic process, this brings into question the importance of NO in terms of its bronchoprotective properties as well as its potential to enhance the inflammatory processes when generated in excess under pro-inflammatory conditions.