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Nitric Oxide in Health and Disease of the Respiratory System

Pulmonary Department, G. Gaslini Institute, Genoa, Italy; Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands; Department of Pediatric Respiratory Medicine, University of Virginia Health System, Charlottesville, Virginia; and Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

ABSTRACT

I. INTRODUCTION

A. Historical View

B. Bioactive Forms of NO

C. Regulation of NOS

D. Localization of NO in the Airways

1. eNOS (NOS III)

2. nNOS (NOS I)

3. iNOS (NOS II)

E. Arginine Uptake and Metabolism

F. Molecular Action of NO

G. Regulation of SNO-Mediated Bioactivities

II. NITRIC OXIDE AND PHYSIOLOGY OF THE RESPIRATORY SYSTEM

A. NO and Lung Development

B. NO and Transcriptional Regulation in the Lung

C. NO and iNANC

D. NO and Airway Smooth Muscle Relaxation

E. NO Against Airway Smooth Muscle Contraction

1. In vivo studies

2. In vitro studies

F. NO and Pulmonary-Bronchial Circulations

1. NO and pulmonary circulation

2. NO and bronchial circulation

G. NO and Mucus-Electrolyte Secretions in the Airways

III. NITRIC OXIDE AND OXIDATIVE STRESS: ''NITROSATIVE STRESS''

A. Formation of RNS

B. Airway Damage by ''Nitrosative Stress''

IV. EXHALED NITRIC OXIDE

A. Exhaled NO and Bronchial Asthma

B. Exhaled NO and Other Respiratory Disorders

V. NITRIC OXIDE AND PATHOPHYSIOLOGY OF THE RESPIRATORY SYSTEM

A. NO and Immune-Inflammatory Responses in the Airways

1. NO and cytokine networks

2. NO and T cells

3. NO and Th2-mediated inflammation in asthma

B. NO and Airway Hyperresponsiveness

C. NO and Cell Proliferation-Survival in the Airways

1. NO and airway remodeling

2. NO and posttransplant obliterative bronchiolitis

3. NO effects on apoptosis

D. NO and Lung Cancer

VI. INHALED NITRIC OXIDE

VII. CONCLUSIONS AND FUTURE PERSPECTIVES

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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A. Historical View

The small, light, and simple molecule nitric oxide (NO) was once regarded only as a noxious environmental pollutant in cigarette smoke, smog (317), and the exhaust from motorcars, destroying the ozone layer and causing acid rain (68). This bad reputation of NO changed when in the 1980s several lines of research showed that NO is an essential molecule in the physiology of the human body.

Early studies demonstrated that endothelial cells are able to release a labile factor, named as endothelium-derived relaxing factor (EDRF), that diffuses to the adjacent muscle layer and relaxes it (124) at least in part stimulating the formation of cGMP (359). Similarly, biochemical experiments showed that nitroglycerin elicits blood vessel relaxation after its conversion to NO with the subsequent formation of cGMP (299). Finally, in 1987, the proof that NO was similar to EDRF (190, 331) was provided. Subsequently, the importance of NO and other nitrogen oxides in the regulation of various body functions, including platelet aggregation (357) and neurotransmission (40), emerged. Eventually, this set of observations was honored by the Nobel prize in 1998.

Shortly after the publication of landmark papers proposing EDRF to be NO, several investigators made observations suggesting that nitrogen oxides are relevant to respiratory biology. First, Pepke-Zaba et al. (339) initiated a successful trial using inhaled NO (ppm concentrations) as a selective pulmonary vasodilator. Simultaneously, Gustafsson et al. (156) measured endogenous NO (ppb concentrations) in the exhaled air of humans and other mammals. Working independently, Gustafsson's group (345) and three other groups (12, 130, 236) found that NO concentrations were higher than normal in patients with asthma, but low in patients with cystic fibrosis; there was great excitement when these parallel findings were reported at the Biology of Nitric Oxide meeting in Cologne in 1992.

The increased NO levels in exhaled air of asthmatic patients might be explained by an overexpression of the enzyme that synthesizes NO (162, 242). NO can be produced by a number of cells in the airways such as endo- and epithelial cells and inflammatory cells. However, these data regarding endogenous NO in the lung represented a series of paradoxes. For example, how could the alveolar space contain NO if it was thought to "sump" out NO by virtue or hemoglobin reactivity? Or more importantly, why are the concentrations measured in expired air three log orders lower than those used to decrease pulmonary vascular resistance? A tremendous amount of research has subsequently been devoted to addressing the troubling paradoxes of pulmonary NO biology; however, many questions remained unanswered. As an example, Beall et al. (30) have recently suggested that concentrations of NO as low at 200 ppb may be relevant to subtle regulation of oxygen uptake in the lungs, but no role has been directly demonstrated for NO gas itself at physiological concentrations. In this regard, it has been argued from the time of the first studies in endogenous nitrogen oxide biology that NO itself may not be the only, or indeed the most important, product of NO synthase (NOS) activation relevant to respiratory physiology (126, 307).

In addition, NO acts also as a neurotransmitter of the inhibitory nonadrenergic noncholinergic (NANC) nerves. In human central and peripheral airways in vitro, NO appears to account for the bronchodilator NANC response (32, 92). Therefore, a physiological function of NO in the airways might be dilatation of bronchial smooth muscle. It has been known for more than half a century that nitrates induce bronchial relaxation (143). NO and NO donors relax human airway smooth muscle in vitro (151, 438), and a bronchodilatory effect of inhaled NO was demonstrated in guinea pigs and humans during methacholine-induced bronchoconstriction (85, 210).

The other way around, inhibition of NO formation increases airway responsiveness to contractile agents in animals and asthmatic patients (315, 365). Again, we face a paradox in pulmonary nitrogen oxide biology here: although the concentrations of exhaled NO are increased in patients with asthma, airway responsiveness is increased instead of suppressed. During the last few years several studies have been performed to assess the relationship between levels of exhaled NO and lung function parameters or other markers of airway inflammation.

B. Bioactive Forms of NO

NO itself has a short half-life in vivo (1–5 s) because of its reactivity with hemoglobin (223, 266, 419) and a broad spectrum of other biological compounds. It has one unpaired electron, making it a free radical that avidly reacts with other molecules such as oxygen, superoxide radicals, or transition metals. NO may be formed and/or bioactivated as nitroxyl (NO^–) or nitrosonium (NO⁺). These chemical species have short half-lives in aqueous solution (<1 s) but are stabilized in biological complexes with thiols (RS^–... ⁺NO), nitrite (O₂N^–... ⁺NO), and other targets and intermediates (404). Here, we will refer to NO·, NO⁺ and NO^– as "NO", unless specified otherwise. NO is an ubiquitous messenger molecule that affects various biological functions, either at low concentrations as a signal in many physiological processes such as blood flow regulation, platelet reactivity, NANC neurotransmission, and central nervous system memory or at high concentrations as cytotoxic and cytostatic defensive mechanisms against tumors and pathogens (for references, see Ref. 298). Many studies demonstrated a significant role for these nitrogen oxides in modulating pulmonary function and in the pathogenesis of various pulmonary diseases (27, 128, 209). Moreover, NO has been detected in exhaled air of animals and humans (156), and the NO concentrations are changed in different inflammatory diseases of the airways such as asthma (12, 126, 345).

Reactions of NO ultimately lead to the nitration (addition of -NO₂), nitrosation (addition of -NO⁺), and nitrosylation (-NO) of most classes of biomolecules. One of the best known interactions of NO leading to cell signaling is the reversible covalent binding, nitrosylation, with the ferrous heme in soluble guanylyl cyclase. Another aspect of NO signaling are S-nitrosothiols (SNO) that appear to be important molecules signaling "NO" bioactivity in the lung. SNOs are products of NOS activation that are present in the airway lining fluid in micromolar concentrations, stored in specific cellular compartments to achieve bioactivity and metabolically regulated to deliver bioactivities both through transnitrosation reactions and through release of NO.

C. Regulation of NOS

NO and related compounds are produced by a wide variety of residential and inflammatory cells in the airways (129). NO itself is generated via a five-electron oxidation of terminal guanidinium nitrogen on the amino acid L-arginine (Fig. 1). The reaction is both oxygen- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and yields the coproduct L-citrulline in addition to nitroxyl (NO^–), in a 1:1 stoichiometry (174, 392). The enzyme system responsible for producing NO, first functionally identified in 1990 by Bult et al. (46), is NOS, which exists in three distinct isoforms: 1) constitutive neuronal NOS (NOS I or nNOS); 2) inducible NOS (NOS II or iNOS); and 3) constitutive endothelial NOS (NOS III or eNOS). Protein purification and molecular cloning approaches have identified the three distinct isoforms of NOS. nNOS, iNOS, and eNOS are products of distinct genes located on different human chromosomes (12, 17, and 7 chromosomes, respectively), each with a characteristic pattern of tissue-specific expression (252). All of the three NOS isoforms are expressed in the airways (108, 162, 242, 374, 397).

View larger version (33K): [in this window] [in a new window]   FIG. 1. Simplified over view on L-arginine uptake and metabolism. L-Arginine is transported into the cell via the cationic amino acid transport (CAT) system and can be metabolized by 2 groups of enzymes. Nitric oxide synthase (NOS) converts L-arginine in two steps to nitric oxide (NO) and L-citrulline with NG-hydroxy-L-arginine as an intermediate. L-Citrulline can be converted by argininosuccinate to L-arginine. Constitutive (c)NOS is activated by an increase in intracellular Ca2+ concentrations. Arginase metabolizes L-arginine to L-ornithine. Lipopolysaccharide (LPS) and several cytokines increases both L-arginine transport and arginase activity. NG-hydroxy-L-arginine decreases the arginase activity. NO can bind thiol groups leading to S-nitrosothiols (R-SNO). As indicated in the text, both NO and S-nitrosothiols have a variety of physiological effects.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Overview of the signal transduction pathway leading to the increased expression of inducible nitric oxide synthase (iNOS). A variety of stimuli cause tyrosine kinase activation with subsequent activation of nuclear transcription factor NFB via phosphorylation and degradation of inhibitory (I)B. NFB will accordingly be translocated to the nucleus, and this will lead to mRNA transcription of the iNOS gene. Translation of iNOS mRNA will take place with assembly of the iNOS protein as a result. L-Arginine will be metabolized to L-citrulline and nitric oxide (NO). As described in the text, NO generated by iNOS has beneficial effects (i.e., host defense) but also a number of harmful effects.

Whereas transcriptional regulation of iNOS has been established for 10 years, no expressional regulation was originally known for the other two isoforms. More recent evidence suggests, however, that the expression of nNOS and eNOS can also be regulated under various conditions. nNOS mRNA transcripts and/or protein have been detected in specific neurons of the central and peripheral nervous systems and in nonneuronal cell types such as airway epithelial cells (114). The subcellular localization of nNOS protein varies among the cell types studied. In neurons, both soluble and particulate protein is found. nNOS expression can be dynamically regulated by various physiological and pathological conditions (114). nNOS mRNA upregulation seems to represent a general response of neuronal cells to stress induced by a large array of physical, chemical, and biological agents such as heat, electrical stimulation, light exposure, and allergic substances. Enhanced nNOS expression is often associated with coinduction of transcription factors such as c-jun (455) and c-fos (422).

While iNOS has been characterized as a soluble (cytosolic) protein, eNOS is targeted to Golgi membranes and plasmalemmal caveolae (small invaginations in the plasma membrane characterized by the presence of the transmembrane protein caveolin). This complex process is probably dependent on myristoylation, palmitoylation, and tyrosine phosphorylation of the enzyme as well as protein-protein interactions with caveolins (292). In endothelial cells it has been demonstrated that the association between eNOS and caveolin suppresses eNOS activity. After agonist activation the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) promotes calmodulin binding to eNOS and the dissociation of caveolin from eNOS. eNOS-calmodulin complex synthesizes NO until [Ca²⁺]_i decreases and then the inhibitory eNOS-caveolin complex reforms (292). Interestingly, estrogen upregulates and activates eNOS in endothelial cells. 17-Estradiol increases NO-dependent dilatation of rat pulmonary arteries and thoracic aorta (142), and estrogen acutely stimulates eNOS in H441 human airway epithelial cells (239). An exciting aspect of this emerging area of study is that estrogen, NO, and caveolae research fields have merged to identify a novel clinical relevant molecular process (468).

D. Localization of NO in the Airways

1. eNOS (NOS III)

Soon after the identification of NO as a messenger molecule generated by endothelial cells, a calcium- and L-arginine-dependent enzyme was identified, and >95% of its activity was sequestered in the particulate fraction of the endothelial (115). Indeed, after the enzyme had been cloned and sequenced (202), and specific antisera for the endothelial isoform of NOS had become available, abundant eNOS immunoreactivity was found in endothelial cells of pulmonary blood vessels. A recent review describes that eNOS is localized to endothelial caveolae by palmitoylation (395).

eNOS is constitutively expressed in human bronchial epithelium (397) and in type II human alveolar epithelial cells (337). Immunoreactivity for eNOS is also localized in the epithelium of human nasal mucosa (219). Ultrastructural studies revealed that eNOS is localized at the basal membrane of ciliary microtubules (458), where it is thought to contribute to the regulation of ciliary beat frequency (197).

2. nNOS (NOS I)

nNOS (NOS I) is localized in airway nerves of humans (78, 106, 152, 242, 271) and animals (77, 78, 152, 242, 271, 428). Substantial species differences are apparent with regard to the extent of innervation and origin of nerve fibers. In human airways, nerve fibers containing nNOS have been shown both by immunohistochemistry and NADPH-diaphorase histochemistry (106, 242, 437). These nerve fibers are present in the airway smooth muscle, where NO is the major mediator for the neural smooth muscle relaxation (32, 258). The density of these nerve fibers decreases from trachea to small bronchi (106), which is associated with a reduced neural bronchodilatation (92, 437) mediated by the inhibitory NANC (iNANC) system (446). Colocalization with vasoactive intestinal polypeptide (VIP) is frequently observed (250). In human airways, NOS-containing nerve fibers are present around submucosal glands (106), although their functional role for the regulation of glandular secretion is not clear yet. In the lamina propria, NO has potent dilatory effects on blood vessels and on the regulation of plasma extravasation (94).

The cell bodies of these neurons innervating human airways are localized predominantly in the local parasympathetic ganglia (78, 107). Additional sources of NOS immunoreactive nerve fibers are found in vagal sensory and sympathetic ganglia (107, 249, 326). NOS immunoreactive neurons are present in vagal sensory ganglia in humans (50, 107, 249) and in rats (7). In sensory neurons, NO could act as a neuromediator both at the central ending and the periphery (382).

In the central nervous system, reports identified nNOS activity in the cytosolic fraction (114). However, a PDZ-domain has been found in the NH₂-terminal nNOS. [The abbreviation PDZ derives from the first three proteins PSD-95/SAP90, Dlg, and ZO-1 in which these domains were identified (244).] This domain is responsible for the membrane attachment of nNOS through an interaction with the postsynaptic density proteins (PSD) 95 and 93 (42). nNOS is also present in nonneuronal tissues like the respiratory epithelium of guinea pig and rat (94, 242) and in normal endothelial cells (267). In the pulmonary arteries and veins of rats, endothelial cells display immunoreactivity in the cytoplasm (268).

3. iNOS (NOS II)

iNOS (NOS II) has been identified as a separate, calcium-independent isoform, which could be detected in brain, lung, and liver of rats after endotoxin treatment (241). In macrophages it has been revealed by cloning and sequencing that iNOS is expressed de novo at the transcriptional level (273, 456). It is now clear that this isoform is not only localized to macrophages (338), but it can be induced in many different cells (105). In the respiratory tract alone, expression of iNOS has been reported in alveolar type II epithelial cells (440), lung fibroblasts (380), airway and vascular smooth muscle cells (150, 418, 459), airway respiratory epithelial cells (2, 337, 374, 441), mast cells (139) endothelial cells (95), neutrophils (35), and chondrocytes (242). The stimuli that cause transcriptional activation of iNOS in these cells varied widely and included endogenous mediators (such as chemokines and cytokines) as well as exogenous factors such as bacterial toxins, virus infection, allergens, environmental pollutants (ozone, oxidative stress, silica), hypoxia, tumors, etc. (Fig. 2) (140, 462, 464). The expression of iNOS in the lung can be prevented by glucocorticoids (157). In respiratory epithelial cells of human lung, a "constitutive" expression of iNOS is observed at mRNA (154) and protein level (242). Under normal conditions, however, some investigators could not detect the expression of iNOS (48). It should be stressed, however, that it is difficult to induce iNOS in human cells in vitro and that there are marked differences in the promoter region of iNOS between humans and rodents. Corticosteroids inhibit rodent iNOS, whereas in humans steroids presumably reduce the inflammatory signals that lead to the induction of iNOS.

In conclusion, all three NOS isoforms are localized in the respiratory system (16) where they may cooperatively regulate airway smooth muscle tone and immunologic/inflammatory responses.

E. Arginine Uptake and Metabolism

Because L-arginine is the only physiological substrate for NOS, regulation of L-arginine availability could determine cellular rates of NO production. L-Arginine is an essential amino acid, which is supplied by the diet and actively transported into the cell. L-Arginine displays affinity for the cationic amino acid transporter in various cell types, but the correlation between L-arginine transport and its availability as a substrate for NO synthesis is not well understood (301, 453).

A high-affinity carrier resembling the cationic amino acid transport (CAT) system y⁺ is likely to be responsible for the transcellular transport of arginine (Fig. 1), with minor roles being played by systems b^o,+, B^0,+, and y⁺L (76). The physiological hallmarks of system y⁺ are the high affinity for amino acids with a positively charged side chain, its independence from the concentration of extracellular Na⁺, and the trans-stimulation of arginine transport by the other cationic amino acids L-lysine and L-ornithine. This system has been detected in many cells, among them macrophages, endothelial cells, platelets, and vascular smooth muscle cells (447). System y⁺ activity is mediated by the CAT family that is composed of four isoforms, CAT-1, CAT-2A, CAT-2B, and CAT-3 (301). NOS inhibitors based on a modification of the arginine structure (with a positive charge) are also transported by system y⁺. Moreover, arginine itself is a proteinogenic amino acid and, once incorporated into proteins, can be posttranslationally N^G-methylated to the NOS inhibitors N^G-monomethyl-L-arginine (L-NMMA) (exogenous) and asymmetric dimethylarginine (ADMA) (endogenous) or deaminated to form citrulline (447).

The activation of L-arginine transport is sensitive to cycloheximide, demonstrating that de novo protein synthesis is essential for enhanced transporter activity. L-Arginine transport in tissues and many different cell types, such as vascular smooth muscle cells and macrophages, can be stimulated by LPS, but is hardly affected by TNF-, IL-1, or IFN- (for an overview, see Ref. 301).

These findings suggest that induction of iNOS and L-arginine transporter activity are dependent on the stimulus used, with an adequate combination of cytokines and/or LPS being responsible for full activation of one or both pathways (Fig. 1). Dexamethasone selectively inhibits the production of NO produced by iNOS whilst having no effect on transport, indicating that the gene for the L-arginine transporter is not sensitive to regulation by glucocorticoids (449). L-Arginine is abundant with a normal dietary intake, but its availability is low owing to extensive protein binding. Oral administration of L-arginine to humans is associated with an increased concentration of NO in exhaled air and was associated with an increase in the concentration of L-arginine and nitrate in plasma (230, 388). These results suggest that an increase in the amount of substrate for NO can increase the formation of endogenous NO.

Arginine can be metabolized by two groups of enzymes. As mentioned above arginine can be converted by NOS to citrulline but can also be catabolized by arginase (Fig. 1).

Arginase exists in two isoforms, liver-type arginase I (165, 220) and nonhepatic type arginase II (36, 302, 435). Arginase I is localized in the cytosol, and arginase II is located in the mitochondrial matrix. iNOS and arginase II are coinduced in LPS-stimulated RAW 264.7 macrophages (304). Moreover, arginase I but not arginase II is coinduced with iNOS in rat peritoneal macrophages and in vivo in rat lung after LPS treatment. In mouse bone marrow-derived macrophages, NOS and arginase activities are regulated by T-helper 1 (Th1) and Th2 cytokines, respectively (297). Moreover, arginase can be induced in the lungs of rats after hyperoxia (355). Allergy is considered to be a Th2-mediated disease, and indeed, arginase activity is increased 3.5-fold in the lungs of guinea pigs after ovalbumin sensitization and challenge (290). Meurs et al. (290) hypothesized that the corresponding airway hyperresponsiveness in these animals is caused by a NO deficiency due to the increased arginase activity (290). Indeed, pretreatment of the tissues with the arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA) suppressed the allergen-induced airway hyperresponsiveness (290). Interestingly, N^G-hydroxy-L-arginine (NOHA) is an intermediate in the biosynthesis of NO (Fig. 1) (36, 45). LPS-treated rat alveolar macrophages produce high amounts of NOHA (166, 169). The inhibition of arginase by NOHA may ensure sufficient high-output production of NO in activated macrophages, which may be important for the killing of microorganisms. On the other hand, a high production of NO is toxic for cells, and arginase I and mitochondrial arginase II prevent NO-mediated apoptosis in activated macrophages. Therefore, a delicate balance between the beneficial and harmful pathophysiological effects of NO exists in the airways, which might be regulated by arginine metabolism.

F. Molecular Action of NO

NO bioactivities are broadly classified as NO mediated/cGMP dependent and cGMP independent. Many bioactivities, such as airway smooth muscle relaxation, appear to use both. Relaxation of human airway smooth muscle by NO, released as a neurotransmitter, may be partially mediated via cGMP (438). However, airway smooth muscle relaxation to NO and other nitrogen oxides has also been shown to be a cGMP-independent process in humans and a variety of other species (127, 200, 341, 421). cGMP-independent bioactivities, ranging from neurotransmission to cell cycle regulation, appear to involve NO reactivity with alternate metal centers and transfer of an NO⁺ (nitrosonium) equivalent from one thiol group to another to up- or downregulate target protein function.

Chemical features of NO radical include its rapid diffusion from the point of synthesis, the ability to permeate cell membranes, the interactions with intracellular molecular sites within both generating and target cells, and its intrinsic instability, all properties that eliminate the need for extracellular NO receptors or targeted NO degradation. The best-characterized target site for NO is the iron bound in the heme component of soluble guanylyl cyclase stimulating conversion of GTP to cGMP and mediating the biological effects attributed to eNOS-derived NO (191). Subsequently, cGMP exerts most of the intracellular actions by coupling to cGMP-dependent protein kinase (PKG). It is generally accepted that cGMP triggers relaxation of smooth muscle by activating two molecular mechanisms: reduction of [Ca²⁺]_i and reduction of the sensitivity of the contractile system to the Ca²⁺. The former is due to the ability of activated PKG to phosphorylate several key target proteins with the final effect of [Ca²⁺]_i reduction. In particular, PKG may stimulate Ca²⁺-activated K⁺ channels (K_Ca), inhibit membrane Ca²⁺ channel activity, activate Ca²⁺-ATPase pump in the plasma membrane and in the sarcoplasmatic reticulum, and inhibit inositol trisphosphate receptor and generation (55). The mechanism of the cGMP-induced Ca²⁺ desensitization is mainly ascribed to the stimulation of myosin light-chain phosphatase activity via inhibition of RhoA-dependent pathway (391). In addition, NO mediates other actions that are independent of guanylyl cyclase and cGMP. The high level of NO released by iNOS has an effect as immune effector molecule in killing tumor cells (170), in halting viral replication (216), and in eliminating various pathogens. In fact, NO has been reported to inhibit the growth of or kill a number of fungi, parasites, and bacteria including Mycobacterium tuberculosis (73). This mechanism may involve, at least in part, inhibition of DNA synthesis by inactivation of ribonucleotide reductase and by direct deamination of DNA (251, 451). Finally, NO appears to signal through its reactivity with cysteine groups, particularly those located at consensus motifs for S-nitrosylation with primary sequence or tertiary structure of a protein (Fig. 1) (see below) (340, 405). One of the general mechanisms of antimicrobial defenses involving NO is S-nitrosylation by NO of cysteine proteases, which are critical for virulence, or replication of many viruses, bacteria, and parasites (390).

Interaction of NO with many molecular targets also may represent a pathway for its breakdown and inactivation. The most important interaction is probably its reaction with superoxide anion (O₂^–) to yield peroxynitrite anion (ONOO^–), which is a potent cytotoxic molecule (356).

G. Regulation of SNO-Mediated Bioactivities

Pulmonary SNO bioactivities are generally those in which functional protein modification is caused by NO transfer to a cysteine thiol (Fig. 1). Specificity of this signaling is achieved by regulation of synthesis, compartmentalization, compositional balance, and catabolism. S-nitrosothiol synthesis may be regulated following NOS activation by proteins such as ceruloplasmin, hemoglobin, and albumin (145, 193, 358) and/or NOS itself (144, 392). Specific compartments of relevance are, for example, the mitochondrial intermembrane space, where S-nitrosylated caspases are sequestered before being released into the reducing environment of the cytosol and thereby activated by reductive cleavage of the SNO bond (277, 278). Compositional specificity is reflected in the requirement of S-nitrosoglutathione (GSNO) to be cleaved to S-nitrosocysteineylglycine, and thereby activated for intracellular transport, by -glutamyltranspeptidase (GGT) (18, 261). S-nitroso-L-cysteine is highly bioactive in S-nitrosylating specific airway epithelial cell proteins, relaxing pulmonary vascular smooth muscle, and increasing neuronal signaling to increased minute ventilation response to hypoxia, in a GGT-independent fashion (261), whereas the D-isomer of S-nitrosocysteine (CSNO) is completely nonfunctional in all of these bioactivities (261, 323). Note in this regard that the L- and D-isomers of CSNO release NO at the same rate. Finally, catabolic regulation is exemplified by the activity of gluthatione-dependent formaldehyde dehydrogenase which, by breaking down GSNO to glutathione disulfide (GSSG) and ammonia, regulates cellular levels of S-nitrosylated protein (264).

	II. NITRIC OXIDE AND PHYSIOLOGY OF THE RESPIRATORY SYSTEM

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A. NO and Lung Development

Spatial and temporal nNOS and eNOS expression patterns occur during development of the lung (218, 460). Quantitative developmental studies of mRNA and protein expression as well as immunohistochemical examination revealed that the eNOS isoform increases during fetal development of the lung (159, 173, 218). In fetal lungs of sheep, eNOS expression was evident in bronchial and proximal epithelia but was absent in terminal and respiratory bronchioles and alveolar epithelium (398). The latter data were confirmed by isoform-specific reverse transcription-polymerase chain reaction assays and NADPH diaphorase histochemistry, which excludes misinterpretation due to immunohistochemistry (64). It was speculated that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time (333). Shaul et al. (396) suggested that the increase in nNOS and eNOS in the lung early in the third trimester in the primate may enhance airway and parenchymal function in the immediate postnatal period.

B. NO and Transcriptional Regulation in the Lung

S-nitrosylation reactions appear to be of particular relevance to regulation of gene expression in the lung (Fig. 1). Several examples are provided. First, SNOs associated with hemoglobin deoxygenation (261, 335) appear to stabilize the -subunit of hypoxia-inducible factor 1 (HIF 1) (330) through increased HIF 1 DNA binding activity, in turn increasing downstream expression of hypoxia-inducible genes such as vascular endothelial growth factor in the pulmonary vascular endothelium. Of note, this system only requires SNO formation through hemoglobin deoxygenation rather than the profoundly low oxygen tension, generally <7 mmHg and not relevant in the airway or pulmonary vasculature, required conventionally in vitro (194) to activate HIF 1. Second, physiological levels of GSNO increase DNA binding of gene regulatory protein SP1 and downstream transcription of housekeeping genes such as that for the cystic fibrosis transmembrane regulatory protein (CFTR), while supraphysiological concentrations (>10 µM) completely inhibit SP1 binding, shutting off transcription of housekeeping genes perhaps to redirect cellular resources to stress response. These observations may have relevance to the effect of high levels of nitrosylating agents in the lung, which paradoxically inhibit wild-type CFTR expression at the transcriptional level (467). Third, high levels of nitrosative stress can inhibit NFB inactivation through direct S-nitrosylation or through S-nitrosylation of IB kinase (280, 324). These signaling mechanisms may serve to control cytokine production under physiological conditions, while increasing cytokine production during periods of nitrosative stress.

C. NO and iNANC

Cholinergic and adrenergic systems control the bronchomotor tone together with the NANC system which mediates contraction [excitatory NANC (eNANC)] or relaxation (iNANC) of airway smooth muscle (39, 408). Recent evidence has shown that NO is a neurotransmitter of iNANC system and that nitrergic neurotransmission is present in several organs including the airways (27). Immunostaining studies demonstrated that nNOS is localized into nerves of guinea pig and human airways (242) which supply vessels, smooth muscle, and lamina propria (108). NOS immunoreactive neurons are found in parasympathetic ganglia and also in sympathetic and sensory (more in jugular than in nodose) ganglia supplying the airways (107, 108). They are more prominent in proximal than in distal airways (437), in agreement with the distribution of iNANC functional responses (108). NO is released from peripheral nerves by nNOS and is activated by calcium entry when the nerve is depolarized (41).

NO mediates approximately one-half of the iNANC (relaxant) response in guinea pig trachea in vitro, and the neuropeptide VIP should be involved in the second half of iNANC relaxant response (258). Of note, VIP-mediated guinea pig airway smooth muscle relaxation is preceded by release of SNOs into the airways (259). The human iNANC response in central and peripheral airways is completely mediated by NO (32, 92). In addition, it has been shown in human airways that iNANC bronchodilator response evoked by electrical field stimulation is associated with a concurrent increase in cGMP content in smooth muscle cells reflecting a cGMP-dependent pathway of neurogenic NO in modulating airway caliber (438). It has also been found that NO-dependent iNANC relaxations are due to the selective activation of K_Ca channels in airway smooth muscle (213). NOS may be colocalized with VIP (250, 399), which can also stimulate NO/SNO production (259, 426). The neurons, which release NO, are probably part of the cholinergic pathway. However, stimulation of the preganglionic cervical vagus nerve in an in vitro guinea pig tracheal tube preparation did not cause NO-mediated bronchodilatation, while activation of postganglionic intrinsic nerves provoked bronchodilatation, suggesting that NO-dependent NANC relaxations of the airways are mediated by postganglionic parasympathetic nerves (442). Recently, it has been shown that a NO-dependent component of noncholinergic parasympathetic nerves modulates airway smooth muscle tone at baseline, pointing out the spontaneous activity of noncholinergic nerves during tidal breathing (225). Fischer et al. (104) provided the first evidence that NOS-immunoreactive neurons intrinsic to the guinea pig esophagus project axons to the adjacent trachealis, showing that these neurons could be the postganglionic parasympathetic neurons mediating iNANC relaxation of the trachealis. Furthermore, inhibition of NOS potentiates cholinergic neural bronchoconstriction (31, 439). However, it does not change neural acetylcholine release (31, 439), suggesting that nNOS-derived NO is a functional antagonist to excitatory cholinergic pathway at the postjunctional, and not the prejunctional, level (175).

Physiological and morphological studies of iNANC nerves indicate that they represent a distinct parasympathetic pathway from the well-characterized cholinergic-parasympathetic pathways innervating the airways (52, 175). Consequently, it seems likely that interactions between these nerve pathways occur postjunctionally and are manifested through their opposing actions on airway smooth muscle. In particular, Canning et al. (51) observed that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, iNANC neurons innervating guinea pig trachealis via activation of both NK₃ and NK₁ receptors (51). It has also been observed that endogenous NO released in association with nerve stimulation regulates the magnitude of eNANC response in guinea pig airways (254). In a recent study it has been observed that the nonadrenergic bronchodilatation induced by capsaicin is suppressed by the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) providing the first evidence of iNANC-derived NO modulation in airway responsiveness of cats in vivo (8).

The fact that in human airways iNANC nerves are the sole neural bronchodilator pathway leads to the hypothesis that any impairment of these nerves such as in inflammatory states has functional consequences for airway patency. Indeed, iNANC responses are significantly reduced from patients with cystic fibrosis, in which there is an intense neutrophilic inflammation of the airways, compared with iNANC responses in normal tissue (27). Furthermore, it has been noted that the circadian variations of the iNANC response may contribute to overnight bronchoconstriction in patients with nocturnal asthma (274). Neural NO-induced relaxation is impaired in guinea pig airways after allergen exposure, without affecting nNOS expression, suggesting a reduced neural NOS activity when allergic inflammation is exacerbated (296).

D. NO and Airway Smooth Muscle Relaxation

The ability of NO to relax smooth muscle has been described in multiple models and muscle types, including airway smooth muscle (55). More than half a century ago, nitrates were supposed to induce bronchial relaxations (143). In 1968 Aviado et al. (19) demonstrated that nebulized nitrovasodilators, but not their administration by intravenous route, reduced baseline lung resistance in anesthetized dogs. However, clinical studies regarding the bronchorelaxant effects of the nitrovasodilators were conflicting (49, 224, 293, 325). Gruetter et al. (151) have shown that nitrovasodilators induce relaxation of isolated airway smooth muscle, activate guanylyl cyclase, and raise cGMP levels. In anesthetized guinea pigs, methacholine-induced bronchoconstriction is reduced by inhaled NO in a concentration-dependent manner from 5 to 300 ppm (85). In addition, a high concentration of NO (300 ppm) causes a small degree of baseline bronchodilatation. Furthermore, in anesthetized and mechanically ventilated rabbits, 80 ppm NO added to the inspired gas prevents increased resistance in response to nebulized methacholine (177). In contrast, there is no effect on pulmonary compliance, suggesting that NO prevented the contraction of the larger airways to a greater extent than the small airways (177). Inhaled NO at a concentration of 80 ppm has no effect in normal human subjects and in chronic obstructive pulmonary disease (COPD) patients, but a small bronchodilator effect in asthmatic patients (178). NO-dependent airway relaxation is partially due to activation of K_Ca channels via guanylyl cyclase and PKG (461). Moreover, these relaxations are due to inhibition of Ca²⁺ release, after stimulation of inositol trisphosphate receptors and ryanodine receptors, from sarcoplasmic reticulum of airway smooth muscle cells mediated via cGMP-dependent mechanisms (214).

Interestingly, there is increasing evidence for another mechanism, in addition to guanylyl cyclase activation, by which NO relaxes human bronchial smooth muscle (24, 127, 199, 341, 421). One of the metabolic pathways for NO also involves its reaction in the presence of thiol to form SNOs (126). SNOs are present in the airways of normal subjects at concentrations sufficient to influence airway tone and have a substantially greater half-life than NO (126). Recently, it has been found that severe asthma is associated with low concentrations of airway SNO, suggesting that the deficiency of such an endogenous bronchodilator mechanism is due to an accelerated degradation of SNO in the lungs of severe asthmatic individuals contributing to severe and refractory bronchospasm (88, 99, 133). Perkins et al. (341) showed that nitrosothiol-induced relaxation is mainly due to cGMP-independent component mediated by reversible oxidation of thiols on unspecified proteins that regulate contraction. Moreover, it has been demonstrated that the activation of K_Ca channels mediates part of NO-induced airway smooth muscle relaxation. NO donor-induced relaxation appeared to result in part from a direct cGMP-independent activation of K_Ca channels by NO, involving trans-nitrosylation reaction that could change the gating of the K_Ca channel (1). In a recent study it has also been found in canine tracheal smooth muscle contracted with KCl that GSNO decreases Ca²⁺ sensitivity by affecting the level of regulatory myosin light-chain phosphorylation. This suggests that myosin light-chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO (328). Furthermore, it has been shown that SNO produced a concentration-dependent decrease in ADP-ribosyl cyclase, a regulatory enzyme of [Ca²⁺]_i in smooth muscle, through a cGMP-independent pathway involving trans-nitrosylation mechanisms (445). Finally, it has been examined whether two redox forms of NO, NO⁺ (liberated by S-nitroso-N-acetylpenicillamine) and NO^– (liberated by 3-morpholinosydnonimine) influence the cytosolic concentration of Ca²⁺ and tone of human main stem bronchi. The authors found that NO⁺ causes release of internal Ca²⁺ in a cGMP-independent fashion, leading to activation of Ca²⁺-dependent K⁺ channels and relaxations, whereas NO^– relaxes the airways through a cGMP-dependent and Ca²⁺-independent pathway (200). In conclusion, the endogenous release of NO as well as the exogenous application of NO donors appear to activate several molecular mechanisms that synergically induce airway smooth muscle relaxation.

E. NO Against Airway Smooth Muscle Contraction

1. In vivo studies

Endogenous NO is also able to modulate excitatory airway responses induced by different mediators in animal models. Nijkamp et al. (315) showed in guinea pigs that aerosolized NOS inhibitors enhanced bronchoconstriction induced by increasing intravenous doses of histamine in vivo, suggesting a modulator role for endogenous NO in airway reactivity. Furthermore, Ricciardolo et al. (366) found a L-arginine/NO-dependent modulation of bradykinin-induced bronchoconstriction in guinea pigs that originates independently from the simultaneous activation of the excitatory neural component: postganglionic cholinergic nerves and capsaicin-sensitive afferent nerves (366). The latter group of investigators also noted that acid inhalation in guinea pigs stimulates a tachykinin- and bradykinin-mediated bronchoconstriction that is limited by endogenous release of NO (367). The NK₁ receptor is likely to be responsible for bronchoprotective NO release in the airways after tachykinin stimulation (370). Interestingly, bronchoconstriction provoked by stimulation of protease activated receptor-2 (PAR-2), after intratracheal instillation or intravenous injection of trypsin or the tethered ligands for PAR-2, was inhibited by tachykinin antagonists and potentiated by NOS inhibitor (368). Furthermore, it has been shown that eNOS^–/– mice were more hyperresponsive to inhaled methacholine and less sensitive to NOS inhibitor compared with wild-type mice, demonstrating that NO derived from eNOS plays a physiological role in controlling airway reactivity (100). In a recent study airway hyperresponsiveness to methacholine was completely abolished in eNOS-overexpressing, ovalbumin-challenged mice compared with control mice in conjunction with a decrease in the number of lymphocytes and eosinophils in the bronchoalveolar lavage fluid (416). In contrast to eNOS it has also been postulated that in mice nNOS could have a role in promoting airway hyperresponsiveness (74, 75).

Different groups of investigators have shown that acute bronchoconstriction induced by allergen inhalation is potentiated by NOS inhibitors in sensitized guinea pigs in vivo, suggesting a modulation by endogenous protective NO on early asthmatic reaction in animal model (286, 342, 343). Other in vivo studies in guinea pigs have shown that the enhanced airway reactivity induced by allergen (6 h after exposure) is not further potentiated by pretreatment with NOS inhibitors (393, 394) and that virus-induced airway reactivity is completely blocked by low doses of inhaled L-arginine (112), suggesting that allergen- or virus-induced airway hyperreactivity is due to the impairment of endogenous release of protective NO. More specifically, it can be postulated that a deficiency in eNOS-derived NO contributes to the increased airway reactivity after early response (EAR) to allergen (4–6 h), whilst a recovery in iNOS-derived NO production aids the reversal of airway reactivity after the late response (LAR: 24–48 h) in guinea pigs. This is supposed by the lack of effect of the specific iNOS inhibitor aminoguanidine on airway reactivity to histamine after EAR and by a significant potentiation of the partially reduced airway reactivity to histamine after the LAR induced by inhalation of the specific iNOS inhibitor aminoguanidine (393). More recently, it has been noted that expression of NOS I is reduced at 6 h, but not at 24 h, after allergen challenge in association with a decrease in constitutive NOS activity and in the amounts of exhaled NO. Together with maximal airway hyperresponsiveness to histamine, this suggests that the transient downregulated NOS I may have a role in airway hyperresponsiveness (387). In agreement with the previous studies, Toward and Broadley (423) found that exposure to inhaled LPS initially inhibited NO synthesis and the reduced NO levels coincided with the period of increased airway reactivity to histamine (1 h after exposure) in guinea pig. In contrast, 48 h after LPS exposure, the bronchoconstrictor response to histamine was attenuated (airway hyporesponsiveness) in association with increased levels of NO metabolites in the bronchoalveolar lavage fluid, suggesting a renewal of NO synthesis probably derived by cytokine-induced NFkB activation of iNOS gene (265), with a bronchial relaxant effect.

For in vivo studies in humans, the reader is referred to section vB.

2. In vitro studies

Bradykinin, endothelin-1, substance P, adenosine, and calcitonin-gene related peptide, applied to the inside of intact tracheal tubes, provoke concentration-dependent relaxations (9, 93, 101–103, 316). The relaxations are reversed into contractions (or contractions are markedly potentiated) by NOS inhibitors, indicating that the relaxant effect in the airways is mediated by the release of endogenous NO (9, 93, 101–103, 316). This effect was mimicked by removal of airway epithelium (111), suggesting that airway epithelium releases NO, which counteracts smooth muscle contraction induced by different spasmogens (9, 93, 101–103, 316). These striking results demonstrate the functional importance of epithelium in airway reactivity, not merely considered as a physical protective barrier between spasmogens and smooth muscle but as a modulator of bronchomotor tone via the release of relaxant substances (so-called epithelium-derived relaxing factors). Treatment of guinea pig trachea in vitro with an inactivator of guanylyl cyclase caused a fivefold increase in the sensitivity to histamine contractile response, indicating the involvement of NO/cGMP pathway in the development of airway hyperresponsiveness (385). Moreover, alterations in guanylyl cyclase activity may account for the strain-related differences in airway reactivity in rats (195). A further study showed that the electrochemical detection of bradykinin-induced NO release in guinea pig airways was fast (duration 2 s), mainly dependent on the epithelium and absent in Ca²⁺-free medium, suggesting that a Ca²⁺-dependent eNOS pathway seems to be involved in the endogenous release of bronchoprotective NO (Fig. 1) (371).

The subsequent step of epithelial-derived NO release is the paracrine effect on airway smooth muscle that is dependent on cGMP increase in the effector cell. In fact, it has been shown that bradykinin raises significantly cGMP levels in guinea pig airways and that this effect is blocked by the pretreatment with NOS inhibitors and in epithelium-denuded preparations. This suggests that cGMP is the final mediator of the bronchoprotection dependent on epithelium-derived NO in this species (102). Meurs et al. (291) demonstrated that polycation-induced airway hyperreactivity to methacholine is dependent on the deficiency of endogenous NO, suggesting that polycationic peptides released by activated eosinophils in the inflamed airways may contribute to the deficiency of bronchoprotective eNOS-derived NO. In a further study these authors found that endogenous arginase activity potentiates methacholine-induced airway constriction by inhibition of NO production in naive guinea pig, presumably by competition with eNOS for the common substrate L-arginine (288). In a recent and elegant study, Ten Broeke et al. (417) showed that calcium-like peptides (CALP1 and CALP2) targeting calcium binding EF hand motif of calcium sensors (calmodulin and calcium channels) may have a role in regulating airway responsiveness by controlling [Ca²⁺]_i and, consequently, modulating the activity of eNOS (Fig. 1) (417). In fact, they observed that CALP2 inhibition of CALP1-induced airway hyperresponsiveness was Ca²⁺ epithelium dependent and NO mediated (417). Interestingly, they found that bradykinin-induced [Ca²⁺]_i increase in epithelial cells was markedly higher after incubation with CALP2. In allergen-challenged guinea pigs, the enhanced contractile response to agonists in tracheal preparations after early reaction was not augmented by NOS inhibition as shown in naive animals, suggesting an impairment of protective NO (70). In a further study the same authors showed that L-arginine administration reduced methacholine-induced contraction in isolated perfused tracheas from guinea pigs, indicating that limitation of the substrate may underlie the reduced eNOS activity and the excessive contractile response (69). Finally, it has also been demonstrated that increased arginase activity contributes to allergen-induced deficiency of eNOS-derived NO and airway hyperresponsiveness after early allergen reaction in guinea pigs, presumably by direct competition with eNOS for L-arginine (290).

F. NO and Pulmonary-Bronchial Circulations

1. NO and pulmonary circulation

Nitrogen oxides can account for the biological activity of EDRF and are involved in the regulation of vascular tone (189, 257). Release of NO from endothelial cells in the pulmonary circulation appears to regulate vascular basal tone and counteract hypoxic vasoconstriction (Fig. 1) (344). Furthermore, NO release is apparently decreased in chronic hypoxia (4). Intravenous infusion of the NOS inhibitor L-NMMA increases pulmonary vascular resistance in normal adults pointing towards a role for endogenous NO in the control of pulmonary vascular tone at baseline (65). In the healthy human, eNOS isoform is present in the endothelium of pulmonary vessels, but its expression is downregulated in patients with primary pulmonary hypertension (136). This suggest that the pulmonary vasoconstriction and the increased smooth muscle layer in the pulmonary vessels, main features of this disease, are associated with impaired expression of eNOS. Interestingly, these abnormalities might be associated with smoking. In a pig model challenge, unfiltered cigarette smoke induced variable responses in the pulmonary circulation, whereas inhalation of filtered smoke caused rapid and consistent pulmonary vasodilatation, probably NO mediated (11). An in vitro study of pulmonary artery endothelial cells incubated with cigarette smoke extract resulted in a time- and dose-dependent decrease in eNOS activity associated with a nonreversible reduction of eNOS protein content and eNOS mRNA. This indicates that chronic exposure of cigarette smoke may contribute to the risk of pulmonary endothelial dysfunction via impairment of eNOS expression (409).

Impaired release of endothelium-derived NO from pulmonary vessels has also been observed in patients with COPD and cystic fibrosis (79). Moreover, isolated pulmonary arteries of patients undergoing heart-lung transplantation for end-stage chronic lung diseases have impaired endothelium-dependent relaxation (67). Recently, it has been demonstrated that overproduction of eNOS-derived NO can inhibit not only the increase in right ventricular systolic pressure associated with pulmonary hypertension, but also remodeling of the pulmonary vasculature and right ventricular hypertrophy induced by chronic hypoxia (Fig. 1) (327). In addition, the lungs of caveolin-1 knock-out mice displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, suggesting an important role for caveolin-1 in endothelium-dependent relaxation of pulmonary vasculature (82). Polymorphisms of the eNOS gene have been associated with high-altitude pulmonary edema, suggesting that a genetic background may underlie the impaired NO synthesis in the pulmonary circulation of this disease contributing to its exaggerated pulmonary hypertension (83).

Interestingly, recent evidence suggests ethyl nitrite is more potent as a selective pulmonary vasodilator in humans and other mammals, and is associated with less withdrawal rebound hypertension, than NO itself (306, 307). This is important because ethyl nitrite is a potent S-nitrosylating agent that releases relatively little NO gas. Consistant with recent observations of Gow et al. (144), this observation suggests that the most relevant reaction leading to pulmonary vascular smooth muscle relaxation may involve S-nitrosylation chemistry.

2. NO and bronchial circulation

Of note, endogenous NO regulates basal bronchial vascular tone, and exogenous NO accounts for most of the bronchial vasodilatation observed after inhalation of cigarette smoke (11). The airway vasculature has also been shown to dilate in vivo when animals are ventilated with NO (59). Finally, endogenous endothelial NO significantly influences acetylcholine-induced bronchovascular dilation (389), but not the vagally induced bronchial vascular dilation in sheep (23).

Conflicting results have been reported about the role of endogenous NO in vascular permeability (247). A recent study in guinea pigs demonstrated that NOS inhibitors inhibit airway microvascular plasma leakage induced by substance P and leukotriene D₄ (LTD₄), but not by histamine, suggesting that endogenous NO plays an important role in plasma extravasation induced by some inflammatory mediators (211). The authors also showed that the substance P- and LTD₄-induced rise in plasma extravasation is increased via endogenous NO in the trachea and main bronchi, but not in the intrapulmonay airways, suggesting differential regulation of transvascular protein flux in anatomically different parts of the airway microvasculature. The inhibition of substance P-induced plasma extravasation by NOS inhibitor is possibly due to the vasoconstriction of perfused vessels and the subsequent decrease in local blood flow at the leaky site. It has also been shown that allergen inhalation in sensitized guinea pigs caused microvascular leakage in all airway portions which was suppressed in a dose-dependent manner by pretreatment with the NOS inhibitor L-NAME, suggesting that endogenous NO increases airway microvascular leakage after airway allergic reaction (295). Similar results have been found after administration of LPS, which was able to provoke a significant plasma leakage in rat trachea inhibited by the NOS inhibitor L-NAME. This effect was paralleled by an increase in iNOS activity in LPS animals, suggesting that iNOS-derived NO is responsible for LPS-induced increase in plasma leakage (33). On the contrary, these authors found that in the trachea of vehicle-treated rats L-NAME significantly increased plasma leakage, suggesting an inhibitor role of NO on plasma leakage under physiological conditions. Thus the possibility that alteration of bronchial blood flow by NOS inhibitors confounds the results on plasma leakage cannot be excluded. Further studies examining blood flow through individual microvascular beds would permit greater information about the precise role of endogenous NO on this important aspect of airway microcirculation relevant to disease such as asthma.

G. NO and Mucus-Electrolyte Secretions in the Airways

NOS inhibitors did not affect mucus glycoprotein secretion tonically, but significantly reduced both methacholine- and bradykinin-induced secretion from feline tracheal isolated submucosal glands (312). In addition, NO generator isosorbide dinitrate significantly increased submucosal gland secretion. Taken together, these results suggest that endogenous NO stimulates airway submucosal gland secretion (312). Other secretagogues, such as platelet activating factor, histamine, and TNF-, enhance release of mucin by guinea pig tracheal epithelial cells, but the stimulatory effect of each is inhibited by precoincubation of the cells with a competitive inhibitor of NOS. This indicates that these mediators provoke mucin secretion via a mechanism involving intracellular production of NO as a critical signaling molecule (3).

Stimulation of airway bovine epithelial cell ciliary beat frequency by isoproterenol, bradykinin, and substance P is dependent on L-arginine/NO pathway (197). Ciliary motility is an important host defense mechanism of airway epithelium, and it is enhanced by the iNOS inducers alveolar macrophage-derived cytokines, such as TNF- and IL-1 (198). The cilia stimulatory effect of TNF- and IL-1 is inhibited by L-NMMA and restored by the addition of L-arginine, suggesting an involvement of iNOS pathway in the regulation of ciliary motility (198). Interestingly, low levels of nasal and exhaled NO in patients with primary ciliary diskinesia (PCD) are related to mucociliary dysfunction, and treatment with NO substrate L-arginine improves mucociliary transport in patients with PCD (269).

Abnormal electrolyte transport produces changes in airway surface liquid volume and composition, inhibits mucociliary clearance, and leads to chronic infection of the airways, as occurs in cystic fibrosis. Modulation of ion channels by NO has emerged recently as a significant determinant of ion channel function (87). NO activates both apical anion channels and basolateral potassium channels via cGMP-dependent pathway (86). Thus NO is a physiological regulator of transepithelial ion movement, and alterations of its generation and action may play an important role in the pathogenesis of lung disorders characterized by hypersecretion of airway surface liquid.

Of note, SNOs have several established effects of potential benefit in the cystic fibrosis airway. These include ventilation-perfusion matching, smooth muscle relaxation, increased ciliary beat frequency, inhibition of amiloride-sensitive sodium transport, augmentation of calcium-dependent chloride transport, augmentation of neutrophil apoptosis, and antimicrobial effects as recently reviewed (403). Additionally, recent evidence suggests that physiological levels of SNOs can increase the expression, maturation, and function of F508 mutant CFTR protein, apparently through S-nitrosylation of trafficking proteins involved in the ubiquitination and degradation of the molecule (14, 179, 466). In this regard, it is of particular interest that metabolism of SNOs appears to be accelerated in the cystic fibrosis airway and that SNO levels are nearly undetectable in the bronchoalveolar lavage fluid of patients with mild cystic fibrosis (146). Augmentation of SNO levels by therapeutic administration of GSNO appears to be well-tolerated in patients with cystic fibrosis and to lead to an improvement in oxygenation (403). Of note, inhaled NO does not improve oxygenation in these patients (360).

	III. NITRIC OXIDE AND OXIDATIVE STRESS: "NITROSATIVE STRESS"

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Reactive oxygen species (ROS) are generated by various enzymatic reactions and chemical processes or they can be directly inhaled. NO can interact with ROS to form other reactive nitrogen species (RNS) (Figs. 2 and 3). ROS, NO, and RNS are essential in many physiological reactions and are important for the killing of invading microorganisms (Fig. 2). However, when airway cells and tissues are exposed to oxidative stress elicited by environmental pollutants, infections, inflammatory reactions, or decreased levels of antioxidants, enhanced levels of ROS and RNS can have a variety of deleterious effects within the airways, thereby inducing several pathophysiological conditions (Fig. 3). ROS and RNS can damage DNA, lipids, proteins, and carbohydrates leading to impaired cellular functions and enhanced inflammatory reactions (Figs. 2 and 3). In this way, ROS and RNS play a prominent role in the pathogenesis of various lung disorders such as adult respiratory distress syndrome (ARDS), interstitial lung disease, cystic fibrosis, COPD, and asthma (37, 110, 141, 182, 362, 431).

View larger version (20K): [in this window] [in a new window]   FIG. 3. Schematic overview of how inhaled substances or proinflammatory mediators contribute to the production of reactive oxygen and nitrogen species in the airways that will finally result in pathophysiological effects. Upon appropriate stimulation, inflammatory cells and a number of airway resident cells can generate superoxide (O2–) via activation of NADPH oxidase or form high amounts of nitric oxide (NO) via an increased expression of iNOS. NO reacts with superoxide to form the potent oxidant peroxynitrite (ONOO–). Peroxynitrite induces the formation of nitrotyrosine residues; however, tyrosine nitration may also be found after exposure of proteins to nitrite (NO2–) in association with hypochlorous acid (HOCl) and myeloperoxidase (MPO) or eosinophil peroxidase (EPO). As mentioned in the different sections in the text, high concentrations of NO formed by iNOS, peroxynitrite, and tyrosine nitration may all cause a variety of pathophysiological effects.

Because NO and superoxide are free radicals, both molecules rapidly react with many different molecules in a biological environment. Of particular interest is the interaction between the two molecules and their reactive downstream metabolites. Enhanced cytotoxicity is possible when NO and superoxide are released simultaneously, which is a likely event during inflammatory responses (Fig. 2). For example, the efficient killing of Salmonella by murine macrophages is dependent on both NADPH oxidase-derived superoxide and iNOS-derived NO. Many of the products formed by the interaction of superoxide and NO are even more reactive than their precursors. The most direct interaction between NO and superoxide is their rapid isostoichiometric reaction to form the potent oxidant peroxynitrite (Fig. 3) (308, 352). The rate constant of this reaction is near the diffusion controlled limit (4–7 x 10⁹ M^–1 · s^–1), and the half-life of peroxynitrite at 37°C and pH 7.4 is 1 s (308, 384). The reaction of peroxynitrite with carbon dioxide is the most important route for degradation of peroxynitrite in biological environments, when carbon dioxide is relatively abundant (430). Many other RNS can emanate from the interaction between NO and superoxide