Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide

Free Radical Biology & Medicine, Vol. 25, Nos. 4/5, pp. 434 – 456, 1998 Published by Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $0.00 1 .00

PII S0891-5849(98)00092-6

Forum CHEMICAL BIOLOGY OF NITRIC OXIDE: INSIGHTS INTO REGULATORY, CYTOTOXIC, AND CYTOPROTECTIVE MECHANISMS OF NITRIC OXIDE DAVID A. WINK

and JAMES

B. MITCHELL

Radiation Biology Branch, National Cancer Institute, Bethesda, MD, USA

Abstract—There has been confusion as to what role(s) nitric oxide (NO) has in different physiological and pathophysiological mechanisms. Some studies imply that NO has cytotoxic properties and is the genesis of numerous diseases and degenerative states, whereas other reports suggest that NO prevents injurious conditions from developing and promotes events which return tissue to homeostasis. The primary determinant(s) of how NO affects biological systems centers on its chemistry. The chemistry of NO in biological systems is extensive and complex. To simplify this discussion, we have formulated the “chemical biology of NO” to describe the pertinent chemical reactions under specific biological conditions. The chemical biology of NO is divided into two major categories, direct and indirect. Direct effects are defined as those reactions fast enough to occur between NO and specific biological molecules. Indirect effects do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress. This report discusses various aspects of the chemical biology of NO relating to biological molecules such as guanylate cyclase, cytochrome P450, nitric oxide synthase, catalase, and DNA and explores the potential roles of NO in different biological events. Also, the implications of different chemical reactions of NO with cellular processes such as mitochondrial respiration, metal homeostasis, and lipid metabolism are discussed. Finally, a discussion of the chemical biology of NO in different cytotoxic mechanisms is presented. Published by Elsevier Science Inc. Keywords—Nitric oxide, Reactive nitrogen oxide species, Superoxide, Mitochondrial respiration, Metal homeostasis, Lipid metabolism

(Fig. 1).1–3 In addition, NO has been shown to be part of the oxidative war chest of the immune system by virtue of involvement in anti-tumor and anti-pathogen host response (Fig. 1).4,5 Though these functions of NO are beneficial in maintaining proper physiological homeostasis, NO also has been found to be involved in a number of different diseases, as well as inflammatory conditions that can ultimately lead to tissue injury. NO has been shown to participate in a large number of pathophysiological conditions such as arthritis, atherosclerosis, cancer, diabetes, numerous degenerative neuronal diseases, stroke, and myocardial infarction to name a few (Fig. 1).3,6 However, there is considerable debate as to the exact function of NO in such diverse pathophysiological states. Though NO and NO-derived chemical species can inhibit enzyme function, alter DNA, and induce lipid peroxidation, NO has antioxidant properties, and the ability to protect cells against cytokine induced injury and apoptosis (see discussion below). The confusion concerning NO’s involvement in tissue injury is further complicated by its multifaceted and often paradoxical action in various cytotoxic mechanisms. NO itself is not a powerful cytotoxic agent; however, it can

INTRODUCTION

The discovery of the endogenous formation of nitric oxide (NO) led to an explosion in research centering on the question, “what role(s) does this free radical molecule play in various biologic events?” Surprisingly, this diatomic free radical has been shown to be involved in numerous regulatory functions ranging from altering the cardiovascular system to modulating neuronal function Address correspondence to: Dr. David A. Wink, Radiation Biology Branch, National Cancer Institute, Bldg. 10, Room B3-B69, Bethesda, MD 20892, USA; Tel: 301-496-7511; Fax: 301-480-2238. Dr. David A. Wink received his Ph.D. in Chemistry at the University of California, Santa Barbara. Following a postdoctoral fellowship as a National Research Service Award recipient at the Massachusetts Institute of Technology in Biochemistry, he joined the Laboratory of Comparative Carcinogenesis at NCI-FCRDC as a Staff Fellow. He then joined the Radiation Biology Branch at NCI in 1995, where today he holds the position of Tenure Track Investigator. Dr. James B. Mitchell received his Ph.D. from Colorado State University in Cellular Radiation Biology in 1978. He came to the NIH and the Radiation Oncology Branch of the NCI in 1979, and became an Independent Investigator in 1984. He served as Chief of the Radiobiology Section and later as Deputy Branch Chief of the Radiation Oncology Branch. In 1993, he was named Branch Chief of the Radiation Biology Branch. 434

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Fig. 1. Multifaceted biological effects of nitric oxide (NO).

render cells susceptible to other cytotoxic agents such as heavy metals, alkylating agents, and radiation (see discussion below). Yet, NO has been shown to be protective against an array of agents that induce oxidative stress, such as hydrogen peroxide (H2O2), alkyl hydroperoxides, and superoxide (see discussion below). NO reacts with some redox metal complexes, yet its reactivity varies from complex to complex (discussed within references 7 and 8). NO reacts with other radicals such as superoxide and lipidderived radicals to form products such as ONOO2 and LOONO, which can further react with other biological targets to influence different physiological and cellular functions. NO can react with oxygen to form a variety of different reactive intermediates that are normally associated with smog and cigarette smoke. These chemical species can alter critical biomolecules such as enzymes and DNA.9 However, NO can also neutralize oxidants associated with oxidative stress and abate reactive oxygen species (ROS)mediated toxicity.10 Quite understandably, the labyrinth of divergent behaviors exhibited by NO has lead to confusion. Indeed, NO researchers have voiced the following: “Is NO good or bad?” or “Can NO be both good and bad?” It is our impression that the answers to these questions are dictated by the unique chemistry of NO in biological systems. Where, when, and how much NO is present or is being produced under a given circumstance determines the biological response. To provide a guide through the diverse reactions NO mediates on biological systems, this review will focus on a discussion of the chemical biology of NO.7,8,11 The chemical biology of NO provides a framework of relevant chemical reactions and provides a perspective that hopefully will allow insight into understanding that the function(s) of NO depends on the specific biology being investigated.

CHEMICAL BIOLOGY OF NO

Figure 2 illustrates two distinct categories of the chemical biology of NO; direct and indirect effects.7,8,11 Direct effects are those reactions in which NO interacts directly with biological molecules. In contrast, indirect effects are derived from the reaction of NO with either superoxide or oxygen, which yields reactive nitrogen oxide species (RNOS) (Fig. 2).7,8,11 The direct and indirect effects of NO reactions can also be separated based on the local concentration of NO produced endogenously or supplied exogenously. Direct effects occur at low NO concentrations (,1mM); whereas, indirect effects of NO involving the formation of RNOS become significant at higher local concentrations of NO (.1mM). Indirect effects can be further subdivided into nitrosation, oxidation, and nitration chemistry (Fig. 2). Oxidation chemistry includes one or two electron removal from substrate, as well as hydroxylation reactions. Nitrosation occurs when an equivalent of NO1 is added to an amine, thiol, or hydroxy aromatic group. For instance, intermediates in the NO/O2 reaction convert thiol peptides to S-nitrosothiol peptides. Lastly, nitration of aromatic groups involve the addition of an equivalent of an NO21. The formation of nitrotyrosine from different RNOS such as ONOO2 is a good example of this chemistry.12,13 Categorizing the chemical reactions of NO into direct or indirect effects allows for consideration of both the amount and duration of time NO is present in a specific environment. Nitric oxide in vivo is derived from the enzyme nitric oxide synthase (NOS).14 –18 There are three isoforms of this enzyme, type I (neuronal nitric oxide synthase, nNOS), type II (inducible nitric oxide synthase, iNOS), and type III (endothelium nitric oxide synthase, ecNOS). Type I and III form a class of NOS that are referred to as the constitutive form (cNOS). cNOS is continuously present in the cell and

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Fig. 2. The chemical biology of nitric oxide (NO).

can be activated by calcium influx that results in calcium binding to the calmodulin receptor to activate the enzyme. The second class is the inducible form or sometimes referred to as iNOS or NOS-2. In some cells, iNOS is expressed after exposure to specific combinations of cytokines. Cell types containing cNOS generate low fluxes of NO for short periods of time; thus, direct effects of NO are the predominant chemistry and indirect effects are limited. However, in the presence of iNOS, production of NO is much greater and indirect effects such as nitrosation, nitration, and oxidation reactions occur. It is clear that the source and level of NO provides a guideline as to what chemistry will most likely occur under a specific biological condition. Another important consideration is the distance of the target from the NO-generating source. Cells or tissues close to an NO source may experience both indirect and direct chemistry. Cells further away from the NO-generating source may only experience direct effects because NO concentration decreases as a result of diffusion and biological consumption. Spatial and temporal factors are therefore important when considering the chemistry responsible for the specific biological effects.

and other free radicals are facile enough to be biologically relevant. Reaction between NO and metal complexes There are three major types of NO reactions with metals: (1) the direct reaction of NO with the metal center, (2) NO redox reaction with dioxygen metal complexes, and (3) high valent oxo-complexes, as shown in Fig. 3. NO may react with a variety of metal complexes to form metal nitrosyls. How rapidly these complexes are formed and their relative stability dictates their biological relevance. NO reacts with some transition metals to form stable metal nitrosyl complexes, a good example being FeONO complexes.

DIRECT EFFECTS OF NO

The important NO reactions in biology are those whose rates are fast enough to be considered physiologically relevant. NO does not rapidly react with thiols or amines; however, the reaction of NO with some metal complexes

Fig. 3. Direct effects of nitric oxide (NO) on metal complexes.

Chemical biology of nitric oxide

NO in the presence aquated ferrous ion forms a ferrous-nitrosyl complex, whereas aquated ferric ion does not [Eq. (1)] (reviewed in reference 19). Feaq(II) 1 NO 3 Feaq(II)ONO

(1)

One of the most facile NO reactions with metalloproteins is that of NO reacting with proteins containing heme moieties. The most relevant reactions of NO with metals in biological systems include those heme proteins such as guanylate cyclase, cytochrome P450, and NOS. The reaction between NO and guanylate cyclase produces an Fe-nitrosyl complex that becomes activated to form cGMP, a key secondary messanger that mediates numerous regulatory functions.20 NO derived from specific cells migrates to other cells/tissue in which NO binds to the heme moiety contained within guanylate cyclase resulting in the removal of the distal histidine and results in a fivecoordinate nitrosyl complex that activates the enzyme.21,22 The NO concentration required to activate guanylate cyclase is relatively low, EC50 of 100 nM.23 The low concentration of NO required for activation of guanylate cyclase is an example of an important direct effect that can be mediated by NO derived from cNOS. The influence of NO on soluble guanylate cyclase has profound effects on vascular tone, platelet function, neurotransmission, and a variety of other intercellular interactions. By far and away, most all of the biological effects of NO are mediated through soluble guanylate cyclase and should be one of the first factors to be considered when trying to decipher different mechanisms involving NO. Another heme/NO interaction is that of NO with cytochrome P450. The cytochrome P450s are a family of enzymes that are involved in the synthesis and catabolism of numerous biomolecules such as fatty acids, steroids, prostaglandins, and leukotrienes.24 There are many cytochrome P450 isoforms that catalyze the synthesis of hormones, activate drugs, and mediate the production of some carcinogens. Adams et al. postulated that endogenously generated NO could function in regulating the synthesis of testosterone.25 In contrast to Fe nitrosyl formation leading to activation of guanylate cyclase, NO inhibits cytochrome P450 activity,26 –28 illustrating how the analogous chemical reaction of NO can result in entirely different biological outcomes as a result of the target with which NO interacts. Cytochrome P450 activity is inhibited by NO in two ways, reversible and irreversible (Fig. 4).26 Reversible inhibition occurs when NO binds to the heme to prevent oxygen binding, thus inhibiting catalysis (a direct effect).26 Irreversible inhibition is mediated by RNOS formed from the autoxidation of NO. This inhibition is abated by either the presence of serum albumin or glutathione.26 Kim et al.

437

proposed that the irreversible inhibition results from heme removal from the protein.29 A plausible mechanism is that NO binds to the heme to form a five-coordinate adduct in which the axial cysteine ligand is removed from the coordination sphere of the iron. If the active site is sufficiently opened, an RNOS can nitrosate or oxidize the cysteine ligand and prevent reattachment to the heme, leading to irreversible inhibition of activity. It should be noted that different isoforms of cytochrome P450 are subject to different thresholds of inhibition. For instance, the cytochrome P450 isoform 2B1 is more susceptible than the 1A1 isoform with respect to both reversible and irreversible inhibition of NO.26 Therefore, different fluxes of NO that depend on distance from NOS, as well as scavengers (glutathione, ascorbate, etc.), may dictate the inhibition profile of cytochrome P450 activity in tissue. Inhibition of cytochrome P450 by NO can exert important pathophysiological sequelae. During chronic infection or septic shock, NO can be produced in copious amounts. Inhibition of liver cytochrome P450s,26,28 in turn, can repress metabolism of drugs27 used to treat the infection, which has the potential to compromise therapy. Yet, NO binding to cytochrome P450 has been shown to release heme and in turn activate hemeoxygenase in hepatocytes.29 The activation of hemeoxygenase serves as a protective mechanism against a variety of pathophysiological conditions.30,31 The interaction of NO with P450 can therefore have both a regulatory and pathophysiological role. NOS, the enzyme responsible for endogenous NO production, can be inhibited by NO analogous to that described above for cytochrome P450. NOS activity is controlled by a number of substances, including tetrahydrobioterin, arginine, and glutathione.14,16 Several studies have shown that NO can attenuate the activity of NOS,32–35 serving as a negative feedback factor regulating NO levels. Comparison of the different isoforms of NOS shows that ecNOS and nNOS are more susceptible to inhibition by NO than iNOS,35 which suggests that significantly higher NO fluxes can be achieved with iNOS than with either ecNOS or nNOS. The difference in NO-mediated inhibition of NOS activity apparently results from the relative reactivity of NO and the stability of the resultant FeONO complex within NOS. It has been shown that oxidation of arginine under catalytic conditions results in the formation of a FeONO complex in nNOS.33,34 The formation of the FeONO complex in nNOS prevents the binding of oxygen to the active site and thus inhibits oxidation of arginine. The formation of FeONO complex in nNOS makes the Km for oxygen linear in the range of physiological oxygen concentrations, which suggests that NOS may serve as an oxygen sensor and an attenuator of oxygen supply to tissue.36 Conversely, the stable FeONO complex restricts the potential concen-

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Fig. 4. Mechanisms for nitric oxide (NO)-mediated inhibition of cytochrome P450.

tration of NO that can be produced by nNOS, suggesting that nNOS, and probably ecNOS, even under hyper-intracellular calcium concentrations, does not produce significant amounts of RNOS. Therefore, the predominate source of RNOS (indirect effects) in vivo is probably iNOS. NO interaction with metal-oxygen and oxo complexes. The reactivity of NO with metals is not limited to just covalent interactions with metal ions. Various metaloxygen complexes and metallo-oxo complexes rapidly react with NO. An important direct effect of NO is the reaction between NO and oxyhemoglobin to form methemoglobin and nitrate [Eq. (2)].37,38 Hb (FeOO2) 1 NO 3 met Hb (Fe(III)) 1 NO32

plexes. Metallo-oxo species are formed from oxidation of metal species or metal-oxygen complexes by agents such as hydrogen peroxide. Such hypervalent metal complexes are powerful oxidants that can lead to cellular damage such as lipid peroxidation.40 NO rapidly reacts with these hypervalent complexes to abate oxidative chemistry mediated by metallo-oxo species10,41,42 [see Eq. (3)]. Fe(2,3)1 1 H2O2 3 Fe(4,5)1AO 1 H2O

(3)

Addition of NO results in the reduction of the hypervalent complex to a lower valent state [Eq. (4)]. Fe41AO 1 NO 3 Fe31 1 NO22

(4)

(2)

This reaction is the primary mechanism by which movement and concentration of NO are controlled in vivo.39 Due to the high concentration of oxyhemoglobin and its rapid reaction with NO, this is one of the primary metabolic fates, as well as a primary detoxification mechanism for NO. Another set of rapid reactions of NO with a biological metal is the reaction of NO with high valent metal com-

The scavenging of deleterious metallo-oxo species is a potentially important reaction by which NO protects tissue from peroxide-mediated damage.10 Nitric oxide interaction with catalase best illustrates the chemistry associated with both nitrosyl formation and metallo-oxo reduction (Fig. 5). Catalase is a heme protein that is critical in protecting cells against hydrogen peroxide damage. Kim et al. demonstrated that cytokine stimulated hepatocytes had reduced catalase ac-

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submicromolar levels, such as those produced by cNOS, NO availability may be in part controlled by this mechanism. Reports have shown that increases in the activity of glutathione peroxidase increases the bioavailability of NO (from ecNOS).46 This implies that hydrogen peroxide by a similar mechanism as shown in Fig. 5, may play a crucial role in regulation of the direct effects of NO in vivo. On the other hand, the mechanism of FeONO formation would be important under conditions in which NO concentrations are higher than H2O2. These two mechanisms may be important in the timing of the NO burst with the peroxide burst under some physiological and pathophysiological conditions. Fig. 5. Nitric oxide and catalase activity.

Reaction of NO with radical species tivity due to the production of NO.43 Examination of the effect of NO donors on hydrogen peroxide-mediated cytotoxicity showed that NO inhibited cellular consumption of hydrogen peroxide.44 Farias-Eisner et al. suggested that NO inhibition of catalase could play a role in the tumoricidal activity of macrophages.45 Though NO inhibits H2O2 consumption, catalase and hydrogen peroxide can consume NO, thus preventing its bioavailability.46,47 There appears to be two reaction mechanisms that are important to understand the relationship between hydrogen peroxide, NO, and catalase. Hoshino et al. showed that NO could bind to the heme moiety forming a ferric nitrosyl with a rate constant of 3 3 107 M21s21 and Kdiss of 1 3 105 M21.48 Analogous to that described for P450 and NOS, an FeONO adduct prevents the binding of hydrogen peroxide to the metal ion thus inhibiting catalase activity. It is estimated that by this mechanism between 10 –15 mM NO inhibits hydrogen peroxide consumption by 80%.45 Because cells that express iNOS have reduced catalase activity, this may suggest that local NO concentrations near or inside these cells maybe as high as 10 mM for prolonged periods of time. The second mechanism by which hydrogen peroxide may attentuate NO levels is different. In the enzymatic mechanism of catalase, hydrogen peroxide first reacts with catalase to form complex I and water (Fig. 5). Complex I then reacts with hydrogen peroxide, forming O2. However, NO can also rapidly react with compound I forming complex II, which then rapidly reacts with an additional NO. This results in the conversion of 2 moles of NO and 1 mole of hydrogen peroxide to 2 moles of HNO2 (Fig. 5) and consumes NO while retarding hydrogen peroxide depletion. Brown has shown that NO can partially inhibit hydrogen peroxide consumption while NO was consumed by catalase/peroxide.47 The Ki for NO in this reaction was 0.18 mM. This finding suggests that at

Another direct effect of NO is its reaction with other radical species. For example, the tyrosyl radical species formed in the catalytic turnover of ribonucleotide reductase reacts with NO.49 –52 Inhibition of this enzyme has been proposed to be a factor in the cytostatic properties of NO, due to the suppression of DNA synthesis. Another example of radical reactions of NO influencing biological outcomes is NO’s ability to radiosensitize cells under hypoxic conditions. Carbon-centered radicals are formed in DNA by ionizing radiation. Such radical species can rapidly react with NO to form C-nitroso adducts, which stabilize the radiation-induced lesion, increase DNA damage, and lead to radiosensitization.53–55 Because the hypoxic cell population in tumors is proposed to be responsible for limiting the effectiveness of radiotherapy, these observations may provide new strategies for cancer treatment.55 Influence of the direct effects of NO on lipid chemistry. Lipid peroxidation results in formation of alkoxy or peroxy radicals that react with NO at near diffusion rates [Eq. (5)].56 LOO• 1 NO 3 LOONO

(5)

Reaction 5 has been proposed to play a role in terminating lipid peroxidation chain reactions by NO, which results in protection of cells against peroxide induced cytotoxicity.10,57,58 Lipid peroxidation induced by oxidants formed as result of exposure to copper, xanthine oxidase, or azo-bis-amidinopropane are terminated by NO.59,60 The chain termination also prevents oxidation of low density lipoprotein in both endothelial61 and macrophage cells.59 The reaction between NO and lipid hydroperoxyl radicals [Eq. (5)] appears to be important in limiting lipid peroxidation in disease processes such as atherosclerosis.

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Fig. 6. Mechanism for nitric oxide (NO)-mediated inhibition of lipid peroxidation.

In addition to NO’s ability to limit lipid peroxidation, NO also influences the normal metabolism of lipids such as the formation of autocoids. Arachidonic acid generated from phospholipase A1 is metabolized by one of two pathways that results in either leukotrienes or prostaglandins.62 Whereas cyclooxygenase (COX) converts arachidonic acid to prostaglandins such as PGE2, thromboxane, or prostacyclin, lipoxygenase converts arachidonic acid to various leukotrienes. Prostaglandins and leukotrienes mediate important physiological processes during a variety of inflammatory conditions and provide a balance of action in numerous physiological and pathophysiological events.62 NO has been shown to inhibit lipoxygenase activity by reacting at the active site with a nonheme iron.63,64 Recent studies suggest that the inhibition of lipoxygenase products results from reaction between hydroperoxide radicals [Eq. (5)] and NO rather than the direct reaction between NO with the ferric metal center.60 These findings are consistent with a reaction between hydroperoxide radicals and NO, which has a rate constant .104 faster than with nonheme ferric ion as discussed above. However, under physiological conditions, the active ferric state of lipoxygenase could be reduced by agents such as superoxide to the ferrous state which then would readily bind to NO (Fig. 6). The formation of FeONO adducts would inhibit the enzyme. Modulation of the redox states of iron-containing enzymes such as lipoxygenase could be a significant mechanism in NO’s ability to modulate autocoid metabolism.

The heme protein COX and related enzymes can be influenced by metal-complex/NO interactions and NO/ radical reactions. Kanner et al. first showed that COX could be inhibited by NO.63 Comparing hemoglobinmediated oxidation of linoleate oxidation, they concluded that linoleate peroxidation was inhibited by NO binding to the heme moiety within COX.63 Another report examined the NO-binding characteristic to prostaglandin H synthase (an enzyme analogous to COX) and found that the ferric state does not efficiently bind NO.65 Yet, the ferrous state bound NO strongly suggesting, as with lipoxygenase, reduction of ferric centers and subsequent reaction with NO can result in nitrosyl formation and enzyme inhibition.65 However, microsomes exposed to NO donors and NO gas (concentrations up to 1 mM) did not inactivate COX activity. These authors concluded that NO did not inhibit COX by direct heme binding.65 Such discrepancies may suggest that there are more complex interactions of NO and COX activity. Contrary to the studies that suggest NO inhibits COX activity, several studies have shown that NO from NOS markedly enhances prostaglandin synthase.66 –72 One possible explanation is that NO is not significantly interacting with the metal centers of COX, but is involved in other regulatory factors. Several reports suggest that superoxide inhibits prostacyclin synthesis in endothelial cells72 and PGE2 in vascular smooth muscle.73,74 Furthermore, superoxide has been shown to be involved in inhibition of prostaglandin H synthase-2 (PGH2)/PGE2 isomerase74 and prostaglandin H synthase.72 A study reported that DMPO, an oxy-radical spin trap, could inhibit the lipid polysaccharide (LPS)-stimulated synthesis of PGH2, possibly suggesting that either superoxide or oxidants derived from Fenton type chemistry may be involved.75 The enhancement of PGE2 synthesis by NO may be due to scavenging of superoxide by the NO/O22 reaction. These reports indicate that the superoxide/NO interaction and the control of superoxide modulation of enzyme activity may play a critical role in prostaglandin metabolism. One possible mechanism to account for COX inhibition by superoxide involves the reduction of the active ferric form to the inactive ferrous by superoxide (Fig. 7). The presence of NO results in scavenging of superoxide and prevents reduction of the active ferric state while converting any ferrous-oxy adducts to the active ferric state [Eq. (3)]. Low levels of NO would be required and the reactions shown in Fig. 7 would be considered direct effects. Therefore, at submicromolar amounts of NO (such as those derived from cNOS), this mechanism would predominate and thereby increase the activity of prostaglandin synthase. However, if higher NO concentrations, such as those produced by iNOS occurred, NO

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Fig. 8. The chemistry of the indirect effects. Fig. 7. Nitric oxide (NO) effect on arachidonic acid metabolism by cyclooxygenase.

could bind directly to the heme center of COX, thus inhibiting activity as described by Kanner et al.63 Stadler et al. showed that NO formed in stimulated Kupffer cells inhibits the formation of the products of COX, which suggests that NO levels are sufficiently high to inhibit COX in cells.64 Their results further suggests that at low levels NO can modulate the redox form of the COX enzyme, but at high NO levels inhibition may occur either by NO binding directly to the heme center or by modification of the enzyme by RNOS. INDIRECT EFFECTS

Unlike direct effects, indirect effects are mediated by RNOS derived from either the NO/O2 or NO/O22 reaction (Fig. 8). Though some reports imply that NO reacts with diamagnetic species such as thiols or other bioorganic molecules, these reactions are far too slow to be significant in biological systems.76,77 Therefore, reactions with thiols and amines have to first proceed through an activation step of NO by molecules such as oxygen or superoxide. The chemistry of the NO/O2 or NO/O22 reaction and their relevance to biological systems will be discussed. NO/O2 reaction The NO/O2 reaction is an important reaction in the atmospheric fate of NO.78 NO is unstable in an oxygen environment. NO forms RNOS, such as nitrogen dioxide

(NO2) and dinitrogen trioxide (N2O3) [Eqs. (6) and (7)], which are known to be injurious to biological tissues.78 2 NO 1 O2 3 2 NO2

(6)

NO2 1 NO 3 N2O3

(7)

In aqueous solution, NO also undergoes autoxidation by a third order rate equation similar to the reaction in the gas phase.79 The resultant intermediates appear to be N2O3 in both the gas phase and aqueous media; however, “free” NO2 formed in the gas phase reaction is not formed in aqueous media (summarized in reference 19). The implication is that a different reactivity pattern exists for the autoxidation reaction between the intermediates formed in the gas phase and aqueous solution. The N2O3 formed in aqueous solution undergoes hydrolysis to form nitrite [Eq. (8)]. N 2 O 3 1 H 2 O 3 2 HNO 2

(8)

The third order nature of the autoxidation reaction gives some insight into the chemical biology of NO. The rate equation for the autoxidation reaction is second order with respect to NO, which means that the half life depends on the concentration of NO. For instance, if the initial concentration of NO is 1 mM, the half life is 800 s; however, if the NO concentration is 100 mM, the half life is 8 s.79 Therefore, given that if there are no scavengers at low concentrations in biological systems, NO will diffuse from its site of origin and thereby decrease in concentration with distance. As the NO concentration dilutes, the lifetime increases, which allows NO to inter-

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act with other biological targets such as guanylate cyclase or oxyhemoglobin. However, if the local NO concentration dramatically increases, formation of the intermediates from the autoxidation reaction increase exponentially. Therefore, at low concentrations of NO the direct effects will predominate, while at higher concentrations the indirect effects mediated by the NO/O2 reaction become preeminent. The rate constant for the autoxidation reaction of NO is not appreciably affected by pH and is similar between room temperature and 37°C (reviewed in references 19 and 79). The rate constant is nearly identical in hydrophobic solvents such as carbon tetrachloride and aqueous solutions80 and is not affected by .10 mM reductants such as GSH, ascorbate, or ferrocyanide.76,81– 84 These observations suggest that the factors which determine the rate of the autoxidation reactions in biological systems are solely dependent on the concentrations of oxygen and NO. The third order rate equation for the NO autoxidation reaction provides insight into where the autoxidation reaction might occur in biological systems. NO and O2 are .20 times more soluble in the lipid fraction of a cell than the aqueous portion. Since the rate constant for autoxidation in the lipid layer is similar to that in aqueous solution, the rate of autoxidation is dramatically accelerated in lipid layers compared to those in the aqueous phase of a biological system (J.R. Lancaster, personal communication). Thus, in biological systems, the lipid layers are predicted to be the primary site for autoxidation. Though RNOS is formed in aqueous solution by different mechanistic paths from those in the gas phase, the NO/O2 reaction in hydrophobic regions produces similar intermediates to that found in the gas-phase [Eqs. (6) and (7)], i.e., NO2. NO/O2 does not appear to involve Eqs. (6) and (7) in the aqueous phase. These reactive intermediates would be expected to be predominant in the lipid layer in vivo. Nitrosation (and possibly nitration) reactions mediated by the NO/O2 reaction in cells would primarily occur in or near the lipophilic membrane and involve Eqs. (6) and (7), which implies that membrane associated proteins would most likely be converted to species such as S-nitrosothiols or nitrosamines by autoxidation reaction. The chemistry of the intermediates formed from the NO/O2 reaction in hydrophobic media is that of NO2 and N2O3.85 Because NO reacts with NO2 at .109 M21s21, and the concentration of NO required to facilitate the NO autoxidation in biological systems is high, the contribution of NO2 is limited to the chemistry of endogenously generated NO.86 Collectively, the data suggest that N2O3 is the predominant RNOS formed from the autoxidation of NO in biological systems. Koppenol calculated the oxidation potential for N2O3 to be 0.8 V,87 which is in

excellent agreement to that observed experimentally by Grisham et al.88 Therefore, N2O3 is a relatively mild oxidant compared with chemical species such as hydroxyl radical. The primary reaction of N2O3 is nitrosation. Nitrosation of amines results in nitrosamines, while nitrosation of thiols result in S-nitrosothiols. Carcinogenic nitrosamines can be formed from some secondary amines by nitrosation mediated through N2O3 and can have deleterious consequences.85 Nitrosation of thiols in proteins has a variety of different effects from inhibiting enzyme activity7,89 to forming S-nitrosothiol adducts that modulate signal transduction.90,91 The selectivity of the intermediates formed in the autoxidation reaction of NO has been determined with respect to biologically relevant molecules. Because N2O3 is hydrolyzed to nitrite with a half-life of 1 ms [Eq. (8)], only substrates that have high enough concentrations and sufficient affinities with RNOS will react.92 The reduced availability of water in hydrophobic compartments decreases the hydrolysis of N2O3 to nitrite [Eq. (8)], thereby, increasing the life-time of N2O3 and allowing other reactions with biomolecules to occur. At neutral pH, peptides with thiols have strong affinity for N2O3.76,92 In addition, buffers such as carbonate and phosphate have affinities less than 400 times that of thiol containing peptides at neutral pH.92 These reactivity patterns suggests that the primary reaction of the NO/O2 in biological aqueous solutions is with thiols, resulting in S-nitrosothiols. S-nitrosothiols, which have a variety of effects including regulation of the cardiovascular system has been detected in vivo,89 which supports the premise that nitrosation does occur from NOS-generated NO. NO/O22 reaction The reaction between superoxide and NO has been shown to occur at near diffusion controlled rates with a rate constant of 7 3 109 M21s21. The product is a powerful oxidant, peroxynitrite [Eq. (9)].93 NO 1 O22 3 ONOO2

(9)

It has been speculated that ONOO2 formation is a primary pathway of NO metabolism.94,95 Though this reaction has a near diffusion controlled rate constant, there are a number of factors which limit the contribution of ONOO2 under various physiological conditions. There are two major determinants of ONOO2 formation.96 The first is the relative amount of NO and superoxide produced. The second is the biological reaction of these radicals with other biological components, which limit

Chemical biology of nitric oxide

the availability of NO and superoxide and constrain the amount of peroxynitrite formed. Superoxide concentrations under normal, nonstressed physiological conditions are less than nanomolar.97 This is due to its short lifetime through dismutation reaction, catalyzed or uncatalyzed, as well as different redox reactions with important biological molecules. Thus, NO produced even at submicromolar concentrations will be in excess. Because superoxide concentration is significantly less than NO, the amount of NO determines the psuedo first order rate constant of reaction between these two radicals [Eq. (9)]. The psuedo first order rate constant determines the half life of the reactions as well as determines the relative reactivity of this reaction with other biologically important reactions. On the other hand, the amount of O22 determines the amount of ONOO2 which is formed. Hence, under normal cellular conditions, ONOO2 formation should be exceedingly low. The second constraint on ONOO2 formation is the reaction of these two radicals with other biological molecules such as superoxide dismutase (SOD) and oxyhemoglobin. For instance, SOD has a similar reaction rate constant to that of the NO/O22 reaction; therefore, micromolar amounts of SOD will compete effectively with NO for superoxide. Normal cellular SOD concentrations are 4 –10 mM,98 which suggests that the flux of NO required to convert 50% of the superoxide to peroxynitrite is 2– 4 mM. However in biological regions where SOD concentrations are less than micromolar, peroxynitrite chemistry may have a role with respect to the NO/O22 reaction. Just as SOD reduces the availability of superoxide, the reaction of NO with heme proteins reduces NO concentration. As discussed above, NO reacts rapidly with oxyhemoglobin (HbO2) which limits NO movement in vivo [Eq. (2)]. Because this reaction is one of the major consumption mechanisms of NO, this reduces the participation of NO/O22, as well as NO/O2, with other biomolecules. Another important determinant of NO concentration is its movement away from the site of generation. Diffusion from the site of formation will significantly dilute NO,39 so that the reaction of NO and superoxide cannot occur except in localized areas. Factors that control the concentration of either radical, such as diffusion, HbO2/NO reaction [Eq. (2)], and O22/ SOD reaction, will limit peroxynitrite formation to regions close to the location of the NO source and, more importantly, to the superoxide source. As discussed in other articles in this issue, peroxynitrite can undergo a variety of chemical reactions that could have potential deleterious consequences. Most of these reactions are mediated by a high energy trans isomer of peroxynitritous acid that is a powerful oxidant.99 Peroxynitrous acid is thought to mediate most of the oxidation and nitration

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reactions of peroxynitrite.99 One reaction that peroxynitrite anion/cis-peroxynitrous acid can undergo is the direct oxidation of thiols.100 The reaction between the cis isomer of peroxynitrite and thiols can result in oxidation of thiol containing proteins; however, this reaction implies that peroxynitrite reactivity within a cell is vastly reduced because of the relatively high GSH concentrations present in most cells. Recent work has suggested that a CO2 adduct of peroxynitrite can be formed that is also a potent oxidizing and nitrating agent.101 This adduct may be the major chemical species that mediates different reactions of ONOO2 in vivo. Another factor that limits peroxynitrite reactivity in some biological circumstances is the reaction between peroxynitrite and either NO or O22 to form nitrogen dioxide [Eqs. (10) and (11)].99,102,103 ONOO2 1 NO 3 NO2 1 NO22

(10)

ONOO2 1 O22 3 NO2 1 O2 1 NO22

(11)

Studies using xanthine oxidase (XO) as an O22 source and NO donors as a source of NO show clearly that the flux of NO is a critical determinate of the oxidation chemistry associated with peroxynitrite.102 NO does not alter the activity of XO and, therefore, XO is an ideal method to study the NO/O22 reaction.104 –107 When the enzymatic production of superoxide remains constant, the oxidation of dihydrorhodamine (DHR) increases as the rate of NO production increases [Eqs. (12) and (13)]. ONOO2 3 NO32

(12)

ONOO2 1 DHR 3 oxidation

(13)

The maximum oxidation occurred when the rate of production of superoxide and NO are equivalent.100 When NO is in excess, the oxidation mediated by peroxynitrite is dramatically reduced. Other studies have shown that oxidation of various biological molecules exhibit the same behavior as DHR. As NO flux was increased in the presence of constant superoxide production, there was a dramatic increase glutathione oxidation.106 However, when the rate of NO formation became greater than superoxide, oxidation decreased. Hogg et al. showed that peroxynitrite is a powerful enough oxidant to initiate lipid peroxidation.109 Studies examining NO/O22 induction of lipid peroxidation show similar behavior as that described for dihydrorhodiamine.102 Maximum lipid peroxidation occurs when the fluxes of superoxide and NO are equivalent.60 However, excess NO suppresses lipid peroxidation similar to that observed for DHR oxidation. NO supression of oxidation under these conditions results from scavenging peroxyni-

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trite [Eq. (10)], as well as terminating the propagation of lipid radicals produced after oxidation. Taken together, studies involving DHR, thiols, and lipids indicate the maximum oxidation chemistry mediated by peroxynitrite occurs only when the fluxes of NO and superoxide are equivalent. Though oxidation mediated by peroxynitrite is reduced by NO, other RNOS are formed. In the presence of excess NO or superoxide, peroxynitrite is proposed to be converted to nitrogen dioxide [Eq. (10)].99,102,103 Nitrogen dioxide rapidly reacts with NO to form the nitrosating species N2O3 [Eq. (7)]. Though nitrosation does not occur directly through peroxynitrite or peroxynitrous acid,107 there are several mechanisms by which nitrosation can occur through the NO/O22 reaction. A good indicator of nitrosation chemistry, in general, is the conversion of 2,3 aminonaphthylene to the fluorescent triazole adduct by NO1 donors. It was demonstrated that induction of superoxide by phorbol ester in rat neutrophils resulted in a decrease of nitrosation.107 Further studies using XO and NO donor model systems showed that increasing O22 in the presence of constant NO production showed no appreciable drop in nitrosative product yield until the radical fluxes were equivalent, which indicates that maximum oxidation by NO/ O22 results in minimal nitrosation. Examination of the thiol nitrosation reaction also shows that S-nitrosothiol formation is reduced by O22.108 It was concluded that oxidative and nitrosative chemistry mediated by the NO/ O22 reaction are mutually exclusive.108 Considering the chemistry of peroxynitrite, the potential locations for peroxynitrite formation and reactivity can be identified. A likely intracellular source are mitochondria, where superoxide is produced as result of aerobic respiration. The NO concentration would be expected to be higher in lipid layers than in the cytosol, suggesting that most ONOO2 formation will occur within the hydrophobic regions of the mitochondria. However, manganese superoxide dismutase (MnSOD), as well as other redox active proteins and antioxidants, play a strong role in controlling the formation and reactivity of peroxynitrite. In addition, if relative NO and superoxide fluxes are imbalanced, conversion of peroxynitrite [Eqs. (7), (8), (10), and (11)] to N2O3 is increased, thus limiting direct ONOO2 chemistry. The primary source for large quantities of superoxide is immune cells through either NADPH oxidase or xanthine oxidase.44,105 Neither enzyme is directly inhibited by NO. Therefore, as NO migrates near the source of O22, it reacts to form peroxynitrite. However, as peroxynitrite moves from its source, it is converted by excess NO to N2O3 (Fig. 9). Thus, the primary chemistry of ONOO2 would be within close proximity of the superoxide source.

Fig. 9. Implications of distance and timing with respect to nitrosative and oxidative stress in the NO/O22 reaction.

Balancing between oxidative and nitrosative stress. There exists a balance between the chemistry of RNOS associated with oxidative stress and that of nitrosative chemistry (Fig. 10). As described above, N2O3 mediates nitrosation reactions derived from the NO/O2 reaction or from the NO/O22 reaction. It is interesting to speculate that the source of N2O3 in hydrophobic layers originates from the NO/O2 chemistry while the source of N2O3 in aqueous solution is derived from the NO/O22 reaction. On the other side of the chemical spectrum, oxidative stress in biological systems can be mediated by the NO/O22 by peroxynitrous acid or by Fenton-type reaction including hydrogen peroxide and redox metals. The oxidative chemistry of peroxide/metals can result in DNA damage and lipid peroxidation, which are abated by NO.58,61 Peroxide oxidation of metalloproteins followed by subsequent degradation is also abated by NO.10,41,42 Therefore, NO may serve to protect against the Fenton type-mediated oxidation. But NO can also lessen oxidative chemistry mediated by peroxynitrite.102,108 Therefore, oxidative stress mediated by

Fig. 10. Free radicals in concert.

Chemical biology of nitric oxide

peroxide/metal and NO/O22 is lessened by excess NO, which implies that NO can function as an antioxidant. In the case of peroxynitrite, NO’s reaction to alleviate oxidative stress results in nitrosative stress. Therefore, a balance exists in the NO chemistry between oxidative and nitrosative stress (Fig. 10). Mechanism of DNA damage. An important target for RNOS is DNA. Chemical alteration of DNA can have important consequences in a variety of cytotoxic and pathological mechanisms. Nitric oxide associated with chronic inflammation has been postulated to be a likely candidate in the genotoxic events associated with the etiology of cancer.110,111 There are three potential chemical mechanisms by which NO can damage DNA. The first is direct reaction of RNOS with the DNA structure. The second is through inhibition of repair processes. The third is to increase production of genotoxic species (alkylating agents and hydrogen peroxide). It was shown that RNOS (N2O3) causes mutations in bacteria and mammalian cells.112–114 Though NO itself does not interact with bioorganic molecules such as DNA112 or amino acids,76 RNOSs (such as N2O3 and peroxynitrite) chemically alter DNA, resulting in a variety of lesions (reviewed in references 7 and 11). It has been shown that NO in an aerobic environment can cause single strand breaks in bacterial or mammalian cells,113,114 as well as deamination of nucleic acids.112,113 It was proposed that the formation of RNOS via the autoxidation of NO is responsible for these lesions. Several studies have shown that DNA exposed to NO under aerobic conditions results in deamination of cytosine, adenine, and guanine.112,113 These deamination products result from nitrosation of the exocyclic amine group forming a primary nitrosamine, which then rapidly deaminates to form a hydroxyl group. NH2OR 1 N2O3 3 RONHNO 1 NO22

(14)

RONHNO 3 RONNOH 3 ROOH 1 N2

(15)

Deamination results in the conversion of cytosine to uracil, guanine to xanthine, methylcytosine to thymine, and adenine to hypoxanthine. It has been proposed that NO contributes to the spontaneous deamination that occurs in vivo.112 Several studies have shown that synthetically generated peroxynitrite can cause various DNA lesions. Peroxynitrite concentrations ranging from 0.05 to 8 mM were used to induce DNA strand breaks in vitro.115,116 Guanine was oxidized by SIN-1 (which decomposes to NO and superoxide) to form HOdG,117 although another study suggests that peroxynitrite did not increase HOdG levels in DNA.118 In addition to oxidation products, the

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nitration product of peroxynitrite, 8-nitroguanine has also been detected.118,119 It has been suggested that oxidative damage to DNA in activated macrophages results from the formation of peroxynitrite.120 Most of the studies with peroxynitrite were done in the presence of large boluses of the synthetically generated compound. Such preparations can be contaminated with excess nitrite and hydrogen peroxide, which may contribute to the observed mutations. The chemistry of peroxynitrite formed in vivo depends on the relative fluxes of NO and superoxide in that microenvironment.102 The amount of peroxynitrite that directly reacts with the biological target would be minimal, if the NO flux is in excess. In fact, XO and peroxide mediated damage to DNA is abated by the presence of NO.121,122 It is presumed that Fenton chemistry mediates DNA strand breaks, which are lessened by NO. Furthermore, hydroxylation reactions are also quenched by the presence of NO.102 Taken together, these protective effects indicate that the involvement of RNOS in modifying DNA directly might be limited in vivo, while NO protection against oxidative stress-mediated DNA damage may be more important. In addition to modification of DNA, NO can form or modulate the activity of other carcinogens. The formation of nitrosamines in vivo has been linked to activated macrophages123,124 and hepatocytes.125,126 These nitrosamines are metabolized to alkylating species, which induce lesions in DNA. In addition to the formation of these lesions, some DNA repair enzymes associated with repair of alkylation of DNA are inhibited by NO.9 It has been shown that NO inhibits DNA alkyl transferase activity both in vitro and in vivo.127 The protein has critical -SH residues that upon exposure to aerobic solutions of NO form S-NO adducts. Such adducts inhibit the transfer of an alkyl group from the O6- position of guanine to the thiol residue within the protein, which results in the potentiation of the toxicity of alkylating agents such as carmustine (BCNU).127 Proteins which contain zinc finger motifs lose their structural integrity upon exposure to NO, resulting in inhibition of enzyme activity.128,129 Reactions between amines and thiols are much more facile than direct alterations to DNA. Therefore, the most likely contribution of NO in the initiation stage of cancer is through the formation of carcinogenic substances and inhibition of DNA repair. DIRECT AND INDIRECT EFFECTS ON EXAMPLES OF CELLULAR METABOLISM

For the most part, we have discussed the influences of NO on cellular processes as either being direct or indirect. In each case, an illustration of the biological effect was described. However, NO can influence some cellular

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functions by both mechanisms. Whether direct effects or indirect effects are present can have profound influence on the biological outcome. For example, the presence of NO can inhibit P450 by either direct or indirect effects. Inhibition of cytochrome P450 by direct effects is reversible and probably plays a regulatory role in processes such as hormone metabolism. Irreversible inhibition, resulting from indirect effects, may represent more pathological consequences. Therefore, to understand the mechanisms of pathological conditions, it is not enough just to know whether NO inhibits a particular cellular function, but to understand whether it is by NO or RNOS. Below are two examples of how NO can act by both direct and indirect effects on mitochondrial function and metal homeostasis. NO inhibition of mitochondrial respiration One of the primary cellular targets for the cytotoxic action of NO has been proposed to be the mitochondrion.2,4,130 Dinitrosyl adducts of aconitase are formed in cells after exposure to NO and may be an important factor in the inhibition of mitochondrial activity leading to cytostasis and cytotoxicity.130 Further studies have shown that NO also de-energizes the mitochondria in a reversible manner,131,132 which involves regulation of intracellular calcium under normal physiological conditions.133 So how does NO inhibit mitochondrial function as part of regulatory processes, yet also mediate cell death? Inhibition of mitochondria mediated by NO appears to have a reversible and irreversible component. Knowles et al. reported that NO derived from S-nitrosoglutathione (GSNO) inhibited mitochondrial respiration by a distinctly different mechanism than ONOO2.134 They suggested that NO derived from GSNO reversibly inhibited respiration, while ONOO2 resulted in irreversible inhibition of respiration. Several studies have shown that NO directly interacts with cytochrome c oxidase to reversibly inhibit respiration.135–140 The interaction at cytochrome c oxidase appears to require low concentrations of NO (submicromolar), characteristic of the NO concentrations resulting from cNOS production. However, under inflammatory conditions, complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate: ubiquinone oxidoreductase) are irreversibly inhibited by NO.137 Under these conditions, RNOS can cause irreversible inhibition. Similar to the mechanism described for cytochrome P450, inhibition of mitochondrial respiration appears to have a reversible component mediated by direct effects and an irreversible component mediated by indirect effects. Modulation of respiration by low amounts of NO near cytochrome c oxidase will determine tissue oxygen gra-

Fig. 11. Nitric oxide (NO) and mitochondrial function.

dients as well as cellular ATP levels. Like other heme proteins, cytochrome c oxidase can react with NO to form a nitrosyl adduct.141 Binding of NO to cytochrome c oxidase may influence the activity of mitochondrial enzymes depending on the oxygen concentration. Under both hypoxic and aerobic conditions, NO is consumed by the mitochondria through direct binding to the cytochrome c oxidase. After the formation of the FeONO, additional electrons channeling down the respiratory chain reduce NO to nitrogenous products.142,143 However, under aerobic conditions, it appears that electrons in the respiratory chain are diverted from the reduction of NO at the cytochrome c oxidase site to form superoxide, which can further react with NO (Fig. 11).144 The partitioning between the reduction of NO and oxygen is dependent on the oxygen tension versus the rate of electron reduction of the nitrosyl cytochrome c oxidase complex. The inhibition by low levels of NO of oxygen consumption by the mitochondria may be important in regulating tissue oxygen. ecNOS (NOS-3) has been found in mitochondria,145 which indicates that this source of NO may be important in different physiological mechanisms. The presence of NOS in mitochondria suggests that the chemistry of NO is well regulated within this organelle. Some reports have proposed that mitochondrodrial NOS may function in the regulation of respiration. At low fluxes, NO de-energizes mitochondria in rat hepatoma cells,131 whereas another report suggests that the influence of NO on mitochondria may cause relaxation of

Chemical biology of nitric oxide

smooth muscle cells and paracymal in both physiological and pathophysiological conditions.146,147 As NO concentrations and time of exposure increases, there is an increase in RNOS formed in the mitochondria (Fig. 11). The source of RNOS under aerobic conditions has been proposed to involve superoxide derived from decoupling of oxygen reduction at the cytochrome c oxidase site, which reacts with NO to form peroxynitrite. However, MnSOD, which exists in the mitochondria at high concentrations (.5 mM), will compete with NO for superoxide, thus limiting formation of peroxynitrite. The amount of NO required to form peroxynitrite results in NO fluxes that are higher than superoxide fluxes. As discussed above, this condition might create an imbalance in the superoxide/NO ratio and would favor the conversion of peroxynitrite to N2O3 by Eqs.(7) and (10). These conditions indicate that the oxidative chemistry mediated by peroxynitrite probably does not have a significant role in mitochondrial dysfunction, rather other RNOS such as NO2 and N2O3 do. Furthermore, according to Eqs. (7)–(10), it is more likely that nitrosative, not oxidative chemistry, would be the predominant indirect effect in mitochondria under high NO fluxes. Most of the mitochondrial studies have been conducted in cell culture or with isolated mitochondria. However, a comparison of cellular and in vivo inhibition of mitochondria suggest that RNOS mediated irreversible inhibition is less important in vivo. Hepatocytes in culture, stimulated with interferon gamma and LPS to activate NOS and generate NO result in inhibition of respiration. In contrast, respiration in cells isolated from animals treated with LPS and interferon gamma was not affected.149,150 This may suggest that oxyhemoglobin and diffusion of NO away from NOS containing cells may determine the extent of mitochondrial inhibition, where RNOS formation is limited and reversible inhibition of respiration is only transient. Metal homeostatics An important function of NO either under physiological or pathophysiological conditions is the regulation of intracellular iron status (see reviews in references 151 and 152). There are different aspects of iron metabolism that NO can affect. NO can influence heme metabolism by releasing free heme, which activates heme oxygenase resulting in catabolism of heme complexes, as well as inhibiting ferrochelatase, an enzyme that places the iron in the porphyrinic complex.29 The inhibition of ferrochelatase reduces heme availability and decreases the amount of active NOS, which may serve as a negative self-regulation of NO formation. In addition to influencing heme metabolism, NO affects the formation of the transferrin receptor and ferritin

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protein which regulate the uptake and storage of cellular iron. The iron responsive elements (IREs) are strands of RNA that are post-transcriptionally regulated (see reviews in references 151 and 152). The iron responsivebinding protein (IRB), which contains an Fe3– 4S4 cluster and has aconitase activity, regulates the IRE synthesis of ferritin and transferrin receptor protein.153 The iron sulfur cluster within the IRB has two forms, apoprotein Fe3S4 and holoprotein Fe4S4, where the fourth iron is in the apical position. If the apical Fe is missing, then binding to the IRE results in the downregulation of ferritin production and upregulation of transferrin receptor. These events result in increased cellular uptake of iron.153 However, if the apical iron is present, then ferritin protein increases while the production of transferrin receptors decreases, resulting in reduction of uptake of iron.153 NO binds to the apical iron to form a nitrosyl complex (Fig. 12), which results in inhibition of aconitase activity of IRB, but does stimulate binding to IREs. It should be noted that peroxynitrite and superoxide inactivate both aconitase activity and the IRB’s ability to bind to the IRE,154,155 thus limiting the uptake of iron. The inactivation of aconitase by either peroxynitrite or superoxide may be a protective mechanism against excessive iron uptake that can limit the iron available to catalyze oxidative chemistry. This may be another mechanism in which NO reduces intracellular oxidative stress. Inactivation of mitrochondrial aconitase activity would also reduce the available electrons to reduce oxygen by the respiration chain. Since NO may increase superoxide/ hydrogen peroxide by inhibiting the respiratory chain (cytochrome c oxidase), the reduction of electron flow may also reduce increased ROS production. The inactivation of aconitase may be protective against intracellular hydrogen peroxide formation. Iron metabolism and availability have tremendous effects on oxidative stress and cell growth. NO may be key in limiting the availability of iron by inhibiting release of iron from ferritin. In mammalian cells, iron release from ferritin by reduction is mediated by NADPH oxidase through the intermediacy of superoxide. The conversion of the ferric to ferrous state makes iron accessible to cells. NADPH oxidase assembly, not activity, is inhibited by NO.105 Reduction in NADPH oxidase in the processes would limit iron availability to the cell. In addition, NO scavenges superoxide, which inhibits the reduction of ferritin bound iron. In addition to inhibition of ribonucleotide reductase, these two mechanisms may play an integral part in cytostatic mechanisms in a variety of disease states (Fig. 12).

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Fig. 12. Effects of nitric oxide (NO) on the metabolism of iron.

CYTOTOXIC VERSUS PROTECTIVE MECHANISMS OF NO

Up to this point, we have been discussing individual effects of NO on specific cellular processes. However, NO has the reputation of not only being injurious but also protective. Nitric oxide can induce cytotoxicity in different cells as well as increase the toxicity of different agents, yet, NO can also be protective against oxidative stress. Whether NO is protective or cytotoxic depends on specific conditions. Effects of NO and NO donors on ROS-mediated cytotoxicity Nitric oxide has been implicated as a cytotoxic agent in a number of different cell types. It was first shown that NO released from macrophages participates in the killing of tumor cells.4,5 However, some studies reveal that NO is not appreciably toxic to a variety of mammalian cells (see review 7). Even under anaerobic conditions, NO shows little or no toxicity in lung fibroblasts.55 In fact, NO has been shown to protect against hydrogen peroxide-mediated cytotoxicity in fibroblast, hepatocyte, endothelial, and neuronal cells.10,44,58 Cells exposed to much higher amounts of NO show only mild toxicity. For instance, to achieve significant toxicity in Chinese hamster V79 lung fibroblast cells or Chinese hamster ovarian cells, 2–5 mM NO delivered over specific time intervals is necessary to decrease the plating efficiency to 30 –50%.10,156 Compared with hydrogen peroxide at similar concentrations, which results in .99% cell killing, NO is not a potent toxic agent. Though NO itself is

not as toxic as other substances, lower amounts of NO can induce apoptotic death in specific cells and enhance the toxicity of other agents. When one considers the reactive nature of NO and RNOS with biological molecules, it is surprising that mammalian cells are so resistant to NO and related chemical species. An important factor in the resistance to NO-mediated toxicity are the detoxification mechanisms for RNOS. As with ROS, GSH is important in detoxifying NO-mediated cell damage. As discussed above, GSH has a high affinity for RNOS.76 If intracellular GSH is depleted, the toxicity of millimolar concentrations of NO is increased 100 times.76,157,158 In addition to GSH’s high reactivity with N2O3, peroxynitrite also rapidly reacts with GSH,100 thus limiting reactions of ONOO2 with intracellular molecules. These observations suggest that GSH plays an important role in detoxification of RNOS by limiting the indirect effects. Therefore, the susceptibility of cells to NO-mediated toxicity is in large part dependent on the amount of intracellular GSH. Though NO is not appreciably toxic to most mammalian cells, NO and RNOS can modulate the toxicity of some agents, such as alkylating agents as discussed above. One example is enhancement of cytotoxicity by heavy metals. It has been shown that N2O3 formation results in labilization of the coordinated metals from zinc finger motifs.128,129 RNOS can preferentially react with thiol rich proteins such as metallothionein. Metallothionein can protect cells against cytotoxicity mediated by RNOS analogous to GSH because RNOS reacts with thiol groups in metallothionein.159 Toxic metals such as cadmium, which have the potential to kill cells, are

Chemical biology of nitric oxide

sequestered in metallothionein. The ability of metallothionein to protect against cadmium cytotoxicity is compromised in the presence of RNOS.129,160 Though NO itself is not appreciably cytotoxic, it can dramatically influence the toxicity of other toxic agents. NO and ROS. Under a variety of biological conditions, both NO and ROS can be produced. The chemical and biological interaction of NO and ROS with various biological molecules has important consequences in the mechanisms of different immunological and pathological conditions. Hydrogen peroxide mediates oxidation of different biological molecules which may result in tissue damage. NO does not react chemically directly with hydrogen peroxide, yet several studies have shown that NO can protect cells against hydrogen peroxide-mediated toxicity. The presence of NO donors, NONOates, dramatically protected against hydrogen peroxide cytotoxicity.104 Extension of these studies have shown that compounds containing thiol-nitrosyl functional groups were also protective against peroxide-mediated toxicity.44 Yet, other nitrovasodilators, such as 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), potentiate hydrogen peroxide toxicity.44,45 The different biological effects resulting from different NO donors contributes to the confusion of NO’s ability to augment or attenuate ROS-mediated toxicity. The effect of NO on the known organic hydroperoxide-mediated oxidation of lipophilic membranes has been examined.57 The NO donor compound, DEA/NO, whose half-life is about 2 min, does not protect against either tert-butyl hydroperoxide or cumene hydroperoxide. However, the NONOate, PAPA/NO whose half-life is 15 min, markedly protects against both tert-butyl hydroperoxide and cumene hydroperoxide. The difference between the NONOates effect on cytotoxicity is the timing of NO delivery. For a given iso-survival effect, exposure to organic peroxides requires up to 2 hr, compared to a 10 –15-min exposure for hydrogen peroxide. Because alkyl hydroperoxides require longer times to penetrate cells and exert their damage, longer sustained fluxes of NO are more protective. Freeman and coworkers have investigated the effect of NO on XOmediated lipid peroxidation and found that NO acts as an antioxidant.60 Can these results be understood in terms of the direct and indirect effects of NO? Kanner et al. first proposed that NO could be an antioxidant.41 When peroxide enters a cell, it quickly reacts with heme proteins to form hypervalent complexes which can lead to lipid peroxidation. Furthermore, these hypervalent complexes can decompose to release intracellular iron that can catalyze peroxide damage to molecules such as DNA. NO can react at near diffusion controlled rate constants with

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these quintessential hypervalent metalloprotein species, restoring these oxidized species to the ferric form [Eq. (4)]. The reduction of these metallo-oxo proteins prevents both their oxidative chemistry and decomposition to release intracellular iron,10,41,42 thus limiting intracellular damage mediated by oxidative stress. In addition to protection against hydrogen peroxidemediated cellular oxidation, NO can induce protective proteins against oxidative stress. In bacterial systems, NO induces the SOX R genes, which results in expression of a number of proteins that protect against oxidative stress.161 Hepatocytes that are pretreated with NO become resistant to hydrogen peroxide insult.162 Therefore, in addition to NO being protective chemically, NO also provides the cellular signal to upregulate a variety of protective genes. Though NO can protect against hydrogen peroxidemediated cytotoxicity in mammalian cells, NO dramatically potentiates the cytotoxicity of hydrogen peroxide in Escherichia coli. When E. coli was exposed to hydrogen peroxide as a bolus or derived from XO, only modest bactericidal activity was observed.163 However, simultaneous exposure of peroxide and NO delivered either as gas or by a NONOate complex resulted in an increase cell kill of four orders of magnitude. Addition of catalase or SOD demonstrated that NO/H2O2 was the chemical species responsible for the bactericidal activity. The observation lead to the conclusion that NO and hydrogen peroxide can form the perfect cocktail for killing E. coli. NO enhances the bactericidal activity of hydrogen peroxide, yet protects the mammalian host from ROS-mediated cell death. Other microorganisms are affected by ROS/NO. Staphylococcal killing by superoxide was abrogated by NO at early time points, yet NO helps sustain killing at longer time intervals.164 Comparing E. coli and Staphylococcal killing by ROS and NO shows that maximal killing depends on different timing of exposure to NO, peroxide, and superoxide These studies give some insight into why the immune system may have different systems to time NO and ROS exposure to specific pathogens. The major differences between mammalian and prokaryotes in response to NO/H2O2 treatment are cellular anatomy and types of metalloproteins utilized for different metabolic processes. Bacteria utilize iron sulfur clusters to a greater extent than mammalian cells.163 These types of proteins are susceptible to degradation mediated by NO or RNOS.151,152 Decomposition of these iron complexes allows the released Fe to bind to DNA, increasing the hydrogen peroxide oxidation in E. coli. The difference in utility of the iron sulfur proteins may be a plausible explanation as to why mammalian cells and bacteria respond differently to NO/ROS.

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Effect of NO/O22 on cytotoxicity. Treatment of cells with peroxynitrite has been shown to result in cell death both in bacterial165 and mammalian cells (see review 95). However, lung fibroblast and neuronal cells treated with a superoxide source, XO, and NO donors showed no appreciable toxicity.104 Other studies have shown that ovarian carcinoma cells exposed to 5 mM SIN-1, a simultaneous superoxide/NO generator, resulted in no appreciable toxicity.45 In fact, cells treated simultaneously with XO and NO releasing compounds resulted in protection against XO mediated toxicity, and no appreciable toxicity due to ONOO2 formation was observed.44 These results suggest that there is a distinct difference between treating cells with bolus millimolar concentrations of peroxynitrite and generating similar amounts from an NO/O2 generating system. Part of the discrepancy between bolus peroxynitrite treatment and peroxynitrite derived from a NO/O22 generating systems can be explained in terms of concentrations. Beckman and co-workers showed that high concentrations of peroxynitrite are necessary to penetrate cells.165 The cell membrane forms a formidable barrier for peroxynitrite penetration to intracellular targets. Generation of NO and superoxide over specific time intervals results in peroxynitrite; however, the short life-time of peroxynitrite in solution does not allow high enough concentrations to accumulate in order to penetrate the cell. The amount of peroxynitrite that could cross the cellular membrane under more biologically relevant conditions, despite production of stoichiometrically high amounts over a prolonged period, is dramatically reduced. Therefore, the cell membrane limits extracellular peroxynitrite contribution to toxicological mechanisms. Another factor to consider with the respect to toxicity mediated directly by peroxynitrite chemistry is the reaction between NO and peroxynitrite to form NO2/N2O3 [Eqs. (7) and (10)]. As was discussed above, competition for superoxide by cellular components such as SOD and redox proteins increase the amount of NO required to form peroxynitrite. Because NO fluxes then become much larger than superoxide, the peroxynitrite formed is converted to potent nitrosating agents. Hence, the chemistry of extracellular formation of ONOO2 by excess NO converts peroxynitrite to nitrite [Eqs. (7), (8), (10), and (11)]. Thus, peroxynitrite-mediated necrotic cell death from exposure of simultaneous NO/superoxide derived from extracellular sources is unlikely. NO and apoptosis. NO can have diverse effects on cytotoxicity; however, this is further complicated when considering apoptosis. Whereas necrotic cell death (unprogrammed) is the result of extensive cellular damage by toxic chemical species, apoptosis-mediated cell death (programmed) is controlled by specific cellular signals.

A survey of the literature shows that NO can either protect the cell from apoptotic death or mediate apoptosis depending on the cell type. NO can protect cells against apoptosis under different conditions. For instance, NO protects hepatocytes in vivo from TNFa- induced apoptosis,166 rat thymocytes from INFg,167 rat ovarian follicles from atretic degeneration,168 and lymphocytes.169,170 On the other hand, NO participates in apoptosis of some cortical neurons,171 neurons in the substantia nigra,172 chondrocytes,173 some thymocytes,174 macrophages,175–178 and pancreatic RIN cells.179 NO induces apoptosis in tumor cells such as mastocytoma,180 sarcoma,181,182 L929 cells,181,183 and melanoma.184 A feature of apoptotic processes is laddering of DNA mediated by endonuclease. The cellular response that leads to apoptotic death varies. For instance, macrophages treated by NO donors increase p53 expression in response to DNA damage, which leads to apoptosis.179 NO-mediated apoptosis of macrophages is inhibited by antagonists of protein kinase A and C activating factors, suggesting these signal transduction proteins are involved the apoptotic pathway of NO.176 Though NO may induce apoptosis, NO can protect cells by several mechanisms. Nitric oxide via cGMP formation is presumed to abrogate apoptosis of cytokinedeprived human eosinophils in peripheral blood.185 Nitric oxide can rescue splenic B lymphocytes from antigen-induced apoptotic death by preventing the drop of bcl-2 levels, both at the mRNA and protein level, through the formation of GMP via guanylate cyclase.186 Yet, it was proposed that NO prevented human B lymphocytes apoptosis by cGMP-independent mechanism associating it with the redox status of thiols.169 Nitric oxide has been shown to prevent dexamethasone-induced apoptosis in thymocytes.174 Interestingly, a report showed that dexamethasone prevented apoptosis of murine mastocytoma cells by inhibiting iNOS expression.180 One key event in several apoptotic mechanisms is the induction of p53.187 Though low NO concentrations induce p53 expression, higher amounts reduce expression.188 This biphasic behavior may be explained in terms of fluxes of NO. At lower concentrations of NO, apoptosis may be mediated by induction of p53.176,179 At higher NO fluxes, p53 maybe inactivated because of the RNOS reaction with zinc finger proteins. This finding would suggest that cells containing cNOS, or a significant distance from an iNOS source, may undergo apoptosis; while cells close to an iNOS source may degrade the p53 protein product formed resulting in protection from apoptosis, which suggests a spatial relationship in cells with regard to the effect of NO on apoptosis. Further experiments showed that cells treated sepa-

Chemical biology of nitric oxide

rately with either GSNO or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), an intracellular superoxide generator, apoptosed. Yet, simultaneous exposure to GSNO and DMNQ resulted in protection.188 Protection probably resulted from the intracellular annihilation of NO and superoxide to form peroxynitrite, which either reacted with intracellular GSH or rearranged to give nitrate. This would dramatically reduce NO and O22 and prevent either radical from mediating apoptosis. This mechanism may detoxify NO and O22 by peroxynitrite formation. Another important interaction relating to cell death is the potential increase in intracellular superoxide/hydrogen peroxide. As described above, NO binds to cytochrome c oxidase, shunting electrons to oxygen to form superoxide/hydrogen peroxide.187 Richter et al. proposed that this increase in intracellular hydrogen peroxide induces DNA damage, thus increasing p53 expression and ultimately resulting in the turning on of the apoptotic pathway.179,189

CONCLUSION

Though NO may have numerous potential reactions, we have tried to provide a template to understand most of the reactions in terms of concentrations and timing. Differentiating the different chemical reactions in such a manner may help to evaluate their participation in vivo. Separation of direct effects and indirect effects can help in defining mechanisms as well to provide insights in devising potential strategies of treatment for different diseases.

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ABBREVIATIONS

AA—arachidonic acid cNOS— constitutive nitric oxide synthase COX— cyclooxygenase DHR— dihydrorhodamine DMNQ—2,3-dimethoxy-1,4-naphthoquinone N2O3— dinitrogen trioxide EDRF— endothelium derived relaxation factor ecNOS— endothelium nitric oxide synthase FAD—flavin adenine dinucleotide FMN—flavin mononucleotide GSH— glutathione GC— guanylate cyclase

H2O2— hydrogen peroxide iNOS— inducible nitric oxide synthase INFg—interferon gamma IRB—iron-responsive-binding protein IRE—iron-responsive elements LOONO—lipid peroxynitrite adducts LPS—lipid polysaccharide MnSOD—manganese superoxide dismutase metHb—methemoglobin SIN-1—3-morpholinosydnonimine nNOS—neuronal nitric oxide synthase NADH—nicotinamide adenine dinucleotide (reduced) NADPH—nicotinamide adenine dinucleotide phosphate (reduced) NO—nitric oxide NOS—nitric oxide synthase NO2—nitrogen dioxide HbO2— oxyhemoglobin ONOO2—peroxynitrite RNOS—reactive nitrogen oxide species ROS—reactive oxygen species SNAP—S-nitroso-N-acetylpenicillamine GSNO—S-nitrosoglutathione SNP—sodium nitroprusside SOD—superoxide dismutase XO—xanthine oxidase DEA/NO—(C2H5)2N[N(O)NO]2Na1) PAPA/NO—(NH31(CH2)3N[N(O)NO]2(CH2)2(CH3)