Nitric oxide in respiratory diseases

Pharmacology & Therapeutics 95 (2002) 259 – 293

Nitric oxide in respiratory diseases B.J. Nevin, K.J. Broadley* Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cathays Park, Cardiff CF10 3XF, UK

Abstract The formation and modulation of nitric oxide (NO) in the lungs is reviewed. Its beneficial and deleterious roles in airways diseases, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis, and in animal models is discussed. The pharmacological effects of agents that modulate NO production or act as NO donors are described. The clinical pharmacology of these agents is described and the therapeutic potential for their use in airways disease is considered. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Nitric oxide; Asthma; COPD; Cystic fibrosis; NO donors; NOS inhibitors Abbreviations: AHPP, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine; AHR, airway hyperreactivity/airway hyperresponsiveness; ARDS, adult respiratory distress syndrome; BAL, bronchoalveolar lavage; BSA, bovine serum albumin; cAMP, cyclic AMP; CFTR, cystic fibrosis transmembrane conductance regulator; cGMP, cyclic GMP; cNOS, constitutive nitric oxide synthase; COPD, chronic obstructive pulmonary disease; COX, cyclo-oxygenase; EAR, early asthmatic reaction; EDRF, endothelium-derived relaxation factor; eNOS, endothelial nitric oxide synthase; EpiDRF, epithelium-derived relaxation factor; FEV1, forced expiratory volume in 1 sec; fMLP, N-formylmethionine leucyl-phenylalanine; GSNO, S-nitrosoglutathione; IFN, interferon; IL, interleukin; iNANC, inhibitory nonadrenergic, noncholinergic; iNOS, inducible nitric oxide synthase; LAR, late asthmatic reaction; L-NAME, N-nitro-L-arginine methyl ester; NANC, nonadrenergic, noncholinergic; L-NIL, L-N-(1-iminoethyl)lysine; L-NMMA, L-N-monomethyl-L-arginine; L-NOARG, L-nitro-N-arginine; LPS, lipopolysaccharide; LT, leukotriene; MBP, major basic protein; NAP, N-acetyl-penicillamine; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; O
2 , superoxide anion; OA, ovalbumin; ODQ, 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one; PAF, platelet-activating factor; PD20BK, provocative concentration of bradykinin producing a 20% fall from baseline; sGaw, specific airways conductance; SIN-1, 3-morpholinosydnonimine-Nethylcarbamide; SNAP, S-nitroso-N-acetylpenicillamine; SNO, S-nitrosothiol; SNO, sodium nitroprusside; SOD, superoxide dismutase; TNF, tumour necrosis factor; VIP, vasoactive intestinal neuropeptide; 1400W, N-(3-(aminomethyl)benzyl)acetamidine.

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Endothelium-derived relaxation factor and nitric oxide . . . . . . . . . . . . . . . . 1.2. Nitric oxide and bronchodilatation. . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Nitric oxide synthesis and investigation . . . . . . . . . . . . . . . . . . . . . . . . Neuronal nitric oxide and inhibitory nonadrenergic-noncholinergic nerve activity . . . . . . Role of nitric oxide in regulating airway smooth muscle tone . . . . . . . . . . . . . . . . 3.1. Nitric oxide and basal airway tone . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Role of nitric oxide in regulating the early asthmatic reaction . . . . . . . . . . . . 3.3. Modulatory role of nitric oxide on airway hyperreactivity . . . . . . . . . . . . . . Nitric oxide and inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Role of nitric oxide in promoting eosinophilia and microvascular hyperpermeability . 4.2. Anti-inflammatory effects of nitric oxide . . . . . . . . . . . . . . . . . . . . . . . 4.3. Role of nitric oxide in regulating the late asthmatic reaction . . . . . . . . . . . . .

* Corresponding author. Tel.: +44-29-2087-5832; fax: +44-29-2087-4149. E-mail address: [email protected] (K.J. Broadley). 0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 2 ) 0 0 2 6 2 - 0

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5.

S-nitrosothiols . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Endothelium-derived relaxation factor and S-nitrosothiols. 5.2. S-nitrosothiols and pulmonary nitric oxide . . . . . . . . 5.3. Ability of S-nitrosothiols to generate bioactive nitric oxide 6. Molecular targets for nitric oxide in smooth muscle . . . . . . . 7. Nitric oxide and histamine release . . . . . . . . . . . . . . . . 8. Other beneficial effects of nitric oxide in the lungs. . . . . . . . 9. Clinical pharmacology of nitric oxide. . . . . . . . . . . . . . . 9.1. Exhaled nitric oxide/S-nitrosothiol . . . . . . . . . . . . . 9.2. Administration of L-arginine . . . . . . . . . . . . . . . . 9.3. Administration of inhaled nitric oxide . . . . . . . . . . . 9.4. Administration of nitric oxide donors . . . . . . . . . . . 9.4.1. Nitroglycerine. . . . . . . . . . . . . . . . . . . 9.4.2. Isosorbide dinitrate . . . . . . . . . . . . . . . . 9.4.3. S-nitrosothiols. . . . . . . . . . . . . . . . . . . 9.4.4. Other nitric oxide donors . . . . . . . . . . . . . 9.5. b2-adrenoceptor agonists and nitric oxide donors . . . . . 9.6. Glucocorticosteroids and nitric oxide . . . . . . . . . . . 10. Clinical applications . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Endothelium-derived relaxation factor and nitric oxide Furchgott and Zawadzki (1980) found that acetylcholine, acting on muscarinic receptors in rabbit thoracic aorta, stimulated the release of a substance(s) that caused relaxation of the vascular smooth muscle. Acetylcholine required the presence of endothelial cells in order for this relaxation to be induced, which suggested the existence of an endothelium-derived relaxation factor (EDRF). The vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of nitric oxide (NO) delivered by superfusion over endothelium-denuded artery and venous strips arranged in cascade (Ignarro et al., 1987). EDRF released from arteries and veins was shown to be biologically indistinguishable from NO with similar half-lives, susceptibility to inactivation by pyrogallol and superoxide anion (O2
), stabilisation by superoxide dismutase (SOD), inhibition by oxyhaemoglobin and K+, and a comparable ability to increase cyclic GMP (cGMP). EDRF was also shown to be chemically identical to NO. Palmer et al. (1987) showed similar results in cultured endothelial cells, as did Hutchinson et al. (1987) in rabbit aorta.

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at high doses delayed the occurrence of bronchial asthma symptoms (Kreye & Marquard, 1979). Intravenous nitroglycerine (glyceryl trinitrate) was shown to relax human tracheal smooth muscle in vivo (Byrick et al., 1983). Nitroglycerine and SNP were found to relax bovine isolated tracheal smooth muscle (Gruetter et al., 1989). Masaki et al. (1989) showed that by bubbling NO gas into water, NO was a potent relaxant of isolated canine tracheal and bronchial smooth muscle and pulmonary artery (Table 1). This supports its relationship with EDRF (Masaki et al.,

Table 1 Pharmacological effects of NO in the airways Target

Effect

Airway smooth muscle

Relaxation: "# airway hyperreactivity depending on degree of activation of iNOS Pro-inflammatory: " eosinophil infiltration " vascular permeability " airway narrowing # apoptosis of eosinophils Anti-inflammatory: # leukocyte chemotaxis, function # endothelial cell relaxation # mast cell activation " apoptosis of eosinophils Host defence: Antiviral, antibacterial, anti-parasitic Pulmonary/bronchial vasodilatation: " clearance of bronchoconstrictors " exudation/oedema and inflammation Regulation of muco-cilliary clearance: "# mucin secretion? " ciliary beat frequency # mucus viscosity

Inflammation/ immunity

1.2. Nitric oxide and bronchodilatation Aviado et al. (1969) showed that inhalation of pentaerythrityl tetranitrate decreased pulmonary resistance in the anaesthetised dog. This suggested a bronchodilator activity for nitrate. Since then, many studies have demonstrated that other nitrate donors show similar activity. In histaminechallenged guinea-pigs, sodium nitroprusside (SNP) aerosol

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Vascular smooth muscle

Epithelial cells

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1989). Superperfusion of NO in the form of S-nitroso-Nacetylpenicillamine (SNAP) was also shown to relax bovine isolated tracheal smooth muscle (Buga et al., 1989). Unlike budesonide, the novel NO-budesonide complex (NCX-1020) reduced methacholine-induced contraction of guinea-pig bronchioles in vitro (Tallet et al., 2001). An EDRF-like substance acting via NO formation was shown to be generated in bovine tracheal smooth muscle (Sheng et al., 1991). From these results, it may be suggested that exogenous NO has bronchodilating properties. Structures of a range of NO donors and related agents are shown in Fig. 1.

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1.3. Nitric oxide synthesis and investigation The terminal guanidine nitrogen atom(s) of L-arginine are the physiological precursors of endothelium-derived NO (Sakuma et al., 1988; Palmer et al., 1988). Endothelial cells contain a cytosolic enzyme, NO synthase (NOS), which appears to be dependent on the presence of Ca2+ (Mayer et al., 1989). Calmodulin is required for Ca2+ to exert this modulatory effect (Busse & Mulsch, 1990). The enzyme converts L-arginine into an EDRF-like compound (Sakuma et al., 1988) in an NADPH-dependent manner (Mayer et al., 1989). This enzyme is involved in the formation of NO and

Fig. 1. Structures of NO donors, including S-nitrosothiols. GEA5145, 1,2,3,4-oxatriazolium,3-(3-chloro-2-methyoxyphenyl)-5-[[(4-phenyl)sulfonyl]amino]hydroxide inner salt; GEA3175, 1,2,3,4-oxatriazolium,3-(3-chloro-2-methyoxyphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-hydroxide inner salt; GEA3268, 1,2,3,4-oxatriazolium,3-(3-chloro-2-methyoxyphenyl)-5-[[(4-methoxyphenyl)ysulfonyl]amino]-hydroxide inner salt.

262 L-citrulline

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from L-arginine (Palmer et al., 1988; Robbins et al., 1993). L-Arginine, O2, and NADPH are co-substrates; flavin adenine dinucleotide, flavin adenine dinucleotide, heme, and tetrahydrobiopterin and calmodulin are cofactors or prosthetic groups of this enzyme (Nathan, 1992; McCall & Vallence, 1992; Nathan & Xie, 1994) (Fig. 2). Isoforms of NOS have been reviewed by Forstermann et al. (1991). The endothelial NOS (eNOS), also known as

NOS-3, is Ca2+/calmodulin-dependent and is a constitutive enzyme (cNOS). cNOS and/or its isoforms may also be found in neuronal NOS (nNOS), also known as NOS-1 (Bredt et al., 1990) and epithelial cells (Asano et al., 1994). The cNOS enzyme is short lived and produces only picomolar quantities of NO. An inducible NOS (iNOS), also known as NOS-2, is found in neutrophils (McCall et al., 1991), eosinophils (Zanardo et al., 1997; Iijima et al., 2001),

Fig. 2. Schematic representation of the NO synthetic pathway and drug interventions. Drug interventions are shown in italics and broken lines. FAD, flavin adenine dinuclotide; FMN, flavin adenine dinucleotide.

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macrophages (Jorens et al., 1991), and epithelial cells (Asano et al., 1994; Hamid et al., 1993; Redington et al., 2001). Unlike cNOS, it produces nanomolar quantities of NO, is long lived, and is Ca2+/calmodulin independent. It is induced by cytokines [interleukin (IL)-1b, tumour necrosis factor (TNF)-a, interferon (IFN)-g] and endotoxin, and its induction is inhibited by glucocorticosteroids (Hamid et al., 1993; Robbins et al., 1994a, 1994b; Hickey et al., 1997; Radomski et al., 1990; Moncada & Higgs, 1991, 1993; Moncada et al., 1991; Morris & Billiar, 1994; Guo et al., 2000). The level of NO in exhaled air is used as a measure of iNOS-derived NO in the airways (see Section 9.1). L -N-monomethyl- L -arginine ( L -NMMA) is a specific inhibitor of NO formation from L-arginine (Rees et al., 1989). It is a potent inhibitor of both cNOS and iNOS (Hasan et al., 1993). L-Nitro-N-arginine (L-NOARG) is also a potent inhibitor of NOS (Rees et al., 1989). N-nitro-Larginine methyl ester (L-NAME) has been used as a nonselective NOS inhibitor, inhibiting eNOS, nNOS, and iNOS (Rees et al., 1990; Belvisi et al., 1991, 1992a, 1992b, 1993; Laszlo et al., 1995; Bernareggi et al., 1997; Iijima et al., 1998; Taylor et al., 1998a, 1998b; De Boer et al., 2001). Tulie et al. (2000) has used it, however, as a selective eNOS inhibitor. Aminoguanidine is equipotent with L-NMMA as an iNOS inhibitor, but 10- to 100-fold less potent as a cNOS inhibitor (Misko et al., 1993). L-N-(1-iminoethyl)lysine (LNIL) has also been shown to be a selective (28-fold difference) iNOS inhibitor (Moore et al., 1994). N-(3-(Aminomethyl)benzyl)acetamidine (1400W) has been shown to be

Table 2 Drug intervention strategies and effects on NO Drug

Effect

NO donors Inhaled NO S-Nitrosothiols

Compensates for deficiency of NO and adds to existing levels of NO for beneficial effects Compensates for deficiency of NO carrier (S-nitrosothiol)

Thiols

Compensates for deficiency of thiols and, therefore, increases S-nitrosothiol levels

L-Arginine

Compensates for deficiency of substrate and, therefore, increases NO (and/or S-nitrosothiol) levels

iNOS inhibitors

Reduces the deleterious effects of iNOS-derived NO

Glucocorticosteroids

Reduces the deleterious effects of iNOS-derived NO

cNOS activators Calmodulin agonists

Increases the beneficial effects of cNOS-derived NO

Peroxynitrite scavengers/xanthine oxidase inhibitors/SOD

Reduces the deleterious effects of peroxynitrite derived from iNOS

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at least 5000-fold more selective for human iNOS than for human cNOS (Garvey et al., 1997). These inhibitors and the precursor L-arginine are stereoselective in their action. They are useful tools in the investigation of the pharmacology of NO. By understanding the NO synthetic pathway and the use of experimental agents that interact with it (Fig. 2, Table 2) (Nathan, 1992; McCall & Vallence, 1992), it is possible to investigate the role and therapeutic potential of NO in the airways.

2. Neuronal nitric oxide and inhibitory nonadrenergicnoncholinergic nerve activity Tetrodotoxin was found to significantly inhibit nonadrenergic, noncholinergic (NANC) relaxation in guinea-pig and human tracheal preparations (Tucker et al., 1990; Belvisi et al., 1992a, 1992b, 1993). These results confirm the existence of a neurogenic component of tracheal relaxation in addition to the classical adrenergic and cholinergic pathways. The NOS inhibitors L-NOARG and L-NAME were found to reduce NANC relaxation in guinea-pig tracheae induced by electrical field stimulation (Tucker et al., 1990; Belvisi et al., 1991, 1993). Similarly, in human tracheal and bronchial smooth muscle, L-NAME was found to reduce NANC relaxation (Belvisi et al., 1992a, 1992b; Ellis, 1992; Bai & Bramley, 1993). Both central and peripheral airways receive this NANC innervation (Ellis, 1992), although this innervation is less evident in the smaller bronchioli (Ward et al., 1992; Fischer et al., 1993). Following nerve stimulation, NO is released. It then modulates cholinergic neurotransmission by a functional antagonism through its bronchodilator action on airway smooth muscle (Belvisi et al., 1991, 1993, 1995b; Ellis, 1992; Brave et al., 1991; Ward et al., 1992). In rat (Sekizawa et al., 1993), but not guinea-pig (Brave et al., 1991) or human airway smooth muscle (Ward et al., 1992), endogenous NO may modulate cholinergic contraction by inhibiting acetylcholine release. In mice, the NO-mediated inhibitory NANC (iNANC) response may be reduced by the release of cyclo-oxygenase (COX) products (Kakuyama et al., 1999). NOS appears to be associated with neurones in the rat lungs (Bredt et al., 1990). NO released following nerve stimulation is formed in neurones, glia, fibroblasts, and muscle or blood cells, all of which occur in close proximity to the neurones (Bredt et al., 1990). These results support the release of NO following nerve stimulation and its role in modulating airway smooth muscle tone. In the guinea-pig colon, acetylcholine, which contracts colon smooth muscle by muscarinic receptor activation, was shown to release NO via muscarinic M1 receptor activation (Iverson et al., 1997). This release of NO represents a negative feedback mechanism on acetylcholine-induced contraction of smooth muscle. An analogous mechanism, however, has not been demonstrated in the lungs.

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In guinea-pig tracheal preparations, it has been suggested that vasoactive intestinal neuropeptide (VIP) is a NANC neurotransmitter (Tucker et al., 1990; Belvisi et al., 1993; Ellis & Farmer, 1989a, 1989b). VIP appears to modulate the production/release of NO. In the rat gastric fundus, VIP released from nerve terminals seems to stimulate the generation of NO (Li & Rand, 1990). In patients with asthma, there appears to be a loss of VIP from pulmonary nerve fibres (Ollerenshaw et al., 1989). Thus, a loss of VIP may impair the modulatory role of NO. However, in human tracheal and bronchial preparations, VIP does not appear to have such a modulatory role and does not appear to function as a NANC neurotransmitter (Belvisi et al., 1992a, 1992b).

3. Role of nitric oxide in regulating airway smooth muscle tone 3.1. Nitric oxide and basal airway tone Unstimulated cell lysates of bovine bronchial epithelial cells demonstrate NOS activity (Robbins et al., 1993). LNAME administered 45 min before contracting the tissue with methacholine enhanced the methacholine-induced contraction in unchallenged guinea-pigs (De Boer et al., 1998). This suggests that endogenous NO has a modulatory role in normoreactive guinea-pig tracheal preparations inhibiting smooth muscle tone. The inhalation of the nonselective NOS inhibitor L-NAME (12 mM, 15 min), but not the selective iNOS inhibitor aminoguanidine (0.1 mM, 3 min), was shown to increase basal airway reactivity to histamine in ovalbumin (OA)-sensitised guinea-pigs (Schuiling et al., 1998a; Iijima et al., 1998). The administration of the eNOS inhibitor L-NAME and the selective nNOS inhibitor Smethyl-L-thiocitrulline, but not aminoguanidine, similarly potentiated airway reactivity to methacholine in healthy, anaesthetised rats (Tulie et al., 2000). Transgenic mice without eNOS showed increased reactivity to methacholine. In transgenic mice, unlike wild-type mice, treatment with LNAME (i.p.) did not potentiate airway reactivity to methacholine. These results suggest that NO derived from eNOS plays an important role in controlling bronchial reactivity in the mouse (Feletou et al., 2001). From the above results, it appears that cNOS-derived, but not iNOS-derived, NO may be associated with the regulation of airway tone in OAsensitised animals. In contrast, however, carboxy-PT10, a NO scavenger, had no effect on acetylcholine-induced bronchoconstriction in healthy, anaesthetised guinea-pigs (Kanazawa et al., 2000). It has also been shown that cNOS-derived NO does not contribute to airway reactivity to carbachol in the healthy rat (Kips et al., 1995). These contrasting results may reflect differences in the sensitisation procedures employed (where used), and may also depend on the animal species considered. Aminoguanidine and L-NAME have been found to have no effect on basal

airway tone in healthy and OA-challenged guinea-pigs and rats (De Boer et al., 1998; Schuiling et al., 1998a, 1998b; Iijima et al., 1998; Kanazawa et al., 2000; Kips et al., 1995; Tulie et al., 2000). The consensus view from these results appears to suggest that cNOS-derived NO, while modulating airway reactivity to spasmogens, does not appear, however, to modulate basal airway tone. Administration of the nonselective NOS inhibitors LNAME or L-NMMA did not affect lung function in asthma patients, suggesting that endogenous NO does not have a role in modulating basal airway tone in asthma (Ricciardolo et al., 1996, 2001; Gomez et al., 1998; Taylor et al., 1998a, 1998b). In patients with mild asthma, the effects of inhaled L-NAME on exhaled NO and airway responsiveness to histamine and 50-AMP was investigated. The lower dose (54 mg) and the higher dose (170 mg) of L-NAME reduced exhaled NO to similar levels: 78 ± 12% and 81 ± 6% respectively. However, the higher dose, but not the lower dose, of L-NAME increased airway responsiveness to both histamine and 50-AMP. This suggested that L-NAME may be exerting its effects by mechanisms independent of NOS inhibition. Since 50-AMP (Mann et al., 1985) and histamine (Holtzman et al., 1980) stimulate airway sensory nerves, iNANC pathways may be important in modulating airway responsiveness to these two spasmogens. It was speculated that the low dose of L-NAME, while maximally inhibiting epithelial iNOS-derived NO (exhaled NO), was not able to penetrate through the epithelium to airway sensory nerves. High-dose L-NAME was able to penetrate into the airway parenchyma, thereby inhibiting nNOS (Taylor et al., 1998b). Similarly, the administration of the nonselective NOS inhibitor L-NMMA, while reducing exhaled NO, did not have an effect on sodium metabisulphite-induced bronchoconstriction and refractoriness in asthma (Hamad et al., 1999). Assuming that nNOS-derived NO antagonises sodium metabisulphite-induced bronchoconstriction, it may also be speculated that nNOS was not inhibited by the dose of L-NMMA used in this study. Ricciardolo et al. (1996) showed that L-NMMA aerosol potentiated bradykinin- and to a lesser extent methacholine-induced bronchoconstriction in asthmatics. Exhaled NO, however, was not measured in this study. The rapid production of NO by bradykinin in the human lungs (Ricciardolo et al., 1996) and by bradykinin and histamine in the guinea-pig tracheae and lungs (Nijkamp et al., 1993; Folkerts et al., 1995; Figini et al., 1996b; Yoshihara et al., 1998; Ricciardolo et al., 2000) has been demonstrated. Leurs et al. (1991) demonstrated that histamine, via H1 receptors, increased cNOS-derived NO production (Fig. 2). In rabbit tracheal smooth muscle, the use of histamine on KCl contraction induced a relaxation of 10% of the initial contraction. Administration of the NOS inhibitor L-NMMA decreased the relaxation to 5% of initial contraction (Gourgoulianis et al., 1998). The release of histamine and leukotriene (LT)C4 mediated an increase in exhaled NO that reduced the increase in insufflation pressure due to antigen challenge

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in the anaesthetised guinea-pig (Persson et al., 1995). Histamine, LTC4, and bradykinin released after antigen challenge to the lungs may increase cNOS-derived NO, enabling it to exert a modulatory role. The tachykinins substance P, neurokinin A, and neurokinin B have also been shown to relax guinea-pig tracheae via NO release. This relaxation was changed to a contraction by pretreatment with the NOS inhibitor L-NMMA (Figini et al., 1996a). Similarly, neurokinin A has been shown to activate cNOS, counteracting the direct neurokinin A-induced bronchoconstriction (Imasaki et al., 2001). These results suggest that when released, tachykinins may elicit a partial relaxation response via cNOS-derived NO release (Fig. 2). Bronchoconstriction induced by cold-air inhalation in the guinea-pig is mediated via bradykinin and tachykinin release. This bronchoconstriction, however, is reduced by the release of endogenous NO by bradykinin. Whether this is important in cold air-induced asthma has yet to be determined (Yoshihara et al., 1998). In mild asthmatics, LNMMA increased bradykinin-induced bronchoconstriction (Ricciardolo et al., 1996). Because of the rapid effect of LNMMA on bradykinin-induced bronchoconstriction (lung function measured 1 – 3 min after increasing bradykinin concentrations), it can be concluded that cNOS-derived NO is produced that inhibits bradykinin-induced bronchoconstriction. A contrasting action of bradykinin, however, has been demonstrated whereby it causes a reduction in exhaled NO in mild asthma patients, together with increased bronchial hyperresponsiveness. Administration of the COX inhibitor L-acetylsalicylic acid attenuated this reduction in exhaled NO and reduced bronchial hyperresponsiveness, suggesting that bradykinin in asthma releases COX products that reduce endogenous NO production and its bronchoprotective effects (Kharitonov et al., 1999). From these results, it may be concluded that bradykinin produces more NO than it inhibits, this being sufficient to exert an inhibitory modulation of bradykinin-induced bronchoconstriction. In severe asthma, however, L-NMMA did not increase bradykinin-induced bronchoconstriction (Ricciardolo et al., 1997), suggesting that NO release by bradykinin depends on the severity of the asthma. Belvisi et al. (1995a) found that there was an increase in cNOS activity in patients with mild asthma. As asthma severity increases, however, cNOS is reduced. In mild asthma, there is sufficient activity to modulate bradykinin-induced bronchoconstriction, but in severe asthma, cNOS-derived NO is reduced or abolished. In comparison with preallergen exposure, a reduction in the PD20BK [provocative concentration of bradykinin producing a 20% fall from baseline forced expiratory volume in 1 sec (FEV1)] was noted in mild atopic asthma 48 hr following allergen challenge. This increase in reactivity was found to correlate with reduced eNOS immunoreactivity and with an increased iNOS immunoreactivity. nNOS immunoreactivity did not change after allergen challenge. The administration of L-NMMA before both preallergen and postallergen bradykinin exposures resulted in similar

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PD20BK values. These values did not differ from the postallergen PD20BK value without L-NMMA. These results suggest that allergen exposure in asthma causes a reduction of eNOS-derived NO production, resulting in increased airway reactivity to bradykinin. nNOS-derived NO production, however, does not appear to be involved (Ricciardolo et al., 2001). Nasal and exhaled NO levels are significantly reduced in patients with cystic fibrosis (Balfour-Lynn et al., 1996; Dotsch et al., 1996; Grasemann et al., 1999b; Thomas et al., 2000), suggesting a deficiency of NO in cystic fibrosis. In cystic fibrosis mutant mice, relaxation of precontracted tracheal segments to electrical field stimulation was reduced. The administration of L-NAME to wild-type mice, but not to cystic fibrosis mice, reduced tracheal relaxation. The administration of an NO solution (by bubbling NO through water) or L-arginine compensated for the relaxation deficiency in cystic fibrosis mice (Mhanna et al., 2001). These results suggest that there is a deficiency of cNOS-derived NO in cystic fibrosis mice. A positive correlation was observed between sputum NO metabolite levels and lung function in stable cystic fibrosis patients (Grasemann et al., 1998; Balint et al., 2001). In cystic fibrosis patients, however, an increase in cNOS activity in lung parenchymal tissue, but a reduction in constitutive iNOS expression, in airway epithelial cells has been reported (Belvisi et al., 1995a; Kelley & Drumm, 1998; Meng et al., 1998). This suggests that iNOS-derived NO may be bronchoprotective in cystic fibrosis. Increased iNOS expression, however, has been reported in the subepithelial tissues of cystic fibrosis lungs (Meng et al., 1998), suggesting that iNOS-derived NO is produced in high quantities in cystic fibrosis. 3.2. Role of nitric oxide in regulating the early asthmatic reaction Inhalation of antigen in atopic asthmatics has been shown to result in an early asthmatic reaction (EAR), reaching a maximum 15 –30 min after challenge, followed by a late asthmatic reaction (LAR) 6– 12 hr after antigen challenge (Pepys & Hutchcroft, 1975; Chung, 1986). An increase in eosinophil numbers in the bronchoalveolar lavage (BAL) of asthmatics and after antigen challenge has been demonstrated (Wardlaw et al., 1988; Robinson et al., 1993). Similarly, in the OA-sensitised guinea-pig, depending on the sensitisation protocol employed, an EAR occurs between 0 and 5 hr following allergen (OA) provocation and an LAR occurs between 3 and 24 hr (Iijima et al., 1998; Lewis et al., 1996; Spruntulis & Broadley, 1999; Suigura et al., 1999). Airway hyperreactivity (AHR) and an increase in eosinophil numbers in the BAL is also noted following antigen challenge (Lewis et al., 1996; Daffonchio et al., 1989; Ishida et al., 1989; Sanjar et al., 1990; Danahay & Broadley, 1998). The antigen-challenged guinea-pig, therefore, provides a useful model of human asthma. It is used to determine the under-

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lying physiology of asthma and to elucidate the pharmacology and efficacy of anti-asthma drugs. Mehta et al. (1997b) demonstrated that within 30 min of OA challenge of sensitised guinea-pigs, NO levels in exhaled air increased, and this was followed by a decrease in NO, with a concomitant increase in airway bronchoconstriction. The administration of L-NAME decreased NO levels to below basal values and increased the degree of bronchoconstriction following OA challenge. This suggested that endogenously produced NO has an important homeostatic role in the early stages of allergic bronchoconstriction. Similar results have been reported by others (Persson & Gustafsson, 1993; Persson et al., 1993a, 1995; Nogami et al., 1998). Middelveld et al. (2000) monitored pigs 90 min after antigen challenge. It was shown that ascaris antigen-induced reactions were significantly increased by pretreating pigs with the NOS inhibitor LNOARG (10 mg/kg i.v.) 35 min before challenge. The increase of NO in exhaled air following OA challenge in the guinea-pig was shown to be abolished by L-NAME. NO levels in exhaled air were shown to rapidly increase, reaching a peak at 15 min, but then decreased below basal levels (Persson et al., 1993a). An increase in the release of NO, at least in the first 30 min of the EAR, appears, therefore, to exert an inhibitory modulatory role. While finding an increase in exhaled NO during the EAR, Iijima et al. (1998) did not find that inhalation of L-NAME (2 mM, 30 min) 30 min before OA challenge increased the EAR or reduced the increase in exhaled NO during the EAR. The dose of L-NAME used in this study may have been too small to affect cNOS. The time course for the observed increase in exhaled NO during the EAR is inconsistent with that required for an increase in iNOS. Basally expressed NOS (cNOS) has been implicated as the source for the increase in expired NO. cNOS-derived NO appears, therefore, to exert a modulatory role during the EAR. Miura et al. (1997) and Samb et al. (2001) demonstrated that the NO component of iNANC relaxation was impaired in isolated trachea of guinea-pigs following chronic OA challenge. This suggests that a deficiency of nNOS-derived NO may contribute to the impaired lung function seen in OA-challenged guinea-pigs. In patients with mild asthma, nebulised L-NAME (170 mg) did not affect the EAR per se, but did reduce exhaled NO during the EAR (Taylor et al., 1998a). This dose of LNAME was effective in potentiating airway responsiveness to 50-AMP and histamine in mild asthma (Taylor et al., 1998b). This suggests that NO does not have a modulatory role in the EAR in patients with mild asthma. In this study, however, a rise in exhaled NO during the EAR was not noted. However, by recognising that spirometry per se reduces exhaled NO levels below baseline, Deykin et al. (1998) reported a rise in exhaled NO (to baseline levels) during the EAR in human asthma. These results suggest that exhaled NO is increased during the EAR. Whether this increase in exhaled NO modulates the EAR has yet to be determined.

3.3. Modulatory role of nitric oxide on airway hyperreactivity In sensitised guinea-pigs, an increase in airway reactivity to inhaled histamine was noted 5 hr (after the EAR) after OA challenge. Inhalation of L-NAME after the EAR had no effect on histamine reactivity. The reactivity to histamine after the LAR (at 23 hr) was shown to be less than that noted after the EAR. Following L-NAME inhalation, however, reactivity was shown to increase to levels seen in the EAR (Schuiling et al., 1998b). Similarly, the administration of the selective iNOS inhibitor aminoguanidine, while not affecting allergen-induced AHR after the EAR, did potentiate the AHR after the LAR to the level of AHR observed after the EAR (Schuiling et al., 1998a). It has been suggested, therefore, that a deficiency of cNOS-derived NO contributes to OA-induced early AHR to histamine after the EAR (Table 1). After the LAR, this deficiency is overcome, although a significant AHR after the LAR does remain. SOD breaks down O
2 to H2O2, thereby reducing peroxynitrite formation from the interaction between NO and O
2 (Oury et al., 1996) (Fig. 2). Asthmatic subjects not on inhaled corticosteroids have been shown to have reduced SOD activity (De Raeve et al., 1997; Henricks & Nijkamp, 2001). In mild, steroid-naı¨ve asthmatics, SOD activity was found to be reduced 10 min following antigen challenge (Comhair et al., 2000). Increased O
2 production by air-space cells in nocturnal asthma has been reported (Jarjour et al., 1992). In allergic rhinitis patients, Calhoun et al. (1992) reported that BALactivated macrophages, but not unfractionated air-space cells, have potentiated O
2 release immediately and 48 hr after antigen challenge. These results suggest increased O
2 production in allergic airways disease. In birch pollen allergic patients, however, Woschnagg et al. (1996) reported reduced oxygen radical production by blood eosinophils during the birch pollen season. Increased severity of asthma is found to be associated with a significant increase in spontaneous O
2 production (Jarjour & Calhoun, 1994). In normoreactive guinea-pig tracheal preparations, the regulatory role of NO is partially counteracted by its reaction with
O
2 . It may be postulated that O2 may reduce the already low levels of cNOS-derived NO during the EAR. In the same study, however, it was demonstrated that a deficiency of NO in hyperreactive tracheal preparations (6 hr after OA challenge) was not related to the reaction of NO with O
2 (De Boer et al., 1998). In tracheae isolated from guinea-pigs, De Boer et al. (1999) demonstrated that the administration of the NO precursor L-arginine dose-dependently reduced the AHR to methacholine after the EAR, supporting a role for NO deficiency in the AHR. In isolated guinea-pig tracheae and in anaesthetised guinea-pigs, L-arginine was similarly shown to prevent virus-induced AHR (Folkerts et al., 1995). A limitation of substrate may underlie a deficiency of NO during the EAR. Miura et al. (1997) found that nNOS-derived NOmediated relaxation was impaired in guinea-pigs chronically exposed to OA. They demonstrated that the impairment of

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the iNANC was most likely due to the inactivation of neuronal NO by O
2 rather than the reduction of nNOS per se (measured as NADPH diaphorase staining). Samb et al. (2001) showed, however, that nNOS expression, measured by western blot analysis, was reduced 6 hr following chronic OA challenge in the guinea-pig. This was accompanied by increased airway responsiveness to histamine. It may be suggested that a deficiency of nNOS-derived NO may contribute to AHR after the EAR. It is debatable whether this deficiency of nNOS-derived NO is due to a deficiency of
L-arginine, the ‘‘mopping up’’ of NO by O2 , or a reduction in nNOS per se. Polycationic proteins such as major basic protein (MBP) released from eosinophils have been shown to damage the epithelium and to cause AHR in isolated guinea-pig trachea (Flavahan et al., 1988). With the use of an antibody to MBP, MBP was shown to contribute to AHR 24 hr after OA challenge in the guinea-pig in vivo (Costello et al., 1999). In isolated guinea-pig tracheal preparations, L-NAME caused an increase in reactivity to methacholine. L-NAME, however, was shown to have no effect on the enhanced methacholine response of poly-L-arginine-treated airways. This suggests that poly-L-arginine decreased cNOS activity. Poly-L-arginine, like MBP, is a polycation. Its reactivity was inhibited by heparin, a polyanion. From these results Meurs et al. (1999) suggested that polycationic peptides may contribute to the deficiency of cNOS-derived NO. MBP damaged the epithelium and inhibited the release of epithelium-derived relaxation factor (EpiDRF) (Motojima et al., 1989; Flavahan et al., 1988) in isolated guinea-pig trachea. MBP may also inhibit Larginine uptake in rat alveolar macrophages (Hammermann et al., 1999). MBP, therefore, may increase AHR by reducing cellular uptake of L-arginine or by damaging the epithelium, thereby reducing EpiDRF NO (Burke-Wolin et al., 1992). Twenty-four hours after OA challenge in the guinea-pig, MBP was shown to cause AHR by antagonising M2 muscarinic receptors (Costello et al., 1999). Loss of epithelial enzymatic function (Devillier et al., 1988) and damage to epithelial barrier function, however, may also contribute to AHR (Barnes, 1987; Small et al., 1990). In common with the early stages after OA challenge, administration of lipopolysaccharide (LPS) to guinea-pigs decreased NO initially, which coincided with AHR. This suggested that a deficiency of NO contributes to the AHR. However, a deficiency of NO alone was not responsible for this LPS-induced AHR because inhibition of NOS by LNAME did not cause AHR (Toward & Broadley, 2000). Airway hyporeactivity to histamine 48 hr after LPS exposure was also noted (Kips et al., 1995; Toward & Broadley, 2000). The induction of iNOS may mediate this response (Kips et al., 1995), since NO levels in BAL fluid are raised at this time (Toward & Broadley, 2000). The selective iNOS inhibitor 1400W was shown in the mouse to completely reverse AHR to methacholine 24 hr after OA challenge (Koari et al., 2000). This suggests that iNOS-derived NO contributes to AHR (Table 1). Twenty-

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four hours following OA challenge, the ex vivo administration of L-NAME to guinea-pig tracheae, however, was found to potentiate airway reactivity to methacholine (De Boer et al., 2001). The administration of the eNOS inhibitor L-NAME and the nNOS selective inhibitor S-methyl-L-thiocitrulline similarly potentiated airway reactivity to methacholine 24 hr following OA challenge in the anaesthetised rat (Tulie et al., 2000). These contrasting results may be due to the different animal models employed. Xanthine oxidase, an enzyme that forms O
2 by the oxidation of purines and of NADH (Sanders et al., 1997) (Fig. 2), was shown to be up-regulated in chronically exposed OA-challenged guinea-pigs (Ikuta et al., 1992). O
2 has been shown to be involved in ozoneinduced AHR in the cat (Takashi et al., 1993). O
2 reacts with NO to form the cytotoxic metabolite peroxynitrite (Blough & Zafiriou, 1985; Radi et al., 1991; Gaston et al., 1994b; Muijsers et al., 1997; Gow et al., 1998; Persinger et al., 2001). Sadeghi-Hashjin et al. (1996) administered peroxynitrite to normal, healthy guinea-pig tracheae, resulting in increased airway responsiveness to histamine or methacholine. When tracheal preparations from OA-challenged guinea-pigs were pretreated with SOD, tracheal hyperreactivity to methacholine was significantly reduced. A similar effect was noted when isolated tracheae were pretreated with L-NAME. This indicates that both O
2 and NO are involved in tracheal hyperreactivity (De Boer et al., 2001). The enzyme NADPH-oxidase may also form O
2 (Marshall et al., 1996). The administration of the selective iNOS inhibitor 1400W or the NADPH-oxidase inhibitor apocynin inhibited AHR 24 hr following repeated OA exposure in conscious, unrestrained mice (Muijsers et al., 2001). From these results, it may be suggested that the cytotoxic metabolite peroxynitrite may be involved in the development of increased airway reactivity in animal models of asthma. O
2 production has been shown to be increased in asthmatic subjects (Jarjour & Calhoun, 1994; De Raeve et al., 1997; Comhair et al., 2000). In subjects with asthma, O
2 generation by isolated neutrophils was found to correlate with increased AHR to inhaled methacholine (Meltzer et al., 1989). In asthmatic patients, Saleh et al. (1998) showed a correlation between protein nitration (measured as nitrotyrosine) and increased airway reactivity to methacholine. Peroxynitrite, derived from increased O
2 and iNOS-derived NO production, may mediate this protein nitration (Fig. 2). Significant protein nitration was observed in lung tissue specimens from asthma patients, while little or no staining of nitrotyrosine was observed in sections from healthy subjects (Saleh et al., 1998; Kaminsky et al., 1999; Dweik et al., 2001). Patients with mild asthma who were not taking corticosteroids were also shown to have increased nitrotyrosine levels in exhaled air and airway epithelial cells in comparison with healthy subjects (Hanazawa et al., 2000; Guo et al., 2000). In asthmatic subjects, a significant correlation between exhaled NO and airway responsiveness to histamine and methacholine has been reported (Salome et al., 1999; Lim et al., 1999). In an alveolar Type II epithelial

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cell line (A549) derived from a lung carcinoma, nitrotyrosine was found to be selectively incorporated into a single protein, a-tubulin. Because microtubules play an important role in determining cell shape, the effect of a-tubulin nitrotyrosination on cell morphology and microtubule structure was investigated. In cells grown with nitrotyrosine, changes in cell morphology and microtubule structure, together with increased permeability to [125I]bovine serum albumin (BSA), an indication of epithelial barrier dysfunction, were reported (Eiserich et al., 1999). Loss of the epithelial protective barrier may result in the exposure of sensory nerve endings to inhaled stimuli causing AHR (Barnes, 1987; Small et al., 1990). Loss of the EDRF NO (BurkeWolin et al., 1992) and loss of enzymatic function (Devillier et al., 1988) in the epithelium may also contribute to AHR. These results suggest that the metabolite of O
2 and NO, peroxynitrite, by forming nitrotyrosine, may interfere with epithelial function, and by doing so, contribute to the increased airway reactivity seen in asthma (Beckman & Koppenol, 1996; Aizawa, 1999). While inhibiting AHR, administration of the selective iNOS inhibitor 1400W or the NADPH-oxidase inhibitor apocynin did not reduce nitrotyrosine staining of lung sections 24 hr following the last OA exposure in conscious, unrestrained mice (Muijsers et al., 2001). This suggests that while O
2 and iNOS-derived NO may contribute to AHR in this animal model of asthma, nitrotyrosine does not appear to be involved. These findings appear to suggest that iNOS-derived NO contributes to AHR after the LAR. This is in conflict with the finding that a deficiency of NO may contribute to AHR and that a recovery of NO levels through iNOS stimulation may reduce or reverse AHR after the LAR (Schuiling et al., 1998a, 1998b). The degree of iNOS stimulation may be important. It may be concluded that some iNOS-derived NO production is beneficial, while hyperproduction leads to protein nitration, which may be deleterious. This is in conflict, however, with the finding that 24 hr after OA challenge, transgenic mice without iNOS and wild-type mice with iNOS showed similar responsiveness to methacholine (De Sanctis et al., 1999). Administration of L-NAME did not reduce AHR 24 hr following chronic OA challenge in the guinea-pig. It did, however, increase airway reactivity to histamine in unexposed animals. iNOS-derived NO, therefore, does not appear to promote AHR 24 hr following chronic OA challenge in this animal model of asthma. Similar increases in exhaled NO during histamine-induced bronchoconstriction were observed in both OA-challenged and unexposed animals. This suggested that a defect in NOrelated bronchodilator activity rather than a deficiency in NO per se contributed to AHR 24 hr following OA challenge (Mehta et al., 1997a). Tulie et al. (2000) reported that the selective iNOS inhibitor aminoguanidine did not affect airway reactivity 24 hr following OA challenge in the guineapig. The selective nNOS inhibitor S-methyl-L-thiocitrulline and the eNOS inhibitor L-NAME, however, did potentiate AHR. iNOS-derived NO, therefore, does not also appear to

have a role in potentiating AHR 24 hr after OA challenge in this animal model of asthma. nNOS-derived NO, however, does appear to be bronchoprotective. Reduced AHR, however, was reported in mice where the nNOS gene is deleted (De Sanctis et al., 1999). nNOS-derived NO was found to be deficient in guinea-pigs 6 hr (Samb et al., 2001) and 1 day (Miura et al., 1997) after chronic OA exposure. It may be suggested that a deficiency of nNOS-derived NO may contribute to AHR in the specific model of allergic asthma employed by De Sanctis et al. (1999).

4. Nitric oxide and inflammation 4.1. Role of nitric oxide in promoting eosinophilia and microvascular hyperpermeability As discussed in Section 3, NO exerts some beneficial effects on airway smooth muscle tone. It modulates basal airway tone, contributes to the recovery of the EAR, and reduces AHR after the LAR in the guinea-pig. NO, however, does appear to have some unwanted effects (Table 1). It may be cytotoxic (Radi et al., 1991; Gow et al., 1998; Hibbs et al., 1987) and neurotoxic (Lipton et al., 1993), and may contribute to inflammation of the airways (Kuo et al., 1992; Xiong et al., 1999). Six hours after OA challenge (after the LAR), L-NAME was shown to reduce eosinophil numbers in the BAL of the guinea-pig. The eosinophil numbers were increased by administrating inhaled L-arginine to the guinea-pigs 2 and 3 hr after OA challenge (Iijima et al., 1998). This suggests that NO may augment eosinophil infiltration in the OAchallenged guinea-pig. L-NAME was shown to reduce eosinophil numbers in the BAL of the rat 48, but not 24, hr after OA challenge (Ferreira et al., 1998). In the mouse, 24 hr after double OA challenge, Koari et al. (2000) demonstrated that continuous infusion (s.c.) of the selective iNOS inhibitor 1400W partially reduces the eosinophil number in stained tracheal segments. The administration of the selective iNOS inhibitor aminoguanidine (100 mg/kg s.c. 23 and 1 hr before and 23 hr following OA challenge) similarly inhibited BAL eosinophil and total cell count 24 hr after OA challenge in the rat (Tulie et al., 2000). In contrast to these results, the administration of inhaled aminoguanidine (5.5 and 23.5 hr following allergen challenge) did not reduce BAL eosinophil cells 24 hr following OA challenge in the guinea-pig (Schuiling et al., 1998a). Similarly, the selective iNOS inhibitor 1400W, while inhibiting AHR, did not reduce BAL eosinophil influx 24 hr following the last OA exposure in mice (Muijsers et al., 2001). The nonselective NOS inhibitor L-NAME, but not the selective iNOS inhibitor 1400W, reduced pulmonary eosinophil influx in the OA-challenged mouse and the Sephadexchallenged rat (Feder et al., 1997; Birrell et al., 2000), suggesting that cNOS-derived NO and not iNOS-derived NO may be pro-inflammatory. These contrasting results,

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however, may be due to the different animal models (and drug regimens) employed. The selective iNOS inhibitor L-NIL (a dose of up to 50 mg/kg i.p 0.5 and 4 hr following challenge), however, did not reduce the eosinophil count in the BAL 24 hr following OA challenge in the mouse. Furthermore, there was no increase in iNOS protein expression or iNOS mRNA in the lungs or nitrite levels in the BAL fluid, suggesting little involvement of iNOS in this animal model of asthma (Feder et al., 1997). Similarly, L-NIL (a dose of up to 100 mg/kg i.p., 2 hr before and 4 and 12 hr following challenge) did not reduce Sephadex-induced airway inflammation in the rat 24 hr following OA challenge (Birrell et al., 2000). In asth
matic subjects, sputum NO metabolite [NO
3 (nitrite), NO2 (nitrate)] levels were found to significantly correlate with sputum eosinophil levels (Jang et al., 1999). Also, in asthma, raised exhaled NO levels were found to significantly correlate with increases in peripheral blood eosinophils and sputum eosinophils (Jatakanon et al., 1998; Salome et al., 1999). These results support a relationship between increasing NO levels and eosinophilia in both asthma and sensitised animal models of asthma. Following repeated OA exposure, in comparison with wild-type rats with iNOS, in transgenic mice without iNOS, the increases in circulatory and pulmonary eosinophils were substantially less than wild-type levels and IFN-g levels were significantly increased (Xiong et al., 1999). This suggests that iNOS-derived NO promotes eosinophil infiltration in the airways via a reduction of IFN-g (Fig. 3). NO, by reducing T helper Type 1 cells, may reduce IFN-g, leading to T helper Type 2 cell expansion and inflammation (for a review, see Barnes & Liew, 1995). De Sanctis et al. (1999), however, failed to show a difference in eosinophils and eosinophil peroxidase levels in the BAL between wild-type and iNOS knock-out transgenic mice, although IFN-g was not measured. With a different protocol for OA sensitisation and challenge employed, it is possible that IFN-g was not decreased in this animal model of asthma. In OA-challenged mice, Trifilieff et al. (2000) suggested that iNOS-derived NO may promote airway inflammation, not by modulating T helper Type 1 and T helper Type 2 cell expansion, but by upregulating chemokines (macrophage inflammatory protein-2, monocyte chemoattractant protein-1). The chemokine eotaxin, however, did not appear to be involved. Eotaxininduced chemotaxis of isolated human eosinophils, however, was reduced by L-NAME administration (Ferreira et al., 2001). In anaesthetised rats, eosinophil migration induced by exogenous bradykinin, platelet-activating factor (PAF), LPS, and carrageenin was reduced by L-NAME. The administration of N-formylmethionine leucyl-phenylalanine (fMLP), PAF, and zymosan-activated serum to isolated rat eosinophils promoted eosinophil chemotaxis. This was markedly inhibited by L-NAME (Ferreira et al., 1996). The nonselective NOS inhibitor L-NAME, the selective iNOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine, and the nNOS and iNOS inhibitor 1-(2-trifluoromethylphe-

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nyl)imidazole similarly inhibited fMLP and LTB4-induced migration of rat isolated peritoneal eosinophils (Zanardo et al., 1997). These findings suggest that iNOS-derived NO may not only promote eosinophilia by reducing IFN-g levels, but may also do so by promoting chemotaxis (Fig. 3). The administration of the NO precursor L-arginine has been shown to cause damage to endothelial cells and basement membrane and to increase plasma extravasation and eosinophilia in the OA-challenged guinea-pig (Iijima et al., 1998). L-Arginine (100 mg/kg s.c.), administered 23 and 1 hr before and 23 hr following OA challenge, similarly potentiated microvascular permeability 24 hr following OA challenge in the OA-challenged rat (Tulie et al., 2000). The NOS inhibitor L-NAME reduced the submucosal oedema, plasma exudation, and eosinophilia (Iijima et al., 1998). L-NAME has been shown to reduce microvascular hyperpermeability in the guinea-pig lungs with a partial, but significant, reduction in eosinophils, together with an expected reduction in exhaled NO. Peroxynitrite produced by the interaction between NO and O
2 appears to be the final molecule inducing airway microvascular hyperpermeability (Suigura et al., 1999) (Table 1, Fig. 3). Xanthine oxidase is involved in the production of O
2 (Demiryurek & Wadsworth, 1999). 4Amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP), a xanthine oxidase inhibitor, and ebselen, a peroxynitrite scavenger, reduced microvascular hyperpermeability during the LAR. This suggests that peroxynitrite causes microvascular hyperpermeability (Suigura et al., 1999). This is further supported by the finding that the selective iNOS inhibitor aminoguanidine and the nonselective NOS inhibitor LNMMA, but not the selective nNOS inhibitor S-methyl-Lthiocitrulline nor L-NAME, reduced microvascular leakage 24 hr following OA challenge in the rat (Tulie et al., 2000). LNAME was shown to significantly, but only slightly, inhibit eosinophil accumulation. AHPP and ebselen had no effect on eosinophil accumulation. This suggests that microvascular hyperpermeability and eosinophil accumulation are independent phenomena, where peroxynitrite appears to be involved in microvascular hyperpermeability, but not in eosinophil accumulation (Suigura et al., 1999). iNOSderived NO, however, may cause eosinophil accumulation by reducing IFN-g and by promoting chemotaxis. Peroxynitrite, by promoting nitrotyrosine formation (Fig. 2), irreversibly inhibits the phosphorylation of tyrosine by kinase. Since tyrosine phosphorylation is important in cell cycle control, tyrosine nitration may result in the impairment of cyclic cascades that control signal transduction processes and that regulate cell cycles (Kong et al., 1996). Peroxynitrite was found to inactivate the anti-inflammatory, receptor-like, T-cell tyrosine phosphatase (CD45) (Takakura et al., 1999; Irie-Sasaki et al., 2001). Whether peroxynitritemediated inhibition of CD45 contributes to inflammation in the airways has not been determined yet. Iijima et al. (2001) demonstrated that in the BAL fluid of OA-challenged mice, NO levels increased with a time course similar to that seen for the increase in BAL eosinophilia. When the nonselective

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Fig. 3. Pharmacological effects of NO (S-nitrosoglutathione) in the airway.

NOS inhibitor L-NAME and the selective iNOS inhibitor LNIL were administered i.p. 0.5 hr before and at 8, 20, 32, and 44 hr after OA challenge, BAL eosinophilia (at 48 hr) was markedly reduced by 61.5% and 37.2%, respectively. This suggested that eosinophil recruitment is partly driven by NO, most notably by iNOS-derived NO. Forty-eight hours after OA challenge, immunolocalisation of iNOS was noted in 80% of BAL eosinophils. This suggested that iNOS-derived NO production by eosinophils and recruitment of eosinophils may be tightly coupled. Forty-eight

hours following OA challenge, there was enhanced iNOS staining in both epithelial cells and peribronchial inflammatory cells associated with increased tissue nitrotyrosine. BAL eosinophils at 48 hr were also shown to stain for nitrotyrosine. The intravenous administration of an anti-IL-5 antibody caused an expected reduction in BAL eosinophilia, with a corresponding reduction in NO. iNOS and nitrotyrosine were reduced to levels seen in unchallenged mice. Forty-eight hours following OA challenge, BAL O
2 levels were also reduced to levels seen in unchallenged mice.

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These results (Iijima et al., 2001) appear to suggest that iNOS-derived NO, possibly a proportion of it derived from the eosinophil, may react with eosinophil-derived superoxide (Pincus et al., 1982) to form peroxynitrite and thereby form nitrotyrosine. While inhibiting AHR, administration of the selective iNOS inhibitor 1400W or the NADPH-oxidase inhibitor apocynin did not reduce nitrotyrosine staining of lung sections 24 hr following the last OA exposure in conscious, unrestrained mice. Therefore, nitrotyrosine formation does not appear to be mediated by iNOS-derived NO production or O
2 generation in this animal model of asthma (Muijsers et al., 2001). By using the NO metabolite nitrate as substrate, eosinophil peroxidase may also cause nitrotyrosine formation (Fig. 2). Isolated eosinophil peroxidase has been shown to increase the consumption of NO by H2O2. This pathway, however, does not involve the formation of peroxynitrite (Wu et al., 1999; Abu-Soud & Hazen, 2000; MacPherson et al., 2001). An association between eosinophil peroxidase and protein nitration was demonstrated in the BAL of asthmatic patients (MacPherson et al., 2001). These findings suggest that O
2 and eosinophil peroxidase, products of the eosinophil, may enhance nitrotyrosine production. In preconstricted rat tracheal rings, the presence of physiologically relevant levels of eosinophil peroxidase and H2O2 inhibited the bronchodilatatory effects of exogenously administered NO, suggesting that eosinophil peroxidase may serve as a catalytic sink for NO, limiting its bioavailability and function (Abu-Soud et al., 2001). A correlation between nitrotyrosine formation and increased reactivity to methacholine in asthmatics has been demonstrated (Saleh et al., 1998). A correlation was found between expired H2O2, sputum eosinophils, and AHR in human asthma (Horvath et al., 1998). These results suggest that eosinophil peroxidase, by ‘‘mopping up’’ NO and by forming nitrotyrosine, may contribute to AHR in human asthma. Other pulmonary cells, however, may also contribute to protein nitration, thereby contributing to AHR and microvascular hyperpermeability. iNOS is also expressed in epithelial cells (Asano et al., 1994; Hamid et al., 1993), neutrophils (McCall et al., 1991), and macrophages (Jorens et al., 1991; Hamid et al., 1993; Wizemann et al., 1994). Pulmonary macrophages, neutrophils, endothelium cells, and smooth muscle cells may also be sources of O
2 (Calhoun et al., 1992; Demiryurek & Wadsworth, 1999). Neutrophil myeloperoxidase, by reacting with nitrate, may also cause protein nitration (Eiserich et al., 1998; Ischiropoulos, 1998; Van der Vilet et al., 1999). This may be of particular importance in cystic fibrosis, where sputum myeloperoxidase levels have been found to correlate with elevated sputum nitrotyrosine levels (Jones et al., 2000). 4.2. Anti-inflammatory effects of nitric oxide L-NAME applied to guinea-pig tracheal mucosa was shown to increase plasma exudation. L-Arginine abolished

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this effect. It was suggested, therefore, that NO suppresses any increase in the permeability of the subepithelial microcirculation (Erjefalt et al., 1994). Similar results have been reported in the feline ileum and rat coronary circulation (Filep et al., 1993; Kubes, 1992). Endogenous NO in vitro may also reduce leukocyte chemotaxis (Kuo et al., 1997) and adherence of leukocytes to the vascular endothelial cell wall (Kubes et al., 1991; De Caterina et al., 1995; Khan et al., 1996; Conran et al., 2001). Endogenously and exogenously administered NO may attenuate mast cell mediator release, resulting in reduced leukocyte adhesion and vascular leakage in inflammatory airways disease (Masini et al., 1991; Valentovic et al., 1992; Kubes et al., 1993; Kurose et al., 1994; Gaboury et al., 1996; Holgate, 1997) (Fig. 3, Table 1). cNOS-derived NO may decrease microvascular leakage by enhancing endothelial cell relaxation, reducing platelet-leukocyte aggregation and decreasing PAF formation in rat mesenteric venules. These effects appear to be mediated via cGMP formation (Kurose et al., 1993). In porcine pulmonary endothelial cells, however, Gupta et al. (2001) demonstrated that cNOS-derived NO modulated H2O2 vascular endothelial barrier dysfunction through a cyclic AMP (cAMP)-dependent signalling mechanism. The anti-inflammatory and pro-inflammatory effects of NO may also be mediated by mechanisms independent of guanylate cyclase activation (Coleman, 2001). These may include the modification of signalling proteins and transcription factors (Raychaudhuri et al., 1999; Thomassen et al., 1999; Schlinder & Bogdan, 2001). Wu et al. (1994) demonstrated that peroxynitrite, by reacting with tissue thiols, relaxed the preconstricted bovine pulmonary arterial smooth muscle by increasing cGMP. Chabot et al. (1997) reported, however, that peroxynitrite relaxed the preconstricted rat pulmonary artery via the activation of poly (adenosine 50-diphosphoribose) synthase and not via a NO-dependent mechanism. Kanazawa et al. (2000) demonstrated that the nebulised NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), exerted bronchoprotective effects against acetylcholine in healthy anaesthetised guinea-pigs via the formation of peroxynitrite. Peroxynitrite appeared to exert this effect by reacting with a thiol to form an S-nitrosothiol (SNO). The administration of exogenous thiol (glutathione) enhanced the bronchoprotective effect of SIN-1. Exogenous addition of peroxynitrite has also been shown to reduce eotaxin-induced eosinophil chemotaxis in vitro (Sato et al., 2000). This effect, however, may not be of significance in vivo. The administration of nanomolar quantities of peroxynitrite were shown to attenuate adhesion and accumulation of neutrophils in the endothelium of the isolated thrombin- or H2O2-stimulated rat mesenteric artery and in isolated rat hearts that were subjected to global ischaemia and reperfusion with rat neutrophils (Lefer et al., 1997). iNOS-deficient mice that were treated with endotoxin (i.v.) showed enhanced leukocyte accumulation in lung tissue compared with wild-type mice similarly treated with endotoxin (i.v.). These results suggest that iNOS-derived NO

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may be beneficial and not deleterious. cNOS-derived NO has been shown to decrease microvascular leakage in cardiac, hepatic, pulmonary, and renal tissues following endotoxin administration to the conscious rat. In contrast, induction of iNOS-derived NO was shown to enhance microvascular leakage (Laszlo et al., 1995; Bernareggi et al., 1997). NO may also enhance apoptosis of eosinophils. In inflammation, however, it may promote eosinophil survival (Beauvais et al., 1995; Beauvais & Joly, 1999). The exogenous administration of NO has also been found to exert anti-inflammatory effects (Table 1, Fig. 3). It has been found to inhibit chemotaxis, adhesion, degranulation, LT production, and O
2 production from human polymorphonuclear leukocytes (neutrophils) in vitro (Clancy et al., 1992; Moilanen et al., 1993; Granger & Kubes, 1996; Schmidt & Walter, 1994). In ischaemia/reperfusion injury of distal microvascular beds in the cat, inhaled NO was shown to reduce oxidant-dependent leukocyte rolling, adhesion and emigration, and endothelial dysfunction (FoxRobichaud et al., 1998). The NO donors, SNP, spermineNO, and SIN-1, similarly reduced leukocyte adherence and emigration in single postcapillary venules in rat mesentery subjected to ischaemia/reperfusion injury (Kurose et al., 1993). Inhaled NO administered to neonates and infants with pulmonary hypertension reduced O
2 production by neutrophils stimulated with Escherichia coli or with fMLP (Gessler et al., 1996). Inhaled NO has also been shown to attenuate neutrophil activation and cytokine release in lungs of patients with adult respiratory distress syndrome (ARDS) (Chollet-Martin et al., 1996). Inhaled NO has been shown to prevent oxygen radical-dependent capillary leak in isolated rat (Guidot et al., 1995) and rabbit (Kavanagh et al., 1994) lungs. Inhaled NO, however, did not show anti-inflammatory properties in microvasculatures of endotoxin-treated (where iNOS is induced) cats (Fox-Robichaud et al., 1998). The inflammatory response in patients with acute lung injury, which is also associated with increased NO production, was similarly unaffected by inhaled NO (Cuthbertson et al., 2000). High levels of NO production may already be exerting maximal immunomodulatory effects. The further administration of NO in the form of inhaled NO, therefore, may not exert anti-inflammatory effects in systems that have increased NO production. However, some systems associated with increased NO production have shown anti-inflammatory effects when exposed to NO donors. Unlike salbutamol, inhalation of the novel NOsalbutamol complex (NCX-950) markedly inhibited the recruitment of neutrophils induced by LPS aerosol in BAL fluid of mice (Corbel et al., 2001). In Pseudomonas aeruginosa-induced sepsis, delayed administration of inhaled NO preserved alveolar-capillary membrane integrity in Yorkshire swine (Bloomfield et al., 1997). Wanikiat et al. (1997) demonstrated that endogenous NO in human neutrophils promoted chemotaxis, but that the administration of exogenous NO attenuated it. It may be speculated that an optimum production of NO may attenuate

inflammation and extravasation. iNOS-derived NO in small quantities (with sufficient thiol concentration) may be bronchoprotective and anti-inflammatory, but that in high concentrations, it may be cytotoxic. As in AHR, whether these inflammatory processes are enhanced or suppressed by NO may depend upon the degree of iNOS activation. Hyperproduction of iNOS-derived NO may be involved in extravasation and inflammation. An optimum production of NO may attenuate inflammation and extravasation, and may also be beneficial in terms of controlling the EAR and AHR after the LAR. The administration of NO, however, may attenuate neutrophil chemotaxis and activation. This may be of particular importance in chronic obstructive pulmonary disease (COPD), where the neutrophil is predominant (Vrugt & Aalbers, 1993). It has been proposed that the relative fluxes of O
2 and NO may act to control the steady-state concentration of peroxynitrite in vivo. As peroxynitrite diffuses into areas of NO (or O
2 ) excess, it decomposes, thereby limiting peroxynitrite formation and its associated deleterious effects (Grisham et al., 1999). It may be suggested that the exogenous administration of NO may decompose peroxynitrite, thereby reducing nitrotyrosine formation and increasing NO levels per se. NO per se may then exert its own immunomodulatory and bronchodilatatory effects. 4.3. Role of nitric oxide in regulating the late asthmatic reaction When the nonselective NOS inhibitor L-NAME (2 mM, 30 min) or the selective inhibitor aminoguanidine (2 mM, 30 min) was administered to the guinea-pig 30 min before and 3 and 4 hr after OA challenge, the LAR (3– 7 hr) was reduced. The administration of the NO precursor L-arginine prolonged the LAR. The development of the LAR was shown to be associated with increases in exhaled NO, suggesting increased production of iNOS-derived NO during the LAR. L-NAME and aminoguanidine were also shown to reduce exhaled NO during the LAR (Iijima et al., 1998). These results suggest that NO may be deleterious in the sense that it may contribute to the LAR. When LNAME (12 mM, 15 min) was administered to the guineapig 5.5 hr after OA challenge, no change in the LAR (8– 23 hr) was noted. Similarly, inhalation of the selective iNOS inhibitor aminoguanidine (0.1 mM, 3 min and 5.5 and 23.5 hr following OA challenge), while potentiating AHR after the LAR (24 hr), did not affect the LAR per se (8 –23 hr) (Schuiling et al., 1998a). It may be concluded that the involvement of NO in the LAR may depend on the animal model used. In mild asthma, the magnitude of the LAR (3– 8 hr) was not affected by the administration of nebulised LNAME (170 mg) 20 min before and 3.5 and 7 hr after allergen challenge. L-NAME, however, did reduce exhaled NO levels. While exhaled NO levels increased from baseline during the LAR, this increase, however, did not reach significance until after the LAR (at 21 hr) (Taylor et al., 1998a). In a different study, however, this increase did reach

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significance at 10 hr. A significant relationship between the size of the late response and the increase in exhaled NO was noted (both measured as areas under the curve between 6 and 10 hr after challenge) (Kharitonov et al., 1995b). Similarly, baseline-exhaled NO levels were found to significantly correlate with the magnitude of the LAR (Deykin et al., 1998). Baseline NO levels in asthmatics, however, were found to be inversely correlated with baseline sGaw (specific airways conductance), but positively correlated with sGaw, 24 hr following antigen challenge (Khatri et al., 2001). These results would suggest that baseline NO may be detrimental to baseline lung function and may also contribute to the LAR per se. A component of it, however, may be bronchoprotective after the LAR. It may be speculated that NO does affect bronchial tone during the LAR in human asthma, and may exert an influence on the allergic inflammation during the LAR. However, further study is required in this area.

5. S-nitrosothiols 5.1. Endothelium-derived relaxation factor and S-nitrosothiols Vasorelaxation by nitrates and nitrites and their ability to activate guanylyl cyclase were shown to act through an intermediate reaction with sulfhydryl groups, such as on cysteine and glutathione, to form SNOs (Gruetter et al., 1980a, 1980b; Ignarro & Gruetter, 1980; Ignarro et al., 1981). Under physiological conditions, NO, or a closely related oxidised derivative, can react with thiol groups to form SNO compounds (Stamler et al., 1992b) (Fig. 3). SNO compounds such as S-nitrosocysteine when added exogenously are potent vasodilators (Ignarro & Gruetter, 1980; Ignarro et al., 1981). EDRF is much more likely to be a nitrosylated compound, a nitrosothiol, than NO itself (Myers et al., 1990). This is supported by evidence that the majority of NO in human plasma is present as SNO, most notably S-nitroso-serum albumin (Stamler et al., 1992a). 5.2. S-nitrosothiols and pulmonary nitric oxide SNO compounds were found to relax isolated and perfused ventilated lung preparations of the guinea-pig (Bannenberg et al., 1995). The SNO compounds S-nitrosoglutathione (GSNO), S-nitroso-cysteine, S-nitroso-N-acetyl-cysteine, and S-nitroso-BSA relaxed human bronchial preparations precontracted with methacholine (Gaston et al., 1994a). GSNO was found to be the predominant SNO compound in airway fluid samples of normal subjects and patients with pulmonary disease. NO itself was not detected (Gaston et al., 1993). It has been suggested, therefore, that NO exists predominantly as GSNO in the human airway. It is thought that the SNO compound(s) acts as the intermedi-

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ate(s) following NO release, which exerts its bronchodilator effect (Fig. 1). 5.3. Ability of S-nitrosothiols to generate bioactive nitric oxide An increase in the polarity and bulk of SNO compounds would decrease SNO penetration across cell membranes. The highly polar SNAP, GSNO, S-nitroso-N-acetyl-cysteine and S-nitroso-cysteine and the bulky and polar S-nitrosocoenzyme A are all found to be potent relaxants of rat aortic rings (Kowaluk & Fung, 1990a). In isolated human bronchial smooth muscle, the high molecular weight SNO compound S-nitroso-BSA was no less potent than the lower molecular weight SNO compounds (Gaston et al., 1994a). The ability of SNO compounds to cross membranes and to enter cells, therefore, does not appear to be required for activity. These compounds have been shown to spontaneously decompose to NO in buffer solution (Bannenberg et al., 1995). It may be postulated that SNO compounds act as NO carriers (Stamler et al., 1992c). These compounds may spontaneously decompose extracellularly where NO then diffuses across the cell membrane. GSNO, which is more stable than S-nitroso-cysteine, has been shown to be equipotent in relaxing human bronchial preparations (Gaston et al., 1994a). The stability in solution of eight different SNO compounds did not correlate with their ability to relax guinea-pig tracheal preparations (Mathews & Kerr, 1993). Also, their ability to stimulate plateletsoluble guanylyl cyclase does not correspond to their stability (Oae et al., 1978). From these results, it does not appear that SNO compounds exert their effects by the spontaneous extracellular liberation of NO. Kowaluk and Fung (1990b) demonstrated that bovine vascular smooth muscle cells exhibited catalytic activity for NO generation from SNAP. In conclusion, it was proposed that an enzymatic degradation process associated with cellular membrane components is the likely mechanism for NO release from SNO compounds. Such a mechanism would allow the local and specific delivery of NO to its target site (Gaston, 1999). In tissue-bathing medium, SOD was found to decrease NO release from SNAP and GSNO (Kowaluk & Fung, 1990b). Xanthine oxidase has been found to catalyse the decomposition of GSNO under aerobic conditions. This is inhibited by SOD (Trujillo et al., 1998). These findings suggest that O
2 catalyses the decomposition of SNO. Nacetyl-penicillamine (NAP), in contrast, was shown to increase NO release from these SNO compounds (Kowaluk & Fung, 1990b). An intermolecular reaction of the thiyl radical of NAP with SNO appears to be responsible for this effect (Oae et al., 1978). In contrast to the inhibitory effect on NO release, the vasorelaxant responses to SNAP and GSNO were enhanced in the presence of SOD and diminished in the presence of NAP (Kowaluk & Fung, 1990b). The inherent instability of SNO compounds and the catalytic effects of O
2

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may result in the early release of NO before it reaches its target site. This may result in the metabolism of the released NO by O
2 to form the cytotoxic metabolite peroxynitrite. Because of this, the beneficial effects of NO, in the form of an SNO, may be limited and reduced. In asthmatics, SNO levels were found to be undetectable in BAL fluid immediately after antigen challenge. However, they were found to be increased 48 hr after antigen challenge. It was suggested that SNO acts as a reservoir for the safe removal of toxic NO metabolites during the late inflammatory response (Mayer et al., 1995). Peroxynitrite, by reacting with glutathione to form GSNO (Fig. 3), stimulates cGMP production and is bronchoprotective (Wu et al., 1994; Kanazawa et al., 2000). Eosinophil peroxidasemediated nitrotyrosine formation may be inhibited by thiols (Wu et al., 1999). NO-mediated cellular injury in rat Type II epithelial cells was attenuated by preincubation with the thiol N-acetylcysteine (Gow et al., 1998). It appears, therefore, that the availability of thiols and the consequent production of SNO is important. It may determine whether the end effect of NO (and peroxynitrite) is beneficial or deleterious. Rather than NO being metabolised to nitrotyrosine before it reaches its target site, SNO may act as a reservoir for NO from which NO is selectively and catalytically released at its target site. SNO formation, by delaying and reducing nitrotyrosine formation, may potentiate NOmediated bronchodilatatory effects.

6. Molecular targets for nitric oxide in smooth muscle EDRF relaxes vascular smooth muscle by increasing the secondary messenger cGMP (Ignarro et al., 1987; Hay et al., 1992). The activation of the enzyme guanylyl cyclase converts GTP to cGMP. NO appears to relax vascular smooth muscle by a cGMP-dependent protein kinasemediated activation of K+ channels (Robertson et al., 1993; Archer et al., 1994; Denninger & Marletta, 1999) (Fig. 3). The relaxant response to endogenously released or exogenously added NO in guinea-pig trachea and the human bronchus is also mediated via the activation of soluble guanylyl cyclase and the formation of cGMP (Ellis, 1997; Vaali et al., 1998). The novel NO-budesonide complex (NCX-1020) reduced methacholine-induced contraction of guinea-pig bronchioles in vitro via a cGMP-dependent mechanism (Tallet et al., 2001). In a guinea-pig tracheal preparation, a sulphonylamide NO donor, GEA 3175 (Fig. 1), was found to induce relaxation by increasing cGMP. cGMP appeared to activate a phosphatase enzyme, which finally opened a Ca2+-activated K+ channel (Rydberg et al., 1997). The relaxing effects of other sulphonylamides, GEA 3268 and GEA 5145, and other NO donors, such as glyceryl trinitrate and SNP (Fig. 1), have also been shown to act by opening Ca2+-activated K+ channels (Hamaguchi et al., 1992; Vaali et al., 1998). K+-channel opening causes hyper-

polarisation, which then causes a decrease in voltage-gated Ca2+ channels, which promotes a relaxation response (Archer et al., 1994; Carvajal et al., 2000). The large Ca2+-activated K+ channels, but not the small conductance K+ channels, appear to be involved (Hamaguchi et al., 1992; Ellis & Conanan, 1994a) (Fig. 3). The selective Ca2+-activated K+ channel blocker iberiotoxin has been shown not to completely block NOinduced guinea-pig tracheal smooth muscle relaxation. It has been proposed, therefore, that NO-induced increases in cGMP may relax smooth muscle by mechanisms in addition to Ca2+-activated K+ channels (Vaali et al., 1998; Ellis & Conanan, 1994a). The guanylyl cyclase inhibitor methylene blue in some cases has been found to only partially inhibit NO-induced relaxation. While, the administration of methylene blue to isolated guinea-pig tracheae inhibited SNP-induced increases in cGMP, it did not inhibit SNP-induced relaxation (Sadeghi Hashjin et al., 1995). Similarly, methylene blue was shown to be unable to completely block S-nitroso-Nacetylcysteine-induced guinea-pig tracheal smooth muscle relaxation (Jansen et al., 1992). Furthermore, there was no correlation between relaxation of airway smooth muscle of the cat, ferret, and guinea-pig by SNP and NO solution and increases in cGMP levels (Mahey et al., 1995). In canine tracheal smooth muscle preparations, methylene blue was shown to decrease SNP-induced cGMP accumulation, but paradoxically increased its relaxation effect (Zhou & Torphy, 1991). In another study, the administration of L-NAME to guinea-pig and cat tracheae, while attenuating electrical field stimulation-induced relaxation (iNANC relaxation), did not alter the increase in cGMP levels (Lindsay et al., 1995). These results suggest a cGMP-independent mechanism for NO-induced relaxation. The specificity of methylene blue for inhibiting guanylyl cyclase, however, has been questioned (Wolin et al., 1990; Kontos et al., 1993). The selective guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (ODQ), however, did not cause total inhibition of the NO donor GEA 3175-induced relaxation of bovine-isolated bronchioles (Hernandez et al., 1998). ODQ failed to inhibit SNP-induced relaxation of isolated guinea-pig tracheae (Hjoberg et al., 1999). Exogenous NO and endogenous EDRF were shown to activate single Ca2+dependent K+ channels in cell-free membrane patches without requiring cGMP (Bolotina et al., 1994). These results support the existence of an additional mechanism, other than guanylyl cyclase activation, for NO-induced smooth muscle relaxation. Cl
channels in the apical membrane of airway epithelium have an important role in the regulation of epithelial fluid and electrolyte transport and, therefore, mucus viscosity. The opening of Cl
channels allows Cl
to passively exit the cell, followed by Na+ flow. The secretion of water follows. In cystic fibrosis, the cAMP-dependent Cl
channel cystic fibrosis transmembrane conductance regulator (CFTR) is dysfunctional, due to a genetic mutation of the

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CFTR-associated gene (Kamosinka et al., 1997). The human alveolar carcinoma cell line A549 does not express CFTR mRNA. It, therefore, acts as an appropriate model for the study of alternative Cl
conductance channels in pulmonary epithelial cell lines. The SNO compounds GSNO and SNAP have been shown to increase Cl
channel conductance in the A549 cell line. The specific guanylyl cyclase inhibitor ODQ has been shown to decrease resting Cl
currents (Anderson et al., 1992). These results suggest that NO, acting on guanylyl cyclase, may play an important role in the regulation of Cl
conductance in epithelial cells and, therefore, mucus viscosity in cystic fibrosis (Fig. 3, Table 1).

7. Nitric oxide and histamine release NO release from rat serosal mast cells was detected by measuring platelet aggregation and guanylyl cyclase activation. In this study, reduced histamine release was found to be associated with increases in cGMP or increased NO release (Masini et al., 1991). This suggests that NO reduces histamine release from mast cells (Table 1). The NO donors glyceryl trinitrate, SNP, and isosorbide dinitrate were shown to inhibit histamine release in bovine lung mince (Valentovic et al., 1992). Conversely, in the guinea-pig lungs, histamine, by acting on histamine H1 receptors, induces cNOS-derived NO production (Leurs et al., 1991) (Fig. 2). Histamine and NO, therefore, appear to modulate each others production. Middelveld et al. (2000) monitored sensitised pigs for 90 min after antigen challenge with Ascaris suum antigen. Airway resistance (0– 90 min) was significantly enhanced by pretreating pigs with the NOS inhibitor L-NOARG, while the histamine aerosol response was unaffected. It was suggested that NO exerts its modulatory role by inhibiting histamine release from mast cells. There was, however, no difference in the total amount of histamine detected in the urine from L-NOARG-treated pigs, although there was delayed clearance in L-NOARG-treated pigs. L-NOARG, by reducing EDRF production, may cause vasoconstriction and may reduce the clearance rate of histamine. By reducing the clearance rate of histamine, the metabolism of histamine may increase. This would explain why increased histamine levels were not detected in the urine of L-NOARG-treated pigs. Reduced bronchial vascular conductance through vasoconstriction could also reduce the clearance of bronchoconstrictive mediators, enabling their effects to persist for a longer time. This may be another mechanism by which inhibition of NO may increase airway responsiveness. Inhaled NO reduced histamine-induced bronchoconstriction in the dog (Lindeman et al., 1995), probably because of its bronchodilatory action. Inhaled NO (20 ppm) was administered to anaesthetised pigs from 30 min before A. suum antigen challenge until the experiment was completed (120 min after allergen challenge) (Middelveld & Alving, 2001). Mast cell activation could not be assessed, as histamine levels in BAL fluid were below the detection limit of the

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assay. Inhaled NO, however, did not result in a significant reduction in the EAR, suggesting that endogenously produced NO, but not exogenously administered NO, inhibits mast cell mediator release and the EAR. Administration of LNAME or L-NMMA increased histamine-induced bronchoconstriction in the guinea-pig (Nijkamp et al., 1993; Persson et al., 1995), suggesting that endogenously produced NO modulates airway tone by its bronchorelaxant action. It was hypothesised that if endogenously produced NO exerted mast cell stabilising properties in vivo, inhibition of its production would increase airway responsiveness to the indirect spasmogen AMP (which exerts its effect indirectly by activating mast cells) to a greater extent than the direct spasmogen histamine. However, airway responsiveness was increased to the same extent for both agents when nebulised L-NAME (170 mg) was administered to asthmatic subjects (Taylor et al., 1998b). This suggests that the mast cell stabilising properties of endogenous NO may not be functionally important in human airways. The modulatory role of NO on bronchial tone may be principally due to its direct bronchodilatory action on airway smooth muscle. It is possible, however, that NOS within mast cells was not inhibited by nebulised L-NAME.

8. Other beneficial effects of nitric oxide in the lungs The canine pulmonary artery was relaxed by NO (Masaki et al., 1989). In rat isolated lungs preconstricted with hypoxia, NO was shown to induce pulmonary vasodilatation (Archer et al., 1990) (Fig. 3, Table 1). Isolated pulmonary artery rings from rats were shown to have substantial inherent tone. This tone appears to be attenuated by an EDRF-like factor, which is most likely NO (Wanstall et al., 1995). These results demonstrated the role of NO in modulating pulmonary vascular tone. Impairment of endothelium-dependent pulmonary artery relaxation may occur in COPD (Dinh-Xuan et al., 1991) and in pulmonary hypertension (Loskove & Frishman, 1995; Leeman & Naeije, 1995; Marin & Rodriguez-Martinez, 1997). Inhalation of NO has been shown to reduce pulmonary vascular resistance in ARDS (Johanningman et al., 2001; Rossaint et al., 1993). In COPD, inhaled NO has been shown to reduce pulmonary artery pressure and pulmonary vascular resistance, but with little effect on arterial oxygenation or pulmonary function (Baigorri et al., 1999; Ashutosh et al., 2000) (Table 3). NO was shown to be spontaneously released by cultured rabbit tracheal epithelial cells. The b2-adrenoceptor agonist salbutamol was shown to dose-dependently increase the ciliary beat frequency of these cells. L-NAME, however, inhibited this effect, suggesting that it was mediated via release of NO. L-Arginine reversed the inhibitory effect of LNAME. L-NAME in itself did not increase ciliary beat frequency. It was suggested, therefore, that NO is involved in the increase of the ciliary beat frequency produced by

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Table 3 Potential therapeutic applications of NO-modulating agents Agent

Application

Effect

NO donors

ARDS COPD Pulmonary hypertension

Pulmonary vasodilatation

Asthma Cystic fibrosis

Improves lung function

Cystic fibrosis Asthma COPD

Improves muco-cilliary clearance? Antiviral, antibacterial, anti-parasitic

Asthma COPD Pulmonary hypertension

Pulmonary vasodilatation

Asthma Cystic fibrosis

Bronchodilatation Improves muco-cilliary clearance?

Asthma COPD

Reduces AHR, inflammation, and oedema Improves airway function Reduces muco-ciliary clearance?

cNOS activators

Selective iNOS inhibitors, peroxynitrite scavengers, xanthine oxidase inhibitors

salbutamol, but that it does not modulate resting ciliary motility (Tamaoki et al., 1995). Similarly, in cultured cilia from bovine alveolar tissue, the nonselective b-adrenoceptor agonist isoprenaline and the tachykinins bradykinin and substance P were shown to up-regulate the ciliary beat frequency by a NO-dependent mechanism (Jain et al., 1993). An increase in ciliary beat frequency would aid in the clearance of harmful substances contained within the mucus mesh (Table 1, Fig. 3). As discussed in Section 6, endogenous NO may decrease mucus viscosity by increasing cGMP-dependent Cl
channel conductance (Fig. 3). iNOS-derived NO production has been shown to increase murine nasal transepithelial chloride secretion (Elmer et al., 1999). The absence of constitutive iNOS-derived NO production in epithelial cells of cystic fibrosis airways may play a role in Na+ hyperabsorption (Kelley & Drumm, 1998; Meng et al., 1998), leading to reduced water secretion and increased mucus viscosity. Increased iNOS expression, however, has been demonstrated in the subepithelial tissues of cystic fibrosis lungs (Meng et al., 1998). Corticosteroid treatment has been shown to reduce exhaled NO levels in cystic fibrosis patients, suggesting that iNOS-derived NO is produced in cystic fibrosis (Linnane et al., 2001). The administration of the NO donors SNP and spermine NONOate did not stimulate Cl
secretion by CFTR or any other Cl
conductance pathway in non-cystic fibrosis or cystic fibrosis tissue primary cultured nasal cells, suggesting that exogenously administered NO has little or no effect on ion transport defects in cystic fibrosis (Ruckes-Nilges et al., 2000).

By using the NOS inhibitor L-NMMA and the NO donor (+/
)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro (FK409), endogenously and exogenously administered NO were shown to act as inhibitors of both basal and neurogenic mucus secretion in ferret trachea in vitro (Ramnarine et al., 1996). In the guinea-pig tracheal preparation, however, it has been demonstrated that TNF-a, histamine, O
2 , and PAF stimulated the secretion of mucin (a glycoprotein), the principal constituent of mucus. This effect was inhibited by L-NMMA, but not D-NMMA, indicating that mucin secretion may be increased by NO (Adler et al., 1995). Inhibition of NOS reduced methacholine- and bradykinin-induced secretion of mucus glycoprotein from isolated feline and human submucosal glands. The NO donor isosorbide dinitrate induced an increase in glycoconjugate secretion (Nagaki et al., 1995). An increase in mucus glycoprotein (mucin) secretion without water secretion would result in an increase in viscosity and a reduction in clearance of mucus. In contrast to these findings, however, it has also been demonstrated that the NO donors isosorbide dinitrate and SNP, the precursor Larginine, and the NOS inhibitor L-NAME do not have an effect on radiolabelled mucin secretion in the rat trachea (Bredenbroker et al., 2001). In order to determine whether NO has a potential role in regulating mucus viscosity in cystic fibrosis, it must first be determined whether or not NO increases or decreases mucin secretion and if it does, whether its net effect is to increase or decrease muco-ciliary clearance. NO is important in nonspecific host defence of the respiratory tract and has toxic effects on bacteria, viruses, and parasites (Granger et al., 1988; Green et al., 1991; Kolb &

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Kolb-Bachofen, 1992; Oswald et al., 1994; Barnes & Liew, 1995; Wei et al., 1995; Sanders et al., 1998; Sanders, 1999) (Fig. 3, Table 1). The absence of constitutive iNOS expression in airway epithelial cells in cystic fibrosis patients may make these patients more susceptible to airway bacterial infection, e.g., P. aeruginosa (Kelley & Drumm, 1998; Meng et al., 1998; Thomas et al., 2000). Increased iNOS expression, however, has been demonstrated in the subepithelial tissues of cystic fibrosis lungs (Meng et al., 1998). Corticosteroid treatment has been shown to reduce exhaled NO levels in cystic fibrosis patients, suggesting that iNOS-derived NO is produced in cystic fibrosis (Linnane et al., 2001). It must be noted, however, that the NO-mediated proinflammatory response to infection may oppose these host defence properties and may contribute to airway obstruction and inflammation (Kolb & Kolb-Bachofen, 1992; Heiss et al., 1994; Barnes, 1996; Flak & Goldman, 1996). From these results, it may be suggested that NO may have a potential role in not only regulating airway smooth muscle tone, but also in host defence, regulating ciliary beat frequency, mucus viscosity, and pulmonary vascular tone (Fig. 3, Table 1). These properties may be important in cystic fibrosis and other pulmonary inflammatory disorders (Table 3).

9. Clinical pharmacology of nitric oxide 9.1. Exhaled nitric oxide/S-nitrosothiol Gerlach et al. (1994) suggested that NO exhaled from healthy human volunteers may be largely derived from NO production in the nasopharynx. NO produced in the lower respiratory tract does not appear to contribute to the NO levels in exhaled air. Nasopharynx-derived NO is inhaled, 50 –70% of which is reabsorbed by the lower respiratory tract. The remaining 30 – 50% is then exhaled. The measurement of exhaled NO, therefore, may give a good indication of inhaled NO levels in the lower airways. Persson et al. (1993b) showed that NO in the exhaled air of healthy subjects is derived from the lower airways. By isolating the nasal airways from the lung by intubation, Massarro et al. (1996) found that exhaled NO in both normal and asthmatic subjects is largely derived from the lower airways and not the nasopharynx. A statistically significant reduction in exhaled NO levels in normal, but not asthmatic, subjects, however, was noted after intubation. From the results of Massarro et al. (1996), it may be suggested that nasopharynx-derived NO production accounts for only a marginal level of NO in the exhaled air of healthy subjects, while in asthmatic patients, it is negligible. Similar results have been reported by Kharitonov et al. (1996a). NO has been detected by chemiluminescence in the exhaled air of normal humans, rabbits, guinea-pigs, and rats (Persson et al., 1994). This indicates that NO is present, which may exert a modulatory role on basal smooth muscle

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tone. NO was shown in humans with mild asthma to reduce airway responsiveness to methacholine. However, it was not involved in the regulation of resting airway tone (Ricciardolo et al., 1996). Exhaled NO and nitrosothiol levels are much greater in asthma and COPD patients than in healthy subjects (Alving et al., 1993; Kharitonov et al., 1994; Massaro et al., 1995, 1996; Baraldi et al., 1997; Agusti et al., 1999; Corradi et al., 1999, 2001; Salome et al., 1999; Hanazawa et al., 2000; Al-Ali & Howarth, 2001; Ansarin et al., 2001; Khatri et al., 2001; Silvestri et al., 2001). Asthma patients exhale more NO than COPD patients (Kanazawa et al., 1998; Ansarin et al., 2001). NO, therefore, appears to have a more significant role in asthma than COPD. Ichinose et al. (2000), while reporting an increase in iNOS immunoreactivity in sputum inflammatory cells, did not report an increase in exhaled NO levels in COPD patients. Levels of exhaled NO in COPD may be influenced by tobacco smoking, infection, and inflammation (Bernareggi & Cremona, 1999). In cystic fibrosis patients, however, exhaled and nasal NO levels are decreased (Balfour-Lynn et al., 1996; Dotsch et al., 1996; Grasemann et al., 1999b; Thomas et al., 2000). Oral prednisolone decreased exhaled NO in children with acute asthma (Baraldi et al., 1997). Inhaled beclomethasone and budesonide in mild asthma were also found to reduce exhaled NO levels (Kharitonov et al., 1994; Dupont et al., 1998; Lim et al., 1999). Similarly, inhaled flunisolone was found to be effective in preventing increases in exhaled NO in allergic asthmatic children re-exposed to allergens (Piacentini et al., 2000). In contrast to corticosteroid-naive asthmatics, corticosteroid-treated asthma patients displayed similar airway epithelial iNOS expression as healthy subjects and nearly undetectable levels of nitrotyrosine staining (Guo et al., 2000). The glucocorticosteroid budesonide and the selective iNOS inhibitor aminoguanidine (Table 2) were used to investigate and confirm the involvement of iNOS in elevating NO in exhaled air of asthmatic patients. It has been proposed that the induction of NOS may be responsible for the raised levels of exhaled NO in asthmatic patients, while the lower levels seen in normal subjects may reflect cNOS activity (Kharitonov et al., 1994, 1996b; Yates et al., 1996). Increased immunostaining for iNOS has been demonstrated in bronchial biopsies obtained from asthmatic patients (Hamid et al., 1993). It has been suggested that cNOS activity is up-regulated in asthma, cystic fibrosis, and obliterative bronchiolitis (Belvisi et al., 1995a; Silkoff et al., 2000). It is possible, therefore, that cNOS is also involved in increasing NO levels in exhaled air in asthma patients. However, it would only have a minor role, as once iNOS is stimulated, it produces much larger quantities of NO. Exhaled NO levels were shown to significantly correlate with AHR to histamine in a population sample of young healthy and asthmatic adults (Salome et al., 1999). While exhaled NO correlated with AHR in corticosteroid-naı¨ve asthma patients, it did not correlate with AHR in steroidtreated patients (Dupont et al., 1998). In steroid-treated asthmatic patients, Horvath et al. (1998) did not report a

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correlation between exhaled NO and airway responsiveness to methacholine. Similarly, others have not shown a correlation between exhaled NO and airway responsiveness in steroid-naı¨ve, steroid-treated, and mixed asthma patients. (Ansarin et al., 2001; Lim et al., 2000). Exhaled NO levels in asthma were found to act as a marker of disease control (symptoms within the last 2 weeks, dyspnoea score, daily use of medications, and reversibility of airflow obstruction), but not asthma severity (history of respiratory failure, fixed airflow obstruction, or a validated asthma severity score) (Sippel et al., 2000). Baseline exhaled NO levels in asthmatics were found to be inversely correlated with baseline sGaw, but positively correlated with sGaw, 24 hr following antigen challenge (Khatri et al., 2001). Similarly, a positive correlation between baseline exhaled NO levels in asthma and maximal reduction in FEV1 after exercise has been reported. In the exercise-induced asthma subgroup, however, exhaled NO remained below baseline 10 min after exercise (Terada et al., 2001). These reports appear to suggest that baseline exhaled NO levels in asthma may be deleterious, but that a component of it may be bronchoprotective after allergen inhalation or exercise. In cystic fibrosis patients, nasal or exhaled NO levels did not correlate with FEV1 (Thomas et al., 2000). In COPD, however, exhaled NO was found to be inversely correlated with FEV1 and other markers of lung function (Maziak et al., 1998; Ansarin et al., 2001). Similarly, an inverse correlation between nitrotyrosine staining in sputum inflammatory cells and FEV1 in COPD patients has been reported (Ichinose et al., 2000). Exhaled NO in COPD patients, but not in asthma or cystic fibrosis patients, therefore, may help to determine disease severity. Sensitised asthmatics exposed to relevant domestic allergens had increased levels of exhaled NO in comparison with sensitised unexposed asthmatics (Simpson et al., 1999), suggesting that exhaled NO may serve as a marker for exposure of sensitised asthmatics to domestic allergens. During the pollen season, natural allergen exposure resulted in an increase of exhaled NO in asthmatic grass pollenallergic children. No significant change in FEV1 during or after the pollen season was noted, however (Baraldi et al., 1999). This result suggested that measurement of exhaled NO may act as an early marker for changes in asthma disease activity. Exhaled NO has been found to be higher in allergic asthmatic children than non-allergic asthmatic children. Exhaled NO was also found to significantly correlate with blood eosinophilia in allergic, but not in non-allergic, asthma patients (Silvestri et al., 2001). NO in exhaled air was found to correlate with sputum and peripheral blood eosinophils in atopic asthmatic and asthmatic patients (Horvath et al., 1998; Salome et al., 1999). Similarly, in corticosteroid-dependent stable childhood asthma, Mattes et al. (1999) reported a correlation between exhaled NO and markers of eosinophilic airway inflammation (sputum eosinophil cationic protein, urinary eosinophil protein X, and

sputum eosinophils). A correlation between exhaled NO and sputum eosinophils was also noted in steroid naı¨ve atopic asthma (Jatakanon et al., 1998). Similarly, a significant correlation between exhaled NO and BAL eosinophils was noted after inhaled budesonide treatment in atopic asthmatic patients (Jatakanon et al., 1999). Exhaled NO was found to increase after the dose of corticosteroid was reduced in asthma patients. No change in spirometry or change in peak flow, however, was noted (Kharitonov et al., 1996c). These results support the view that NO in asthma may have proinflammatory effects. Exhaled NO may provide useful information on the degree of eosinophilic inflammation in corticosteroid-treated asthma and, therefore, may be of use in assessing the efficacy and compliance of corticosteroid use in asthma patients. In mild asthma, low-dose theophylline significantly reduced sputum, BAL, and biopsy eosinophils without affecting exhaled NO levels, suggesting that exhaled NO is derived from an inflammatory pathway different from that of eosinophil influx (Lim et al., 2001). Furthermore, the effects of inhaled corticosteroids is doserelated, budesonide, plateauing at a relatively low dose of 400 mcg daily (Lim et al., 1999). In inhaled beclomethasone-treated atopic asthma patients, there was no correlation between exhaled NO and eosinophil levels in mucosal biopsy specimens. There was also, however, no correlation between exhaled NO and eosinophil levels in steroid-naı¨ve asthmatic patients (Lim et al., 2000). In a study where a mild exacerbation of asthma was induced by switching patients from a high dose of inhaled corticosteroid to a low dose of inhaled corticosteroid, exhaled NO, unlike sputum eosinophils, did not act as a marker for predicting loss of asthma control (Jatakanon et al., 2000). However, where there was a complete withdrawal of inhaled corticosteroid, exhaled NO and sputum eosinophil levels similarly predicted the loss of asthma control (Jones et al., 2001). Exhaled NO could serve as a useful marker for monitoring disease severity in COPD. This, however, may be influenced by tobacco smoking, infection, and underlying inflammatory physiology. It appears that exhaled NO levels may not always reflect the extent of inflammation in asthma and may not always serve as a useful marker for corticosteroid efficacy or use. In one study, exhaled NO levels were, in fact, higher in asthma patients who required oral prednisolone than those not receiving such treatment. This result, however, was possibly due to the development of steroid resistance (Stirling et al., 1998). A relationship between exhaled NO levels and mucosal airway inflammation, however, has been reported in asthma patients who have persistent symptoms, despite corticosteroid use (Payne et al., 2001). The measurement of exhaled NO, therefore, may be of use in determining asthma patients who have persistent symptoms associated with airway eosinophilia. Different phenotypes of asthma may exist where the involvement of NO in inflammation and the effects of corticosteroids on NO may vary. The role of exhaled NO in pulmonary disease has also been reviewed by others

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(Bernareggi & Cremona, 1999; Ashutosh, 2000; Berlyne & Barnes, 2000; Hunt & Gaston, 2000; Marshall & Stamler, 2000; Kharitonov & Barnes, 2001; Yates, 2001). 9.2. Administration of L-arginine In asthma and normal human subjects, there is an increase in the activity of endogenous NOS in response to administration (oral and inhaled) of the substrate for NOS, L-arginine (Kharitonov et al., 1995a; Sapienza et al., 1998) (Table 2). Sapienza et al. (1998) showed that these effects are more pronounced in asthmatics. This suggests a decreased substrate availability in asthmatics. They also noted that the effect is dose-related in asthma patients, but not in normal subjects. This supports the view that a deficiency of L-arginine occurs in asthma. Since, the increase in exhaled NO is likely to be derived from increased iNOS activity, the ability of iNOS to produce NO may be limited by a deficiency of substrate. The ability of iNOS, at optimal levels, to modulate airway reactivity in asthma, therefore, may be limited. cNOS-derived NO and its ability to modulate airway tone may be similarly affected. Depletion of L-arginine in nNOS-transfected human kidney 293 cells resulted in the switching of NOS from NO production to O
2 production, leading to nitrotyrosine formation and cellular injury (Xia et al., 1996). In OAchallenged guinea-pigs, inhalation of L-arginine, at 2 and 3 hr after OA challenge, decreased airway conductance immediately and extended the duration of the LAR (Iijima et al., 1998). These results suggest that the administration of Larginine may provide excess NO, which through its proinflammatory action will amplify the late bronchoconstriction. Excess substrate, L-arginine, has been shown in vitro to inhibit cNOS (Su et al., 1997). Since cNOS-derived NO inhibits bronchoconstriction in asthma (De Boer et al., 1998), excess L-arginine, by inhibiting cNOS, may amplify bronchoconstriction. L-Arginine may increase iNOS-derived NO production, nitrotyrosine formation and associated inflammation, oedema, and airway narrowing (Saleh et al., 1998; Iijima et al., 1998; Xiong et al., 1999). Sapienza et al. (1998) demonstrated, however, that the non-substrate amino acid L-alanine produced a similar fall in FEV1 as L-arginine. In contrast to L-arginine, however, L-alanine did not produce a significant increase in exhaled NO. This indicates that there may be instead a nonspecific inflammatory component to bronchoconstriction by both L-arginine and L-alanine. The administration of L-arginine reduced virus-induced AHR (Folkerts et al., 1995) and AHR after the EAR (De Boer et al., 1999). Oral L-arginine (50 mg/kg) administered to asthmatic patients 90 min prior to inhaled histamine challenge did not affect FEV1 and exhaled NO. There was a slight, but significant, decrease in airway response to histamine, but no change in the PC20 (provocative concentration to cause a 20% fall in FEV1), of inhaled histamine (De Gouw et al., 1999). In cystic fibrosis mutant mice, Mhanna et al. (2001) demonstrated that the administration

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of L-arginine compensated for a reduced relaxation response in precontracted tracheal segments following electrical field stimulation. This suggested that a deficiency of NO in cystic fibrosis may be corrected by L-arginine administration. Administration of L-arginine (500 mg/kg) to cystic fibrosis patients, while increasing nasal and exhaled NO levels (but not to normal levels), did not improve lung function (Grasemann et al., 1999a). It may be concluded that L-arginine may have a therapeutic use in pulmonary diseases in which there is a defective production of NO, but not where there is an already excess level of NO (Tables 2 and 3). It appears, however, that the involvement of L-arginine deficiency in asthma is only marginal and insignificant. Cystic fibrosis patients, while being deficient in L-arginine, do not appear to functionally respond to L-arginine administration. 9.3. Administration of inhaled nitric oxide Inhaled NO (50 – 500 ppm) was shown to reduce histamine-induced bronchoconstriction in the dog via a cGMPdependent mechanism (Lindeman et al., 1995). Inhaled NO (300 ppm) was shown to reduce baseline pulmonary resistance in the guinea-pig. In the same study, 5 –300 ppm of NO gas was shown to dose-dependently reduce methacholine (i.v.)-induced increases in pulmonary resistance and also to increase lung compliance (Dupuy et al., 1992). In the rabbit, inhaled NO (80 ppm) blocked nebulised methacholineinduced increases in airway resistance. However, it did not alter baseline pulmonary resistance (Hogman et al., 1993a). Hyperreactive subjects may be defined as those who have normal baseline FEV1 and sGaw, but display hyperreactivity to methacholine provocation (Hogman et al., 1993b). Asthmatic patients differ from hyperreactive patients in that they do not have normal baseline FEV1 and sGaw before challenge. In patients with hyperreactive airways, inhaled NO was shown to reduce the decrease of sGaw in response to methacholine. Inhalation of NO caused a small increase in basal sGaw in asthmatic patients (Hogman et al., 1993b). Inhalation of NO (100 ppm) by mild asthmatic patients was also shown to result in a minor, but significant, relaxation of airway tone against methacholineinduced bronchospasm (Kacmarek et al., 1996). Inhalation of NO was found to cause no change in basal sGaw in patients with COPD (Hogman et al., 1993b). Inhaled NO (20 ppm, 20 min), while causing a reduction in pulmonary vascular resistance, did not affect COPD symptoms and pulmonary function (Baigorri et al., 1999). Similarly, inhaled NO did not have any effect on lung function in cystic fibrosis patients (Ratjen et al., 1999). From these results, inhalation of NO was shown to act as a weak bronchodilator in patients with reversible airway obstruction. Why it does not have a bronchodilatatory effect in COPD and cystic fibrosis is not fully understood. It may be due to a different inflammatory physiology. In COPD and cystic fibrosis, there could have been increased oedema or increased mucus viscosity that may have prevented inhaled

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NO from exerting its bronchodilatatory effect (Hjoberg et al., 1999; Hogman et al., 1998). The above results support the claim that NO is a bronchodilator and that its use may be of benefit to those with reversible airway disease (Table 3). 9.4. Administration of nitric oxide donors 9.4.1. Nitroglycerine Nitroglycerine (glyceryl trinitrate) has been shown to spontaneously release NO in buffer solution (Feelisch & Noack, 1987) and to increase cGMP in bovine tracheal smooth muscle (Hamaguchi et al., 1992). In cultured vascular smooth muscle cells and endothelial cells from various species, it was demonstrated that nitroglycerine, by generating NO, increases cGMP (Feelisch & Kelm, 1991). Nitroglycerine is a potent relaxant of guinea-pig tracheal smooth muscle (Jeong et al., 1978). Before cardiopulmonary bypass, intravenous nitroglycerine (4.0 + 0.3 mg/kg) administered to 12 patients without reversible airway obstruction significantly decreased the pressure within a water-filled tracheal cuff (Byrick et al., 1983). This demonstrated the ability of nitroglycerine to relax human tracheal smooth muscle. Sublingual nitroglycerine (1.2 mg) administered to 10 patients with acute asthma did not significantly improve FEV1 and forced vital capacity (Kennedy et al., 1981). Three patients experienced transient, but severe, hypotension. Similarly, in asthmatic patients receiving 0.4 mg of nitroglycerine sublingually (Miller & Schultz, 1979), the changes in FEV1 were insignificant. Some of the patients noted headache and flushing. In contrast, Goldstein (1984) reported that 0.15 mg of sublingual nitroglycerine given to a 12-year-old steroid-treated asthmatic boy significantly improved FEV1 and also improved symptoms 5 min later. This boy was then given a transdermal patch (25-mg nitroglycerine) daily, while his steroid dose was tapered to nothing. Only very mild intermittent dizziness with no orthostatic hypotension was noted. Five minutes after administration, 0.2 mg of inhaled nitroglycerine produced a moderate improvement in FEV1 in patients with moderate asthma (Rolla et al., 1995b). This was significantly higher when patients were pretreated with the vasoconstrictive agent noradrenaline. This suggests that bronchodilatation induced by nitroglycerine is limited by bronchial vasodilatation (Table 1). NO-induced vasodilatation may increase the clearance of bronchoconstrictive agents. This beneficial effect, however, may be offset by an increase in vasodilatation-associated exudation, resulting in airway narrowing. Although immediate effects may be observed, no significant difference in FEV1 was noted 10 min after nitroglycerine inhalation. The short-acting effect of this dose of nebulised nitroglycerine limits its usefullness. 9.4.2. Isosorbide dinitrate Isosorbide dinitrate has been shown to spontaneously release NO in buffer solution (Feelisch & Noack, 1987). A

correlation of isosorbide dinitrate-induced increases in cGMP levels and NO formation has also been demonstrated (Feelisch & Kelm, 1991). It has been suggested that isosorbide dinitrate induces vasodilatation in endothelialdenuded rabbit aortic rings by a cGMP-dependent mechanism (Dona et al., 1995). The administration of 5-mg sublingual isosorbide dinitrate to patients with bronchial asthma resulted in a decrease in mean respiratory resistance and an increase in both mean vital capacity and FEV 1 5 min after administration (Okayama et al., 1984). These effects lasted for 60 min. Headache as a side-effect was noted. 9.4.3. S-nitrosothiols In comparison with normal subjects, tracheae of asthmatic children in respiratory failure were shown to have substantially lower levels of SNOs and thiols (obtained by tracheal suctioning) than normal subjects (Gaston et al., 1998). Gaston et al. (1998) suggested that a low concentration of SNO may be explained by increased catabolism of SNO compounds to NO. This is supported by the increase of NO in exhaled air of asthma patients. Dweik et al. (2001) similarly reported lower SNO and increased nitrite levels in BAL fluid from asthmatics in comparison with healthy controls, suggesting increased catabolism of SNOs in asthmatic patients. It was suggested that the increase in catabolism of SNOs is a consequence of increased oxidative stress and inflammation in asthma (Gaston et al., 1998; Dweik et al., 2001). In support of this, it has been shown that the ratio of SNO to NO in airway aspirate fluid may be dependent on NO concentration, oxygen tension, thiol concentration, pH, and redox state (Gaston et al., 1993). A route of enzymatic degradation of SNO compounds was shown earlier (Section 5.3) to be via O
2 generation from xanthine oxidase. Thirty minutes following a final OA or phosphate-buffered saline (control) challenge in vivo, guinea-pig isolated tracheal rings were set up and constricted with methacholine. GSNO gave a reduced relaxation response in the OA-challenged group compared with the control group. Two soluble proteins associated with increased catabolic activity of GSNO and reduced relaxation response to GSNO were found in the OA-challenged group, suggesting that increased enzymatic activity may mediate increased catabolism and reduction in relaxation response to GSNO in this animal model of asthma (Fang et al., 2000). Whether this is important in human asthma has yet to be determined. The induction of iNOS in asthma may lead to an increase in SNO synthesis. This up-regulation of SNO synthesis is, however, more than offset by an increase in its catabolism. The increase in production of SNOs may decrease the thiol concentration. This explains the reduction in thiol concentrations seen in asthmatic patients (Gaston et al., 1998). Smith et al. (1993), however, reported increased levels of the thiol glutathione in BAL from patients with asthma. In comparison with normal and mild asthma patients, Corradi et al. (2001) found increased nitrosothiol

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levels in exhaled air in patients with severe asthma and cystic fibrosis. Increased nitrotyrosine and nitrite in exhaled breath condensate in cystic fibrosis has also been noted (Ho et al., 1998; Balint et al., 2001). Corticosteroid treatment has been shown to reduce exhaled NO levels in cystic fibrosis patients, suggesting iNOS-derived NO production in cystic fibrosis (Linnane et al., 2001). SNO levels in BAL were shown to be decreased in cystic fibrosis, while the metabolites of NO, nitrate and nitrite, were normal (Grasemann et al., 1999b). In cystic fibrosis patients, a reduction of NO levels in exhaled air and NO metabolites in sputum has also been reported (Balfour-Lynn et al., 1996; Dotsch et al., 1996; Grasemann et al., 1998). It appears, therefore, that in cystic fibrosis, the relationship between exhaled NO, NO metabolite levels, and SNO levels is more complex than that seen in asthma. An increase in exhaled NO has been reported in COPD (Kanazawa et al., 1998; Agusti et al., 1999; Corradi et al., 1999; Ansarin et al., 2001). An increase in plasma NO metabolites nitrate and nitrite and a reduction in plasma thiols has also been reported in patients undergoing hospital treatment for COPD and chronic respiratory insufficiency (Atzori et al., 1997). An increase in the production of NO, associated with inflammation, and an increase in the metabolism of NO and SNO, associated with oxidative stress, may explain these results in COPD. As discussed in Section 5.3, SNOs are thought to act as NO carriers from which NO is enzymatically released at cellular membrane components, from where it acts on airway smooth muscle. Therefore, a change in NO/SNO ratio in pulmonary disease may decrease the ability of NO to reach its target and, therefore, decrease its bronchodilatory effect. This may be important in pulmonary disease. The administration of SNOs may be of use in compensating for the increase in catabolism of these compounds (Richardson & Benjamin, 2002). SNOs have been shown to relax isolated guinea-pig tracheal and isolated human bronchial preparations (Jansen et al., 1992; Gaston et al., 1993, 1994a; Bannenberg et al., 1995). Bannenberg et al. (1995) demonstrated that perfusion of GSNO and other structurally different SNOs via the pulmonary circulation or inhalation of GSNO resulted in a reduction in methacholine-induced bronchoconstriction in guinea-pig isolated, ventilated lungs. Similarly, the bronchoprotective effects of SIN-1 against acetylcholine in healthy anaesthetised guinea-pigs was enhanced by exogenous glutathione administration (Kanazawa et al., 2000). A reduction in thiols has been observed in asthma (Gaston et al., 1998). This suggests that the administration of thiols, such as glutathione and cysteine, may increase endogenous levels of SNOs in asthma and other pulmonary diseases (Table 2). 9.4.4. Other nitric oxide donors Other NO donors have also been investigated. SIN-1 has been shown to relax guinea-pig trachea and human bronchus (Ellis & Conanan, 1994a, 1994b; Ellis, 1997). It has also been shown to be bronchoprotective against exogenous

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acetylcholine in anaesthetised guinea-pigs (Kanazawa et al., 2000). This is thought to be mediated via peroxynitrite formation, followed by the formation of SNO-like compounds. In asthma and other pulmonary diseases, peroxynitrite is thought to be at an excessive deleterious level. Therefore, the further addition of peroxynitrite in the form of a NO donor such as SIN-1 may be undesirable. The sulphonylamides GEA 3175, GEA 3268, and GEA 5145 were shown to relax bovine, guinea-pig, and rat isolated bronchioles and guinea-pig trachea (Vaali et al., 1996; Rydberg et al., 1997; Hernandez et al., 1998). They have been shown to be more potent than the conventional NO donors SNP, SIN-1, and SNAP (Vaali et al., 1996; Hernandez et al., 1998). 9.5. 2-adrenoceptor agonists and nitric oxide donors b2-Adrenoceptor agonists such as salbutamol exert their bronchodilating properties by stimulating cAMP production. NO is thought to relax smooth muscle by increasing cGMP. Heaslip et al. (1987) demonstrated that cAMP- and cGMP-dependent mechanisms induce relaxations of the guinea-pig tracheae that are functionally additive. Therefore, it may be suggested that co-administration of a b2-adrenoceptor agonist and a NO donor may produce an additive bronchodilating response. Indeed, Rolla et al. (1995a), in moderate asthmatics, showed that co-administration of inhaled salbutamol and inhaled nitroglycerine (0.2 mg) gave an additive bronchodilating effect over salbutamol administration alone. The duration of this additive effect post nitroglycerine inhalation was only studied for the first 15 min after exposure. In another experiment, 6 mg of nebulised nitroglycerine was administered to 18 patients with severe asthma. FEV1, forced vital capacity, and peak expiratory flow were increased by nitroglycerine administration. The bronchodilating effects of nitroglycerine remained significantly greater compared with placebo 5, 15, and 30 min after administration. Co-administration of nitroglycerine and salbutamol gave additive bronchodilating effects over salbutamol administration alone. The administration of nitroglycerine alone, however, was shown to be a weak bronchodilator in comparison with salbutamol alone. Fifteen of the 18 patients reported mild to moderate headache, and 4 reported dizziness. A postural change in heart rate and a reduction in systolic blood pressure were also noted (Sharara et al., 1998). Inhalation of b2-adrenoceptor agonist immediately after NO inhalation gave a slightly greater increase in sGaw over b2-agonist administration alone (Hogman et al., 1993b). Inhalation of a novel salbutamol-NO complex (NCX-950) showed greater bronchodilator activity than the parent salbutamol hemisulphate in inhibiting histamine-induced bronchoconstriction in conscious, healthy guinea-pigs (Toward et al., 2001). These findings further support the suggestion that the co-stimulation of cGMP and cAMP may give additive bronchodilatory effects over cAMP stimulation alone.

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In summary, it appears that NO donors may have an important role as bronchodilators. Due to the superior bronchodilatory effects of b2-adrenoceptor agonists, the administration of NO donors alone appears unlikely. Their concomitant use with current bronchodilator therapy, however, may prove useful. NO donors with a prolonged duration of action with minimal side-effects may be of particular benefit. 9.6. Glucocorticosteroids and nitric oxide Bronchial biopsy specimens obtained from asthma patients taking inhaled corticosteroids and b2-adrenoceptor agonists showed reduced immunoreactivity for iNOS compared with asthma patients taking b2-adrenoceptor agonists without corticosteroids (Redington et al., 2001). Similarly, inhaled flunisolone [4 puffs (1000 mg) daily] and inhaled b2adrenoceptor agonist [metaproterenol (orciprenaline) 2600 mg daily] for 3 weeks reduced iNOS expression in airway epithelium of normal human airways. Airway epithelial cells were obtained by bronchial brushing, and iNOS expression was measured by northern analysis (Guo et al., 1995). From these results, it may be suggested that iNOS expression is reduced by glucorticosteroid therapy. In support of this, it has been demonstrated that the steroid dexamethasone may inhibit cytokine stimulation of iNOS mRNA in human and murine cultured epithelial cells. This inhibition is thought to occur at a transcriptional level (Robbins et al., 1994a, 1994b; Guo et al., 2000) and may involve inhibition of activated nuclear factor-kB (Barnes & Adcock, 1997). In IL-1b-stimulated mesangial cells, however, the dexamethasone-induced reduction in iNOS gene transcription was offset by a prolongation in the half-life of iNOS mRNA. A demonstrable reduction in iNOS mRNA translation and increased degradation of iNOS protein, however, was noted, suggesting that it was through these two mechanisms by which dexamethasone inhibits iNOS protein expression (Kunz et al., 1996). While glucocorticosteroids appear to inhibit iNOS expression, it is important to note, however, that glucocorticosteroids do not inhibit cNOS expression (Radomski et al., 1990). Inhaled beclomethasone dipropionate or budesonide were shown to reduce increases of exhaled NO in asthma to levels seen in normal subjects (Kharitonov et al., 1994, 1996b; Alving et al., 1995). Inhaled fluticasone propionate, flunisolide, and budesonide were also shown to significantly reduce exhaled NO in asthma patients (Alving et al., 1995; Kharitonov et al., 1996b; Van Rensen et al., 1999; Piacentini et al., 2000). Oral prednisolone (1 mg/kg daily) similarly reduced exhaled NO in children with acute asthma (Baraldi et al., 1997). There was no significant difference in increased exhaled NO levels and other inflammatory markers between COPD patients using corticosteroids and those not using corticosteroids (Keatings et al., 1997; Ansarin et al., 2001). Similarly, in cystic fibrosis, there was no significant difference in decreased exhaled NO or

increased nitrotyrosine levels in breath condensate between patients taking corticosteroids and those not taking corticosteroids (Balint et al., 2001). These contrasting results may have been due to different underlying inflammatory processes occurring in COPD, cystic fibrosis, and asthma. Hanazawa et al. (2000) reported an increase in exhaled nitrotyrosine in mild asthmatic patients. These patients were not taking corticosteroid therapy. In comparison with normal subjects, moderate and severe asthmatic patients, who were taking inhaled or oral corticosteroid therapy, were shown to have reduced nitrotyrosine levels in exhaled air. Similarly, Saleh et al. (1998) demonstrated that there was a significant reduction in nitrotyrosine immunoreactivity in bronchial biopsies of asthmatic patients who were taking budesonide. Glucocorticosteroids reduce leukocyte infiltration (Duddridge et al., 1993) and increased airway responsiveness (Burke et al., 1992) in asthmatic subjects. Nitrotyrosine formation has been associated with AHR (Saleh et al., 1998; Van Rensen et al., 1999) and inflammation (Iijima et al., 2001). Glucocorticosteroids by inhibiting the induction of iNOS reduce NO and nitrite production. Inhaled corticosteroids have also been shown to reverse the reduced SOD activity in asthma (De Raeve et al., 1997). This leads to reduced nitrotyrosine formation. It is possible that the success of glucocorticosteroids in asthma may be partially due to their ability to inhibit the induction of iNOS. These results support the view that glucocorticosteroids may exert a modulatory role on excess NO levels in asthma (Table 2). The ultimate goal in this area is to develop a safe iNOS inhibitor as an alternative to glucocorticosteroids (Barnes, 1993). This would allow the evaluation of the therapeutic effects of inhibiting excess iNOS-derived NO in human asthma.

10. Clinical applications The bronchodilatory effects of NO (and/or SNO) donors in conjuction with concurrent b2-adrenoceptor agonist therapy may be beneficial in asthma and cystic fibrosis. NO appears to also play a role in the regulation of mucus viscosity and muco-ciliary clearance, and thus, NO and NO donors may be particularly useful in cystic fibrosis. The pulmonary vasodilatory properties of NO may be of use in ARDS and other conditions associated with pulmonary hypertension (Table 3). However, its vasorelaxant properties by causing oedema and airway narrowing may limit its bronchodilatatory action. While some iNOS-derived NO may be beneficial through bronchodilatory action, excess iNOS-derived NO may cause inflammation, exudation, and consequent airway narrowing. The deleterious effects of iNOS-derived NO appears to be caused by its ability to form peroxynitrite and nitrate tyrosine residues (nitrotyrosine) and its ability to reduce IFN-g. The partial inhibition of the induction or activity of iNOS may provide a method of attaining an

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optimum level of NO. Potential drug targets with the aim of attaining this optimum level are summarised in Fig. 2 and Table 2. Whether the ability of glucocorticosteroids to improve asthma symptoms is partially due to their ability to inhibit iNOS is unknown. Inhibitors of the induction or activity of iNOS may have a role in the treatment of the inflammatory component of pulmonary disease. Xanthine oxidase forms O
2 by oxidation of purines and of NADH (Sanders et al., 1997). O
2 may then react with NO to form peroxynitrite (Fig. 2). The development of SOD mimetics to remove O
2 (Salvemini et al., 1999), peroxynitrite scavengers, or xanthine oxidase inhibitors may assist in limiting the unwanted effects of NO via this route. Xanthine oxidase inhibitors may also reduce the O
2 induced decomposition of SNO compounds (Fig. 2). The administration of thiols may limit the cytotoxic effects of excess peroxynitrite (Fig. 3). Where there is a deficiency of cNOS-derived NO, the administration of L-arginine or the development of calmodulin agonists (Villain et al., 2000) or selective cNOS inducers may be useful. The administration of L-arginine, however, has not been proven successful in asthma or in cystic fibrosis. Whether the chronic administration of NO promotes or attenuates inflammation in asthma and COPD has yet to be determined. Further work is also required with regard to the involvement of NO in the EAR and the LAR. Drugs that act on the NO synthetic and metabolic pathway may be used to investigate NO levels in the lungs. The ultimate goal is to obtain optimum levels of NO in the lungs, where the beneficial effects of NO are utilised while minimising its deleterious pro-inflammatory effects. It may be then that the true potential of NO therapy in asthma, COPD, cystic fibrosis, and other pulmonary diseases may be realised and understood.

Acknowledgements We wish to thank Nicox S.A., Sophia Antipolis, France for financial support with a studentship to B.J.N. and to Dr. Jean-Luc Burgaud of Nicox S.A. for his encouragement and for suggesting this review.

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