ORIGINAL RESEARCH COMMUNICATION
NADPH Oxidase 2 Mediates Intermittent Hypoxia-Induced
Mitochondrial Complex I Inhibition:
Relevance to Blood Pressure Changes in Rats
Shakil A. Khan,1Jayasri Nanduri,1Guoxiang Yuan,1Brian Kinsman,1Ganesh K. Kumar,1
Joy Joseph,2Balaraman Kalyanaraman,2and Nanduri R. Prabhakar1
Previous studies identified NADPH oxidases (Nox) and mitochondrial electron transport chain at complex I as
major cellular sources of reactive oxygen species (ROS) mediating systemic and cellular responses to intermittent
hypoxia (IH). In the present study, we investigated potential interactions between Nox and the mitochondrial
complex I and assessed the contribution of mitochondrial ROS in IH-evoked elevation in blood pressure. IH
treatment led to stimulus-dependent activation of Nox and inhibition of complex I activity in rat pheochromocy-
toma (PC)12 cells. After re-oxygenation, Nox activity returned to baseline values within 3h, whereas the complex I
activity remained downregulated even after 24h. IH-induced complex I inhibition was prevented by Nox inhibi-
tors, Nox2 but not Nox 4 siRNA, in cell cultures and was absent in gp91phox-=Y(Nox2 knock-out; KO) mice. Using
pharmacological inhibitors, we show that ROS generated by Nox activation mobilizes Ca2+flux from the cytosol to
activity, which results in elevated mitochondrial ROS. Systemic administration of mito-tempol prevented the
sustained but not the acute elevations of blood pressure in IH-treated rats, suggesting that mitochondrial-derived
ROS contribute to sustained elevation of blood pressure. Antioxid. Redox Signal. 14, 533–542.
of breathing (*10–15s in adults), resulting in periodic de-
creases in arterial blood O2or intermittent hypoxia (IH). Pa-
tients with recurrent apneas develop several comorbidities,
including hypertension, sympathetic activation, and abnor-
malities of breathing. (24). Studies on adult and neonatal ro-
dent models suggest that reactive oxygen species (ROS)
mediate many of the systemic responses to IH, including
hypertension, sympathetic activation [reviewed in refs. (29,
35)], alterations in carotid body function (26, 27, 30), increase
in catecholamine secretion from the adrenal chromaffin cells
(14, 33), and changes in breathing (25). In addition, ROS sig-
factors and second messengers in IH-treated cell cultures (38,
39). Together, these studies suggest that ROS signaling me-
diates systemic and cellular responses to IH.
Membrane or cytosolic oxidases are the major sources
for enzymatic generation of ROS (18). Recent studies impli-
leep disordered breathing manifested as recurrent
apneas is characterized by transient, repetitive cessations
cated NADPH oxidases (Nox), especially Nox2 (also called
gp91phox), in IH-evoked changes in sleep behavior (40), sen-
sory long-term facilitation of the carotid body (27), and
plasticity of respiratory behavior (22). Besides oxidases, mi-
tochondrial electron transport chain complexes also generate
ROS (17, 23). For instance, mitochondrial complex I (E.C
220.127.116.11, proton-translocating NADH:ubiquinone oxidoreduc-
tase), which is responsible for the oxidation of NADH by
ubiquinone, is a major source of mitochondrial ROS (41). We
complex I, but not the complex III in IH-treated rodents (27)
and cell cultures (38). However, the mechanism(s) by which
IH inhibits complex I activity have not been examined. Given
that mitochondrial complex I activity can be altered by the
redox state (32), we hypothesized that Nox activation and the
resulting ROS mediates IH-induced inhibition of complex I.
We tested this possibility in IH-treated cell cultures and mice
deficient in Nox function. Our results demonstrate that ROS
generated by Nox, especially Nox2, inhibit complex I activ-
ity via Ca2þ-dependent S-glutathionylation of complex I
1Department of Medicine, Center for Systems Biology of O2Sensing, University of Chicago, Chicago, Illinois.
2Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin.
ANTIOXIDANTS & REDOX SIGNALING
Volume 14, Number 4, 2011
ª Mary Ann Liebert, Inc.
importantly, our data further showed that selective scaveng-
ing of mitochondrial ROS prevents IH-induced sustained but
not transient elevation of blood pressure in rats.
Materials and Methods
Exposure of cell cultures to IH
PC12 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% horse serum, 5%
fetal bovine serum (FBS), penicillin (100U=mL), and strep-
tomycin (100mg=mL) under 10% CO2and 90% air (20% O2)
at 378C. Before all experiments, the cells were serum starved
for 16h in antibiotic-free medium to avoid any confounding
effects of serum. In the experiments involving treatment
with drugs, cells were preincubated for 30min with either
drug or vehicle. Cell cultures were exposed to IH (1.5% O2
for 30s followed by 20% O2for 5min at 378C) as described
previously (38, 39). O2levels were monitored by an elec-
trode (Lazar) placed in the tissue culture medium and am-
bient O2levels were monitored by an O2analyzer (Beckman
Measurement of complex I activity
Complex I activity (NADH-ubiquinone reductase) was
measured as rotenone (50mM)-sensitive rate of NADH oxi-
dation and expressed in nanomoles of NADH oxidized per
min per mg protein as previously described (38). Protein
analysis was performed using Bio-RAD protein assay kit.
PC12 cells (5?105) were plated on collagen type IV (BD
Biosciences)-coated culture dish and were grown for 24h. The
transfection was performed with siRNAs specific for Nox2,
Nox4, or scrambled (control) sequences (Santa Cruz Bio-
technology; Cat # sc-61838 (Nox2), sc-61887 (Nox4), and sc-
37007 Scrambled; 100pmol=mL) using a DharmaFECT-2
(Dharmacon Research) according to the manufacturer’s rec-
ommendations. After transfection, cells were grown in the
medium containing serum and antibiotics for 48h before
subjecting to either 60 cycles of IH or normoxia.
Measurement of NADPH oxidase activity
Membrane-enriched fractions from PC12 cells and brain
were isolated as described (19). NADPH oxidase activity
in the membrane-enriched protein fractions was measured
using cytochrome c reduction assay as described (20). Briefly,
the assay medium contained 100mg membrane protein, cy-
tochrome c, and NADPH at a final concentration of 150 and
100mM, respectively, and 25mM modified HEPES buffer (pH
7). The assay was performed in the presence and absence of
superoxide dismutase (200 units=mL) at 378C for 30min.
Cytochrome c reduction was measured by reading absor-
bance at 550nm on a microplate reader. The amount of SOD
inhibitable NADPH oxidase activity was calculated using
extinction coefficient of 21mmol=(L cm) and expressed in
Measurement of aconitase activity
Mitochondrial fractions were isolated from cells or brain
extracts by differential centrifugation as described (15). Aco-
nitase enzyme activity was determined in the mitochondrial
fractions as described (38) and expressed as nanomoles of
isocitrate formed per minute per milligram of protein.
Measurement of S-glutathionylation
Cells were lysed in Mannitol=Tris=EDTA (MTE) buffer and
mitochondrial fractions were isolated by differential centri-
fugation. The mitochondrial pellet was solubilized in 20mM
N-dodecyl-b-d-maltopyranoside (1% final concentration) and
centrifuged at 72,000 g for 30min. Immune complexes were
precipitated from the supernatant using anticomplex I
monoclonal antibody (Mitosciences, Eugene, OR) and Protein
A=G agarose beads. Complexes were eluted from the beads
using SDS buffer without mercaptoethanol and analyzed on
10% SDS-PAGE nonreducing gels and transferred to a poly-
vinylpyrrolidone difluoride membrane (Immobilon-P; Milli-
pore). Membranes were incubated with antireduced form of
glutathione (GSH) mouse monoclonal antibody (Virogen) at
1:1000 dilution of stock in Tris-buffered saline- Triton x-100
containing 3% nonfat milk after overnight blocking in 5%
milk. Membranes were treated with goat anti-mouse sec-
ondary antibody conjugated with horseradish peroxidase
(dilution 1:2,000; Chemicon) and immune complexes were
system (Amersham BioSciences). The membranes were ex-
posed to Kodak XAR films.
Measurement of GSH and oxidized glutathione
GSH and oxidized glutathione levels were determined in
PC12 cells using Glutathione assay kit (#703002; Cayman
Chemicals). The sulfhydryl group of GSH reacts with DTNB
(5,50-dithio-bis-2-nitrobenzoic acid, Ellman’s reagent) to pro-
duce a yellow-colored 5-thio-2-nitrobenzoic acid (TNB)
product, the absorbance of which is measured at 405nm. The
rate of TNB production is directly proportional to the con-
centration of GSH. Quantification of oxidized from of gluta-
thione (GSSG) exclusive of GSH is done by first derivatizing
GSH with 2-vinylpyridine as described in the kit.
Exposure of mice and rats to IH and measurement
of blood pressure
Experiments were approved by the Institutional Animal
Care and Use Committee of the University of Chicago and
were performed on adult male rats (200–300g; Sprague
Dawley), wild type (Wt, C57BL=6), hemizygous gp91phox–=Y
(from Jackson Laboratories; weights 20–25gm). Rats were
treated with mito-tempol (10mg=Kg; intraperitoneal [IP]), a
selective scavenger of mitochondrial ROS or apocynin
(10mg=kg; IP) every day before 8h regimen of IH treatment
for 10 days. Unrestrained, freely moving animals housed in
feeding cages were exposed to intermittent hypoxia for 10
days (IH; 8h=day) as previously described (25–27). Briefly,
animals were placed in a specialized chamber, which was
flushed with alternating cycles of pure nitrogen and com-
pressed air.Duringhypoxia, inspired O2levelsreached 5%O2
(nadir). The gas flows were regulated by timer-controlled
solenoid valves. Ambient O2and CO2levels in the chamber
were continuously monitored by an O2=CO2analyzer (Series
9500; Alpha Omega Instrument). Control experiments were
performed on animals exposed to alternating cycles of com-
534KHAN ET AL.
pressed room air instead of hypoxia in the same chamber.
Blood pressures were monitored by the tail cuff method in
unanaesthetized rats using a noninvasive BP system (AD In-
struments) as described (28). Rats were placed in the re-
strainer provided by the manufacturer and were allowed to
acclimate for at least 1h before blood pressure measurements.
Baseline blood pressures were determined for 3 consecutive
days in the morning (9 a.m.) and in the evening (5 p.m.).
Subsequently, rats were exposed to IH for 10 days. Blood
pressures were determined at the end 10th day within 1 and
15h after terminating IH.
All chemicals were of analytical grade and obtained from
commercial sources. Mito-tempol was prepared in the labo-
ratories of Drs. Joseph and Kalyanaraman.
The data were expressed as mean?SEM from at least three
individual experiments. Two-way analysis of variance with
repeated measures followed by Tukey’s test was used to
evaluate the statistical significance and p-values <0.05 were
Time course of IH on Nox and mitochondrial
complex I activities
Nox and mitochondrial complex I activities were deter-
mined in PC12 cells treated with increasing durations of IH.
Nox activity increased progressively in membrane fractions
with increasing the duration of IH from 10, 30, and 60 cycles
(Fig. 1A). Analysis of Nox activity using whole cell also re-
vealed similar increase in response to IH (data not shown).
Analysis of complex I activity in mitochondrial fractions
showed progressive decrease with increasing the duration of
IH (Fig. 1B). After re-oxygenation, Nox activity returned to
baseline values within 3h (Fig. 1A), whereas complex I ac-
tivity remained downregulated significantly even after 24hof
re-oxygenation (p<0.05; Fig. 1B).
Nox=ROS signaling mediates IH-evoked
complex I inhibition
To assess whether Nox activation mediates IH-evoked
complex I inhibition, PC12 cells were treated with two
structurally distinct Nox inhibitors, apocynin (500mM) or
4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride
(300mM). Cells treated with Nox inhibitors were challenged
with 60 cycles of IH (i.e., IH60; the maximum duration of
IH used in this study). Both Nox inhibitors prevented
IH60-evoked Nox activation and complex I inhibition (Fig.
Among the several isoforms of Nox (1), Nox2 mediates IH-
evoked cellular responses in PC12 cells (39) and systemic re-
sponses in mice (27). The Nox inhibitors used in the above
experiments cannot distinguish between different Nox iso-
forms. Therefore, siRNA approach was employed to assess
the contribution of Nox 2 to IH-evoked complex I inhibition.
Nox2 and Nox4 proteins were upregulated in IH-treated cells
and this response was absent in cells transfected with Nox2
and Nox4 siRNAs (Fig. 2C; top panel). More importantly, IH-
evoked inhibition of complex I activity was absent in cells
treated with Nox2 but not Nox4 siRNA (Fig. 2C; bottom
To further establish a role for Nox2 in IH-induced complex
1 inhibition, experiments were performed on age-matched
wild-type (Wt) and hemizygous gp91phox–=Ymice (i.e., Nox2
knock-out mice) treated with 10 days of IH, a duration chosen
based on previous study (28). Nox and complex I activities
were analyzed in brain stem samples from both groups of
mice, We chose the brain stem region because it provides
adequate amount of tissue for biochemical assays and neu-
rons in this region mediate increased sympathetic tone, which
is a hallmark of IH [reviewed in ref. (29)]. Nox activity was
significantly elevated in IH-treated Wt mice but not in Nox2
KO mice (Fig. 2D). As in PC12 cells, mitochondrial complex I
activity was inhibited in IH-treated Wt mice and remarkably
this response was absent in Nox2 KO mice (Fig. 2E).
Ca2þmediates IH-evoked complex I inhibition
Ca2þinhibits mitochondrial complex I activity (32). IH ac-
tivates Nox and the ensuing ROS elevates basal [Ca2þ]ilevels
ibility of Nox and mitochondrial
complex I activities during IH. (A)
Nox activity was determined in
membrane fractions of PC12 cells
treated with increasing durations
of IH. Reversibility of the response
was determined by placing IH60-
treated cells under normoxia for
varying periods. (B) Complex I
activity was determined in mito-
chondrial fractions of PC12 cells
treated with different durations
of IH and after re-oxygenation.
Data are mean?SEM from four
independent experiments run in
triplicate. **p<0.01 and *p<0.05
compared to normoxic controls, respectively. IH60, cells exposed to 60 cycles of intermittent hypoxia; N, normoxia; Nox,
NADPH oxidase; n.s., not significant; PC12, pheochromocytoma 12.
Time course and revers-
MITOCHONDRIAL COMPLEX I INHIBITION BY INTERMITTENT HYPOXIA535
in PC12 cells (39). We therefore reasoned that elevated cyto-
solic [Ca2þ]iand the resulting Ca2þentry into mitochondria
inhibits complex I. To test this possibility, cells were treated
with increasing concentrations (1, 3, and 10mM) of 1,2-bis(o-
aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA-
AM), an intracellular Ca2þ-chelator, and as shown in Figure
3A, 10mM BAPTA-AM completely prevented IH-induced
complex I inhibition. Likewise, 10mM ruthenium red (RR) an
inhibitor of mitochondrial Ca2þuniporter, which mediates
Ca2þentry into mitochondria, also prevented complex I inhi-
bition in IH60-treated cells (Fig. 3A). Treating control cells
with Ca2þionophore (1.4mM ionomycin) inhibited the com-
plex I activity, and 10mM RR or BAPTA-AM prevented this
effect (Fig. 3B).
To further assess the effects of Ca2þ, complex I activity was
determined in the presence of varying concentrations of Ca2þ.
Increasing the concentration of Ca2þfrom 0–80mM progres-
sively inhibited complex I activity in control cells with a Kiof
5.3þ0.4mM and this effect was not seen in IH-treated cells
(Fig. 4A). Analysis of complex I activity as a function of
NADH concentration in the presence of 20mM Ca2þ(4xKi)
revealed a *2-fold reduction in Vmaxand Km(Fig. 4B). The
baseline Vmaxand Kmvalues in IH60-treated cells were sig-
nificantly lower than control cells but quite similar to those in
Ca2þ-treated control cells (Vmax: IH60¼20.92?0.14 vs. Con-
59.24?3.68 vs. Controlþ20mM Ca2þ¼69.50?2.02mM). Ad-
dition of 20mM Ca2þhad no further effect on either Vmaxor
Kmin IH60-treated cells (Fig. 4C).
IH increases S-glutathionylation
of mitochondrial complex I subunits
Ca2þ-induced inhibition of the complex I activity can be
reversed by sulfhydryl reducing agents, suggesting that oxi-
dative modification of sulfhydryl residues leads to inhibi-
tion of complex I (34). S-glutathionylation of 75-kDa protein
of complex I subunit represents one such sulfhydryl modifi-
cation reaction (12). We monitored S-glutathionylation of
75-kDa and *50kDa complex I proteins in control and IH60-
treated cells. Mitochondrial complex I proteins were im-
munoprecipitated and probed with anti-glutathione antibody.
apocynin or 300mM AEBSF on Nox and complex I activities in IH60-treated PC12 cells. (C) Effect of silencing Nox2 and Nox4
RNAs (siRNAs) on IH60-evoked complex I inhibition. Top panel represents Western blots of Nox2 and Nox4 protein ex-
pressions in cells treated with normoxia (lane 1), IH60(lane 2), IHþscr RNA (lane 3), IH60þNox2 siRNA (lane 4), and
IH60þNox4 siRNA (lane 5). Bottom panel presents average data (mean?SEM) of complex I activity. (D, E) Effect of 10 days of
IH treatment (IH10D) on Nox and complex I activities in brainstem tissue samples from wild-type and gp91phox-=Yknock-out
mice (n¼5 mice in each group). Data are mean?SEM from three to five independent experiments. **p<0.01 compared to
normoxic controls. AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; scr, scrambled; Wt, wild type.
Nox mediates IH-induced complex I inhibition. (A, B) Effects of two structurally distinct Nox inhibitors 500mM
536KHAN ET AL.
S-glutathionylation of 75- and 50-kDa subunits of complex I
increased by 2.5- and 3.5-fold, respectively, in IH60-treated
cells compared with control cells (Fig. 5A; panel 1). Similar
increases in S-glutathionylation of complex I subunits were
also seen in control cells challenged with a Ca2þionophore
(1.4mM ionomycin) (Fig. 5A, panel 2). RR (10mM) prevented
the increase in S-glutathionylation of complex I subunits in
1 and 2).
IH-evoked S-glutathionylation of complex I subunits was
absent in PC12 cells treated with 500mM apocynin or man-
ganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin, a ROS scav-
enger or transfected with Nox2 siRNA (Fig. 5A; panels 3 and
4). Further, IH treatment also increased S-glutathionylation of
mitochondrial complex I subunits in brain stem tissues from
Wt mice and this effect was absent in tissues from Nox2 KO
mice (Fig. 5A, panel 5). In addition, treatment of cells with N-
acetyl-cysteine, a precursor of glutathione, also prevented IH-
induced S-glutahtionylation of complex I subunits (Fig. 5A,
Glutathione prevents IH-induced complex I inhibition
S-glutathionylation requires oxidation of glutathione to
glutathione disulfide (GSSG). Therefore, we determined
whether IH affects the ratio of GSSG=GSH and whether ad-
dition of GSH reverses the inhibition of complex I activity in
IH60-treated cells. As shown in Figure 5B, ratio of GSSG=GSH
increased by *2-fold in IH60-treated cells compared with
controls. Addition of GSH to mitochondrial fractions from
IH60cells restored the complex I activity in a concentration-
dependent manner with a complete recovery occurring at
5mM GSH (Fig. 5C). Since oxidative modification of sulfhy-
dryl residues leads to inhibition of complex I, reducing agents
like dithiothreitol (DTT) should prevent IH-induced complex
1 inhibition. Indeed, treatment of cells with 1mM DTT pre-
vented inhibition of complex I by IH (Fig. 5D).
Analysis of the complex I activity as a function of NADH in
the presence and absence of 5mM GSH showed that GSH
increased the Vmaxof the reaction by 65% in IH60-treated cells,
whereas it did not alter Vmaxin the control cells (Fig. 6A, B).
On the other hand, GSH did not affect the Kmof NADH both
in the control as well as IH60-treated cells (Fig. 6A, B).
IH-induced complex I inhibition increases
We determined whether IH-evoked complex I inhibition
increases ROS generation in the mitochondria. The aconitase
bition. (A) Effect of Ca2þchelator BAPTA-AM (10mM
BAPTA) and RR, an inhibitor Ca2þuniporter (10mM) on
complex I activity in IH60-treated PC12 cells. (B) Effect of
ionomycin (1.4mM), a Ca2þionophore on complex I activ-
ity in normoxic PC 12 cells with and without RR. **p<0.01
compared to controls; BAPTA, 1,2-bis(o-aminophenoxy)eth-
ane-N,N,N0,N0-tetraacetic acid; RR, ruthenium red.
Ca2þis required for IH-evoked complex 1 inhi-
I activity in control PC 12 cells (o-o) but not in IH exposed cells (&-&) (inset: plot of the reciprocal of complex I activity (1=V)
versus Ca2þconcentration. Intercept represents the concentration required to produce 50% inhibition of the enzyme, that is,
Ki. (B, C) Mitochondrial fractions were assayed for complex 1 activity with increasing NADH concentrations (0–300mM) in
control (N) and IH60-treated cells in the absence (o-o) or presence (&-&) of 20mM Ca2þ. Inset represents the Eadie-Hofstee
plot of rate (V) versus V=S (S is the NADH concentration). Vmaxand Kmwere calculated from the intercept and slope,
respectively. Each data point represents the mean?SEM from three independent experiments.
Kinetic analysis of the effects of Ca2þon complex I activity. (A) Increasing Ca2þconcentrations inhibited complex
MITOCHONDRIAL COMPLEX I INHIBITION BY INTERMITTENT HYPOXIA537
enzyme activity was monitored in the mitochondrial fractions
as an index of ROS generation (10). Mitochondrial aconitase
activity progressively decreased as the duration of IH in-
creased from 10 to 60 cycles and remained significantly in-
hibited even after 24h of re-oxygenation (Fig. 7A). IH-evoked
inhibition of aconitase activity was prevented by Nox inhib-
itor (apocynin), Ca2þchelator (BAPTA-AM), RR, an inhibitor
of mitochondrial Ca2þuniporter, as well as by N-acetyl-
inhibition of mitochondrial aconitase activity was absent in
cells transfected with Nox2 but not with Nox4 or scrambled
siRNAs (Fig. 7C).
Mitochondrial ROS contributes to IH-induced
sustained elevation in blood pressure
Previous studies reported that antioxidant treatment pre-
vents IH-evoked hypertension in rodents [reviewed in refs.
(29, 35)], suggesting a role for ROS signaling. To address the
role of ROS generated by Nox and mitochondria in IH-
evoked hypertension, rats were treated with apocynin
(10mg=Kg; IP), a Nox inhibitor or mito-tempol (10mg=Kg;
IP), a selective scavenger of mitochondrial ROS (21) each day
before 8h regimen of IH treatment for 10 days. Blood
pressures were monitored *1 and *15h after terminating
the IH regimen in unsedated rats. The results are summa-
rized in Figure 8A. In vehicle-treated IH rats, mean blood
pressures were elevated measured after *1 as well as *15h
after terminating IH. Mito-tempol treatment abolished sus-
tained elevations in blood pressures measured at *15h
without affecting the blood pressure elevations seen within
1h after terminating IH. In contrast, apocynin treatment
prevented both early (within 1h) and sustained (after 15h)
elevations in blood pressure (Fig. 8A). Results similar to
apocynin treatment were also obtained in Nox-2 KO mice
(data not shown).
Mitochondrial aconitase and Nox activities were deter-
mined in brain stem samples from rats treated with IH alone
or in combination with mito-tempol or apocynin. In mito-
tempol-treated rats IH-evoked decreases in mitochondrial
increased S-glutathionylation of 75- and 55-kDa subunits of the complex I in IH60and ionomycin (Iono; 1.4mM)-treated PC12
cells and blockade of the responses by RR (10mM; panels 1 and 2). Blockade of IH60-induced S-glutathionylation of complex I
subunits by Nox inhibitor apocynin (Apo; 500mM) and antioxidant (MnTMPyP; 50mM; panel 3) and by Nox2 siRNA (panel 4).
S-glutathionylation of complex I subunits in brain stem cell lysates from wild-type (Wt) and gp91phox-=Y mice treated with
either 10 days of normoxia or IH (panel 5). Note the absence of increased S-glutathionylation of complex I subunits in tissues
lysates from IH-treated gp91phox-=Ymice. Blockade of IH60-induced S-glutathionylation of complex I subunits by N-acetyl-
cysteine (2mM NAC), a precursor of glutathione (panel 6). (B) Ratio of oxidized from of glutathione=GSH was determined in
normoxic (N) and IH60-treated PC12 cells as described in Materials and Methods. (C) IH-induced inhibition of complex I
activity is prevented in the presence of 2 and 5mM GSH or (D) 2mM dithiothreitol (DTT). Data represent the mean?SEM
from three to five independent experiments **p<0.01 compared to normoxic controls (N). GSH, reduced form of glutathione;
MnTMPyP, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin; NAC, N-acetyl-cysteine.
IH increases S-glutathionylation of mitochondrial complex I subunits. (A) Representative immunoblots showing
538 KHAN ET AL.
absent, whereas increased Nox activity was unaffected
IH-evoked decrease in mitochondrial aconitase and complex
I activities as well as increased Nox activity (Fig. 8B–D).
The present study reveals three novel and mechanistically
linked findings: (i) IH-evoked mitochondrial complex I inhi-
bition requires ROS generation by Nox2 and S-glutathiony-
lation of complex I subunits, (ii) inhibition of complex I
increases mitochondrial ROS, and (iii) mitochondrial ROS
contributes to sustained but not transient elevations in blood
pressure in IH-treated rats.
We previously reported that IH activates Nox (26) and
inhibits mitochondrial complex I activity (27) in the rat ca-
rotid body. However, carotid bodies being too small in size
(weighing 50–80mg) precluded analysis of potential interac-
tions between Nox and complex I. PC12 cells respond sim-
ilarly to IH with increased Nox and decreased mitochondrial
complex I activities (38, 39) (Fig. 1), which prompted us to
assess the interactions between Nox and mitochondrial
complex I in this cell line. We further verified the data from
PC12 cell cultures with brain stem tissue from intact mice or
rats and found that IH increased Nox and decreased com-
plex I activities in both preparations. However, Nox activity
measured in brainstem tissue was lower than in PC12 cells.
It is likely that Nox is expressed in certain population of
neurons or a subset of glial cells, whereas PC12 cells are a
homogenous population. The heterogeneity of brain stem
tissue might account for the relatively lower complex I and
Nox activities compared to PC12 cell cultures. Further, we
also found similar changes in Nox and complex 1 activity in
cerebral cortical tissue samples from IH-treated rats and
mice, suggesting that these responses can be seen in other
neural tissues as well.
activity in the mitochondrial fractions from PC12 cells treated with normoxia (N) or to increasing durations of IH as indicated
(IH cycles). For reversibility studies, PC12 cells exposed to IH60were placed in normoxia for indicated duration (h). (B) Effect
of apocynin (500mM), BAPTA (10mm), RR (10mm), and MnTMPyP (50mM), NAC (500mM) on mitochondrial aconitase
activity in IH60-treated PC12 cells. (C) IH-induced inhibition of mitochondrial aconitase activity was absent in PC12 cells
transfected with Nox2 siRNA but not with Nox4 siRNA or scr RNAs. Data are mean?SEM from three to four independent
experiments. **p<0.01 compared to normoxia. ROS, reactive oxygen species.
IH increases mitochondrial ROS. Mitochondrial aconitase activity was determined as index of ROS. (A) Aconitase
complex I inhibition by restoring
Vmax. Complex I activity was deter-
mined as a function of NADH con-
centration with (&-&) and without
(o-o) addition of 5mM GSH in control
(N) (A) and IH60-treated PC12 cells
(B). Insets represent Eadie-Hofstee
plots of rate (V) versus V=S (S is the
NADH concentration). Vmaxand Km
are calculated from the intercept and
GSH prevents IH-evoked
MITOCHONDRIAL COMPLEX I INHIBITION BY INTERMITTENT HYPOXIA 539
The following observations demonstrate that activation of
two structurally distinct inhibitors of Nox prevented IH-
evoked inhibition of complex I; (b) genetic silencing of Nox2
but not Nox 4 abrogated complex I inhibition in IH-treated
cells, and (c) complex I inhibition by IH was absent in tissues
from Nox2 KO mice. Consistent with previous studies (3, 5, 7,
13) we found that activation of Nox increases mitochondrial
ROS. It has been proposed that mitochondrial permeability
transition pore (mPTP) plays a critical role in mitochondrial
redox signaling (6, 11, 21). The possibility that IH-induced
mitochondrial ROSmightbeduetochanges inmPTPrequires
further investigation. The finding that Nox activation by IH
causes mitochondrial complex I inhibition under IH is remi-
niscent of mitochondrial dysfunction by Nox activation by
angiotensin (37) and nitoglycerine (36). Nox activation by IH
was transient and reversible, whereas complex I inhibition
and the ensuing ROS triggers prolonged ROS generation by
mitochondria via inhibition of complex I activity. In other
words, these data indicate that IH evokes ROS-induced ROS
mechanism. Taken together these observations demonstrate a
functional cross-talk between Nox and mitochondrial com-
plex I activity under IH leading to mitochondrial dysfunction.
Whether increased mitochondrial ROS impacts Nox activa-
tion as proposed recently (16, 31) also plays a role in IH re-
main to be investigated.
Our results provide further insight into how Nox activation
by IH inhibits complex I. Yuan et al. (39) reported that ROS
generated by Nox leads to persistent elevation of baseline
[Ca2þ]iin IH-treated PC12 cells. The following observations
demonstrate that Ca2þis critical for evoking complex I inhi-
bition in IH-treated cells: (a) BAPTA-AM, a Ca2þchelator or
RR, an inhibitor of mitochondrial Ca2þuniporter prevented
IH-evoked complex I inhibition; (b) ionomycin, a Ca2þiono-
was prevented by RR; and (c) Ca2þinhibited the complex I
activity in control cells, and this effect was occluded in IH-
treated cells. It can be argued that RR may inhibit Ca2þrelease
from the sarcoplasmic reticulum as evidenced in normal car-
diomyocytes (4). However, RR at the concentrations that pre-
vented complex I inhibition in IH-treated cells had no impact
on complex I activity in control PC12 cells. Taken together,
critical for complex I inhibition in IH-treated cells.
How might Ca2þinhibit the mitochondrial complex I ac-
tivity? Previous studies identified two catalytically distinct
forms of mitochondrial complex I, including an active A-form
and the other deactivated D-form (8). Ca2þand other divalent
the complex I with altered substrate affinity (9). IH decreased
the substrate affinity of the complex I similar to that seen in
control cells treated with Ca2þ, suggesting that IH causes
Ca2þ-dependent conformational change in complex I similar
to that reported with ischemia and re-perfusion in rat heart
preparation (32). Previous studies have shown that the de-
redox modifications (8). Indeed IH caused redox modulation
tration of mitochondrial ROS scav-
enger mito-tempol or apocynin, a Nox
inhibitor on blood pressure, mito-
chondrial aconitase, and NOX activity
in rats exposed to 10 days IH. Adult
rats were exposed to 10 days of IH
and were treated daily with either
saline (IH) or mito-tempol (10mg=
kg=day; intraperitoneal) or apocynin
(10mg=kg=day; intraperitoneal). Con-
trol experiments were performed on
rats exposed to normoxia (N). (A)
Mean blood pressure values mea-
sured *1 and *15h after terminating
10 days of IH with and without
mito-tempol or apocynin treatment.
(B, C) Treatments with mito-tempol
or apocynin prevented IH-induced
decrease in mitochondrial aconitase
and complex 1 activities. (D) IH-
evoked increase in Nox activity was
unaffected by mito-tempol but pre-
vented by apocynin treatment. Data
presented are mean?SEM from six
rats in each group. **p<0.01.
Effects of systemic adminis-
540KHAN ET AL.
of modification of complex I as evidenced by increased
S-glutathionylation of 75 and 50kDa subunits both in cell
cultures and in the brain tissues from IH-treated mice. The
findings that IH-evoked S-glutathionylation can be blocked
by a Ca2þchelator as well as RR and mimicked by a Ca2þ
ionophore further support the notion that IH-induced S-
glutationylation involves a Ca2þ-dependent conformational
change of complex I subunits.
S-glutathionylation, in addition to conformational change
of the complex I, also requires elevated GSSG levels (2). Not
only did IH elevate GSSG as evidenced by increased ratio of
GSSG=GSH, but more importantly, addition of GSH (reduced
form) abrogated complex I inhibition and restored the Vmaxof
the reaction in IH-treated cells. These results demonstrate
that S-glutathionylation is the critical signaling event re-
sponsible for IH-evoked complex I inhibition. The findings
that IH-evoked S-glutathionylation could be prevented by a
Nox inhibitor as well as by genetic silencing of Nox 2 in
cell cultures and was absent in IH-treated Nox2 KO mice
suggest that Nox 2 activation by IH and the resulting ROS
IH increased mitochondrial ROS, as evidenced by de-
creased mitochondrial aconitase activity, an in vivo marker of
ROS. This increase in mitochondrial ROS was due to complex
I inhibition, because preventing complex I inhibition abro-
gated the changes in mitochondrial aconitase activity. What
might be the physiological significance of increased mito-
chondrial ROS by IH? One of the hallmarks of IH is the ele-
vated blood pressures [reviewed in (29) and the present
study]. Although ROS signaling has been implicated in IH-
induced hypertension (28, 35), the relative importance of mi-
that treatment with mito-tempol, a mitochondrial ROS scav-
enger, selectively abolished sustained but not the acute ele-
vations of blood pressure in IH-treated rats, suggesting a role
for mitochondrial ROS in IH-evoked hypertension. On the
other hand, Nox inhibition by apocynin prevented both the
transient as well as sustained elevations in blood pressures.
Although cellular mechanisms by which mitochondrial ROS
contribute to sustained elevations in blood pressure in IH-
treated rats remain to be investigated, our results suggest a
role for mitochondrial ROS generated by complex I inhibition
resulting from Nox activation in mediating IH-induced long-
lasting elevations of blood pressures.
This work was supported by National Institutes of Health–
National, Heart, Lung, and Blood Institute Grants HL-90554,
HL-76537, and HL-86493.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Nanduri R. Prabhakar
Department of Medicine
Center for Systems Biology of O2Sensing
University of Chicago
Emergency Medicine, MC5068
5841 South Maryland Ave.
Chicago, IL 60637
Date of first submission to ARS Central, March 25, 2010; date
of final revised submission, July 8, 2010; date of acceptance,
July 10, 2010.
AEBSF¼4-(2-aminoethyl) benzenesulfonyl fluoride
GSH¼reduced form of glutathione
GSSG¼oxidized from of glutathione
IH60¼cells exposed to 60 cycles of intermittent
ROS¼reactive oxygen species
542KHAN ET AL.