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Regulation of Oxidant Production in Acute Lung Injury^*

^* From the University of Utah and Salt Lake City Veterans Administration Medical Center, Salt Lake City, UT.

Correspondence to: John R. Hoidal, MD, University of Utah Medical Center, 50 N Medical Dr, Salt Lake City, UT 84124

	Introduction

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
Free molecular oxygen probably appeared on the earth's surface some 2 x 10⁹ years ago as a result of photosynthetic microorganisms acquiring the ability to split water.¹ It is the most abundant element in the earth's crust, and the second most abundant element in the biosphere. Oxygen is an unusual molecule in that it has two unpaired electrons, with parallel spins. It is therefore a biradical. To overcome spin restriction, oxygen prefers to accept electrons one at a time, and the sequential addition of electrons leads to the formation of reactive oxygen intermediates (ROI), including superoxide anion (O₂-), hydrogen peroxide (H₂O₂), hydroxyl radical (OH), and products of myeloperoxidase. ROIs are produced continuously in living cells in numerous biological processes. In rats, an average of about 10¹² oxygen molecules are processed by each cell daily under basal conditions, and the leakage of partially reduced oxygen molecules is about 2%.² Under basal conditions, human cells produce about one tenth the ROI of rats or 2 x 10⁹ O₂^- and H₂O₂ molecules per cell per day.

	The Role of Oxidants in ARDS

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
ARDS patients have increased production of oxidants in their lungs as indicated by an increased concentration of H₂O₂ in expired air,³ a deficiency in alveolar epithelial lining fluid glutathione with a greater percentage in the oxidized form⁴ and potentially toxic levels of peroxynitrite.⁵ In addition urinary F2 isoprostanes, vasoconstrictors that are markers of nonenzymatic lipid oxidation, are notably elevated in ARDS subjects (Garrett Fitzgerald, personal communication, January, 1997).

Several mechanisms have been identified by which ROIs can cause lung dysfunction. H₂O₂ and related ROIs react with many cellular components, oxidizing proteins, lipids, DNA bases, enzymes for intermediary metabolism, and extracellular matrix components including collagen and hyaluronic acid.^{6 7} The production of epoprostenol, isoprostanes, and lipoxygenase products is altered, causing defects in platelet aggregation and vasorelaxation.^{8 9 10} Depending on the extent of oxidant stress, cells exposed to ROIs will undergo apoptosis or necrosis.¹¹ In endothelial and epithelial cells, oxidant injury may also impair macromolecular barrier function, leading to pulmonary edema.¹² As one possible mechanism, in posthypoxic endothelial cells, O₂^- may directly affect the actin cytoskeleton by changing the redox state of actin regulatory proteins or of actin itself, thereby inducing actin polymer formation.¹³ Finally, H₂O₂ and O₂^- can function as second messengers within cells to initiate production of potent chemotaxins¹⁴ or can increase leukocyte adhesion to endothelium via activation of nuclear factor-kappaB (NF-B)-mediated transcription of integrin genes.¹⁵ Thus, localized production of ROIs may initiate a cascade that culminates in a fulminant inflammatory response, tissue destruction, and organ malfunction.

	Sources of ROIs in the Lung

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
While there is ample evidence implicating ROIs in many clinical and experimental examples of acute lung injury, key questions that still need to be addressed include the following: what are the endogenous (non-phagocyte) sources of ROI within the lung? and how are they regulated? The endogenous sources that contribute the generation of ROI in the lung likely include the molybdenum hydroxylases (xanthine dehydrogenase/oxidase [XDH/XO] and aldehyde oxidase), the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH) oxidoreductases (including the phagocytic cell oxidase, cytochrome P450, and nitric oxide synthase), the mitochondrial electron transport chain, and arachidonic acid metabolizing enzymes (such as cyclooxygenase). The best-studied sources for ROI in the lung are NADPH oxidase of phagocytic cells, XDH/XO, and mitochondrial respiration. This review will focus predominantly on NADPH oxidase and on XDH/XO. It will emphasize the multicomponent and highly regulated nature of these ROI generating systems. Finally, examples of paradoxic features of the systems will be provided.

	NADPH as a Source of ROI in the Lung

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
The NADPH oxidase of phagocytes is a multisubunit complex that generates O₂^- in one-electron reduction of O₂ using electrons supplied by NADPH.¹⁶ The oxidase consists of two membrane proteins, gp91 and p22, that together form a unique cytochrome with a redox midpoint potential of -245 mV and a reduced minus oxidized difference spectrum of 558 and several cytosolic components, including p47 and p67 which are essential, a small G protein (known as Rac2 in humans), and p40-phox.¹⁷ In phagocytes, the oxidase is activated by assembly of the cytosolic proteins with the membrane components. Heritable defects of either gp91, p22, p47, or p67 are the basis for chronic granulomatous disease, a disorder of white blood cell function characterized by recurrent, severe bacterial and fungal infections.¹⁶

During the past few years, evidence has accumulated that an enzyme complex similar to the phagocytic cell oxidase is present and exerts a variety of functions in nonphagocytic cells, including vascular endothelial, smooth muscle cells, fibroblasts, and the carotid body. Both the endothelium and vascular smooth muscle contain a membrane-bound oxidase that utilizes NADH and NADPH as substrates for electron transfer to O₂ which appears similar to the NADPH oxidase of neutrophils.^{18 19} In cultured vascular smooth muscle cells, the oxidase is a significant source of O₂^- formation.²⁰ For example, in calf pulmonary and coronary artery smooth muscle, this oxidase accounts for the majority of the O₂^- generated.^{18 21} Importantly, it utilizes a cytochrome b558 electron transport system.¹⁸ There are, however, important differences between the non-phagocyte and the phagocyte oxidase. These include the delayed time course for activation, low output, and in some studies, the preference for NADH rather that NADPH of the nonphagocyte oxidase. The low-output property does not detract from the importance of the vascular oxidase as an initiator of endothelial or smooth muscle dysfunction and it may function as a signaling system for gene activation.²² In addition, as we will describe, the nonphagocyte oxidase appears to be induced in acute lung injury.

To date, and to our knowledge, there has not been a comprehensive molecular characterization of the nonpha-gocyte NADPH oxidase. Griendling and coworkers²⁰ provided evidence that p22 is a critical component of the O₂^- generating vascular NADPH oxidase and suggested a central role for this oxidase system in vascular hypertrophy. However, it is unlikely that p22 serves as a complete oxidase on its own, since it can function only as a one-electron acceptor and lacks substrate binding sites and flavin binding sites.¹⁶ The other subunit of the electron transport element of the vascular NADPH oxidase has been more elusive. Recent immunohistochemical studies have suggested expression of gp91 in vascular smooth muscle cells,¹⁹ but this has not been a consistent finding.²³ Since the substrate specificity of the neutrophil NADPH oxidase resides in gp91, it is likely that it is this subunit that determines the unique properties of the vascular oxidase.

Our laboratory has initiated studies to characterize the NADPH oxidase of nonphagocytic cells and to assess its expression in tissue from the lungs of subjects with ARDS. We found that human endothelial cells derived from large vessel (aorta, pulmonary artery, and umbilical vein [HUVEC]) and lung microvascular endothelial cells express transcripts for gp91, p22, and p67. Transcripts for gp91 and p22 also are detected in human pulmonary smooth muscle cells (PASMC). The p47 component of the oxidase is not found in either endothelial or smooth muscle cells, suggesting the NADPH oxidase system in the vasculature differs from that in the phagocyte. Vascular NADPH oxidase also appears to differ in its regulation compared with the phagocytic system. The phagocytic NADPH oxidase is regulated by inflammatory cytokines such as tumor necrosis factor-, interleukin-1ß (Il-1ß), and lipopolysaccharide. However, these agents do not modulate transcription of the NADPH oxidase components in HUVEC or PASMC. The expression of gp91 transcripts in HUVEC and PASMC was increased by members of the IL-6 family of cytokines with oncostatin M being the most effective. In addition, hypoxia (3% oxygen) increases the rate of gp91 expression in HUVEC. Neither p22 nor p67 transcripts in vascular cells are affected by the IL-6 family of cytokines or hypoxia suggesting that they are constitutively expressed in HUVEC and PASMC. Thus the NADPH oxidase system and its regulation is different in vascular cells compared with phagocytes and the main pathway for modulation appears to be via gp91. By immunohistochemistry, we observe prominent staining for gp91 in the airway epithelium and less intense staining in microvascular endothelial and PASMC in the lungs of ARDS subjects. Except for prominent staining in the macrophages, staining of lung cells is minimal or absent in control lungs. This suggests that expression of gp91 is up-regulated in response to lung injury. The observation that circulating oncostatin M is increased several hundred-fold in patients with septic shock²⁴ provides a possible link between the isolated cell responses to agonists and the findings in the ARDS subjects.

	Novel Functions of Nonphagocytic NADPH Oxidase: NADPH Oxidase as an Oxygen Sensor and Iron Uptake System

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
Most studies investigating the role of NADPH oxidase in disease pathogenesis have focused on its ability to generate ROI to promote tissue injury. However, recent information indicates that its significance may be due not only to its ability to generate toxic oxidants, but also its ability to function as an oxygen sensor and possibly as an iron uptake system.

Studies in the carotid body,²⁵ pulmonary neuroepithelial cells,²⁶ and PASMC²¹ provide evidence that NADPH oxidase is a potential candidate as an oxygen sensor. The model proposed is that the NADPH oxidase is coupled to an H₂O₂-sensitive K⁺ channel. Hypoxia alters the function of the oxidase resulting in closure of the K⁺ channel, which in turn initiates membrane depolarization. The mechanism, source, and even the direction of change in O₂^- generation (increased or decreased) with hypoxia have not been established. However, immunohistochemistry using specific antibodies has identified p22, gp91, p47, p67 and Rac 2 in neuroepithelial cells of rabbit fetal lungs as well as in glomus cells of rat carotid bodies. We submit that this system may contribute to the pulmonary vasoconstriction that is prominent in many clinical forms of acute lung injury.

Speculatively, we suggest that a third function of the vascular NADPH oxidase central to the pathobiochemistry of acute lung injury is to serve as a ferric reductase in a transferrin-independent iron uptake system. The primary mechanism controlling the concentration of iron in biological systems is the regulation of iron uptake. Iron uptake through a transferrin-dependent system has been well established and occurs primarily in erythroid precursors and proliferating cells. In addition, a transferrin-independent pathway is widely preserved throughout phylogeny and is independent of cellular iron requirements or growth state.²⁷ In order to acquire iron from the extracellular environment by the transferrin-independent system, cells must convert extracellular ferric chelates to their ferrous counterparts.²⁸ The ferric reductase in mammalian cells has not been identified. However, there are striking similarities between the yeast ferric reductase system and the NADPH oxidase system. In the yeast Saccharomyces cerevisiae, reduction of ferric ions is primarily attributable to the FRE1 protein. FRE1, like gp91, is a glycosylated hemoprotein with a b cytochrome spectrum. The C-terminal 402 amino acids of FRE1 show 62% similarity with gp91.²⁹ A similar degree of homology is seen between gp91 and the ferric reductase of the fission yeast Schizosaccharomyces pombe, Frp1.³⁰ It has been suggested that these three proteins may constitute a distinct family of flavocytochromes capable of moving electrons across the plasma membrane.³⁰ We propose, based on these striking similarities, that an as yet unrecognized function of the NADPH oxidase system of endothelial and smooth muscle cells may be as a ferric reductase in the transferrin-independent system of iron uptake.

	XDH/XO as a Source of ROI in the Lung

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
XDH/XO, a rate-limiting enzyme in purine metabolism, is a member of molybdenum-containing hydroxylases and a homodimer with a subunit Mr of about 150,000. Each subunit contains four redox active centers: two iron-sulfur, one FAD, and one molybdopterin. XDH catalyzes the final two reactions of purine metabolism to produce uric acid. XDH can be readily converted to XO by reversible sulfhydryl oxidation or by irreversible proteolytic modification. XO uses molecular oxygen as its electron carrier and produces ROI, either O₂^- or H₂O₂.

Most studies investigating the role of XDH/XO in disease pathogenesis have emphasized that the significance of XDH/XO as a mechanism for generation of ROI in tissues is determined by the rate of conversion of XDH to XO. However, recent molecular and biochemical approaches have provided new insights into the regulation of XDH/XO. Thus, besides the simple conversion of XDH to XO, additional mechanisms may regulate the enzyme leading to enhanced ROI generation.

Recent studies have demonstrated that several cytokines can up-regulate XDH/XO gene expression. Our laboratory first demonstrated transcriptional regulation of the enzyme, finding that interferon- selectively induced gene activation in pulmonary microvascular cells and in rat lungs in vivo.³¹ We subsequently demonstrated transcriptional regulation of XDH/XO in epithelial cells by cytokines and steroids in a pattern analogous to the profile seen by acute phase reactants in response to injury, trauma, or infection.³² Although the acute phase response is generally believed to be a response to protect the host, the functional role of XDH/XO upregulation as part of this reaction could be deleterious due to the increased generation of ROS. More recently, transcriptional activation of XDH/XO has been shown in response to hypoxia³³ or following lipopolysaccharide exposure in vivo.³⁴ The importance of these studies rests on their demonstration that XDH/XO gene expression is regulated in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events, such as hypoxia.

New information has also been obtained on posttranslational regulation of XDH/XO. We have recently demonstrated that hypoxia not only transcriptionally regulates XDH/XO (as suggested by Hassoun and colleagues³³ ), but also enhances enzyme activity by as yet undefined posttranslational processes that do not involve conversion of XDH to XO.³⁵ Posttranscriptional regulation by iron³⁶ and posttranslational regulation by nitric oxide³⁷ have also been established. Moreover, the recent evidence that human XDH/XO can undergo activation-deactivation cycles at the molybdenum redox center³⁸ further emphasizes the potential importance of posttranslational regulation of XDH/XO.

An additional advance is the new evidence that XDH/XO acts not only on purines but also on reducing substrates, including NADH with resultant O₂^- formation. Recent studies have shown that XDH/XO can oxidize NADH producing O₂^- at 0.23 µmol/min/mg, a substantial rate.³⁹ These findings suggest an alternative mechanism for ischemia-induced reperfusion injury. Ischemia elevates intracellular NADH levels leading to O₂^- generation by XDH/XO on reoxygenation of tissues. Such a mechanism does not depend on XDH to XO conversion (a controversial issue). Moreover, the NADH oxidation does not involve the molybdenum center of the enzyme (Fig 1) and therefore would not be inhibited by purine analogs such as allopurinol or oxypurinol, or by a supplementation with tungstate. This observation is particularly noteworthy in light of the recent demonstration of an as yet uncharacterized NADH oxidase as an important source of ROI in lung cells and reperfusion injury^{29 30} and the striking increase in cellular NADH that occurs during ischemia.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Electron transfer pathways within XDH. XDH as a NADH oxidoreductase.

	The Paradox: Uric Acid the Protector Causes Gout

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 
An intrigue in medicine is the diverse and discrepant roles assigned to many biochemical systems. XDH/XO is an example of an enzyme with such diverse roles. In the above discussion, we emphasized the importance of XDH/XO as a source of ROI, particularly when the enzyme is in its oxidase form, and suggested that the enzyme may be important in the pathobiochemistry of acute lung injury. However, a major metabolite of XDH/XO is uric acid. In humans and higher primates, the enzymatic catabolism of adenine- and guanine-based purines proceeds only as far as uric acid. Most mammals, in contrast, excrete allantoin and urea as the major nitrogen-containing degradatives of purines. Consequently, the plasma levels of uric acid encountered in these species are < 10% of those found in man. The distinction arises because the enzyme urate oxidase, responsible for transforming uric acid to allantoin, is no longer expressed in man. Historically, the enhanced uric acid in man has been regarded merely as an evolutionary "quirk," assigning to uric acid nothing more than the role of a "waste product." The disinterest has arisen because uric acid and its salt, urate, were generally believed to be biologically unreactive. However, the physiologic handling of uric acid by humans displays several characteristics not readily compatible with a waste product. For example, only uric acid appears in the systemic circulation after ingestion of purines. Furthermore, in the kidney, uric acid and urate are primarily filtered and additionally secreted, and the majority (90%) is normally reabsorbed and returned to the blood.

There is now substantial evidence that uric acid functions as an antioxidant in man and has been cited as a major factor in lengthening life span and decreasing age-specific cancer rates during primate evolution by providing improved protection against ROI. Studies indicate that soluble urate can scavenge powerful oxidants, including OH, singlet oxygen, and oxo-heme oxidants. Recent investigation on the antioxidant properties of urate has focused on its ability to form stable coordination complexes with both ferrous and ferric ions and, in so doing, protect against iron-catalyzed oxidation. It has been suggested that this compound may be one of the major iron-binding agents in human blood. In isolated organ preparations, urate protects against reperfusion damage induced by activated granulocytes, prevents oxidative inactivation of endothelium enzymes (cyclooxygenase, angiotensin-converting enzyme), and preserves the ability of the endothelium to mediate vascular dilatation in the face of oxidative stress. Urate also appears to be a major antioxidant in human airway secretions. Thus, rather than an enzyme responsible for generation of a waste product, XDH appears to catalyze the formation of a valuable physiologic agent.

The uric acid concentration in man, that is several times higher than those found in most other mammals, contributes to a Faustian compact. That is, uric acid functions as an antioxidant in man and has been cited as a possible basis for evolutionary increases in life span and decreasing cancer rates. However, crystalline deposition of this same compound is the histopathologic hallmark of the "King of Diseases," gout, whose descriptions can be traced to the dawn of recorded medical history. Recent studies suggest that the basis for this apparent paradox relates to the change in antioxidant properties of the molecule imposed by a change in its physical state. In solution, the iron-binding properties of urate are due to its ability to form stable coordination complexes that protect against iron-catalyzed oxidation. However, in crystalline form, the complexation of the coordination sites on the iron is incomplete leaving one or more sites open to function as a Fenton catalyst to produce OH, a potent oxidant.

Recent studies suggest that the inflammation and cell activation that occur in gout may be directly attributed, in part, to the ability of crystalline urate to chelate iron and promote OH generation.

In summary, XDH presents a series of paradoxes highlighted by its contrasting roles as an antioxidant or a prooxidant enzyme. The form of the enzyme as well as the physical state of its product, uric acid, are the determinants.

	References

TOP Introduction The Role of Oxidants... Sources of ROIs in... NADPH as a Source... Novel Functions of Nonphagocytic... XDH/XO as a Source... The Paradox: Uric Acid... References

 

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