Free Radical Biology & Medicine, Vol. 33, No. 11, pp. 1440 –1450, 2002
Copyright © 2002 Elsevier Science Inc.
Printed in the USA. All rights reserved
0891-5849/02/$–see front matter
PII S0891-5849(02)01112-7
Serial Review: Nitric Oxide in Mitochondria
Guest Editors: Christoph Richter and Matthias Schweizer
NITRIC OXIDE INHIBITION OF MITOCHONDRIAL RESPIRATION AND ITS
ROLE IN CELL DEATH
GUY C. BROWN
and
VILMANTE BORUTAITE
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
(Received 18 June 2002; Revised 26 August 2002; Accepted 3 September 2002)
Abstract—NO or its derivatives (reactive nitrogen species: RNS) inhibit mitochondrial respiration in two different
ways: (i) an acute, potent, and reversible inhibition of cytochrome oxidase by NO in competition with oxygen; and, (ii)
irreversible inhibition of multiple sites by RNS. NO inhibition of respiration may impinge on cell death in several ways.
Inhibition of respiration can cause necrosis and inhibit apoptosis due to ATP depletion, if glycolysis is also inhibited
or is insufficient to compensate. Inhibition of neuronal respiration can result in excitotoxic death of neurons due to
induced release of glutamate and activation of NMDA-type glutamate receptors. Inhibition of respiration may cause
apoptosis in some cells, while inhibiting apoptosis in other cells, by mechanisms that are not clear. However, NO can
induce (and inhibit) cell death by a variety of mechanisms unrelated to respiratory inhibition. © 2002 Elsevier Science Inc.
Keywords—Free radicals, Nitric oxide, Mitochondria, Cell death, Apoptosis, Necrosis, Permeability transition,
Respiration
INTRODUCTION
5), e.g., during ischemia [14]. 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 (unclear, but
probably 0.1–100 nM) is relatively unreactive, and most
of its physiological actions are mediated by NO binding
to Fe2⫹ in the heme of soluble guanylate cyclase causing
activation and cGMP production [6]. 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 another NO to give N2O3. NO2 may oxidize
or nitrate (add an NO2⫹ group to) a variety of molecules
(including tyrosine), while N2O3 can nitrosate/nitrosylate
(add an NO⫹ group to) amines or thiols. NO reacts at the
diffusion-limited rate with O2⫺ to produce peroxynitrite
(ONOO⫺), which can oxidize/nitrate other molecules or
decay and produce other damaging species (possibly the
hydroxyl radical OH• and NO2). NO may indirectly
(possibly via N2O3) nitrosate thiols (e.g., in proteins or
glutathione) to give S-nitrosothiols (RSNO, e.g., S-nitroso-glutathione and S-nitroso-albumin). The NO⫹
group is directly transferable between different S-nitro-
Nitric oxide (NO) and its derivatives (reactive nitrogen
species, RNS) inhibit mitochondrial respiration and either stimulate or inhibit cell death depending on conditions. This review outlines these effects and explores the
two responses. Related reviews on NO inhibition of
mitochondrial respiration [1–5], NO effects on cell death
[6 – 8], or both [9 –11] have been published elsewhere.
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 inflammation and produces
higher levels of NO for a longer period [6]. There may
also be a mitochondrial isoform (mtNOS), but its origin
and status is still unclear [12,13]. NO may also be
produced nonenzymatically from nitrite at low pH (⬍ pH
This article is part of a series of reviews on “Nitric Oxide in
Mitochondria.” The full list of papers may be found on the homepage
of the journal.
Address correspondence to: Dr. Guy C. Brown, University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge
CB2 1QW, UK; Tel: ⫹44 1223 333342; Fax: ⫹44 1223 333345;
E-Mail: [email protected].
1440
NO, mitochondrial respiration, and cell death
Fig. 1. Main actions of nitric oxide (NO) and reactive nitrogen species
on mitochondria. NO specifically and reversibly inhibits cytochrome
oxidase (complex IV); peroxynitrite (ONOO⫺) inactivates multiple
respiratory complexes (I, II, III, IV), the ATP synthetase (ATPase),
creatine kinase, and aconitase. ONOO⫺ stimulates proton leak through
the mitochondrial inner membrane. Nitrosothiols (RSNO) inhibit complex I.
sothiols, a process known as transnitrosation or transnitrosylation. S-Nitrosated or tyrosine-nitrated proteins
may have altered function. S-Nitrosothiols break down
and release NO in the presence of either light, reduced
thiols, or metal ions, such as Cu⫹.
NO and each of its derivatives have different properties (and have different properties at different concentrations), which act by different means, but particular species may (or may not) be interconvertible in particular
conditions. The field of NO biology is bedevilled by
research where biological samples are exposed to NO
donors (substances releasing NO or RNS) or NO-releasing cells, where the levels of NO or its derivatives are
unclear and their relation to physiological levels ignored.
NO INHIBITION OF MITOCHONDRIAL RESPIRATION
NO inhibits mitochondrial respiration by different
means: (i) NO itself causes rapid, selective, potent but
reversible inhibition of cytochrome oxidase; and, (ii)
RNS cause slow, nonselective, weak but irreversible
inhibition of many mitochondrial components [1,2,15]
(Fig. 1).
Reversible NO inhibition of cytochrome oxidase
The reversible NO inhibition of cytochrome oxidase
occurs at nanomolar levels of NO [16 –18], so that NO is
1441
potentially a physiological regulator of respiration
[2,19 –22]. NO inhibits cytochrome oxidase apparently
by two different means involving NO binding to two
different components of the oxygen-binding site; both
cases block oxygen binding. The oxygen-binding site
consists of two metals, the iron of heme a3 and the
copper of the CuB center, and oxygen binds between
them (and is rapidly reduced by them) when both metals
are reduced (a32⫹ and Cu⫹). NO can either (i) bind to
reduced cytochrome a3 to give cytochrome a32⫹-NO, or
(ii) bind and reduce oxidized CuB2⫹ to give CuB⫹-NO⫹
and the NO⫹ can rapidly hydrate to give nitrite (NO2⫺)
[23–29]. Both forms of inhibition are rapid and reversible due to (i) debinding of NO, and (ii) debinding of
nitrite. The first form of inhibition is competitive with
oxygen and reversible by light, whereas the second is
not, and these characteristics may be used to distinguish
between them [27]. It seems that, at least in vitro, both
forms of inhibition may occur simultaneously, but the
first form is favored at high levels of cytochrome reduction and low oxygen, whereas the second form is favored
by the opposite conditions [27]. However, the inhibition
observed in cells in response to NO or iNOS expression
appears to be largely competitive with oxygen
[18,26,30,31] and reversible by light [32], suggesting
that inhibition due to reversible binding to heme a32⫹
may be more important in cells.
Mitochondrially associated NO synthases (i.e., eNOS,
nNOS, or iNOS bound to mitochondria) or mtNOS (a
possibly distinct NOS) may produce NO locally at mitochondria and, thus, has the potential to regulate respiration [12,13,33; Guilivi, this series]. For example, NO
production from individual mitochondria was recently
shown, though absent in nNOS-knockout mice; thus,
possibly this mtNOS is another splice variant of nNOS
[33]. In cardiomyocytes from dystrophin-deficient mice,
where this mtNOS was the only apparent form of NOS
present, there was beat-to-beat formation of NO, which
apparently inhibited cell shortening via the NO inhibition
of cytochrome oxidase [33].
Endogenously produced NO may tonically inhibit respiration at cytochrome oxidase in some cells [25,26].
Inhibition is generally in competition with oxygen, so
that NO can dramatically increase the apparent Km of
respiration for oxygen [1,17–19]. In synaptosomes (isolated nerve terminals), half-inhibition of respiration occurred at 250 nM NO when the oxygen level was about
150 ␮M (roughly the arterial level), but the Ki was about
50 nM NO at 30 ␮M O2 (a median tissue level) [18].
According to this data, then, the presence of 50 nM NO
would raise the apparent Km of respiration for oxygen
from below 1 to 30 ␮M O2—well into the physiological
range. Thus, NO can make cells effectively hypoxic at
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G. C. BROWN and V. BORUTAITE
relatively high oxygen levels and potentially sensitize
tissues to hypoxic damage. A variety of cells (including
macrophages, microglia, astrocytes, endothelial cells,
and aorta), when inflammatorily activated to express
iNOS, have been shown to produce sufficient NO to not
only inhibit their own respiration but also that of surrounding cells via reversible NO inhibition of cytochrome oxidase [30,31,34]. Recently, NO inhibition of
cytochrome oxidase and its reversal by light was implicated in regulating the frequency at which fireflies emit
bursts of light [35].
Whether NO can regulate mitochondrial respiration in
vivo, either physiologically or pathologically, remains an
important unresolved problem [2,3]. Hemoglobin and
myoglobin both have very high capacities and rates of
NO scavenging, which may protect tissues from significant NO inhibition of respiration in vivo [36,37]. The
sensitivity of soluble guanylate cyclase to NO appears to
be at least two orders of magnitude higher than that of
cytochrome oxidase [38], which might indicate that the
latter is not a significant physiological target for NO.
However, inhibition of NOS in vivo has been shown to
cause substantial increases in organ and whole-body
oxygen consumption, apparently not due to changes in
blood flow, consistent with a tonic inhibition of mitochondrial respiration in vivo [5,39]. On the other hand, it
is still not known whether such effects of NO on oxygen
consumption are mediated by cGMP, cytochrome oxidase, or other processes [2].
Irreversible inhibition of respiration by NO
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 [15,20,40,41]. One of the
more rapid effects is an inactivation of complex I, possibly due to S-nitrosation of the complex [20,32], followed by inhibition of aconitase and complex II, possibly
due to removal of iron from iron-sulphur centers [42– 44]
under conditions where peroxynitrite may be formed
[44]. Peroxynitrite can inhibit complex I, complex II/III,
cytochrome oxidase (complex IV), the ATP synthase,
aconitase, Mn-SOD, creatine kinase, and probably many
other proteins [1,15,45]. 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) [46].
The mechanism(s) by which NO and RNS inhibit
complex I remain unclear. The S-nitrosothiols, S-nitrosoglutathione and S-nitrosoacetylpenacillamine, can rap-
idly inactivate complex I when added to isolated mitochondria, even in conditions when little or no NO is
released, and such inhibition is reversed by light or
reduced thiols (glutathione or DTT), suggesting that the
inactivation is mediated by transnitrosation [32]. However, the inhibition of complex I induced by peroxynitrite
(which is a poor nitrosating agent) is also partly reversed
by light and reduced thiols [32] (as is the inhibition
induced by NO treatment of cells [20]), suggesting that
mechanisms other than nitrosation might be involved. It
has been suggested recently that peroxynitrite inhibits
complex I by tyrosine nitration [47] and NO-induced
inhibition of complex I in isolated mitochondria was
prevented by peroxynitrite scavengers [40]. An alternative (but not exclusive) target might be one or more
iron-sulphur centers in complex I. High concentrations of
NO can destroy iron-sulphur centers by binding and
displacing the iron, and there is EPR evidence for such
damage in complex I [48,49]. Damage must start with
NO binding/reacting with the iron and/or cysteine residues that bind the iron, and this initial phase of inhibition
might be reversible by light or reduced thiols. Whatever
the mechanism, NO inhibition of complex I may be
important in cell dysfunction or death. However, NOinduced thiol depletion seems to precede inhibition of
complex I, and reduced thiols reverse the inhibition [20].
There is some controversy concerning inhibition of
complex II and complex III by peroxynitrite: some authors show that peroxynitrite (and NO) inhibit complex
II with little or no effect on complex III [15,49], whereas
others find complex III inhibited and complex II unaffected by peroxynitrite [50].
Peroxynitrite has relatively little effect on the Vmax of
cytochrome oxidase when added to mitochondria at levels that inhibit the other complexes [15]. However, it
does have various damaging effects on isolated cytochrome oxidase, including particularly increasing the Km
for oxygen [51]. High concentrations (⬎1 ␮M) of NO
(possibly via NO2 or N2O3) can also induce an irreversible rise in Km for oxygen both in isolated cytochrome
oxidase or in cells treated with NO [51,52].
Peroxynitrite and possibly NO itself inhibit cytosolic
and mitochondrial aconitase, the latter being a component of the Kreb’s cycle and, thus, required for cellular
respiration. Aconitase has a 4Fe4S iron-sulphur center,
including one particular iron atom that can be removed
by peroxynitrite and other oxidants [44], and in some
conditions NO itself may S-nitrosate the iron-sulphur
center [53]. Mitochondrial creatine kinase aids the export
of ATP from mitochondria in muscle and nerves and is
inhibited by S-nitrosothiols, probably by transnitrosation
of a critical thiol [54 –56].
NO, mitochondrial respiration, and cell death
NO-induced ROS, RNS, and MPT
Apart from inhibiting respiration, NO has two other
effects on mitochondria relevant to the induction of cell
death: (i) induction of ROS and RNS production from
mitochondria, and (ii) induction of mitochondrial permeability transition (MPT) by RNS.
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 H2O2 production by inhibiting
mitochondrial respiration, while at higher levels it inhibits H2O2 production by scavenging the precursor superoxide, resulting in peroxynitrite production [21,57]. NO
may also apparently react with ubiquinol (QH2) to produce NO⫺ (which may react with O2 to produce
ONOO⫺) and ubisemiquinone (QH•) (part of which may
react with O2 to produce O2⫺) [58]. Thus, NO may cause
O2⫺, H2O2, and ONOO⫺ production, but in other conditions it may produce NO⫺, NO2, or N2O3. Both NO
and O2 are more soluble in lipid bilayers than aqueous
solution and, thus, reach higher concentrations within
cell membranes that cell cytosol; as the reaction rate
between NO and O2 is proportional to the square of the
NO concentration and proportional to the O2 concentration, this reaction is much more rapid within cell membranes, including mitochondrial membranes, than in the
aqueous phases [59]. Therefore, part of the ability of
mitochondria to break down added NO is due to this
simple reaction within the bilayer [59], which produces
both NO2 and N2O3. Mitochondria can also increase NO
breakdown by reactions with superoxide, ubiquinol, and
possibly cytochrome oxidase [29,57– 60], and this might
contribute to the regulation of NO levels in cells.
Reversible NO inhibition of respiration may result in
local peroxynitrite production (due to local superoxide
production), causing irreversible inhibition of respiration
and further oxidant production—a vicious cycle that
might contribute to cell death [40].
In addition to stimulating H2O2 production, NO or
RNS can also inhibit catalase, deplete cellular glutathione, and inhibit glutathione peroxidase, thus increasing
H2O2 levels in cells [6,21,61]. Indeed, NO and H2O2
have been found to be synergistic in killing cells, possibly in part by superoxide dismutase-catalyzed peroxynitrite production [62]. NO may also release iron from
iron-sulphur centers and ferritin, potentially causing further oxidative stress [6].
RNS, S-nitrosothiols, or ROS cause MPT in isolated,
calcium-preloaded mitochondria [63– 65]. MPT is a dramatic increase in permeability of the inner mitochondrial
membrane to small (up to 1.5 kDa) molecules (for reviews see [66 – 69]). Mitochondrial membrane potential
1443
and matrix calcium are known to determine the ability of
other compounds to induce MPT; thus, inhibition of
respiration by NO and subsequent decrease in membrane
potential should favor MPT opening. On the other hand,
NO itself can inhibit MPT due to the direct inhibition of
respiration, preventing calcium accumulation in mitochondria [70]. cGMP (which is formed by guanylyl
cyclase after stimulation by NO) may also inhibit MPT
in cells via protein kinase G [71]. However, oxidants
(such as tert-butyl hydroperoxide and phenylarsine oxide) at high concentrations can induce MPT even in the
absence of calcium, and this effect is probably related to
direct reaction of these compounds with functional thiols
[72,73]. Therefore, NO at high concentrations can promote MPT probably due to either (i) the production of
peroxynitrite, nitrosothiols, or NO2/N2O3; or, (ii) depletion/oxidation of glutathione levels. NO/RNS may directly oxidize the protein thiols that regulate opening of
the MPT pore [74,75].
MPT plays an important role in both necrotic and
apoptotic cell death. MPT dissipates the protonmotive
force, causing uncoupling of oxidative phosphorylation,
and reversal of the ATP synthase, potentially hydrolyzing cellular ATP, resulting in necrosis. MPT also causes
rapid swelling of the mitochondria, such that the outer
membrane can be ruptured releasing intermembrane proteins like cytochrome c. However, MPT-related cytochrome c release in cells can occur by other mechanisms
that do not involve mitochondrial swelling and membrane rupture. Release of cytochrome c and matrix components, such as NADH, inhibits respiration, potentially
causing necrosis. But release of cytochrome c and other
apoptogenic intermembrane proteins, such as AIF and
SMAC/Diablo, potentially triggers apoptosis [76,77].
Transient MPT opening may be a physiological process
and usually does not cause cell damage, while longer,
sustained MPT opening may cause either apoptosis or
necrosis [78,79]. 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 favoring necrosis) [67].
Calcium has been suggested to cause cytochrome c release from mitochondria and subsequent apoptosis by
stimulating mtNOS to produce peroxynitrite, which then
induces MPT or related processes [13].
NO-INDUCED CELL DEATH
NO-induced cell death is important in two contexts:
(i) host cells kill pathogens using NO; and, (ii) in a
variety of inflammatory, ischemic, and neurodegenerative diseases, NO kills host cells.
NO and RNS kill cells by a variety of different mechanisms (that are still not clearly defined), which differ
1444
G. C. BROWN and V. BORUTAITE
depending on the cell type and conditions. Two main
types of NO-induced cell death can be distinguished: (i)
energy depletion-induced necrosis, and (ii) oxidant-induced apoptosis. In addition, in neurons, NO induces
excitotoxic cell death.
NO-induced necrosis
Energy depletion-induced necrosis results from NO
and/or its derivatives causing ATP depletion via four
different mechanisms: (i) inhibition of mitochondrial respiration, (ii) induction of MPT, (iii) inhibition of glycolysis at glyceraldehyde-3-phosphate dehydrogenase, and
(iv) activation of poly-ADP ribose polymerase (PARP;
or synthetase, PARS).
It is important to note that inhibition of mitochondrial
respiration in cultured cells or in vivo, induced for example by anoxia, cyanide, or carbon monoxide, is alone
sufficient to cause rapid death of a wide range of cells,
including particularly neurons and cardiomyocytes; thus,
NO inhibition of respiration in these cells should also be
sufficient to cause rapid death. However, respiratory
inhibition does not kill other cell types, such as skeletal
muscle or cell lines derived from tumor cells, due to their
high glycolytic capacity. An important implication of
NO raising the Km of cytochrome oxidase for oxygen is
that NO should sensitize cells to hypoxia-induced cell
death; however, this has not been experimentally tested.
NO does not inhibit glycolysis directly; however,
peroxynitrite directly oxidizes the active-site cysteine
residue of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), causing irreversible inhibition [80], and Snitrosothiols can apparently S-nitrosate the active-site
cysteine residue causing reversible inhibition, but this
can further react with NADH or glutathione to cause
irreversible inhibition [81]. Reduced thiols like glutathione can prevent such inhibition, and the dehydrogenase
is not rate limiting for glycolysis so that effective inhibition of glycolysis may only occur when glutathione is
substantially depleted in the cell. On the other hand,
NO/RNS can cause slow depletion of cellular glutathione
[20,82], potentiating the inhibition of glycolysis, respiration at complex I, and caspases and thereby converting
NO-induced apoptosis into necrosis.
In addition, NO, or more likely RNS, can oxidize
the active-site cysteine in GAPDH, inhibiting its dehydrogenase activity and inducing an acyl phosphatase activity in the enzyme [83]. This results in uncoupling of glycolytic flux from ATP synthesis by
substrate level phosphorylation. NO-producing macrophages or cells exposed to NO donors exhibit reduced GAPDH activity, increased glycolysis, and decreased ATP content and turnover [83– 85], potentially
leading to ATP depletion-induced necrosis.
In cells exposed to NO, glycolytic generation of ATP
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 sufficient 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 [86,87]). Therefore, in many cells
necrosis induced by NO (or NO-producing cells) is prevented by glucose [87,88]. If, however, MPT is chronically stimulated by NO derivatives, then respiration may
be inhibited, oxidative phosphorylation uncoupled, and
glycolytic ATP hydrolyzed by reversal of the ATPase;
thus, necrosis might proceed even in the presence of
rapid glycolysis. However, there is to date little direct
evidence that MPT is involved in NO-induced necrosis.
The rate at which ATP is being consumed by the cell also
may be important in determining the sensitivity to energy
depletion-induced necrosis, as suggested by the synergy
between NO and impulse frequency in neuronal death
[89].
Another potential cause of inhibition of glycolysis is
depletion of NAD⫹ due to activation of PARP. PARP is
a nuclear protein, which, when activated by DNA strand
breaks (possibly caused by peroxynitrite or NO2/N2O3),
catalyzes multiple ADP-ribosylation (using NAD⫹ as
substrate) of proteins including particularly itself [90]. If
DNA damage is extensive, then PARP activity is so high
that cytosolic NAD⫹ is depleted (causing inhibition of
glycolysis) and adenine nucleotides may also be depleted
(either because they are substrates for NAD⫹ 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 [90 –92]. But activation of PARP may contribute to
energy depletion of the cell, possibly in synergy with
inhibition of respiration, inhibition of glycolysis, and/or
activation of MPT (Fig. 2). Recently, it has been suggested that PARP activation can by some undefined
means cause mitochondrial permeabilization and release
of factors such as AIF to cause a form of apoptosis that
is not caspase mediated [93].
NO-induced apoptosis
NO can induce apoptosis in cells or conditions where
respiratory or glycolytic ATP production is sufficient.
Although specific respiratory inhibitors can induce apoptosis in such conditions, induction is weaker and slower
so respiratory inhibition cannot fully explain NO-induced apoptosis [65,87]. NO-induced apoptosis is mediated by downstream caspases (such as caspase-3) and is
blocked by caspase inhibitors [65,87,94]. Cytochrome c
NO, mitochondrial respiration, and cell death
1445
Fig. 2. Possible pathways leading to NO-induced necrotic cell death mediated by ATP depletion.
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 nonmitochondrial pathways [94 –
96].
How NO induces apoptosis is still poorly characterized [7,8], but the main routes may include: (i) MPT
[7,65,97]; (ii) oxidation of mitochondrial phospholipids
[98 –100]; (iii) upregulation of proapoptotic proteins,
such as p53 [100,101] and Bax, and downregulation of
antiapoptotic Bcl-2 [102]; (iv) activation of MAP kinase
pathways [103]; or, (v) activation of the endoplasmic
reticulum stress-response pathway [104,105] (Fig. 3).
NO by itself usually does not induce MPT; however,
peroxynitrite, S-nitrosothiols, NO2, and/or N2O3 may
Fig. 3. Possible pathways leading to NO-induced apoptotic cell death. PL ⫽ phospholipids; ER ⫽ endoplasmic reticulum.
1446
G. C. BROWN and V. BORUTAITE
cause apoptosis by directly activating MPT, leading to
cytochrome c release and caspase activation, and these
events can be blocked with the MPT inhibitor cyclosporin A [7,65,97]. Apoptosis induced by “pure” NO
donors, such as the NONOates, is less susceptible to
inhibition by cyclosporin A but is inhibited by antioxidants (such as N-acetyl-L-cysteine, catalase, superoxide
dismutase, ascorbate, and Trolox) and thus may require
NO-induced ROS or RNS [65,94,106].
NO treatment of cells can cause oxidant-induced degradation of the mitochondrial phospholipid cardiolipin
(probably due to peroxynitrite- or NO2-induced peroxidation), which is associated with irreversible inhibition
of the respiratory chain and apoptosis [98]. Cytochrome
c is normally bound to cardiolipin on the inner mitochondrial membrane, but peroxidation causes the release of
cytochrome c [99]. Addition of peroxynitrite to isolated
mitochondria causes lipid peroxidation and thiol crosslinking associated with increased proton and ion permeability, depolarization, and rapid swelling, which in the
absence of calcium may be not mediated by MPT [46]. 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 [13]. Mitochondrial lipid peroxidation may be an important event in mitochondria-mediated apoptosis [107], but there is no direct evidence that
lipid peroxidation mediates NO-induced apoptosis.
NO can upregulate Bax protein levels in cells and
cause translocation of Bax from cytosol to mitochondrial
outer membranes [102,103]. The mechanism by which
NO initiates Bax translocation to mitochondria is unclear. NO-induced p38 MAP kinase activation has been
shown to mediate Bax activation and translocation [103].
It was reported also that incubation of cells with mitochondrial uncouplers or with inhibitors of the respiratory
chain can trigger Bax translocation to mitochondria, suggesting that a decrease of mitochondrial membrane potential is enough to induce Bax translocation [108,109],
which leads to apoptosis if ATP levels are maintained.
Thus, it is possible that NO triggers Bax association with
mitochondria by inducing a collapse of membrane potential due to respiratory inhibition. Respiratory inhibitors (and hypoxia) can induce apoptosis in cell lines
[65,87,108,110], possibly via a partial fall in ATP [111],
an increase in ROS production, or the decrease of mitochondrial membrane potential, which may either activate
MPT or induce Bax translocation to mitochondria, or
otherwise activate Bax or Bak [103,108,110]. However,
it is clear that NO can and usually does induce apoptosis
by mechanisms unrelated to respiratory inhibition.
NO can also inhibit apoptosis induced by other
agents. Several mechanisms have been suggested: (i)
Fig. 4. Possible pathway of NO-induced neuronal excitotoxic cell
death. ⌬⌿P ⫽ plasma membrane potential; NMDA-R ⫽ N-methyl-Daspartate receptor.
cGMP-mediated blockage upstream of cytochrome c release (cGMP has been shown to inhibit MPT in the
presence of protein kinase G [71]), (ii) cGMP-mediated
inhibition of ceramide synthesis, (iii) S-nitrosylation of
caspases, (iv) energy depletion, (v) mitochondrial hyperpolarization, (vi) activation of MAP kinase pathways,
(vii) activation of transcription factors NF-␬B and/or
AP-1, and (viii) increased expression of heat shock proteins and Bcl-2 [8]. Recently, there have been reports
that NO can cause an increase in mitochondrial membrane potential in some cells (astrocytes, peritoneal macrophages) but not in others (rat cortical neurons, thymocytes) [112–114]; the increase has been suggested to
inhibit apoptosis [112,113,115]. How NO increases mitochondrial membrane potential is unknown; it may be
caused by a secondary stimulation of glycolytic ATP
production, used by the ATPase to maintain mitochondrial membrane potential [112,113,115].
NO-induced excitotoxicity
Neurons are exceptionally sensitive to NO or NOproducing cells [116]. One reason is that NO inhibition
of neuronal respiration causes glutamate release and subsequent excitotoxic death of neurons [34] (Fig. 4). NO,
hypoxia, and specific respiratory inhibitors all cause
NO, mitochondrial respiration, and cell death
rapid ATP depletion of neurons and glutamate release,
apparently due to ATP limitation of the Na⫹ pump
leading to decline of the plasma membrane potential and
Na⫹ gradient, causing reversal of the Na⫹-coupled glutamate uptake carrier [34,117]. High concentrations of
extracellular glutamate (the main excitatory neurotransmitter in the brain) can kill neurons via activation of
glutamate receptors, in particular the NMDA receptor,
and such neuronal death is known as excitotoxic death.
Respiratory inhibitors can strongly potentiate such death
by depolarizing the plasma membrane, as depolarization
is required together with glutamate for activation of
NMDA receptors, which once activated act as ion channels allowing both Na⫹ and Ca2⫹ into the neuron [118].
Astrocytes and microglia (brain macrophages) become inflammatorily activated in most CNS pathologies,
including during infection, trauma, aging, after ischemia
or stroke, and most inflammatory and neurodegenerative
diseases [119]. These activated glia express iNOS and
produce high levels of NO capable of inhibiting respiration both within themselves and surrounding neurons
[34,120]. Thus, activated glia coincubated with neurons
cause rapid inhibition of neuronal respiration at cytochrome oxidase, which then results in rapid glutamate
release from the neurons and in turn causes excitotoxic
death of the neurons [34,121]. This may be one important means by which brain inflammation, present in most
CNS pathologies, causes neuronal death.
In some cases, inhibition of nNOS blocks glutamate-induced death of neurons, and cell death has
been attributed to NO and calcium-induced mitochondrial depolarization, although mechanisms remain unclear [116,122–124]. NO and NO-producing astrocytes cause irreversible damage to respiratory
complexes of neuronal mitochondria [9], but this now
appears to be secondary to glutamate release and excitotoxicity [125]. Activation of PARP may also be
involved in the neurotoxicity of NO [91].
CONCLUSIONS
To summarize, NO inhibits mitochondrial respiration
in two ways: (i) reversible inhibition at cytochrome oxidase by NO, and (ii) irreversible inhibition or inactivation of complex I, complex II/III, cytochrome oxidase,
the ATP synthase, creatine kinase, and aconitase by
peroxynitrite or S-nitrosothiols. The reversible inhibition
of cytochrome oxidase is caused either by NO binding to
reduced heme a3 in competition with oxygen (reversible
by light or removing the bulk NO) or by NO binding to
oxidized CuB resulting in the formation of an inhibitory
nitrite ion. Although NO from constitutive forms of NOS
or mitochondrial NOS can regulate mitochondrial respiration in vitro and in cultured cells, it is still unclear
1447
whether this occurs significantly in vivo. iNOS produces
higher levels of NO for a longer period, causing potent
inhibition of respiration at cytochrome oxidase followed
by irreversible inhibition of other mitochondrial components.
There are three main roles of mitochondria in NOinduced cell death: (i) NO inhibition of respiration can (if
glycolysis is insufficient to compensate) induce necrosis
(or excitotoxicity in neurons) and inhibit apoptosis via
ATP depletion, (ii) RNS-induced MPT may induce
apoptosis or necrosis, and (iii) RNS/ROS-induced signal transduction, ER stress, lipid peroxidation, or
DNA damage may activate the mitochondrial pathway
to apoptosis. [41]
Acknowledgements — Our own work in this field has been supported
by the Royal Society, Wellcome Trust, Medical Research Council, and
British Heart Foundation.
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ABBREVIATIONS
eNOS— endothelial nitric oxide synthase
FADD—Fas-associated death domain
GAPDH— glyceraldehyde-3-phosphate dehydrogenase
iNOS—inducible nitric oxide synthase
MAP kinase—mitogen-activated protein kinase
MPT—mitochondrial permeability transition
NMDA—N-methyl-D-aspartate
nNOS—neuronal nitric oxide synthase
PARP—poly-ADP ribose polymerase
RNS—reactive nitrogen species
ROS—reactive oxygen species