Role of neutral endopeptidase in exercise-induced bronchoconstriction in asthmatic subjects.

The membrane-bound metalloproteinase, neutral endopeptidase (NEP), is a degrading enzyme of both bronchoconstrictor and bronchodilator peptides within the airways. To examine the role of NEP in exercise-induced bronchoconstriction (EIB) in asthmatic subjects, we used inhaled thiorphan, a NEP inhibitor, as pretreatment to a 6-min standardized exercise challenge. Thirteen clinically stable asthmatic subjects participated in this double-blind, placebo-controlled, crossover study that was performed on 2 days separated by 48 h. Thiorphan was administered by two inhalations of 0.5 ml containing 1.25 mg/ml. Subsequently, exercise was performed on a bicycle ergometer at 40-50% of predicted maximal voluntary ventilation while inhaling dry air (20 degrees C, relative humidity 6%). The airway response to exercise was measured by forced expiratory volume in 1 s (FEV1) every 3 min, up to 30 min postexercise challenge, and was expressed both as the maximal percent fall in FEV1 from baseline and as the area under the time-response curve (AUC) (0-30 min). The acute effects of both pretreatments on baseline FEV1 were not different (P > 0.2), neither was there any difference in maximal percent fall in FEV1 between thiorphan and placebo (P > 0.7). However, compared with placebo, thiorphan reduced the AUC by, on average, 26% [AUC (0-30 min, +/-SE): 213.6 +/- 47.7 (thiorphan) and 288.6 +/- 46.0%fall.h (placebo); P = 0.047]. These data indicate that NEP inhibition by thiorphan reduces EIB during the recovery period. This suggests that bronchodilator NEP substrates, such as vasoactive intestinal polypeptide or atrial natriuretic peptide, modulate EIB in patients with asthma.

Role of nitric oxide in the airway response to exercise in healthy and asthmatic subjects.

A role of nitric oxide (NO) has been suggested in the airway response to exercise. However, it is unclear whether NO may act as a protective or a stimulatory factor. Therefore, we examined the role of NO in the airway response to exercise by using N-monomethyl-L-arginine (L-NMMA, an NO synthase inhibitor), L-arginine (the NO synthase substrate), or placebo as pretreatment to exercise challenge in 12 healthy nonsmoking, nonatopic subjects and 12 nonsmoking, atopic asthmatic patients in a double-blind, crossover study. Fifteen minutes after inhalation of L-NMMA (10 mg), L-arginine (375 mg), or placebo, standardized bicycle ergometry was performed for 6 min using dry air, while ventilation was kept constant. The forced expiratory volume in 1-s response was expressed as area under the time-response curve (AUC) over 30 min. In healthy subjects, there was no significant change in AUC between L-NMMA and placebo treatment [28.6 +/- 17.0 and 1.3 +/- 20.4 (SE) for placebo and L-NMMA, respectively, P = 0.2]. In the asthmatic group, L-NMMA and L-arginine induced significant changes in exhaled NO (P < 0.01) but had no significant effect on AUC compared with placebo (geometric mean +/- SE: -204.3 +/- 1.5, -186.9 +/- 1.4, and -318.1 +/- 1.2%. h for placebo, L-NMMA, and L-arginine, respectively, P > 0.2). However, there was a borderline significant difference in AUC between L-NMMA and L-arginine treatment (P = 0.052). We conclude that modulation of NO synthesis has no effect on the airway response to exercise in healthy subjects but that NO synthesis inhibition slightly attenuates exercise-induced bronchoconstriction compared with NO synthase substrate supplementation in asthma. These data suggest that the net effect of endogenous NO is not inhibitory during exercise-induced bronchoconstriction in asthma.

Effect of oral L-arginine on airway hyperresponsiveness to histamine in asthma.

BACKGROUND: Nitric oxide (NO) may exert protective properties within the airways of asthmatic patients. It was postulated that airways obstruction in asthma may be associated with endogenous NO deficiency caused by limited availability of NO synthase substrate. METHODS: In a double blind, crossover study 14 asthmatic patients received pretreatment with oral L-arginine (50 mg/kg body weight) or placebo prior to histamine challenge. Histamine challenge was performed until a 50% fall in forced expiratory volume in one second (FEV(1)) occurred and the response was expressed as the provocative concentration causing a 20% fall in FEV(1) (PC(20)) and as the dose-response slope (maximal % fall in FEV(1)/cumulative dose (micromol)). RESULTS: Pretreatment with L-arginine did not affect PC(20) histamine (mean change in doubling dose 0.18 (95% confidence interval (CI) -0.36 to 0.71), p = 0.5) but the dose-response slope to histamine was slightly reduced (mean change: 0.7 (95% CI 0.6 to 0. 9), p = 0.016). CONCLUSIONS: Oral L-arginine does not influence airway hyperresponsiveness to histamine as reflected by PC(20), although the dose-response slope is slightly reduced in patients with asthma. This indicates only marginal, clinically unimportant limitation of NO synthase substrate in asthma.

Decreased mortality among contemplative monks in The Netherlands.

Although it is generally believed that a simple lifestyle may promote health, a recent study among Trappist and Benedictine monks in the Netherlands reported an increase in general morbidity (Am J Epidemiol 1993;138:569-73). As this increased morbidity might be the consequence of an increased life expectancy, we studied the level of mortality among these contemplative monks. Standardized mortality ratios (SMRs) and death rates were calculated from 1,523 monks whose data were abstracted from the monastery rolls of the period 1900-1994. For this period the SMR was 0.88 (95% confidence interval (CI) 0.81-0.95). A lower SMR was present in almost all of the age categories and in all except one of the monasteries. There were two distinct subperiods. In the pre-World War II era, the SMR was 1.25 (95% CI 1.04-1.49), but it lowered to 0.76 (95% CI 0.69-0.85) after World War II. From 1950 on, mortality in the monasteries remained lower than in the general population, even after correction for the generally higher educational level of the monks. The death rates showed a continuing decline from 1900 to 1950. After 1950, the mortality among monks declined further at a time when the secular trend of decreasing mortality leveled off in the general male population. The higher mortality before World War II was largely unexplained. From the 1950s onward, the mortality among monks was lower, presumably because of the epidemic of lung cancer and cardiovascular disease in the general population. Taken together, the present and earlier data suggest that, among contemplative monks, a simple lifestyle is associated with an extension of life in which they suffer from nonfatal morbidity.

Relationship between exhaled nitric oxide and airway hyperresponsiveness following experimental rhinovirus infection in asthmatic subjects.

Exhaled nitric oxide (NO) is elevated in asthmatics, and varies with disease severity. We postulated that a respiratory virus infection increases exhaled NO levels in asthma, and examined the relationship between the virus-induced changes in exhaled NO and in airway hyperresponsiveness to histamine. In a parallel study, seven patients underwent experimental rhinovirus 16 (RV16) inoculation at days 0 and 1, whilst seven patients received placebo. Exhaled NO was measured at baseline (day 0) and at days 1, 2 and 3 after inoculation. Histamine challenges were performed prior to (day -7) and after inoculation (day 3), and were expressed as provocative concentration causing a 20% fall in forced expiratory volume in one second (FEV1) (PC20). Following RV16 infection there was a significant increase in NO at days 2 and 3 as compared to baseline (median change (range): 4.2 (7.5) parts per billion (ppb), p=0.03, and 3.0 (10.1) ppb, p=0.02, respectively). Furthermore, PC20 decreased significantly following RV16 infection (mean+/-SD change in doubling dose: -0.65+/-0.54, p=0.02), whereas PC20 did not change in the placebo group (p=0.1). There was a significant correlation between the RV16-induced changes in exhaled NO levels at day 2 and the accompanying changes in PC20 at day 3 (rank correlation coefficient (rs): 0.86, p=0.01). Hence, the greater the increase in exhaled NO, the smaller the decrease in PC20. We conclude that rhinovirus infection increases exhaled nitric oxide levels in asthmatics, and that this increase is inversely associated with worsening of airway hyperresponsiveness to histamine. These results suggest that viral induction of nitric oxide synthase within the airways may play a protective role in exacerbations of asthma.

Exhaled nitric oxide (NO) is reduced shortly after bronchoconstriction to direct and indirect stimuli in asthma.

Exhaled NO is increased in patients with asthma and may reflect disease severity. We examined whether the level of exhaled NO is related to the degree of airway obstruction induced by direct and indirect stimuli in asthma. Therefore, we measured exhaled NO levels before and during recovery from histamine and hypertonic saline (HS) challenge (Protocol 1) or histamine, adenosine 5'-monophosphate (AMP), and isotonic saline (IS) challenge (Protocol 2) in 11 and in nine patients with mild to moderate asthma, respectively. The challenges were randomized with a 2-d interval. Exhaled NO and FEV1 were measured before and at 4, 10, 20, and 30 min after each challenge. NO was measured during a slow VC maneuver with a constant expiratory flow of (0.05 x FVC)/s against a resistance of 1 to 2 cm H2O. Baseline exhaled NO levels were not significantly different between study days in Protocol 1 (mean +/- SD: 4.8 +/- 1.8 ppb [histamine] versus 5.4 +/- 2.1 ppb [HS], p = 0.4) or in Protocol 2 (7.9 +/- 4.7 ppb [histamine], 8.3 +/- 5.2 ppb [AMP], and 7.2 +/- 3.7 ppb [IS], p = 0.7). A significant reduction in exhaled NO was observed directly after HS (mean +/- SEM: 39.2 +/- 3.9 %fall) and AMP challenge (32.3 +/- 7.3 %fall) (MANOVA, p < 0.001), respectively, whereas exhaled NO levels tended to decrease after histamine challenge. Isotonic saline challenge did not induce changes in exhaled NO (p = 0.7). There was a positive correlation between %fall in FEV1 and the %fall in exhaled NO after histamine, HS, and AMP challenge as indicated by the mean slope of the within-subject regression lines (p <= 0.04). We conclude that acute bronchoconstriction, as induced by direct and indirect stimuli, is associated with a reduction in exhaled NO levels in asthmatic subjects. This suggests that airway caliber should be taken into account when monitoring exhaled NO in asthma.

Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma.

Sputum induced by inhalation of nebulized hypertonic saline is increasingly used to monitor airways inflammation in asthma. The aim of this study was to assess the repeatability of measuring cellular and soluble markers of inflammation in whole sputum samples as obtained by sputum induction in patients both with mild and moderate-to-severe asthma. Twelve patients with mild, atopic asthma without inhaled steroid treatment and nine patients with moderate-to-severe, atopic asthma treated with inhaled steroids were studied on two separate days at least 2 days apart. Whole sputum samples, induced by inhalation of hypertonic (4.5%) saline, were homogenized, and analysed for differential cell counts and for concentrations of albumin, fibrinogen, interleukin-8 (IL-8), and eosinophil cationic protein (ECP). Repeatability was expressed as intraclass correlation coefficient (Ri), and as coefficient of repeatability (CR) in percentage cells or in doubling concentration. Samples from two patients with mild asthma contained more than 80% squamous cells and were excluded from analysis. The repeatability for cell differential counts in both groups combined was: for neutrophils, Ri = 0.57 and CR = 31.0; for eosinophils, Ri = 0.85 and CR = 12.4; and for lymphocytes, Ri = 0.76 and CR = 6.9. The repeatability of the fluid phase measurements was: for albumin, Ri = 0.71 and CR = 3.2; for fibrinogen, Ri = 0.88 and CR = 2.8; for IL-8, Ri = 0.66 and CR = 2.2; and for ECP, Ri = 0.82 and CR = 1.1. We conclude that the repeatability of cellular and soluble markers of inflammation in induced sputum from patients with mild and moderate-to-severe asthma is satisfactory. Hence, induced sputum, processed by using the whole expectorated sample, seems to be a valuable method to monitor airway inflammation in asthma.

Effects of experimental rhinovirus 16 infection on airway hyperresponsiveness to bradykinin in asthmatic subjects in vivo.

Disturbance of the balance between excitatory and inhibitory activity of the airway sensory nerves has been implicated in asthma pathogenesis, particularly during exacerbations of the disease. The objective of this study was to examine the effect of experimental rhinovirus 16 (RV16) infection on airway responsiveness to bradykinin, a potent sensory nerve stimulus, in asthma. Thirteen atopic, mildly asthmatic subjects participated in a parallel, placebo-controlled study. A total dose of 2.6 to 5.6 x 10(4) TCID50 RV16 (n = 7) or its diluent (n = 6) was inoculated on 2 consecutive days (Days 0 and 1). Histamine and bradykinin challenges were performed before (Days-7 and-6) and after (Days 3 and 4) inoculation. The response was measured by FEV1 and partial flow-volume curves, and it was expressed as PC20FEV1 and PC40V40p, respectively (changes expressed in doubling dose: DD). Before inoculation, PC20FEV1 and PC40V40p to histamine were not significantly different between the groups (p > or = 0.22), whereas PC20FEV1 and PC40V40p to bradykinin tended to be higher in the RV16 group (p = 0.11 and p = 0.06, respectively). PC20FEV1 and PC40V40p to histamine decreased significantly in the RV16 group (mean change +/- SEM: -0.65 +/- 0.20 DD, p = 0.02 and -0.98 +/- 0.28 DD, p = 0.01, respectively), but not in the placebo group (p > or = 0.26). PC40V40p to bradykinin increased significantly in the placebo group (+2.46 +/- 0.92 DD, p = 0.04), with a similar trend for PC20FEV1 (+1.50 +/- 0.62 DD, p = 0.06), whereas there were no significant changes in the RV16 group (p > or = 0.77). These changes in PC40V40p to histamine and bradykinin were significantly different between the groups (p = 0.02). We conclude that repeated bradykinin challenge over a 10-d interval induces tachyphylaxis in asthmatic subjects in vivo and that experimental RV16 infection abolishes such tachyphylaxis to bradykinin while it enhances airway responsiveness to histamine. These results do not favor a predominant role of airway sensory nerves in rhinovirus-induced exacerbations of asthma.

Allergen-induced impairment of bronchoprotective nitric oxide synthesis in asthma.

BACKGROUND: Endogenous nitric oxide protects against airway hyperresponsiveness (AHR) to bradykinin in mild asthma, whereas AHR to bradykinin is enhanced by inhaled allergens. OBJECTIVE: Hypothesizing that allergen exposure impairs bronchoprotective nitric oxide within the airways, we studied the effect of the inhaled nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on AHR to bradykinin before and after allergen challenge in 10 subjects with atopic asthma. METHODS: The study consisted of 3 periods (1 diluent and 2 allergen challenges). AHR to bradykinin (PD(20)BK) was examined before and 48 hours after allergen challenge, both after double-blinded pretreatment with L-NMMA or placebo. The accompanying expression of the various NOS isoforms (ecNOS, nNOS, and iNOS) was examined by means of immunohistochemistry in bronchial biopsies obtained after diluent and allergen challenge. RESULTS: After placebo, AHR to BK worsened after allergen challenge in comparison with before allergen challenge (PD(20)BK, 70.8 nmol [range, 6.3-331] and 257 nmol [35.5-2041], respectively; P =.0004). After L-NMMA, preallergen and postallergen PD(20)BK values (50.1 nmol [1.8-200] vs 52.5 nmol [6.9-204]; P =.88) were similarly reduced (P <.01) and not different from the postplacebo/postallergen value (P >.05). After allergen challenge, the intensity of staining in bronchial epithelium decreased for ecNOS (P =.03) and increased for iNOS (P =.009). These changes in immunostaining were correlated with the accompanying worsening in AHR to BK (R(s) = -0.66 and 0.71; P <.04). CONCLUSIONS: These data indicate that allergen exposure in asthma induces increased airway hyperresponsiveness to bradykinin through impaired release of bronchoprotective nitric oxide associated with downregulation of ecNOS. This suggests that new therapeutic strategies towards restoring the balance among the NOS isoforms during asthma exacerbations are warranted.