Basic and Behavioural Sciences Indian J Pediatr 1998; 65 : 333-345 Nitric Oxide : Biological Role and Clinical Uses Mathur S. Kannan ~,2, Sixto Guiang- and Dana E. johnson: Departments of Veterinary PathoBiology I and Pediatrics e, University of Minnesota Colleges of Veterinary Medicine 1 and Medicine:, 1971 Commonwealth Ave., St. Paul, MN 55108, LISA Abstract : Nitric oxide is a product of the conversion of L-arginine by the enzyme mtric oxide synthase. Nitric oxide is involved in a variety of physiological situations and is produced by many different cell types. It is involved in neurotransmission, maintenance of vascular smooth muscle tone, and cytotoxicity. Nitric oxide has been suggested to play ~n anti-inflammatory role by inhibiting the expression of the genes for inflammatory cytokines. The pathophysiologicai role of nitric oxide is also evident in a variety of diseases, including septic shock, asthma, reperfusion injury, etc. Nitric oxide, by stimulating the production of cyclic GMP, relaxes smooth muscles of the cardiovascular, respiratory, gastrointestinal, and genito-urinary systems. Recent studies have provided important information on the use of inhaled nitric oxide for the management of several diseases characterized by the presence of abnormal pulmonary vascular tone, such as persistent pulmonary hypertension of the newborn. This review'addresses the biology and clinical uses of inhaled nitric oxide. (Indian J Pediatr 1998; 65:333-345) Key words : L-arginine; Neurotransmission; Anti-inflammatory. The enzyme nitric oxide synthase (NOS) catalyzes the two-step oxidation of L- arginine to L-citrulline and free nitric oxide (NO)L NO is known to be involved in a va- riety of physiological roles, including neurotransmission, maintenance of vascu- lar smooth muscle tone, and cytotoxicity. Such diverse physiological functions dic- tate that the production of NO is regulated depending on the location of the enzymes responsible for its synthesis and the factors that induce their activity. In this review, we will discuss the biosynthesis of NO, the mechanisms by which NO causes smooth Reprint requests : M.S. Kannan, B.V. Sc., M.Sc., Ph.D., Department of Veterinary PathoBiology, College of Veterinary Medicine, 1971 Common- wealth Ave., St. Paul, MN 55108, USA, Fax : 612- 625-5203, E-mail : kanna00i@maroon.tc.umn.edu muscle relaxation, the experience to date with the use of inhaled NO m treating per- sistent pulmonary hypertension of tile newborn, and some recommendations re- garding its use. Regulation of NO Biosynthesis Furchgott and Zawadzki: showed that vascular relaxation induced by acetylcho- line is mediated by a factor released from the endothelium, which thev termed ell- dothelium-derived relaxing factor (EDRF) which was shown to be NO derived from vascular endothelium ill response to a vari- ety of stimuli, including shear stress:L NO is synthesized from L-arginine by NOS. During its synthesis, NOS is translated as a monomer, and ill the presence of tetrahyd- robiopterin it forms a dimer. In the pres-
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`334 MATHUR $. KANNAN ETAL Vol. 65, N~). 3, 1998 Fig. 1. Monomer I nac~i.ve I)ime~ Nitric Oxide Synthesis [ @@ "~~ Tetra hydrobiopterin ] k.,mo,.,,o+Cn" I Active l)imer Nitric oxide synthase (NOS) is synthe- sized as a monomer. In the presence of the co-factor tetrahydrobiopterin, the two NOS monomers form an inactive dimer. In the presence ~f calmodulin and calcium (required for the calcium- independent isoformg), the inactive Jimer is converted to an active dimer. Fhe active dimer enzyme converts L-ar- ginine in the presence of molecular oxy- gen to NO and citrulline. ence of calmodulin and molecular oxygen, NOS converts L-arginine to NO, with cit- rulline as a byproduct (Fig. 1). At least three isoforms of NOS have been described : neuronal NOS (n-NOS or NOS1), en- dothelial NOS (e-NOS or NOS3), and in- ducible NOS (i-NOS or NOS2) 4. Several co- factors are involved in the synthesis of NO including calmodulin, FAD, FMN, NADPH, and tetrahydrobiopterin. NOS contains heine moiety in its structure. Bredt et al ~ first isolated n-NOS from the brain. Although n-NOS has considerable homology in terms of amino acid sequence to the other NOS isoforms, the amino ter- minal region of this enzyme ha> a long se- quence of amino acids whose precise func- tion is as yet undefined, n-NOS is found in several parts of the brain, but appears to be more abundant in the cerebelltm~ and as a result it has been speculated that NO may mediate motor function and coordination ~. n-NOS is also found in several peripheral neurons. Activation of n-NOS in the pe- ripheral neurons and the subsequent re- lease of NO results in relaxation of smooth muscle 7. For example, NO released from peripheral neurons regulates the function of smooth muscles of the gastrointestinal, respiratory, and vascular systems. NO re- lease is also blown to mediate penile erec- tion. n-NOS and e-NOS are constitutively ex- pressed in neurons and endothelial cells re- spectively ~. Under resting conditions, these enzwnes do not swlthesize appreciable quantities of NO. However, when intracellular calcium levels rise in response to excitation of the cells possessing these enzymes, the resultant calcium-calmodulin complex activates NOS, resulting in NO re- lease. When the intracelluIar calcium levels fall, the activity of the enzyme decreases. Therefore, the constitutive NOS isoforms are described as calcium-dependent, since their activity is tightly regulated by the level of intracellular calcium, i-NOS, on the contrary, is not expressed to a significant extent by cells under resting conditions. In- duction of i-NOS activity in inflammatory cells such as macrophages usually results in the production ot large quantities of NO*. NO production by macrophages may be involved in host defense mechanisms. When i-NOS is induced, for example in re- sponse to inflammatory cytokines, immune complexes or LPS, it binds to calmodulin and produces NO even in the absence of
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`Vol. 65, No. 3, 1998 NITRIC OXIDE : BIOLOGICAL ROLE AND CLINICAL USES 335 calcium ~~ Induction of i-NOS synthesis by LPS involves transcription and the NF-kB family of transcription factors plays a key role in this process~L i-NOS has been de- scribed as calcium-independent, since its activity is not dependent on the level of intracellular calcium. Mechanisms of NO Action in Smooth Muscle Cells By interacting with the heme moiety of the enzyme soluble guanylyl cyclase, NO stimulates the production of cyclic GMP 12. The resultant elevation of intracellular cGMP leads to activation of the cGMP-de- pendent protein kinase and phosphorylation of some key intracellular proteins, including ion channels. In vascu- lar smooth muscle, recent studies have in- dicated that elevation of intracellular cGMP by NO and NO donors results in ac- tivation of caclium-activated K + channels, membrane hyperpolarization and relaxa- tion of vascular tone 13. The nitrovasodilators used in the treatment of hypertension and as coronary vasodilators act in a manner to decrease vascular resist- ance and thus effect greater blood flow to the affected organ. Previous studies from our laboratory have indicated that cGMP- dependent activation of the calcium-acti- vated K § channels by NO released from neurons within the airways and s-nitroso- n-acetylpenillamine (SNAP), a NO donor, is also involved in the relaxation of airway smooth muscle 14. Not all the effects of NO and NO donors in smooth muscle are mediated via eleva- tion of intracellular cGMP. In a recent study, Bolotina et al ~s showed that NO di- rectly activates calcium-dependent K + channels in vascular smooth muscle cells, causing relaxation. Exposure of aortic rings to NO caused an elevation of intracellular cGMP and relaxation. Pre-exposure to methylene blue, an inhibitor of the soluble guanylyl cyclase, did prevent the rise in the intracellular level of cGMP, without signifi- cantly affecting relaxation. However, pre- exposure to charybdotoxin, a toxin derived from the scorpion Leiurus quinquestriatus herbraeus, and a selective inhibitor of the calcium-dependent K § channels, reduced the relaxation response to NO. These re- sults provided strong evidence for cGMP- independent pathways for vascular relaxa- tion by NO involving direct activation of K § channels. In porcine airway smooth muscle, our earlier studies also support a cGMP-independent activation of the calci- um-dependent K§ by NO donors and neurally released NO in the relaxation response. However, unlike vascular smooth muscle cells, activation of the calci- um-dependent K + channels may not neces- sarily be associated with membrane hyper- polarization in airway smooth muscle cells. Therefore, other mechanisms may be in- volved in the relaxation response to NO donors in airway smooth muscle cells, such as changes in local intracellular calcium concentration, decreased calcium sensitivi- ty of the contractile proteins, etc. Elucida- tion of these mechanisms may help us un- derstand the basis for cellular specificity of NO action. The effects of NO and NO donors are also characterized by inhibition of agonist stimulated elevation of intracellular cal- cium 1~. In smooth muscles of airways and vasculature, contractions to agonists in- volves elevation of intracellular calcium concentration above the basal level. Under normal, unstimulated conditions, smooth muscle cells maintain a relatively low
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`330 MATHUR S. KANNAN ET AL Vol. e~5, No. 3, 1998 intraceiluiar caicmm level, usually in the range of 100-150 nM. In recent studies, we have shown that calcium release from intracellular structures known as the sarcoplasmic reticulum plays an important role in the maintenance of steady-state cal- cium levels during agonist stimulation :7. This steady-state level of calcium is critical for the maintenance of force during agonist stimulation and intracellular calcium levels can reach 1000 mM or higher during the steady-state response to agonists in several ~mooth muscle cells. Inhibition of this steady-state level of calcium is expected to lead to smooth muscle relaxation and in the airways, such relaxation may result in decreased resistance to airflow. In airway smooth muscle, NO, NO donors, and ]32- adrenoceptor agonists that are used in the symptomatic treatment of asthma as bronchodilators, all cause relaxation by re- ducing intraceilular calcium levels during agonist stimulatioW",J'~.~% Furthermore, the effects of NO and NO donors are mimicked Dy exogenous membrane-permeant ana- logue of cGMP and that of 132-adrenoceptor agonists by exogenous membrane- permeant analogue of cAMP. However, our recent studies have indicated that the mechanisms by which NO and NO donors lower intracellular calcium levels in airway smooth muscle cells are diffelent from those induced by salbutamol, a 132- adrenoceptor agonist. NO and NO donors, by increasing intracellular cGMP levels, decrease calcium release from the sarcoplasmic reticulum 16,:~, while ]32- adrenoceptor agonist salbutamol increases intracellular cyclic AMP levels and thus decreases calcium influx through voltage- gated calcium channels and augments cal- cium efflux from the cell 19. The net effect of inhibition of calcium release from the sarcopiasmic reticulum, inhibition of cal- cium influx and augmented calcium efflux is lower intracellular calcium level and smooth muscle relaxation. The results of the studies outlined above clearly point to multiple mechanisms by which cyclic nucleotides, which mediate the effects of NO, NO donors, and 132- adrenoceptor agonists, cause smooth mus- cle relaxation. Among the mechanisms as- sociated with NO action are cGMP-de- pendent activation of K + channels, mem- brane, hyperpolarization, reduction of intracellular calcium levels by inhibition of calcium release, calcium influx, and cal- cium efflux, and cGMP-independent mechanisms involving activation of K ~ channels. One or more of these mecha- nisms may be operative in a given cell. A ttxorough understanding of these various mechanisms is essential towards designing therapeutic strategies for the management of asthma, hypertension, etc. Anti-inflammatory Role of NO There is clear evidence of NO in inflam- mation. It is known to inhibit the produc- tion of several pro-inflammatory cytokines, including IL-6 and IL-8:". This action of NO appears to be independent of cGMP eleva- tion. Evidence seems to indicate that NO inhibits transcription of these pro-inflam- matory cytokine genes. The genes that en- code the messenger RNAs for these pro-in- flammatory cytokines contain DNA bind- ing motifs in their promoter regions for the transcription factor NF-KB :~ In a variety of cells, including macrophages and vascular endothelial cells, activation of NF-KB is an essential critical first step in the transcrip- tion of cytokine genes involved in inflam- mation. The binding of NF-KB to the pro-
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`Vol. 05, No. 3, 1998 NITRIC OXIDE : BIOLOGICAL ROLE AND CLINICAL USES 337 rooter regions of the genes encoding the messenger RNAs for these cytokines initi- ates transcription. It has been shown that NF-KB is located in the cytoplasm in an in- active form, bound to an inhibitor}, subunit known as IKB as well as an inhibitory prosequence2k Activation of NF-KB re- quires phosphorylation of these inhibitory subunits and the eventual translocation of the free NF-KB into the nucleus 22. Although phosphorylation can be induced by several kinases within the cells, the role of tyrosine kinases in the phosphorylation of IKB is be- coming increasing clear zz. NO, by inhibit- ing NF-KB translocation and by increasing the expression of IKB 23, can prevent tran- scription of pro-inflammatory cytokine genes and thus can play a protective role in inflammation. Furthermore, agents that in- terfere with phosphorylation of these in- hibitory subunits and thereby prevent NF- KB translocation, may also possess anti-in- flammatory properties. In this regard, tyrosine kinase inhibitors are being investi- gated as potential anti-inflammatory agents. In experimental mvdels of sepsis, Tyrphostin AG 556, a tyruaine kinase in- hibitor, has been shown to decrease tumour necrosis factor (TNF) production, improve survival and reduce cardiopu[monary dys- function:4.2L NOdonors such as S-nitroso- glutathione (GSNO) have been shown to inhibit not only NF-KB binding tO. promoter regions in the DNA, but to also incluce IKB transcription and overxpression of the IKB protein 2~. The latter mechanism would, lead to increased binding of NF-KB in the cyto- plasm, maintaining it in an inactive form, thereby preventing transcription of the cytokine genes. The anti-inflammatory role of NO is evi- dent in some pathological situations such as atherosclerosis. Although atherogenesis has a complex pathophysiology, NO has been shown clearly to inhibit a variety of steps that contribute to vascular injury in atherogenesis, including vascular smooth muscle cell proliferation and platelet adhe- sion to endothelial cells. In addition, NO prevents the interaction of leukocvtes with vascular endothelial cells bv inhibiting chemotaxis and adhesion2~L Recent studies have shown that NO decreases cytokine-in- duced induction of vascular endothelial cell adhesion molecule (VCAM-1), endothelial leukocyte adhesion molecule (ELAM-1), and intracellular adhesion mol- ecule (ICAM-I):~L These surface receptors on inflammatory cells such as the eosinophils and endothelial cells are essen- tial for the adhesion and diapedeMs of cells to sites of inflammation. NO, by inhibiting the expression of these adhesion molecules, can also exert an anti-inflannnatory effect in the host. Pathophysiological Roles of NO There is unequivocal evidence of NO as an effector of the inflammatory response. The mechanisms bv which NO causes tox- icity in the host are complex, but genera- tion of reactive oxygen intermediates (ROIs) and its reaction with superoxide to generate peroxynitrite are potential mecha- nisms2~L Peroxynitrite is more toxic than NO because of its potential for direct cytotoxicity through protein tyrosine nitra- tion. Clearly, these actions of NO may be independent of cGMP elevation in the ef- fector cells. The role of NO as a toxin has been delineated in several clinical entities. These include septic shock, various inflam- matory diseases, atherosclerosis, reperfusion injury of the myocardium, acute renal failure, glomerulonephritis, and
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`338 MATHUR ~. KANNAN ET AL Vol. (~5, No. 3, 1998 asthma. Endotoxic shock : The vascular endotheli- um can be stimulated by LPS to express the calcium-independent i-NOS 27. This is known to occur in bacterial sepsis, where LPS derived from gram negative bacteria ~tm~ulates the production of the pro-in- riamrnatory cytokines IL-1 and IL-6, TNF-R and ]nterferon-y. These pro-inflammatory cytokmes can in turn stimulate the expres- sion of i-NOS in the vascular endothelium, resulting in enhanced uninterrupted pro- duction of NO in blood vessels. There is ev- Ldence of increased NO production in pa- :ients with sepsis. The hemodynamic changes leading to systemic hypotension, :efractoriness to vasoconstrictor agents and Alock du.'ing sepsis arise from overproduc- tmn of NO- Selective inhibitors of the calci- um-independent i-NOS are being devel- oped in an attempt to reverse the hemody- namic changes associated with shock. These inhibitors should have minimal, if an~, effects on other isoforms of NOS. Fherefore, their actions should be restricted to the cardiovascular system for most part. Asthma : Biopsied airway tissues from asthmatics appear to show increased unmunoreactive i-NOS 2s. Furthermore, the level 0f NO gas in exhaled air is consider- ably greater in asthmatics than in normal individuals > . Treatment of asthmatics with corticosteroids has been shown to decrease exhaled NO gas levels 30. The selective in- hibitor of i-NOS, aminoguanidine, has also been shown to significantly reduce exhaled NO in asthmatics, but not in normal hu- man volunteers 30. However, i-NOS inhibi- tors do not appear to improve spirometry in asthmatics. Similarly, in experimental animals challenged with allergen, there ap- pears to be an association between in- creased serum IgE level, eosinophilia and i- NOS levels in the lung. Pre-treatment of these animals with corticosteroids also re- sults in a significant reduction of i-NOS ac- tivity within the lungs. It has been sug- gested that increased i-NOS activity within the lungs and the apparent relationship be- tween i-NOS activity and eosinophilia may be contributing factors in the pathogenesis of reactive airway disease. Therefore, the palliative effects of corticosteroids and i- NOS inhibitors in asthma may be related to inhibiton of i-NOS induction and eosinophilia, and the inflammation associ- ated with damage to respiratory mucosa due to release of eosinophil granular con- tents. Endogenous Roles of NO : As a Neurotransmitter The smooth muscles of many organs are relaxed by neurotransmitters released from inhibitory nerves. There are two principal types of inhibitory neural controls of smooth muscles, i.e., adrenergic and non- adrenergic-noncholinergic (NANC). Adrenergic nerves release norepinephrine from the post-synaptic nerve terminals Norepinephrine released from these nerves can activate both ~- and [~-adrenoceptors. In vascular smooth muscles, activation of the cq-adrenoceptors results in vasconstric- tion, while activation of the [32-adrenocep- tors causes vasodilatation. Smooth muscles of airways, gastrointestinal tract, and pos- sibly other organs are relaxed by NO re- leased from NANC nerves 7. In fact, in many smooth muscles, NO appears to be the principal, if not the exclusive, mediator of NANC nerves. In the vasculature, NO released from the endothelial cells causes vasodilatation under both physiological and pathophysiological situations. Recent
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`Vol. 65, No. 3, 1998 NITRIC OXIDE : BIOLOGICAL ROLE AND CL1NICAL USES 339 studies from our laboratory and others have shown that NO is an important medi- ator of the NANC nerves in airway smooth muscleL It also appears that NO-releasing NANC nerves innervate the pacemaker and the mycoardium and have negative in- otropic control 6. Furthermore, NO has pro- found effects in many secretory cells, such as pancreatic ~ cells, adrenals, and salivary gland, where it is known to stimulate secre- tion 6 . Effects of Inhaled NO The ability of NO to cause localized va- sodilatation makes it a potentially ideal pulmonary vasodilator when used in inha- lation therapy. Since its first reported use in 1991, NO has been tried for a wide variety of clinical conditions characterized by the presence of abnormal pulmonary vascular tone. In the dose range of 2-80 ppm by vol- ume, inhaled NO has been shown to result in selective pulmonary vasodilatation in both animal and human subjects 3~. In hu- man patients, it appears that inhaled NO selectively inhibits hypoxic pulmonary va- soconstriction, with minimal systemic he- modynamic consequences. In anesthetized sheep subjected to hypoxia (FiO2 of 0.06), addition of 40-80 ppm of NO to the inhaled gas was shown to reverse the resulting pul- monary vasoconstriction. The changes in pulmonary vascular resistance and pulmo- nary blood flow in response to inhaled NO can be achieved without any significant changes in systemic vascular resistance, cardiac output or blood pressure. Other studies have indicated that the pulmonary vasodilatory effects of NO gas in the con- text of hypoxic vasoconstriction in anesthe- tized lambs is indeed mediated via cGME since cGMP levels were found to be elevat- ed in the lung tissue of these animals. Fur- thermore, the pulmonary vasodilatory ef- fects of NO appear to be independent of prostacyclin (PGI2) release, since arachido- nate cyclo-oxygenase inhibitors have no ef- fect on NO-induced vasodilatation. In healthy human volunteers subjected to acute hypoxia (FiO2 0.12) for 30 mins, addi- tion of 40 ppm NO to inhaled gas also re- versed the pulmonary hypertension, with- out any significant systemic hemodynamic effects. The onset of complete vasodilata- tion in these volunteers was rapid and complete vasodilatation was achieved within a short time after exposure to 10 ppm of NO gas. It is because of this selec- tive pulmonary vasodilating capability that inhaled NO has become a promising thera- peutic modality for a large number of clini- cal entities such as Persistent Pulmonary Hypertension of the Newborn (PPHN) and pulmanory hypertension associated with cardiopulmonary bypass surgery. In children having congenital heart dis- ease and exhibiting diminished endothelial function, as evidenced by decreased va- sodilatory response to acetylcholine, in- haled NO causes sustained vasodilata- tion 32. It is well known that acetylcholine- induced vasodilatation is mediated by NO released from the vascular endothelium 2. These observations support the conclusion that inhaled NO has the ability to dilate constricted puhnonary vessels even in the presence of endothelial injury, wihout sig- nificant systemic cardiovascular side ef- fects. These observations also support the notion that NO administered by inhalation may be useful in improving gas exchange and therefore in the treatment of PPHN. In infants with PPHN, there is evidence of decreased NO production. In such in- fants, inhaled NO has been shown to im-
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`340 MATHUR S. KANNAN ET ,'IL Vol. 65, No. 3, 1998 prove oxygenation by increasing pulmo- nary blood flow and matching ventilation ~.0 perfusio~3L In several clinical studies 3~, the systemic and pulmonary vascular re- sistance changes have been compared in response to inhaled NO and intravenous PGI> Inhaled NO causes significant reduc- tion of pulmonary vascular resistance and this effect appears to be clinically signifi- cant in ventilated patients. Inhaled NO ap- pears to improve arterial oxygen tension (PaO:) and decrease ventilation pressures m PPHN. In PPHN, the systemic arterial hypox- emia results from increased pulmonary vascular resistance and the consequent R-->L shunting of deoxygenated blood. In such patients, there is improved systemic oxygenation in response to inhaled NO. In patients with severe pulmonary hyperten- sion, I.V. PGI2 or its stable analogue, ilo- prost is also known to result in significant pulmonary vasodilatation, leading to im- proved cardiac output and arterial oxygen- ation. However, there is also significant re- duction of systemic vascular resistance as- sociated with PGI2 administration. In this regard, inhaled NO may offer some advan- tages over PGI2 in improving gas exchange, with minimal systemic cardiovascular side effects. Much of the enthusiasm for the clinical use of NO stems from the hope that in- haled NO would significantly reduce the present mortality associated with PPHN and decrease the need for high risk inva- sive ther.apies such as Extracorporeal Membrane Oxygenation (ECMO). The first clinical application of many of the pub- lished clinical studies involving inhaled NO has been in the treatment of PPHN. The syndrome of PPHN may be idiopathic with a chest X-ray which is essentially clear, the so called black lung or idiopathic PPHN. Often, PPHN may be associated with other underlying pulmonary patholo- gies such as respiratory distress syndrome, meconium aspiration syndrome, group B streptococcal sepsis, and pneumonia or pulmonary malformations such as congen- ital diaphragmatic hernia. All of these dis- ease entities have different pathophysio- logic mechanisms of lung injury, but are similar in that they may be associated with a final common pathway of increased pul- monary vascular tone, resulting in refracto- ry hypoxia. The early reports on the use of inhaled NO clearly demonstrated improve- ment in gas exchange after initiation of therapy. Since these early reports, multiple clinical trials have been published with consistent improvement in oxygenation and gas exchange in the above mentioned clinical entities. There have been two multicentre randomized, blinded placebo-controlled clinical trials of inhaled NO for the treat- ment of PPHN. These two trials demon- strated similar results. The largest of these two trials conducted by the Neonatal In- haled Nitric Oxide Study Group 34, con- sisted of 235 infants greater than 34 weeks gestation with severe respiratory failure re- quiring mechanical ventilation and who had an oxygenation index of at least 25. These infants were randomized to receive either inhaled NO at 20 ppm or placebo. The infants in the NO group had improved oxygenation with higher PaO:, lower oxy- genation indices, and Iower alveolar-arte- rial oxygen gradients 30 rain after initiation of NO when compared to controls. More importantly, there was a lower risk of death (relative rise of 0.75; 95')',, confidence interval : 0.57-0.91), and they received ECMO less often (39% vs. 55% for con-
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`goi. o5, No. 3, 1998 NITRIC OXIDE : BIOLOGICAL ROLE AND CLINICAL USES 341 trols). Not all the infants had a beneficial effect of inhaled NO. Only 66% of infants in the NO group had a complete or partial response as compared to 26% in the control group. Higher doses of NO (80 ppm) didn't improve oxygenation in 76% of the infants who were non-responders to lower doses of inhaled NO. The multicentre trial by Roberts et al 3~ consisted of 58 infants randomized to re- ceive either 80 pprn NO or placebo. Similar to the large multicentre trial conducted by the Inhaled Nitric Oxide Study Group, more infants in the NO group had im- proved oxygenation : 55% vs. 7% in the contro] group,, with higher PaO2, lower oxygenation index, along with a decreased use of ECMO (odds ratio 3.8; 95% CI 1.1- 13.1). In both these trials, there was a signifi- cant number of infants who did not show an improvement in oxygenation in re- sponse to high dose of inhaled NO, i.e., 80 ppm. Although the factors regulating effi- cacy of inhaled NO are unclear, diffusability of NO through the lung adventitia may play a significant role in its ability to alter pulmonary vascular resist- ance. Another factor is the ability of NO to reach the distal airways where the bulk of the pressure changes occur. Methods of im- proving lung aeration with various strate- gies of mechanical ventilation and exogenous surfactant therapy have im- proved efficacy of inhalation NO therapy. In a randomized crossover controlled trial by Kinsella et al (see 36) comparing high frequency oscillatory ventilation to inhaled NO for acute respiratory failure in near term newborns, only 28% of infants had a beneficial response to inhaled NO alone. However, there was improvement in oxy- genation in infants who did not have an initial response to inhaled NO by the addi- tion of high frequency oscillatory ventila- tion. In infants treated with inhaled NO, follow-up studies for 1 to 2 years have not demonstrated any additional medical or developmental problems compared to in- fants of similar degree of illness treated with standard therapy including high fre- quency oscillatory ventilation, alkalosis and ECMO. Congenital Diaphragmatic Hernia : The use of inhaled NO has not been suc- cessful in treating all clinical entities associ- ated with PPHN. Congenital diaphragmat- ic hernia is associated with persistent ele- vation of pulmonary vascular resistance. Frequently, there is puhnonary vascular re- modelling in response to intrauterine pul- monary blood flow through a hypoplastic vascular bed in addition to pulmonary ar- terial vascular spasm. There are reported cases documenting improvement in oxy- genation and gas exchange in infants with congenital diaphragmatic hernia. Leveque et aP 7 reported improved gas exchange and oxygenation in infants with congenital dia- phragmatic hernia in conjunction with a delayed repair of the defect. Karamanouki- an et al 3~ reported no sustained improve- ment in oxygenation and gas exchange in infants with lung hypoplasia. All infants eventually required ECMO. Infants who subsequently had return of pulmonary hy- pertension following ECMO were then re- sponsive to inhaled NO. The exact mecha- nism and the factors responsible for the re- sponsiveness to NO may involve diffusa- bility of NO through the adventitia to the pulmonary vessels. In the lamb model of surgically created diaphragmatic hernia, the ability of NO to decrease pulmonary vascldar resistance and increase blood flow
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`342 MATI-IUR S. KANNAN ETAL Vol. 65, No. 3, 1998 can 0e greatly augmented with the instilla- tion of exogenous surfactant 39. Again the proposed mechanism of synergy between inhaled NO and surfactant therapy is that the surfactant allows recruitment and sta- bilization of lung volume, allowing inhaled NO to diffuse adequately to its site of ac- tion m the pulmonary blood vessels. In a btudy conducted by the Neonatal Inhaled Nitric Oxide Study Group, 53 infants with congenital diaphragmatic hernia were ran- 3omized to receive either 20 ppm NO or placebo. Although, there was some tran- sient improvement in gas exchange in 56% of the infants receiving NO, the overall ef- fect was transient. There was no difference in the primary outcome of death or initia- tion of ECMO (82% control vs. 96% treat- nlent group). In fact, more of the infants in the inhaled NO group went on to ECMO. Pulmonary Hypertension Associated with Congenital Heart Disease and Cardi- opulmonary Bypass : Inhaled NO has been used successfully in the setting of pulmo- nary hypertension following cardiothoracic ~urgery. Following cardiopulmonary by- pass, dysfunction of the pulmonary vascu- lar endothelium results in a decrease in en- dothelial-derived NO, thereby causing an imbalance between endogenously pro- duced vasodilators and vasoconstrictors. This results in an increased pulmonary vascular tone and pulmonary hyperten- sion. Inhaled NO mav make up for the de- ficiency in the endogenous production of NO by the vascular endothelium and shift vascular tone back in favour of vasodilata- tion. Inhaled NO has been found to be effec- tive following lung transplantation where the grafted lung undergoes ischemic and reperfusion injury. In a small fraction of lung transplant patients with puhncnary hypertension following acute grafted lung dysfunction, inhaled NO at a concentration of 80 ppm. decreased puhnonary artery pressure with a concomitant decrease in the intrapu!monary shunt fraction. Infants and children with congenital heart disease are at risk of developing ab- normal elevation in pulm