Am J Physiol Lung Cell Mol Physiol 283: L671–L677, 2002;
10.1152/ajplung.00177.2002.
EB2002 featured topic
Oxygen-dependent signaling in pulmonary vascular
smooth muscle
USHA RAJ1 AND LARISSA SHIMODA2
Department of Pediatrics, Harbor-University of California at Los Angeles Research and Education
Institute, University of California at Los Angeles School of Medicine, Torrance, California 90502;
and 2Division of Pulmonary and Critical Care Medicine, Department of Medicine,
Johns Hopkins School of Medicine, Baltimore, Maryland 21224
1
Raj, Usha, and Larissa Shimoda. Oxygen-dependent signaling in
pulmonary vascular smooth muscle. Am J Physiol Lung Cell Mol Physiol
283: L671–L677, 2002; 10.1152/ajplung.00177.2002.—The pulmonary
circulation constricts in response to acute hypoxia, which is reversible on
reexposure to oxygen. On exposure to chronic hypoxia, in addition to
vasoconstriction, the pulmonary vasculature undergoes remodeling, resulting in a sustained increase in pulmonary vascular resistance that is
not immediately reversible. Hypoxic pulmonary vasoconstriction is physiological in the fetus, and there are many mechanisms by which the
pulmonary vasculature relaxes at birth, principal among which is the
acute increase in oxygen. Oxygen-induced signaling mechanisms, which
result in pulmonary vascular relaxation at birth, and the mechanisms by
which chronic hypoxia results in pulmonary vascular remodeling in the
fetus and adult, are being investigated. Here, the roles of cGMP-dependent protein kinase in oxygen-mediated signaling in fetal pulmonary
vascular smooth muscle and the effects of chronic hypoxia on ion channel
activity and smooth muscle function such as contraction, growth, and
gene expression were discussed.
fetal and neonatal pulmonary circulation; potassium channels; hypoxiainducible factor 1; pulmonary vascular remodeling
IN THE FETUS,
hypoxia is a physiological stimulus for the
pulmonary vasculature to be constricted and the resistance to be high. However, at birth, the factors that
promote vasoconstriction in utero must be downregulated, and the mechanisms that dilate the vasculature
must become instantly operative. Oxygen is a powerful
stimulus that sets into motion a variety of events that
result in vasorelaxation. The mechanisms by which the
fetal pulmonary vascular smooth muscle cell (PVSMC)
responds immediately to oxygen may be unique to the
fetus. Sustained exposure to hypoxia in utero and after
birth leads to vasoconstriction as well as remodeling of
pulmonary vessels, resulting in persistent pulmonary
hypertension, which is not immediately ameliorated by
oxygen. The mechanisms of vascular remodeling in the
Address for reprint requests and other correspondence: U. Raj,
Harbor-UCLA Research and Education Institute, 1124 W. Carson
St., Bldg. RB-1, Torrance, CA 90502-2064 (E-mail: [email protected]).
http://www.ajplung.org
fetus and in the adult may be very different. The issues
summarized in this report were considered at a featured
topic session held during the Experimental Biology 2002
Meeting in New Orleans, LA.
OXYGEN-DEPENDENT SIGNALING IN FETAL PVSMC:
ROLE OF cGMP-DEPENDENT PROTEIN KINASE1
Control of pulmonary vasomotor tone in the perinatal
period. In the transition from fetal to neonatal life,
there is an immediate fall in pulmonary vascular resistance, with an almost 8- to 10-fold increase in blood
flow (10, 32), which is brought about by active vasodilation of the pulmonary vasculature, induced primarily
by exposure to oxygen, the mechanical effects of expansion of the lungs, stretch of the pulmonary blood vessels due to increased blood flow, and the creation of an
air-liquid interface. Increased synthesis of vasodilator
1
Presented by Usha Raj.
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society
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Fig. 1. Depiction of intracellular signaling pathways in pulmonary vascular smooth muscle. NO, nitric oxide;
PKG, cGMP-dependent protein kinase;
PKA, protein kinase A; EDNO, endothelium-derived nitric oxide; ANF,
atrial natriuretic factor; AC, adenylyl
cyclase; GC, guanylyl cyclase; MLC,
myosin light chain; MLC-p, phosphorylated myosin light chain.
agonists such as endothelium-derived nitric oxide
(NO), prostacyclin, prostaglandin E2, and bradykinin
results in increased production of intracellular cGMP
and cAMP in vascular smooth muscle. Intracellular
accumulation of these nucleotides results in smooth
muscle relaxation via a variety of mechanisms (Fig. 1).
The relative importance of these two nucleotides in
inducing pulmonary vasodilation in the perinatal period was investigated by Dhanakoti et al. (6). They
found that ovine newborn pulmonary arteries were
more sensitive to relaxation induced by cGMP than by
cAMP (Fig. 2). Data from several laboratories seem to
indicate that the endothelium-derived NO-guanylyl cyclase-cGMP pathway is perhaps the most important
mechanism of vasodilation in the pulmonary circulation during the transition from fetal to newborn state
(1, 13, 34, 37, 46). Studies in fetal and neonatal ovine
pulmonary vessels indicate that cGMP-dependent pro-
Fig. 2. Relaxation responses of intact pulmonary arteries of newborn
lambs to 8-bromoguanosine 3⬘,5⬘-cyclic monophosphate (8-Br-cGMP)
and 8-bromoadenosine 3⬘,5⬘-cyclic monophosphate (8-Br-cAMP). Active tension (g/dry wt of tissue) of the arteries was first raised by
endothelin-1 (3 nM), followed by exposure to increasing concentrations (0.01–300 ␮M) of 8-Br-cGMP or 8-Br-cAMP, respectively.
Changes in tension induced by both agents are recorded over a 4-h
period. A representative trace is shown.
AJP-Lung Cell Mol Physiol • VOL
tein kinase (PKG) is critically important for mediating
the action of cGMP in the transitional (fetal to neonatal) pulmonary circulation (6, 12) (Fig. 3). It has also
been shown that cAMP can cross activate PKG, indicating that PKG also mediates cAMP-induced relaxation in the pulmonary vasculature.
Importance of PKG type 1 in the normal fetal to
neonatal transition. In animal models of neonatal pulmonary hypertension, the NO-cGMP pathway has
been shown to be impaired (7–9, 24, 35, 38, 43). Furthermore, in some models of hypoxia-induced pulmo-
Fig. 3. Effects of racemic isomer of ␤-phenyl-1, N2-etheno-8-bromoguanosine-3⬘,5⬘-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS)
and KT-5720 on 8-Br-cGMP-induced relaxation of pulmonary arteries of newborn lambs. Experiments were performed during contraction to endothelin-1 (3 nM). The control tensions (g/mg dry wt of
tissue) raised by endothelin-1 were 1.12 ⫾ 0.13, 1.05 ⫾ 0.11, and
1.03 ⫾ 0.06 for untreated (control) or treated with Rp-8-Br-PETcGMPS or KT-5720, respectively. Changes in tension are expressed
as percent contraction to endothelin-1. Data are shown as means ⫾
SE; n ⫽ 5 for all except KT-5720, n ⫽ 4 animals/group. *Significant
difference (P ⬍ 0.05) from control.
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nary hypertension in the neonatal (2, 42) and adult
(18) animal, the vasodilator response of pulmonary
vessels to cGMP is specifically impaired. Other reports
in the literature indicate that hypoxia attenuates
cGMP-mediated effects in vascular smooth muscle (25,
40). It is possible that the impaired pulmonary vascular responses to cGMP in these animal models of neonatal pulmonary hypertension may be explained by
impaired PKG activity. Hofmann et al. (26, 33) have
developed knockout mice bearing homozygous PKG
type 1 null mutations (in both ␣ and ␤) to eliminate
PKG type 1 gene expression and abolish NO-cGMPdependent vascular relaxation. Notably, in this knockout, there is a very high amount of fetal loss, with most
dying immediately after birth. In survivors, other
mechanisms of vasodilation must be in play.
Oxygen effect on cGMP-PKG-mediated pulmonary
vasodilation. Because, at birth, an increase in oxygen
tension is an important biological stimulus, the role of
oxygen in upregulating the cGMP-PKG pathway in
fetal PVSMC is being actively investigated. We have
found that fetal pulmonary vessels exposed to oxygen
have an augmented relaxation response to cGMP when
preconstricted compared with fetal vessels that have
not been exposed to oxygen (Fig. 4). This augmented
relaxation response to cGMP is mostly abolished if
PKG kinase activity is blocked, suggesting that the
effect of oxygen is predominantly on PKG activity.
Oxygen can increase PKG activity by affecting the 1)
catalytic rate/amount of cGMP binding, 2) dissociation
rate of bound cGMP, 3) autophosphorylation of PKG, 4)
intracellular location of PKG, 5) oxidation of thiol
groups in PKG protein via generation of oxygen radicals, 6) inhibition of substrate binding, 7) proteolytic
rate of PKG, 8) conformational change, 9) kinetics of
ATP binding, 10) activation of an inhibitor of PKG, 11)
binding of other regulatory factors to allosteric site(s),
and 12) complexing with other protein factors. These
mechanisms need to be investigated.
Oxygen effect on pulmonary vasoactivity via reactive
oxygen and nitrogen species. Biologically relevant reactive oxygen species (ROS) include superoxide anion
Fig. 4. Intrapulmonary vessels incubated in low or high oxygen
tensions were preconstricted with endothelin-1 and then exposed to
increasing concentrations of cGMP, as described for Fig. 3. The
responsiveness to cGMP was significantly augmented by exposure of
the vessels to a high oxygen tension, indicating that signaling mechanisms downstream from cGMP were augmented by oxygen. * Significant difference (P ⬍ 0.05) from low oxygen tension.
AJP-Lung Cell Mol Physiol • VOL
(O2⫺), hydrogen peroxide (H2O2), and hydroxy radical.
Approximately 1–3% of all tissue oxygen consumption
occurs through the partial reduction of oxygen to O2⫺,
with this species present at a steady-state concentration of ⬃10 pM, increasing to 0.01–0.1 ␮M in pathological states (11). Under physiological conditions, antioxidant enzymes such as superoxide dismutase
(SOD), glutathione peroxidase, and catalase modulate
ambient steady-state levels of ROS (27). In vascular
smooth muscle, recent studies show that the majority
of O2⫺ produced is attributable to NAD(P)H oxidases.
The cardiovascular NAD(P)H oxidases are low-output,
slow-release enzymes with biochemical characteristics
that differ considerably from those of neutrophil
NADPH oxidase. Also, the vascular enzymes appear to
have a moderate constitutive activity that is absent in
phagocytes. The kinetics of activation on cellular stimulation are also unique. O2⫺ is produced in minutes to
hours in endothelial cells, vascular smooth muscle
cells, and fibroblasts, in contrast to the almost instantaneous release seen in neutrophils (16). In lungs, O2⫺
inhibits cGMP-mediated vasodilation. In contrast,
H2O2 augments vasodilation via activation of soluble
guanylyl cyclase (3).
ROS and reactive nitrogen species in PVSMC. Biologically relevant nitrogen species include NO and peroxynitrite. Endothelial and vascular smooth muscle
cells produce both NO and O2⫺ (16, 17). These two
radicals can recombine to form peroxynitrite. NO reacts more rapidly with O2⫺ than SOD and can scavenge
O2⫺ very efficiently. Although SOD concentration in
cells is 1–10 ␮M, the rapid reaction of NO with O2⫺
assures that peroxynitrite is always formed (11). Normally, in the cell, peroxynitrite is converted to nitrosoglutathione, mainly by reduced glutathione. These
thiol intermediates may subsequently regenerate NO
and cause vasodilation (21, 31). When cellular glutathione levels are low, as in hypoxia, peroxynitrite is
likely to accumulate. Peroxynitrite is a powerful oneand two-electron oxidant that can cause DNA damage.
Protein tyrosine nitration by peroxynitrite may interfere with phosphorylation/dephosphorylation signaling
pathways or alter protein function (4). In pulmonary
arterial smooth muscle cells, production of ROS and
NO synthase may dramatically increase during hypoxia (20, 23). Recent studies identify a novel mitochondrial NO synthase (15). A drop in cellular PO2 may
stimulate NO production by this enzyme or lead to
liberation of NO from heme-binding sites in cytoplasm
and mitochondrial matrix (14), and this NO may
readily react with O2⫺ to form peroxynitrite. Increased
production of ROS in hypoxia may decrease intracellular glutathione levels and lead to further peroxynitrite
accumulation, due to reduced conversion of peroxynitrite by glutathione into thiol intermediates (5). The
net effect might be that during hypoxia, there is
greater accumulation of peroxynitrite than under normoxic conditions.
Previously, ROS have been viewed as toxic, leading
to DNA damage and lipid oxidation. Recent data suggest that this concept may be incorrect. It is believed
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that ROS are produced in a controlled fashion and have
critical cellular functions as second messengers. Activation of signaling cascades and redox-sensitive transcription factors leads to induction of many genes in
vascular cells (16, 19, 41). No one has studied the role
of ROS and/or reactive nitrogen species in modulating
PKG activity, protein levels, and gene expression. Preliminary data were presented to show that in fetal
PVSMC, increased levels of peroxynitrite are produced
during hypoxia, which decrease PKG protein levels.
Additionally, data were also presented to show that
exogenous peroxynitrite (1 pM) decreased PKG protein
levels in fetal PVSMC by ⬃30%, suggesting that peroxynitrite is involved in regulation of PKG protein
levels during hypoxia.
Future research directions. The focus of future studies will be on understanding the mechanisms by which
oxygen mediates intracellular signaling in PVSMC.
The effects of oxygen on systemic vs. PVSMC appear to
be different, an area of future investigation. Furthermore, the effects of oxygen in PVSMC in the developing
fetus and newborn appear to be different from that in
adult PVSMC, the mechanisms of which need to be
determined.
EFFECT OF HYPOXIA ON ION HOMEOSTASIS
IN PVSMC2
Prolonged exposure to decreased oxygen tension, as
occurs with many pulmonary diseases, results in pulmonary hypertension, significantly worsening prognosis. Structural remodeling, characterized by smooth
muscle cell proliferation, intimal thickening, and extension of smooth muscle into previously nonmuscular
arterioles (30), and active contraction of vascular
smooth muscle, evidenced by acute reduction in pulmonary arterial pressure in response to vasodilatory
agents (22), are commonly observed after long-term
hypoxic exposure and contribute to the increase in
pulmonary arterial pressure. Despite extensive characterization of the structural and functional changes
that occur in pulmonary arteries in response to hypoxia, the exact cellular mechanisms underlying hypoxic pulmonary vasoconstriction, pulmonary arterial
smooth muscle cell (PASMC) hypertrophy and hyperplasia, and subsequent development of pulmonary hypertension remain poorly understood.
Abnormalities in PASMC are likely to contribute to
pathogenesis of hypoxic pulmonary hypertension. Because ion homeostasis regulates numerous smooth
muscle cell functions, including contraction, growth,
and gene expression, investigative efforts have focused
on elucidating pathways involved in alterations in intracellular K⫹, Ca2⫹, and H⫹ handling in response to
chronic hypoxia. To study the effects of prolonged in
vivo hypoxia on PASMC function, a murine model of
hypoxic pulmonary hypertension was developed (44).
Adult male C57B6 mice were placed in a chamber
gassed with either room air (normoxic controls) or 10%
2
Presented by Larissa Shimoda.
AJP-Lung Cell Mol Physiol • VOL
O2 for 3 wk. Single PASMCs were obtained by enzymatic digestion of intrapulmonary arteries. Using
whole cell patch-clamp techniques, we determined that
PASMCs from animals exposed to chronic hypoxia exhibit an attenuation of voltage-gated K⫹ (KV) channel
activity (Fig. 5), similar to results originally demonstrated by Smirnov et al. (36). The observed decrease in
KV current could be due to either a change in channel
regulation or a change in channel expression. PCR
analysis revealed a significant reduction in the expression of KV1.2 and KV1.5 channel ␣-subunits in pulmonary arteries from chronically hypoxic animals, suggesting that hypoxia acts to decrease KV current by
repressing KV channel gene expression. Experiments
are currently ongoing to determine the effect of chronic
hypoxia on the expression of other KV channel subunits
and to identify the mechanisms by which a decrease in
O2 can inhibit KV channel expression.
Under normal conditions, KV channels are the major
regulators of resting membrane potential (Em) in
PASMC (45). The observed reduction in KV channel
activity corresponded to a depolarization of the Em
from ⫺40 to ⫺11 mV (Fig. 5C). These results are
consistent with those first reported by Suzuki and
Twarog (39) in the hypoxic rat model of pulmonary
hypertension.
Em plays a major role in regulating intracellular
Ca2⫹ concentration ([Ca2⫹]i). Thus inhibition of KV
channels and subsequent PASMC depolarization are
likely to correspond to a change in cytosolic Ca2⫹.
Indeed, in PASMC loaded with 5 ␮M fura 2-AM, a
Ca2⫹-sensitive dye, and subjected to fluorescent microscopy, resting [Ca2⫹]i was significantly elevated af-
Fig. 5. Effect of chronic hypoxia on voltage-gated K⫹ (KV) currents
and membrane potential (Em). A: representative traces of KV current, measured at test potentials from ⫺50 to ⫹30 mV. Current was
measured in whole cell configuration in voltage-clamp mode in the
presence of 100 nM charybdotoxin to inhibit Ca2⫹-activated K⫹
currents. B: average peak KV current normalized to cell capacitance
(current density) in pulmonary arterial smooth muscle cells
(PASMC) from normoxic mice (n ⫽ 8) and chronically hypoxic mice
(n ⫽ 5). C: bar graph represents resting Em measured in PASMC
from normoxic (n ⫽ 8) and chronically hypoxic mice (n ⫽ 9). Em was
measured in the whole cell configuration during current-clamp mode
(I ⫽ 0). *Significant difference from normoxic value (P ⬍ 0.05).
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Fig. 6. Effect of chronic hypoxia on resting intracellular Ca2⫹ concentration ([Ca2⫹]i) in PASMC. A: bar
graph represents resting [Ca2⫹]i measured with
fura 2-AM in PASMC from normoxic (n ⫽ 10) and
chronically hypoxic mice (n ⫽ 13). B: representative
traces demonstrate the effect of removal of extracellular Ca2⫹ and application of nifedipine, a voltagegated Ca2⫹ channel antagonist, or lanthanum, an
inhibitor of nonselective cation channels, on resting
[Ca2⫹]i in PASMC isolated from chronically hypoxic
mice. *Significant difference from normoxic value
(P ⬍ 0.05).
ter exposure to chronic hypoxia (Fig. 6A). The hypothesis is that this would be due to activation of voltagegated Ca2⫹ channels, with hypoxia inducing a shift in
Em to a range where these channels would open and
allow Ca2⫹ influx into the cell. Consistent with this
hypothesis, removal of extracellular Ca2⫹ caused a
rapid decrease in Ca2⫹. Surprisingly, application of
nifedipine (10⫺6 M) and lanthanum (10⫺5 M), inhibitors of voltage-gated Ca2⫹ and nonselective cation
channels, respectively, had no effect on resting [Ca2⫹]i
(Fig. 6B). These results suggest that while influx of
extracellular Ca2⫹ is required for maintaining the observed elevated basal [Ca2⫹]i, the mechanism by which
this occurs involves Ca2⫹ handling mechanisms other
than voltage-gated Ca2⫹ and nonselective cation channels.
Elevated [Ca2⫹]i has been shown to contribute to cell
contraction, proliferation, and changes in gene expression. In addition to [Ca2⫹]i, intracellular pH (pHi) has
been shown to play a similar modulatory role in smooth
muscle cell function. The pHi was measured in PASMC
from normoxic and chronically hypoxic mice using the
pH-sensitive fluorescent dye 2⬘,7⬘-bis(2-carboxyethyl)5(6)-carboxyfluorescein-AM (5 ␮M). Basal pHi was
found to be significantly elevated in PASMC after exposure to chronic hypoxia (Fig. 7A). Although several
transporters participate in pH homeostasis, studies by
Quinn et al. (28) clearly demonstrated that activation
of Na⫹/H⫹ exchange was required for PASMC proliferation in response to growth factors and that inhibitors
of Na⫹/H⫹ exchange reduced pulmonary vascular remodeling in chronically hypoxic rats (29). With the use
of the ammonium pulse technique, Na⫹/H⫹ exchange
activity, measured as the Na⫹-dependent recovery
from NHCl4-induced acidosis, was found to be significantly increased in PASMC from chronically hypoxic
mice (Fig. 7B). These data suggest that increased
Na⫹/H⫹ exchange activity in response to chronic hypoxia could account for the increase in basal pHi.
In summary, the above data indicate that prolonged
exposure to alveolar hypoxia results in marked alterAJP-Lung Cell Mol Physiol • VOL
Fig. 7. Effect of chronic hypoxia on intracellular pH (pHi). Basal pHi,
monitored in PASMC isolated from normoxic (n ⫽ 117) and chronically hypoxic mice (n ⫽ 53), was significantly higher after exposure
to chronic hypoxia. B: bar graph showing the effect of chronic hypoxia
on Na⫹/H⫹ exchanger activity. Na⫹/H⫹ exchanger activity was measured as the rate of Na⫹-dependent recovery from NHCl4-induced
acidosis. PASMC from chronically hypoxic mice (n ⫽ 25) had significantly higher Na⫹/H⫹ exchanger activity compared with PASMC
isolated from normoxic mice (n ⫽ 36). In cells from both normoxic
and chronically hypoxic mice, addition of ethylisopropyl amiloride
(EIPA; 10⫺5 M), an inhibitor of Na⫹/H⫹ exchanger, significantly
reduced Na⫹/H⫹ exchanger activity. All experiments were performed
in the absence of bicarbonate. *Significant difference from normoxic
value (P ⬍ 0.001); **significant difference from control value (P ⬍
0.001).
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ations in PASMC ion homeostasis. These changes in
intracellular K⫹, Ca2⫹, and H⫹ concentrations create
conditions that promote PASMC contraction and proliferation and are likely to contribute to the pathogenesis of hypoxic pulmonary hypertension.
U. Raj was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-59435 and HL-47804. Coinvestigators on
the work presented by Dr. Raj were Dr. Yuansheng Gao, Dr. Sri
Dhanakoti, and Fred Sander. L. Shimoda was supported by American Heart Association Scientist Development Grant AHA9930255N
and by NHLBI Grant HL-67919.
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