Published online before print June 7, 2001, doi: 10.1161/hh1201.091960
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© 2001 American Heart Association, Inc.
Cellular Biology |
Model for Hypoxic Pulmonary Vasoconstriction Involving Mitochondrial Oxygen Sensing
From the Department of Medicine, The University of Chicago, Chicago, Ill.
Correspondence to Paul T. Schumacker, PhD, Department of Medicine, MC6026, The University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637. E-mail pschumac{at}medicine.bsd.uchicago.edu
Abstract
Abstract—We
tested whether mitochondria function as the O2
sensor underlying hypoxic pulmonary vasoconstriction (HPV). In
buffer-perfused rat lungs, rotenone, myxothiazol, and
diphenyleneiodonium, which inhibit mitochondria in the proximal region
of the electron transport chain (ETC), abolished HPV without
attenuating the response to U46619. Cyanide and antimycin A inhibit
electron transfer in the distal region of the ETC, but they did not
abolish HPV. Cultured pulmonary artery (PA) myocytes contract
in response to hypoxia or to U46619. The hypoxic response was
abolished while the response to U46619 was maintained in mutant
(
0) PA myocytes lacking a mitochondrial
ETC. To test whether reactive oxygen species (ROS) derived from
mitochondria act as signaling agents in HPV, the antioxidants
pyrrolidinedithiocarbamate and ebselen and the Cu,Zn superoxide
dismutase inhibitor diethyldithiocarbamate were used. These
abolished HPV without affecting contraction to U46619, suggesting that
ROS act as second messengers. In cultured PA myocytes, oxidation of
intracellular 2',7'-dichlorofluorescin diacetate (DCFH) dye increased
under 2% O2, indicating that myocytes increase
their generation of H2O2
during hypoxia. This was attenuated by myxothiazol, implicating
mitochondria as the source of increased ROS during HPV. These results
indicate that mitochondrial ATP is not required for HPV, that
mitochondria function as O2 sensors during
hypoxia, and that ROS generated in the proximal region of the
ETC act as second messengers in the response.
Key Words: reactive oxygen species • hypoxia • redox signaling • pulmonary circulation • oxidants
Hypoxic pulmonary vasoconstriction (HPV) diverts blood flow away from the lung during fetal development and optimizes lung gas exchange after birth by enhancing the matching of blood flow and ventilation. Excised lungs retain the HPV response.1 2 3 4 5 6 Rings of pulmonary artery (PA) constrict under low O2 conditions7 8 even if denuded of endothelium.9 10 Even isolated PA myocytes contract during hypoxia,11 indicating that the O2 sensor is intrinsic to those cells.
Although HPV has been well characterized, the underlying mechanism of O2 sensing is not established. Among the putative O2 sensors that have been proposed, mitochondria have been discounted because the Km of cytochrome oxidase for O2 is too low to permit detection of physiological hypoxia.12 Moreover, inhibition of cytochrome oxidase with cyanide failed to abolish HPV.2 However, our studies have implicated mitochondria in the O2 sensing underlying functional and transcriptional responses to hypoxia in other cells.13 14 15 Those data suggest that mitochondria generate reactive oxygen species (ROS) in response to low PO2, which constitutes an O2-dependent signal.14 15 Oxidant signaling during hypoxia appears to originate at complex III, which could continue to function despite inhibition of complex IV with cyanide. The present study sought to determine whether mitochondria also function as the O2 sensor during HPV and whether ROS generated by mitochondria function as second messengers in that response.
Materials and Methods
Isolated Perfused Lung
Lungs from Sprague-Dawley rats (Harlan Sprague
Dawley, Indianapolis, Ind) were isolated as described
previously.16 Lungs and
heart were removed en bloc and the PA and left atrium were cannulated
and perfused (8 mL/min) with a buffered salt solution containing BSA
(0.5% wt/vol) and indomethacin (10 mg/L).
Perfusate was maintained at 38°C, pH 7.4, and bubbled with
5% O2, 5% CO2, and 90%
N2. Lungs were ventilated with a humidified
mixture of 21% O2, 5%
CO2, and 74% N2
(normoxia) at 54 breaths/min, tidal volume of 2 to 3 mL, and
end-expiratory pressure of 3 cm H2O. Left
atrial and PA pressures were continuously recorded. All animals
were housed and cared for under National Research Council guidelines
for care and use of laboratory animals.
HPV in Isolated Lungs
Angiotensin II (10 nmol/L) was added to
the perfusate, and HPV was induced by switching from normoxia
to hypoxia (2% O2, 5%
CO2, 93% N2).
Hypoxia-induced changes in pulmonary vascular impedance
are represented as the change in PA pressure during
constant flow, compared with normoxia, in
cm H2O. Two hypoxic challenges were averaged to
define the baseline response before experimental intervention. The
experimental agents were added to the reservoir and recirculated, after
which two more hypoxic challenges were administered and averaged. In
the continued presence of the agents, the stable
thromboxane A2 analogue U46619 (5
ng/mL) was added to the reservoir to determine the vasoconstrictor
response to this receptor-mediated agonist.
PA Myocytes
PA microvessel myocytes were isolated after the
method of Marshall et al.7
Myocytes were plated on collagen-coated coverslips and grown until 70%
or 15% confluent, for 2',7'-dichlorofluorescin diacetate (DCFH) or
contraction studies, respectively. Mutant
(0) PA myocytes were generated from
wild-type cells by incubation in ethidium bromide (25 ng/mL) for 2
weeks.17 This inhibits
replication of mitochondrial DNA, which encodes critical subunits of
the ETC.18 The absence of
cytochrome oxidase subunit II was confirmed by polymerase chain
reaction. Myocytes on coverslips were placed in a flow-through chamber
on an inverted microscope and studied under controlled
[O2] at 37° using Hoffman-modulation optics.
Cell contraction was assessed from changes in cell length after 30
minutes and was expressed as percent decrease from the original length
at t=0, {[(original
length)-(length at 30 minutes)/(original
length)]x100}.11
Measurement of ROS
ROS generation in PA myocytes was assessed using
DCFH-DA (5 µmol/L, Molecular Probes). In the
presence of H2O2, this
probe is oxidized to 2',7'-dichlorofluorescein (DCF), which
was quantified using fluorescence imaging (excitation: 488 nm,
emission: 535 nm) and reported as percent of initial values, after
subtracting background (Universal
Imaging).
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Results
HPV in Isolated Perfused Lung
Figure 1A
shows a representative PA pressure
tracing for an isolated rat lung during alveolar hypoxia or in
response to U46619. Hypoxia (2% O2, 5%
CO2, 93% N2) increased
PA pressure by 8.3±0.6 cm H2O
(Hypoxia,
Figure 1B). Administration of drug vehicle (0.1% DMSO) had
no effect on the HPV response (7.2±0.6 cm H2O)
compared with hypoxia. Addition of U46619 (5 ng/mL) increased
PA pressure by 17.8±2.5 cm H2O, confirming the
ability to respond to a receptor-mediated vasoconstrictor. Washout and
replacement with fresh perfusate containing
angiotensin II had no effect on HPV (8.3±0.5
cm H2O). These results confirm the suitability
of this model for studies of HPV.
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Mitochondria as O2
Sensors During HPV
To determine the requirement for electron transport in
HPV, the flavoprotein inhibitor diphenyleneiodonium (DPI)
was added to the isolated lung perfusate before
hypoxia. DPI (10 µmol/L) blunted HPV compared with controls
(6.6±0.6 cm H2O) without affecting the
response to U46619 (11±3.8 cm H2O)
(Figure 2A). In cultured PA myocytes, hypoxia
elicited contraction compared with normoxia; this response was
attenuated in the presence of DPI
(Table 1).
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|
To further clarify the mitochondrial ETC requirement for
HPV, the inhibitor rotenone was added to the
perfusate before hypoxia. Rotenone at 5 µg/mL
inhibited HPV compared with controls (6.2±0.6
cm H2O)
(Figure 2B
), but it also abolished the response to U46619
(1.2±0.2 cm H2O). Rotenone at 50 ng/mL
inhibited the rate of O2 uptake by isolated
pulmonary cells by 78%, and it abolished HPV (8.0±1.0
cm H2O)
(Figure 2C) without attenuating the response to U46619
(13.2±1.5 cm H2O). At 5 ng/mL, rotenone had no
effect on HPV or the response to U46619
(Figure 2D). In cultured PA myocytes, rotenone at 50 ng/mL
abolished the contractile response to hypoxia without affecting
the response to U46619
(Table 1).
Myxothiazol inhibits complex III by blocking electron
transfer to the Rieske iron-sulfur center in the
bc1
complex.19 Myxothiazol at 50
ng/mL inhibited lung mitochondrial O2 uptake by
99%. It also attenuated HPV (1.6±0.2 cm H2O)
compared with controls (12.2±0.8 cm H2O),
without affecting the response to U46619 (17±0.6
cm H2O)
(Figure 3A). These effects were reversed after washout with
fresh perfusate (11.6±1.9 cm H2O). In
cultured PA myocytes, myxothiazol attenuated the contractile response
to hypoxia without affecting the response to U46619
(Table 1).
|
Antimycin A inhibits the oxidation of cytochrome
b562 at
complex III.19 Antimycin A
(10 ng/mL) inhibited HPV but also attenuated the response to U46619
(not shown). At 1 ng/mL, antimycin A attenuated mitochondrial
O2 uptake by 97% without inhibiting HPV
(8.9±3.7 cm H2O) compared with controls
(7.4±1.4 cm H2O) and without affecting the
response to U46619 (13.8±2.5 cm H2O)
(Figure 3B). In PA myocytes, antimycin A elicited contraction
during normoxia
(Table 1). Cyanide inhibits complex IV of mitochondria.
Cyanide at 10 µmol/L inhibited mitochondrial
O2 uptake by 89% and augmented HPV (10.2±0.8
cm H2O) compared with controls (7.6±0.5
cm H2O) without affecting the response to
U46619 (14.8±1.2 cm H2O)
(Figure 3C).
To clarify the requirement for mitochondria in HPV, mutant
0 cells were generated from wild-type rat
PA myocytes. Contraction of 0 cells under
hypoxia (2% O2) was attenuated compared
with controls, while the response to U46619 was preserved
(Table 1).
Increased ROS Signaling in HPV
To determine whether increases in ROS are required for
HPV, the thiol reductant pyrrloidinedithiocarbamate (PDTC) was added
before hypoxic challenge of isolated lungs. As an
antioxidant,15 20
PDTC appears to enhance
H2O2 clearance by
reducing oxidized glutathione. PDTC (5 µmol/L) attenuated HPV to
2.6±0.7 cm H2O compared with controls
(7.3±0.8 cm H2O)
(Figure 4A), without affecting the response to U46619
(6.8±1.3 cm H2O). The HPV response was
restored (6.2±0.7 cm H2O) after the PDTC was
washed out and perfusate was replaced. At 10 µmol/L, PDTC
also blocked HPV (2.1±0.5 cm H2O) compared
with controls (7.4±1.1 cm H2O), without
affecting the response to U46619 (9.2±1.2
cm H2O)
(Figure 4B). However, HPV was not restored (2.4±0.5
cm H2O) after washout of this higher
concentration. In cultured PA myocytes, PDTC inhibited the contractile
response to hypoxia
(Table 1).
|
Ebselen, a synthetic glutathione peroxidase, was added to
the perfusate before hypoxic challenge to clarify the role of
H2O2 in HPV. Ebselen (50
µmol/L) attenuated HPV (1.0±0.1 cm H2O)
compared with controls (6.4±0.4 cm H2O)
(Figure 4C), without altering the response to U46619
(8.1±1.1 cm H2O). The HPV response was not
restored after washout (1.4 cm H2O). In
cultured PA myocytes, ebselen also attenuated contraction during
hypoxia
(Table 1).
To clarify the relative importance of superoxide versus
H2O2 in HPV, a cytosolic
Cu,Zn superoxide dismutase (SOD) inhibitor,
diethyldithiocarbamate (DDC), was added to the perfusate before
hypoxic challenge. By inhibiting the formation of
H2O2 in the cytosol, this
should attenuate HPV if
H2O2 is required for
signaling, but could enhance the response if superoxide itself was
involved. DDC (1 mmol/L) blunted HPV (2.1±0.5
cm H2O) compared with controls (6.5±0.8
cm H2O)
(Figure 4D) without inhibiting the response to U46619
(18.3±0.8 cm H2O). Inhibition of HPV by DDC
was reversed after washout (6.2±0.5 cm H2O).
To test whether peroxide is sufficient to cause vasoconstriction,
H2O2 (100 µmol/L) was
added during normoxia. This increased PA pressure by 4.1±0.4
cm H2O
(Table 2). When administered to normoxic cultured PA
myocytes, H2O2 also
elicited contraction
(Table 1).
|
Some mitochondrial inhibitors induce
pulmonary vasoconstriction during
normoxia.1 2 In our
study, DPI (10 µmol/L), rotenone (50 and 5 µg/mL), antimycin A (10
ng/mL), and cyanide (10 µmol/L) caused transient (<10 minutes)
increases in PA pressure during normoxia
(Table 2). However, myxothiazol (50 ng/mL) had no effect.
The transient increase in PA pressure induced by cyanide was attenuated
by pretreatment with wither ebselen or myxothiazol. Neither PDTC,
ebselen, DDC, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid
(DIDS), nor apocynin altered PA pressure during
normoxia.
Mitochondrial Generation of ROS During
Hypoxia
To determine the source of ROS during hypoxia,
cultured PA myocytes were studied using DCFH dye at 37°C in a
flow-through chamber. When the [O2] bubbling
the media was switched from 16% to 2% O2, DCF
fluorescence increased
(Figure 5A). Return to 16% O2 was
associated with a return toward baseline in each case, which likely
reflects leakage of oxidized dye from the cells. The response to
hypoxia was repeated in the same field of cells before and
after addition of myxothiazol. At 100 ng/mL, myxothiazol attenuated the
increase in DCFH oxidation during hypoxia
(Figure 5B).
|
Superoxide generated in the mitochondria would require an
anion channel to reach the cytosol. If so, then an
inhibitor of anion channels should attenuate cytosolic ROS
signaling during hypoxia. To test this, we evaluated the anion
channel inhibitor DIDS in perfused lungs. DIDS (200
µmol/L) inhibited HPV (2.8±0.7 cm H2O)
compared with hypoxia alone (9.4±0.7
cm H2O)
(Figure 3D), without affecting the response to U46619
(13.4±2.4 cm H2O). HPV was restored after
washout (8.4±1.1 cm H2O). In PA myocytes, DIDS
blunted contraction during hypoxia without affecting the
response to U46619. DIDS also attenuated the contractile response to
antimycin A seen during normoxia
(Table 1).
Alternative Sources of ROS
To test the involvement of NADPH oxidase in
HPV,4 7 the
inhibitor apocynin was added before hypoxia.
Apocynin (3 mmol/L) abolished HPV compared with controls (6.3±0.7
cm H2O), but it also abolished the response to
U46619 (0.6±0.2 cm H2O). At a lower
concentration (300 µmol/L) that suppresses the respiratory burst of
alveolar
macrophages,21
apocynin failed to inhibit HPV (6.6±0.6
cm H2O) compared with controls (9.5±1.0
cm H2O), but did not inhibit the response to
U46619 (12.7±2.3
cm H2O).
Discussion
Role of Mitochondria as the
O2 Sensor in HPV
We used parallel studies in intact lungs and in
cultured PA myocytes to test whether mitochondria function as the
O2 sensor underlying HPV. In perfused lungs,
inhibitors of the proximal region of the mitochondrial ETC
including DPI, rotenone, and myxothiazol abrogated the
hypoxia-induced increase in PA pressure without affecting the
response to U46619. In PA myocytes, these inhibitors also
abolished contraction during hypoxia. By contrast, more distal
inhibitors of the ETC failed to abolish HPV. In this
regard, cyanide augmented the HPV response, and antimycin A caused
constriction of PA myocytes during normoxia. Thus, the response to
hypoxia requires electron transport but does not require
mitochondrial ATP because all of the inhibitors block
oxidative phosphorylation, yet only the proximal
inhibitors selectively abolish HPV.
To use a nonpharmacological approach, we generated mutant
0 cells from wild-type PA myocytes. These
cells lack mitochondrial DNA, which encodes a number of essential
subunits in the ETC
complexes.17 These cells do
not respire and depend on glycolytic ATP, yet they are morphologically
indistinguishable from wild-type cells. We observed that
0 PA myocytes retain the contractile
response to U46619 but fail to respond to hypoxia. These
findings support the conclusion that mitochondria function as the
O2 sensor underlying HPV. Interestingly, we
previously found that 0 Hep3B selectively
lose the ability to activate the transcription factor HIF-1
during hypoxia, yet they retained the ability to respond to
other stimuli such as cobalt
chloride.15 Collectively,
these observations suggest that a similar mitochondrial
O2 sensing mechanism may be responsible for HPV
and for HIF-1–mediated transcriptional activation during
hypoxia.
ROS as Second Messengers in HPV
Our results suggest that the mitochondrial ETC acts as
an O2 sensor during hypoxia by releasing
ROS that function as signaling messengers
(Figure 6). Superoxide generation is known to occur at the
ubisemiquinone site of complex III via univalent electron transfer to
O2.22
The antioxidants PDTC, ebselen, and DDC blocked the response to
hypoxia without affecting the U46619 response, suggesting that
ROS and, in particular,
H2O2 act as second
messengers. The predicted increase in oxidant signaling during
hypoxia was confirmed using the intracellular probe DCFH in
cultured PA myocytes during hypoxia. Myxothiazol attenuated the
increase in fluorescence during hypoxia,
consistent with its expected inhibition of ubisemiquinone
generation at complex III. These results further implicate ROS
generated by the mitochondrial ETC in the signaling
process.
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Previous studies also implicate increased ROS generation in the response to hypoxia. Monaco et al23 showed that HPV was augmented when catalase was inhibited with aminotriazole. Weissmann et al5 observed that SOD and 4,5-dihydroxy-1,3-benzenedisulfonic acid (to accelerate H2O2 generation from superoxide) did not affect HPV, suggesting that H2O2, rather than superoxide, is involved. By contrast, nitrobluetetrazolium, which traps superoxide and prevents H2O2 formation, attenuated HPV. Finally, our data and previous studies show that H2O2 constricts the pulmonary circulation during normoxia.24 These findings are consistent with a role for increased H2O2 as a signaling molecule involved in HPV.
Mechanism of ROS Generation During
Hypoxia
Our previous studies demonstrated that hypoxia
affects cytochrome oxidase, causing it to cycle at a more reduced
state.25 We had suggested
that the increase in reduction state of that complex should cause a
similar redox change at more proximal ETC sites, which might explain
the increase in ROS generation at complex III during
hypoxia.14 However,
we later observed that hypoxia still increased ROS generation
when electron transport at the distal end of complex III was inhibited
by antimycin A.15 In the
presence of an inhibitor, more proximal ETC complexes
become fully reduced while those at more distal locations become
oxidized. The observations that hypoxia augmented ROS signaling
during antimycin A, and that antimycin A and cyanide failed to abolish
the hypoxic constriction, suggest that the
O2-sensing site must be located upstream from
the antimycin A inhibition site. Moreover, the
O2 sensor must still be able to function if the
ETC chain is fully reduced.
During normoxia, ETC inhibitors acting at sites
distal to ubisemiquinone (eg, cyanide, azide, or antimycin A) tend to
augment ROS generation by increasing the reduction state of the
ubiquinone
pool.14 15 19 26
During hypoxia, our model suggests that the biophysical process
for ROS generation from that site is amplified, even when the complex
is fully reduced. By contrast, if the complex becomes fully oxidized by
ETC inhibition at a more proximal site (eg, rotenone, DPI, or
myxothiazol) then ROS generation and O2 sensing
are abolished by the lack of electrons. In accordance with this model
(Figure 6), distal inhibitors of the ETC such as
cyanide induced constriction during normoxia, through a mechanism that
could be inhibited by ebselen (an antioxidant) or myxothiazol (a more
proximal ETC inhibitor). Similarly, Rounds and
McMurtry1 previously found
that antimycin A elicits vasoconstriction in normoxic lungs, a response
we reproduced
(Table 2). Also, Archer et
al2 found that cyanide
induced vasoconstriction during normoxia and augmented HPV without
affecting the response to angiotensin II or KCl.
Collectively, these observations are consistent with our
proposed model, but the mechanism by which hypoxia amplifies
ROS generation at complex III is not yet known.
An alternative explanation for the increase in ROS signaling during hypoxia involves the regulation of superoxide egress from mitochondria to cytosol. Superoxide would presumably require an anion channel to escape from the matrix. The inner membrane anion channel (IMAC) could conceivably function as that pathway. If the IMAC were an O2-sensitive channel that increased its conductance during hypoxia, the egress of superoxide from the matrix could increase even if the rate of mitochondrial superoxide generation were to decrease during hypoxia. This could explain why ROS signaling in the cytosol is increased during moderate hypoxia, and why DIDS, which inhibits mitochondrial IMAC,27 28 attenuated HPV and abolished PA myocyte contraction during hypoxia. It should be noted that DIDS is also a thiol-reactive compound; however, pretreatment with DIDS had no effect on H2O2-induced vasoconstriction in the lung (see online data supplement available at http://www.circresaha.org). In either case, it appears that superoxide enters the cytosol where it is dismuted to H2O2 by cytosolic Cu,Zn SOD. Inhibition of SOD by DDC abrogated the hypoxic responses, indicating that superoxide conversion to H2O2 is required for the response.
Downstream Signaling in HPV
Smooth muscle contraction during HPV requires an
increase in cytosolic [Ca2+]. Although
H2O2 appears to act as a
signaling messenger in the sequence leading to calcium activation, the
details of that pathway are not fully understood. One possibility is
that potassium channels become inhibited through redox-mediated
signaling, resulting in membrane depolarization and the opening of
voltage-dependent calcium
channels.2 9 10 11 29 30 31
However, other studies show that PA myocytes demonstrate increases in
cytosolic [Ca2+] and cell contraction in
the presence of 4-aminopyridine, a pharmacological
inhibitor of these Kv
channels.32 Their results
indicate that HPV can be elicited by a mechanism that does not require
closure of Kv channels. One possibility is that
H2O2 might cause
oxidation of mitochondrial pyridine nucleotides, resulting
in mitochondrial Ca2+
release.33 However, further
studies are required to address this theory.
Drug-Induced Contraction During
Normoxia
In previous studies, the mitochondrial
inhibitor rotenone attenuated HPV but also produced a
transient increase in PA pressure immediately after
administration.1 Rounds and
McMurtry1 suggested that this
was due to an inhibition of ATP production, whereas Archer et
al2 suggested that a shift in
the cytosolic redox status elicits contraction. We also observed a
small transient increase in PA pressure on addition of rotenone,
cyanide, or DPI. However, myxothiazol had no such effect, so not all
ETC inhibitors elicit vasoconstriction. We suggest that the
transient responses to some compounds reflect nonspecific effects on
the pulmonary circulation. By contrast, their effects on the
subsequent response to hypoxia reflect their influence on
HPV.
Alternative ROS Sources
Marshall et
al7 suggested that
hypoxia accelerates ROS generation by a membrane-bound NADPH
oxidase, based on their observation that DPI inhibited the oxidant
signal and the contractile response to hypoxia. DPI inhibits a
wide range of flavoproteins including NADPH oxidase, mitochondrial
complex I,34 glutathione
reductase, nitric oxide synthase, and prostaglandin
synthetase.34 35
Therefore, the site of inhibition responsible for the attenuation of
HPV is not known. It is conceivable that DPI abolished HPV by
inhibiting mitochondrial complex I and abolished their
chemiluminescence signal through a separate effect on NAD(P)H oxidase.
Grimminger et al4 also found
that DPI attenuated HPV without affecting the vascular response to
U46619, consistent with our findings. To address the possible
involvement of NADPH oxidase in HPV, we used apocynin, a selective
inhibitor of the neutrophil form of this
enzyme.21 At concentrations
that preserved the response to U46619, apocynin failed to attenuate
either HPV or PA myocyte contraction during hypoxia. In
homozygous knockout animals lacking the
gp91phox
subunit of the NADPH oxidase complex, Archer et
al36 found that the response
to hypoxia was preserved, further suggesting a lack of
involvement of that system in HPV.
In summary, our results demonstrate that HPV requires mitochondrial electron transport proximal to the ubisemiquinone site but does not require the entire mitochondrial ETC to be functional. ROS generated by mitochondria appear to function as second messengers during hypoxia and contribute to the signal transduction process leading to smooth muscle cell contraction in HPV.
Acknowledgments
This research was supported by NIH Grants HL32646, HL35440, HL66315, and HL10405. The authors gratefully acknowledge the technical assistance of Carol Mathieu, Dr Ningfang Chen, and Dr Matthew Mack in these studies.
Footnotes
Original received December 27, 1999; resubmission received December 21, 2000; revised resubmission received April 23, 2001; accepted April 24, 2001.
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