Life, 52: 189–195, 2001
c 2001 IUBMB
Copyright °
1521-6543/01 $12.00 + .00
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 insufŽ 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: 189–195, 2001
Keywords
Nitric oxide; mitochondria; cell death; apoptosis; necrosis; permeability transition; respiration; peroxynitrite.
INTRODUCTION
Nitric oxide-induced cell death is important in two contexts
A) The nonspeciŽ 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 in ammation 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: [email protected]
1
Abbreviations: eNOS, iNOS, and nNOS—endothelial, 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.
and the actions of NO on mitochondria. A number of related
reviews have been published elsewhere (1–7).
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 in ammation, 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 OH² 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
189
190
BROWN AND BORUTAITE
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 (11–13),
so that NO is potentially a physiological regulator of respiration (3, 14–17). 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 ¡ (18–20). 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, 12–14). 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 sufŽ 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
iron–sulphur centers (25–27). 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 speciŽ 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 speciŽ 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 (QH² ) (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 production—a
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 (30–33), 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 (36–38). MPT is a dramatic increase in permeability to all small molecules [for reviews see (39–42)]. MPT
191
NO, MITOCHONDRIA, AND CELL DEATH
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 deŽ 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 2. Possible pathways leading to NO-induced necrosis.
Figure 3. Possible
apoptosis.
pathways
leading
to
NO-induced
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 sufŽ 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
192
BROWN AND BORUTAITE
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
(54–56).
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 sufŽ cient to compensate for respiratory inhibition, then NO can induce apoptosis. Although speciŽ 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 (61–63).
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 Ž 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 insufŽ cient to compensate) induce necrosis (or
NO, MITOCHONDRIA, AND CELL DEATH
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).
16.
17.
18.
19.
ACKNOWLEDGMENTS
Our own work in this Ž eld has been supported by the Royal
Society, Wellcome Trust, Medical Research Council, and British
Heart Foundation.
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