Nitric oxide, hypoxia, and superoxide: the good, the bad, and the ugly!
http://www.100md.com
《胸》
Correspondence to:
Dr R A Dweik
Department of Pulmonary, Allergy, and Critical Care Medicine, The Cleveland Clinic Foundation, 9500 Euclid Avenue/A90, Cleveland, Ohio 44195, USA; dweikr@ccf.org
A possible role for NO in ARDS
Abbreviations: ARDS, acute respiratory distress syndrome; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; KMO2, Michaelis constant; NADPH, reduced nicotinamide-adenine dinucleotide phosphate; NO, nitric oxide; NOS, nitric oxide synthase; O2–, superoxide; ONOO–, peroxynitrite; ROS, reactive oxygen species; SOD, superoxide dismutases
Keywords: nitric oxide; hypoxia; superoxide; pulmonary artery; acute respiratory distress syndrome (ARDS)
Nitric oxide (NO) is endogenously synthesised by nitric oxide synthases (NOS) which convert L-arginine to L-citrulline and NO. Three NOS isoforms (types I, II and III) have been identified and all of them are expressed in the human lung.1–8 NOS I (nNOS) and III (eNOS) are constitutively expressed in tissues and are dependent on increases in intracellular calcium for enzyme activation while NOS II (iNOS) is an inducible form that is calcium independent (table 1).9 All NOS isoforms require oxygen, NADPH, FAD, FMN, tetrahydrobiopterin, and calmodulin for activity.1,9 NO is recognised to have a key role in virtually all aspects of lung biology and has been implicated in the pathophysiology of lung diseases.1,4,6,10–15 It is involved in pulmonary neurotransmission, host defence and bacteriostasis, airway and vascular smooth muscle relaxation, pulmonary capillary leak, inflammation, mucociliary clearance, airway mucus secretion, and cytotoxicity.4,6,14
Table 1 Nitric oxide synthase enzymes
Cellular sources of NO in the lung include epithelial cells, endothelial cells of pulmonary arteries and veins, inhibitory non-adrenergic non-cholinergic neurones, smooth muscle cells, mast cells, mesothelial cells, fibroblasts, neutrophils, lymphocytes, and macrophages.4,6,14 Specifically, NOS I is located in inhibitory non-adrenergic non-cholinergic neurones in the lung while NOS III is found in endothelial cells and the brush border of ciliated epithelial cells.1,5,7 NOS II is found in the epithelial cells of the airway. Although NOS II may be induced in several types of cells in response to cytokines, endotoxin, or reactive oxygen species, it is continuously expressed in normal human airway epithelium at basal airway conditions.8,16
Once produced, NO is freely diffusible and enters target cells activating soluble guanylate cyclase to produce guanosine 3',5'-cyclic monophosphate (cGMP) which mediates most of the physiological effects of NO on smooth muscle including vasodilation and bronchodilation.2,11 NO reaction products may also mediate other physiological and pathological functions in the lungs and many other organ systems. Due to the high reactivity, NO participates in a wide variety of reactions at different sites within the cell, lung tissue, extracellular fluids, and intravascular compartments. Primary reactions that may involve NO intracellularly and extracellularly include its reaction with oxygen, superoxide, haemoglobin, another molecule of NO, enzymes containing iron-sulfur centres, heme-containing proteins, and thiol proteins.12 Notably, NO undergoes a direct bimolecular reaction with superoxide (O2–) yielding peroxynitrite (ONOO–) at a rate that is even faster than the dismutation of O2– by superoxide dismutases (SOD), which puts NO at the epicentre of oxidative metabolism and inflammation.
REGULATION OF NO SYNTHESIS BY OXYGEN
All NOS isoforms require the presence of oxygen for activity.9 Although it is recognised that oxygen is a substrate for NOS, its effects on the regulation of NOS activity are more complex than a simple enzyme-substrate interaction.4,17 Interestingly, the effect of hypoxia on NO levels in the airway is primarily a result of airway and alveolar oxygen tension rather than vascular oxygen tension.18,19 One proposed mechanism(s) for oxygen regulation of NOS activity is outlined in fig 1. NOS activity during the steady state includes an active cycle (A) that generates NO and an inactive cycle (B) that involves formation and decay of a heme-NO complex. In the active cycle, oxygen binding to ferrous heme (Fe2+) is limiting for enzyme activity. In contrast, resolution of the inactive cycle and entry into the active cycle is oxygen-dependent due to effects on the stability of the heme-NO complex. This includes a reaction between the heme-NO complex and oxygen which results in loss of the heme-NO complex (fig 1).17
Figure 1 Simplified proposed mechanism of oxygen regulation of NOS enzyme kinetics. NOS activity during the steady state includes an active cycle (A) that generates NO and an inactive cycle (B) that involves formation and decay of a heme-NO complex. In the active cycle, oxygen binding to ferrous heme (Fe2+) is limiting for enzyme activity. In contrast, resolution of the inactive cycle and entry into the active cycle is oxygen-dependent due to effects on the heme-NO complex stability. This includes a reaction between the heme-NO complex and oxygen, which results in loss of the heme-NO complex.
The oxygen concentration in intact tissues ranges from 1 to 150 μM,4,17,20 with the highest levels found in the lung. Airway epithelial cells are unique in their exposure to oxygen since, above a thin layer of epithelial lining fluid, the airway cells are exposed directly to air containing 21% oxygen. Based on oxygen solubility and the low differential oxygen gradient between overlying fluid to intracellular endoplasmic reticulum (1–2 μM),4 the levels of oxygen in airway epithelial cells may actually approach 260 μM. Thus, the Michaelis constant (KMO2) determined for NOS II (135 μM), but not NOS III (4 μM) or NOS I (400 μM), is well within the physiological range of oxygen concentrations in lung epithelial cells. Importantly, KMO2 for NO synthesis in the intact human lung (190 μM) is similar to NOS II KMO2 in vitro.3,4,21
REGULATION OF NOS GENE EXPRESSION BY OXYGEN
The immediate effects of short term changes in oxygen concentration on the activity of NOS enzymes are probably due to the effects of oxygen on NOS enzyme kinetics. However, prolonged hypoxia can have significant effects on the gene expression of the different NOS isoforms.2,3,21–23 These transcriptional effects may vary among species or among organ systems in the same species.2,3,21 For example, while hypoxia produces a progressive decline in constitutive NOS mRNA levels in bovine pulmonary artery endothelial cells,22–24 chronic hypoxia upregulates constitutive NOS expression in rabbit heart25 and rat lung pulmonary arteries.26 Chronic hypoxia also increases NOS expression and NOS activity in rat carotid bodies.27
In this issue of Thorax, Muzaffar et al28 describe the effect of hypoxia on the expression of endothelial nitric oxide synthase (NOS III) and gp91phox (the active catalytic subunit of NADPH oxidase), and the formation of superoxide in pig pulmonary artery segments, pulmonary artery smooth muscle cells, and pulmonary artery endothelial cells. They incubated pulmonary artery segments (with and without intact endothelium) and cells (endothelial and smooth muscle cells) in the absence of ambient oxygen for 2 hours and measured the formation of superoxide by ferricytochrome c reduction. They also measured the expression of proteins by Western blotting and immunocytochemistry. The absence of oxygen in the ambient air promoted the formation of superoxide in the studied tissues and cells. Various enzyme inhibitors were used to determine the source of superoxide production. They also pre-incubated the cells with several inflammatory mediators to determine if they could enhance the effects of hypoxia. A summary of the findings is that hypoxia upregulates NADPH oxidase and NOS III resulting in increased production of superoxide, NO, and peroxynitrite in their system.
A major component missing in the model studied by Muzaffar and colleagues is the role of NOS II. In humans NOS II is continuously expressed in the airway epithelium, is a major source of NO in the lung, and appears to be the most responsive to hypoxia in the physiological range.2,3,21 Due to the free diffusion of NO and the close apposition of airways to pulmonary vessels, endogenous NO production in the airways can have significant effects on the pulmonary vessels. The authors comment on both eNOS (NOS III) and iNOS (NOS II) throughout the paper, but their system does not seem to be appropriate for the study of NOS II which is mainly expressed in the airway epithelium (which the authors did not study) and not in the endothelium or smooth muscle (reported here). The cells they studied do not express NOS II in detectable levels at baseline and that does not change with hypoxia. So, the additional use of NOS II inhibitors does not add much. While the authors emphasise the relevance of their findings to acute respiratory distress syndrome (ARDS), the link is rather speculative. They studied healthy piglets and evaluated their pulmonary artery rings or cells in isolation from the rest of the lung. Although they used pre-incubation with some inflammatory markers as a suggestion as to what happens in ARDS, it would have been more appropriate to study rings from piglets with and without ARDS. The weak link to ARDS, however, does not diminish the relevance of the findings.
NO-SUPEROXIDE INTERACTION
Free radicals/reactive oxygen species (ROS) may be toxic in two ways. They can interact with metal or organic redox centres and promote irreversible oxidation reactions inactivating the target metabolic process, or they can initiate reactions which then become self-sustaining through the generation of propagating radicals. In either case, this can result in deleterious effects on the cell. The most effective protection against oxidant mediated tissue damage is to scavenge the initiating radical.15,29 Although NO is itself a radical, many of the same chemical and physical properties of NO that allow it to exert oxidant effects can also result in antioxidant actions. The role of NO as an oxidant or an antioxidant probably depends on the local tissue milieu. In an environment where the oxidant load is low, the highly reactive properties of NO give the molecule oxidant properties. However, in situations where the oxidant load is high (as in asthma and ARDS), NO plays an antioxidant role by scavenging superoxide and other ROS. NO undergoes a direct bimolecular reaction with O2– yielding ONOO– at almost diffusion limited rates (rate constant (k) = 6.7 – 19 x 109/M/s).12 The rate constant is over 3.5 times faster than the dismutation of O2·– by SOD. By rapidly consuming superoxide, NO produces ONOO–/ONOOH, a far less reactive oxidant than superoxide that can be further metabolised to innocuous products like NO3– (fig 2).12,30
Figure 2 Simplified proposed model of NO reaction with superoxide (O–). NO undergoes a direct bimolecular reaction with O2– yielding peroxynitrite (ONOO–) at almost diffusion limited rates (k = 6.7 – 19 x 109/M/s). The rate constant is over 3.5 times faster than the dismutation of O2– by superoxide dismutases. By rapidly consuming superoxide, NO produces ONOO–/ONOOH, a far less reactive oxidant than superoxide that can be further metabolised to innocuous products like NO3–. Tyr-NO2, nitrotyrosine.
Muzaffar and colleagues speculate that the upregulation of two enzymes with opposite effects may represent a protective mechanism to counteract the effect of hypoxia or a pathological mechanism leading to the progression of ARDS. There are no data in their study to favour one explanation over the other. However, based on models in other diseases and on our knowledge of the biology of NO and reactions in the lung, one would suspect that, if hypoxia upregulated NADPH oxidase without simultaneously upregulating NOS expression, the increased release of superoxide (O2–) would go unchecked resulting in serious tissue injury. By reacting with superoxide to form peroxynitrite (ONOO–), NO produced by NOS serves as a scavenger of superoxide resulting in a net antioxidant effect (fig 2). The simultaneous upregulation of NOS is therefore probably a protective feature. Although the link to ARDS remains speculative, these findings have potential implications for a wide variety of lung diseases from asthma to pulmonary hypertension12,13,15,31,32 in which oxidative stress and/or hypoxia have a role.
FOOTNOTES
Dr Dweik is supported by the National Institutes of Health grant NIH-HL68863.
REFERENCES
Dweik RA, Laskowski D, Ozkan M, et al. High levels of exhaled nitric oxide (NO) and NO synthase III expression in lesional smooth muscle in lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 2001;24:414–8.
Dweik RA, Erzurum SC. Effects of nitric oxide and cyclic GMP on smooth muscle cell proliferation. In: Moss J, ed. LAM and other diseases characterized by smooth muscle cell proliferation (Lung Biology in Health and Disease). New York: Marcel Dekker, 1999:333–49.
Dweik RA, Erzurum SC. Regulation of nitric oxide (NO) synthases and gas phase NO by oxygen. In: Marczin N, Kharitonov SA, Yacoub MH, Barnes PJ, eds. Disease markers in exhaled breath (Lung Biology in Health and Disease). New York: Marcel Dekker, 2003:235–46.
Dweik RA, Laskowski D, Abu-Soud HM, et al. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 1998;101:660–6.
Dweik RA, Guo FH, Uetani K, et al. Nitric oxide synthase in the human airway epithelium. Zhongguo Yao Li Xue Bao 1997;18:550–2.
Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 1994;78:915–8.
Kobzik L, Bredt DS, Lowenstein CJ, et al. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am J Respir Cell Mol Biol 1993;9:371–7.
Guo FH, Uetani K, Haque SJ, et al. Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J Clin Invest 1997;100:829–38.
Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999;1411:217–30.
Dweik RA. The promise and reality of nitric oxide in the diagnosis and treatment of lung disease. Cleve Clin J Med 2001;68:486, 488, 490, 493.
Dweik RA. Pulmonary hypertension and the search for the selective pulmonary vasodilator. Lancet 2002;360:886–7.
Dweik RA, Comhair SA, Gaston B, et al. NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response. Proc Natl Acad Sci USA 2001;98:2622–7.
Khatri SB, Hammel J, Kavuru MS, et al. Temporal association of nitric oxide levels and airflow in asthma after whole lung allergen challenge. J Appl Physiol 2003;95:436–40.
Schmidt HH, Walter U. NO at work. Cell 1994;78:919–25.
Ozkan M, Dweik RA. Nitric oxide and airway reactivity. Clin Pulmon Med 2001;8:199–206.
Guo FH, De Raeve HR, Rice TW, et al. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc Natl Acad Sci USA 1995;92:7809–13.
Abu-Soud HM, Rousseau DL, Stuehr DJ. Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension. J Biol Chem 1996;271:32515–8.
Ide H, Nakano H, Ogasa T, et al. Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide. J Appl Physiol 1999;87:1629–36.
Grimminger F, Spriestersbach R, Weissmann N, et al. Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. J Appl Physiol 1995;78:1509–15.
Vanderkooi JM, Erecinska M, Silver IA. Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am J Physiol 1991;260:C1131–50.
Dweik RA. Nitric oxide production in the lung and its regulation by oxygen. In: Marczin N, Yacoub MH, eds. Disease markers in exhaled breath: basic mechanisms and clinical applications (NATO Science Series). Amsterdam, Netherlands: IOS Press, 2002:11–17.
Liao JK, Zulueta JJ, Yu FS, et al. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Invest 1995;96:2661–6.
Melillo G, Musso T, Sica A, et al. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med 1995;182:1683–93.
Phelan MW, Faller DV. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J Cell Physiol 1996;167:469–76.
Baker JE, Holman P, Kalyanaraman B, et al. Adaptation of hearts to chronic hypoxia increases tolerance to subsequent ischemia by increased nitric oxide production. Adv Exp Med Biol 1998;454:203–17.
Sato K, Rodman DM, McMurtry IF. Hypoxia inhibits increased ETB receptor-mediated NO synthesis in hypertensive rat lungs. Am J Physiol 1999;276:L571–81.
Di Giulio C, Di Muzio M, Sabatino G, et al. Effect of chronic hyperoxia on young and old rat carotid body ultrastructure. Exp Gerontol 1998;33:319–29.
Muzaffar S, Shukla N, Angelini GD, et al. Acute hypoxia simultaneously induces the expression of gp91phox and endothelial nitric oxide synthase in the porcine pulmonary artery. Thorax 2005;60:305–13.
Freeman BA, White CR, Gutierrez H, et al. Oxygen radical-nitric oxide reactions in vascular diseases. Adv Pharmacol 1995;34:45–69.
Dweik RA. Nitric oxide reactions in the asthmatic airway. In: Marczin N, Yacoub MH, eds. Disease markers in exhaled breath: basic mechanisms and clinical applications (NATO Science Series). Amsterdam, Netherlands: IOS Press, 2002:159–66.
Ghamra ZW, Dweik RA. Primary pulmonary hypertension: an overview of epidemiology and pathogenesis. Cleve Clin J Med 2003;70 (Suppl 1) :S2–8.
Kaneko FT, Arroliga AC, Dweik RA, et al. Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension. Am J Respir Crit Care Med 1998;158:917–23.(R A Dweik)
Dr R A Dweik
Department of Pulmonary, Allergy, and Critical Care Medicine, The Cleveland Clinic Foundation, 9500 Euclid Avenue/A90, Cleveland, Ohio 44195, USA; dweikr@ccf.org
A possible role for NO in ARDS
Abbreviations: ARDS, acute respiratory distress syndrome; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; KMO2, Michaelis constant; NADPH, reduced nicotinamide-adenine dinucleotide phosphate; NO, nitric oxide; NOS, nitric oxide synthase; O2–, superoxide; ONOO–, peroxynitrite; ROS, reactive oxygen species; SOD, superoxide dismutases
Keywords: nitric oxide; hypoxia; superoxide; pulmonary artery; acute respiratory distress syndrome (ARDS)
Nitric oxide (NO) is endogenously synthesised by nitric oxide synthases (NOS) which convert L-arginine to L-citrulline and NO. Three NOS isoforms (types I, II and III) have been identified and all of them are expressed in the human lung.1–8 NOS I (nNOS) and III (eNOS) are constitutively expressed in tissues and are dependent on increases in intracellular calcium for enzyme activation while NOS II (iNOS) is an inducible form that is calcium independent (table 1).9 All NOS isoforms require oxygen, NADPH, FAD, FMN, tetrahydrobiopterin, and calmodulin for activity.1,9 NO is recognised to have a key role in virtually all aspects of lung biology and has been implicated in the pathophysiology of lung diseases.1,4,6,10–15 It is involved in pulmonary neurotransmission, host defence and bacteriostasis, airway and vascular smooth muscle relaxation, pulmonary capillary leak, inflammation, mucociliary clearance, airway mucus secretion, and cytotoxicity.4,6,14
Table 1 Nitric oxide synthase enzymes
Cellular sources of NO in the lung include epithelial cells, endothelial cells of pulmonary arteries and veins, inhibitory non-adrenergic non-cholinergic neurones, smooth muscle cells, mast cells, mesothelial cells, fibroblasts, neutrophils, lymphocytes, and macrophages.4,6,14 Specifically, NOS I is located in inhibitory non-adrenergic non-cholinergic neurones in the lung while NOS III is found in endothelial cells and the brush border of ciliated epithelial cells.1,5,7 NOS II is found in the epithelial cells of the airway. Although NOS II may be induced in several types of cells in response to cytokines, endotoxin, or reactive oxygen species, it is continuously expressed in normal human airway epithelium at basal airway conditions.8,16
Once produced, NO is freely diffusible and enters target cells activating soluble guanylate cyclase to produce guanosine 3',5'-cyclic monophosphate (cGMP) which mediates most of the physiological effects of NO on smooth muscle including vasodilation and bronchodilation.2,11 NO reaction products may also mediate other physiological and pathological functions in the lungs and many other organ systems. Due to the high reactivity, NO participates in a wide variety of reactions at different sites within the cell, lung tissue, extracellular fluids, and intravascular compartments. Primary reactions that may involve NO intracellularly and extracellularly include its reaction with oxygen, superoxide, haemoglobin, another molecule of NO, enzymes containing iron-sulfur centres, heme-containing proteins, and thiol proteins.12 Notably, NO undergoes a direct bimolecular reaction with superoxide (O2–) yielding peroxynitrite (ONOO–) at a rate that is even faster than the dismutation of O2– by superoxide dismutases (SOD), which puts NO at the epicentre of oxidative metabolism and inflammation.
REGULATION OF NO SYNTHESIS BY OXYGEN
All NOS isoforms require the presence of oxygen for activity.9 Although it is recognised that oxygen is a substrate for NOS, its effects on the regulation of NOS activity are more complex than a simple enzyme-substrate interaction.4,17 Interestingly, the effect of hypoxia on NO levels in the airway is primarily a result of airway and alveolar oxygen tension rather than vascular oxygen tension.18,19 One proposed mechanism(s) for oxygen regulation of NOS activity is outlined in fig 1. NOS activity during the steady state includes an active cycle (A) that generates NO and an inactive cycle (B) that involves formation and decay of a heme-NO complex. In the active cycle, oxygen binding to ferrous heme (Fe2+) is limiting for enzyme activity. In contrast, resolution of the inactive cycle and entry into the active cycle is oxygen-dependent due to effects on the stability of the heme-NO complex. This includes a reaction between the heme-NO complex and oxygen which results in loss of the heme-NO complex (fig 1).17
Figure 1 Simplified proposed mechanism of oxygen regulation of NOS enzyme kinetics. NOS activity during the steady state includes an active cycle (A) that generates NO and an inactive cycle (B) that involves formation and decay of a heme-NO complex. In the active cycle, oxygen binding to ferrous heme (Fe2+) is limiting for enzyme activity. In contrast, resolution of the inactive cycle and entry into the active cycle is oxygen-dependent due to effects on the heme-NO complex stability. This includes a reaction between the heme-NO complex and oxygen, which results in loss of the heme-NO complex.
The oxygen concentration in intact tissues ranges from 1 to 150 μM,4,17,20 with the highest levels found in the lung. Airway epithelial cells are unique in their exposure to oxygen since, above a thin layer of epithelial lining fluid, the airway cells are exposed directly to air containing 21% oxygen. Based on oxygen solubility and the low differential oxygen gradient between overlying fluid to intracellular endoplasmic reticulum (1–2 μM),4 the levels of oxygen in airway epithelial cells may actually approach 260 μM. Thus, the Michaelis constant (KMO2) determined for NOS II (135 μM), but not NOS III (4 μM) or NOS I (400 μM), is well within the physiological range of oxygen concentrations in lung epithelial cells. Importantly, KMO2 for NO synthesis in the intact human lung (190 μM) is similar to NOS II KMO2 in vitro.3,4,21
REGULATION OF NOS GENE EXPRESSION BY OXYGEN
The immediate effects of short term changes in oxygen concentration on the activity of NOS enzymes are probably due to the effects of oxygen on NOS enzyme kinetics. However, prolonged hypoxia can have significant effects on the gene expression of the different NOS isoforms.2,3,21–23 These transcriptional effects may vary among species or among organ systems in the same species.2,3,21 For example, while hypoxia produces a progressive decline in constitutive NOS mRNA levels in bovine pulmonary artery endothelial cells,22–24 chronic hypoxia upregulates constitutive NOS expression in rabbit heart25 and rat lung pulmonary arteries.26 Chronic hypoxia also increases NOS expression and NOS activity in rat carotid bodies.27
In this issue of Thorax, Muzaffar et al28 describe the effect of hypoxia on the expression of endothelial nitric oxide synthase (NOS III) and gp91phox (the active catalytic subunit of NADPH oxidase), and the formation of superoxide in pig pulmonary artery segments, pulmonary artery smooth muscle cells, and pulmonary artery endothelial cells. They incubated pulmonary artery segments (with and without intact endothelium) and cells (endothelial and smooth muscle cells) in the absence of ambient oxygen for 2 hours and measured the formation of superoxide by ferricytochrome c reduction. They also measured the expression of proteins by Western blotting and immunocytochemistry. The absence of oxygen in the ambient air promoted the formation of superoxide in the studied tissues and cells. Various enzyme inhibitors were used to determine the source of superoxide production. They also pre-incubated the cells with several inflammatory mediators to determine if they could enhance the effects of hypoxia. A summary of the findings is that hypoxia upregulates NADPH oxidase and NOS III resulting in increased production of superoxide, NO, and peroxynitrite in their system.
A major component missing in the model studied by Muzaffar and colleagues is the role of NOS II. In humans NOS II is continuously expressed in the airway epithelium, is a major source of NO in the lung, and appears to be the most responsive to hypoxia in the physiological range.2,3,21 Due to the free diffusion of NO and the close apposition of airways to pulmonary vessels, endogenous NO production in the airways can have significant effects on the pulmonary vessels. The authors comment on both eNOS (NOS III) and iNOS (NOS II) throughout the paper, but their system does not seem to be appropriate for the study of NOS II which is mainly expressed in the airway epithelium (which the authors did not study) and not in the endothelium or smooth muscle (reported here). The cells they studied do not express NOS II in detectable levels at baseline and that does not change with hypoxia. So, the additional use of NOS II inhibitors does not add much. While the authors emphasise the relevance of their findings to acute respiratory distress syndrome (ARDS), the link is rather speculative. They studied healthy piglets and evaluated their pulmonary artery rings or cells in isolation from the rest of the lung. Although they used pre-incubation with some inflammatory markers as a suggestion as to what happens in ARDS, it would have been more appropriate to study rings from piglets with and without ARDS. The weak link to ARDS, however, does not diminish the relevance of the findings.
NO-SUPEROXIDE INTERACTION
Free radicals/reactive oxygen species (ROS) may be toxic in two ways. They can interact with metal or organic redox centres and promote irreversible oxidation reactions inactivating the target metabolic process, or they can initiate reactions which then become self-sustaining through the generation of propagating radicals. In either case, this can result in deleterious effects on the cell. The most effective protection against oxidant mediated tissue damage is to scavenge the initiating radical.15,29 Although NO is itself a radical, many of the same chemical and physical properties of NO that allow it to exert oxidant effects can also result in antioxidant actions. The role of NO as an oxidant or an antioxidant probably depends on the local tissue milieu. In an environment where the oxidant load is low, the highly reactive properties of NO give the molecule oxidant properties. However, in situations where the oxidant load is high (as in asthma and ARDS), NO plays an antioxidant role by scavenging superoxide and other ROS. NO undergoes a direct bimolecular reaction with O2– yielding ONOO– at almost diffusion limited rates (rate constant (k) = 6.7 – 19 x 109/M/s).12 The rate constant is over 3.5 times faster than the dismutation of O2·– by SOD. By rapidly consuming superoxide, NO produces ONOO–/ONOOH, a far less reactive oxidant than superoxide that can be further metabolised to innocuous products like NO3– (fig 2).12,30
Figure 2 Simplified proposed model of NO reaction with superoxide (O–). NO undergoes a direct bimolecular reaction with O2– yielding peroxynitrite (ONOO–) at almost diffusion limited rates (k = 6.7 – 19 x 109/M/s). The rate constant is over 3.5 times faster than the dismutation of O2– by superoxide dismutases. By rapidly consuming superoxide, NO produces ONOO–/ONOOH, a far less reactive oxidant than superoxide that can be further metabolised to innocuous products like NO3–. Tyr-NO2, nitrotyrosine.
Muzaffar and colleagues speculate that the upregulation of two enzymes with opposite effects may represent a protective mechanism to counteract the effect of hypoxia or a pathological mechanism leading to the progression of ARDS. There are no data in their study to favour one explanation over the other. However, based on models in other diseases and on our knowledge of the biology of NO and reactions in the lung, one would suspect that, if hypoxia upregulated NADPH oxidase without simultaneously upregulating NOS expression, the increased release of superoxide (O2–) would go unchecked resulting in serious tissue injury. By reacting with superoxide to form peroxynitrite (ONOO–), NO produced by NOS serves as a scavenger of superoxide resulting in a net antioxidant effect (fig 2). The simultaneous upregulation of NOS is therefore probably a protective feature. Although the link to ARDS remains speculative, these findings have potential implications for a wide variety of lung diseases from asthma to pulmonary hypertension12,13,15,31,32 in which oxidative stress and/or hypoxia have a role.
FOOTNOTES
Dr Dweik is supported by the National Institutes of Health grant NIH-HL68863.
REFERENCES
Dweik RA, Laskowski D, Ozkan M, et al. High levels of exhaled nitric oxide (NO) and NO synthase III expression in lesional smooth muscle in lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 2001;24:414–8.
Dweik RA, Erzurum SC. Effects of nitric oxide and cyclic GMP on smooth muscle cell proliferation. In: Moss J, ed. LAM and other diseases characterized by smooth muscle cell proliferation (Lung Biology in Health and Disease). New York: Marcel Dekker, 1999:333–49.
Dweik RA, Erzurum SC. Regulation of nitric oxide (NO) synthases and gas phase NO by oxygen. In: Marczin N, Kharitonov SA, Yacoub MH, Barnes PJ, eds. Disease markers in exhaled breath (Lung Biology in Health and Disease). New York: Marcel Dekker, 2003:235–46.
Dweik RA, Laskowski D, Abu-Soud HM, et al. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 1998;101:660–6.
Dweik RA, Guo FH, Uetani K, et al. Nitric oxide synthase in the human airway epithelium. Zhongguo Yao Li Xue Bao 1997;18:550–2.
Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 1994;78:915–8.
Kobzik L, Bredt DS, Lowenstein CJ, et al. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am J Respir Cell Mol Biol 1993;9:371–7.
Guo FH, Uetani K, Haque SJ, et al. Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J Clin Invest 1997;100:829–38.
Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999;1411:217–30.
Dweik RA. The promise and reality of nitric oxide in the diagnosis and treatment of lung disease. Cleve Clin J Med 2001;68:486, 488, 490, 493.
Dweik RA. Pulmonary hypertension and the search for the selective pulmonary vasodilator. Lancet 2002;360:886–7.
Dweik RA, Comhair SA, Gaston B, et al. NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response. Proc Natl Acad Sci USA 2001;98:2622–7.
Khatri SB, Hammel J, Kavuru MS, et al. Temporal association of nitric oxide levels and airflow in asthma after whole lung allergen challenge. J Appl Physiol 2003;95:436–40.
Schmidt HH, Walter U. NO at work. Cell 1994;78:919–25.
Ozkan M, Dweik RA. Nitric oxide and airway reactivity. Clin Pulmon Med 2001;8:199–206.
Guo FH, De Raeve HR, Rice TW, et al. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc Natl Acad Sci USA 1995;92:7809–13.
Abu-Soud HM, Rousseau DL, Stuehr DJ. Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension. J Biol Chem 1996;271:32515–8.
Ide H, Nakano H, Ogasa T, et al. Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide. J Appl Physiol 1999;87:1629–36.
Grimminger F, Spriestersbach R, Weissmann N, et al. Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. J Appl Physiol 1995;78:1509–15.
Vanderkooi JM, Erecinska M, Silver IA. Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am J Physiol 1991;260:C1131–50.
Dweik RA. Nitric oxide production in the lung and its regulation by oxygen. In: Marczin N, Yacoub MH, eds. Disease markers in exhaled breath: basic mechanisms and clinical applications (NATO Science Series). Amsterdam, Netherlands: IOS Press, 2002:11–17.
Liao JK, Zulueta JJ, Yu FS, et al. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Invest 1995;96:2661–6.
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