Prostaglandins E 2 and F 2 Reduce Exhaled Nitric Oxide in Normal and Asthmatic Subjects Irrespective of Airway Caliber Changes

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1 Prostaglandins E 2 and F 2 Reduce Exhaled Nitric Oxide in Normal and Asthmatic Subjects Irrespective of Airway Caliber Changes SERGEI A. KHARITONOV, MARIA A. SAPIENZA, PETER J. BARNES, and K. FAN CHUNG Department of Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London, United Kingdom Cyclooxygenase products modulate the expression of nitric oxide synthase (NOS) in certain cell types. We determined the effect of prostaglandins (PG) E 2 and F 2 on exhaled nitric oxide (NO) concentrations measured by chemiluminescence. Inhaled PGE 2 and PGF 2 significantly reduced exhaled NO. After the highest dose of PGE 2 (100 g), NO concentrations fell from ppb to ppb (p 0.001), and from ppb to ppb (p 0.001), whereas after PGF 2, it fell from ppb to ppb (p 0.001), and from ppb to ppb (p 0.001) in normal (n 7) and asthmatic (n 8) subjects, respectively. Although the prostaglandins did not change FEV 1 in normal subjects, PGE 2 caused an increase in asthmatics (from L to L, p 0.05) and PGF 2 caused a transient reduction in FEV 1 from L to L (p 0.05). To further determine the relationship between bronchoconstriction and exhaled NO levels, we examined the effect of inhaled methacholine which did not change exhaled NO concentrations in normal and asthmatic subjects despite a greater than 20% fall in FEV 1 in asthmatics. Therefore, PGE 2 and PGF 2 reduce exhaled NO, an effect not related to airway caliber changes but which may result from an inhibition of nitric oxide synthase (NOS), particularly inducible NOS (inos). Kharitonov SA, Sapienza MA, Barnes PJ, Chung KF. Prostaglandins E 2 and F 2 reduce exhaled nitric oxide in normal and asthmatic subjects irrespective of airway caliber changes. AM J RESPIR CRIT CARE MED 1998;158: Nitric oxide (NO) is formed from L-arginine by nitric oxide synthase (NOS) in many cells within the respiratory tract and may play an important role in the pathophysiology of airway diseases (1 4). NO can be detected in exhaled air (5) and is increased in patients with asthma (6 9). Concentrations of exhaled NO are reduced by treatment with inhaled steroids (6). The source of the increase in exhaled NO in asthma is not entirely clear but could be from the airway epithelium where there is an increased expression of the inducible nitric oxide synthase (inos) (10, 11). Apart from the presence of inos which can be induced by the action of proinflammatory cytokines, other isoforms of NOS have been detected in the human respiratory tract (12 15) which could also contribute to exhaled NO levels. Thus, constitutive isoforms of NOS producing small amounts of NO also exist in endothelial and epithelial cells (enos) and in neurons (nnos). However, their exact contribution to exhaled NO levels is not known. Nonspecific inhibitors of NOS such as N-monomethyl-L-arginine (L-NMMA) and N-nitro-L-arginine methylester (L-NAME) (Received in original form July 15, 1997 and in revised form September 24, 1997) Supported in part by the British Lung Foundation (UK). Correspondence and requests for reprints should be addressed to Professor K. Fan Chung, Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK. Am J Respir Crit Care Med Vol 158. pp , 1998 Internet address: reduce the levels of exhaled NO in normal and asthmatic subjects, but this observation does not indicate whether the inos isoform is the most predominant form in asthma (16, 17). Products of the cyclooxygenase pathway may play a role in the pathophysiology of asthma. Prostaglandins such as PGF 2 and PGE 2 are known to modulate airway caliber. Thus, PGF 2 induces bronchoconstriction while PGE 2 causes a small degree of bronchodilatation (18, 19). In addition, PGE 2 inhibits exercise-induced bronchoconstriction (20) and allergen-induced early- and late-phase responses (21), and also prevents aspirininduced bronchoconstriction in aspirin-sensitive asthma. PGE 2 has also been shown to prevent the induction of inos in certain cell lines (22). We reasoned that prostaglandins may modulate the level of exhaled NO by inhibiting of inos, particularly in patients with asthma. Because PGE 2 and PGF 2 have divergent effects on airway caliber, we determined the relationship of airway caliber changes to those of exhaled NO. To further determine the relationship of airway caliber to exhaled NO, we examined the effect of methacholine-induced bronchoconstriction. METHODS Patients Eight (1 female) atopic asthmatic patients and seven (all male) normal subjects volunteered for this study (Table 1). None of them were current cigarette smokers and two normal volunteers were ex-smokers of more than 5 yr. Asthmatic subjects had a history of wheezing or

2 Kharitonov, Sapienza, Barnes, et al.: PGE 2 and PGF 2 and Exhaled NO 1375 Subjects TABLE 1 CHARACTERISTICS OF SUBJECTS Sex (F/M) Age (yr) Atopy* FEV 1 (% pred) PC 20 FEV 1 (mg/ml) Control, n 7 0/ none Asthma, n 8 1/ Definition of abbreviation: PC 20 provocative concentration of methacholine producing a 20% fall in FEV. * Positive immediate skin test to one or more allergens. Geometric mean SEM. chest tightness and were not taking corticosteroid therapy. Two of them had stopped using bronchodilators for more than 3 mo; and the other occasionally used inhaled 2 -agonists as required. None of the patients were aspirin-sensitive. All patients had positive cutaneous responses (skin prick test 3 mm wheal) to at least one common aeroallergen (cat dander, house dust mite, grass pollen). All had FEV 1 values 80% of predicted normal and values of provocative concentration of methacholine required to produce a 20% fall in FEV 1 (PC 20 ) of 4 mg/ml. There was no history of upper respiratory tract infection for at least 4 wk prior to the study and none of the subjects had received antibiotics in the previous week. Patients did not consume any caffeine for 2 h or inhaled 2 -agonists for 8 h prior to challenge. All subjects gave their written informed consent to the study which was approved by the ethics committee of the Royal Brompton Hospital. Protocol All subjects attended the laboratory on three different days separated by at least 7 d apart in order to receive inhaled challenges in random order with methacholine, PGE 2, and PGF 2. All challenges were performed between 9:00 and 12:00 h. The subject and the experimenter who measured FEV 1 and NO were not aware of the inhaled substances. Six asthmatic and five normal subjects also attended on an additional day to receive four nebulizations of 0.9% NaCl separated by 15-min intervals in order to mimic the four concentrations of prostaglandins. Methacholine Challenge A standard procedure was employed for bronchial challenge with methacholine. After baseline FEV 1 assessment, subjects inhaled five breaths of 0.9% saline control via a hand-held nebulizer (Dosimeter MB3; MEFAR Electromedical, Bovezzo, Italy) with an output of 10 l per breath. Aerosols were inhaled from end-tidal volume to full inspiratory capacity. Subjects were trained to take 3 s to reach full inspiratory capacity. Methacholine (Sigma Chemical Company, Poole, Dorset, UK) concentrations ranged from to 32 mg/ml and 5 inhalations at each concentration were administered (inhalation time 1 s, breath-holding time 6 s), and FEV 1 was measured 2 min after the last inhalation, until there was a fall in FEV 1 of 20% compared with the control inhalation (0.9% NaCl solution) or until the maximal concentration was inhaled. The PC 20 value was calculated by linear interpolation of the logarithmic dose response curve. Prostaglandin Challenge Subjects were given increasing concentration of PGE 2 (ProstinE 2 ; Upjohn SA, Puurs, Belgium) or PGF 2 (Prostin F 2 ; Upjohn SA) aerosols at 25-min intervals. After a 15-min rest, three baseline measurements of FEV 1 were made at intervals of 1 min followed by inhalation of 0.9% NaCl and three FEV 1 measurements were repeated with 1-min interval in between. Exhaled NO was measured before and after saline inhalations, and the mean of the two measurements was taken as baseline value. Provided FEV 1 had not fallen by 10% of the baseline value, PGE 2 or PGF 2 challenge was performed with doubling concentrations of freshly prepared PGE 2 or PGF 2 at concentrations of 6 g/ml, 12.5 g/ml, 25 g/ml, and 50 g/ml. Prostaglandins (2 ml) were delivered continuously from a nebulizer giving doses of 12 g, 25 g, 50 g, and 100 g (23). FEV 1 was measured every 5 min up to 15 min after each dose and every 5 min for the following 15 min during spontaneous recovery. Measurement of Exhaled NO Exhaled NO was measured by chemiluminescence analyzer (Model LR2000; Logan Research, Rochester, Kent, UK), with sensitivity from 1 part per billion (ppb) to 5,000 ppb of NO, accuracy 0.3 ppb, and response time of 2 s to 90% of full scale. In addition, the analyzer also measured CO 2 (range 0 to 10% CO 2, accuracy 0.1%, response time 200 ms to 90% of full scale), expiration flow and pressure, and exhaled volume in real-time. The analyzer was fitted with a biofeedback display unit to provide a visual guidance for the subject to maintain the pressure and exhalation flow within a given range (3 0.4 mm Hg and 5 to 6 L/min) for end-exhaled NO measurements, hence, improving test repeatability and enhancing patient cooperation. Pressure created in the mouthpiece, and subsequently in the reaction chamber, varied insignificantly and therefore caused negligible change ( 0.1 ppb) in NO readings. The sampling rate through the reaction chamber of the analyzer was 250 ml/min for all measurements. 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% CO 2 (BOC). Ambient air NO level was recorded and the absolute zero was adjusted prior to all measurements. For the end-exhaled NO measurements, subjects exhaled slowly from TLC over 15 to 20 s at an exhalation flow rate 5 to 6 L/min. NO was sampled from a side-arm attached to the mouthpiece. The mean value of the last 100 measurements, acquired at 0.04-s intervals, was taken from the point corresponding to the plateau of end-exhaled CO 2 reading (5 to 6% CO 2 ), and representing the lower respiratory tract sample. Results of the analyses were computed and graphically displayed on a plot of NO and CO 2 concentrations, pressure, and flow against time. Statistics Results were expressed as means SEM, apart from PC 20 results which were expressed as geometric means and geometric SEM. Comparisons between groups were made by repeated measured two-way analysis of variance (ANOVA) with Bonferroni s correction for multiple comparisons. A p value 0.05 was considered significant. RESULTS Effect of Methacholine Challenge in Normal and Asthmatic Subjects There was no change in exhaled NO and FEV 1 during and after methacholine challenge in normal subjects. Despite the reduction in FEV 1 induced by a PC 20 concentration of methacholine, there was no change in exhaled NO in patients with asthma (Figure 1). Effect of PGE 2 and PGF 2 Inhalation in Normal Subjects Inhalation of 0.9% NaCl prior to PGE 2 or PGF 2 inhalation did not change either exhaled NO concentrations or FEV 1 values (Figure 2). In addition, four consecutive nebulizations of 0.9% NaCl separated by 15-min intervals had no effect on exhaled NO. All subjects experienced a short-lasting episode of cough associated with retrosternal soreness after the first inhalation of prostaglandins. Exhaled NO levels were significantly (p 0.001) reduced after PGE 2 inhalation (from ppb to ppb 15 min after 12 g and to ppb after 100 g) and remained low for up to 25 min during the recovery period ( ppb, p 0.001). There was a nonsignificant increase in FEV 1 after PGE 2 inhalation. PGF 2 inhalation also caused a significant reduction in exhaled NO from ppb to ppb at 15 min after 12 g, and to ppb after 100 g. Exhaled NO remained low for up to 30 min during the recovery period (4.3

3 1376 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 1. Concentration response curve of exhaled nitric oxide (NO) (closed circles) and FEV 1 (open circles) to increasing concentrations of inhaled methacholine in normal (A) and asthmatic subjects (B). *p 0.05 and **p compared with baseline values. Figure 2. Concentration response curve of exhaled NO (closed circles) and FEV 1 (open circles) to increasing concentrations of inhaled PGE 2 (A) and PGF 2 (B) in normal subjects. The time-course of the responses is shown for the first 15 min after the highest concentrations of prostaglandins. 0.7 ppb, p 0.001). There was no change in FEV 1 throughout the study. Effect of PGE 2 and PGF 2 Inhalation in Asthmatic Subjects Inhalation of 0.9% NaCl prior to PGE 2 or PGF 2 inhalations did not change either exhaled NO concentrations or FEV 1 (Figure 3). In addition, four consecutive nebulizations of 0.9% saline did not change exhaled NO. Patients with asthma also experienced a short-lasting episode of cough (associated with retrosternal soreness and tightness of the chest after the first inhalation of prostaglandins). NO levels were significantly (p 0.001) reduced after PGE 2 inhalation (from ppb to ppb 15 min after 12 g and to ppb after 100 g), an effect that persisted for up to 30 min during the recovery period ( ppb, p 0.001). There was a small but significant (p 0.05) increase in FEV 1 from L at baseline to L at 15 min after 25 g, with no further increase after the 50 g and 100 g doses of PGE 2. FEV 1 remained significantly elevated for up to 25 min after the last dose of PGE 2. There was also a significant reduction in exhaled NO after PGF 2 inhalation (from ppb to ppb 15 min after 12 g and to ppb after the dose of 100 g). Exhaled NO remained low for up to 30 min after inhalation ( ppb, p 0.001). Although there was a significant reduction in FEV 1 at 5 to 10 min after the initial doses of PGF 2, there was no further change. DISCUSSION We have shown that inhaled prostaglandins PGE 2 and PGF 2 rapidly reduce exhaled NO concentrations in both normal and asthmatic subjects while causing bronchodilatation and bronchoconstriction respectively in patients with asthma. Taken together with the finding that bronchoconstrictor doses of methacholine did not alter exhaled NO levels, we conclude that PGE 2 - and PGF 2 -induced changes of airway caliber are independent of changes in concentrations of exhaled NO. The fall in exhaled NO induced by both prostaglandins occurred in both normal and asthmatic subjects. Although the percentage fall from baseline values of exhaled NO were similar in both groups, there was a greater absolute fall in asthmat-

4 Kharitonov, Sapienza, Barnes, et al.: PGE 2 and PGF 2 and Exhaled NO 1377 Figure 3. Concentration response curve of exhaled NO (closed circles) and FEV 1 (open circles) to increasing concentrations of inhaled PGE 2 (A) and PGF 2 (B) in asthmatic patients. The time-course of the responses is shown for the first 15 min after the highest concentrations of prostaglandins. PGE 2 induced significant bronchodilation, whereas PGF 2 caused significant bronchoconstriction. Both prostaglandins induced a fall in NO. *p 0.05 and **p compared with baseline values. ics because they started from a higher basal value of exhaled NO. The increased concentrations of exhaled NO in asthmatics may result from an increased expression of the inos in airway epithelial cells and possibly alveolar macrophages (10, 13, 24, 25). Analogues of L-arginine such as L-NMMA and L-NAME induce a fall in exhaled NO in both normal and asthmatic subjects (6, 16, 17) but these inhibitors of NOS are not specific for any particular NOS. However, the more specific inhibitor of inos, aminoguanidine, only inhibits the higher levels of exhaled NO in patients with asthma but not in normal subjects (17). Because PGE 2 and PGF 2 inhibited exhaled NO in both normal subjects and asthmatics, it is likely that these prostaglandins were inhibiting both inducible and endogenously expressed NOS. It is unlikely that the changes in exhaled NO we observed are linked to or are the consequence of changes in airway caliber. Bronchoconstriction induced by methacholine in asthmatic patients did not result in significant changes in the levels of exhaled NO. Conversely, bronchodilatation with a -adrenergic agonist also does not affect exhaled NO levels (26). Furthermore, the effects of PGE 2 and of PGF 2 on airway caliber in the asthmatic subjects were divergent, with PGE 2 causing bronchodilatation and PGF 2 bronchoconstriction, whereas both prostaglandins induce a fall in exhaled NO concentrations. In both normal and asthmatic subjects, we observed significant decreases in exhaled NO at the lowest dose of prostaglandins that we used (12 g), indicating that the sensitivity of exhaled NO to the prostaglandins was unlikely to be different in the two groups despite the airway caliber responses in the asthmatics. Conversely, our data also do not link changes in exhaled NO concentrations to airway caliber, which is in agreement with studies in which breathing up to 80 parts per million of exogenous NO did not affect specific airway conductance in normal subjects (27) and had only a very weak bronchodilatory effect in asthmatic subjects (28). Thus, in contrast to the pulmonary vascular smooth muscle, airway smooth muscle is less responsive to inhaled NO. The functional consequences of lowering endogenous NO production, particularly in patients with asthma, are unknown. Although this may not modulate airway caliber directly, other effects may occur such as changes in bronchial mucosal blood flow and reduction of airway microvascular leakage (29). The mechanism by which the prostaglandins E 2 and F 2 inhibit NOS activity, hence leading to a fall in exhaled NO, is not known. In studies of renal mesangial cells, inhibition of endogenous products of the cyclooxygenase pathway by indomethacin led to an increase in NO production induced by interleukin-1 (IL-1 ) stimulation, whereas exogenous PGE 2 reversed this effect (30). These observations indicate that PGE 2 is an inhibitor of inos. The changes in exhaled NO that we observed in vivo were very rapid, occurring within a few minutes of inhalation, indicating that the effects of the prostaglandins are likely to occur through a direct effect on the NO synthases, reflecting an enzyme induction. Our current data indicate that both PGE 2 and PGF 2 had similar effect in reducing the level of exhaled NO, possibly an effect occurring through stimulation of similar prostaglandin receptors. Interactions between NO and prostaglandins have been described in various inflammatory models (31). NO has been shown to be able to directly activate both the constitutive and inducible forms of cyclooxygenase enzyme, thus leading to the overproduction of prostaglandins (32, 33). Conversely, inhibitors of NOS such as L-NMMA and aminoguanidine can attenuate PGE 2 production through inhibition of inducible cyclooxygenase (33, 34). Our data indicate that prostaglandins E 2 and F 2 can in turn inhibit the production of NO. 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