Modulation of nitric oxide pathways: Therapeutic potential in asthma and chronic obstructive pulmonary disease

European Journal of Pharmacology 533 (2006) 263 – 276 www.elsevier.com/locate/ejphar

Review

Modulation of nitric oxide pathways: Therapeutic potential in asthma and chronic obstructive pulmonary disease Anthony E. Redington ⁎ Department of Respiratory Medicine, Hammersmith Hospital, Du Cane Road, London W12 0HS, United Kingdom Accepted 13 December 2005 Available online 7 February 2006

Abstract Nitric oxide (NO) is present in the exhaled breath of humans and other mammalian species. It is generated in the lower airways by enzymes of the nitric oxide synthase (NOS) family, although nonenzymatic synthesis and consumptive processes may also influence levels of NO in exhaled breath. The biological properties of NO in the airways are multiple, complex, and bidirectional. Under physiological conditions, NO appears to play a homeostatic bronchoprotective role. However, its proinflammatory properties could also potentially cause tissue injury and contribute to airway dysfunction in disease states such as asthma and chronic obstructive pulmonary disease (COPD). This article will review the physiological and pathophysiological roles of NO in the airways, discuss the rationale for the use of drugs that modulate NO pathways – nitric oxide synthase inhibitors and NO donors – to treat inflammatory airway diseases, and attempt to predict the likely therapeutic benefit of such agents. © 2006 Elsevier B.V. All rights reserved. Keywords: Asthma; Chronic obstructive pulmonary disease; Nitric oxide

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exhaled NO and airway expression of NOS isoforms . . . . . . . . . . . . 3.1. Normal airways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Chronic Obstructive Pulmonary Disease (COPD) . . . . . . . . . . . 4. NO in airway physiology and pathophysiology . . . . . . . . . . . . . . . 4.1. Airway tone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Airway responsiveness . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Airway inflammation . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Proinflammatory properties of NO . . . . . . . . . . . . . . 4.3.2. Animal studies using NOS inhibitors . . . . . . . . . . . . 4.3.3. Studies in transgenic and gene-deficient animals. . . . . . . 5. Therapeutic potential for NOS inhibitors and NO donors in airways disease References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Tel.: +44 208 453 2150; fax: +44 208 453 2450. E-mail address: [email protected]. 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.069

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1. Introduction It is now 15 years since Gustafsson et al. (1991) reported that nitric oxide (NO) is detectable in exhaled breath. During that time, information has accumulated rapidly describing the expression of this molecule in the respiratory tract in health and disease. Although measurement of exhaled NO is now close to approval as a clinical test, however, the significance of many of the observations made remains poorly understood. NO is a freely diffusible redox-based signalling molecule that plays a homeostatic role in multiple physiological processes. As a toxic free radical with antimicrobial and tumouricidal properties, it is also important in host defence but has the potential to cause tissue injury. Which of its many properties predominates in any particular circumstance and whether the effects of NO in the airways should be regarded as primarily beneficial or harmful are still unanswered questions. Asthma and chronic obstructive pulmonary disease (COPD) are both diseases characterized by airflow limitation, airway hyperresponsiveness, and airway inflammation. The inflammatory response in allergic asthma is typified by immunoglobulin E overproduction, mast cell and eosinophil infiltration and activation in large and small airways, and increased production of Th2 cytokines (interleukin (IL)-4, IL-5, IL-9, and IL-13) by CD4+ lymphocytes (Busse and Lemanske, 2001). In COPD, inflammation is present in the central airways, peripheral airways, and lung parenchyma and involves in particular neutrophils and CD8+ lymphocytes (Saetta et al., 2001). In both cases, inflammation is believed in part to underlie the clinical expression of the disease and this has provided the rationale for the development of therapeutic agents that interfere with specific inflammatory pathways. This article will review our current understanding of the physiological and pathophysiological roles of NO in the airways and consider whether attempts to modulate NO pathways might prove a worthwhile therapeutic strategy in inflammatory airways diseases such as asthma and COPD. 2. Biosynthesis of NO NO can be synthesized enzymatically by oxidation of the amino acid L-arginine to L-citrulline (Moncada and Higgs, 1993). In humans and other mammalian species, three isoforms of the enzyme nitric oxide synthase (NOS), encoded by separate genes, have been cloned and characterized (Charles et al., 1993; Geller et al., 1993; Janssens et al., 1992; Marsden et al., 1992; Nakane et al., 1993; Sherman et al., 1993). Neuronal NOS (nNOS) or NOS1 and endothelial NOS (eNOS) or NOS3 are constitutively expressed enzymes whose activity is regulated by intracellular calcium and calmodulin. Generation of NO by these two isoforms occurs rapidly (within seconds) but is short-lived and relatively small quantities (in the picomolar range) of NO are produced. In contrast, the activity of inducible NOS (iNOS) or NOS2 is independent of calcium but is transcriptionally regulated by cytokines and other proinflammatory stimuli. Maximum induction of iNOS takes several hours, is prolonged, and generates much higher (nanomolar) levels of NO.

The paradigm of constitutive and inducible NOS isoforms has been modified from its original conception (Förstermann et al., 1998). In particular, although nNOS and eNOS are constitutively expressed – and indeed are sometimes collectively referred to as constitutive NOS (cNOS) – it is becoming increasingly clear that their activity can be regulated by various factors. Conversely, as will be discussed below, iNOS is constitutively expressed at certain sites. 3. Exhaled NO and airway expression of NOS isoforms 3.1. Normal airways Nitric oxide (NO) is normally present in the exhaled breath of many mammalian species, including humans, rats, mice, guinea-pigs, rabbits, and elephants (Gustafsson et al., 1991; Leone et al., 1994; Lewandowski et al., 1996). When measured by standardized methods based on chemiluminescence (American Thoracic Society, 2005), the fractional concentration of orally exhaled NO (FENO) in healthy subjects is typically between 5 and 25 parts per billion (ppb). There was initial speculation that the primary source of exhaled NO might in fact be autoinhalation of endogenous NO synthesized in the nasopharynx (Gerlach et al., 1994; Lundberg et al., 1994; Münch et al., 1994). However, direct bronchoscopic sampling of lower airway gas clearly demonstrated that NO in exhaled breath is indeed derived from the lower airways (Kharitonov et al., 1996a; Massaro et al., 1996). All three NOS isoforms are constitutively expressed in human airways. Using in situ hybridization and immunohistochemistry, iNOS has been localized to airway epithelial cells in resected lung tissue and in specimens obtained bronchoscopically (Guo et al., 1995; Hamid et al., 1993; Kobzik et al., 1993; Lane et al., 2004; Watkins et al., 1997). Ciliated cells, basal cells, and secretory cells are all sites of epithelial iNOS expression within the epithelium (Guo et al., 1995). Epithelial expression of iNOS is reduced in more distal airways (Kobzik et al., 1993) and is normally absent from peripheral lung (Guo et al., 1995). Although macrophages can express iNOS when activated, there appears to be little or no basal iNOS expression by alveolar macrophages (Guo et al., 1995, 2000; Lane et al., 2004; Tracey et al., 1994). Immunostaining for nNOS has been localized to nerves of the inhibitory nonadrenergic noncholinergic (iNANC) system in airway smooth muscle (Ward et al., 1995) and in the submucosa (Kobzik et al., 1993). Extraneuronal nNOS immunostaining has been described in the airway epithelium (Ricciardolo et al., 2001) and in endothelial cells (Kobzik et al., 1993; Lührs et al., 2002). Murine airway smooth muscle cells express nNOS in vivo (Shan et al., 2005), as do human airway smooth muscle cells in culture (Hamad and Knox, 2001; Patel et al., 1999). Giaid and Saleh (1995) localized eNOS mRNA and immunoreactivity to the airway epithelium in surgical specimens of normal human lung. Endothelial cells in vessels of all sizes also expressed the enzyme. In another study, however, no mRNA for either nNOS or eNOS could be detected in resected human airway specimens (Watkins et al., 1997).

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There has been some controversy regarding the relative importance of constitutive and inducible NOS isoforms as enzymatic sources of the NO detectable in exhaled breath. It was initially believed that one or both of the constitutive isoforms were responsible. Oral prednisolone treatment, for example, was reported not to alter FENO in healthy volunteers (Yates et al., 1995). Further evidence derives from the comparative effects of selective and nonselective NOS inhibitors. When administered by inhalation to healthy subjects, the G L-arginine analogues N -monomethyl-L-arginine (L-NMMA) G and N -nitro-L-arginine methyl ester (L-NAME) – which inhibit all NOS isoforms – were reported to reduce FENO whereas aminoguanidine – which exhibits modest selectivity for iNOS over other isoforms – had no significant effect (Yates et al., 1995, 1996). However, the inhibitory potency of aminoguanidine is relatively weak (Misko et al., 1993). In a more recent study, oral administration of SC-51, a prodrug of the more potent and more selective iNOS inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL), reduced FENO from around 6 ppb to b2 ppb in healthy subjects (Hansel et al., 2003). This suggests that iNOS is in fact the enzyme principally responsible for generating the NO detectable in exhaled air of healthy subjects. The small residual component may derive from the activity of constitutive NOS isoforms. There appear to be important species differences in the airway expression of NOS isoforms and in the enzymatic source of exhaled NO. On the basis of experiments with various NOS inhibitors, for example, Vaughan et al. (2003) reported that virtually all exhaled NO in isolated perfused rabbit lung was produced by eNOS. Consistent with this finding, eNOS was the principal NOS isoform detectable immunohistochemically in the airway and alveolar epithelium and in the vascular endothelium, with very little expression of either nNOS or iNOS at any site. Studies in mice with targeted disruptions of the eNOS (Cook et al., 2003) and nNOS (Cook et al., 2003; De Sanctis et al., 1997) genes have estimated that nNOS contributes approximately 40% and iNOS 60% of the NO detectable in lower-airway exhaled air in the basal state. 3.2. Asthma Numerous studies have shown that the FENO is elevated in untreated asthma in adults (Alving et al., 1993; Kharitonov et al., 1994; Persson et al., 1994) and children (Lundberg et al., 1996; Nelson et al., 1997). Several of these reports have found correlations between FENO and other variables including airway hyperresponsiveness (Al-Ali et al., 1998; Dupont et al., 1998; Jatakanon et al., 1998) and induced sputum eosinophilia (Jatakanon et al., 1998). Further rises in FENO in subjects with asthma occur during allergen-induced late phase responses (Deykin et al., 1998; Kharitonov et al., 1995a) and during experimental rhinovirus infection (de Gouw et al., 1998). Levels are also elevated during naturally occurring asthma exacerbations and fall with response to treatment (Massaro et al., 1995). Both cross-sectional (Garnier et al., 1996; Kharitonov et al., 1994) and interventional (Baraldi et al., 1997; Kharitonov et al., 1996b,c; Silkoff et al., 1998; Yates et al., 1995) studies have

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shown that corticosteroid treatment in adult and paediatric asthma is associated with a reduction in FENO towards normal. Furthermore, an FENO of ≥ 10 ppb is predictive of oral corticosteroid responsiveness (Little et al., 2000). In asthmatic children, FENO is also reduced by the leukotriene receptor antagonist montelukast (Bisgaard et al., 1999) and by the monoclonal anti-immunoglobulin E antibody omalizumab (Silkoff et al., 2004). There are also many reports of increased iNOS expression in asthma. Hamid et al. (1993) detected immunoreactive iNOS in the airway epithelium in bronchoscopic biopsy specimens from 22 of 23 subjects with asthma but only 2 of 20 healthy nonsmoking control subjects. Immunostaining for iNOS was also present in the subepithelial inflammatory infiltrate in the specimens from asthmatic subjects. Using in situ hybridization and quantitative immunohistochemistry, Redington et al. (2001) confirmed an increase in both iNOS mRNA and protein expression in untreated asthma. In a separate group of asthmatic subjects receiving regular maintenance treatment with inhaled corticosteroids, in contrast, expression of iNOS mRNA and protein did not differ from nonasthmatic control subjects. Similar findings in relation to iNOS mRNA and protein were reported by Saleh et al. (1998) in a crossover study of 10 asthmatic subjects treated with budesonide 1600 μg daily or placebo. Guo et al. (1995) found that treatment of healthy volunteers with the inhaled corticosteroid flunisolide 1000 μg daily for 3 weeks reduced epithelial cell expression of iNOS mRNA in vivo. Finally, Guo et al. (2000) described increased iNOS mRNA and protein in lysates of epithelial cells obtained by bronchial brushing from non-steroid-treated asthmatics compared with healthy control subjects, but no difference in steroid-treated asthmatics. The elevated FENO in untreated asthma is therefore likely to reflect induction of iNOS in airway epithelial cells. This is supported by the rapid, prolonged (N 72 h), and profound (∼95% inhibition) fall in FENO in asthmatic subjects following a single orally administered dose of SC-51, the prodrug of the iNOSselective inhibitor L-NIL (Hansel et al., 2003). Expression of iNOS by airway epithelial cells is regulated by exposure to proinflammatory cytokines. Stimulation of A549 or BEAS-2B respiratory epithelial cell lines and primary airway epithelial cells with cytokine combinations including interferon-gamma (IFN-γ), interleukin (IL)-1β, and tumour necrosis factor-alpha (TNF-α) increases iNOS gene and protein expression and also NO synthesis (Asano et al., 1994; Hamid et al., 1993; Robbins et al., 1994a,b; Watkins et al., 1997). IFN-γ appears essential in this response and IFN-γ-induced iNOS expression is synergistically increased by IL-4 (Guo et al., 1997). Regulation of iNOS occurs at a transcriptional level, particularly via the transcription factor Stat-1, although post-translational events may also operate (Guo et al., 2000). Robbins et al. (1994a,b) reported that dexamethasone inhibited cytokine-induced iNOS mRNA and protein expression by cultured human and murine airway epithelial cells. In contrast, Donnelly and Barnes (2002) found that iNOS expression by human primary airway epithelial cells was insensitive to dexamethasone, and suggested that the in vivo effects of corticosteroids on iNOS may be indirectly mediated.

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Nonenzymatic synthesis may also contribute to the elevated FENO in asthma independently of changes in NOS activity. Hunt et al. (2000) drew attention to the potential for NO production by acidification of nitrite. These authors showed that the pH of exhaled breath condensate collected from patients hospitalized with acute asthma was approximately two log-orders lower than that of control subjects, but normalized with corticosteroid treatment and clinical recovery. In vitro experiments suggested that the degree of acidification observed would be sufficient to generate NO from nitrite present in the airway epithelial lining fluid. In asthmatic subjects with mild stable disease, however, Ojoo et al. (2005) demonstrated that the FENO was elevated compared with healthy control subjects whereas the exhaled breath condensate pH and levels of nitrite and nitrate in exhaled breath condensate were unaltered. Consistent with this, Kostikas et al. (2002) described a reduction in exhaled breath condensate pH in moderate, but not mild, asthma. A low exhaled breath condensate pH, and by inference a contribution of airway acidification to FENO, appears therefore only to be relevant in the case of more severe asthma. In addition to increased generation by enzymatic or other means, FENO may be influenced by consumptive processes. NO is a highly reactive molecule and the NO detectable in exhaled air is likely to represent only a fraction of that generated. In the airways, NO can react with oxygen or reactive oxygen species to generate nitrite and nitrate or with superoxide anions to yield peroxynitrite. In addition, a substantial proportion of the NO generated reacts oxidatively with endogenous thiols, predominantly glutathione, to form stable NO adducts known as Snitrosothiols (SNOs) (Gaston et al., 1993). In vitro, these compounds are potent relaxants of human (Gaston et al., 1993, 1994) and animal (Bannenberg et al., 1995; Jansen et al., 1992) airway smooth muscle. Compared with healthy controls, concentrations of SNOs in the airways are substantially lower both in individuals with mild asthma (Dweik et al., 2001) and in ventilated children with asthmatic respiratory failure (Gaston et al., 1998). Depletion of endogenous SNOs may therefore contribute to bronchoconstriction in asthma. Accelerated breakdown of SNOs might also be implicated in the high levels of exhaled NO in the disease. 3.3. Chronic Obstructive Pulmonary Disease (COPD) In comparison with asthma, there have been fewer studies of FENO in COPD and their findings have been less consistent. Initial studies in fact appeared contradictory in their conclusions (Clini et al., 1998; Corradi et al., 1999; Maziak et al., 1998; Rutgers et al., 1999), but interpretation was limited by the nonstandardized methods used to measure NO, small sample sizes, differences in smoking status, and variations in disease severity. However, there is now general agreement that FENO is increased in stable ex-smokers with COPD, although the degree of elevation is modest compared with the very marked increases seen in some individuals with asthma. Partitioning of exhaled NO in COPD by measurement at different flow-rates has suggested that it originates from the alveolar compartment rather than the airways (Brindicci et al., 2005). In some studies,

correlations have been reported between FENO and various physiological measures of disease severity (Ansarin et al., 2001; Clini et al., 1998). Further rises in FENO have been described during acute exacerbations of COPD (Agustí et al., 1999) whereas treatment with an inhaled corticosteroid has been shown to reduce FENO in COPD (Ferreira et al., 2001). There have also been fewer studies of NOS isoform expression in COPD than in asthma. In a study of resected peripheral lung tissue from individuals with severe COPD, Maestrelli et al. (2003) reported that immunoreactive iNOS was expressed by alveolar macrophages, airway smooth muscle cells, and by cells in the alveolar walls identified as type 2 pneumocytes. The number of iNOS-positive type 2 pneumocytes was greater than in a control group of smokers without airflow obstruction. Alveolar macrophages have also been reported to express eNOS in COPD (van Straaten et al., 1998). 4. NO in airway physiology and pathophysiology 4.1. Airway tone A potentially important property of NO in relation to airway disease is its ability to relax airway smooth muscle. This effect is in part mediated by activation of soluble guanylyl cyclase and increased formation of cyclic guanosine 3′5′ monophosphate (cGMP), although cGMP-independent pathways are increasingly being recognized. In early animal studies, inhalation of 300 ppm NO produced a modest reduction in pulmonary resistance in anaesthetized guinea-pigs (Dupuy et al., 1992) but concentrations in the range 3–300 ppm had no effect on baseline airway mechanics in anaesthetized rabbits (Högman et al., 1993a, 1994). Attempts to demonstrate bronchodilator properties of exogenous NO in humans have also produced less than impressive results. Högman et al. (1993b) found that breathing 80 ppm NO for 10 min had no effect on specific airway conductance (sGAW) in healthy individuals or subjects with COPD. In asthmatic subjects, an increase in sGAW was observed but this effect was very modest in comparison with that of the β2-adrenoceptor agonist terbutaline. Pfeffer et al. (1996), in contrast, could detect no bronchodilator effect of inhaled 40 ppm NO in children with mild asthma. Roger et al. (1996) similarly found no effect of 40 ppm NO on respiratory resistance in either healthy subjects or subjects with COPD. A single dose of the NOS substrate L-arginine administered orally to healthy subjects (Kharitonov et al., 1995b) or to subjects with mild asthma (Kharitonov et al., 1995c) resulted in transient elevations of FENO. However, there were no associated changes in airway calibre as measured by the forced expiratory volume in one second (FEV1). Sapienza et al. (1998) reported similar increases in FENO when L-arginine was administered by inhalation to healthy and, to a greater extent, asthmatic subjects. In that study, an acute fall in FEV1 was observed, but this effect appeared nonspecific as a comparable change was also seen following inhalation of L-alanine. Conversely, administration of either the nonselective NOS inhibitors L-NAME and L-NMMA (Yates et al., 1995, 1996) or the selective iNOS inhibitor SC-51

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(Hansel et al., 2003) produced rapid and substantial falls in FENO in both normal and asthmatic subjects. Again, however, no accompanying changes in FEV1 were seen. Collectively, these findings make it unlikely that NO plays a significant role as an endogenous regulator of basal airway tone. 4.2. Airway responsiveness In contrast, a substantial body of evidence has accumulated to indicate an important role for NO in the regulation of airway responsiveness. Several studies have shown that exogenous NO can modulate airway responses to methacholine. In anaesthetized rabbits, inhalation of either 80 ppm NO (Högman et al., 1993a) or 300 ppm NO (Högman et al., 1994) protects against methacholineinduced increases in respiratory resistance. Similar effects have been demonstrated in human subjects. Inhalation of 80–100 ppm NO attenuates methacholine-induced increases in sGAW in healthy subjects (Sanna et al., 1994) and subjects with hyperreactive airways (Högman et al., 1993b) and also methacholine-induced falls in FEV1 in subjects with mild asth-ma (Kacmarek et al., 1996). In anaesthetized spontaneously breathing guinea-pigs, Nijkamp et al. (1993) showed that pretreatment with aerosolized L-NAME or L-NMMA increased airway responsiveness to histamine. A similar effect of NOS inhibition on histamine and methacholine responsiveness in vitro was observed with intact – but not epithelium-denuded – specimens of guinea-pig trachea. The ability of endogenous NO to modulate motor responses to another contractile agonist bradykinin was also demonstrated in guinea-pigs both in vivo (Ricciardolo et al., 1994) and using isolated tracheal preparations (Figini et al., 1996; Schlemper and Calixto, 1994). In other experiments, Folkerts et al. (1995) showed that airway hyperresponsiveness caused by intratracheal inoculation of parainfluenza virus in guinea-pigs, which is associated with epithelial damage, can be prevented by pretreatment with aerosolized L-arginine. Together, these studies suggest that epithelium-derived NO plays a protective role against bronchoconstriction evoked by a variety of stimuli in guinea-pigs. In elegant experiments using a microsensor to detect NO, Ricciardolo et al. (2000) directly demonstrated bradykinin-induced NO release in guinea-pig tracheal preparations and confirmed that the epithelium was the principal although not the only source. Studies in humans have confirmed a bronchoprotective role for endogenous NO in subjects with mild asthma. de Gouw et al. (1999) found that orally administered L-arginine reduced airway reactivity (i.e. the slope of the dose–response curve) to histamine, although this effect was fairly small. Ricciardolo et al. (1996) reported that pretreatment with inhaled L-NMMA in subjects with mild asthma potentiated airway narrowing induced by inhaled bradykinin, reducing the provocative dose producing a 20% fall in FEV1 (PD20) by 3.6 doubling dilutions. A comparable, although quantitatively smaller, effect of L-NMMA on methacholine-induced bronchoconstriction was also demonstrated. Similarly, Taylor et al. (1998a) reported increased airway responsiveness to histamine and adenosine-5′-

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monophosphate in patients with mild asthma after pretreatment with nebulized L-NAME, although in both cases the magnitude of the effect was only approximately one doubling dilution. These studies demonstrate that endogenous NO release protects against both directly and indirectly acting bronchoconstrictor stimuli in mild asthma. There has been debate about the mechanisms underlying bronchoprotection by endogenous NO and in particular the cellular source of NO and the NOS isoforms involved. Both the rapidity and the calcium dependence of the response in guineapig airways in vitro point to the involvement of one or both constitutive NOS isoforms rather than iNOS (Ricciardolo et al., 2000). One possible source would be nerves of the iNANC system, which express nNOS (Ward et al., 1995) and use NO as a neurotransmitter either alone (Belvisi et al., 1992a,b) or as a co-transmitter with vasoactive intestinal peptide (Li and Rand, 1991). Studies in nNOS-deficient mice have produced conflicting findings. Electrical field stimulation activates iNANC pathways in isolated airways and inhibits cholinergic contractile responses by a mechanism involving endogenous NO release (Ward et al., 1993). Kakuyama et al. (1999) showed that pretreatment with both L-NAME and indomethacin potentiated cholinergic contractions in airways from wild-type mice but not those from nNOS-deficient animals. Hasaneen et al. (2003) reported that methacholine-induced contractions were consistently greater in isolated tracheal strips from nNOSdeficient mice than those from wild-type controls, although the difference between groups was not statistically significantly. Surprisingly, however, De Sanctis et al. (1997) found that intact nNOS-deficient mice, which had lower FENO levels than their wild-type counterparts, in fact had slightly reduced airway responsiveness to methacholine. On the other hand, the various studies in guinea-pigs (Figini et al., 1996; Folkerts et al., 1995; Nijkamp et al., 1993; Ricciardolo et al., 1994, 2000; Schlemper and Calixto, 1994) strongly point to the airway epithelium as the major source of endogenous bronchoprotective NO. As discussed above, airway epithelial cells are capable of expressing all three NOS isoforms. Feletou et al. (2001) found that mice with a targeted disruption of the eNOS gene exhibited hyperresponsiveness to inhaled methacholine that was unaffected by pretreatment with L-NAME. Although NO plays a bronchoprotective role in milder forms of asthma, this appears not to be the case in more severe disease. Ricciardolo et al. (1997) studied 10 subjects with atopic asthma who, despite regular treatment with inhaled corticosteroids, had FEV1 measurements of only 65–75% predicted and methacholine provocative concentration producing a 20% fall in FEV1 (PC20) values b 1 mg/mL. The degree of hyperresponsiveness to bradykinin was greater than that seen in mild asthma but pretreatment with aerosolized L-NMMA did not potentiate bradykinin-induced airway narrowing. A loss of bronchoprotection by NO has also been reported after acute allergen challenge in asthma. Ricciardolo et al. (2001) performed inhalation challenge with house dust mite extract in 10 subjects with mild atopic asthma. Compared with diluent control, allergen exposure produced an increase in bradykinin responsiveness but the magnitude of the increase was not altered by

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pretreatment with L-NMMA. In bronchoscopic biopsy specimens obtained 48 h after exposure, there was increased epithelial expression of iNOS, reduced epithelial expression of eNOS, and no change in epithelial nNOS expression. Expression of NOS isoforms at sites other than the airway epithelium was not reported. Insights into possible mechanisms underlying the loss of bronchoprotection in severe asthma and after allergen exposure have been provided by a series of experiments in sensitized guinea-pigs in which ovalbumin challenge results in early and late phase bronchoconstrictor responses. In this model, both ex vivo (de Boer et al., 1996) and in vivo (Schuiling et al., 1998a) studies have shown that a deficiency of endogenous cNOSderived NO contributes to the development of airway hyperresponsiveness after the early reaction. Induction of iNOS does not occur until later time-points in this model (Schuiling et al., 1998b). Although a transient reduction in nNOS protein expression has been described (Samb et al., 2001), recent attention has centred on reduced substrate availability as an explanation for NO deficiency. The importance of substrate limitation in this model is demonstrated by the ability of exogenous L-arginine to prevent ovalbumin-induced ex vivo tracheal hyperresponsiveness (de Boer et al., 1999). Two separate pathways have been implicated in reduced substrate availability. First, heparin has the ability to reverse airway hyperresponsiveness induced both by the polycation polyL-arginine (Meurs et al., 1999) and by ovalbumin (Maarsingh et al., 2004). These findings point to the involvement of endogenous polycations, especially eosinophil-derived major basic protein, which have the ability to inhibit cellular uptake of L-arginine and thereby reduce its availability for NO synthesis (Hammermann et al., 1999). Secondly, experiments using the highly specific arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) have shown that ovalbumin-induced methacholine hyperresponsiveness is dependent on the induction of arginase activity, which presumably competes with constitutive NOS isoforms for the common substrate (Meurs et al., 2002). Overexpression of arginase has been confirmed by DNA microarray analysis of whole lung RNA in murine models of experimental asthma (Zimmermann et al., 2003). Recent studies in human disease have also identified dysregulated arginase expression. In particular, greater numbers of arginase-immunoreactive cells in bronchoalveolar lavage fluid and arginase mRNA+ cells in bronchial biopsy specimens (Zimmermann et al., 2003) and increased serum arginase activity (Morris et al., 2004) have been described in asthmatic individuals. Other studies using allergen challenge models have focussed on the possible role of iNOS induction in the development of airway hyperresponsiveness. In sensitized rats and mice (Eynott et al., 2002; Liu et al., 1997; Renzi et al., 1997; Trifilieff et al., 2000; Yeadon and Price, 1995), a single ovalbumin challenge leads to a rapid increase in iNOS mRNA and/or protein expression and an increase in airway responsiveness to methacholine. Pretreatment with N-(3-(aminomethyl)benzyl)acetamidine (1400 W), which is a potent and selective iNOS inhibitor (Garvey et al., 1997), or with SC-51, the prodrug of L-NIL, has been reported to prevent ovalbumin-induced hyperresponsive-

ness (Eynott et al., 2002; Koarai et al., 2000; Muijsers et al., 2001). However, studies in gene-deficient animals have shown that iNOS is not essential for the development of airway hyperresponsiveness. Both Xiong et al. (1999) and De Sanctis et al. (1999) reported no difference between iNOS-deficient animals and wild-type controls in ovalbumin-induced hyperresponsiveness to methacholine. De Sanctis et al. (1999) did, however, find that nNOS-deficient animals developed smaller increases in methacholine responsiveness after ovalbumin challenge then either iNOS null mice or wild-type controls. Consistent with this, Tulic et al. (2000) described the potentiation of ovalbumin-induced hyperresponsiveness in rats following pretreatment with S-methyl-L-thiocitrulline, a 100-fold selective nNOS inhibitor. Collectively, these studies suggest an important role for nNOS in limiting allergen-induced airway hyperresponsiveness in these models. Finally, dysregulated metabolism of SNOs in asthma may contribute to altered airway responsiveness. In individuals with mild allergic asthma, SNOs are undetectable in bronchoalveolar lavage fluid at baseline but increase to reach levels found in healthy control subjects 48 h after segmental allergen bronchoprovocation (Dweik et al., 2001). Further insights have recently been provided by a study of mice with a targeted deletion of the gene for the enzyme S-nitrosoglutathione reductase (GSNOR). Que et al. (2005) showed that GSNOR deficient mice had lower baseline methacholine responsiveness than wild-type controls. The increase in SNO levels in lung homogenates following ovalbumin challenge was substantially greater in GSNOR deficient animals. Moreover, there was little increase in methacholine responsiveness in the GSNOR null mice compared with a marked increase in wild-type mice. Levels of nitrite increased to a similar degree in both groups, indicating that dysregulated SNO homeostasis was independent of NOS activity. These findings suggest that by maintaining airway patency endogenous SNOs may be important regulators of airway responsiveness during allergen exposure. 4.3. Airway inflammation 4.3.1. Proinflammatory properties of NO At high concentration, NO has a number of proinflammatory effects, including increased vascular permeability, cytotoxicity, and inflammatory cell infiltration. A role for these properties in host defence is suggested by the increased susceptibility of iNOS-deficient mice to infection with certain viruses (Karupiah et al., 1998; MacLean et al., 1998), bacteria (MacMicking et al., 1995, 1997), and protozoan parasites (Diefenbach et al., 1998; Wei et al., 1995). In the context of airway inflammatory diseases such as asthma and COPD, however, such effects may have a deleterious impact on airway function and may contribute to disease progression. Under physiological conditions, endogenous NO release appears in fact to tonically suppress microvascular permeability in the airway mucosa. Erjefält et al. (1994) showed that topical application of L-NAME to guinea-pig tracheal mucosa in vivo resulted in a rapid plasma exudative response. Increased plasma leakage has also been demonstrated in rat trachea following

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intravenous administration of L-NAME (Bernareggi et al., 1997). In contrast, excess NO production in asthma and COPD may increase vascular permeability leading to plasma exudation and mucosal oedema, features that likely contribute to airflow obstruction and airway hyperresponsiveness (Brown et al., 1995). NO is a potent vasodilator in both the pulmonary (Frostell et al., 1993) and bronchial (Alving et al., 1992) circulations. Airway microvascular leakage induced by intravenous lipopolysaccharide (LPS) in anaesthetized rats is inhibited by pretreatment with intravenous L-NAME (Bernareggi et al., 1997). Similarly, L-NAME is able to block plasma exudation into guinea-pig airways induced by electrical stimulation of the vagus nerves (Kuo et al., 1992), by intravenous injection of substance P or leukotriene D4 (Kageyama et al., 1997), and by antigen exposure in sensitized animals (Miura et al., 1996). The specific NOS isoform(s) involved in these responses have not been characterized in detail. However, a recent study in eNOS-deficient mice has indicated a critical role for eNOS activation in vascular leakage during acute inflammation (Bucci et al., 2005). At high concentrations, NO exhibits cytotoxic effects on a number of cell types, including respiratory epithelial cells (Heiss et al., 1994). The cytotoxic properties of NO have been attributed to the formation of peroxynitrite, a highly reactive intermediate generated by the rapid reaction of NO and superoxide anions. Peroxynitrite causes epithelial desquamation when applied directly to isolated guinea-pig trachea and can induce airway hyperresponsiveness in guinea-pigs both in vitro and in vivo (Sadeghi-Hashjin et al., 1996). At one time, the detection of 3-nitrotyrosine was regarded as a specific marker of protein oxidation by peroxynitrite. Several reports of increased 3-nitrotyrosine immunostaining in bronchial tissue from asthmatics were accordingly interpreted as evidence of peroxynitrite-induced damage (Guo et al., 2000; Kaminsky et al., 1999; Saleh et al., 1998). More recently, however, other mechanisms of protein tyrosine nitration have been identified that are likely to be more relevant. In particular, peroxidases such as myeloperoxidase (Eiserich et al., 1998; van der Vliet et al., 1997) and eosinophil peroxidase (Wu et al., 1999) can efficiently nitrate protein tyrosyl residues through mechanisms independent of peroxynitrite. Studies comparing the extent of 3-nitrotyrosine immunostaining after ovalbumin challenge in eosinophil peroxidase-deficient and iNOS-deficient mice have clearly shown that eosinophil peroxidase is in fact the dominant source of nitrotyrosine formation in this model of allergic inflammation (Brennan et al., 2002; Duguet et al., 2001). Additionally, NO has a number of actions on inflammatory cells that may directly promote allergic inflammation. Nonselective NOS inhibitors suppress in vitro chemotaxis of human peripheral blood eosinophils (Thomazzi et al., 2001), monocytes (Belenky et al., 1993a) and neutrophils (Belenky et al., 1993b; Kaplan et al., 1989). NO donors can prolong the survival of cytokine-deprived human eosinophils by inhibiting apoptosis (Beauvais et al., 1995). Earlier studies suggested that NO could also inhibit the differentiation and proliferation of cloned murine Th1 cells and their ability to secrete IL-2 and IFN-γ but in contrast had no effect on Th2 cell differentiation or IL-4

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production (Taylor-Robinson et al., 1994). It was therefore speculated that an excess of NO might promote a Th2 pattern of inflammation by altering the Th1/Th2 balance in the airways (Barnes and Liew, 1995). More recent work, however, has suggested that relatively low concentrations of NO inhibit proliferation of both Th1 and Th2 cells and have similar effects on cytokine production by the two subsets (van der Veen et al., 1999). 4.3.2. Animal studies using NOS inhibitors A number of investigators have examined the possible antiinflammatory effects of NOS inhibition using the model of pulmonary inflammation induced by challenge with ovalbumin in sensitized rodents. In mice, Feder et al. (1997) reported that intraperitoneal administration of L-NAME, L-NMMA, or aminoguanidine before challenge produced dose-dependent reductions in the numbers of eosinophils in bronchoalveolar lavage fluid and lung tissue obtained 24 h after ovalbumin exposure whereas the more selective iNOS inhibitor L-NIL was without effect. In that study, there was no demonstrable induction of iNOS mRNA or protein following ovalbumin challenge. The authors concluded that NO was involved in the development of lung eosinophilia in this model but that it was not generated by iNOS. Ferreira et al. (1998) also found that L-NAME markedly reduced the influx of eosinophils (but not neutrophils) into bronchoalveolar lavage fluid and tissue after intratracheal ovalbumin injection in sensitized rats but the effects of selective NOS inhibitors were not studied. Tulic et al. (2000) investigated the effects of NOS inhibitors on ovalbumin-induced inflammation in Piebald–Virol–Glaxo rats. Pretreatment with either L-NMMA or aminoguanidine was reported to reduce total cell numbers and numbers of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid. In contrast, pretreatment with the selective nNOS inhibitor S-methyl-L-thiocitrulline was without effect. Using a protocol shown to produce a marked increase in iNOS expression, Trifilieff et al. (2000) studied the effects of L-NAME and two other agents, S-ethylisothiourea (EIT) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), regarded as selective for iNOS. All three compounds were reported to suppress infiltration of both eosinophils and neutrophils into bronchoalveolar lavage fluid. In addition, AMT pretreatment reduced expression of mRNA for the chemokines, macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. In both these studies, the authors concluded that inflammatory cell influx after allergen challenge was partially dependent on NO produced mainly by iNOS. However, interpretation is critically dependent on the relative selectivity of the NOS inhibitors used. The selectivity of aminoguanidine for iNOS over other isoforms is modest (Misko et al., 1993) and doubts have also been expressed about the true selectivity of EIT and AMT for iNOS (Birrell et al., 2003). Eynott et al. (2002) used a rat model in which ovalbumin challenge in sensitized animals was shown to increase exhaled NO and expression of immunoreactive iNOS in the lungs. Pretreatment with SC-51, the prodrug of L-NIL, reduced FENO and

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reduced the ovalbumin-induced increase in bronchoalveolar lavage fluid neutrophils but did not significantly alter the influx of eosinophils. Several other investigators have examined the effect of the compound 1400 W which exhibits greater potency and selectivity as an iNOS inhibitor than either aminoguanidine or L-NIL (Garvey et al., 1997). In sensitized mice, Koarai et al. (2000) reported that continuous infusion of 1400 W by an osmotic minipump before and during ovalbumin challenge inhibited the resulting influx of eosinophils by approximately 30%. Birrell et al. (2003), however, were unable to demonstrate an inhibitory effect of 1400 W on the increase in lung tissue eosinophils induced either by intratracheal administration of Sephadex beads or by aerosol challenge with ovalbumin in rats. In contrast, L-NAME produced dose-dependent reductions in eosinophil numbers and also in tissue levels of TNF-α, eotaxin and IL-13. Similarly, Muijsers et al. (2001) were also unable to show an effect of 1400 W on the bronchoalveolar lavage fluid eosinophilia resulting from repeated ovalbumin challenge in sensitized mice. The apparent discrepancies between these various studies may relate to many factors. These include differences between species and strains of animal, the different sensitization and challenge protocols, the possibility of contamination of commercial ovalbumin preparations with bacterial LPS, and the relative selectivity of the different NOS inhibitors used. Taken together, however, they appear most consistent with the view that it is constitutive NOS isoforms rather than iNOS that are important in the development of eosinophilic inflammation. 4.3.3. Studies in transgenic and gene-deficient animals To examine the potential proinflammatory effects of iNOS overexpression, Hjoberg et al. (2004) constructed an externally regulatable transgenic mouse containing a reverse tetracycline transactivator under control of the CC10 promoter and murine iNOS cDNA under control of a tetracycline operator. In this system, addition of doxycycline to the drinking water of the animals leads to a sustained induction of iNOS in epithelial cells of large and small airways and a small elevation of mean FENO from 10 to 18 ppb. Compared with either wild-type mice or transgenic mice not receiving doxycycline, administration of doxycycline for up to 3 weeks led to no discernable proinflammatory effects as assessed by lung histology (haematoxylin and eosin, periodic acid Schiff, and trichrome stains), bronchoalveolar lavage total and differential cell counts, and bronchoalveolar lavage fluid protein. This study provides clear evidence that overexpression of iNOS has no proinflammatory effect per se in murine airways. Other investigators have studied ovalbumin-induced airway inflammation in mice with targeted deletions of various NOS genes. As with the studies of NOS inhibitors discussed above, there have been conflicting findings. Xiong et al. (1999) used a protocol for sensitization and challenge that produced a severe tissue inflammatory response in wild-type animals. Features included very pronounced (N 90%) bronchoalveolar lavage fluid eosinophilia, gross alterations in the structural integrity of the airway wall, microvascular leakage and mucosal oedema, and

mucous occlusion of the airway lumen. In iNOS-deficient mice, the number of bronchoalveolar lavage fluid eosinophils was reduced by 55–60% and the histological grading of pulmonary inflammation was less severe. Increased IFN-γ production was apparently responsible for the suppression of eosinophilia in iNOS-deficient mice. De Sanctis et al. (1999) examined mice with deletions of the genes for iNOS, eNOS, nNOS, and both eNOS and nNOS. Similar increases in total pulmonary NOS activity were seen after ovalbumin sensitization and challenge in wild-type mice and in mice deficient in eNOS, nNOS or both, whereas there was no increase in total NOS activity in iNOSdeficient animals. However, no differences between groups were evident in inflammatory markers in bronchoalveolar lavage fluid or lung tissue following challenge of sensitized animals with either aerosolized ovalbumin or PBS control. The discrepancies between these two reports may in part relate to the different sensitization and challenge protocols, as a far greater degree of eosinophilia was achieved in the former study. Keller et al. (2005) have also drawn attention to the possibility of contamination of ovalbumin with LPS as a factor in the findings of Xiong et al. In a more recent study, Iijima et al. (2005) studied ovalbumin-induced lung inflammation in nNOS-deficient mice. In wild-type animals, ovalbumin challenge produced not only an increase in iNOS mRNA, as expected, but also an increase in nNOS mRNA and in cNOS activity. Following ovalbumin chal-lenge, the number of eosinophils, but not neutrophils, in bronchoalveolar lavage fluid was reduced by about half in nNOS-deficient mice compared with their wildtype controls. Furthermore, there was a failure to upregulate iNOS in response to ovalbumin in these nNOS-deficient animals. These findings appear to point to some form of interaction occurring between nNOS and iNOS in this model, although the molecular mechanism of this interaction is unknown. 5. Therapeutic potential for NOS inhibitors and NO donors in airways disease There has been speculation for several years that measurement of FENO might be helpful in the diagnosis and monitoring of airway inflammation. The potential value of this approach in clinical practice is indeed now becoming clearer. In a recent study, for example, Smith et al. (2005) showed that regular monitoring of FENO in chronic asthma allowed maintenance of clinical control despite a substantial reduction in the dose of inhaled corticosteroid. Compared with measurements of eosinophil numbers in induced sputum or airway responsiveness to methacholine, it is the relative ease with which gaseous NO can be noninvasively measured in exhaled breath that make it attractive as a marker of underlying airway inflammation. However, the evidence that upregulation of iNOS is a critical component in the complex inflammatory cascade and that inhibition of iNOS would therefore be a worthwhile therapeutic strategy in airway inflammatory disease remains limited. The study of Hjoberg et al. (2004) in fact provides strong evidence that iNOS overexpression in isolation does not result in airway inflammation, although

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proinflammatory interactions between NO and other components (e.g. reactive oxygen species) in vivo remain possible. The contradictory results of the various animal studies, both those using pharmacological approaches and those in gene-deficient mice, are confusing and cannot easily be reconciled. In any case, the relevance of these models to human disease is uncertain. There are important species differences in the expression and regulation of iNOS, presumably relating to the significant sequence variations that have been described between human, murine, and rat iNOS gene promoters (Chu et al., 1998; Wen et al., 2000). Furthermore, the deficiency of endogenous bronchodilator SNOs in asthma might be worsened by inhibition of iNOS. In vivo studies to assess possible effects of iNOS inhibition on airway inflammation in humans have not yet been reported. However, Taylor et al. (1998b) showed that pretreatment with nebulized L-NAME had no influence on the magnitude of the late-phase bronchoconstrictor response following allergen inhalation challenge in subjects with allergic asthma despite a substantial (N70%) reduction in FENO at that time. Although no markers of inflammation were reported, the failure of NOS inhibition to modulate the late-phase response in this study is not encouraging. The late-phase response is regarded as closely associated with airway inflammatory events and in many instances this model accurately predicts the likely clinical benefit of anti-asthma agents. Although iNOS has been the principal NOS isoform considered as a therapeutic target, increasing importance is becoming attached to the role of the “constitutive” NOS isoforms in airways disease. The observations that nNOS appears to be involved in the development of allergen-induced hyperresponsiveness and that eNOS may regulate microvascular permeability in inflammation are interesting and warrant further exploration. Indeed, associations between polymorphisms of both the eNOS (Lee et al., 2000; Yanamandra et al., 2005) and nNOS (Grasemann et al., 1999) genes and the asthma phenotype have recently been described. On the other hand, endogenous NO derived from nNOS inhibits proliferation of cultured human airway smooth muscle cells, acting via both cGMP-dependent and cGMP-independent mechanisms (Hamad and Knox, 2001; Patel et al., 1999). Chronic blockade of this activity might therefore increase airway dysfunction by promoting hypertrophy and hyperplasia of airway smooth muscle (Redington and Howarth, 1997). Interpretation of many studies of NOS inhibition is limited by the relative lack of selectivity of the agents used. However, progress is being made in the development of novel inhibitors that are both potent and highly selective for individual NOS isoforms (Salerno et al., 2002). Studies combining these novel agents with direct measurement of inflammatory markers, for example in induced sputum, should in time provide more definitive information regarding the significance of iNOS/NO dysregulation in asthma and COPD, the potential of iNOS and other NOS isoforms as targets for therapeutic intervention, and any toxicity associated with this strategy. The body of evidence supporting a bronchoprotective role for endogenous NO and the apparent loss of this broncho-

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protection in asthma has also stimulated interest in the possible therapeutic benefits of NO. Despite the practical obstacles, a study in ambulatory patients with severe COPD has shown that long-term delivery of gaseous NO is achievable (Vonbank et al., 2002). No clinical outcomes were reported but this study did demonstrate that supplemental inhaled NO could be administered safely and effectively over a period of 3 months. An alternative strategy to increase the bioavailability of NO is the use of NO donors. Despite their drawbacks, organic nitrates and sodium nitroprusside have been used in cardiovascular therapeutics for many years. A recent novel approach has centred on the addition of NOdonor moieties to existing pharmacological agents. Thus NOreleasing derivatives of both prednisolone (Paul-Clark et al., 2000) and budesonide (Nevin and Broadley, 2004) and an NO-releasing salbutamol (Lagente et al., 2004) have been described. Further studies of these agents in models of airway inflammation and perhaps in clinical disease are awaited with interest. Finally, the recent studies implicating arginase overexpression as an important factor in loss of bronchoprotective NO point to arginase pathways as a potential novel therapeutic target. References Agustí, A.G.N., Villaverde, J.M., Togores, B., Bosch, M., 1999. Serial measurements of exhaled nitric oxide during exacerbations of chronic obstructive pulmonary disease. Eur. Respir. J. 14, 523–528. Al-Ali, M.K., Eames, C., Howarth, P.H., 1998. Exhaled nitric oxide; relationship to clinicophysiological markers of asthma severity. Respir. Med. 92, 908–913. Alving, K., Fornhem, C., Weitzberg, E., Lundberg, J.M., 1992. Nitric oxide mediates cigarette smoke-induced vasodilatory responses in the lung. Acta Physiol. Scand. 146, 407–408. Alving, K., Weitzberg, E., Lundberg, J.M., 1993. Increased amount of nitric oxide in exhaled air of asthmatics. Eur. Respir. J. 6, 1368–1370. American Thoracic Society, 2005. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am. J. Respir. Crit. Care Med. 171, 912–930. Ansarin, K., Chatkin, J.M., Ferreira, I.M., Gutierrez, C.A., Zamel, N., Chapman, K.R., 2001. Exhaled nitric oxide in chronic obstructive pulmonary disease: relationship to pulmonary function. Eur. Respir. J. 17, 934–938. Asano, K., Chee, C.B.E., Gaston, B., Lilly, C.M., Gerard, C., Drazen, J.M., Stamler, J.S., 1994. Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 91, 10089–10093. Bannenberg, G., Xue, J., Engman, L., Cotgreave, I., Moldéus, P., Ryrfeldt, Å., 1995. Characterization of bronchodilator effects and fate of S-nitrosothiols in the isolated perfused and ventilated guinea pig lung. J. Pharmacol. Exp. Ther. 272, 1238–1245. Baraldi, F., Azzolin, N.M., Zanconato, S., Dario, C., Zacchello, F., 1997. Corticosteroids decrease exhaled nitric oxide in children with acute asthma. J. Pediatr. 131, 381–385. Barnes, P.J., Liew, F.Y., 1995. Nitric oxide and asthmatic inflammation. Immunol. Today 16, 128–130. Beauvais, F., Michel, L., Dubertret, L., 1995. The nitric oxide donors, azide and hydroxylamine, inhibit the programmed cell death of cytokine-deprived human eosinophils. FEBS Lett. 361, 229–232. Belenky, S.N., Robbins, R.A., Rubinstein, I., 1993a. Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro. J. Leuk. Biol. 53, 498–503.

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