Respiratory Physiology & Neurobiology 132 (2002) 81 – 91

www.elsevier.com/locate/resphysiol

O2 sensing in hypoxic pulmonary vasoconstriction: the

mitochondrial door re-opens
Gregory B. Waypa, Paul T. Schumacker *
Department of Medicine MC6026, The Uni6ersity of Chicago, 5841 South Maryland A6enue, Chicago, IL 60637, USA
Accepted 9 April 2002

Abstract

The identity of the O2 sensor underlying the hypoxic pulmonary vasoconstriction (HPV) response has been sought
for more than 50 years. Recently, the mitochondria have again come into sharp focus as the cellular organelle
responsible for triggering the events that culminate in pulmonary artery constriction. Studies from different
laboratories propose two disparate models to explain how mitochondria react to a decrease in PO2. One model
proposes that hypoxia slows or inhibits mitochondrial electron transport resulting in the accumulation of reducing
equivalents and a decrease in the generation of reactive oxygen species (ROS). This is proposed to activate a
redox-sensitive pathway leading to pulmonary vasoconstriction. A second and opposing model suggests that hypoxia
triggers a paradoxical increase in mitochondrial ROS generation. This increase would then lead to the activation of
an oxidant-sensitive signaling transduction pathway leading to HPV. This article summarizes the potential involve-
ment of mitochondria in these two very different models. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Blood, flow, pulmonary; Hypoxia, pulmonary vasoconstriction; Mitochondria, reactive oxygen species; Oxygen, reactive
species, HPV; Perfusion, pulmonary

1. Introduction in health and disease states, and toward unravel-

Hypoxic pulmonary vasoconstriction (HPV) di-
verts blood flow away from the lung during in
utero development and it helps to optimize lung
gas exchange after birth by enhancing the match-
ing of blood flow and ventilation. Much effort has
been directed toward characterizing this response
* Corresponding author. Tel.: +1-773-702-6790; fax: + 1-
773-702-4736
E-mail address: pschumac@medicine.bsd.uchicago.edu (P.T.
Schumacker).
ing the underlying cellular mechanism by which
the cells in the pulmonary artery activate the
response. A paramount and still unresolved ques-
tion relates to the underlying mechanism by which
cells detect low PO2 conditions and convert this
into a biological signal. It is rightly believed that
an understanding of this mechanism will enhance
the base of knowledge related to the physiology
and pathophysiology of the pulmonary circula-
tion. An understanding of the mechanism of tis-
sue oxygen sensing in the lung could also help to
lay the foundation for our understanding of oxy-
gen sensing in other tissues. Arguably, progress

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PII: S1569-9048(02)00051-4
82 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
has been made toward identifying the O2 sensor in
HPV. However, a diverse collection of candidate
O2 sensors are still being evaluated by different
laboratories, and definitive studies in support of
any single model have not yet appeared. Not
surprisingly, no consensus of opinion has emerged
behind any single mechanism. Several excellent
review articles dealing with HPV have appeared
recently, and the reader is directed to those for a
more comprehensive analysis of the field (Mar-
shall et al., 1994; Michelakis et al., 1995; Ward
and Robertson, 1995; Weir and Archer, 1995;
Michelakis et al., 1997; Ward and Aaronson,
1999; Archer et al., 2000; Jones et al., 2000; Jones
and Morice, 2000; Sweeney and Yuan, 2000;
Coppock et al., 2001; Michelakis and Weir, 2001;
Sylvester, 2001; Weissmann et al., 2001).
The present report is intended to provide a
more narrow analysis of information and ideas
pointing to mitochondria as the O2 sensor during
HPV. The notion that mitochondria might func-
tion as O2 sensors is an old one. From a teleolog-
ical standpoint it is reasonable that an organelle
where most of the O2 is consumed would be the
site of O2 sensing. However, smooth muscle cells
do not contain large numbers of mitochondria.
Also, certain mitochondrial inhibitors failed to
block the HPV response in early experiments.
This led investigators to wonder how that or-
ganelle could be the site of O2 sensing if the
downstream responses were preserved in experi-
ments where mitochondrial function was crippled.
Nevertheless, more recent studies have provided a
clearer explanation of how mitochondria might be
involved. In addition, they provide an explanation
of why certain mitochondrial inhibitors block
HPV while others do not. Two different models of
mitochondrial O2 sensing recently have come to
the forefront. One model proposes that hypoxia
slows or inhibits mitochondrial electron transport
resulting in the accumulation of reducing equiva-
lents and a decrease in the generation of reactive
oxygen species (ROS). A second and contrasting
model suggests that hypoxia triggers a paradoxi-
cal increase in mitochondrial ROS generation. It
therefore appears that the mitochondrial door is
re-opening in the field of O2 sensing in HPV. The
present article will attempt to summarize the cur-
rent views on mitochondrial O2 sensing and to
reconcile these points.
2. The HPV response
HPV was first described in 1946 by von Euler
and Liljestrand, in a paper characterizing the
effects of interventions on pulmonary arterial
pressure in the cat (von Euler and Liljestrand,
1946). They found that pulmonary arterial pres-
sure increased when the lungs were ventilated with
low inspired O2 mixtures and decreased when
ventilated with pure O2. They recognized that
these responses reflected changes in pulmonary
vascular resistance, and they speculated that a
vasoconstrictor response to hypoxia could help to
improve gas exchange efficiency by diverting
blood flow away from poorly ventilated lung re-
gions and toward areas with better oxygenation.
Much later, this prediction was shown to be true,
especially in damaged or diseased lungs where
hypoxic and normoxic regions co-exist. We now
know that the HPV response is retained in excised
lungs (Rounds and McMurtry, 1981; McMurtry,
1984; Archer et al., 1993; Grimminger et al., 1995;
Weissmann et al., 1995, 1998) and hypoxia-in-
duced contraction can even be seen in rings of
pulmonary arteries subjected to low O2 conditions
in an organ bath (Marshall et al., 1996; Zhao et
al., 1996). Constriction is preserved if the vessels
are denuded of endothelium (Gelband and Gel-
band, 1997; Archer et al., 1998), although the
response is quite markedly attenuated. This has
led to the conclusion that the endothelium am-
plifies, but does not initiate HPV. Given that the
pulmonary endothelium can amplify the HPV re-
sponse, particularly during conditions of sus-
tained hypoxia (Ward and Robertson, 1995;
Robertson et al., 2001), it is probably more ap-
propriate to consider the intact pulmonary artery
when studying HPV than to focus narrowly on
the pulmonary arterial myocyte. Nevertheless, iso-
lated pulmonary arterial (PA) myocytes do con-
tract under hypoxia (Zhang et al., 1997),
indicating that PA smooth muscle cells possess
the ability to detect hypoxia and to activate a
contractile response. If both smooth muscle cells
 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
and endothelium contribute to the HPV response,
it is interesting to ask whether the same O2 sensor
is functioning in both cell types, or whether the
endothelium is merely permissive for the normal
vasoconstriction response to hypoxia.
It has now been well established that the HPV
response in pulmonary arteries is biphasic (Dipp
et al., 2001; Leach et al., 2001). An initial tran-
sient constriction response (phase 1) is followed
by a slow sustained constriction response (phase
2) (Fig. 1). Phase 1 has been shown to occur
independently of an intact endothelium (Zhang et
al., 1997) and is thought to be initiated by the
release of calcium from intracellular stores in PA
myocytes, resulting in the closure of voltage-de-
pendent potassium channels (KV), membrane de-
polarization, and the opening of voltage-gated
L-type calcium channels (Gelband and Gelband,
1997; Archer et al., 1998; Morio and McMurtry,
2002). The influx of Ca2 + through the voltage-
gated L-type Ca2 + channels initiates the calmod-
ulin-mediated activation of myosin light chain
kinase, resulting in actin– myosin interaction and
PA myocyte contraction. Phase 1 is also referred
to as the acute HPV response because it begins
within seconds after hypoxic challenge. By con-
Fig. 1. An overview of hypoxic pulmonary vasoconstriction
integrating phase 1 and phase 2 of the response. The trace
shows a typical example of the biphasic response of the
pulmonary vasculature to hypoxia. This figure is based on
previously published work (Ward and Aaronson, 1999), with
permission.
83
trast, phase 2 occurs within minutes after hypoxic
challenge and is associated with chronic hypoxia.
Phase 2 requires an intact endothelium which
suggests that an endothelial-derived promoting
factor may be involved (Liu et al., 2001). En-
dothelin-1 (ET-1) is a potent vasoconstrictor that
may function as the amplifier of hypoxia-induced
vasoconstriction. In this regard, the expression
and release of ET-1 by endothelial cells has been
shown to increase during hypoxia (Bodi et al.,
1995; Hu et al., 1998; Yamashita et al., 2001).
Furthermore, some studies suggest that phase 2
can be attenuated with BQ-123, an antagonist of
endothelin-1 receptor subtype A (ETA-receptor)
(Liu et al., 2001; Sato et al., 2001). However,
other studies suggest that phase 2 is not affected
by endothelin-1 receptor blockade with bosentan,
a antagonist of both the ETA- and ETB-receptor
subtypes, and that phase 2 contraction of small
pulmonary arteries in rats does not involve en-
dothelin-1 (Lazor et al., 1996). In either case,
phase 1 and phase 2 are mechanistically distinct
contributors to the HPV response. It is conceiv-
able that a similar O2 sensor mechanism underlies
the different responses to hypoxia in PA myocytes
and PA endothelium, but this has not been
demonstrated.
3. Historical basis for implicating mitochondria as
the O2 sensor
The idea that mitochondria may function as the
O2 sensor during HPV is an old one. Early studies
focused on the premise that O2, acting as a sub-
strate for biochemical reactions, could impart a
rate-limiting effect during hypoxia that somehow
resulted in the contraction of PA myocytes. This
was appealing from a teleological standpoint since
the mitochondria are the primary consumers of
O2 in the cell. In accordance with this reasoning,
it was suggested that the inhibition of mitochon-
drial electron transport and oxidative phosphory-
lation could mimic the HPV response. Indeed, in
1981 Rounds and McMurtry reported that certain
inhibitors of electron transport and oxidative
phosphorylation, including azide, cyanide, dini-
trophenol, antimycin A, and rotenone, all mim-
84 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
icked the hypoxic pressor response in isolated,
blood-perfused lungs (Rounds and McMurtry,
1981). They suggested that hypoxia-induced de-
creases in mitochondrial electron transport could
compromise the energy state of the cell, thereby
initiating HPV. However, later studies in isolated
lungs by Buescher et al. (1991) and in isolated
pulmonary arteries by Leach et al. (2000) showed
that the energy state of the cell does not decrease
during hypoxia in either of these systems
(Buescher et al., 1991; Leach et al., 2000). There-
fore, one may conclude that HPV is not triggered
by a decrease in the energy state as a consequence
of hypoxia-induced inhibition of mitochondrial
electron transport. Classically, mitochondrial res-
piration has been found to remain unchanged
until intracellular PO2 falls below a critical
threshold of  5 mmHg (Jones and Kennedy,
1982, 1986). It therefore seems unlikely that en-
ergy supply in PA myocytes would become O2-
limited at oxygen tensions in the range of 10– 40
mmHg where HPV is activated. Some investiga-
tors have underscored this by point by noting that
mitochondria cannot serve as the an O2 sensor
because the Km of cytochrome oxidase is too low
to permit detection of hypoxia in the physiologi-
cal range (Bunn and Poyton, 1996). Such reason-
ing appeared to shut the door on the
mitochondria as a putative oxygen sensor in the
response to hypoxia
4. Does hypoxia decrease mitochondrial ROS
generation?
Renewed interest in the mitochondria as an O2
sensor has been stimulated by speculation that
hypoxia could act by slowing down or inhibiting
the rate of ROS production. In 1993, Archer et al.
reported that hypoxia increased PA pressure and
simultaneously caused a decrease in whole lung
chemiluminescence. Their study therefore sug-
gested that HPV and hypoxia could be linked by
a decrease in ROS production. The same labora-
tory reported that antimycin A and rotenone,
agents that inhibit mitochondrial electron trans-
port, caused a decrease in measured chemilume-
nescence and an increase in PA pressure (Archer
et al., 1993). They suggested that the decrease in
available O2 during hypoxia resulted in the inhibi-
tion of mitochondrial electron transport, an accu-
mulation of reducing equivalents such as
NAD(P)H, and a decrease in ROS production.
According to this model, inhibitors of mitochon-
drial electron transport ought to mimic HPV by
augmenting NAD(P)H levels and decreasing
ROS. In support of this idea, Reeve et al. re-
ported that the mitochondrial complex I inhibitor
rotenone decreased chemilumenescence and in-
creased vascular tone in isolated perfused lungs
(Reeve et al., 2001). Their data is consistent with
a model in which complex I (NADH:ubiquinone
oxidoreductase) would function as an O2 sensor
by maintaining a basal level of superoxide and
H2O2 production during normoxia. During hy-
poxia, the ROS produced by this system would
decrease and NADH concentrations would rise.
These changes would shift the state of cytosolic
redox toward a more reduced level (Mohazzab et
al., 1995; Zhu et al., 1999). The change in redox
state would then result in the inhibition of K+
current through redox-sensitive 4-aminopyradine
(4-AP)-sensitive KV channels (Reeve et al., 1995).
Inhibition of currents through KV-channels would
then trigger membrane depolarization, opening of
L-type, voltage gated Ca2 + channels and subse-
quent myocyte contraction (Archer et al., 1998,
2000). However, it has been reported that antago-
nism of KV channels did not prevent HPV, sug-
gesting that this scheme may not be correct
(Hasunuma et al., 1991; Sham et al., 2000). More-
over, the importance of the change in ROS rela-
tive to the change in cytosolic NAD/NADH ratio
is not clear in this model.
5. Does hypoxia increase mitochondrial ROS
generation?
In opposition to the theory that hypoxia results
in a shift towards a reduced state, other labs have
suggested that a paradoxical increase in ROS
generation occurs during hypoxia. For example,
Marshall et al. (1996) found an increase
chemilumenescence during hypoxia in isolated PA
myocytes (Marshall et al., 1996). Similar increases
 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
in oxidant signaling have also been reported by
other laboratories in a variety of cell types (Chan-
del et al., 1998; Duranteau et al., 1998; Chandel et
al., 2000; Killilea et al., 2000; Liu et al., 2001;
Waypa et al., 2001). In cultured PA myocytes, we
observed an increase in oxidant signaling during
hypoxia using the intracellular probe 2%,7%-dichlor-
ofluorescin-diacetate (DCFH-DA). This probe en-
ters cells and can be oxidized to the fluorescent
compound 2%,7%-dichlorofluorescein (DCF) in the
presence of ROS including H2O2, hydroxyl radi-
cals and nitric oxide, but not by superoxide di-
rectly. It should therefore be regarded as a
sensitive but nonspecific indicator of oxidant
stress.
Although some confusion exists with respect to
the cellular mechanisms of DCFH oxidation dur-
ing hypoxia, other studies provide independent
support for the conclusion that hypoxia-induced
increases in ROS are involved in HPV. For exam-
ple, pharmacological antioxidants block the hy-
poxic response in isolated, buffer-perfused lungs
(Waypa et al., 2001), suggesting that increases in
ROS generation are required for HPV. In addi-
tion, when H2O2 formation in the cytosol was
blocked during hypoxia by inhibiting Cu,Zn su-
peroxide dismutase (SOD), the HPV response was
abrogated indicating that H2O2 is an important
downstream signaling agent (Waypa et al., 2001).
Other studies also implicate increased H2O2 gener-
ation in the HPV response. For example, Monaco
and Burke-Wolin (1995) showed that HPV was
augmented when catalase was inhibited with
aminotriazole. Catalase normally degrades H2O2,
so this supports the notion that hypoxia-induced
ROS generation mediates HPV. Weissmann et al.
(1998) observed that SOD and 4,5-dihydroxy-1,3-
benzenedisulfonic acid (to accelerate H2O2 genera-
tion from superoxide) did not affect HPV,
confirming that H2O2, rather than superoxide, is
involved (Weissmann et al., 1998). Furthermore,
nitrobluetetrazolium, which traps superoxide and
prevents H2O2 formation, attenuated HPV. Other
studies show that H2O2 constricts the pulmonary
circulation during normoxia (Waypa et al., 2000).
Collectively, these findings are consistent with a
role for increased H2O2 as a signaling molecule
involved in HPV.
85
Marshall et al. (1996) suggested that hypoxia
accelerates ROS generation by a membrane-
bound NADPH oxidase, based on their observa-
tion that diphenyleneiodonium (DPI) inhibited
the oxidant signal and the contractile response to
hypoxia (Marshall et al., 1996). Grimminger et al.
(1995) also found that DPI attenuated HPV
(Grimminger et al., 1995). DPI inhibits a wide
range of flavoproteins including NADPH oxidase,
mitochondrial complex I, glutathione reductase,
nitric oxide synthase, and prostaglandin syn-
thetase (Holland et al., 1973; Majander et al.,
1994). Therefore, the site of inhibition by DPI
responsible for the attenuation of HPV is not
known. To clarify the possible involvement of an
NAD(P)H-oxidase in HPV, two separate groups
used apocynin, an inhibitor of the neutrophil
form of this enzyme (Stolk et al., 1994; Grim-
minger et al., 1995; Waypa et al., 2001). Apocynin
failed to attenuate either HPV or PA myocyte
contraction during hypoxia. The possible involve-
ment of NADPH-oxidase was further explored by
Archer et al. (1999), who studied HPV in ho-
mozygous knockout animals lacking the
gp91phox subunit of the NADPH oxidase com-
plex. They found that the response to hypoxia
was preserved, suggesting a lack of involvement of
that system in HPV (Archer et al., 1999). How-
ever, because of the specificity of this knockout,
these results do not rule out that possibility that
other NAD(P)H-like oxidase systems could still
be involved in HPV.
6. Mitochondrial as the source of ROS
generation during hypoxia
Mitochondria have long been known to gener-
ate ROS (Parsons et al., 1966) although these
oxidants have classically been viewed as toxic
byproducts of the electron transport pathway,
possibly contributing to the effects of aging (So-
hal and Weindruch, 1996). More recently, mito-
chondrial ROS have been implicated as
intracellular signaling agents. Our laboratory pre-
viously reported that mitochondria increase ROS
generation during hypoxia and that site-specific
inhibition of electron transport could attenuate
86 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91

Fig. 2. Schematic diagram showing mitochondrial ROS production at complex III. Solid arrows show electron transfer steps; solid
bars show sites of electron transport inhibition. Normal mitochondrial electron transport involves the movement of reducing
equivalents generated in the Krebs cycle through complex I or II, then through complex III and IV (cytochrome oxidase). The Q
cycle converts the dual electron transfer in complex I and II into single electron transfer steps in complex IV. Ubisemiquinone (a
free radical) created in this process can generate superoxide. This process appears to be amplified during hypoxia.

hypoxia-induced ROS generation (Chandel et al.,
1998; Duranteau et al., 1998). These observations
suggest that a similar mechanism could be occur-
ring in the pulmonary arteries during HPV. Dur-
ing normoxia, mitochondrial electron transport
inhibitors such as cyanide or antimycin A, which
inhibit sites distal to complex III, tend to augment
ROS generation (Fig. 2). The likely explanation
for these findings is that ROS production occurs
primarily within complex III. In the Q cycle, a
free radical (ubisemiquinone) is normally gener-
ated during the electron transport process. This
radical can potentially donate its unpaired elec-
tron to O2, thereby generating superoxide. During
hypoxia, our model suggests that the process of
ROS generation from that site is amplified. By
contrast, when mitochondrial electron transport is
inhibited at a more proximal site by inhibitors
such as rotenone, DPI, or myxothiazol, the
ubiquinone pool becomes fully oxidized and ROS
generation at complex III is abrogated. According
to this model, the ROS production at Complex III
increases during hypoxia, and these oxidants trig-
ger signal pathways culminating in Ca2 + activa-
tion and contraction during HPV. Distal
inhibitors of the ETC such as cyanide induce
constriction during normoxia, presumably by aug-
menting ROS production. This conclusion is sup-
ported by the observation that the
vasoconstriction in response to cyanide could be
inhibited by ebselen (an antioxidant) or myxothia-
zol (a more proximal ETC inhibitor).
Recently, Leach et al. (2001) reported an ele-
gant series of studies in isolated pulmonary arter-
ies that are consistent with this model. They
found that rotenone and myxothiazol could atten-
uate hypoxia-induced increases in intracellular
calcium ([Ca2 + ]i) activation and subsequent vaso-
constriction. By contrast, inhibition of complex
IV with cyanide augmented the hypoxic response
but had no effect on [Ca2 + ]. Interestingly, in PA
vessels that had been treated with rotenone, they
were able to restore HPV by using succinate to
shuttle electrons into complex III via complex II,
effectively bypassing the rotenone inhibition of
Complex I (Leach et al., 2001) (Fig. 2). This was
consistent with earlier data from Rounds and
McMurtry, who had found that antimycin A elic-
its vasoconstriction in normoxic lungs (Rounds
and McMurtry, 1981). Also, Archer et al. (1993)
found that cyanide induced vasoconstriction dur-
ing normoxia and augmented HPV without af-
fecting the response to angiotensin II or KCl
(Archer et al., 1993). Collectively, these findings
 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
are consistent with a role for increased mitochon-
drial ROS in HPV. A caveat is that these studies
rely on pharmacological inhibitors, which may
have unexpected effects. However, r0 PA my-
ocytes, which lack a functional mitochondrial
electron transport chain, selectively lost the re-
sponse to hypoxia while they retained the ability
to contract in response to exogenous H2O2 or
U46619 in our study (Waypa et al., 2001). These
observations are consistent with the proposed
model, but the mechanism by which hypoxia am-
plifies ROS generation at complex III is not yet
known.
An alternative explanation for the increase in
ROS signaling during hypoxia involves the regula-
tion of superoxide egress from mitochondria to
cytosol. Superoxide generated inside the mito-
chondria would presumably require an anion
channel to escape to the cytosol, because charged
species cannot easily diffuse through the mem-
brane lipid bilayer. The mitochondrial inner mem-
brane anion channel (IMAC) is a nonspecific
anion channel that could conceivably function as
such a pathway. If the IMAC were O2-sensitive
such that it increased its conductance during hy-
poxia, the egress of superoxide from the matrix
could increase even if the rate of mitochondrial
superoxide generation were to decrease during
hypoxia. This might explain why ROS signaling in
the cytosol is increased during moderate hypoxia,
and why 4,4%-diisothiocyanatostilbene-2.2%-disul-
ponic acid (DIDS), an anion channel inhibitor
which inhibits mitochondrial IMAC (Beavis and
Davatol-Hag, 1996; Terada, 1996), attenuated
HPV and abolished PA myocyte contraction dur-
ing hypoxia (Waypa et al., 2001). It should be
noted that DIDS is also a thiol-reactive com-
pound; however, pretreatment with DIDS had no
effect on H2O2-induced vasoconstriction in the
lung. In either case, it appears that superoxide
enters the cytosol where it is dismuted to H2O2 by
cytosolic Cu,Zn SOD.
7. Role of mitochondria in phase 1 of the HPV
response
Phase 1 of HPV is transient and occurs in PA
87
myocytes independently of the endothelium. This
indicates that the O2 sensor responsible for trig-
gering phase 1 must be intrinsic to the PA my-
ocyte. The HPV response may also involve one or
more 4-AP-sensitive KV channels. Inhibition of
Kv channels would promote membrane depolar-
ization, the opening of voltage-gated L-type Ca2 +
channels, and subsequent vasoconstriction
(Archer et al., 1993, 1998, 2000; Urena et al.,
1996; Gelband and Gelband, 1997; Zhang et al.,
1997; Barman, 1998; Weir et al., 1998). However,
other studies bring into question the requirements
of the KV channel for the HPV response. As
mentioned previously, HPV was shown to remain
intact after inhibition of KV channels with 4-AP
(Hasunuma et al., 1991; Sham et al., 2000). Fur-
thermore, HPV was significantly blunted, but not
completely abolished, in SWAP mice which lack
the KV1.5 channel (Archer et al., 2001). This
suggests that KV channels may function to aug-
ment the HPV response, rather than to trigger it
directly.
It is possible that hypoxia causes the activation
of Ca2 + as an early step in phase I. The release of
Ca2 + from intracellular stores seems to be re-
quired for HPV (Gelband and Gelband, 1997;
Morio and McMurtry, 2002) and it may be the
source of that initial signal. Post et al. (1995)
reported that release of Ca2 + from internal stores
causes inhibition of 4-AP-sensitive KV channels
and subsequent membrane depolarization (Post et
al., 1995). These results are consistent with a
model of HPV in which hypoxia triggers the
release of Ca2 + from internal stores, resulting in
KV channel inhibition, membrane depolarization,
and the opening of voltage-gated L-type Ca2 +
channels.
Possible sources of intracellular Ca2 + during
hypoxia included inositol 1,4,5-triphosphate (IP3)-
sensitive sarcoplamic reticulum (SR) stores and
ryanodine-sensitive SR stores. Gelband and Gel-
band (1997) and Robertson et al. (2001) found
that hypoxic contraction of isolated pulmonary
arteries was inhibited by thapsigargin, which
blocks the uptake of Ca2 + into intracellular
stores. These observations suggest that hypoxia
triggers the release of Ca2 + from IP3-sensitive SR
88 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
stores. Along similar lines, exogenous H2O2 has
been shown to activate phospholipase C (PLC) in
a time- and concentration-dependent manner
(Wang et al., 2001). This suggests the possibility
that mitochondrial ROS could trigger intracellu-
lar Ca2 + release by activating PLC. PLC in turn
would metabolize phosphatidylinositol 4,5-bis-
phosphate (PIP2) to inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol (DAG). IP3 could then
release Ca2 + from IP3-sensative Ca2 + stores.
However, studies demonstrating this mechanism
have not been published.
Hypoxia has been shown to trigger the release
of Ca2 + from ryanodine-sensitive SR stores (Gel-
band and Gelband, 1997; Dipp and Evans, 2001;
Dipp et al., 2001; Liu et al., 2001; Wilson et al.,
2001; Morio and McMurtry, 2002). Recently,
Wilson et al. (2001) reported that hypoxia in-
creases cyclic ADP-ribose accumulation in iso-
lated rabbit PA and Ca2 + release from
ryanodine-sensitive SR stores. Richter et al.
(1995) reported that H2O2 could cause oxidation
of mitochondrial pyridine nucleotides, resulting in
increased ADP-ribose production, monoADP-ri-
bosylation of mitochondrial proteins, and calcium
release (Richter et al., 1995). These results suggest
a mechanism in which mitochondrial ROS gener-
ation could trigger Ca2 + release from IP3- and/or
ryanodine-sensitive SR stores. The resulting in-
crease in intracellular Ca2 + could then trigger
HPV by inhibiting KV-channels. The increase in
[Ca2 + ]i resulting from membrane depolarization
would trigger calmodulin-mediated activation of
myosin light chain kinase, actin– myosin interac-
tion, and contraction.
8. Role of mitochondria in phase 2 of the HPV
response
Phase 2 of HPV involves a slow sustained vaso-
constriction that is dependent on the endothelium.
An endothelium-derived vasoconstrictor may
therefore be required for phase 2. ET-1, a 21-
residue peptide synthesized and secreted by en-
dothelial cells, has been proposed to be the
vasoconstrictor involved in phase 2 of the HPV
response. ET-1 expression and release from cul-
tured endothelium increases during hypoxia (Bodi
et al., 1995; Hu et al., 1998; Yamashita et al.,
2001), and BQ-123, an ETA antagonist, attenu-
ates phase 2 of HPV in isolated vessels and whole
lungs (Sato et al., 2000; Liu et al., 2001). This
suggests that an O2 sensor must also exist within
the PA endothelium. However, the relationship
between the O2 sensor in the myocytes and the
sensor in endothelium is not known.
Recently, Hu et al. (1998) reported that the
proximal promoter of the ET-1 gene contains a
hypoxia-inducible factor 1 (HIF-1) binding site on
the antisense DNA strand positioned at −118 to
− 125 base pairs upstream from the transcription
start site (Hu et al., 1998). HIF-1 has been shown
to be the principal O2-responsive factor underly-
ing increased expression of glycolytic enzymes,
glucose transporters, and other genes during hy-
poxia (Semenza, 2001a,b,c). This suggests a mech-
anism in which hypoxia could regulate ET-1
expression and release by endothelial cells
through HIF-1 activation. Several groups have
found evidence that hypoxia-induced mitochon-
drial ROS generation activates HIF-1 during hy-
poxia (Chandel et al., 1998, 2000; Agani et al.,
2000; Semenza, 2000). Furthermore, hypoxia in-
creases ROS generation at complex III of the
mitochondria in endothelial cells (Pearlstein et al.,
2002). This suggests that the mitochondria in PA
endothelium could contribute to phase 2 by in-
creasing ROS generation, resulting in increased
ET-1 expression and release, which in turn would
contribute to PA myocyte contraction. It seems
reasonable to predict that such a response could
be important for the enhanced contraction and
even vascular remodeling during periods of sus-
tained or chronic hypoxia, but more definitive
experiments are needed to test this hypothesis
directly.
9. Conclusion
It is clear that a consensus has yet to be reached
regarding the O2 sensing mechanism underlying
the HPV response. However, mitochondria have
once again stepped into the spotlight as a likely
O2 sensor involved in the initiation of HPV. This
 G.B. Waypa, P.T. Schumacker / Respiratory Physiology & Neurobiology 132 (2002) 81–91
model is attractive given that the mitochondria
are the primary consumers of oxygen in the cell.
However, recent studies by different laboratories
suggest two opposing explanations of how the
mitochondria could trigger a biological response
to hypoxia. At this point, more definitive experi-
ments are needed to provide a resolution to these
differences.
Acknowledgements
This work was supported by NHLBI grants
HL66315 and HL10405.
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