Jacek Rysz

Robert A Stolarek

Rafal Luczynski

Agata Sarniak

Anna Wlodarczyk

Marek Kasielski

Dariusz Nowak
email: dnowak@zdn.am.lodz.pl

Prof Dariusz Nowak

Increased hydrogen peroxide concentration in the exhaled breath condensate of stable COPD patients after nebulized N-acetylcysteine

Keywords: N-acetylcysteine, hydrogen peroxide, nitrites, nitrates, chronic obstructive pulmonary disease, exhaled breath condensate COPD chronic obstructive pulmonary disease, EBC exhaled breath condensate, GSNO

Background: The oxidative burden in the airways is a hallmark of chronic obstructive pulmonary disease (COPD). Aims: This prospective, cross-over, placebo (PL) controlled study was designed to investigate the effect of N-acetyl-L-cysteine (NAC) on hydrogen peroxide (H 2 O 2 ), nitrites/nitrates (NO 2 -+NO 3 -) and thiol (RSH) concentrations in exhaled breath condensate (EBC) in stable COPD patients (n=19, aged 52.6±15.6 years, 10 females, mean FEV 1 95.2±23.8%, FEV 1 /FVC 69.1±11.4%). Methods: H 2 O 2 , NO 2 -+NO 3 -and RSH concentrations in EBC were determined with homovanillic acid, NADPH-nitrite reductase assays and Ellman's reaction, respectively. Results: Thirty minutes after nebulization, H 2 O 2 concentration increased if levels after NAC (0.45±0.25 µM) and PL (0.17±0.17 µM) were compared in COPD patients (p=0.002). This increased H 2 O 2 level in EBC was no longer observed either after ninety minutes: 0.16±0.09 µM (PL 0.17±0.15 µM) or three hours: 0.12±0.07 µM (PL 0.21±0.23 µM) (p=0.5 and p=0.2, respectively). The levels of NO 2 -and NO 3 -did not differ between NAC and PL. There was no significant difference in RSH levels between nebulized NAC and PL. After nebulized NAC, however, exhaled RSH increased from 1.42±1.69 µM (0 min) to 2.49±2.00 µM (30 min,) and 1.71±1.83 µM (180 min) (p=0.009 and p=0.03, respectively, compared with 0 min). Conclusions: These data demonstrate that nebulized NAC transiently increases exhaled H 2 O 2 level, whereas it has no effect on other oxidative parameters.

Introduction

The oxidative burden in the airways is a hallmark of chronic obstructive pulmonary disease (COPD). Generation of reactive oxygen (ROS) and nitrogen species (RNS) coincides with increased lipid peroxidation, alveolar injury, increased endothelial permeability and extracellular matrix turnover, extensive mucus secretion and accumulation of activated macrophages and polymorphonuclear leukocytes [START_REF] Rahman | Oxidative stress in pathogenesis of chronic obstructive pulmonary disease: cellular and molecular mechanisms[END_REF]. Redox sensitive inflammatory transcription factors upregulated in the oxidative milieu may alter chromatin structure accounting for attenuation of inhaled steroid efficiency in COPD patients [START_REF] Adcock | Redox regulation of histone deacetylases and glucocorticoid-mediated inhibition of the inflammatory response[END_REF]. The increased nitrotyrosine and heme oxygenase and myeloperoxidase activities are specifically associated with COPD exacerbation [START_REF] Tsoumakidou | Nitrosative stress, heme oxygenase-1 expression and airway inflammation during severe exacerbation of COPD[END_REF]. The progressive deterioration of lung function and oxidative damage of lung parenchyma has an insidious time pattern and antioxidant approaches to COPD therapy have long now been considered as a likely beneficial option [START_REF] Rahman | Oxidative stress in pathogenesis of chronic obstructive pulmonary disease: cellular and molecular mechanisms[END_REF].

Nitric oxide (NO) is generated by sequestrated polymorphonuclear leukocytes and activated macrophages in the course of chronic inflammatory reaction in the lungs of COPD patients [START_REF] Ichinose | Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways[END_REF].

Its oxidation is terminated with nitrites and nitrates as stable end products. NO readily reacts with superoxide to from peroxynitrite (ONOO -) anion. It may initiate a set of reactions leading to extensive oxidative damage of histone deacetylase, antiproteases, surfactant, membrane lipids and the injury of alveolar epithelium [START_REF] Barnes | Corticosteroid resistance in chronic obstructive pulmonary disease: inactivation of histone deacetylase[END_REF]. Also, ONOO -formation leads to nitration and oxidation of protein sulfhydryl groups of the reduced cysteine and intracellular glutathione [START_REF] Forman | Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers[END_REF] along with

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the inhibition of antioxidant enzymes in a dose-dependent manner [START_REF] Lawler | Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors[END_REF]. The levels of NO in exhaled air were demonstrated to correlate with accumulation of inflammatory cells and the functional indices of COPD exacerbation [START_REF] Rutgers | Markers of nitric oxide metabolism in sputum and exhaled air are not increased in chronic obstructive pulmonary disease[END_REF]. Exhaled breath condensate (EBC) is considered suitable for the assessment of airway inflammatory reactions [START_REF] Mutlu | Collection and analysis of exhaled breath condensate in humans[END_REF] and nitrates levels in EBC were demonstrated to differ specifically in various diseases including asthma, COPD or community acquired pneumonia [10].

Several lines of evidence demonstrate the increased levels of H 2 O 2 in the airways of COPD patients [START_REF] Rahman | Oxidative stress in pathogenesis of chronic obstructive pulmonary disease: cellular and molecular mechanisms[END_REF]. The increased H 2 O 2 concentration in EBC was found in stable COPD patients [START_REF] Benedetto | Long-term oral N-acetylcysteine reduces exhaled hydrogen peroxide in stable COPD[END_REF][START_REF] Nowak | Increased content of thiobarbituric acid-reactive substances and hydrogen peroxide in the expired breath condensate of patients with stable chronic obstructive pulmonary disease: no significant effect of cigarette smoking[END_REF] and in the acute exacerbation [START_REF] Dekhuijzen | Bast A Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease[END_REF]. The role of H 2 O 2 may likely extend far beyond relatively stable ROS end product and oxidative injury parameter. H 2 O 2 may act as a second messenger, sometimes in concert with NO, and may regulate redox state of cysteine residues of signaling proteins including tyrosine kinase, PTEN and Ras [START_REF] Forman | Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers[END_REF]. The mean EBC H 2 O 2 level in COPD patients were 10 times higher than in healthy subjects, although it varied in a wide range [START_REF] Nowak | Increased content of thiobarbituric acid-reactive substances and hydrogen peroxide in the expired breath condensate of patients with stable chronic obstructive pulmonary disease: no significant effect of cigarette smoking[END_REF][START_REF] Dekhuijzen | Bast A Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease[END_REF]. The oral administration of NAC was demonstrated to decrease exhaled H 2 O 2 in COPD patients [START_REF] Benedetto | Long-term oral N-acetylcysteine reduces exhaled hydrogen peroxide in stable COPD[END_REF][START_REF] Kasielski | Long-term administration of N-acetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease[END_REF]. Albeit inhaled NAC was shown to reduce even baseline levels of exhaled H 2 O 2 in healthy subjects thirty minutes after its administration, the apparent increase of expired H 2 O 2 was noted three hours later indicating either pro-oxidant effects or a rebound, oxidative response to antioxidant deposition in the airways [START_REF] Szkudlarek | Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects[END_REF].

NAC is a readily diffusible reductant aminothiol and a precursor of glutathione (GSH). The milimolar concentrations of intracellular GSH provide efficient scavenging of ROS and RNS.

NAC scavenges hydroxyl radical and rapidly reacts with hypochlorous acid, inhibits oxidation of myeloperoxidase targets [START_REF] Van Antwerpen | Thiol-containing molecules interact with the myeloperoxidase/H2O2/chloride system to inhibit LDL oxidation[END_REF], exhibiting only a trace of affinity to H 2 O 2 and none to superoxide anion [START_REF] Aruoma | The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid[END_REF]. NAC is able to upregulate gene expression at a transcriptional level facilitating cytoskeleton rebuilding and cell cycle arrest [START_REF] Edlundh-Rose | Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation[END_REF]. In the animal model, NAC appeared to hamper

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the progressive destruction of lung parenchyma and emphysema elicited by the specific inhibition of tyrosine kinase activity of VEGF receptors and attenuated the oxidative stress [START_REF] Voelkel | Emphysema: an autoimmune vascular disease?[END_REF].

Albeit NAC in various route of administration indeed demonstrated appreciable benefits in COPD patients [START_REF] Kinnula | Focus on antioxidant enzymes and antioxidant strategies in smoking related airway diseases[END_REF], the antioxidant effects of nebulized NAC have not been investigated so far in the setting of stable, mild COPD. Since intravenous GSH in healthy volunteers increased the level of cysteine in the expired air [START_REF] Fukagawa | Plasma methionine and cysteine kinetics in response to an intravenous glutathione infusion in adult humans[END_REF], it seems likely that inhaled NAC could increase airway GSH levels and improve antioxidant defense.

Materials and Methods

Reagents and medications

Homovanillic acid, NADPH-nitrate reductase (EC 1.6.6.

Patients and study protocol

The study included 19 patients with COPD (10 women, aged 65±13; 9 men, aged 67±12) from municipal hospital COPD patient registry (Table 1

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The study subjects were asked to attend the lab twice and were randomly chosen in double blind manner to receive assigned study medication package containing either nebulized Fluimucil (300 mg in 3ml) or PL (3 ml) within two weeks' time. The dosage and dilution of nebulized Fluimucil preparation was according to common clinical practice and manufacturer's recommendations (Zambon Group, Italy) . The study protocol required lab staff to prepare solution for nebulization in a separate room and the delivery of the drug itself was conducted with the patient wearing a nose clip. EBC was collected before, 30, 90 and 180 min after either Fluimucil or PL inhalation.

Lung function tests were performed according to the American Thoracic Society standards [START_REF] Thoracic | Standardization of spirometry: 1994 update[END_REF],

initially during preselection phase, immediately before the first and just after the last EBC collection. The experiments started always between 8 30 and 9 00 and finished 13 20 -13 50 . Each patient involved in the study gave informed consent and the study protocol was thoroughly reviewed and approved by the Institutional Review Board at Medical University of Lodz.

Functional lung tests

Master-Laboratory Screen (Jaeger Toennies, Wuerzburg, Germany) was used for lung functional tests including FVC (forced vital capacity), FEV 1 (forced expiratory volume in the first second), DLCO c (single breath carbon monoxide diffusing capacity corrected for hemoglobin and alveolar volume), RV He (residual volume, helium dilution technique) according to the standard procedure [START_REF] Thoracic | Standardization of spirometry: 1994 update[END_REF][START_REF]Single-breath carbon monoxide diffusing capacity (transfer factor). Recommendations for a standard technique -1995 Update[END_REF] and the results were expressed as percentage of predicted values [START_REF] Thoracic | Standardization of spirometry: 1994 update[END_REF][START_REF] Carpo | Standarized single breath normal values for carbon monoxide diffusing capacity[END_REF].

Collection of exhaled breath condensate

The EBC specimens for H 2 O 2 assay were collected as previously described [START_REF] Szkudlarek | Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects[END_REF]. The amylase levels in EBC specimens were assayed as a control of salivary contamination with Vitros 250 assay (enzymatic test, lower detection limit 2 U/l, Johnson&Johnson, Ortho Clinical Diagnostics,

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USA). The patient respiratory rate during EBC collection ranged from 14 to 19 breaths per minute. The procedure of EBC collection was 20 minutes and the mean volume of EBC was 5 ml. The EBC samples were processed in the assays for H 2 O 2 and thiol content or were stored at -80ºC prior to nitrites/nitrates assays.

Measurement of H 2 O 2 in exhaled breath condensate

The concentration of H the study subjects, only four results were below the method sensitivity and they were assumed a half of detection limit for the purposes of data analysis.

Nitrites and nitrates assay in exhaled breath condensate

The modified method was used for the nitrites (NO 2 - ) and nitrates (NO 3

-

) assay with NADPHnitrite reductase [START_REF] Dziedzic | Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3-in breath condensate[END_REF]. EBC (60 µl) was added in duplicate into 96 well plates with control wells Total NO 2 - was calculated with the values of absorbance directly from NO 2 - standard curve for the concentrations from 0.5 to 12.5 µM. The results were expressed as the sum of NO 2 - converted from NO 3 - plus NO 2 - originally detected. Concentration of NO 3 - was calculated by subtracting NO 2 - concentration before reduction from total NO 2 - concentration after the reduction. The daily variability nitrites/nitrates assay in EBC was below 7% as assessed for up to 10 days.

Assay of thiol compounds in exhaled breath condensate

The equal volume (500 µl) of EBC, 0.3 M Na 2 PO 4 and Ellman's reagent were mixed on ice and the absorbance was read at 412 nm (Ultrospec III, Pharmacia LKB spectrophotometer). Control samples were supplemented with deionized water instead of EBC. Standard curve was prepared with serial dilutions of 200 µM glutathione solution form 0.1 to 200 µM. The assays were ran in duplicate. The assay sensitivity was 0.25 µM and the reproducibility 4.7% [START_REF] Szkudlarek | Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects[END_REF].

Statistical analysis

Results

Effect of nebulized N-acetylcysteine on lung function parameters in COPD patients

The baseline characteristics of the study subjects is presented in Table

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were subtracted from respective H 2 O 2 levels after NAC to determine if there were actual differences between PL and NAC (Fig. 1). The levels of relative increase of exhaled H 2 O 2 were significantly higher 30 min after nebulized NAC (p=0.002). The time course of H 2 O 2 exhalation for up to three hours after the nebulized NAC and PL in COPD patients is presented in Table 2.

Effect of N-acetylcysteine on the time course of nitrites and nitrates exhalation in COPD patients

The EBC level of nitrites and nitrates (NO 2A and2B.

Effect of N-acetylcysteine on the time course of thiol exhalation in COPD patients

With regard to the changes at the consecutive time points, after nebulized NAC the EBC concentration of thiols increased from 1.42±1.69 µM (0 min) to 2.49±2.00 µM (30 min, p=0.009 if compared with 0 min) and 1.71±1.83 µM (180 min, p=0.03 if compared with 0 min). After nebulized PL, the EBC thiol concentration varied from 1.75±1.56 µM (0 min) to 1.73±1.82 µM

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(30 min) and 2.13±1.96 µM (180 min) (p>0.05, in each time point) (Table 3). The differences in RSH levels between nebulized NAC and PL were not significant. For detailed comparisons, see Figure 3. The thiols content variability evaluated in the same subjects before either PL or NAC inhalation was 16%. In statistical terms, the differences between the same patients before PL and NAC nebulization were not significant.

Correlation between exhaled breath condensate parameters

Both 30 min. and 180 min. after nebulized NAC, there was significant, positive correlation between the EBC concentration of nitrates and RSH: r=0.67 and r=0.80; (p=0.02 and p=0.003), respectively. Further, the EBC concentrations of RSH and H 2 O 2 were significantly interrelated (r=0.78; p=0.003) 180 min after NAC nebulization. None of these relationships were noted after nebulized PL in the same subjects.

Discussion

The range of H 2 O 2 concentrations detected in our study patients is consistent with their clinical characteristics of mild or stage 0, stable COPD. The stable COPD subjects in our study presented with exhaled H 2 O 2 levels below those reported in moderate and severe COPD stage [START_REF] Benedetto | Long-term oral N-acetylcysteine reduces exhaled hydrogen peroxide in stable COPD[END_REF][START_REF] Kasielski | Long-term administration of N-acetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease[END_REF] or COPD exacerbation [START_REF] Dekhuijzen | Bast A Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease[END_REF]. EBC levels of H 2 O 2 in patients with more advanced, moderate COPD were previously shown to be 10 times higher than in healthy controls [START_REF] Nowak | Increased content of thiobarbituric acid-reactive substances and hydrogen peroxide in the expired breath condensate of patients with stable chronic obstructive pulmonary disease: no significant effect of cigarette smoking[END_REF]. They are approximately twice higher than H 2 O 2 levels in COPD patient included into the present study and the current COPD H 2 O 2 levels are still 4 times higher than those reported in healthy subjects [START_REF] Nowak | Increased content of thiobarbituric acid-reactive substances and hydrogen peroxide in the expired breath condensate of patients with stable chronic obstructive pulmonary disease: no significant effect of cigarette smoking[END_REF]. Oral N-acetylcysteine was demonstrated to decrease exhaled H 2 O 2 in stable COPD patients [START_REF] Benedetto | Long-term oral N-acetylcysteine reduces exhaled hydrogen peroxide in stable COPD[END_REF][START_REF] Kasielski | Long-term administration of N-acetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease[END_REF] as in our former study [START_REF] Szkudlarek | Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects[END_REF]. The contribution of auto-oxidation in NAC solution would be most probably only minor after half an hour of its deposition in the airways. Therefore, it seems more credible that the temporary and short-lived H 2 O 2 increase is related with redox interactions in the airways of COPD patients.

The clinical attempts to evaluate the redox effects of drugs commonly encounter similar obstacles related with the biased understanding of the redox reactions and their relativity with respect to physical and chemical parameters. Also, it may be over simplistic to assume that NAC may have pro-oxidant side effects [START_REF] Kinnula | Focus on antioxidant enzymes and antioxidant strategies in smoking related airway diseases[END_REF]. The redox potential of cysteine/cystine couple is reportedly high enough to oxidize corresponding glutathione GSH/GSSG couple and act not as an oxidant but as a critical intracellular redox control of protein activity [START_REF] Jones | Cysteine/cystine couple is a newly recognized node in the circuitry for biologic redox signaling and control[END_REF]. NAC may increase H 2 O 2 levels mediating the increase of intracellular NO and provide an excess of thiol groups that redirect NO away from cellular targets and enhance ROS generation [START_REF] Borutaite | Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion[END_REF][START_REF] Motterlini | Endothelial Heme Oxygenase-1 Induction By Hypoxia Modulation By Inducible Nitric-Oxide Synthase And S-Nitrosothiols[END_REF]. Additionally, ROS may be generated in the aerobic lung environment in the reactions involving native or thiol bound NO and glutathione or cysteine [START_REF] Kikugawa | Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks[END_REF]. Fortunately, these experiments have already been conducted with cysteine. Hydroxyl radical •OH and thiyl radicals •RS are generated by cysteine gradually decomposing S-nitrosogluthatione [START_REF] Kikugawa | Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks[END_REF] and are capable of forming H 2 O 2 .

NAC may initiate and interfere with other specific reactions in the airways of COPD patients.

Thiol compounds (RSH), unlike ionized thiolates (RS -), do not react with H 2 O 2 [START_REF] Forman | Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers[END_REF] and it is unlikely non-ionized NAC would directly alter H 2 O 2 levels. Also, reaction of superoxide anion

•O 2 -with RSH is probable in the airways but quite unlikely significant at the intracellular level due to rapid dismutation of •O 2 - [START_REF] Forman | Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers[END_REF]. S-nitrosoglutathione and NAC derivative not only release NO but effectively increase the extend and selectivity of NO action. RSNO stabilizes NO reactivity and increases its half-life redirecting its target actions into more selective thioregulatory functions upon proteins [START_REF] Stamler | S-nitrosothiols and the bioregulatory actions of nitrogen oxides through reactions with thiol groups[END_REF]. The reaction between RNS and NAC initially involved the intermediates on the redox path to nitrates and nitrites. NAC may react with either nitrogen dioxide radical (•NO 2 ) as well as directly with nitric oxide or nitrogen trioxide (N 2 O 3 ) to from S-nitrosthiol [START_REF] Forman | Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers[END_REF]. Further, NAC may inhibit the heme oxygenase mediated oxidation and increase iron levels in COPD. The lung parenchyma overload with iron in smoking COPD patients may accelerate not only Fenton's reaction but also nitrosothiol formation in the reaction

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introsonim ion intermediate. Ionized thiol adduct with iron are characterized with the increased reactivity compared with native thiols [START_REF] Forman | Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers[END_REF]. Proximity of a redox metal to a target thiol may be an important factor in thiol nitrosylation. Finally, the forms of ionized thiolates (RS -) and thiyl radicals (•RS), including not only NAC but also GSH pools, in aerobic conditions from superoxide anion, eventually decomposing to hydrogen peroxide detected in our COPD patients.

The existing data on interrelation between lung functional and oxidative parameters in subjects with COPD provide evidence on the reverse relation between FEV 1 values and nitrotyrosine formation [START_REF] Ichinose | Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways[END_REF]. In the experimental setting with nebulized thiols, GSNO increased expired NO for up to 30 minutes in cystic fibrosis patients [START_REF] Snyder | Acute effects of aerosolized S-nitrosoglutathione in cystic fibrosis[END_REF]. Reduced and oxidized glutathione and nitrosothiols were increased approximately twice in COPD patients but exhaled NO did not correlate with nitrosothiols [35]. NAC may function as an NO antagonist by providing an excess of thiol groups that redirect NO [START_REF] Motterlini | Endothelial Heme Oxygenase-1 Induction By Hypoxia Modulation By Inducible Nitric-Oxide Synthase And S-Nitrosothiols[END_REF]. Nitrates and nitrites levels in EBC were related with increased exhaled levels of H 2 O 2 [36] and their increased levels in asthmatic patients were similar as those reported hereby [START_REF] Dziedzic | Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3-in breath condensate[END_REF]. Nitrates were elevated in smokers, whereas The difference between nitrite (Fig. 2A) and nitrate (Fig. 2B) concentration in exhaled breath The difference between thiol (RSH) concentration in exhaled breath condensate (EBC) of COPD patients before -initial, 30 min and 180 min. after 3 ml nebulized N-acetylcysteine and placebo.

Relative RSH concentration is a difference between individual levels after nebulized NAC and PL. Underlined are median of thiol concentration increase after NAC with subtracted placebo values at respective time points. * p=0.009 vs. initial 39.

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  s c r i p t containing the same volume of deionized water. Control samples received 60 µl PBS solution (pH 7.5). Then, 30 µl of NADPH-nitrite reductase (250 U/ml), and 10 µl of NADPH 0.625 mg/ml solution were added into each well. The plates were incubated for 30 minutes at room temperature and protected from light, mixed with Griess solutions and absorbance was read at wavelength 562 nm with microplate reader EL 340 Bio-Tek Instruments (Winooski, VT, USA).

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  Data are expressed as arithmetic mean ± standard deviation or median and range [Me, R]. The normality of distribution was assessed with either Shapiro-Wilk's or Kolmogorov-Smirnov test.The temporal pattern of variable changes and the differences between parameters after nebulized NAC and PL were analyzed either with Student t or Wilcoxon matched-pair test. The differences between groups were analyzed with Mann-Whitney U test or Friedman ANOVA test. The Pearson correlation coefficient was used to assess the data interrelation. In all cases, a p value ≤ 0.05 was considered significant.

  were not reportedly increased in COPD patients[10]. Also, nebulization alone may accelerate droplet formation at epithelial lining fluid. Due to both decreased mucus viscosity and increased mucociliary clearance, more droplets from lower airways epithelial lining are blown away and moved upwards.The existing clinical data on inhaled NAC confirm insignificant effects on forced expiratory parameters even if NAC did appear to improve exercise-induced desaturation in pulmonary fibrosis[37]. NAC recently turned out ineffective in reducing annual number of exacerbation and did not prevent from deteriorating FEV 1 in COPD patients in a randomized, placebo-controlled, three-year study[38].

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  Apparently, the clinical significance of NAC nebulization in this group of patients merely exceeds placebo effects. Although the relevance of these observations remains yet to be established, this study provides some novel evidence on pro-oxidant effects of nebulized NAC. Albeit these effects were short-lived and moderate in COPD patients, they may be specifically associated with increased thiol oxidative turnover further accelerated by NAC deposition in airways.

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A c c e p t e d m a n u s c r i p tFigure 1 .A c c e p t e d m a n u s c r i p tFigure 2

 12 Figure 1. Relative EBC H 2 O 2 concentration in COPD patients after nebulized N-

2 -

 2 condensate (EBC) of COPD patients before (initial), 30 min, 90 min and 180 min after inhalation of 3 ml nebulized N-acetylcysteine or placebo. Relative nitrite (NO calculated by subtracting individual levels after nebulized PL form NAC levels. No significant differences were found in the time patterns.

A c c e p t e d m a n u s c r i p tFigure 3 .

 3 Figure 3. Relative concentrations of thiols in exhaled breath condensate of COPD patients after

A c c e p t e d m a n u s c r i p t propellant

  alone (3 ml of sterile, deionized water with addition of 2.7 mM EDTA, 75 mM NaOH, pH adjusted to 6.6 with HCl) was a sterile filtered, pyrogen free solution. NAC and placebo (PL)

	aerosol was generated with 3 ml Fluimucil or PL sterile solution, respectively, placed inside
	automated (output 0.3 ml/min) De Vilbiss 700 nebulizer (Sunrise Medical, Wollaston, Great
	Britain) wired up with pneumatic inhalator AP-50 De Vilbiss. Mean size of 50% aerosol
	particles or mass median aerodynamic diameter did not exceed 4 µm.
	The reagents were prepared freshly in deionized, pyrogen free water (resistance > 18 MΩcm,
	HPLC Water Purification System USF ELGA, England) and kept on ice prior to use.
	Griess solutions: (A) 58.07 mM sulphanilamide in 2 M sulfuric acid and (B) 38.58 mM
	naphthylethylenediamide dihydrochloride in deionized water were protected from light and
	stored at 4°C. Elman's reagent was prepared with 0,04 % DTNB -5,5 dithio-bis (2-nitrobenzoic
	acid) in 10% citrate solution. All remaining reagents were of analytical grade.
	Fluimucil (300 mg of N-acetylcysteine dissolved in 3 ml H 2 O injection grade, 2.7 mM EDTA,
	616.7 mM NaOH) was commercially available from Zambon, Vicenza (Italy). Placebo-Fluimucil

2) from Aspergillus species, NaNO 2 , naphthylethylenediamide dihydrochloride, nicotinamide adenine dinucleotide phosphate reduced form (NADPH), peroxidase from horseradish type II (HRP; EC1.11.1.7), reduced glutathione (GSH) and sulphanilamide were obtained from Sigma Chemicals Co., St. Louis, MO. USA.

acetylcysteine on time course of H 2 O 2 exhalation in COPD patients

  1. Lung functional parameters including FVC, FEV 1 , MEF25, PEF and FEV 1 /FVC were not affected with either nebulized NAC or PL. Lung diffusing capacity DLCO c was 86.4±17.3% before and 84.9±15.5% 180 min after nebulized NAC and was similar in case of PL nebulization: 87.3±12.9 and 86.8±13.4 %, respectively. At baseline, the mean H 2 O 2 concentration in COPD patients was 0.20±0.12 µM before nebulized NAC and 0.34±0.76 µM before nebulized PL, respectively (p>0.05). Thirty min after NAC inhalation, the mean H 2 O 2 concentration reached 0.45±0.25 µM (vs. PL 0.17±0.17 µM; p<0.05, if individual levels were compared, for details see Fig.1). The H 2 O 2 level in EBC after 90 min was 0.16±0.09 µM (vs. PL 0.17±0.15 µM). After three hours, H 2 O 2 level was 0.12±0.07 µM and was similar if compared with the respective values after PL inhalation: 0.21±0.23 µM (Table2). There were no differences in the mean levels of exhaled H 2 O 2 between NAC and PL at any of the study time points. However, exhaled H 2 O 2 was increased if the exhaled H 2 O 2 levels after

	Effect of N-

nebulized PL were considered a background, confounding factor and were subtracted from exhaled H 2 O 2 levels after nebulized NAC (Fig.

1

). The H 2 O 2 levels in any given patient after PL

  in parallel with the significant levels of H 2 O 2 generated in NAC solution used for nebulization under in vitro conditions[START_REF] Szkudlarek | Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects[END_REF]. Inhaled NAC entirely inhibited H 2 O 2 exhalation half an hour and increased H 2 O 2 approximately twice three hours after its nebulization in healthy, non-smoking subjects[START_REF] Szkudlarek | Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects[END_REF]. Conversely, in the current patients with COPD, NAC increased exhaled H 2 O 2 half an hour later above the reference levels returning to baseline at later time points. If the increased H 2 O 2 level in the current study were due to the auto-oxidation in NAC solution, the increase could likely be noticeable not only 30 min. after nebulized NAC, but also on further time points,

. Previously, the decreased exhaled H 2 O 2 levels were found 30 min after nebulized NAC A c c e p t e d m a n u s c r i p t

  The ionized part of nebulized NAC would immediately react with H 2 O 2 and decrease H 2 O 2 levels. That could contribute to the decrease of H H 2 O 2 due to initiation of the net of redox reaction favoring the formation of H 2 O 2 from superoxide anion. Notably, the halftime of S-nitrosogluthatione (GSNO) of approximately 30 min. would be ideal to support this interpretation, as at this time increased H 2 O 2 was found in our patients. Further, if this hypothetical redox chemistry were correct, half an hour after nebulized NAC we would in fact observe thiol-induced washout of excessive H 2 O 2 . In order to verify this, additional in vitro experiments on the interactions between NAC and GSNO would have to be designed. It would have been crucial for their validation to demonstrate that NAC is able to trigger redox reactions with nitrosothiols in the presence of oxygen and generate ROS.

2 O 2 in healthy subjects half an hour after inhaled NAC. However, if nebulized NAC were to from S-nitrosothiols in the reaction between NO and NAC, that could lead to the increased A c c e p t e d m a n u s c r i p t exhaled

Table 1 Characteristics of study subjects COPD patients

 1

	Number		19
	gender M/F		9/10
	age (yrs)		52.6±15.6
	Current smokers (pack years)		14 (20±14)
	BMI [kg/m 2 ]		26.9±3.7
	HGB [g/dl]		13.9±1.8
	WBC [×10 3 /µl]		7.22±2.47
		Before nebulization	After nebulization
	Lung functional parameters	NAC	PL	NAC	PL
	FEV 1 %	95.2±23.8	97.4±22.9	95.3±21.8	97.7±22.7
	FVC %	112.7±15.6 113.3±13.3 111.1±12.0 114.7±13.
	FEV 1 /FVC %	69.1±11.4	70.3±11.4	70.4±12.1	69.9±12.6
	PEF %	63.2±24.3	75.2±22.8	74.8±22.9	71.9±23.5
	MEF 25 %	65.7±24.9	68.2±26.7	70.7±26.7	69.0±26.5
	RV He %	112.0±43.9 124.1±31.6 127.3±30.1 120.9±35.0
	DLCO c %	86.4±17.3	87.3±12.9	84.9±15.4	86.8±13.4
	The study included 9 patients at stage 0 COPD, 7 with mild and 3 with moderate COPD. Lung
	functional parameters are expressed as percentage of the predicted value; FVC-forced vital
	capacity, FEV 1 -forced expiratory volume in the first second, DLCO c -single breath carbon
	monoxide diffusing capacity corrected for hemoglobin and alveolar volume, RV He -residual
	volume, BMI-body mass index, NAC-N-acetyl-L-cysteine, PL-placebo.

Table 2 . Hydrogen peroxide in exhaled breath condensate of COPD patients after inhalation of N-acetylcysteine and placebo H 2 O 2 levels (µM) in EBC Inhaled solution

 2

	(3 ml)
	Before	30 min after 90 min after	3h after
	0.20±0.29	0.45±0.25 #	0.16±0.09	0.12±0.07
	NAC (300 mg)
	[0.13; 1.33]	[0.46; 0.88]	[0.15; 0.41]	[0.11; 0.34]
	0.17±0.16	0.17±0.17	0.17±0.15	0.21±0.23
	Placebo
	[0.13; 0.7]	[0.11; 0.73]	[0.13; 0.58]	[0.14; 0.97]

# p=0.087 vs. before (border of significance), for detailed comparison with placebo effects see Figure 1. Median (Me) and range (R) are provided in brackets [Me; R]

Table 3 . Effect of N-acetylcysteine and placebo on the time course of thiol exhalation in

 3

	COPD patients
			EBC thiol levels (µM)
	Inhaled solution (3 ml)
		Before	30 min after	3 h after
	NAC	1.42±1.69	2.49±2.00*	1.71±1.83 #
		[ 0.73; 4.58]	[2.27; 5.66]	[0.95; 5.24]
	Placebo	1.75±1.56	1.73±1.82	2.13±1.96
		[1.28; 5.02]	[1.06; 5.89]	[1.94; 6.77]
	There was no significant difference in thiol levels between nebulized N-acetylcysteine (NAC)

and placebo. If the consecutive time points were compared, EBC concentration of thiols increased after nebulized NAC after 30 min (p=0.009 if compared with 0 min) and 180 min (p=0.03 if compared with 0 min). Mean values± standard deviation are presented. Median (Me) and range (R) are provided in brackets [Me; R] * p=0.009 vs. baseline (before), # p=0.03 vs. baseline; for detailed comparison with placebo see Figure 3.

Acknowledgements

This study was supported by Ministry of Education and Science grant no. PBZ-KBN-094/P06/2003/01 and Medical University of Lodz institutional grant no. 503-0079-1.

 A c c e p tThere was no significant differences between these parameters at the study time points.


Jürg Hamacher
email: hamacher@greenmail.ch

Katja Eichert

Clemens Braun

Thomas Grebe

Andreas Strub

Rudolf Lucas

Manfrid Eltze

Albrecht Wendel

Montelukast exerts no acute direct effect on NO synthases

Keywords: Montelukast, Nitric oxide synthases, Leukotrienes, Lung vascular resistance

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The leukotrienes LTC 4 , LTD 4 and LTE 4 , collectively called cysteinyl leukotrienes (CysLTs), are potent proinflammatory lipid mediators derived via the 5-lipoxygenase pathway from arachidonic acid and play a central role in inflammation, bronchoconstriction, oedema formation and airway remodelling of asthmatics [START_REF] Busse | Leukotriene pathway inhibitors in asthma and chronic obstructive pulmonary disease[END_REF][START_REF] Busse | Leukotrienes and Inflammation[END_REF][START_REF] Drazen | Leukotrienes as Mediators of Airway Obstruction[END_REF][START_REF] Peters-Golden | Do anti-leukotriene agents inhibit asthmatic inflammation?[END_REF]. The selective antagonist of the G protein-coupled cysteinyl leukotriene-1 (CysLT 1 ) receptor, montelukast, has been shown to have a high binding affinity at LTD 4 receptors (IC 50 9x10 -10 M; [START_REF] Jones | Pharmacology of montelukast sodium (Singulair), a potent and selective leukotriene D4 receptor antagonist[END_REF][START_REF] Seidenberg | Montelukast -an antileukotriene treatment for asthma. Changing the asthma-treatment paradigm[END_REF][START_REF] Markham | Inhaled salmeterol/fluticasone propionate combination: a review of its use in persistent asthma[END_REF] and to functionally to antagonize LTD 4 -evoked contraction of guinea-pig trachea with high potency (pA 2 9.7; ( 8)).

Pharmacodynamically it mainly acts by blocking poiesis and chemoattraction of inflammatory cells like eosinophils, but also effects on cytokine and growth factor patterns and actions during the process of airway inflammation have been described (4;9-11). However, not all aspects of actions of the CysLT 1 receptor antagonists are fully understood, as their mode of action might be more complex than previously assumed (4;12-14).

In a study with asthmatics, LTE 4 inhalation did not increase exhaled nitric oxide (NO) content, despite occurrence of airway narrowing and sputum eosinophilia [START_REF] Deykin | Exhaled nitric oxide following leukotriene E(4) and methacholine inhalation in patients with asthma[END_REF] and a concomitant expression of NO synthases (NOS) in eosinophils (16;17), suggesting a more complex relationship between NO production and the CysLTs during eosinophilic airway inflammation. A number of studies in asthmatic subjects treated with montelukast have shown that such LTD 4 receptor blockade is associated with lower levels of exhaled NO [START_REF] Bisgaard | NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast[END_REF][START_REF] Bratton | Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: a preliminary study[END_REF][START_REF] Wilson | Antiasthmatic effects of mediator blockade versus topical corticosteroids in allergic rhinitis and asthma[END_REF][START_REF] Wilson | Evaluation of Salmeterol or Montelukast as Second-Line Therapy for Asthma Not Controlled With Inhaled Corticosteroids[END_REF][START_REF] Wilson | A comparison of topical budesonide and oral montelukast in seasonal allergic rhinitis and asthma[END_REF][START_REF] Ghiro | Effect of montelukast added to inhaled corticosteroids on fractional exhaled nitric oxide in asthmatic children[END_REF][START_REF] Sandrini | Effect of montelukast on exhaled nitric oxide and nonvolatile markers of inflammation in mild asthma[END_REF][START_REF] Berkman | The effect of montelukast on bronchial provocation tests and exhaled nitric oxide levels in asthmatic patients[END_REF][START_REF] Straub | The effect of montelukast on lung function and exhaled nitric oxide in infants with early childhood asthma[END_REF][START_REF] Straub | The effect of montelukast on exhaled nitric oxide and lung function in asthmatic children 2 to 5 years old[END_REF]. In a murine allergic asthma model in which inflammation was induced by injection of ovalbumin, no evidence for a change in inducible NOS (iNOS) mRNA levels following montelukast treatment was observed [START_REF] Sade | Effect of montelukast pretreatment on inducible nitric oxide synthase mRNA expression in the lungs of antigen-challenged allergic mice[END_REF]. However, lung iNOS expression was modulated in a rat allergic inflammation model, but activity remained unchanged. In contrast, montelukast as well as dexamethasone significantly decreased iNOS expression and activity in a probably

CysLT-free assay of cytokine stimulated rat lung epithelial cells, suggesting that montelukast might be able to directly or indirectly modulate iNOS function, at least in chronic models, enabling pathways to be detectable which are not related to its acute and known action as a LTD 4 receptor antagonist [START_REF] Offer | A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model[END_REF].

The obvious question arising from these studies is whether such a relationship between CysLTs and exhaled NO is due to a direct effect of montelukast on NO levels via NOS inhibition, or whether such effect is indirectly mediated in response to its anti-inflammatory activity, or both. Since the structures of known NOS inhibitors are heterogeneous, a theoretical prediction of a potential NOS inhibition by montelukast is virtually impossible. We

A c c e p t e d m a n u s c r i p t

addressed that issue by using different experimental in vitro approaches: First, the interaction between montelukast and increased NO levels after lipopolysaccharide (LPS) challenge was studied in an ex-vivo CysLT-independent rat lung perfusion and ventilation model, which offers the detection of NOS-mediated changes in lung vascular resistance [START_REF] Uhlig | Functional and fine structural changes in isolated rat lungs challenged with endotoxin ex vivo and in vitro[END_REF][START_REF] Uhlig | Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin[END_REF][START_REF] Chlopicki | Biphasic response to lipopolysaccharide from E. coli in the isolated ventilated blood-perfused rat lung[END_REF].

Montelukast was used at a concentration of 3x10 -8 M, which is more than 100-fold higher than its potency to antagonize LTD 4 -evoked trachea contraction (pA 2 9.7; (8)). Furthermore, functional in vitro experiments on aortic rings from the same species were performed to assess any direct inhibitory effect of montelukast on the inducible and endothelial isoenzyme (i-and eNOS) as well as the possibility to inhibit iNOS expression [START_REF] Rees | Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock[END_REF][START_REF] Eltze | Potency and selectivity of inhibitors of inducible and endothelial nitric oxide synthase in rat aorta correlate with human isozymes (abstract)[END_REF][START_REF] Fleming | Incubation with endotoxin activates the L-arginine pathway in vascular tissue[END_REF]. Additionally, field stimulated rabbit cavernosum strips were used to exclude inhibition of neuronal NOS (nNOS) (36;37). Finally, montelukast was investigated for its potential inhibition of isolated human iNOS enzyme [START_REF] Strub | The novel imidazopyridine 2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) is a highly selective inhibitor of the inducible nitric-oxide synthase[END_REF]. The highest concentrations of montelukast investigated in these assays (up to 10 -4 M) significantly exceeded those generally measured as plasma concentrations (3x10 -8 M) following oral administration of 10 mg/day montelukast sodium as therapeutic regimen [START_REF] Zhao | Pharmacokinetics and bioavailability of montelukast sodium (MK-0476) in healthy young and elderly volunteers[END_REF].

Materials and methods:

Isolated perfused rat lung: Vascular resistance

Lungs of Wistar rats (female, 200-250 g; Harlan-Winkelmann, Borchen, Germany) were isolated after terminal intraperitoneal anaesthesia with 160 mg/kg pentobarbital sodium and ventilated and perfused as previously described in detail (30;31;40). Wistar rats were chosen despite their relative insensitivity to LTD 4 as the experimental setting focused on NO synthase and not on CysLT effects and was established with this strain. Lungs were perfused at constant hydrostatic pressure (12 cm H 2 O) through the pulmonary artery with Krebs-Henseleit solution containing 2% BSA, 0.1% glucose, and 0.3% HEPES at 37°C, which resulted in a flow rate of approximately 35 ml/min. The pH of the perfusate before entering the lung was kept at 7.25 to 7.35 by automatic bubbling of the buffer with CO 2 as soon as the pH exceeded this range. The total amount of recirculating buffer was 100 ml. The relationship between vascular pressure P V and flow Q is given by the equation

P V = R V x Q A c
P = 1/C L x V T + R L dV/dt
where P is chamber pressure, C L pulmonary compliance, V T tidal volume, and R L airway resistance, reflecting the pressure that forces the volume V at the velocity dV/dt into the lung.

All parameters were normalized to the time point at the end of the preconditioning perfusion, which was at 40 min post-lung isolation. Perfusion and ventilation was continued for another 60 min. Data were recorded on a computer using the Mathlab Software package (MathWorks, Inc., Nattick MA, USA).

Isolated rat thoracic aorta: iNOS and eNOS inhibition

Thoracic aortae were obtained from Wistar rats (male, 250-300 g; Charles River, Kisslegg, Germany) killed by cervical dislocation. Aortic rings of 1.5 mm length were mounted under 1 g tension in 10 ml organ baths filled with warmed (37 o C), oxygenated (95% O 2 /5% CO 2 )

Krebs solution (mM): NaCl 120.0, KCl 5.5, CaCl 2 2.5, MgCl 2 1.2, NaH 2 PO 4 1.2, NaHCO 3 25.0 and glucose 11.0, additionally containing 10 -5 M indomethacin for the experiments of iNOS inhibition. Isometric tension changes were measured by force-displacement transducers. In case of iNOS inhibition, the endothelium was removed by gently rubbing the intima surface before cutting the vessel into rings, which were than placed in Petri dishes containing RPMI-1640 medium with LPS (200 ng/ml, Salmonella abortus equi) and incubated at 37 °C for 16 h.

Aortic rings were considered denuded when a maximal concentration of the muscarinic receptor agonist, arecaidine propargyl ester (APE, 3x10 -6 M), caused relaxation of less than 5% of the precontracted vessel.

Rat aortic rings were precontracted by 3x10 -7 M phenylephrine (EC 80-90 of its own maximal effect), after that 3x10 -6 M APE (EC 100 ) was added to cause rapid relaxation of the tissue within 5 min, before the cumulative administration of the test drug was started to evoke

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contraction due to inhibition of eNOS. Likewise, for measuring iNOS inhibition, LPS-treated aortic rings were challenged with 3x10 -7 M phenylephrine, which caused a partial contraction

(5-10% compared with untreated tissue). Thereafter, due to inhibition of iNOS, cumulative administration of test drugs restored the contraction of the hyporeactive tissue. The nonselective NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) at 10 -5 M was finally added to define the maximal contractile effect of test drug related to that elicited by AMT (100%). All results were expressed as a percentage of the AMT-induced maximal response (34;35;38).

Isolated rat aorta: iNOS expression

Similarly, the inhibition of iNOS expression can be assessed in rat aortic rings rendered hypoactive with LPS alone (200 ng/ml, Salmonella abortus equi) and by co-incubation with the test drug during LPS exposure (16 h). After this treatment, the restoration of contractility of the tissue to cumulatively administered phenylephrine (10 -8 -3x10 -5 M) by active test drugs, e.g. dexamethasone (10 -8 and 10 -7 M) or cycloheximide (10 -6 and 10 -5 M), can be assessed in comparison with concomitantly run control rings untreated with LPS and test drug (41).

Field stimulated rabbit corpus cavernosum: nNOS inhibition

Adult New Zealand White rabbits (male, 2.

Human iNOS activity assay

The enzyme reaction using isolated human iNOS was performed in 96-well microtiter Fplates at L-arginine concentrations of 3 to 5x10 -6 M essentially as described previously (38;42). After adding [ 3 H]L-arginine (150,000 dpm/well) the assay mixture was incubated for 60 min at 37 o C after that it was stopped and analysed for [

Drugs and chemicals

Montelukast was a gift from Merck Frosst Canada. The NOS inhibitors AMT (2-amino-5,6-

dihydro-6-methyl-4H-1,3-thiazine hydrochloride), L-NMMA (N ω -monomethyl-L-arginine monoacetate), L-NIL (L-N ω -(1-iminoethyl)lysine hydrochloride) and L-NAME (N ω -nitro-L-
arginine methyl ester) were purchased from Alexis (Läufelingen, Switzerland). APE (arecaidine propargyl ester) and phenylephrine hydrochloride were from Tocris (Köln, Germany). Cycloheximide and dexamethasone were from Sigma (Munich, Germany).

Pentobarbital sodium was from Merial (Hallbermoos, Germany). LPS from Salmonella minnesota or Salmonella abortus equi was purchased from Sigma (Munich, Germany).

Bovine serum albumin (BSA), fraction V was from Serva (Heidelberg, Germany), HEPES from ICN Biomedicals Inc. (Ohio, USA).

Statistical analysis

Data are given as mean and standard error of the mean (mean ± SEM) unless otherwise indicated. In the rat lung perfusion experiments, two-way ANOVA was used to differentiate between drug effects with Bonferroni-post hoc analysis, considering p ≤ 0.05 as significant.

-log(IC 50 )M values for half-maximal restoration of contraction of LPS-or APE-pretreated rat aortic rings (inhibition of i-and eNOS, respectively) or inhibition of relaxant responses to EFS in rabbit corpus cavernosum (inhibition of nNOS) were calculated by a linear curve fitting model and were related to their individual maximal inhibitory effects.

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Results

Isolated perfused rat lung

The isolated perfused rat lung model was used to assess the involvement of vascular NO in response to LPS. Infusion of LPS alone (100 µg/ml of Salmonella minnesota) did not increase vascular resistance normalized to the end of the preconditioning perfusion of 40 min and expressed as R V /R V40min (Fig. 1). However, when LPS was co-administered with the NOS inhibitor L-NMMA (10 -4 M), a significant increase in vascular resistance occurred, which was maximal at 40 min after combined drug administration, demonstrating the involvement of NO synthesis to keep the vascular system dilated after LPS treatment. In contrast, a combination of LPS and montelukast (3x10 -8 M) did not cause vasoconstriction in this model, suggesting that montelukast has no inhibitory effect on NO synthesis. Furthermore, no change of vasotonus by administration of montelukast itself is observed (Fig. 1).

On the other hand, perfusion of the rat lung with LPS increased airway resistance R L , decreased pulmonary compliance C L and tidal volume V T , starting at about 30 min after LPS administration (not shown). The time lag of 30 min is probably due to induction of cyclooxygenase-2 (COX-2), as supported by the ability of the COX-2 inhibitor CGP-28238 to prevent LPS-induced thromboxane release and hence bronchoconstriction in this model [START_REF] Uhlig | Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin[END_REF].

Administration of LPS together with L-NMMA (10 -4 M), did not affect the LPS-induced bronchoconstriction, revealing that synthesis of NO does not participate in this response.

Also montelukast (3x10 -8 M) did not impair the increase in airway resistance evoked by LPS, whereas montelukast, L-NMMA or vehicle, each being administrated alone, did not show any effect on airway resistance (not shown). For completion, previous findings have also shown that neither inhibition of 5-lipoxygenase with AA-861 nor blockade of LTC 4 /LTD 4 receptors with MK-571 prevent the LPS-induced bronchoconstriction [START_REF] Uhlig | Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin[END_REF].

Similarly, neither LPS alone, nor its combination with montelukast (3x10 -8 M) or L-NMMA (10 -4 M) significantly affected the lung weight, as compared to those treated with L-NMMA, montelukast or vehicle alone (not shown).

Effect of montelukast on i-and eNOS in rat aorta and nNOS in rabbit corpus cavernosum

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The reference NOS inhibitors, L-NIL, L-NMMA and L-NAME, caused concentrationdependent reversal of contraction to phenylephrine of rat aortic rings rendered hypoactive with LPS (iNOS) or relaxed by the muscarinic receptor agonist APE (eNOS), and attenuated nitrergic relaxations in response to EFS in rabbit corpus cavernosum (nNOS). Concentrationresponse curves of these compounds are depicted in Fig. 2, their -log(IC 50 )M values being summarized in Table 1. The rank order of potency of the standard inhibitors observed for inhibition of iNOS in rat aorta (L-NIL > L-NMMA > L-NAME) was different from that observed for the compounds displayed both on eNOS in rat aorta and nNOS in rabbit corpus cavernosum (L-NAME > L-NIL > L-NMMA). As derived from their -log(IC 50 )M values on these three NOS isoenzymes, L-NIL is selective for i-over e-and nNOS, L-NAME is selective for both e-and n-over iNOS, whereas L-NMMA behaved non-selective. However, none of the three NOS isoenzymes was inhibited by montelukast up to already supraphysiological concentrations of 3x10 -5 -10 -4 M (Fig. 2).

Effect of montelukast on iNOS expression

In a further series of experiments, the contraction of the rat aorta to cumulative administration of phenylephrine (10 -8 -3x10 -5 M) 16 h after LPS treatment alone or in the additional presence of the protein synthesis inhibitor cycloheximide, the glucocorticoid dexamethasone or the test drug montelukast was investigated (Fig. 3). Phenylephrine caused concentration-dependent contraction of the tissue, which could be nearly totally prevented treatment of the tissue with LPS for 16 h. Cycloheximide (10 -6 and 10 -5 M) in the presence of LPS, concentration-dependently restored the contractile response to phenylephrine nearly to levels obtained in untreated control tissue, whereas dexamethasone (10 -8 and 10 -7 M) in the presence of LPS restored at least 50% of maximal contraction to the agonist. However, montelukast up to 10 -5 M (lower concentration of 10 -6 and 10 -7 M were also ineffective but not depicted in Fig. 3) when co-incubated with LPS, did not prevent the endotoxin-induced vascular hypocontractility in rat aortic rings, demonstrating its failure to inhibit the induction/expression or synthesis of NOS.

Effect of montelukast on human iNOS activity

Finally we analysed if montelukast has a direct inhibitory effect on isolated human iNOS by inhibition amounted to less than 20% (Fig. 4).

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Discussion

Since the CysLT 1 receptor antagonist, montelukast, has been shown to reduce NO levels in exhaled air of asthmatics, the present in vitro experiments aimed to assess whether this drug is capable (a) to influence NO synthesis after endotoxin challenge in perfused rat lung, (b) to directly inhibit different NOS isoenzymes (i-, e-and nNOS) derived from rat, rabbit and human, and (c) to affect iNOS expression in rat vasculature.

In an ex-vivo rat lung perfusion and ventilation model we could demonstrate that vasodilatation in response to added endotoxin (LPS) maintained by stimulation of NO synthesis, did not change even with a more than physiological concentration of montelukast. This is consistent with effects observed with endotoxin on airways, showing that neither inhibition of 5-lipoxygenase with AA-861, nor LTC 4 /LTD 4 receptor blockade by MK-571 prevented the LPS-induced increase in airway resistance [START_REF] Uhlig | Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin[END_REF]. Similarly, previous studies have shown that, while L-NMMA (10 -4 M) without LPS did not cause any change in vascular resistance in perfused rat lung as compared to controls, infusion of L-NMMA together with LPS leads to a similar production of the stable eicosanoid degradation products, thromboxane B 2 und 6-keto-PGF 1α , as LPS alone [START_REF] Uhlig | Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin[END_REF]. Therefore, changes in vascular resistance in this setting seem not to be critically dependent on the thromboxane-prostacyclin system. As a result of the present study, the relaxing effect of NO on the vascular system in rat perfused lung can be observed roughly 40-50 min after LPS administration attributable to permanent NO synthesis by inducible NOS (iNOS), which can be converted into vasoconstriction by concomitant administration of a NOS inhibitor, like L-NMMA, but not by montelukast.

In order to further corroborate these results with montelukast, additional in-vitro studies in thoracic aorta from the same species were performed to assess its potential inhibition of iNOS and eNOS and to affect iNOS expression in this tissue. It has been previously shown that incubation with LPS activates iNOS in rat aorta to generate NO, which induces a permanent hyporeactivity to α 1 -adrenoceptor agonists, and that inhibitors of iNOS are capable to restore the contractile response to the agonists to control [START_REF] Fleming | Incubation with endotoxin activates the L-arginine pathway in vascular tissue[END_REF]. Thus, iNOS and eNOS inhibition by drugs was tested as reversal of contraction to the α 1 -adrenoceptor agonists phenylephrine of rat aortic rings rendered hypoactive by either overnight treatment with LPS or acutely by muscarinic receptor stimulation with APE [START_REF] Eltze | Potency and selectivity of inhibitors of inducible and endothelial nitric oxide synthase in rat aorta correlate with human isozymes (abstract)[END_REF]. On the other hand, NO synthetized by a cytosolic, constitutive isoform of NOS (nNOS) like that found in brain

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neuronal tissue, is the predominant nonadrenergic noncholinergic (NANC) relaxant neurotransmitter released in rabbit corpus cavernosum in response to EFS [START_REF] Ignarro | Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle[END_REF]. Previously, these functional in-vitro assays for i-, e-and nNOS and their inhibition by unselective or isoenzyme-selective inhibitors had been compared with human isoenzymes revealing that both rat aorta and rabbit corpus cavernosum can be used to assess selectivity for inhibitors observed at human isoenzymes (34;37;38). Particularly, in rat aorta L-NIL has been characterized as being 14-fold selective for iNOS over eNOS (-log(IC 50 )M 6.0 vs. 4.9), exactly matching the 17-fold selectivity assessed for human iNOS over eNOS (-log(IC 50 )M 6.2 vs. (34;38)]. Furthermore, a selectivity of L-NAME for nNOS over iNOS could be demonstrated using rabbit corpus cavernosum and rat aorta (11-fold) and human isozymes (25-fold) [START_REF] Eltze | The inducible: neuronal nitric oxide sythase (i/n-NOS) selectivity of NOS-inhibitors in rat aorta versus rabbit corpus cavernosum or rat gastric fundus correlates with human i-and nNOS (abstract)[END_REF].

A possible inhibition of iNOS expression or its synthesis was investigated in rat aortic rings and demonstrated by the ability of dexamethasone or cycloheximide, respectively, to partly restore contraction to phenylephrine of tissue previously treated and rendered hypoactive with LPS [START_REF] Rees | Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock[END_REF]. Again, montelukast up to 10 -5 M did not show any effect. To answer the question whether montelukast has an inhibitory effect on human iNOS, we performed experiments on isolated human iNOS enzyme. Montelukast up to the highest concentration investigated of 10 -4 M does not inhibit the enzymatic activity. Likewise, experiments on respective human e-and nNOS isoenzymes yielded the same results (not shown). Taken together these assays clearly gave no evidence of any direct inhibitory effect of montelukast on NOS isoenzymes within the concentration range from 10 -7 to 10 -4 M, far exceeding that achieved during drug therapy [START_REF] Zhao | Pharmacokinetics and bioavailability of montelukast sodium (MK-0476) in healthy young and elderly volunteers[END_REF]. Therefore, alternative explanantions for the decreased NO levels measured in expired air from asthmatic subjects after treatment with montelukast should be considered [START_REF] Bisgaard | NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast[END_REF][START_REF] Bratton | Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: a preliminary study[END_REF][START_REF] Wilson | Antiasthmatic effects of mediator blockade versus topical corticosteroids in allergic rhinitis and asthma[END_REF][START_REF] Wilson | Evaluation of Salmeterol or Montelukast as Second-Line Therapy for Asthma Not Controlled With Inhaled Corticosteroids[END_REF][START_REF] Wilson | A comparison of topical budesonide and oral montelukast in seasonal allergic rhinitis and asthma[END_REF][START_REF] Ghiro | Effect of montelukast added to inhaled corticosteroids on fractional exhaled nitric oxide in asthmatic children[END_REF][START_REF] Sandrini | Effect of montelukast on exhaled nitric oxide and nonvolatile markers of inflammation in mild asthma[END_REF][START_REF] Berkman | The effect of montelukast on bronchial provocation tests and exhaled nitric oxide levels in asthmatic patients[END_REF][START_REF] Straub | The effect of montelukast on lung function and exhaled nitric oxide in infants with early childhood asthma[END_REF][START_REF] Straub | The effect of montelukast on exhaled nitric oxide and lung function in asthmatic children 2 to 5 years old[END_REF].

Montelukast has been shown to reduce iNOS expression in the lung tissue of ovalbumin challenged rat after 4 weeks of treatment while the iNOS activity remained unchanged. In addition, montelukast as well as dexamethasone significantly decreased iNOS expression and activity in a probably CysLT-free assay of rat lung epithelial cells stimulated with cytokines. This finding suggests that montelukast might be able to directly or indirectly modulate iNOS function as an anti-inflammatory compound after long time exposure to allergen challenged animals [START_REF] Offer | A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model[END_REF]. This is consistent with the finding, that reduced levels of

A c c e p t e d m a n u s c r i p t

exhaled NO can be measured only after some time of chronic treatment with montelukast, which parallels its effect of other surrogate markers of inflammation such as blood eosinophils. Thus, the effect of montelukast on exhaled NO levels observed during asthma therapy excludes direct effects on NOS enzymes, but most probably is secondary to its expected effect as antagonist of CysLTs.

Since no acute inhibitory effect on NO synthesis, neither directly on the three NOS isoenzymes, nor a suppression of the expression of iNOS was found, alternative explanantions for the decreased NO levels measured in expired air from asthmatic subjects after treatment with montelukast must be considered [START_REF] Bisgaard | NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast[END_REF][START_REF] Bratton | Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: a preliminary study[END_REF][START_REF] Wilson | Antiasthmatic effects of mediator blockade versus topical corticosteroids in allergic rhinitis and asthma[END_REF][START_REF] Wilson | Evaluation of Salmeterol or Montelukast as Second-Line Therapy for Asthma Not Controlled With Inhaled Corticosteroids[END_REF][START_REF] Wilson | A comparison of topical budesonide and oral montelukast in seasonal allergic rhinitis and asthma[END_REF][START_REF] Ghiro | Effect of montelukast added to inhaled corticosteroids on fractional exhaled nitric oxide in asthmatic children[END_REF][START_REF] Sandrini | Effect of montelukast on exhaled nitric oxide and nonvolatile markers of inflammation in mild asthma[END_REF][START_REF] Berkman | The effect of montelukast on bronchial provocation tests and exhaled nitric oxide levels in asthmatic patients[END_REF][START_REF] Straub | The effect of montelukast on lung function and exhaled nitric oxide in infants with early childhood asthma[END_REF][START_REF] Straub | The effect of montelukast on exhaled nitric oxide and lung function in asthmatic children 2 to 5 years old[END_REF]. They may be explained by changes in quantities or sites of NO generation, binding or more distal processes. Those effects might occur later than assessed in our experimental settings, e.g. with prolonged drug administration, and might therefore be due to the drug to antagonize the effect of CysLTs which might reduce airway inflammation.

A further possibility is that CysLTs may have biological effects exceeding allergic disease. In conclusion, none of the functional in-vitro assays in our study gave evidence for a direct acute inhibition of the different NOS isoenzymes (i-, e-and nNOS) and of iNOS expression by montelukast, even up to high concentrations and far exceeding those observed during drug therapy [START_REF] Zhao | Pharmacokinetics and bioavailability of montelukast sodium (MK-0476) in healthy young and elderly volunteers[END_REF]. Its observed effect to reduce exhaled NO in asthmatics is therefore probably secondarily mediated, presumably indirectly due to effects on inflammatory mediators, cytokines, growth factors, receptors, and their partially convergent actions ultimately occurring to reduce inflammation during asthma (4).

4 1. 2 ,

 42 5-3.0 kg; Dunkin Hartley, Charles River, Kisslegg, Germany) were sacrificed by intravenous pentobarbital injection (60 mg/kg) and exsanguination. Four longitudinal strips (1.5x1.5x6 mm) were dissected from each corpus cavernosum and mounted in organ baths under a resting tension of 1 g to measure isometric tension changes in Krebs solution (mM: NaCl 118.3, KCl 4.7, CaCl 2 1.9, MgSO 4 1.2, KH 2 PO NaHCO 3 25.0, Na-EDTA 0.03 and glucose 11.1), additionally containing 10 -6 M atropine and 5x10 -6 M guanethidine to exclude a possible participation of cholinergic or adrenergic responses, respectively, to electrical field stimulation (EFS). The nutrient solution was aerated with a mixture of 95% O 2 and 5% CO 2 maintaining pH 7.4 at 37 o C. The strips were precontracted by 10 -5 M phenylephrine (EC 80-90 of its own maximal response); after that, relaxant nonadrenergic, noncholinergic (NANC) responses were evoked by EFS (15 V, 0.3 ms, 4 Hz, for 5 s, every 2 min) using a pair of platinum electrodes, one located at the bottom of the organ bath and directly connected with the tissue, and the other ring electrode placed at the top of the bathing fluid. After stabilization of EFS-evoked relaxant responses within 30 to 45 min, the test drug was added in a cumulative manner. The potential inhibition of nNOS A c c e p t e d m a n u s c r i p t could be followed by inhibition of tissue relaxation mediated by synthesis and release of neuronal NO (36-38).

  3 H]L-citrulline content in a Micro-Beta scintillation counter (PerkinElmer Wallac, Turku, Finland). -log(IC 50 )M values for test drugs were calculated from concentration-response curves by Prisma 3.0 (GraphPad Inc., San Diego, CA).

measuring [ 3 HA c c e p t e d m a n u s c r i p t

 3 ]L-arginine conversion to [ 3 H]L-citrulline. Whereas the reference NOS inhibitors, L-NIL, L-NMMA and L-NAME, caused a concentration-dependent inhibition of human iNOS (-log(IC 50 )M 6.29, 5.91 and 4.40, respectively), montelukast up to 3x10 -5 M was without any inhibitory effect. Even at the highest concentration investigated (10 -4 M),

5. 0 )

 0 , whereas L-NAME showed selectivity for eNOS over iNOS in rat aorta (-log(IC 50 )M 5.6 vs. 4.0; 40-fold selectivity) and on human isozymes (-log(IC 50 )M 5.7 vs. 4.4; 20-fold selectivity) [this study;

Fig. 1 .

 1 Fig. 1. Change of vascular resistance normalized to that measured at 40 min (R V /R V40 min ) in the isolated perfused rat lung after drug administration at 40 min into the perfusate. LPS (100 µg/ml; control n = 14) together with L-NMMA (10 -4 M; n = 3) causes vasoconstriction, whereas no vasoconstriction occurs with LPS administered together with montelukast (3x10 -8 M; n = 4). Given are means ± SEM of n = 3-14. *; p<0.0001 for LPS + L-NMMA vs. LPS + montelukast at the time points indicated.

Fig. 2 .

 2 Fig. 2. Cumulative concentration-response curves for NOS inhibitors in comparison with montelukast to restore contraction to 3x10 -7 M phenylephrine of rat aortic rings rendered hypoactive with LPS (inhibition of iNOS, top), or relaxed with 3x10 -6 M APE (inhibition of eNOS, middle), and to inhibit nitrergic relaxation to EFS in rabbit corpus cavernosum (inhibition of nNOS, bottom). Given are means ± SEM of n = 10-16.

Fig. 3 .

 3 Fig.3. Cumulative concentration-response curves of phenylephrine on untreated rat aortic rings (Control), treated for 16 h with LPS alone (LPS) or in the additional presence of either cycloheximide (10 -6 , 10 -5 M), dexamethasone (10 -8 , 10 -7 M) or montelukast (shown is the missing effect of 10 -5 M; lower concentrations of 10 -7 to 10 -6 M were also ineffective and not depicted). Given are means ± SEM of n = 6-8.

Fig. 4 .

 4 Fig. 4. Inhibition of human iNOS by NOS inhibitors in comparison with montelukast.log(IC 50 )M values of 6.29, 5.91 and 4.40 were obtained for L-NIL, L-NMMA and L-NAME, respectively, whereas the value for montelukast amounted to be less than 3.00. Given are means ± SEM of n = 6 determinations.

A c c e p t e d m a n u s c r i p t

  Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4

 4 Figure 4

c e p t e d m a n u s c r i p t where

  R V represents vascular resistance. In case of present constant-pressure perfusion (P V = constant), perfusion flow rate Q is measured to calculate vascular resistance R V . The lungs were suspended by the trachea and ventilated by negative pressure ventilation (inspiratory

	pressure: -7 cm H 2 O, expiratory pressure: -2 cm H 2 O) with 80 breaths/min resulting in a tidal
	volume of approximately 2 ml. Every 5 min a deep inspiratory breath (-20 cm H 2 O) was
	performed. A weight transducer was integrated into the chamber lid and allowed continuous
	measurement of lung weight. Additionally, parameters of lung mechanics were analyzed by
	applying the following formula

Table 1

 1 Potencies of drugs for inhibition of iNOS and eNOS in rat thoracic aorta and of nNOS in field stimulated rabbit corpus cavernosum. Given are -log(IC 50 )M values (with % maximal inhibitory effect in brackets) for the drugs derived from their individual half-maximal effects

	(means ± SEM, n = 10-16)
			-log(IC 50 )M
		iNOS	eNOS	nNOS
	___________________________________________________________________
	L-NIL	6.03 ± 0.03 (90)	4.90 ± 0.03 (94)	4.58 ± 0.08 (74)
	L-NMMA	4.78 ± 0.06 (49)	4.73 ± 0.04 (72)	4.19 ± 0.06 (66)
	L-NAME	4.01 ± 0.03 (38)	5.56 ± 0.02 (100)	5.05 ± 0.03 (96)
	Montelukast	<4.0	<4.5	<4.0

Acknowledgements

A Merck Medical School Grant supported the isolated rat lung perfusion assays. The work of J. Hamacher was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR 321/2-1; research group "Endogenous tissue injury: Mechanisms of autodestruction").