Department of Pediatrics, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Nitric oxide (NO) synthesis is markedly
augmented in states of inflammation, largely due to the expression of
inducible nitric oxide synthase (iNOS). Although NO has
anti-inflammatory consequences under basal conditions, it remains
enigmatic as to why NO displays proinflammatory characteristics in
chronic inflammation. Either the anti-inflammatory actions are weak and
of little consequence or, alternatively, other factors influence the
role of NO in chronic inflammation. We propose that the answer to this
enigma lies in the conversion of NO to other higher oxides of nitrogen
(NO2, nitrogen dioxide;
N2O3,
dinitrogen trioxide; and
ONOO, peroxynitrite).
Emerging therapeutic strategies may be independent of NO synthesis;
e.g., antioxidants have no direct interaction with NO but attenuate the
levels and activity of higher nitrogen oxides. Thus, whereas iNOS may
be a marker for the proinflammatory actions of NO, the species that
mediate tissue injury/dysfunction in inflammation are likely to be
nitrogen oxides other than NO.
peroxynitrite; apoptosis; oxidant; cytokine
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INTRODUCTION |
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NITRIC OXIDE (NO) is a free radical; it has an unpaired electron in its outer orbit, and this is the basis of its biological activity, allowing for electrochemical interactions between NO and metals, e.g., the active site of guanylate cyclase. However, as a free radical species, its reactivity is quite weak. This poor reactivity, combined with its lipophilicity, allows NO to be remarkably diffusible (8, 23). In vivo, this diffusion may be largely regulated by its reaction with hemoglobin, as determined by the extent of tissue vascularity (11).
Species that will react with NO · are actually limited. They
are basically oxygen, transition metals, and other radicals. Often,
discussions on the biological consequences of NO formation encompass
all the redox forms of NO (NO ·,
NO+, and
NO), which may cause
serious confusion. Furthermore, there is a need to consider the
biological results of products generated from the oxidation of NO. NO
is a precursor to a family of reactive compounds, collectively called
reactive nitrogen species, in a manner analogous to the fate of
superoxide and its family of reactive oxygen species. Table
1 highlights some of the reactive nitrogen species to be
considered and their potential effects. One misconception is that NO
(NO ·) reacts with amines (to form nitrosamines) and thiols
(to form nitrosothiols). These are nitrosation reactions and are
mediated by nitrosonium (NO+);
nitrosylation is the reaction with NO ·, and nitrosylation of
thiols and amines does not occur biologically, although the nitrosylation of transition metals is common. Thus, in discussions of
the consequences of NO reactivity, the mixing and matching of
NO · and NO+ and
NO
is not appropriate
because these forms react with quite different substrates and result in
divergent products and consequences. It should also be noted that a
major source of NO+ biologically
is dinitrogen trioxide
(N2O3),
which breaks down into NO+ and
nitrite (NO
2). Acidification of
nitrite to form
N2O3,
and hence a nitrosating species, is the basis of the griess reaction, a
common assay to measure NO production.
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The interaction of NO with superoxide is an example of its reactivity
with other free radical species (1). In this case, the product is
peroxynitrite (ONOO), a
potent and toxic oxidizing species. Peroxynitrite is not a radical
because the unpaired electrons of its precursors are now paired.
However, peroxynitrite is a far more cytotoxic species than either of
its parents, NO or superoxide. The same analogy holds true for reactive
oxygen species. Superoxide is a relatively innocuous free radical.
However, the species derived from superoxide (collectively termed
reactive oxygen species) can be far more injurious [hydroxyl
radical (OH ·),
H2O2,
lipid peroxides, and so forth]. NO could be viewed as the mother
of a similar family of reactive species, the reactive nitrogen species,
some of which are radicals. The advantage of forming these species at a
distance from the original source is that the cellular site of NO
formation will not be subjected to the same chemical stress as would
happen if these more toxic species were formed directly. This strategy is the basis of cellular host defense and facilitates the survival of
the cells generating these toxic species while still facilitating the
cells' role in host defense.
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INDUCIBLE NITRIC OXIDE SYNTHASE AS A MARKER FOR NO-MEDIATED TISSUE INJURY |
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Experiments that have demonstrated that NO production results in anti-inflammatory consequences have almost uniformly been acute in nature (7). In terms of sources of NO production, this means that the constitutive, calcium-independent forms of nitric oxide synthase (cNOS) are responsible for NO synthesis. Because these isoforms (neuronal and endothelial) release NO in a manner that is regulated primarily by intracellular calcium concentrations, the production is reduced compared with inducible NOS (iNOS) and perhaps, more importantly, not sustained.
Regulation of iNOS is primarily at the level of transcription; once expressed, iNOS produces NO independent of intracellular calcium. Thus, in acute experiments, there is invariably insufficient time for iNOS to be expressed. As a consequence, only the contributions of cNOS are evident. The effects of bacterial endotoxin (lipopolysaccharide, LPS) on gut injury may best characterize this divergence in the biological roles of NO with time (isoform expression). When administered intravenously, LPS produces marked gut injury within 3 h of administration due to the activation of circulating leukocytes, vasculature, and resident immune-related cells in the gut. In a 3-h protocol, inhibitors of NOS exacerbate injury and NO donors reduce the degree of gut damage. Clearly, NO is protective under these circumstances. If, however, the protocol is extended to 6 h, sufficient time is now available for the expression of the otherwise quiescent iNOS and a different pattern emerges (9). LPS administration still results in gut injury, but if the NOS inhibitors are administered at the 3-h mark, half-way through the protocol when iNOS is being expressed, then they are protective. In other words, after 3 h the same therapy that previously exacerbated injury is now protective. NO has gone from being protective to being injurious (or has it?).
The explanation for this stark contrast in effects has been that after 3 h iNOS has been expressed and more NO is being produced, and when NO levels are elevated NO flips from being protective to being injurious. We believe that this explanation is partly correct. Although iNOS expression is most certainly a key element, it remains incredibly difficult to reconcile that NO goes from being helpful to being harmful with a relatively minor change in production. Something is missing in this explanation. We propose that it is inappropriate to consider a radical species, like NO, in isolation. Unless one accounts for the oxidative end products of NO, then one is considering only a small component of a family of molecules that are being generated.
In terms of the application of this knowledge base to therapeutics, the influence of time for applying the best and appropriate therapy must be appreciated. For example, in reperfusion injury, NO donors at the onset of reperfusion would indeed be helpful (7). On the other hand, in a chronic inflammation (gastritis, inflammatory bowel disease, arthritis, and so forth), inhibition of NO production appears to be beneficial (5, 14-16).
Certainly, it is a reasonable generalization that the deleterious effects of nitrogen oxides are in states characterized by iNOS expression and hence elevated NO production. The expression of iNOS can be useful to infer a deleterious role of nitrogen oxides. One exception to that "rule" is stroke, during which neuronal NOS behaves in a similar manner to iNOS because of the profoundly deranged intracellular calcium levels. It is no mere coincidence that the expression of iNOS coincides with cell injury both in terms of time and location (5, 12, 15, 16). Combined with the cytoprotective effects of selective iNOS inhibitors, one can infer that iNOS contributes to tissue injury, commensurate with its role in killing tumors and invading microorganisms. However, this host defense role is not due to the enzyme itself, and it is questionable that this enzyme's initial product, NO, is the mediator.
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THE PREGNANCY EXCEPTION |
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The expression of iNOS is not always correlated with tissue injury. Perhaps the best example of this is pregnancy, during which iNOS is expressed (constitutively) in the maternal uterus, placenta, and fetal organs (2, 17-19). Here is an example in which the nomenclature breaks down, since iNOS is constitutively expressed in pregnancy. Unlike inflammation, the expression of iNOS in pregnancy is not associated with cell death; apoptosis is not colocalized with iNOS sites of expression.
In the fetus, it appears that iNOS plays a role that would otherwise be assigned to cNOS, since iNOS, together with cNOS, regulates vascular tone in the fetal circulation (2, 19). The need to utilize iNOS for NO production in the fetus may be due to hypoxic conditions of the fetus, thereby reducing NO production, but iNOS is also expressed in the immediate postpartum period, when the newborn is normoxic. Competition for L-arginine for tissue growth may also be an explanation for the utilization of iNOS. In uterine, placental, and fetal tissue explants, NO production is in the micromolar range (17), compatible with chronic inflammation; however, whether this is indicative of in vivo production is not quite clear.
How the fetus manages to express iNOS and not suffer any deleterious consequences may be critical to our understanding of the role of nitrogen oxides in tissue injury. One critical observation is that, despite the presence of iNOS, nitrotyrosine formation (an index of peroxynitrite formation) is absent in pregnancy unless it is accompanied by a secondary perturbation, e.g., LPS administration or diabetes (17, 18). In pregnancy, the generation of nitrating species is linked to apoptosis and dysfunction and the expression of iNOS is not.
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OXIDATION OF NO TO OTHER TOXIC NITROGEN OXIDES |
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As discussed in the introduction, NO has limited reactivity but it does
react with oxygen. The final product of this reaction is nitrite, but
the intermediates are NO2 and
N2O3,
highly reactive and short-lived species. It is the reaction with oxygen
that explains the degradation of NO in aerobic solutions. This reaction
is second order, with two NO molecules reacting with each oxygen
molecule (Fig. 1). Consequently, the
half-life of NO, as determined by this reaction, depends on the NO
concentration: NO half-life is shorter at high concentrations and
longer at low concentrations.
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Thus any site that concentrates NO and oxygen should facilitate their reaction, and the half-life of NO should be reduced. Are there sites that would tend to focus the reaction of NO and oxygen in biology? The answer is yes: membranes. The hydrophobic interior of membranes is lipophilic, and both oxygen and NO concentrate in lipophilic environments. Thus one would predict that the reaction of NO and oxygen is accelerated in the hydrophobic milieu of membranes and indeed it is accelerated, ~310-fold (10).
This is critical not simply because it defines a locus for the biological degradation of NO but because it raises the issue as to whether the products of this reaction (NO and O2) contribute to cellular responses. One could state that for the same rate of NO synthesis the rate of NO2 and N2O3 production is 310-fold greater in membranes than in the cytosol. Taken together with an increased NO synthesis during inflammation due to iNOS expression, the potential rate of NO2 and N2O3 formation in inflammation could be accelerated 15,000-fold in membranes compared with the cytosol under basal conditions. Finally, we have a potential explanation as to why a modest increase in NO synthesis (50-fold) can result in a total reversal of biological actions. An increased production by four orders of magnitude of toxic species is far more likely to account for the cytotoxicity attributed to nitrogen oxides than that from modest increases in the synthesis of a weakly reactive free radical, NO.
One could predict that these toxic species would be generated in a
heterogeneous manner. Organelles that have a high membrane content
would be particularly sensitive, e.g., mitochondria. The myelin sheath
of neuronal cells would also be a prime target for this effect.
Structures that are perimembrane or transmembrane in location would be
particularly sensitive, and, because of the chemical reactivity of
NO2 and
N2O3,
amines, thiols, and metals would be focal points of reactivity.
However, whether the products of these reactions are evident in
inflammation remains to be clearly shown. Grisham and colleagues (6)
have indicated that activated neutrophils do accelerate nitrosamine
formation commensurate with iNOS expression. Interestingly, nitrosamine
formation can be attenuated by antioxidants [vitamin C or
5-aminosacylic acid (5-ASA), neither of which directly interacts with
NO itself (see ANTIOXIDANTS AS ANTI-INFLAMMATORY
AGENTS)]. Antioxidants interfere with the
reactivity of nitrogen oxides that are nitrosating species, and this
may explain their therapeutic benefit. However, nitrosation is not necessarily a prelude to dysfunction and cell death. Indeed, Fiorucci and colleagues (4) have recently described that a NO-donating nonsteroidal anti-inflammatory drug class can prevent gastric injury
and can prevent the apoptosis induced by inflammatory signals like
tumor necrosis factor- because of an interference of caspase activity that involves nitrosation of a critical thiol group. Nitrosothiols should not be regarded as mere reservoirs of
NO+, to be released at a later
date, but should be regarded as a means of modifying function. The
outcome of nitrosation is a viable area of future research and drug development.
The redox form of NO appears to be a critical determinant of the
cellular response to NO. In general,
NO+ is generally regarded as being
protective, but
N2O3,
a potent nitrosating agent, can be highly toxic. The nitroxyl,
NO, the one-electron
reduction product of NO, is highly reactive and toxic (25). Conversion
of nitroxyl to NO by ferricyanide can prevent DNA damage and apoptosis,
highlighting the critical influence that the redox state has on the
biological consequences of NO synthesis.
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PEROXYNITRITE |
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Peroxynitrite is the product of NO and superoxide
(O2), a reaction that is
extremely rapid and approaches diffusion-limited kinetics. Because of
the intense reactivity of peroxynitrite, it is ephemeral and difficult
to measure in vivo. Whereas peroxynitrite can induce cell damage (1,
20) and apoptosis (12, 21, 22) in vitro, its role in vivo is less
clear. There are issues surrounding the requirement of stoichiometric relationships of NO and superoxide for this reaction and their reactivity of the substrates with the end-product (24). The standard
index of peroxynitrite formation in vivo is the formation of nitrated
tyrosine residues. The formation of nitrotyrosine has been observed in
numerous states of chronic inflammation (12, 16, 17). We noted that the
administration of NOS inhibitors prevents the increase in nitrotyrosine
formation with inflammation (16), which was critical because NO cannot
directly nitrate any substrate. Nitration involves the addition of
nitronium (NO+2), a reaction
that is possible for peroxynitrite but not NO ·. There are
other pathways to nitrotyrosine formation in inflammation (3);
therefore, we still need to use this index with caution. Nevertheless,
it is an important clue that higher oxides of nitrogen are being formed
in inflammation via NO but with different reactivities.
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ANTIOXIDANTS AS ANTI-INFLAMMATORY AGENTS |
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If NO is an important mediator of tissue injury in inflammation, then
therapies that are effective in treating these disorders should modify
NO levels or reactivity. Certainly glucocorticoids are the best example
of this type of strategy; glucocorticoids prevent the expression of
iNOS by inhibiting the activity of nuclear factor-B. Are there other
anti-inflammatory strategies that operate through NO or nitrogen
oxides? Misoprostal, the PGE analog, does not, but the best candidates
may be antioxidants because of the free radical nature of NO. The first
line of therapy for gut inflammation is 5-ASA (mesalamine). Although
mesalamine has been found to possess a myriad of actions that may
account for its anti-inflammatory properties, most of these occur at
high concentrations. The precise mechanism of action is unknown. We
tested whether mesalamine directly reacts with NO or with peroxynitrite
(21). We noted that this antioxidant had no effect on NO levels but was
a potent inhibitor of peroxynitrite toxicity. This was due, in part, to
a direct consumption of peroxynitrite by mesalamine as well as indirect mechanisms. Mesalamine was able to attenuate peroxynitrite-induced apoptosis when administered 30 min after peroxynitrite administration. Peroxynitrite has a half-life of <1 s in physiological pH, indicating that mesalamine can negate the cellular sequelae of peroxynitrite that
lead to apoptosis.
We have extended this concept to a number of other studies that addressed the interactions of antioxidants with NO vs. peroxynitrite. Uniformly, antioxidants reduce peroxynitrite levels and cellular toxicity (21, 22). However, antioxidants do not affect NO levels. An exception is vitamin C. Although ascorbate does not directly interact with NO (determined in the absence of oxygen), in the presence of oxygen, vitamin C was consumed. However, NO levels were not lowered; rather, the decline with time (due to its reaction with oxygen) was attenuated (unpublished results). In other words, compared with the normal consumption of NO by O2, ascorbic acid raised NO levels while still reacting with a product of this reaction. A schematic representation of the potential interactions is depicted in Fig. 1. We propose that, although NO does not interact with ascorbate, higher nitrogen oxides, e.g., NO2 and N2O3, will react. The relative increase in NO levels in the presence of ascorbate reflects the recycling of N2O3 into NO or the elimination of NO2 as a substrate to form N2O3. This concurs with studies by Grisham et al. (6), who demonstrated that ascorbic acid negated the formation of nitrosamines from endogenous NO.
Antioxidants are consumed in inflammation (13), and the utility of antioxidants as therapeutic agents in these conditions is well appreciated. The failure of antioxidants to lower NO levels suggests that other factors are involved in the tissue damage and its attenuation by vitamin C and that NO itself is not the cause of cell injury. Similarly, because antioxidants uniformly negate peroxynitrite levels and toxicity and interact with the higher oxides of nitrogen generated by the reaction of NO and O2, it is far more likely that the deleterious effects of NO in inflammation are due to the conversion of NO to other nitrogen oxides. These should be amenable to therapeutic intervention. In a long-term clinical study performed in patients with Helicobacter pylori gastritis, we noted that administration of ascorbic acid reduced the degree of nitrotyrosine staining in mucosal biopsies and reduced epithelial apoptosis (12). This effect was seen without clearance of the underlying infection or a significant alteration of iNOS expression. In other words, the production of NO in mucosal inflammation was unaffected but ascorbic acid attenuated an index for nitrogen oxide (peroxynitrite) formation and cell death, suggesting a causal link distal to NO itself. Whether therapies that target nitrogen oxides but yet do not lower NO levels per se facilitate the expression of the underlying anti-inflammatory actions of NO concomitant with an elimination of toxic reactions is unknown.
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SUMMARY |
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There is ample evidence that tissue injury in inflammation is promoted by nitrogen oxides and in some cases may be a link between chronic inflammation and cancer. NO production is elevated following the expression of iNOS, and inhibitors of iNOS confer protection in chronic inflammation. However, the mere expression of iNOS and increased NO levels are insufficient explanations for this role reversal from basal conditions in which NO is protective. In pregnancy, during which iNOS is expressed without compromised function, we learn that the expression of iNOS for a sustained period is not associated with tissue injury. Antioxidants do not interact with NO, a result indicative of the poor reactivity of NO despite its classification as a radical. Antioxidants will protect tissue from the injurious effects of other nitrogen oxides, peroxynitrite, nitrogen dioxide, and N2O3. These higher oxides of nitrogen are the likely chemical mediators of tissue injury in inflammation. We need to move on from the concept that NO · is "good" at low concentrations and "bad" at high concentrations. Perhaps a more reasonable concept is that the biological consequences of NO are dictated by its metabolic fate; in other words, the form of nitrogen oxide(s) determines biological outcomes. A better understanding of the chemical pathways and localization of NO oxidation can lead to new therapeutic approaches in the treatment of inflammation and cancer, ones that are directed and less likely to be associated with side effects.
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FOOTNOTES |
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* Third in a series of invited articles on Nitric Oxide.
Address for reprint requests and other correspondence: M. J. S. Miller, Dept. of Pediatrics, MC-88, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: mark_miller{at}ccgateway.amc.edu).
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