Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer.
ABSTRACT Nitrite (NO(2)(-)) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II-IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.
- SourceAvailable from: Matthew James Rowland[Show abstract] [Hide abstract]
ABSTRACT: The nitric oxide synthase pathway plays a pivotal role in brain blood flow regulation•Disruption to this pathway occurs after injury, and contributes to secondary insult•Nitric oxide donor agents show promise as therapy for secondary brain injuryExperimental Neurology 10/2014; · 4.62 Impact Factor
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ABSTRACT: Abstract Cancer stem cells (CSCs) represent a subpopulation of tumor cells endowed with self-renewal capacity and are considered as an underlying cause of tumor recurrence and metastasis. The metabolic signatures of CSCs and the mechanisms involved in the regulation of their stem cell-like properties still remain elusive. We utilized nasopharyngeal carcinoma (NPC) CSCs as a model to dissect their metabolic signatures and found that CSCs underwent metabolic shift and mitochondrial resetting distinguished from their differentiated counterparts. In metabolic shift, CSCs showed a greater reliance on glycolysis for energy supply compared with the parental cells. In mitochondrial resetting, the quantity and function of mitochondria of CSCs were modulated by the biogenesis of the organelles, and the round-shaped mitochondria were distributed in a peri-nuclear manner similar to those seen in the stem cells. In addition, we blocked the glycolytic pathway, increased the ROS levels, and depolarized mitochondrial membranes of CSCs, respectively, and examined the effects of these metabolic factors on CSC properties. Intriguingly, the properties of CSCs were curbed when we redirected the quintessential metabolic reprogramming, which indicates that the plasticity of energy metabolism regulated the balance between acquisition and loss of the stemness status. Taken together, we suggest that metabolic reprogramming is critical for CSCs to sustain self-renewal, deter from differentiation and enhance the antioxidant defense mechanism. Characterization of metabolic reprogramming governing CSC properties is paramount to the design of novel therapeutic strategies through metabolic intervention of CSCs.Cell cycle (Georgetown, Tex.) 10/2014; · 5.24 Impact Factor
- Polyhedron 01/2014; 67:471-480. · 2.05 Impact Factor
The Journal of Experimental Medicine
Vol. 204, No. 9, September 3, 2007 2089-2102 www.jem.org/cgi/doi/
Ischemia/reperfusion (I/R) injury is character-
ized by several cellular events, including the
release of tissue enzymes, oxidative modifi cation
of essential proteins and lipids, and a dysregu-
lated infl ammatory response, which ultimately
leads to tissue necrosis and apoptosis ( 1, 2 ). On
a subcellular level, the mitochondrion is vital
to tissue viability, and mitochondrial damage
plays a central role in the progression of pa-
thology after I/R. During I/R, mitochondrial
ATP synthesis is decreased (leading to deple-
tion of tissue high-energy phosphate stores) ( 3 ),
enzymes of the respiratory chain are damaged
(leading to diminished inner membrane po-
tential) ( 4, 5 ), the permeability transition pore
is opened ( 6 ), and upon reperfusion, reactive
oxygen species (ROS) generation is increased.
The mechanisms underlying cytoprotection of
a large number of I/R therapeutic agents in-
volves the regulation of mitochondrial function
either through the modulation of membrane
potential, ROS formation, or the activity of
the ATP-sensitive potassium channel. Accu-
mulating data suggest that modulation of mito-
chondrial function is particularly important in
ischemic preconditioning, where cytoprotec-
tion is evident from hours to days after the
nonlethal ischemia-activated cell-survival pro-
grams ( 5, 7 ).
The circulating molecule nitrite (NO 2 ? ) has
been shown to mediate potent cytoprotection
after I/R injury in the heart, liver, and brain
when administered during ischemia or imme-
diately before reperfusion ( 8 – 11 ). For example,
Mark T. Gladwin:
Abbreviations used: ALT,
alanine aminotransferase; iNOS,
inducible NO synthase; I/R,
reactive oxygen species; SNO,
D.J. Lefer and M.T. Gladwin contributed equally to this work.
Nitrite augments tolerance to ischemia/
reperfusion injury via the modulation
of mitochondrial electron transfer
Sruti Shiva, 1 Michael N. Sack, 2 James J. Greer, 4 Mark Duranski, 4
Lorna A. Ringwood, 1 Lindsay Burwell, 5 Xunde Wang, 1
Peter H. MacArthur, 1 Amir Shoja, 4 Nalini Raghavachari, 1 John W. Calvert, 4
Paul S. Brookes, 5 David J. Lefer, 4 and Mark T. Gladwin 1,3
1 Vascular Medicine Branch and 2 Cardiology Branch, National Heart Lung Blood Institute and 3 Critical Care Medicine
Department, National Institutes of Health, Bethesda, MD 20892
4 Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461
5 Department of Anesthesiology, University of Rochester Medical Center, Rochester, NY 14642
Nitrite (NO 2 ? ) is an intrinsic signaling molecule that is reduced to NO during ischemia and
limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain.
Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator
of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct
acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both
acute and delayed (24 h before ischemia) exposure to physiological concentrations of
nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion
injury. This cytoprotection is associated with increases in mitochondrial oxidative
phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed
by reoxygenation were directly protected by nitrite administered both in vitro during
anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-
dependently modifi es and inhibits complex I by posttranslational S-nitrosation; this dampens
electron transfer and effectively reduces reperfusion reactive oxygen species generation
and ameliorates oxidative inactivation of complexes II – IV and aconitase, thus preventing
mitochondrial permeability transition pore opening and cytochrome c release. These data
suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury
and may represent an effector of the cell-survival program of ischemic preconditioning and
the Mediterranean diet.
NITRITE PROTECTS FROM I/R INJURY BY REGULATING MITOCHONDRIA | Gladwin et al.
cytoprotection when administered during ischemia or im-
mediately before reperfusion ( 8, 9 ), it is not known whether
nitrite can temporally replicate delayed preconditioning. To
test this, mice were administered one bolus intraperitoneal
injection of nitrite (48 nmol) or saline and subjected to either
myocardial infarction or hepatic ischemia 24 h later ( Fig.
1 A ). After hepatic I/R, serum alanine aminotransferase
(ALT) levels, representing hepatic injury, increased (525 ?
40 U/liter) compared with sham surgical controls (50 ? 10
U/liter). Nitrite administered both acutely and 24 h before
ischemia attenuated the increase in ALT (210 and 220 ? 30
U/liter, respectively) to a similar degree ( Fig. 1 B ). Simi-
larly, nitrite administration acutely or 24 h before ischemia
signifi cantly reduced myocardial infarct size (18 ? 4 and
35 ? 14%, respectively) compared with saline-treated mice
(53 ? 5%; Fig. 1, C and D ). Thus nitrite exerts potent cyto-
protective eff ects after I/R injury temporally analogous to
Nitrite-dependent protection occurs
at the mitochondrial level
To elucidate the mechanism of cytoprotection in the heart
and liver, we examined mitochondria isolated from the liver
of mice after hepatic I/R and polarographically measured
their respiratory rate. State 3 respiratory rates, using succinate
as a substrate, correlated inversely with the measured ALT
levels. Mice subjected to sham surgery in the presence and
absence of nitrite had the lowest ALT levels and the highest
mitochondrial respiratory rates (34 – 40 nmol O 2 /min/mg of
protein), whereas those subjected to I/R showed an ? 50%
decrease in respiratory rate (18 ? 4 nmol O 2 /min/mg). Ni-
trite administration (48 nmol) during or 24 h before ischemia
increased state 3 succinate-dependent mitochondrial respira-
tion (25 ? 2 and 27 ? 2 nmol O 2 /min/mg, respectively) after
I/R ( Fig. 2 A ).
In an attempt to evaluate whether this protection occurs
at the mitochondrial level versus an eff ect on cellular injury
resulting in secondary, indirect mitochondrial protection, we
examined isolated rat mitochondria in the absence of other
cellular organelles subjected to an in vitro model of I/R injury.
In these experiments, mitochondria isolated from rat liver were
used because of their robust activity and abundant quantity in
comparison with mitochondria isolated from mice. The mito-
chondria (1 mg/ml) were stimulated to respire in state 3
through complex II by the addition of 5 mM succinate and
1 mM ADP until all the oxygen in the chamber was consumed.
After 30 min of anoxia, the mitochondria were reoxygenated,
washed, and resupplied with substrate. The mitochondrial re-
spiratory rate was decreased by ? 50% in comparison with the
preanoxic respiratory rate and compared with control mito-
chondria suspended in normoxic buff er for 30 min ( Fig. 2,
B and C ). This diminished respiratory rate recapitulates that
seen in vivo and implicates mitochondrial damage induced by
anoxia/reoxygenation. The isolated mitochondrial experi-
ments were then repeated after one intraperitoneal injection of
either nitrite (480 nmol) or saline to rats 24 h before hepatic
in murine models of myocardial and hepatic infarction,
nanomole doses (1.2 – 48 nmol) of nitrite reduced infarction
volume and apoptosis by ? 50% compared with controls
( 8, 11 ); similar eff ects have been observed in the isolated per-
fused rat heart ( 10 ). More recently, in a rat model of stroke,
48 – 480 nmol of nitrite reduced cerebral infarct volume by
? 75% and enhanced neurological functional recovery ( 9 ).
Although the eff ects of nitrite are undoubtedly potent, the
mechanisms orchestrating cytoprotection are unknown.
Emerging data demonstrate that nitrite is an important
endocrine reservoir of NO that is reduced to bioactive NO
along a physiological pH and oxygen gradient by several
mechanisms, including enzymatic reduction by hemoglobin,
myoglobin ( 12–14 ), components of the mitochondrial respi-
ratory chain ( 15 ), and xanthine oxidoreductase ( 16 ), as well
as nonenzymatically by acidic disproportionation ( 17, 18 ).
Although NO is known to protect tissues from I/R injury,
its therapeutic window is limited in terms of dose, source,
and duration of exposure ( 19 – 23 ). In addition, the ability
of the enzyme NO synthase to generate NO during I/R is
compromised because of the requirement for oxygen as a
substrate. Conversely, the conditions during I/R when oxy-
gen is limiting and the tissue becomes acidotic are optimal
for the reduction of nitrite to NO. Consistent with the idea
of nitrite-dependent NO generation during I/R, the NO
(PTIO) reversed nitrite-dependent cytoprotection in all pre-
viously published studies ( 8 – 10 ).
NO is a well-characterized regulator of mitochondrial
function, with nanomolar concentrations reversibly inhibit-
ing cytochrome c oxidase ( 24, 25 ), regulating ROS forma-
tion ( 26, 27 ), initiating biogenesis ( 28, 29 ), and limiting
apoptotic cytochrome c release ( 30, 31 ). Given the central
role of mitochondria during I/R, as well as the concept that
its modulation may enhance ischemic tolerance during
preconditioning, we hypothesized that (a) the cytoprotective
eff ects of nitrite mimic and may mediate the ischemic pre-
conditioning cell-survival program and that (b) the cytopro-
tective eff ects of nitrite occur at the level of the mitochondrion,
by enhancing reperfusion respiration and energetics and
limiting ROS-mediated cellular dysfunction. Finally, con-
sidering the fact that nitrate intake in the Mediterranean
diet is associated with both nitrite uptake into plasma and
a reduction in cardiovascular morbidity and mortality, we
evaluated whether dietary nitrite may modulate the organ
response to I/R.
Nitrite mimics ischemic preconditioning in vivo
Transient nonlethal ischemia, termed the ischemic precon-
ditioning “ trigger, ” evokes cellular resilience to I/R ( 32 ).
Importantly, there exist two temporal windows of I/R tol-
erance: acute, or classical, ischemic preconditioning, lasting
minutes to a few hours after the ischemic trigger, and delayed
ischemic preconditioning, evident 1 – 3 d after the transient
ischemic trigger. Although nanomole doses of nitrite mediate
JEM VOL. 204, September 3, 2007
Nitrite does not regulate mitochondrial biogenesis
Because the half-life of nitrite in blood is only 11 min ( 33 ),
we considered that nitrite may mimic delayed preconditioning
through the transcriptional regulation of nuclear-encoded mito-
chondrial functioning genes ( 34, 35 ). Moreover, recent studies
have shown that NO activates the mitochondrial biogenesis
regulatory program ( 28, 29 ). To explore this possibility, we
evaluated the gene expression profi les in response to daily nitrite
administration for 1, 2, 3, and 9 d before mRNA extraction.
The gene expression profi les of the nuclear genes encoding
mitochondrial biogenesis regulatory proteins, antioxidant en-
zymes, uncoupling proteins, and respiratory chain enzymes were
quantifi ed. Although nitrite-dependent protection was main-
tained in the mitochondria throughout this exposure time ( Fig.
3, A and B ), nitrite-treated rats showed no change in mRNA
expression of genes encoding transcriptional regulators of the
mitochondrial biogenesis program (PGC-1 ? and NRF2), respi-
ratory proteins (cytochrome c oxidase, cytochrome c, and ade-
nine nucleotide translocase), or in antioxidant defense mediators
mitochondrial isolation. Although the preanoxic complex
II-dependent respiratory rate was similar with and without
prior nitrite administration (85 ? 12 and 80 ? 10 nmol O 2 /
min/mg, respectively), mitochondria from the nitrite-treated
rats had signifi cantly higher postanoxic respiration rates (67 ?
12 nmol O 2 /min/mg) versus saline-treated rats (45 ? 6 nmol
O 2 /min/mg; Fig. 2 C ). Examination of the pre- and postan-
oxic rates demonstrates that the saline group sustained a 48 ?
6% drop in respiration rate, whereas the nitrite group decreased
by only 20 ? 4% ( Fig. 2 D ).
To exclude mitochondrial uncoupling as a major compo-
nent in diff erential postanoxia respiratory rates, the rate of
ATP formation was determined. Preanoxic rates of ATP pro-
duction were similar with or without nitrite administration.
However, after anoxia, mitochondria from the saline-treated
rats recovered 52 ? 4% of ATP synthesis, whereas mitochon-
dria from nitrite-treated rats recovered 78 ? 2% ( Fig. 2 E ).
These data suggest that nitrite-mediated delayed cytoprotec-
tion occurs at the level of the mitochondrion.
Figure 1. Nitrite mediates both acute and delayed cytoprotection after I/R injury to the heart and liver. (A) Model of hepatic ischemia and
myocardial infarction in which nitrite or saline was administered either 24 h before or during ischemia. (B) Plasma ALT levels 5 h into reperfusion in mice
after sham surgery, sham surgery with nitrite treatment, ischemia with acute nitrite treatment, or ischemia with nitrite preconditioning (PC). (C) Infarct
size as a percentage of area at risk 24 h after myocardial infarction in the absence of nitrite (I/R), with nitrite treatment 5 min before reperfusion (acute),
or nitrite treatment 24 h before myocardial infarction (PC). (D) Representative sections of myocardium stained with Evan ’ s blue and triphenyltetrazolium
chloride 24 h after infarction in mice receiving no nitrite (I/R ? vehicle), acute nitrite treatment, or nitrite preconditioning. *, P ? 0.05 in comparison with
the I/R ? vehicle group; **, P ? 0.01 versus the I/R ? vehicle group.
NITRITE PROTECTS FROM I/R INJURY BY REGULATING MITOCHONDRIA | Gladwin et al.
delayed cytoprotection cannot be completely excluded with-
out further study.
Acute in vitro nitrite treatment protects mitochondria
from I/R injury
The lack of evidence for nitrite-dependent regulation of
mitochondrial protein expression suggested that the protec-
tive eff ect of nitrite on mitochondria was most likely caused
by the posttranslational modifi cation of existing mitochon-
drial proteins. We hypothesized that if posttranslational
modifi cation was indeed responsible, mitochondrial protec-
tion of respiration and ATP synthesis should be observed
even with acute nitrite treatment of mitochondria in vitro.
To test this, we subjected isolated mitochondria from un-
treated rats to 30 min of anoxia followed by reoxygenation
(catalase, superoxide dismutase, and uncoupling protein 2) in
comparison with saline-treated rats ( Fig. 3 C ). Mitochondrial
protein expression ( Fig. 3 D ) and mitochondrial number and
morphology (unpublished data) remained constant despite nine
consecutive days of nitrite treatment. These data show that
nitrite-dependent protection is not mediated by changes in
gene transcription or translation that directly modulate the
mitochondrial biogenesis program. Similarly, a single dose of
nitrite administered 5 h before the extraction of hepatic tissue
demonstrated no signifi cant up-regulation of genes encoding
cyclooxygenase-2 and inducible NO synthase (iNOS; 1.66 ?
0.02 fold change [P ? 0.08] and 1.335 ? 0.09 fold change
[P ? 0.23], respectively; n ? 10). As cyclooxygenase-2 and
iNOS are established mediators of delayed preconditioning
( 36 ), a partial role of this signaling cascade in nitrite-dependent
Figure 2. Nitrite preconditioning protects from ischemic damage at the mitochondrial level. (A) Respiratory rates (in the presence of succinate
and ADP) of liver mitochondria isolated from mice 5 h after being subjected to hepatic I/R in the presence and absence of acute and preconditioning nitrite.
(B) In vitro model of mitochondrial anoxic/reoxygenation. Red trace is mitochondria subjected to anoxia. Blue trace is mitochondria subjected to normoxia for
30 min. (C) Representative respiration traces of 1 mg/ml of mitochondria isolated from rats preconditioned with saline (continuous lines) or 480 nmol nitrite (dashed
lines) before (green) and after (blue) anoxia. (D and E) Postanoxic respiration (D) and ATP synthesis (E) rates expressed as a percentage of preanoxic rate for
mitochondria isolated from rats preconditioned with saline or nitrite. All experiments are means ? SEM of at least n ? 3 independent experiments. *, P ? 0.01.
JEM VOL. 204, September 3, 2007
It is well established that modulation of mitochondrial
ATP-dependent potassium (K ATP ) channels during I/R can
aff ect tissue viability. To determine whether nitrite mediates
cytoprotection by regulating K ATP channels, we tested the
eff ects of nitrite treatment during anoxia/reoxygenation in
the presence of diazoxide, a K ATP channel opener, and gliben-
clamide, an inhibitor of this channel. In our model, opening
of the K ATP channel with 200 ? M diazoxide was protective,
with a 63 ? 6% recovery of respiration after anoxia in com-
parison with 42 ? 5% in the control. Likewise, inhibition of
the K ATP channel with 100 ? M glibenclamide resulted in a
signifi cant decrease in postanoxic recovery of respiration (22 ?
3%). Although both of these pharmacologic agents had inde-
pendent eff ects on postanoxic respiration rates, neither drug
inhibited nitrite-dependent protection of mitochondria ( Fig.
5 A ), suggesting that the K ATP channel was not the target for
in the presence and absence of nitrite ( Fig. 4 A ). As predicted,
nitrite-treated (10 ? M) mitochondria had both higher com-
plex II – dependent state 3 respiration (78 ? 12 nmol O 2 /
min/mg; Fig. 4, A and B ) and ATP production (16 ? 6 rel-
ative light units/s; Fig. 4, C and D ) rates than control mito-
chondria (54 ? 4 nmol O 2 /min/mg and 9.8 relative light
units/s, respectively) after anoxia/reoxygenation. Interest-
ingly, nitrite-dependent protection from the anoxia-induced
drop in respiration rate showed a biphasic dose – response
curve (0 – 100 ? M), with peak protection occurring at a
concentration of 12.5 ? M ( Fig. 4 E ). This biphasic response
is identical in shape and concentration to previously ob-
served dose – response curves for nitrite-mediated cytopro-
tection in vivo, and the concentration of 12.5 ? M nitrite
conferring peak protection to the mitochondria parallels the
tissue concentration of nitrite shown to orchestrate peak
protection in vivo ( 8 ).
Figure 3. Nitrite-dependent cytoprotection does not modulate mitochondrial biogenesis. Rats were given one intraperitoneal injection (480 nmol)
of saline or nitrite daily for nine consecutive days. (A and B) Recovery of respiration (A) and ATP (B) generation rates of mitochondria isolated from
these rats after 30 min of anoxia in vitro (white, saline; green, nitrite). (C) Relative expression of genes in the livers of nitrite-treated rats on day 9 pre-
sented as the fold change in gene expression compared with saline-treated rats. (D) Protein expression of the 39-kD subunit of complex I, cytochrome c,
and ATPase subunit B in the livers of rats after 9 d of nitrite or saline treatment. All experiments are means ? SEM of at least n ? 3 independent experi-
ments. *, P ? 0.01.
NITRITE PROTECTS FROM I/R INJURY BY REGULATING MITOCHONDRIA | Gladwin et al.
not enhanced, by nitrite treatment during anoxia/reoxy-
genation ( Fig. 5 B ). Interestingly, the same eff ect was ob-
served in the postanoxic respiration rate of mitochondria
isolated from rats treated with 480 nmol nitrite 24 h before
isolation, though there was no signifi cant eff ect of nitrite
treatment on preanoxic respiration rate ( Fig. 5 C ). This
trend was recapitulated in mitochondria isolated from mice
that were subjected to hepatic I/R in vivo ( Fig. 5 D ). Spectro-
photometric measurement of complex I – dependent oxi-
dation of NADH confi rmed that nitrite treatment (0 – 100 ? M)
of isolated mitochondria during anoxia concentration-
dependently inhibited the enzymatic activity of complex I
( Fig. 5 E ), whereas we saw no substantial eff ect on the isolated
activities of complex II or IV (unpublished data).
NO can S-nitrosate critical thiols on complex I, leading
to an inhibition of its activity ( 38 – 40 ). This inhibition of
complex I is proposed to exert cytoprotection during I/R,
Nitrite inhibits complex I after I/R
Although we have observed a nitrite-dependent protection
of the complex II respiratory rate after anoxia/reoxygen-
ation, it is important to also consider the eff ects of nitrite
on complex I – dependent respiration, because complex I is
known to be a primary site of both injury and ROS pro-
duction after I/R ( 37 ). To test the eff ects of nitrite on com-
plex I, we measured glutamate/malate-supported (5 mM
each) state 3 respiration in isolated mitochondria before and
after anoxia/reoxygenation. After anoxia/reoxygenation,
both complex I – and II – supported respiration showed a de-
crease in respiration rate, though complex I showed a much
greater decrease in rate (62 ? 4 vs. 35 ? 3% recovery),
consistent with the increased susceptibility of complex I to
I/R damage. However, in contrast to observed eff ects on
complex II respiration, respiration through complex I was
inhibited (20 vs. 35% recovery in control mitochondria),
Figure 4. Acute nitrite treatment protects mitochondria against I/R injury. (A) Model of in vitro mitochondrial damage showing preanoxic (green)
and postanoxic rates in the absence (blue) and presence (red) of nitrite. Arrow denotes the addition of 10 ? M nitrite. Respiration rates (B) and ATP gen-
eration rates (C) before anoxia (green) and after anoxia in the presence of saline (blue) and nitrite (red). (D) Quantifi cation of rate of ATP generation before
and after anoxia. (E) Recovery of respiration rate with increasing concentrations (0 – 100 ? M) of nitrite added during anoxia. All experiments are means ?
SEM of at least n ? 3 independent experiments. *, P ? 0.01. RLU, relative light units.
JEM VOL. 204, September 3, 2007
of protein, suggesting that S-nitrosation of mitochondrial
proteins could be responsible for nitrite-dependent cytopro-
tection. Consistent with this thesis, isolated mitochondria
from rats treated with 480 nmol nitrite 24 h before isolation
had detectable S-nitrosation (4.8 ? 3 pmol/mg), whereas no
signifi cant S-nitrosation could be measured in saline-treated
controls ( Fig. 6, C and D ).
To further characterize the target of S-nitrosation, nitrite-
treated (20 ? M) mitochondria were fractionated by Superose
6 gel fi ltration, and SNO levels were measured by reductive
chemiluminescence in each fraction. Signifi cant S-nitrosation
( ? 10 pmol) of the 900-kD mitochondrial fraction was observed
in the nitrite-treated mitochondria, which includes complex I
proteins and activity ( 39 ), whereas no S-nitrosation was
detected on this fraction in control mitochondria ( Fig. 6 E ).
potentially through the modulation of ROS production. We
found that 200 ? M of the NO scavenger PTIO reversed the
nitrite-dependent inhibition of complex I respiration after
in vitro anoxia/reoxygenation, suggesting that the modifi ca-
tion and subsequent inhibition of complex I was NO depen-
dent ( Fig. 5 F ).
To identify the type of NO-dependent posttranslational
modifi cation, we measured protein S-nitrosothiols (SNOs),
iron-nitrosyl, and N -nitrosamine complexes from isolated
mitochondria treated with 0 – 100 ? M nitrite during anoxia/
reoxygenation using gas-phase ozone-based reductive chemi-
luminescence with both triiodide and copper/cysteine reduc-
tants ( Fig. 6, A and B ). Both reductive methods confi rmed that
nitrite treatment increased mitochondrial protein S-nitrosation
in a concentration-dependent manner from 0.05 – 160 pmol/mg
Figure 5. Nitrite treatment inhibits complex I. (A) Recovery of complex II – dependent respiratory rate in mitochondria that were untreated (Ctrl),
treated with 10 ? M nitrite alone, 200 ? M diazoxide (Diaz), diazoxide and nitrite, 150 ? M glibenclamide (Glib), or glibenclamide and nitrite during anoxia.
(B) Recovery of respiration of mitochondria respiring on succinate (complex II) or glutamate/malate (complex I) and ADP in the absence (green) and pres-
ence (red) of 10 ? M nitrite during anoxia/reoxygenation. (C) Absolute respiratory rates before and after anoxia of mitochondria from rats treated with
nitrite (green) or saline (red) 24 h earlier. (D) Respiratory rates of mitochondria isolated from mice 5 h after they were subjected to hepatic I/R in vivo.
(E) Complex I activity of isolated mitochondria treated with increasing concentrations of nitrite. (F) Recovery of complex I respiration rate in untreated
mitochondria or treatment with 20 ? M nitrite, 100 ? M PTIO, or nitrite and PTIO. All experiments are means ? SEM of n ? 3 experiments. *, P ? 0.01.
NITRITE PROTECTS FROM I/R INJURY BY REGULATING MITOCHONDRIA | Gladwin et al.
by this complex ( 39, 43, 44 ). To determine whether nitrite-
dependent inhibition of complex I diminishes mitochondrial
ROS generation, we subjected mitochondria to 30 min of
anoxia in the presence and absence of nitrite 10 ? M and
measured H 2 O 2 production by the oxidation of amplex red at
reoxygenation. After I/R, H 2 O 2 production was increased in
isolated mitochondria (9.9 ? 0.5 nmol/min/mg) in compari-
son with preanoxic levels (3 ? 0.5 nmol/min/mg). How-
ever, this increase was attenuated (4.5 ? 1.4 nmol/min/mg)
by nitrite treatment ( Fig. 7 A ).
The Fe-S containing mitochondrial enzyme aconitase is
highly susceptible to oxidative damage. We measured the ac-
tivity of this enzyme as an index of oxidative protein damage,
Although nitrite appeared to S-nitrosate additional targets on
the electron transport chain, these data suggest that nitrite-
dependent S-nitrosation and NO-mediated thiol oxidation of
complex I are responsible for the inhibition of complex I ac-
tivity after anoxia-reoxygenation both in vivo and in vitro.
Nitrite-dependent complex I inhibition limits reperfusion
ROS generation and aconitase inactivation
Complex I is a primary site of mitochondrial ROS produc-
tion, with superoxide generation by forward and reverse
electron transport ( 41, 42 ). Inhibition of complex I may con-
fer cytoprotection potentially because of the limitation of ox-
idative protein damage by the inhibition of ROS production
Figure 6. Nitrite S-nitrosates complex I. (A) Representative chemiluminescence trace of nitrite-treated mitochondria injected into triiodine before
and after pretreatment with mercuric chloride (+Hg). (B) Quantitation of S-nitrosation in mitochondria from A (red) and an identical experiment using
copper (I)/cysteine as a reductant instead of triiodine (blue). (C) Representative copper (I)/cysteine – based chemiluminescence trace of mitochondria iso-
lated from rats treated with 480 nmol of saline or nitrite 24 h earlier. (D) Quantifi cation of traces similar to those shown in C. (E) Protein was extracted
from rat liver mitochondria and loaded onto a Superose 6 size-exclusion column. The SNO and complex I activity of each fraction was then determined and
are represented as picomoles of SNO per fraction and complex I activity of the neat fractions, respectively. Mitochondria subjected to ischemia without
NO 2 ? treatment resulted in no SNO detection (not depicted). All experiments are means ? SEM of at least n ? 3 independent experiments. *, P ? 0.01.
JEM VOL. 204, September 3, 2007
which the I/R-induced increase in oxidative damage was
diminished by nitrite treatment either acutely or 24 h before
the ischemic episode (48 nmol; Fig. 7 C ).
Nitrite treatment prevents opening of the permeability
transition pore and cytochrome c release
Opening of the mitochondrial permeability transition pore has
been linked to the release of cytochrome c and is important
with a lowered activity indicating greater oxidative damage.
Consistent with a nitrite-dependent decrease in ROS pro-
duction, mitochondria subjected to anoxia/reoxygenation
sustained more oxidative damage (13 ? 1.1 mU/mg of protein)
than control mitochondria (28 ? 4 mU/mg), whereas those
treated with nitrite during anoxia were signifi cantly protected
(18 ? 1.8 mU/mg; Fig. 7 B ). These eff ects translated to en-
tire livers taken from mice 5 h after in vivo hepatic I/R, in
Figure 7. Nitrite decreases oxidative damage in mitochondria. (A) Representative traces of ROS production measured by amplex red in mitochon-
dria without substrate (light blue) in the presence of glutamate/malate and ADP before anoxia/reoxygenation (green), and after anoxia/reoxygenation in
the presence (red) and absence (blue) of 20 ? M nitrite. (B) Aconitase activity of mitochondria during normoxia and after anoxia/reoxygenation in the
presence and absence of 20 ? M nitrite. (C) Aconitase activity in liver tissue from mice 5 h after they were subjected to hepatic I/R or sham surgery in the
presence and absence of 48 nmol of acute or preconditioning nitrite. (D) Representative traces of calcium-induced pore opening of mitochondria sub-
jected to anoxia/reoxygenation in the presence of 20 ? M nitrite. Traces are in the absence of calcium (black), in the absence of nitrite treatment (red), or
with nitrite treatment (blue). (E) Western blot analysis of cytochrome c remaining in mitochondria from the conditions shown in D. All experiments are
means ? SEM of at least n ? 3 independent experiments. *, P ? 0.01; #, P ? 0.05.
NITRITE PROTECTS FROM I/R INJURY BY REGULATING MITOCHONDRIA | Gladwin et al.
oxidative protein damage to the electron transport chain, cy-
tochrome c release, and cellular and tissue infarction.
Interestingly, NO production has been shown to evoke
acute (classical) preconditioning and is required in the de-
layed ischemic preconditioning cell-survival program ( 19, 49 ).
This occurs through increased endothelial NOS activity after
acute ischemic preconditioning and via induction of iNOS
24 h later for remote preconditioning ( 50 ). Relevant to the
current study, nitrite levels increase in a biphasic manner
after both acute and delayed ischemic preconditioning, with
maximal increases observed during the I/R tolerant periods
( 50 ). In this study, we show that administration of nitrite at
either of these preconditioning time points confers potent
cytoprotection in vivo and modulates mitochondrial func-
tion. In addition, previous studies have shown complex I
inhibition ( 51 ), as well as an increase in mitochondrial
S-nitrosation ( 39 ), after ischemic preconditioning of the rat
heart. Given these data, it is intriguing to consider that ni-
trite could serve as both an endogenous reservoir for NO
production and an eff ector molecule mediating the cell-
survival eff ects of both acute and delayed ischemic precon-
ditioning. Consistent with such NO-dependent signaling,
in initiating the mitochondrial apoptotic pathway. Because
oxidants have been shown to sensitize the permeability
transition pore ( 45, 46 ), we determined whether the cyto
protective eff ects of nitrite could in part be caused by nitrite-
dependent protection against pore opening after I/R. Iso-
lated mitochondria were subjected to 30 min of anoxia in
the presence or absence of 20 ? M nitrite, 10 ? M calcium
was added to stimulate pore opening during reperfusion, and
mitochondrial swelling was spectrophotometrically moni-
tored as a measure of pore opening. In the presence of
calcium, mitochondrial pore opening was facilitated, as in-
dicated by a rapid drop in absorbance. However, in mito-
chondria treated with nitrite during anoxia, pore opening
was delayed after the addition of calcium and occurred at
a considerably slower rate and to a lesser extent than in the
absence of nitrite ( Fig. 7 D ).
To assess whether nitrite limits cytochrome c release, the
mitochondria were centrifuged 1 min after the addition of
calcium, and the amount of cytochrome c remaining in the
mitochondria was measured. In control mitochondria, cal-
cium-induced pore opening elicited the loss of cytochrome c
from control mitochondria, whereas nitrite-treated mitochon-
dria were protected from cytochrome c release ( Fig. 7 E ).
Oral nitrite administration confers resistance to hepatic
and cardiac I/R injury
Several chemical agents, including rotenone and amobarbi-
tol, are known to inhibit complex I and confer protection to
mitochondria after I/R. However, these agents are not prac-
tical therapeutic candidates and certainly are not naturally
occurring compounds. Conversely, nitrite forms from the
reduction of dietary nitrate by oral bacterial fl ora and is a po-
tential cardioprotective agent in the nitrate-rich Mediterra-
nean diet ( 47, 48 ). To evaluate whether oral nitrite would
promote preconditioning cytoprotection after I/R injury,
we next tested oral dosing of nitrite. To begin to explore this,
nitrite or saline vehicle was administered to mice via oral
gavage 24 h before in vivo hepatic or cardiac I/R. Both liver
and heart I/R were evaluated 24 h after a single oral gavage
of 200 ? l of 100 mg/liter sodium nitrite. This would corre-
spond to 9.6 ? mol of sodium nitrite per kilogram. Because
the total body load of nitrite after ingestion of a nitrate-rich
meal (i.e., 100 g of spinach) amounts to ? 1 ? mol/kg ( 47 ),
our dose is consistent with the daily nitrite intake on the
Mediterranean diet. This regimen replicated the cytoprotec-
tion evoked by the earlier intraperitoneal administration of
nitrite used in the previous experiments performed in this
study ( Fig. 8, A and B ).
These data demonstrate that nanomole doses of nitrite mediate
both acute cytoprotection and mimic delayed preconditioning
in the heart and liver in vivo. Mechanistically, both acute and
delayed nitrite-dependent cytoprotection occur at the mito-
chondrial level, with the inhibition of mitochondrial complex I
by S-nitrosation limiting mitochondrial ROS generation,
Figure 8. Hepatic and myocardial I/R injury are attenuated in mice
after oral nitrite therapy. (A) The effects of oral nitrite preconditioning
(PC) on the severity of hepatic I/R injury (measured as aspartate amino-
transferase [AST] and ALT) in mice. Mice received nitrite via oral gavage at
24 h before 45 min of hepatic ischemia and 5 h of reperfusion. Numbers
for each group are shown inside the bars. (B) Bar graph of oral nitrite
preconditioning and myocardial I/R injury in mice. Mice received oral
nitrite at 24 h before 30 min of ischemia and 24 h of reperfusion. The
myocardial area-at-risk (AAR) per total left ventricle (LV) was not signifi -
cantly different between study groups (NS). The myocardial infarct size
(Inf) per area-at-risk was signifi cantly (P ? 0.0001) reduced in the oral
nitrite preconditioning group when compared with the oral vehicle group.
Eight animals were investigated in each group.
JEM VOL. 204, September 3, 2007
The chemistry of nitrite-dependent nitrosation of com-
plex I will also require further study, considering the fact
that the NO radical will not directly nitrosate reduced thiol
(one-electron oxidation is required). There are several pos-
sibilities that might be unique to the mitochondria. First, di-
rect nitrosation of thiols by nitrous acid: the inner membrane
space has an estimated pH of ? 7.2, with the matrix having
a pH of ? 7.9. Although minimal nitrous acid should form
at pH 7.2 (the pKa of nitrite is ? 3.2), any nitrous acid that
formed would occur in the inner membrane space and po-
tentially at complexes I, III, IV, and V, which actively pump
protons. The hydrophobic transmembrane environment of
these complexes would stabilize both nitrous acid, N 2 O 3 ,
and the formed SNO. Second, NO production from nitrite
reduction could secondarily react with nitrogen dioxide or
superoxide formed from mitochondrial oxidation pathways
and could form nitrosating intermediates such as N 2 O 3 or
ONOO ? . Third, we have recently found that nitrite binds
to and reacts with metheme proteins, such as methemoglo-
bin, to produce an NO 2 radical – like intermediate that can
then react with NO to form N 2 O 3 (58); N 2 O 3 would di-
rectly nitrosate complex I. This would provide a metal-based
pathway to S-nitrosation by nitrite. Although these pathways
remain theoretical, the mechanisms of S-nitrosation by nitrite
are currently an active area of research in the NO fi eld.
It is tempting to speculate that nitrite is the endogenous
molecule that regulates ischemic ROS formation at com-
plex I after reperfusion. Such an innate mechanism could repre-
sent an evolved response to limit tissue injury during birth
(for the fetus and mother), traumatic hemorrhage, and ex-
treme exercise. From a therapeutic standpoint, nitrite would
represent an ideal candidate to confer mitochondrial and, by
extension, cellular resilience to I/R injury through the partial
inhibition of complex I. The therapeutic potential of nitrite
is underscored as follows: (a) this anion is a chemically stable
endogenous reservoir for NO, (b) the required therapeutic
concentration is in the nanomolar range, and (c) it is also
readily available in diet (i.e., the Mediterranean diet) and, as
shown in our study, its protective eff ect is clearly observed
with oral intake.
Consistent with an innate physiological role for nitrite in
modulating the stress response, the cytoprotective eff ects of
nitrite in the heart and liver are measurable at nitrite doses
? 1.2 nmol in murine models of myocardial infarction and
hepatic I/R ( 8 ). These doses increase plasma nitrite levels by
? 10% and are consistent with increases observed after the in-
gestion of a standard leafy green salad ( 47, 48 ) or after regular
moderate exercise ( 59 – 61 ). Furthermore, mice with dimin-
ished basal plasma nitrite concentrations are more susceptible
to I/R injury, an eff ect that is attenuated by administration of
exogenous nitrite ( 11 ). These data suggest that a diet rich in
nitrate and nitrite may have profound cytoprotective eff ects
and, most provocatively, could constitute the “ active ” ingre-
dient of the cardioprotective Mediterranean diet ( 47, 48 ).
In conclusion, we have shown that nitrite potently me-
diates cytoprotection after I/R of the mammalian heart and
PTIO, a direct NO scavenger, has been observed to inhibit the
cytoprotective eff ects of nitrite in cardiac ( 10, 20 ), liver ( 20 ),
and brain ( 9 ) I/R.
NO itself has been shown to directly protect cultured rat
neonatal cardiomyocyte mitochondria when administered in
time periods compatible with both classical and delayed pre-
conditioning ( 52, 53 ). Previous studies show that NO modu-
lates mitochondrial Ca 2+ handling that in turn diminishes
reoxygenation-associated Ca 2+ overload ( 52, 53 ). In this
manuscript, we demonstrate that nitrite administration di-
rectly modifi es the electron transfer chain complex I activity
in association with S-nitrosation of this complex. This is asso-
ciated with blunted reperfusion/reoxygenation ROS-mediated
injury. The putative mechanisms that could confer this pheno-
type include the direct inhibition of complex I – generated
ROS or via the modest reduction in the mitochondrial mem-
brane potential that may result from the partial inhibition of
complex I activity ( 5 ).
An apparent paradox is evident with respect to complex I
inhibition and ROS generation. In brief, in an aerobic envi-
ronment with adequate oxidative phosphorylation, substrate,
and reducing equivalents, the direct inhibition of complex I
with either rotenone ( 54 ) or by preformed SNOs ( 40 ) results
in complex I – derived ROS production. Conversely, when
complex I is inhibited in the context of I/R by rotenone, the
production of ROS is decreased during the oxidation of
complex I substrate, in parallel with the preservation of mito-
chondrial content and the rate of oxidation through cyto-
chrome c oxidase ( 55 ). This mitochondrial “ protective ”
response to complex I inhibition during ischemia has been
replicated by the use of amobarbital (a reversible inhibitor of
complex I) ( 43 ), by S-nitrosoglutathione – dependent transni-
trosation in the Langendorff perfused heart ( 39 ), and in this
study, by the naturally occurring anion nitrite. Collectively,
these data suggest that transient inhibition of complex I in the
I/R milieu can have protective eff ects associated with re-
duced ROS production during reperfusion. In contrast,
chronic inhibition of complex I under an otherwise physio-
logic milieu can be cytotoxic and associated with elevated
ROS production ( 56 ).
Interestingly, nitrite appears to be unique among the
aforementioned agents in that it does not inhibit complex I
activity during normal physiology but only inhibits complex I
activity in the setting of I/R injury. These data suggest that
the modifi cation of complex I by these near physiological
concentrations of nitrite is dynamic and only operational in
the setting of ischemia. The mechanisms whereby low-dose
nitrite-mediated modifi cation of complex I is “ fi ne-tuned ”
to have no appreciable inhibitory eff ect under normal ho-
meostatic conditions but suffi cient inhibitory eff ects in the
context of hypoxic or redox stress are currently unknown
and require further investigations. However, such dynamic
inhibition of complex I allows for sustained mitochondrial
oxidative phosphorylation through complex II during I/R
while limiting ROS generation, mitochondrial calcium over-
load, and the release of cytochrome c ( 37, 52, 57 ).
NITRITE PROTECTS FROM I/R INJURY BY REGULATING MITOCHONDRIA | Gladwin et al.
100 ? M NADH in the presence of 10 ? M coenzyme Q 1 in the presence
and absence of 25 ? M rotenone.
Detection of S-nitrosation. To measure S-nitrosation, 10 mg of isolated
mitochondria treated with nitrite was lysed with a solution of 1% NP-40 and
100 ? M diethylenetriamene pentaacetate (DTPA). Half of the sample was
immediately injected into copper/cysteine-based reductive chemilumines-
cence, which is specifi c for S-nitrosated protein adducts. The other half of
the protein was divided into three parts: one left untreated, one treated with
10% acidifi ed sulfanilamide to eliminate nitrite, and one treated with 5 mM
mercuric chloride and 10% acidifi ed sulfanilamide (v/v) to eliminate nitrite
and SNOs. The three fractions were injected into a vessel containing triio-
dine and connected inline to an NO chemiluminescence detector (Seivers),
as previously described ( 64 ).
ROS generation. H 2 O 2 generation was spectrophotometrically assayed by
monitoring the oxidation of amplex red to product resofurin at 585 nm us-
ing the Amplex Red Hydrogen Peroxide/Peroxidase assay kit (Invitrogen).
Mitochondrial fractionation. 3 mg of mitochondrial protein was extracted
in 60 ? l of buff er (10 mM Hepes, pH 7.4, 1 mM EDTA, 100 ? M DTPA,
1.6% lauryl-maltoside). 25 ? l of sample ( ? 1.25 mg of protein) was loaded
onto a 1 ? 24 cm Superose 6 size-exclusion column, run at a fl ow rate of
0.5 ml/min, with the running buff er (10 mM Hepes, pH 7.4, 1 mM EDTA, 100
? M DTPA). 500- ? l fractions were collected, and 475 ? l was immediately
subjected to chemiluminescent SNO analysis, as described in Detection of
S-nitrosation, with the remaining 25 ? l being used for protein determination
by a bicinchoninic acid–based protein assay. The column was calibrated by run-
ning high-range molecular mass standards (68 – 669 kD; GE Healthcare).
Aconitase activity. Mitochondria were lysed by three cycles of freeze/
thaw, and aconitase activity was measured by spectrophotometrically moni-
toring the formation of NADPH at 340 nm using the Bioxytech Aconitase-
340 kit (Oxis Research).
Permeability transition pore opening and cytochrome c release. After
being subjected to anoxia/reoxygenation in the presence or absence of
nitrite, 1 mg/ml of mitochondria was given fresh substrate and spectrophoto-
metrically monitored at 540 nm at 37 ° C. After 100 s, 10 ? M calcium
chloride was added to each cuvette, and monitoring was continued. 1 min
after the addition of calcium, pore opening was suspended by the addition of
500 ? M EGTA and 20 ? M cyclosporine A, and the mitochondria were
centrifuged at 10,000 g for 5 min. The mitochondrial pellets were subjected
to Western blot analysis using an anticytochrome c antibody (Santa Cruz
Oral nitrite studies. Sodium nitrite was dissolved in saline at a concentra-
tion of 100 mg/liter. 24 h before either hepatic or myocardial ischemia, mice
were administered 200 ? l of the 100 mg/liter sodium nitrite stock solution
or vehicle (saline) orally with the aid of a 20-gauge oral gavage needle. The
hepatic and myocardial I/R injuries were performed as described in Hepatic
I/R and Myocardial infarction.
We would like to thank Dr. Xeuxing Yu at the National Heart Lung Blood Institute
Histology Core Facility for his assistance with the processing and analysis of tissue
for electron microscopy. We would also like to thank Tish Murphy, Toren Finkel, and
Robert Balaban for helpful comments and suggestions about the experimental data.
These studies were supported by grants from the National Institutes of Health
(RO1 HL-60849 to D.J. Lefer and F32 DK-077380-01 to J.W. Calvert) and the
American Diabetes Association (7-04-RA-59 to D.J. Lefer).
David J. Lefer and Mark T. Gladwin are named on a provisional patent for the
use of sodium nitrite in cardiovascular disease. The authors have no further
confl icting fi nancial interests.
Submitted: 26 January 2007
Accepted: 2 July 2007
liver at the mitochondrial level through the transient in-
hibition of complex I and subsequent limitation of oxida-
tive damage. The acute and remote temporal windows of
nitrite-dependent cytoprotection suggest that nitrite may be
a central mediator in ischemic preconditioning. Collectively,
these studies reveal a global role for nitrite in the regulation
of ischemic responses at the subcellular level. Finally, its ef-
fi cacy as an oral preparation at near physiologic doses suggests
a role for nitrite as a dietary cardioprotective agent.
MATERIALS AND METHODS
Chemicals. All reagents were obtained from Sigma-Aldrich unless other-
Animals. 8 – 10-wk-old male C57BL6/J mice (The Jackson Laboratory)
were used in accordance with the Animal Care and Use Committee of AE-
COM. Male Sprague-Dawley rats (250-500 g; Harlan) were used in accor-
dance with the Animal Care and Use Committee of the National Heart
Lung Blood Institute.
Hepatic I/R. Mice were anesthetized with 100 mg/kg ketamine and 8 mg/kg
xylazine injected intraperitoneally. A midline laparotomy incision was
performed, and mice were heparinized (100 ? g/kg intraperitoneally) to prevent
blood clotting. Microaneurysm clamps were used to completely clamp the
hepatic artery and portal vein, causing ischemia of the left lateral and median
lobes of the liver, as previously described ( 8 ). The liver was kept moist with
normal saline for 45 min, after which the microaneurysm clamp was removed
to allow reperfusion. Mice were sutured, and serum was collected 5 h later
for measurement of liver transaminase levels.
Myocardial infarction. Mice were anesthetized by injection of 50 mg/
kg ketamine and 50 mg/kg pentobarbital and were then intubated with
polyethylene tubing connected to a rodent ventilator, after which the re-
spiratory rate was set to 122 breaths per minute. A median sternectomy
was performed, and the left main coronary artery was completely ligated
witha 7-0 silk suture mounted on a tapered needle, as previously described
( 8 ). After 30 min, the suture was removed, and animals were reperfused
for 24 h.
Mitochondrial isolation and respiration. Liver mitochondria were iso-
lated by diff erential centrifugation in a buff er (250 mM sucrose, 10 mM Tris,
1 mM EGTA, pH 7.4) at 4 ° C, as previously described ( 62 ). To measure
respiration of isolated mitochondria, 1 mg/ml of protein was suspended in
respiration buff er (120 mM KCl, 25 mM sucrose, 10 mM Hepes, 1 mM
EGTA, 1 mM KH 2 PO 4 , 5 mM MgCl 2 ) in a stirred, sealed chamber fi t with
a Clark-type oxygen electrode (Instech Corp.) connected to a data recording
device (DATAQ Systems).
In vitro anoxia/reoxygenation. The in vitro anoxia/reoxygenation pro-
tocol was adapted from Ozcan et al. ( 63 ). State 3 respiration was initiated,
and mitochondria were allowed to consume oxygen until the chamber be-
came anoxic. The mitochondria were left in this anoxic state for 30 min. To
reoxygenate, mitochondria were centrifuged and resuspended in oxygenated
buff er containing fresh substrate and ADP and allowed to respire once again.
Postanoxic respiratory rate was expressed as a percentage of preanoxic rate
and called recovery of respiration.
ATP generation. ATP synthesis in isolated mitochondria suspended in
respiration buff er with 15 mM succinate and 0.5 mM ADP was measured by
monitoring the luminescence of luciferase/luciferin over time using the
ATP detection kit from Invitrogen.
Complex I activity. Complex I activity was determined in isolated mito-
chondria by spectrophotometrically (340 nm) monitoring the oxidation of
JEM VOL. 204, September 3, 2007
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