Nitric oxide (NO) is an important determinant of local blood flow and is formed by the action of NO synthase (NOS) on the semi-essential amino acid L-arginine in the presence of molecular oxygen. Inhaled NO (iNO) results in preferential pulmonary vasodilatation and lowers pulmonary vascular resistance (PVR), augments hypoxic pulmonary vasoconstriction (HPV), and improves oxygenation. These effects are harnessed in the therapeutic applications of iNO to patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), to those with acute right ventricular failure (RVF) complicating cardiac surgery or acute pulmonary embolism, or in acute sickle chest crisis. Despite dramatic physiological improvements that are often seen during the therapeutic use of iNO, there remains a lack of evidence concerning any beneficial effect on outcomes. This 'Bench-to-bedside' review focuses on the mechanisms of action of iNO and its clinical applications, with particular attention to ALI and ARDS. Alterations in endogenous NO production and the use of exogenous intravenous NO donors in acute inflammatory conditions are beyond the scope of this review.

The licensed indication of iNO is restricted to persistent pulmonary hypertension in neonates, yet most iNO is administered for unlicensed indications. Pharmaceutical iNO is available at a very high cost, and in light of this and concerns over potential adverse effects of iNO, international guidelines have been developed. An advisory board under the auspices of the European Society of Intensive Care Medicine and the European Association of Cardiothoracic Anaesthesiologists published its recommendations in 2005 1. Although this valuable project was sponsored by the manufacturer of iNO (INO Therapeutics, now part of Ikaria Holdings, Clinton, NJ, USA), the board stated that the sponsor had no authorship or editorial control over the content of the meetings or any subsequent publication.

iNO is administered most commonly to invasively ventilated patients, although other routes are possible. To minimise the admixture of high concentrations of oxygen with NO (risk of nitrogen dioxide [NO₂] formation), the NO/nitrogen mixture is introduced into the inspiratory limb of the ventilator tubing as near to the patient as possible. It is obligatory to monitor the NO and NO₂ concentrations, and although concentrations of iNO administered clinically should not cause methemoglobinaemia, guidelines recommend that methemoglobin levels be measured regularly. iNO administration reduces endogenous NO production, and therefore rapid withdrawal of iNO can cause a significant rebound pulmonary hypertension, but in clinical practice, this can be avoided by gradual withdrawal 2.

There is marked variation in response to iNO between patients 2 and in the same patient at different times. After prolonged use, there is a leftward shift in the dose-response curve such that, without regular titration against a therapeutic goal, there is a risk of excessive iNO administration, associated with toxicity and loss of the therapeutic effect 3. A survey of 54 intensive care units in the UK revealed that the most common usage was in treating ARDS, followed by pulmonary hypertension 4, in keeping with results of a European survey 5. By contrast, a survey of therapeutic iNO usage in adult patients from a single US centre (2000 to 2003) demonstrated that the most common application was in the treatment of RVF in patients after cardiac surgery and then, in decreasing order, orthotopic heart transplantation, ventricular assist device placement, medical patients (mostly with refractory hypoxaemia), orthotopic lung transplantation, and for hypoxaemia in other surgery 6.

ALI and its extreme manifestation, ARDS, are characterised by hypoxaemia despite high inspired oxygen (PaO₂/FiO₂ [arterial partial pressure of oxygen/fraction of inspired oxygen] ratios of less than 300 mm Hg [40 kPa] and less than 200 mm Hg [27 kPa], respectively) in the context of a known cause, evidence of pulmonary oedema, and the absence of left atrial hypertension suggestive of a cardiogenic mechanism 7. Pathologically, there is alveolar inflammation and injury leading to increased pulmonary capillary permeability and resultant accumulation of alveolar fluid rich in protein and inflammatory cells. This is manifest clinically as hypoxaemia, ventilation-perfusion mismatch, physiological shunting, atelectasis, and reduced compliance.

Since 1993, when the first investigation on the effects of iNO on adult patients with ARDS was published 8, there have been several randomised controlled trials (RCTs) examining the role of iNO in ALI/ARDS (Table 1). The first systematic review and meta-analysis 9 scrutinised five RCTs and found no beneficial effect on mortality or ventilator-free days, but given wide confidence intervals, the authors concluded that the effects were uncertain. More recently, a meta-analysis considered 12 RCTs that included a total of 1,237 patients 10 and came to conclusions that were more definitive: no benefit was seen on mortality but there was improved oxygenation at 24 hours (13% improvement in PaO₂/FiO₂ ratio) at the cost of increased risk of renal dysfunction (relative risk 1.50, 95% confidence interval 1.11 to 2.02). Indeed, the authors highlight a trend to increased mortality in patients receiving iNO and suggest that it not be used in ALI/ARDS. However, findings from meta-analyses of many small underpowered RCTs have significant limitations and should be viewed as hypothesis-generating, not authoritative. Further elucidation of why iNO may fail to improve patient outcomes stems from understanding recent advances in our knowledge of the biology of iNO, particularly those actions that occur outside the pulmonary vasculature.

NO is a naturally occurring colourless and odourless gas. In biological solutions, it is highly diffusible in water, with a half-life of seconds. NO was regarded mainly as an environmental pollutant prior to its identification as an endothelium-derived relaxing factor and an important determinant of local blood flow 11. NO has an unpaired electron and, as such, reacts very rapidly with other free radicals, certain amino acids, and transition metal ions. In biological solutions, it is stabilised by forming complexes.

The canonical source of endogenous NO is the action of NOS on the semi-essential amino acid L-arginine in the presence of molecular oxygen. Neuronal NOS was the first isoform to be identified, followed by inducible NOS (iNOS or NOS2), and finally endothelial NOS (eNOS or NOS3). iNOS is calcium-independent and generates higher concentrations of NO 12 than the other isoforms do. Its activity is implicated in the pathogenesis of the vasoplegia that characterises septic shock.

Exogenous NO is administered by controlled inhalation or through intravenous administration of NO donors such as sodium nitroprusside or glyceryl trinitrate. Traditionally, iNO was thought to work exclusively in the lung, and thus be free from remote or non-pulmonary effects, through immediate inactivation by circulating haemoglobin (Hb). However, appreciation of the remote effects of iNO has highlighted the importance of the actions of NO on circulating targets (Figure 1).

First, proteins including Hb and albumin contain reduced sulphur (thiol) groups that react reversibly with NO. Previously, NO was considered to react with oxyhaemoglobin to form methemoglobin and nitrate or heme iron nitrosyl Hb and thereby lose all vasodilating properties. However, a stable derivate that retains vasodilatory properties is formed by a reaction resulting in nitrosylation of a conserved cysteine residue of the β subunit of Hb: S-nitrosylated-Hb (SNO-Hb). This reaction is favoured in the presence of oxyhaemoglobin, whereas binding of NO to the heme iron predominates in the deoxygenated state 13. As such, circulating erythrocytes may effectively store and release NO peripherally in areas of low oxygen tension, augmenting microvascular blood flow and oxygen delivery via hypoxic vasodilation of systemic vascular beds 14. Thus, in isolation, NO can act as an autocrine or paracrine mediator but when stabilised may exert endocrine influences 15.

Second, in addition to de novo synthesis, supposedly inert anions nitrate (NO₃^-) and nitrite (NO₂^-) can be recycled to form NO. Indeed, it has been suggested that nitrite mediates extra-pulmonary effects of iNO 16. In the absence of molecular oxygen (hypoxic environment), NOS cannot produce NO and deoxyhaemoglobin catalyses NO release from nitrite, thus potentially also providing a hypoxia-specific vasodilatory effect. Given that effects of iNO are mediated in part by S-nitrolysation of circulating proteins, therapies aiming at directly increasing S-nitrosothiols have been developed. In a small observational study, inhaled ethyl nitrite safely reduced PVR without systemic side effects in persistent pulmonary hypertension of the newborn 17. In animal models, pulmonary vasodilatation was maximal in hypoxia and had prolonged duration of action after cessation of administration 18.

When inhaled with high concentrations of oxygen, gaseous NO slowly forms the toxic product NO₂. Other potential reactions include nitration (addition of NO²⁺), nitrosation (addition of NO⁺), or nitrosylation (addition of NO). Furthermore, NO may react with reactive oxygen species such as superoxide to form reactive nitrogen species (RNS) such as peroxynitrite (ONOO^-), a powerful oxidant that can decompose further to yield and hydroxyl radicals. NO is therefore NO₂ potentially cytotoxic, and covalent nitration of tyrosine in proteins by RNS has been used as a marker of oxidative stress.

NO activates soluble guanylyl cyclase by binding to its heme group, and consequently cyclic guanosine 3'5'-monophosphate (cGMP) is formed, in turn activating its associated protein kinase. This protein kinase decreases the sensitivity of myosin to calcium-induced contraction and lowers the intracellular calcium concentration by activating calcium-sensitive potassium channels and inhibiting the release of calcium from the sarcoplasmic reticulum. These changes cause smooth muscle cells (SMCs) to relax. iNO causes relaxation of SMCs in the pulmonary vasculature with a resultant decrease in PVR. The right ventricle (RV) is exquisitely sensitive to afterload, and if RV function is impaired, it may respond favourably to the decreased afterload, improving cardiac output. iNO must be used with caution in the presence of left ventricular impairment as the decrease in PVR may permit increased right ventricular output to a greater extent than the left ventricle can accommodate and this may excessively increase the left atrial pressure, causing or exacerbating pulmonary oedema. Similarly, pulmonary oedema can result from disproportionate vasodilatation of the pre-capillary compared with post-capillary vasculature, causing an increased transpulmonary gradient.

iNO augments the normal physiological mechanism of HPV and improves ventilation-perfusion matching and systemic oxygenation (Figure 2). In the absence of hypoxaemia being caused by ventilation-perfusion mismatching and HPV, the beneficial effects of iNO on oxygenation are severely limited. Indeed, experimental data confirm that intravenously administered vasodilators worsen oxygenation by counteracting HPV 3. Further signs of the extent of non-pulmonary effects of iNO are increased renal blood flow and improved hepatic tissue oxygenation 14.

Neutrophils are important cellular mediators of ALI. Limiting neutrophil adherence experimentally and production of oxidative species and lytic enzymes reduce lung injury. In neonates, prolonged iNO diminished neutrophil-mediated oxidative stress 19, and in animal models, neutrophil deformability and CD18 expression were reduced 20 with resultant decreases in adhesion and migration 21. These changes limit damage to the alveolar-capillary membrane and the accumulation of protein-rich fluid within the alveoli. Platelet activation and aggregation, microthrombosis, and intra-alveolar deposition contribute to ALI. iNO attenuates the procoagulant activity in animal models of ALI 22 and a similar effect is seen both in patients with ALI 23 and in healthy volunteers 2324. In patients with ALI, decreased surfactant activity in the alveoli contributes to impaired pulmonary function and is of prognostic significance 25.

Although a major cause of diminished surfactant activity is the presence of alveolar exudate, iNO may have deleterious effects on the function of surfactant proteins through the alteration in their structure by reactions with RNS 26. Finally, prolonged exposure to NO in experimental models impairs cellular respiration 27 and may contribute to cytopathic dysoxia.

The failure of iNO to improve outcome in ALI/ARDS is therefore potentially due to several factors. First, patients with ALI/ARDS do not die of refractory hypoxaemia but of multi-organ failure. The actions of NO are mainly considered to have their beneficial effects on oxygenation and are not expected to improve the outcome of multi-organ failure. Indeed, any beneficial effects of iNO on oxygenation may be abrogated by detrimental systemic effects mediated by downstream products of iNO. Second, ALI/ARDS is a heterogeneous condition with diverse causes, potentially requiring specific interventions to affect outcome. Finally, the use of iNO without frequent dose titration risks inadvertent overdose with increased unwanted systemic effects without further cardiopulmonary benefits.

ALI: acute lung injury; ARDS: acute respiratory distress syndrome; Hb: haemoglobin; HPV: hypoxic pulmonary vasoconstriction; iNO: inhaled nitric oxide; iNOS: inducible nitric oxide synthase; NO: nitric oxide; NO₂: nitrogen dioxide; NOS: nitric oxide synthase; PaO₂/FiO₂: arterial partial pressure of oxygen/fraction of inspired oxygen; PVR: pulmonary vascular resistance; RCT: randomised controlled trial; RNS: reactive nitrogen species; RV: right ventricle; RVF: right ventricular failure; SCD: sickle cell disease; SMC: smooth muscle cell.

The authors declare that they have no competing interests.

This article is part of a review series on Gaseous mediators, edited by Peter Radermacher.

Other articles in the series can be found online at http://ccforum.com/series/gaseous_mediators