1 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 2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland 21224
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ABSTRACT |
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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; hypoxia-inducible factor 1; pulmonary vascular remodeling
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INTRODUCTION |
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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 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.
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OXYGEN-DEPENDENT SIGNALING IN FETAL PVSMC: ROLE OF cGMP-DEPENDENT PROTEIN KINASE1 |
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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 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 protein 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.
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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 pulmonary 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-cGMP-dependent
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.
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Oxygen effect on pulmonary vasoactivity via reactive oxygen and
nitrogen species.
Biologically relevant reactive oxygen species (ROS) include superoxide
anion (O
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
O
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.
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EFFECT OF HYPOXIA ON ION HOMEOSTASIS IN PVSMC2 |
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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%
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.
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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 µM fura 2-AM, a Ca2+-sensitive dye, and subjected to
fluorescent microscopy, resting [Ca2+]i was
significantly elevated after exposure to chronic hypoxia (Fig.
6A). The hypothesis is that
this would be due to activation of voltage-gated 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
(106 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.
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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 µM). 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.
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In summary, the above data indicate that prolonged exposure to alveolar hypoxia results in marked alterations 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.
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ACKNOWLEDGEMENTS |
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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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FOOTNOTES |
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1 Presented by Usha Raj.
2 Presented by Larissa Shimoda.
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: raj{at}gcrc.rei.edu).
10.1152/ajplung.00177.2002
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