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Editorial Views  |   July 2001
New Mechanisms for Inhaled NO: Release of an Endogenous NO Inhibitor?
Author Notes
  • Professor and Chair, Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland. rajohns@jhmi.edu
Article Information
Editorial Views
Editorial Views   |   July 2001
New Mechanisms for Inhaled NO: Release of an Endogenous NO Inhibitor?
Anesthesiology 7 2001, Vol.95, 3-5. doi:
Anesthesiology 7 2001, Vol.95, 3-5. doi:
THE endogenous signaling molecule, nitric oxide (NO), plays a significant role in many physiologic and pathophysiologic processes in the lung. 1,2 In addition to its role as an endothelial-dependent vasodilator, NO has been shown to regulate ciliary motility, to participate in angiogenesis and vasculogenesis, and to prevent leukocyte adhesion and platelet adhesion. Produced in excess, NO may contribute to the vascular remodeling of pulmonary hypertension, to inflammatory lung injury, and to reactive airway disease.
Exogenous inhaled NO has been widely investigated in the therapy of lung diseases associated with increased pulmonary vascular resistance. 2 In this regard, inhaled NO has been shown to be effective in reducing the use of extracorporeal membrane oxygenation in neonates with persistent pulmonary hypertension of the newborn. Inhaled NO has significant promise as effective therapy in several other diseases of the lung, particularly those in which the increased pulmonary vascular resistance is likely to reverse within a period of a few days.
The role of endogenous NO in pulmonary vasoconstriction is complex and is dependent on the specific physiologic circumstances. The disease-free, normoxic lung has very little NO synthase expressed in the small resistance vessels. 3 Consistent with this lack of expression, numerous studies using inhibitors of NO synthase failed to demonstrate an increase in normal lung vascular resistance, suggesting that NO does not play a significant role in the normally low pulmonary vascular resistance. 1 
Nitric oxide content is regulated by oxygen in a complex manner. NO content is dependent on its rate of production from NO synthase and its stability once produced. Low- and high-oxygen environments can each regulate NO production and stability. 1,4–6 NO requires two molecules of oxygen as substrate in addition to l-arginine. The Michaelis constant (Km) of the eNOS for oxygen, 7.7 μm, suggests that NO production would be significantly reduced with a partial pressure of oxygen (Po2) of less than 30 mmHg. 4 Because of the second order kinetics of the chemical interaction between NO and oxygen, in an environment with high oxygen content, NO rapidly combines with oxygen to form nitric acid, making it unavailable for vascular action. NO also combines with superoxide radical to form peroxynitrite. 5,6 Thus, lowering oxygen tension prolongs the half-life of NO in counterbalance to its reduction of NO synthesis through limitation of oxygen substrate. High oxygen tension provides adequate oxygen substrate but accelerates NO metabolism to peroxynitrite and nitric acid. In addition, prolonged exposure to low oxygen tension up-regulates the endothelial and inducible isoforms of NO synthase in the lung. 3 
In multiple human and animal studies, when acute hypoxia reduces lung oxygen to a physiologically relevant degree, the net effect of all these interactions seems to be an increase in NO available for vascular action. 1 In this context, when inhibitors of NO synthase are administered to the hypoxic lung, the vasoconstriction normally seen with hypoxia is increased because of the inhibition of NO. 1 NO is thus an important modulator of hypoxic pulmonary vasoconstriction, but it plays a small role in regulating pulmonary blood flow during normoxia.
Hambraeus-Jonzon et al.  7 have made the interesting observation that in anesthetized humans, inhaled NO to a single hyperoxic lung increases the blood flow to this lung, but only if the other lung is hypoxic. This increase in regional blood flow caused by unilateral inhaled NO did not occur in the absence of regional hypoxia when both lungs were either normoxic or hyperoxic. This observation suggested a much more complex mechanism of action for inhaled NO, involving an interaction between the hyperoxic lung regions receiving inhaled NO and the hypoxic lung regions not directly reached by inhaled NO. In work published in the current issue of Anesthesiology, Hambraeus-Jonzon et al.  8 investigated the mechanism of this response in a pig model. Their work provides strong evidence that inhaled NO to the hyperoxic lung releases a blood-borne mediator that inhibits NO synthase in the hypoxic lung that is not receiving inhaled NO. Inhaled NO is traditionally thought to improve oxygenation by dilating vessels in ventilated lung areas and thereby redistributing blood flow to these ventilated areas and away from the nonventilated regions, with a resulting decrease in shunt fraction. The current findings suggest an entirely novel mechanism by which inhaled NO may improve shunt and oxygenation. In addition to dilating vessels in the ventilated lung regions, it seems that NO somehow results in the release of a factor that constricts vessels in regions that do not receive inhaled NO.
This new work by Hambraeus-Jonzon et al.  8 used both a single pig model and a cross-circulation model to suggest strongly the existence of a blood-borne mediator. In the single pig study, delivery of inhaled NO to only the normal right lung resulted in a reduction of blood flow and a decrease in exhaled NO from the isolated hypoxic left lower lobe. Thus, the delivery of NO to one area of the lung resulted in an effect on an area to which NO was not delivered. This response in the single piglet model could potentially be explained by changes in shear stress. It is established that NO production from the endothelium is stimulated by shear stress. Decreasing shear would reduce NO production. Administration of inhaled NO to the normal lung could vasodilate that lung, shunt blood away from the left lower lobe, decrease left lower lobe blood flow, decrease shear stress, and therefore decrease NO synthase activity and exhaled NO. An alternate explanation would be a blood-borne mediator that potentially inhibits NO synthase or acts via  another contractile mechanism. To address this question, the investigators used a cross-circulation model that administered inhaled NO to a normal pig and cross-circulated that animal’s blood to another pig with an open chest and an isolated, hypoxic left lower lobe. As in the single-pig model, an increase in pulmonary vascular resistance to the left lower lobe and a decrease in exhaled NO occurred. In addition, a modest decrease in NO synthase activity was demonstrated through biochemical studies. These data are clearly consistent with the authors conclusion that inhaled NO releases a blood-borne mediator that down-regulates endogenous NO production in lung regions that do not receive inhaled NO, and more so in hypoxic than hyperoxic regions.
These studies are remarkable for two reasons. First, they suggest that our previous simplistic understanding of inhaled NO acting simply through vasodilation is much more complex. They also suggest a novel regulatory pathway for NO signaling through NO-stimulated production of an endogenous NOS inhibitor. At this time, the authors have not further characterized this response or attempted to isolate the factor involved. It would be interesting to know whether blood from animals with inhaled NO shows vasoconstriction in an isolated vascular ring. Would blood from the lung receiving inhaled NO alter an NO synthase activity assay? Is there response with serum, or is whole blood required? What is the role of hemoglobin versus  other serum proteins? What is the biochemical nature of the factor?
Endogenous inhibition of NO synthase has been reported, and a few of these inhibitors have been identified and characterized. 9–17 However, none have been shown to be released by NO. l-arginine analogs that have been chemically modified at the terminal guanidino nitrogen group, such as l-NMMA, have been used as synthesized products to inhibit NO synthase. l-NMMA and other methylated l-arginine analogs have also been shown to be synthesized endogenously. 9–13 Among these, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have been shown to be most abundant. ADMA is an inhibitor of NO synthase, whereas SDMA is inactive. ADMA is produced by a family of N  -methyl transferases that methylate l-arginine residues within specific proteins. ADMA is subsequently released after proteolytic cleavage of these proteins. ADMA also undergoes specific enzymatic metabolism by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Endothelial dysfunction has been observed when DDAH activity has been inhibited and increased ADMA concentrations are present. Several studies have now implicated ADMA as a factor in atherosclerosis, hypercholesterolemia, end-stage renal failure, hypertension, and heart failure. 9–13 Recently, ADMA has been shown to increase endothelial oxidative stress and potentiate monocyte adhesion to the endothelium. 9 Whether exogenous NO can enhance the production of ADMA is unknown but can be readily investigated.
Another endogenous inhibitor of NO synthase, first described in regard to the neuronal NOS, is the protein PIN-1. 16,17 PIN-1 is homologous to dynein and was first shown to inhibit the neuronal NO synthase. Subsequently, PIN has been shown to inhibit all three isoforms of NO synthase. 17 However, there is currently no evidence that PIN-1 plays a significant role in vascular responses of NO.
The current study results could also be explained by feedback inhibition of NO synthase by NO. Our laboratory and others have shown that NO itself is an inhibitor of NO synthase. 14 This is because of the high affinity of NO for the iron and other sites (e.g.  , cysteine 93) in protoporyphin heme groups and the binding of NO to the protoporyphin heme present in NO synthase isoforms. In the current studies, inhaled NO was only given to the normoxic lung and was not given to the hypoxic lung, where the inhibition of NO production and vasoconstriction was observed. It is possible that the NO is actively transported to hypoxic regions where it is released and able to inhibit NO synthase. Interesting recent work by Gow and Stamler 18 and by Gow et al.  19 has suggested that NO bound to hemoglobin plays a physiologic role in oxygen delivery and that NO bound to hemoglobin facilitates oxygen transport. This work suggests that NO binds to hemoglobin (in the R state; fully ligand bound) in the high-oxygen pulmonary circulation, enhancing oxygen binding. Then, in low-oxygen tissues, NO is released from hemoglobin (T state; partially nitrosylated) and simultaneously enhances the release of oxygen to the tissues (negative cooperativity). 17–19 This released NO has been shown to exchange between hemes and cysteines of other proteins. 18–22 Transfer to the heme of NO synthase would inhibit NO production. This would be consistent with the current study, in which the endogenous inhibitor response was observed primarily in the hypoxic lung lobe.
The models used by Hambraeus-Jonzon et al.  8 are complex and unsuited to isolation, identification, or biochemical characterization of a novel mediator. Initially, these complex physiologic studies need to be confirmed and complemented by bioassay for the blood-borne factor of question. If a blood-borne factor is confirmed, then the next and exciting steps will be to isolate, identify, and characterize that factor and to understand how NO enhances its production. Studies can address the three potential inhibitory pathways discussed but must also consider the possibility of novel factors that may be involved in this fascinating physiologic observation.
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