Life, 52: 189195, 2001 c Copyright 2001 IUBMB 1521-6543/01 $12.00 + .

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IUBMB

Critical Review
Nitric Oxide, Mitochondria, and Cell Death
Guy C. Brown and Vilmante Borutaite
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom

Summary NO or its derivatives (reactive nitrogen species: RNS) have three types of actions on mitochondria: 1) reversible inhibition of mitochondrial respiration at cytochrome oxidase by NO, and irreversible inhibition at multiple sites by RNS; 2) stimulation of mitochondrial production of superoxide, hydrogen peroxide, and peroxynitrite by NO; and 3) induction of mitochondrial permeability transition (MPT) by RNS. Similarly there are three main roles of mitochondria in NO-induced cell death: a) NO inhibition of respiration can induce necrosis (or excitotoxicity in neurons) and inhibit apoptosis if glycolysis is insuf cient to compensate, b) RNSor oxidant-induced signal transduction or DNA damage may activate the mitochondrial pathway to apoptosis, and c) RNS-induced MPT may induce apoptosis or necrosis. IUBMB Life, 52: 189195, 2001 Keywords Nitric oxide; mitochondria; cell death; apoptosis; necrosis; permeability transition; respiration; peroxynitrite.

and the actions of NO on mitochondria. A number of related reviews have been published elsewhere (17).

INTRODUCTION Nitric oxide-induced cell death is important in two contexts A) The nonspeci c (innate) immune response uses NO produced by in ammatory-activated host cells to kill a wide range of pathogens. B) NO may also kill healthy host cells and therefore contribute to pathology, particularly during the chronic inammation of in ammatory and neurodegenerative diseases, but also acutely during and after ischaemia. This review discusses the mechanisms by which NO induces cell death, in particular, the role of mitochondria in NO-induced apoptosis and necrosis. It also brie y outlines NO biochemistry
Received 31 July 2001; accepted 4 September 2001. Address correspondence to Guy Brown. Fax: (44) 1223 333345. E-mail: gcb@mole.bio.cam.ac.uk 1 Abbreviations: eNOS, iNOS, and nNOSendothelial, inducible, and neuronal nitric oxide synthases; FADD, Fas-associated death domain; MAP kinase, mitogen-activated protein kinase; MPT, mitochondrial permeability transition; NMDA, N-methyl-D-aspartate; RNS, reactive nitrogen species; ROS, reactive oxygen species.

NO Biochemistry NO is synthesized from L-arginine by three isoforms of NO synthase, two of which (eNOS and nNOS) are constitutively expressed and are acutely regulated by calcium/calmodulin and phosphorylation, while the third (iNOS) is induced during inammation, and produces higher levels of NO for a longer period (1). There may also be a mitochondrial isoform (mtNOS), but its origin and status is still unclear (8, 9). NO may also be produced nonenzymatically from nitrite at low pH (<pH 5), for example, during ischaemia. NO diffuses very rapidly both through water and membranes, so NO can easily diffuse from one cell to the next. NO itself at physiological concentrations (probably 1 100 nM) is relatively unreactive, and most of its physiological actions are mediated by NO binding to Fe2C in the haem of soluble guanylate cyclase causing activation and cGMP production (1). However, NO may be converted to a number of more reactive derivatives, known collectively as reactive nitrogen species (RNS). At high concentrations (or within membranes), NO reacts directly with oxygen to produce NO2 , which rapidly reacts with a further NO to give N2 O3 . NO2 may oxidise or nitrate (add an NO2C group) a variety of molecules, while N2 O3 can nitrosate/nitrosylate (add an NOC group) amines or thiols. NO reacts at the diffusion-limited rate with O2 to produce peroxynitrite (ONOO ), which can oxidise or nitrate other molecules, or can decay producing other damaging species (possibly the hydroxyl radical OH and NO2 ). NO may indirectly (possibly via NO2 /N2 O3 ) nitrosate thiols (e.g. in proteins or glutathione) to give S-nitrosothiols (RSNO), which may alter protein function. The NOC group is directly transferable between different S-nitrosothiols. S-nitrosothiols can also release NO in the presence of either light or reduced thiols. It is important to realise that NO and each of its derivatives have different properties (and have different properties at different concentrations), which act by different means, but particular
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species may (or may not) be interconvertible in particular conditions.

NO Actions on Mitochondria NO or its derivatives have three main, direct actions on mitochondria that are relevant to cell death: a) inhibition of mitochondrial respiration, b) stimulation of superoxide, hydrogen peroxide and peroxynitrite production, and c) induction of permeability transition. NO inhibits mitochondrial respiration by different means: 1) NO itself causes rapid, selective, potent, but reversible inhibition of cytochrome oxidase, and 2) RNS cause slow, nonselective, weak, but irreversible inhibition of many mitochondrial components (2, 3, 10) (Fig. 1). The reversible NO inhibition of cytochrome oxidase occurs at nanomolar levels of NO (1113), so that NO is potentially a physiological regulator of respiration (3, 1417). NO inhibits by reacting with either the reduced or oxidised binuclear (oxygen-binding ) site of cytochrome oxidase to give either cytochrome a3 2C -NO or cytochrome a3 3C NO2 (1820). Endogenously produced NO may tonically inhibit respiration at cytochrome oxidase in some cells (21, 22). Inhibition is in competition with oxygen, so that NO can dramatically increase the apparent Km of respiration for oxygen (2, 1214). Thus, NO can make cells effectively hypoxic at relatively high oxygen levels, and potentially sensitise tissues to hypoxic damage. A variety of cells (including macrophages, microglia, astrocytes, and endothelial cells), when in ammatory activated to express iNOS, have been shown to produce suf cient NO to not only inhibit their own respiration but also that of surrounding cells via reversible NO inhibition of cytochrome oxidase (14, 21, 23). Cells exposed to NO (or NO-producing cells) show immediate but reversible inhibition of respiration at cytochrome oxidase. However, after several hours of exposure to NO an irreversible inhibition develops, probably due to conversion of NO to RNS that inhibit respiration at multiple sites (15). One of the more rapid effects is an inactivation of complex I, possibly due to S-nitrosylation of the complex (15, 24), followed by inhibition of aconitase and complex II, possibly due to removal of iron from ironsulphur centers (2527). Peroxynitrite can inhibit complex I, complex II, cytochrome oxidase, the ATP synthase, aconitase, Mn-SOD, creatine kinase, and probably many other proteins (2, 10, 28). Peroxynitrite is a strong oxidant and can also cause DNA damage, induce lipid peroxidation, and increase mitochondrial proton (and other ion) permeability (probably by lipid peroxidation or thiol cross-linking) (29). The mitochondrial respiratory chain can produce superoxide, which dismutates to hydrogen peroxide, and inhibition of the respiratory chain may enhance the production of these ROS. At moderate levels NO can acutely increase O2 and H2 O2 production by inhibiting mitochondrial respiration, whereas at higher levels it inhibits H2 O2 production by scavenging the precursor superoxide, resulting in peroxynitrite production (16, 30). NO may also apparently react with ubiquinol (QH2 ) to

Figure 1. Main actions of nitric oxide (NO) and reactive nitrogen species (RNS) on mitochondria. NO speci cally and reversibly inhibits cytochrome oxidase (complex IV); RNS inactivate multiple respiratory complexes (I, II, IV), the ATP synthetase (ATPase), aconitase, and Mn-superoxide dismutase (MnSOD). RNS activate the permeability transition pore (PTP), which may be formed from the ATP/ADP-translocator (ANT), cyclophilin D (CP), creatine phosphokinase (CrK), and the voltage-dependent anion channel (VDAC). Activation of PTP may lead to cytochrome c (Cyt.c) release either due to swellinginduced rupture of the outer membrane or through speci c pores. Cytochrome c release also may be induced by insertion of proapoptotic BH3 proteins into the outer membrane. produce NO (which may react with O2 to produce ONOO ) and ubisemiquinone (QH ) (part of which may react with O2 to produce O2 ) (31). Thus, NO may cause O2 , H2 O2 , and ONOO production, but in other conditions it may produce NO , NO2 , or N2 O3 . Reversible NO inhibition of respiration may then result in local peroxynitrite production causing irreversible inhibition of respiration and further oxidant productiona vicious cycle that might contribute to cell death. Mitochondria increase NO breakdown by reactions with: a) superoxide, b) ubiquinol, c) cytochrome oxidase, and d) oxygen in the membranes (3033), and this might contribute to the regulation of NO levels in cells. In addition to stimulating H2 O2 production, NO or RNS can also inhibit catalase, deplete cellular glutathione and inhibit glutathione peroxidase, thus increasing H2 O2 levels in cells (1, 16, 34). Indeed, NO and H2 O2 have been found to be synergistic in killing cells, possibly in part by superoxide dismutase-catalysed peroxynitrite production (35). NO may also release iron from iron-sulphur centers and ferritin potentially causing further oxidative stress (1). Addition of S-nitrosothiols, RNS, or ROS to isolated mitochondria preloaded with calcium causes mitochondrial permeability transition (3638). MPT is a dramatic increase in permeability to all small molecules [for reviews see (3942)]. MPT

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dissipates the protonmotive force, causing uncoupling of oxidative phosphorylation, and reversal of the ATP synthase, potentially hydrolysing cellular ATP, resulting in necrosis. MPT also causes rapid swelling of the mitochondria, such that the outer membrane can be ruptured, releasing intermembrane proteins, such as cytochrome c. Release of cytochrome c and matrix components such as NADH inhibits respiration, thus potentially causing necrosis. But release of cytochrome c and other apoptogenic intermembrane proteins such as AIF and Smac/DIABLO potentially triggers apoptosis (43, 44). Transient MPT opening may be a physiological process, and usually does not cause cell damage, whereas longer, sustained MPT opening may cause either apoptosis or necrosis (45, 46). The mode of cell death after MPT opening is likely to depend on additional factors such as activation of Bid/Bax/Bad pathway or availability of ATP (ATP depletion probably favouring necrosis) (44). NO itself generally inhibits MPT due to the direct inhibition of respiration preventing calcium accumulation, but very high concentrations can promote MPT probably due to formation of NO2 /N2 O3 (47). RNS may directly oxidise thiols of the adenine nucleotide translocator, a key component of the MPT pore (48).

NO-Induced Cell Death NO and RNS kill cells by a variety of different mechanisms (that are still not clearly de ned), which differ depending on the cell type and conditions. Two main types of NO-induced cell death can be distinguished: 1) energy depletion-induced necrosis, and 2) oxidant-induced apoptosis (Fig. 2). Energy depletion-induced necrosis results from NO and/or its derivatives causing ATP depletion via four different mechanisms: a) inhibition of mitochondrial respiration, b) induction of mitochondrial permeability transition, c) inhibition of glycolysis at glyceraldehyde-3-phosphat e dehydrogenase , and d)

Figure 3. Possible apoptosis.

pathways

leading

to

NO-induced

Figure 2. Possible pathways leading to NO-induced necrosis.

activation poly-ADP ribose polymerase (PARP, or synthetase: PARS). Inhibition of glyceraldehyde-3-phosphat e dehydrogenase can be induced by peroxynitrite or S-nitrosothiols (but not directly by NO), and in the latter case appears to be due to S-nitrosylation of an active-site cysteine residue, which is subsequently ADP ribosylated (49). However, reduced thiols such as glutathione can prevent such inhibition, and the dehydroge nase is not rate limiting for glycolysis, so that effective inhibition of glycolysis may only occur when glutathione is severely depleted in the cell. On the other hand, NO/RNS can cause slow depletion of cellular glutathione (15). In cells exposed to NO, glycolysis is critical to cell survival because moderate levels of NO invariably inhibit mitochondrial respiration and thus mitochondrial ATP production. In the absence of glucose or suf cient glycolytic capacity, moderate levels of NO cause necrosis simply due to respiratory inhibition and consequent ATP depletion [and apoptosis is prevented by the lack of ATP (50)]. Thus, in many cells necrosis induced by NO is prevented by glucose (51, 52). If, however, MPT is chronically stimulated by NO derivatives then respiration may be inhibited, oxidative phosphorylation uncoupled, and glycolytic ATP hydrolysed by reversal of the ATPase, thus necrosis might proceed even in the presence of rapid glycolysis. However, there is as yet little direct evidence that MPT is involved in NO-induced necrosis. The rate at which ATP is being consumed by the cell may also be important in determining the sensitivity to energydepletion induced necrosis, as suggested by the synergy between NO and impulse frequency in neuronal death (53). PARP is a nuclear protein, which when activated by DNA strand breaks (which may be caused by peroxynitrite or

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NO2 /N2 O3 ) catalyses multiple ADP-ribosylation (using NADC as substrate) of proteins including particularly itself (54). If DNA damage is extensive, then PARP activity is so high that cytosolic NAD C is depleted (potentially inhibiting glycolysis) and adenine nucleotides may also be depleted (either because they are substrates for NADC synthesis or because glycolysis is inhibited). Thus, PARP inhibitors or PARP knockouts can protect against NO-induced cell death in some cell types and conditions; however, these inhibitors are ineffective in other cells and conditions (5456). Neurons are exceptionally sensitive to NO or NO-producing cells (57). One reason is that NO inhibition of neuronal respiration causes glutamate release and subsequent excitotoxic death of neurons (58). Activation of PARP may also be involved in the neurotoxicity of NO (55). NO and NO-producing glia cause irreversible damage to respiratory complexes of neuronal mitochondria (5), but this may be secondary to glutamate release and excitotoxicity. Glutamate is the main neurotransmitter in the brain, but high extracellular levels cause neuronal death (known as excitotoxicity) mainly through activation of one type of glutamate receptor (the NMDA receptor). In some cases, inhibition of nNOS blocks glutamate-induced death of neurons, and cell death has been attributed to NO and calcium-induced mitochondrial depolarisation, although mechanisms remain unclear (57, 59, 60). If glycolysis is suf cient to compensate for respiratory inhibition, then NO can induce apoptosis. Although speci c respiratory inhibitors can induce apoptosis in such conditions, induction is weaker and slower, so respiratory inhibition cannot explain NO-induced apoptosis (38). NO-induced apoptosis is accompanied by activation of downstream caspases (such as caspase-3), and is blocked by caspase inhibitors (38, 51, 61). Cytochrome c is generally released, suggesting that NO-induced apoptosis is normally mediated by mitochondria, but in some cell types early activation of caspase-8 or caspase-2 is observed indicating that NO-induced apoptosis may be triggered not through mitochondrial pathway (6163). How NO induces apoptosis is still poorly characterised, but three main routes have been suggested: 1) MPT, 2) upregulation of p53, and 3) MAP kinase pathways (4, 6). NO by itself usually does not induce MPT; however, peroxynitrite, S-nitrosothiols, NO2 , and/or N2 O3 may cause apoptosis by directly activating MPT, leading to cytochrome c release and caspase activation, thus these events can be blocked with the MPT inhibitor cyclosporin A (4, 38). Apoptosis induced by pure NO donors such as the NONOates is less susceptible to inhibition by cyclosporin A, but is inhibited by antioxidants, such N-acetylL-cysteine, catalase, Mn(III)tetrakis(4-benzoic acid)porphyrin (Mn-TBAP), superoxide dismutase, ascorbate, and Trolox and thus may require NO-induced ROS or RNS (38, 61, 64). NO can cause p53 phosphorylation and accumulation, either via DNA damage-activated kinases, proteasome inhibition, or MAP kinases, and this can lead to apoptosis as demonstrated by the nding that p53-antisense oligonucleotides can block

NO-induced death in some cells (65, 61). NO can cause DNA damage, possibly via peroxynitrite, NO2 , N2 O3 , free iron, or ROS (67 ). NO may stimulate ROS production in cells, possibly by inhibition of catalase, depletion of glutathione, and inhibition of the respiratory chain. Preventing NO-induced ROS can block NO-induced p53 activation and apoptosis (68). How p53 induces apoptosis is not clear, but the mechanisms may involve transcription-dependent and -independent processes: a) p53-mediated activation of expression of several pro-apoptotic proteins (such as Bax, Noxa, etc.) that translocate to mitochondria; b) direct targeting of phosphorylated p53 to mitochondria possibly causing cytochrome c release; or c) causing caspase-8 activation by FADD-independent mechanism (6, 69, 70). Several different MAP kinase pathways can be activated by NO (or RNS or ROS), and there is evidence that inhibiting these pathways can block (or in some cases enhance) NO-induced apoptosis. MAP kinase pathways may increase the ratio of proto anti-apoptotic BH3 -domain proteins (e.g., Bax/Bcl-2) or induce Bid cleavage and translocation to mitochondria leading to the release of cytochrome c (6, 71). NO-induced activation of p38 MAP kinase has been shown to promote Bax translocation to mitochondria and cause cell death, which is not blocked by caspase inhibitors (72). NO treatment of cells can cause oxidant-induced degradation of the mitochondrial phospholipid cardiolipin (probably due to peroxynitrite or NO2 /N2 O3 -induced peroxidation), associated with irreversible inhibition of the respiratory chain and apoptosis (73). Cytochrome c is normally bound to cardiolipin on the inner mitochondrial membrane, but peroxidation causes the release of cytochrome c (74). Addition of peroxynitrite to isolated mitochondria causes lipid peroxidation and thiol cross-linking associated with increased proton and ion permeability, depolarisation and rapid swelling, which in the absence of calcium are not mediated by MPT (29). It has also been reported that addition of calcium to isolated mitochondria stimulates mtNOS, causing peroxynitrite production and (cyclosporin-insensitive) cytochrome c release associated with peroxidation of mitochondrial lipids (9). Mitochondrial lipid peroxidation may be an important event in mitochondria-mediated apoptosis (75), but there is no direct evidence that lipid peroxidation mediates NO-induced apoptosis. NO can also inhibit apoptosis induced by other agents. Several mechanisms have been suggested: a) cGMP-mediated blockage upstream of cytochrome c release, b) cGMP-mediated inhibition of ceramide synthesis, c) S-nitrosylation of caspases, d) energy depletion, e) mitochondrial hyperpolarisation, f) activation of MAP kinase pathways, g) activation of transcription factors NF-kB and/or AP-1, and h) increased expression of heat shock proteins and Bcl-2 (6).

Mitochondria in NO-Induced Cell Death To summarise, there are three main roles of mitochondria in NO-induced cell death: a) NO inhibition of respiration can (if glycolysis is insuf cient to compensate) induce necrosis (or

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excitotoxicity in neurons) and inhibit apoptosis, b) RNS-induced MPT may induce apoptosis or necrosis and c) RNS/ROSinduced signal transduction or DNA damage may activate the mitochondrial pathway to apoptosis. Other suggested roles include: mitochondrial lipid peroxidation-induced cytochrome c release (9, 73, 75), NO inhibition of respiration blocking MPT (47), NO hyperpolarising the mitochondrial membrane potential (76 ), mitochondria producing superoxide or hydrogen peroxide (especially after respiratory inhibition) synergising with NO to induce death (10), and NO sensitising cells to hypoxic inhibition of respiration (14). ACKNOWLEDGMENTS Our own work in this eld has been supported by the Royal Society, Wellcome Trust, Medical Research Council, and British Heart Foundation. REFERENCES
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