Nitric Oxide, the Biological Mediator of the Decade: Fact Or Fiction?

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SERIES 'CLINICAL PHYSIOLOGY IN RESPIRATORY INTENSIVE CARE' Edited by A. Rossi and C. Roussos Number 14 in this Series Nitric oxide, the biological mediator of the decade: fact or fiction?

S. Singh, T.W. Evans

Nitric oxide, the biological mediator of the decade: fact or fiction? S. Singh, T.W. Evans. Unit of Critical Care, National Heart & ERS Journals Ltd 1997. Lung Institute, Royal Brompton Hospital, ABSTRACT: Nitric oxide (NO), an atmospheric gas and free radical, is also an London, UK. important biological mediator in animals and humans. Its enzymatic synthesis by constitutive (c) and inducible (i) isoforms of NO synthase (NOS) and its reactions Correspondence: T.W. Evans with other biological molecules such as reactive oxygen species are well charac- Royal Brompton Hospital Sydney Street terized. NO modulates pulmonary and systemic vascular tone through its vasodila- London SW3 6NP tor property. It has antithrombotic functions and mediates some consequences of UK the innate and acute inflammatory responses; cytokines and bacterial toxins induce widespread expression of iNOS associated with microvascular and haemodynam- Keywords: Acute respiratory distress syn- ic changes in sepsis. drome Within the lungs, a diminution of NO production is implicated in pathological hypoxic pulmonary vasoconstriction states associated with pulmonary hypertension, such as acute respiratory distress nitric oxide syndrome: inhaled NO is a selective pulmonary vasodilator and can improve ven- nitric oxide synthase tilation-perfusion mismatch. However, it may have deleterious effects through mod- pulmonary hypertension ulating hypoxic pulmonary vasoconstriction. Inhibitors of NOS may be of benefit reactive oxygen species in inotrope-refractory septic shock, but toxicity of newly developed selective iNOS inhibitors have prevented clinical trials of efficacy. Received: September 2 1996 An expanding literature on the origins and measurement of NO in exhaled breath Accepted after revision December 30 1996 implicates NO as a potentially useful marker of disease activity in respiratory tract SS is a British Heart Foundation Research inflammation in the future. Fellow in the Unit of Critical Care, National This report reviews some aspects of research into the clinical importance of Heart & Lung Institute, Royal Brompton nitric oxide. Hospital, London. Eur Respir J 1997; 10: 699Ð707.

The ability of mammalian cells to synthesize an endo- is converted to L-citrulline in a stereospecific reaction cat- thelially-derived relaxant factor (EDRF) was first demon- alysed by a family of nitric oxide synthases (NOS) (fig. strated in 1980. In 1987, it was shown that the actions 1). These are complex haemoproteins, with properties of EDRF and nitric oxide (NO) were substantially simi- similar to cytochrome P450, and contain both oxidative lar [1Ð3], and during the subsequent decade progress in and reductive domains. NOS require a number of co- understanding the biological role of NO has been re- factors, such as tightly-bound flavoproteins and tetrahy- markable. Thus, NO was regarded initially only as an drobiopterin, while the production of NO itself requires atmospheric pollutant present in vehicle exhaust emis- the co-substrates oxygen (O2) and nicotinamide adenine sions and cigarette smoke. More recently, NO seems set dinucleotide phosphate (NADPH). Mechanistic studies to be identified as an almost ubiquitous biological medi- are consistent with the hypothesis that the reductase do- ator, implicated in the pathogenesis of diseases as diverse main of NOS provides reducing equivalents from NADPH as hypertension, asthma, septic shock and dementia; and to the haem domain, where oxidation of L-arginine occurs as a potential marker of clinical diseases, that may prove [4]. Three major isoforms of NOS have so far been iden- amenable to therapeutic manipulation. tified: endothelial NOS (eNOS or type 3) and neuronal NOS (nNOS or type 1), which are expressed constitu- NO production and metabolism tively and are collectively termed constitutive nitric oxide synthase (cNOS); and a macrophage-derived form that Nitric oxide is synthesized from the terminal guanidine is not normally expressed but is induced (iNOS or type nitrogen of the semiessential amino acid L-arginine, which 2) by endotoxin and inflammatory mediators, such as Previous articles in this series: No. 1: S.K. Pingleton. Enteral nutrition in patients with respiratory disease. Eur Respir J 1996; 9: 364Ð370. No. 2: R. Dhand, M.J. Tobin. Bronchodilator delivery with metered-dose inhalers in mechanically-ventilated patients. Eur Respir J 1996; 9: 585Ð595. No. 3: N. Ambrosino. Noninvasive mechanical ventilation in acute respiratory failure. Eur Respir J 1996; 9: 795Ð807. No. 4: P. Pelosi, S. Crotti, L. Brazzi, L. Gattinoni. Computed tomography in adult respiratory distress syndrome: what has it taught us? Eur Respir J 1996; 9: 1055Ð1062. No. 5: H. Burchardi. New strategies in mechanical ventilation for acute lung injury. Eur Respir J 1996; 9: 1063Ð1072. No. 6: V.M. Ranieri, M. Dambrosio, N. Brienza. Intrinsic PEEP and cardiopulmonary interactions in patients with COPD and acute ventilatory failure. Eur Respir J 1996; 9: 1283Ð1292. No. 7: A. Corrado, M. Gorini, G. Villella, E. De Paola. Negative pressure ventilation in the treatment of acute respiratory failure: an old noninvasive technique reconsidered. Eur Respir J 1996; 9: 1531Ð1544. No. 8: A. Torres, M. El-Ebiary, N. Soler, C. Montón, N. Fàbregas, C. Hernández. Stomach as a source of colonization of the respiratory tract during mechanical ventilation: association with ventilator-associated pneumonia. Eur Respir J 1996; 9: 1729Ð1735. No. 9: J. Mancebo. Weaning from mechanical ventilation. Eur Respir J 1996; 9: 1923Ð1931. No. 10: D. Georgopoulos, C. Roussos. Control of breathing in mechanically ventilated patients. Eur Respir J 1996; 9: 2151Ð2160. No. 11: T. Vassilakopoulos, S. Zakynthinos, Ch. Roussos. Respiratory muscles and weaning failure. Eur Respir J 1996; 9: 2383Ð2400. No. 12: G.U. Meduri. The role of the host defence response in the progression and outcome of ARDS: pathophysiological correlations and response to glucocorticoid treatment. Eur Respir J 1996; 9: 2650Ð2670. No. 13: H.E. Fessler. Heart-lung interactions: applications in the critically ill. Eur Respir J 1997; 10: 226Ð237. 700 S. SINGH, T.W. EVANS

+ H2NNH2 H2N NHOH H2N O The inducible isoform was originally isolated from lipo- polysaccarrhide/interferon-gamma (IFN-γ)-treated murine macrophages [11], but has since been identified in many NADPH NADPH cell types, including vascular smooth muscle, vascular en- NH NH NH dothelium, endocardium, myocardium, hepatocytes, fib- + •N=O roblasts, astrocytes and immune cells. All three isoforms of NOS have been detected in the human respiratory O2 O2 tract [12, 13]. Following an appropriate stimulus, such as γ β +H N COO– +H N COO– +H N COO– IFN- , interleukin (IL)-1 , tumour necrosis factor-alpha 3 3 3 (TNF-α), endotoxin or exotoxin, iNOS is induced in the L-arginine N-hydroxyarginine L-citrulline Nitric relevant cell type by gene transcription, probably via oxide the transcription factor nuclear factor-kappa B (NF-κB). Fig. 1. Ð Chemical reactions involved in the synthesis of nitric oxide. This process can be inhibited by glucocorticoids [14]. L-arginine is converted to L-citrulline and nitric oxide. The reaction Inducible NOS generates up to 1,000 times more NO is catalysed by nitric oxide synthase (NOS) in the presence of oxygen and the co-substrate nicotinamide adenine dinucleotide phosphate than cNOS, and cellular production continues for hours. (NADPH). NOS requires several co-factors, including flavones and The effects of NO produced by iNOS are, therefore, more tetrahydrobiopterin. pronounced and widespread [15], an important concept which determines whether its effects are beneficial or cytokines [5, 6] (table 1). The genes for these isoforms detrimental to health in a particular clinical circumstance. have been localized to chromosomes 7 (eNOS), 12 Nitric oxide is a free radical, by virtue of its unpaired (nNOS) and 17 (iNOS) [7]. electron; this makes it highly reactive with other ra- The constitutive enzymes produce limited amounts of dicals, such as superoxide [16]. Its half-life at physio- NO and are activated by calcium and calmodulin. Agonists logical concentrations is measured in seconds [4], and Ð activate cNOS via a rapid increase in intracellular cal- it decomposes in aqueous solutions to nitrite (NO2 ) and Ð cium concentration, resulting in NO release within sec- nitrous oxide (NO3 ); a reaction catalysed by transition onds or minutes. Endothelially-derived NOS is membrane metals, such as iron. There is, consequently, a rapid in- bound [8], and has been identified in vascular endothe- activation of NO by haemoglobin to methaemoglobin, Ð Ð lial cells, platelets, myocardium and endocardium. Neu- NO2 and NO3 , via an intermediary compound nitroso- ronal type NOS is cytosolic [9], and is found in skeletal haemoglobin. muscle as well as in nervous tissue, while cNOS has In the presence of superoxide anion, NO combines also been identified in neutrophils and mast cells [10]. three times faster than superoxide dismutase (a scaven- Inducible NOS has a 50% homology to cNOS; it is ger of superoxide), leading to the formation of peroxy- cytosolic and calcium-independent, perhaps due to its nitrite (OONOÐ); an unstable, strongly pro-oxidant species tight (noncovalent) binding to calmodulin, which pre- with toxic effects on many molecules (i.e. nucleic acids, vents calcium-binding at physiological concentrations. lipids and proteins) [17] (fig. 2). The cellular effects of

Table 1. Ð Properties of constitutive and inducible isoforms of nitric oxide synthase NOS isoform Type 1 (nNOS) Type 2 (eNOS) Type 3 (iNOS) constitutive constitutive inducible Location Chromosome number 12 7 17 Cellular compartment Cytosolic Membrane-bound Cytosolic Cell type Neurones Endothelium Immune cells Skeletal muscle Platelets Vascular smooth muscle Endocardium Endothelium Myocardium Endo/myocardium Hepatocytes Astrocytes Fibroblasts Activation Calcium-dependent Calcium-dependent Calcium-independent Regulation Induction prevented by corticosteroids and protein synthesis inhibitors Response to Constitutive Constitutive Induction by LPS/cytokine stimulation e.g. glutamate/receptor e.g. ACh/receptor leads to massive NO interaction leads to immediate interaction leads to production after 2Ð6 h NO release by neuronal den- immediate NO release by drites vascular endothelium Nonselective L-arginine analogues L-arginine analogues L-arginine analogues inhibitors Selective Aminoguanidine inhibitors Under development NOS: nitric oxide synthase; nNOS: neuronal NOS; eNOS: endothelial NOS; iNOS: inducible NOS; ACh: acetylcholine; LPS: lipopolysaccharide. STATUS OF NITRIC OXIDE AS BIOLOGICAL MEDIATOR 701

– – + •NO + O2 ONOO + H ONOOH endothelium produces NO at a basal rate, maintaining background vasodilatation in small arteries and arteri- Peroxynitrite anion oles in animals and humans. By contrast, veins synthe- ONOOH •OH + NO NO – + H+ size very little NO basally. Systemic injection of L-NMMA 2 3 increases blood pressure in experimental animals and Hydroxyl radical Nitrate healthy volunteers, but produces little change in venous Fig. 2. Ð Interactions of nitric oxide with reactive oxygen species. pressure [22]. It seems that pulmonary vascular endothe- Ð ¥NO: nitric oxide; O2 : superoxide anion; NO2: nitrogen dioxide. lial cells produce NO continuously, and that hypoxia re- duces this release [23]. Furthermore, the normal human NO are, in the main, attributable to its activation of gua- pulmonary vasculature expresses cNOS abundantly and nylyl cyclase after binding to its haem moiety. This enz- its diminution is inversely correlated with total pulmo- yme catalyses the conversion of guanylate triphosphate nary vascular resistance (PVR) [24]. Such studies impli- (GTP) to cyclic guanylate 3'5'-monophosphate (c-GMP). cate NO as an important mediator of pulmonary blood The rise in cGMP concentration, an intracellular messen- flow and, consequently, of ventilation-perfusion (V'/Q') ger, results in smooth muscle relaxation and many of the matching within the lungs. This recent work extends the alterations in cellular function produced by NO. Other findings of earlier studies in vitro, that demonstrated a intracellular second messenger systems, such as protein reduced vasodilator response to acetylcholine in pulmo- kinase activation, inositol triphosphate inhibition and in- nary arterial vessels following pretreatment with NOS tracellular calcium release, have been proposed as effec- inhibitors [25Ð27]. Thus, it is now clear that increased tors of NO function at the cellular level. Indeed, NO is vascular resistance or vasospasm need not be due to an thought to form intermediary complexes with thiols, such increase in vasoconstrictor effects, but may reflect a loss as cysteine; the formation of sulphydryl complexes may of NO-mediated basal vasodilator tone [28]. This may prolong the half-life of extracellular nitric oxide, facili- underlie the intricacy of alterations in local and region- tating its role in cell-cell communication prior to inacti- al microvascular perfusion in pathological states, such vation [18]. as pulmonary hypertension. Thus, through processes at the cell surface, NOS iso- In addition to its direct haemodynamic effects, NO forms are activated either via a second messenger, such inhibits the adhesion [29], activation and aggregation as calcium, (cNOS) or in the case of iNOS through gene of platelets [30, 31], thereby conferring antithrombotic transcription. In the presence of appropriate substrates, properties on the endothelial surface. Furthermore, the these enzymes catalyse the formation of NO, which is antiaggregatory effect of NO is synergistic with that of either inactivated by its reaction with haemoglobin or prostacyclin, another endothelium-derived vasoactive me- albumin, acts as a biological mediator via its effect on diator. Nitric oxide may also be involved in modulat- guanylate cyclase, or forms toxic radical derivatives thro- ing negative inotropic effects [32], as it affects cardiac ugh its reaction with reactive oxygen species (ROS). relaxation during diastole [33]. NOS inhibition NO in lung function Nitric oxide synthase inhibitors (NOSIs) have been vital tools in determining the physiological and patho- Nitric oxide mediates the nonadrenergic, noncholi- physiological roles of NO. Furthermore, they are on the nergic (NANC) neural inhibitory responses of human verge of clinical use as pressor agents in inotrope-refrac- airways [34], either directly or via stable NO carrying tory septic shock [19, 20]. NOSIs are analogues of L- intermediates, such as nitrosocysteine. NANC pathways arginine and act as false substrates for the enzyme, thus represent the only neural bronchodilator mechanism in blocking endogenous NO formation. A number of non- human airways [35], and NO is thought to act as a "bra- selective NOSIs, such as N-monomethyl-L-arginine (L- king" mechanism to cholinergic bronchoconstriction, rat- NMMA) and N-nitro-L-arginine methylester (L-NAME) her than having any direct bronchodilator effect per se. have been developed. Their effects are overcome by L- Nitric oxide derived from resident immune cells may also arginine. Agents more selective for iNOS inhibition, such contribute to host defence or inflammation in the lungs as aminoguanidine, have been investigated in the noncli- [36]. nical setting, but are currently unavailable for clinical use. NO as a neurotransmitter Proposed functions of NO in homeostasis In addition to the neural bronchodilatation mediated by Physiological roles for NO have been proposed fol- NO, it seems to be involved in neurally-mediated vasodi- lowing a number of experimental studies, including the latation in the pulmonary circulation. Studies using elec- localization of NOS isoforms to various tissues, in vary- trical field stimulation suggest that adenosine triphosphate ing distributions, by immunohistochemical and bioche- (ATP) from sympathetic neurones causes the release of mical techniques or messenger ribonucleic acid (mRNA) endothelially-derived NO, which acts on the pulmonary detection. The functional effects of stimulating or inhibi- vasculature causing relaxation [37], whilst NOS inhibiting the L-arginine-NO pathway have also proved inval- tion produces an increased adrenergic vasoconstrictor res- uable in this respect. ponse. Neurones staining for NOS have been identified NO and the cardiovascular system in the urinary tract and gastrointestinal system, as well as the vascular bed and bronchi. Nitric oxide produced by microvascular endothelial cells Through its smooth muscle relaxing propensity, NO is a major determinant of resting vascular tone [21]. The is thought to play important roles in adaptive relaxation 702 S. SINGH, T.W. EVANS of the stomach to accommodate food, and in sphincter tected in patients with essential hypertension, diabetes relaxation, as suggested in animal studies [38, 39]. In mellitus, heart failure and overt atheroma [53, 54]. the central nervous system, NO has been proposed as a In the gastrointestinal tract, infantile hypertrophic mediator of memory function [40], electrocortical acti- pyloric stenosis and achalasia are associated with loss vation (important as an alerting response in the control of nitrergic nerves [55], in keeping with findings in mice of arousal) [41], and as a modulator of pain perception deficient in the gene for nNOS, which have grossly dis- [42, 43]. tended stomachs and hypertrophy of the pyloric circular muscle [38]. Abnormalities of peripheral nitrergic nerves are also found in erectile dysfunction [39]. In the cen- NO and the immune system tral nervous system, relatively little has been learned Endogenous NO produced by iNOS in activated murine from human studies, but excess NO appears to protect macrophages is important in host defence [44], and ap- against epilepsy in rats and could contribute to pro- pears to play a crucial role in controlling infection in grammed cell death in the central nervous system. In vivo, as demonstrated indirectly by experiments in mice Huntington's chorea, NOS-staining neurones are spared infected with Leishmania. Nitric oxide is also involved for an as yet unknown reason [56]. in mononuclear cell-mediated killing of Mycobacterium The role of NO in the respiratory system and in sep- tuberculosis and other pathogens in rodents [45], is toxic sis has been studied in some depth. Endotoxaemia and to tumour cell lines in vitro, and may selectively inhibit a number of cytokines stimulate the induction of iNOS a subset of thymus-derived lymphocytes (type-1 T-helper [57]. In severe sepsis and other inflammatory states, dur- (Th1)) [46]. Definitive confirmation of these roles in hu- ing which widespread induction of iNOS occurs, very mans is awaited. In the upper respiratory tract (URT), large amounts of NO (up to 1,000 times the physiologi- NO appears to be important in maintaining ciliary func- cal concentrations) are produced. Thus, increased plasma tion [47], and may have a role in sterilizing the mucosa concentrations of nitrate and cGMP have been demon- of URT, thus reducing susceptibility to subsequent lower strated in septic patients, and increased conversion of respiratory tract infection. L-arginine to nitrate following treatment with IL-2 [58]. The uncontrolled production of NO is thought to con- Nitric oxide also contributes to the acute inflamma- tribute to the vascular hyporeactivity and myocardial de- tory response mediated by endotoxin, cytokines or physi- pression that cause systemic hypotension seen in these cochemical stress, through the activation of iNOS. The circumstances [59Ð61]. resultant NO production causes vasodilatation, plasma exu- The adult respiratory distress syndrome (ARDS) rep- dation [48], and activation of innate, nonadaptive immune resents the pulmonary manifestation of a global inflam- mechanisms. This, consequently, has implications for the matory process, in which widespread induction of iNOS role of NO in inflammatory disease states, and provides probably occurs in the injured lung [62]. It is defined by a therapeutic target for suppression of inflammation by refractory hypoxaemia in the presence of radiographic manipulation of the NO pathway. evidence of pulmonary infiltrates [63], and is characterized by high permeability alveolar oedema and a loss NO in other homeostatic mechanisms of hypoxic pulmonary vasoconstriction (HPV), which produces a marked increase in V'/Q' mismatch [64]. The NO is linked to homeostasis in many physiological sys- uncontrolled release of iNOS-derived NO may contribute tems. In the gastrointestinal tract, in addition to its role to its pathogenesis. Additionally, a loss of local vascu- in modulating gut motility in mice, the possibility of NO lar regulation, vasoconstriction and mechanical block- being a defence mechanism against ingested organisms age of vessels all cause a reduction in the cross sectional has been proposed [49]. In the endocrine system, endoge- area of the pulmonary vascular bed, an increase in PVR, nous NO appears to be involved in regulating renin pro- pulmonary hypertension and consequent right ventricu- duction and sodium homeostasis in the kidney [50], in lar dysfunction, which can lead to impaired left ventri- so far as it has been shown to determine sensitivity to the cular function [65, 66]. pressor effects of a sodium challenge [51]. In pregnan- Pulmonary hypertension, whether primary or second- cy, NO may be responsible for the associated vasodila- ary, is characterized by increased thickening of the walls tation, reduced systemic vascular resistance, and increased of pulmonary arteries, narrowing of the pulmonary arte- cardiac output [52]. Oestrogens increase the expression rial luminal diameter and increased PVR [67, 68]. Endo- of eNOS. Nitric oxide may also provide a basal tocolyt- thelially-derived vasoactive factors mediate pulmonary ic tone, and a decrease in the activity of the L-arginine- vascular control [69]. Exogenous NO, administered by NO pathway appears to coincide with the onset of labour. inhalation, has successfully reduced PVR and improved refractory hypoxaemia in children with persistent pulmo- Nitric oxide and disease nary hypertension of the newborn (PPHN) [70]. Reduced expression of NOS has been demonstrated in the pulmo- Excess or deficient NO has recently been linked to nary arteries of patients with primary and secondary pul- numerous pathological conditions, representing an ex- monary hypertension [24]. An inverse correlation was tension of the roles discussed in homeostasis. Within shown between diminished eNOS mRNA expression the cardiovascular system, diminished NO production and the morphological severity of pulmonary hyperten- is implicated in conditions associated with increased sion. These findings represent an elegant addition to the vascular tone, vasospasm and thrombosis due to enhan- existing literature, although the question of whether ced adhesion of platelets and white cells to the vessel endothelial dysfunction (i.e. diminished basal NO pro- wall. Abnormalities of arterial blood flow in the human duction) leads to the development of pulmonary hyper- forearm following infusions of L-NMMA have been de- tension (as a result of the subsequent vasoconstriction, STATUS OF NITRIC OXIDE AS BIOLOGICAL MEDIATOR 703 in situ thrombosis and vascular smooth muscle prolifera- shown in PPHN [83]. Clearly, a prerequisite would be tion caused by the lack of NO [71]) or is the consequ- sufficient NOS present for the substrate; a deficiency of ence of secondarily elevated pulmonary arterial pressures NOS, as demonstrated in patients with pulmonary hy- remains unresolved [72]. Nevertheless, progression of pertension, would suggest the use of inhaled NO as an the disease is accompanied by further endothelial dis- alternative therapy. The potential therapeutic effect of ruption that itself promotes disease progression. providing L-arginine supplements in parenteral nutrition, In normal airways, NO limits bronchoconstriction to fuel NOS in septic patients, has been raised [84], through the nitrergic neural bronchodilator mechanism although, in this situation, an abundance of NO prevails via prevailing cNOS expression. By contrast, in asthma, through the induction of iNOS. an inflammatory disorder of airways, increased levels of exhaled NO [73] are thought to be produced following induction of iNOS, and bronchial biopsy material Inhaled NO displays increased expression of iNOS compared with Inhaled NO has been used in the intensive care sett- that from nonasthmatics [74]. Furthermore, exhaled NO ing to treat a number of disorders, including ARDS, pri- concentrations increase during the inflammatory late mary pulmonary hypertension, PPHN, and pulmonary response to allergen [75] and in acute exacerbations of hypertension in congenital heart disease, together with asthma [76]; and are reduced by inhaled and oral glu- post/perioperative pulmonary hypertension in paediatric cocorticoids known to inhibit iNOS induction, but not and adult cardiac surgery and pulmonary hypertension cNOS mRNA expression [77]. Interestingly, NO produc- associated with exacerbations of chronic obstructive pul- tion does not appear to reflect asthma severity directly monary disease (COPD) [85Ð88]. nor relate directly to airway hyperresponsiveness to me- The concept that inhaled NO produces selective pul- thacholine challenge. Taken together, this suggests that monary vasodilatation is an attractive one. A lack of the role of NO in asthma is more likely to be as a mar- pharmacological specificity for the pulmonary circula- ker of cytokine-mediated airway inflammation than as tion becomes less relevant, so long as the agent is in- a direct detrimental influence on airway function. Whether activated prior to its reaching the systemic circulation. NO contributes to the airway narrowing (secondary to Conventional intravenous vasodilator therapy to reduce vasodilatation and plasma exudation) in the late response to allergen is unknown. PVR and improve hypoxaemia and right ventricular Further work demonstrating increased exhaled NO dysfunction in patients with pulmonary hypertension is levels in a rodent model of endotoxic sepsis syndrome, limited by two problems. Firstly, the global pulmonary and in patients with bronchiectasis (NO levels correlate vasodilatation induced by intravenous agents may abo- with disease extent) [78] suggests that exhaled NO is lish the protective effect of HPV in underventilated al- V Q likely to be an important marker of airway inflamma- veoli, causing further deterioration in '/ ' matching tion in pulmonary disease. and worsening hypoxaemia. Secondly, the doses required to produce a beneficial effect in the pulmonary vascular bed frequently cause systemic vasodilatation and car- Therapeutic potential of NO diovascular instability. The action of inhaled NO, which NO donors is rapidly inactivated by haemoglobin, is thus confined to the area of deposition, recruiting blood to functional Amyl nitrate was used in the treatment of asthma as lung units to which the inspired gas has access, with- long ago as 1866 [79], and is widely quoted as the earli- out deleterious systemic effects. This should lead to a est therapeutic use of a NO donor in respiratory disease. simultaneous improvement in V '/Q ' matching, a reduc- NO is also the active moiety of glyceryl trinitrate and tion in shunt fraction and a fall in PVR [89]. Indeed, nitroprusside, which act as nitrovasodilators. Glyceryl early animal studies using inhaled NO (at concentrations trinitrate preferentially dilates veins, and its relative veno- of 5Ð80 parts per million (ppm)) in acute hypoxic and selectivity can be explained by the low basal output of pharmacologically-induced pulmonary hypertension de- NO in venous smooth muscle, with an upregulation of monstrated rapidly reversible pulmonary vasodilatation, guanylate cyclase enabling an enhanced response to exo- with no systemic effects [90, 91]. genously administered NO [80]. Other drugs increase The effect of inhaled NO in human volunteers during the production of endogenous NO, such as inhibitors of hypoxia was similar, except that much lower concen- angiotensin-converting enzyme (ACE) that block the trations (10 ppm) of NO were required for maximum breakdown of bradykinin, which in turn stimulates endo- effect [92]. In a retrospective trial of inhaled NO in thelial release of NO. This may contribute to their anti- ARDS, concentrations of 18 ppm for 40 min produced hypertensive properties. significant reductions in mean pulmonary artery pres-

sure, and an increase in arterial oxygen tension (Pa,O2) L-arginine to fraction of inspired oxygen (FI,O2) ratio, enabling FI,O2 to be reduced by 15% [93]. Similar results have The therapeutic potential of L-arginine, the substrate been reported at lower concentrations (100 parts per bil- for NOS, in fuelling NO production is now being real- lion (ppb) to 18 ppm) [94, 95], although no study has ized; reports suggest that arginine prevents the onset of yet demonstrated a clear reduction in mortality or mor- atheroma in experimental models, and restores certain bidity. A trend towards reduced mortality has been shown aspects of endothelial function in humans [81]. It has in a subgroup of inotrope/vasopressor resuscitated sep- also been shown to improve endothelially-derived vaso- tic patients with ARDS, who responded to inhaled NO dilator function in animal models of pulmonary hyper- (>20% rise in arterial oxygen tension (Pa,O2) and/or tension [82], and a deficiency of L-arginine has been >15% fall in mean pulmonary artery pressure) [96]. A 704 S. SINGH, T.W. EVANS response to NO was associated with increased right found hypotension seen in experimental models of sep- ventricular function, other cardiac indices and improved tic shock. In patients with septic shock supported by oxygen delivery; while lack of response to NO and high- large doses of inotropes, L-NMMA and L-NAME were er mortality were characteristic of patients with depres- first shown to restore blood pressure by increasing PVR sed cardiac reserves. [19]. Similar results were demonstrated in a series of However, there is also some evidence of impaired gas 15 patients with septic shock, but there was an associ- exchange after the use of inhaled NO from small clinical ated fall in cardiac output, and no conclusions could be trials of patients with ARDS [97, 98]. Trials are cur- drawn regarding morbidity and mortality. Methylene rently ongoing to address outcome, although death may blue, a guanylyl cyclase inhibitor, has been used with be an insensitive end-point for ARDS, due to the influ- similar haemodynamic effects in small studies. A major ence of its diverse aetiologies on mortality [99]. drawback of such nonselective NOSIs is the detrimental There is also a growing body of evidence of the bene- effect of inhibiting constitutive (endothelial) NOS, poten- ficial role of inhaled NO in PPHN. Outside the intensive tially leading to systemic hypertension and vasospasm care setting, use of inhaled NO in patients with COPD [103, 104]. Aminoguanidine is 10Ð100 times more selec- and secondary pulmonary hypertension produced pulmo- tive for iNOS inhibition than L-NMMA and L-NAME, nary vasodilatation, although Pa,O2 remained unchanged but toxicity remains a major drawback to its use in clin- [88]. The potential for preventing, or reducing, right ven- ical trials in septic shock. The development of selective tricular dysfunction in this context, akin to that seen in iNOS inhibitors, with the beneficial effects of inhibit- long-term oxygen therapy, is an enticing prospect if the ing excessive NO production in inflammatory disorders safety of long-term inhaled NO can be addressed and without the systemic effects of inhibiting cNOS, is in appropriate delivery systems designed. progress. However, inhaled NO therapy may well be detrimental in chronic respiratory failure [100]. Thus, worsening Exhaled NO pulmonary gas exchange with inhaled NO (40 ppm) has been demonstrated in 13 advanced COPD patients, due Exhaled NO has been proposed as a simple, nonin- to a reversion of HPV and subsequent deterioration of vasive means of measuring airway inflammation [77, V'/Q' matching. The authors speculated that differences 105]. Because of its nonspecific increase in inflamma- in NO responsiveness between COPD and ARDS may be tion due to asthma, bronchiectasis and respiratory tract due to the underlying mechanism of hypoxaemia. It seems infections [78]; it has been suggested that serial mea- that patients with prominent intrapulmonary shunt con- surements, rather than absolute values may be useful in tributing to hypoxaemia, as in ARDS, are more likely monitoring whether treatment is adequate, or indeed whe- to benefit from the selective pulmonary vasodilatatory ther newer anti-inflammatory inhaled medications are effect of inhaled NO. By contrast, COPD is characteriz- effective. This would require the development of small- ed by broad heterogeneity in V'/Q' but negligible shunt, er, inexpensive NO analysers. and inhaled NO might inappropriately dilate pulmonary vessels in chronically underventilated alveolar lung units, Toxicity and safe use of NO thus worsening V '/Q ' matching. High concentrations of inhaled NO are likely to have Inhaled NO also has a beneficial role in reducing the toxic effects resulting from the formation of nitrogen dio- morbidity associated with high altitude pulmonary oede- xide (NO2, which dissolves to form toxic nitrous oxide), ma [101]. An improvement in hypoxia-induced pulmo- peroxynitrite radicals (in the presence of superoxide), nary hypertension and arterial oxygenation is thought to and methaemaglobinaemia (interaction of nitrate and occur through a favourable action of inhaled NO on re- haemaglobin, causing a reduced oxygen carrying capac- distribution of pulmonary blood flow from oedematous ity). The deleterious pro-oxidant outcome versus the to nonoedematous alveolar units. cytoprotective antioxidant outcome seems critically depen- It is clear that the potential benefit to be gained by dent on the prevailing oxygen concentrations and rela- the clinical use of inhaled NO may be tempered by its tive concentrations of individual ROS. NO combines detrimental effects in certain conditions. This necessi- with oxygen in high FI,O2s to produce potentially toxic tates careful risk/benefit analysis prior to use and iden- NO2, and levels in inhaled gas should, therefore, be mea- tification of the lowest effective clinical doses for each sured using fuel cell analysers and soda lime to remove indication. Indeed, concerns over toxicity have led to an excess. Inhalation circuits also accurately measure inspi- interest in alternative short-acting inhaled vasodilator, red NO concentrations by chemiluminescence or electro- such as nebulized prostacyclin. A recent study in ARDS chemical analysers. Although there is evidence to suggest has suggested that inhaled NO and nebulized prostacy- that NO is an effective scavenger of ROS [106] and clin have similar efficacy [102]. may inhibit xanthine oxidase [107] (and so reduce ROS); under hypoxic conditions, NO reacts with superoxide to NOS inhibition form peroxynitrite [108], which damages lipids, surfac- tants, nucleic acids and proteins through its pro-oxidant NOSIs have been invaluable in unravelling the patho- capacity. However, frequently quoted toxicity studies have physiological effects of NO, and have been proposed as been carried out principally in nondiseased animals therapeutic interventions in inflammatory conditions exposed to inhaled NO [109]. Thus, no histological, ultra- where excessive production of NO by iNOS is delete- structural or gravimetric evidence of pulmonary toxicity rious, such as septic shock, ARDS and asthma. Substrate was evident after exposure of rats to 1,000 ppm NO for analogues, such as L-NMMA and L-NAME, reverse the 30 min [116], rabbits to 43 ppm for 6 days [111], or local inflammation-associated vasodilatation and prolong-term studies in mice for 6 months [112]. This lack STATUS OF NITRIC OXIDE AS BIOLOGICAL MEDIATOR 705 of demonstrable pulmonary toxicity may have little rele- 11. Nathan C. Nitric oxide as a secretory product of mam- vance to the clinical setting, in which the propensity of malian cells. FASEB J 1992; 6: 3051Ð3064. inflamed lungs to produce ROS in the presence of high 12. Hamid Q, Springall DR, Riveros-Moreno V, et al.

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