Developmental changes in endothelin expression and activity in
the ovine fetal lung
D.
Dunbar Ivy1,
Timothy D.
le Cras2,
Thomas
A.
Parker3,
Jeanne P.
Zenge3,
Malathi
Jakkula2,
Neil E.
Markham2,
John P.
Kinsella3, and
Steven H.
Abman2
Pediatric Heart Lung Center and Sections of
1 Pediatric Cardiology,
2 Pediatric Pulmonary Medicine, and
3 Pediatric Neonatology, University of Colorado
School of Medicine and The Children's Hospital, Denver, Colorado
80218
 |
ABSTRACT |
Mechanisms that regulate endothelin (ET) in the perinatal
lung are complex and poorly understood, especially with regard to the
role of ET before and after birth. We hypothesized that the ET system
is developmentally regulated and that the balance of ETA
and ETB receptor activity favors vasoconstriction. To test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and adult sheep. Lung preproET-1 mRNA levels, tissue ET peptide levels, and cellular localization of ET-1 expression were determined by Northern blot analysis, peptide assay, and immunohistochemistry in distal lung tissue
from fetal lambs between 70 and 140 days (term = 145 days), newborn
lambs, and ewes. Lung mRNA expression for the ETA and ETB receptors was also measured at these ages. We found
that preproET-1 mRNA expression increased from 113 to 130 days
gestation. Whole lung ET protein content was highest at 130 days
gestation but decreased before birth in the fetal lamb lung.
Immunolocalization of ET-1 protein showed expression of ET-1 in the
vasculature and bronchial epithelium at all gestational ages.
ETA receptor mRNA expression and ETB receptor
mRNA increased from 90 to 125 and 135 days gestation. To determine
changes in activity of the ETA and ETB
receptors, we studied the effect of selective antagonists to the
ETA or ETB receptors at 120, 130, and 140 days
of fetal gestation. ETA receptor-mediated vasoconstriction
increased from 120 to 140 days, whereas blockade of the ETB
receptor did not change basal fetal pulmonary vascular tone at any age
examined. We conclude that the ET system is developmentally regulated
and that the increase in ETA receptor gene expression
correlates with the onset of the vasodilator response to
ETA receptor blockade. Although ETB receptor
gene expression increases during late gestation, the balance of ET
receptor activity favors vasoconstriction under basal conditions. We
speculate that changes in ET receptor activity play important roles in
regulation of pulmonary vascular tone in the ovine fetus.
endothelin receptors; pulmonary hypertension; persistent pulmonary
hypertension of the newborn; fetus; pulmonary circulation
 |
INTRODUCTION |
PULMONARY VASCULAR RESISTANCE (PVR) is elevated in the
normal fetal lung because pulmonary blood flow accounts for
<8-10% of the combined ventricular output of blood from the
heart (15). Mechanisms responsible for the maintenance of high PVR in
the fetus may include physical factors such as lack of an air-liquid interface or ventilation, relative low oxygen tension, decreased vasodilator activity, or perhaps increased vasoconstrictor activity (3,
12, 33). Endothelium-derived products, including vasodilator stimuli
such as nitric oxide and prostacyclin, and vasoconstrictor stimuli,
such as leukotrienes and endothelin (ET)-1, contribute to vascular tone
in the fetal lung (3, 8, 10, 12, 17, 33, 37). These endothelial
products may contribute not only to basal tone in the fetal lung but
may also modulate responses to physiological stimuli, such as increases
in pressure and shear stress (1, 8, 18). Intrauterine mechanisms of
altered pulmonary vascular structure and function may be an important determinant of successful postnatal adaptation.
The ETs are a family of isopeptides consisting of ET-1, ET-2, and ET-3.
ET-1 is the best-characterized member of the ET family, is a potent
vasoactive peptide with comitogenic effects on vascular smooth muscle,
and is produced primarily by the vascular endothelium in the normal
lung circulation (7, 42). ET-1 or ET-2 is 10 times more abundant than
ET-3 in the adult rat lung (27), and ET-1 is 2.5 times more potent than
ET-2 in the pulmonary vasculature (28). The actions of ET-1 depend on a
complex cascade of signal transduction pathways. ET-1 is initially
synthesized as a 203-amino acid prepropeptide (preproET-1) that is then
cleaved by an endopeptidase to proET-1 ("Big ET-1"). Big ET-1, a
38-amino acid peptide, is then converted to ET-1 by an ET-converting
enzyme, which is a membrane-bound metalloprotease present in
endothelial and smooth muscle cells (41). The activity of ET-1 depends
on activation of at lease two distinct ET receptors (ETA
and ETB) that may mediate different ET activities in
endothelium and vascular smooth muscle cells (2). The ETA
receptors are located on smooth muscle cells and mediate
vasoconstriction in most vascular beds. ETA receptors have
a high affinity for ET-1 and ET-2, with much lower affinity for ET-3
(16). ETB receptors have equal affinity for ET-1, ET-2, and
ET-3; are mostly present on endothelial cells; and may release nitric
oxide with stimulation (17). Some studies have shown ETB
receptors on vascular smooth muscle mediating vasoconstriction (31);
however, ETB receptors are present only on the
endothelium in the ovine fetal lung (17). Furthermore, recent studies
suggest that the ETB receptor may act as a clearance
receptor for circulating ET-1 (13).
In the normal fetal lung at 1-2 wk before birth, ET-1 is present
and contributes to high PVR (17, 26, 30, 37). The effects of ET-1 in
the ovine fetal lung are dependent on stimulation of ETA
and ETB receptors, which mediate vasoconstriction and
vasodilation, respectively (17). However, the primary role of ET-1 and
its receptors in regulation of vascular tone in the ovine fetus remains incompletely understood. Several studies have suggested that the predominant role of endogenous ET-1 in the normal ovine fetus is
vasodilation (4, 40), whereas other studies have suggested that the
predominant role of ET-1 is vasoconstriction (17, 37, 38). We
hypothesized that from mid to late gestation ET levels increase and
that the balance of ET receptor activity favors vasoconstriction. To
test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and ewe. Northern blot analysis of the mRNA for ppET-1, ET peptide
levels, and cellular localization of ET-1 expression by
immunohistochemistry was performed on lung tissue from the fetus,
newborn lamb, and ewe. Analysis of the mRNA for the ETA or
ETB receptors was performed at similar ages. Physiological
studies utilizing selective antagonists to the ETA or
ETB receptors were performed in fetal lambs at 120, 130, and 140 days gestation.
 |
METHODS |
Northern blot analysis.
To determine the ontogeny of gene expression of ppET-1 and the
ETA and ETB receptors in the ovine fetal lung,
we performed Northern analysis of whole lung homogenates. At different
ages, the ewe was sedated with pentobarbital sodium. Distal pieces of whole lung were rapidly frozen in liquid nitrogen (19). Total RNA was
purified from 48 lung samples from separate animals with TRI Reagent
(Molecular Research Center, Cincinnati, OH) and the method of
Chomczynski and Sacchi (6). Lung samples were obtained from separate
animals of the following gestational ages: 70 days (n = 3), 90 days (n = 5), 113 days (n = 4), 118 days (n = 4), 125 days (n = 4), 130 days (n = 4), 135 days
(n = 4), and 140 days (n = 4). Samples from newborn
lungs were obtained at 1 day (n = 4), 5 days (n = 4),
and 13 days (n = 4) as well as samples of lung from postpartum
ewes (n = 4). The RNA was quantified by measuring the
absorbance at 260 nm. Twenty micrograms of total RNA per lung were
analyzed with standard Northern blot and hybridization techniques using
cDNA probes as previously described (19). Ovine ppET-1, human
ETA, and human ETB cDNA probes were labeled
with [
-32P]dCTP with random-primer labeling
(RTS Random Primer DNA Labeling System; GIBCO BRL, Life Technologies,
Gaithersburg, MD). Dr. Thomas Quertermous (Vanderbilt University School
of Medicine, Nashville, TN) kindly provided the 522-bp sheep-specific
ppET-1 cDNA probe (11). Dr. Scott Magnuson (Abbott Laboratories, Abbott
Park, IL) kindly provided the 1,350-bp human ETA and human
ETB cDNA probes. An 18S rRNA oligonucleotide was labeled
with terminal deoxytransferase and
[
-32P]dCTP. After hybridization, blots were
washed at room temperature in 1× saline-sodium citrate (SSC) and
0.1% SDS (low stringency) and then at 65°C in 0.4× SSC and
0.1% SDS (high stringency). Imaging and quantification of mRNA signals
was performed with a Molecular Dynamics Storm 860 phosphorimager.
Normalization to 18S rRNA levels was used in quantification of mRNA signals.
ET peptide ELISA methods.
To determine the ontogeny of ET protein expression in the fetal lung,
we performed analysis of whole lung homogenates. Twenty-four lung
samples were studied from the following ages: 70-90 days (n = 4), 118 days (n = 4), 130 days (n = 4),
and 140 days gestation (n = 4) as well as the 1 day newborn
(n = 4) and postpartum ewe (n = 4). Frozen tissue (100 mg) was homogenized in 1 ml of 1 M acetic acid-0.1% Triton X-100
(Sigma, St. Louis, MO) and immediately boiled for 7 min as previously
reported (25). C2 columns (Amersham, Arlington Heights, IL) were
equilibrated with 2 ml of methanol followed by 2 ml of deionized water.
Forty microliters of homogenate were applied to the column and washed
with 5 ml of 0.1% trifluoroacetic acid (TFA). The sample was eluted
with 2 ml of 80% methanol-0.1% TFA, and the volume was reduced for 5 h (to near dryness) on a Speed-Vac concentrator (Savant, Farmingdale,
NY). Samples were reconstituted to 250 µl, and 100 µl of sample and
standard were applied to replicate wells of the ET-1 peptide ELISA kit
(Cayman Chemical, Ann Arbor, MI) according to the manufacturer's
instructions. The antibody used in this assay cross-reacts with ET-2
and ET-3. Briefly, samples and an acetylcholinesterase-linked ET
antibody were incubated overnight at 4°C in a 96-well plate. After
the samples were washed and Ellman's reagent was applied, enzyme
activity was quantified by determination of optical density units at
410 nm with a DynaTech MR 700 plate reader (Bio-Tek, Winooski, VT).
Immunolocalization of ET-1 protein.
To determine localization of ET-1 protein in the fetal lung, we
performed immunohistochemistry. Sheep lung tissue was prepared, and
immunolocalization was performed as previously described (14, 35).
Three to five sections were studied from the following ages: 70 days
gestation, 114 days gestation, 130 days gestation, 140 days gestation,
and 1 day newborn. At autopsy, the lung was inflated by slow infusion
of agarose. The pulmonary vasculature was perfused with saline,
followed by buffered Formalin at 30-40 cmH2O pressure.
Paraffin sections (5 µm) were serially mounted onto Superfrost Plus
slides (Fisher Scientific, Fair Lawn, NJ). The slides were
deparaffinized in 100% Hemo-De and then rehydrated by immersion in
100% ethanol, 95% ethanol-5% water, 70% ethanol-30% water, and
then 100% deionized water. Endogenous peroxidase activity in the
tissue sections was blocked by 2% hydrogen peroxidase-60% methanol.
The slides were washed in 1× PBS. Sections were blocked with
Super Block (diluted 1:10 vol/vol in 1× PBS; Sky Tek, Logan, UT),
washed in 1× PBS (once for 5 min), and then incubated with ET-1
antibody (QED Bioscience) diluted 1:300 or mouse IgG, negative control
(Jackson Laboratories, West Grove, PA), in horse serum for 60 min.
After the unbound antibody was washed off (3 times for 5 min in
1× PBS), specific ET-1 antibody binding was detected with a
biotin-labeled anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) diluted at 1:200 in 2.5 ml of 1× PBS plus 50 µl of horse serum. After the excess secondary antibody was washed off in
1× PBS, the slides were incubated in streptavidin-peroxidase solution and developed with diaminobenzidine (DAB) and hydrogen peroxide, with nickel chloride for enhancement (Vector). The
NiCl2 enhancement DAB color development reaction was
stopped by rinsing in water, then the slides were dehydrated in 70%
ethanol-30% water; 95% ethanol-5% water; 100% ethanol; and finally
100% Hemo-De before coverslips were applied. An observer blinded to
the gestational age (T. D. Le Cras) determined intensity of staining.
The staining intensity was evaluated as absent, strong, or weak.
Surgical preparation
. To determine whether activity of the ETA and
ETB receptors changes during mid to late gestation, we
infused selective antagonists to the ETA and
ETB receptors at 120, 130, and 140 days gestation. All
procedures and protocols were previously reviewed and approved by the
Animal Care and Use Committee at the University of Colorado Health
Sciences Center. Eleven mixed breed (Columbia-Rambouillet) pregnant
ewes between 116 and 118 days gestation (term = 145 days) underwent
surgery with methods previously described (17). Ewes were sedated with
intravenous pentobarbital sodium (2-4 g) and anesthetized with 1%
tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes remained
sedated with pentobarbital sodium but breathed spontaneously throughout
the surgery. Under sterile conditions, the fetal lamb's left forelimb
was delivered through a uterine incision. A skin incision was made
under the left forelimb after local infiltration with lidocaine
(2-3 ml, 1% solution). A left axillary to sternal thoracotomy
exposed the heart and great arteries. A catheter was inserted into the
main pulmonary artery (MPA) by direct puncture through purse-string
sutures. The MPA catheter was inserted between the ductus arteriosus
and the pulmonic valve. A left atrial (LA) catheter was inserted in the
medial portion of the left atrial appendage. An ultrasonic flow
transducer (6 mm; Transonic Systems, Ithaca, NY) was placed around the
left pulmonary artery (LPA) to measure blood flow to the left lung. The
thoracotomy incision was closed in layers. The ewe was allowed to
recover. The flow transducer cables were attached to an internally calibrated flowmeter (Transonics) for continuous measurements of LPA
flow. The absolute values of flows were determined from phasic blood
flow signals obtained during baseline periods as previously described
(17, 21). A correction factor between end-diastolic flow and the
internally calibrated zero point on the Transonics flowmeter was added
to the mean flow on the Transonics flowmeter. The value obtained from
this method correlates with previously determined measures of LPA flow
in the late-gestation ovine fetal lung (24). Calculation of resistances
is reported as left lung PVR [PVR
(mmHg · ml
1 · min) = (mean MPA pressure
LA pressure)/LPA flow]. Study
measurements included pulmonary arterial pressure, aortic pressure, LA
pressure, LPA flow, and arterial blood gas tensions. The aortic, MPA,
and LA catheters were connected to a computer-driven system to record pressures and flow (Biopac, Santa Barbara, CA). The pressure transducer was calibrated with a mercury column manometer. Blood samples for pH,
arterial PCO2, and arterial
PO2 were drawn from the aorta and
measured at 39.5°C with a Radiometer OSM-3 blood gas analyzer and
hemoximeter (Radiometer, Copenhagen, Denmark).
Dose-response studies of BQ-123 in the late-gestation fetal lamb have
been reported (17). A dose-response study was performed during infusion
of BQ-788 in seven animals at 135 ± 3 days (Alexis Biochemicals, San Diego, CA). To determine the degree of blockade of
the ETB receptor with BQ-788, a selective ETB
receptor agonist, SFX-S6c, was infused after the BQ-788 doses. Serial
arterial blood gas tensions and hemodynamic parameters were recorded
throughout baseline, drug infusion, and recovery periods. SFX-S6c (1 µg over 10 min) (17) was infused after random doses of BQ-788 (1, 10, and 100 µg infused over 10 min) in the LPA in seven animals.
To determine changes in activity of the ETA and
ETB receptors during late gestation, infusions of the
ETA-selective antagonist BQ-123 and the
ETB-selective antagonist BQ-788 were performed at 120 ± 2 days gestation, 130 ± 3 days gestation, and 140 ± 3 days gestation
in four animals. The infusions were performed in random order on
consecutive days. BQ-123 and BQ-788 (Alexis Biochemicals) were
dissolved in DMSO. The molecular weights of BQ-123 (633.7) and BQ-788
(663.7) are similar. A dose of 10.0 µg was chosen because this was
the lowest dose of BQ-788 that blocked SFX-S6c vasodilation. After 30 min of baseline measurements, drugs were infused in the LPA, and
hemodynamic measurements were continuously monitored for 30 min. The
peak effect was recorded.
Statistical analysis.
Data are presented as means ± SE. For hemodynamic parameters,
statistical comparisons were made with ANOVA for repeated measures and
Fisher's least significant difference test for post hoc comparisons. For all other comparisons, nonparametric analysis was performed. The
Kruskal-Wallis test was performed to detect overall group differences
in Northern blot analysis for preproET-1 mRNA, ETA receptor
mRNA, and ETB receptor mRNA as well as ET lung protein content. Because a group difference was found with each variable, pairwise comparisons were made with Dunn's procedure, with an experimentwise type I error rate of 0.05.
 |
RESULTS |
ET-1
. Northern blot analysis of the mRNA for ppET-1 revealed a single
2.3-kb transcript as previously reported (19). We found that lung
ppET-1 mRNA levels increased from 113 to 130 days gestation (Fig.
1). Lung ppET-1 levels were higher in the
13-day newborn than in the 113-day fetal lung. ET peptide levels in
whole lung homogenates increased from 118 to 130 days gestation but
fell at 140 days gestation before birth. ET peptide levels were greater at 130 days gestation than in the newborn lamb (Fig.
2).


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Fig. 1.
Maturation-related changes in steady-state preproendothelin-1 (ppET-1)
mRNA expression in the normal ovine fetal lung. A:
representative Northern blots. Nos. on top, days gestation;
NB, newborn; M, maternal. B: expression of ppET-1 mRNA was
greater at 130 days gestation and 13 days after birth than in the
113-day fetal lung.
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Fig. 2.
Whole lung endothelin (ET) peptide content in the normal ovine fetal
lung. ET peptide content was higher at 130 days fetal gestation than at
118 and 140 days gestation as well as in the newborn lamb.
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Immunolocalization of ET-1 peptide revealed ET-1 peptide staining in
the lung vasculature, bronchial epithelium, and isolated pneumocytes
(Fig. 3). At 70 and 114 days, strong
staining was seen in the bronchial epithelium with weaker staining of
isolated pneumocytes and the vasculature. At 130 days, 140 days, and in the newborn lamb, strong staining was seen in the bronchial epithelium, vascular endothelium, and isolated pneumocytes with weaker staining in
the vascular media.

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Fig. 3.
Immunolocalization of ET-1 peptide in the normal ovine fetal lung. ET-1
peptide immunostaining is noted in the vascular endothelium (open
arrows) and bronchial epithelium (solid arrows). At 70 and 114 days,
strong staining was seen in the bronchial epithelium, with weaker
staining of isolated pneumocytes and the vasculature. At 130 days, 140 days, and in the newborn lamb, strong staining was seen in the
bronchial epithelium, vascular endothelium, and isolated pneumocytes,
with weaker staining in the vascular media.
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ETA receptor
. Northern blot analysis of the mRNA for the ETA
receptor revealed a 5.2- and 4.2-kb transcript as previously described
(25). We found that ETA receptor mRNA levels were greater
at 125 and 135 days than in the 90-day fetal lung (Fig.
4).


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Fig. 4.
Maturation-related changes in steady-state endothelin A receptor
(ETA) mRNA expression in the normal ovine fetal lung.
A: representative Northern blots. Nos. on top, days
gestation. B: expression of ETA receptor mRNA was
higher at 125 and 135 days than at 90 days in the fetal lamb.
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ETB receptor.
Northern blot analysis of the mRNA for the ETB receptor
revealed a single 5.0-kb transcript as previously described (25). We
found that ETB receptor mRNA levels were greater at 125 and 135 days than in the 90-day fetal lung (Fig.
5). Lung ETB receptor mRNA
levels were greater in the 13-day newborn lung than at 90 days
gestation.


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Fig. 5.
Maturation-related changes in steady-state endothelin B receptor
(ETB) mRNA expression in the normal ovine fetal lung.
A: representative Northern blots. Nos. on top, days
gestation. B: expression of ETB receptor mRNA was
higher at 125 and 135 days gestation than at 90 days in the fetal lamb.
ETB receptor mRNA was also higher in the 13-day newborn
than the 90-day fetus.
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Hemodynamic data
. Baseline values for hemodynamic and blood gas variables at
120, 130, and 140 days are shown in Table
1. Mean pulmonary arterial pressure was
greater at 140 than at 120 or 130 days. LPA blood flow was greater at
140 than at 120 days. PVR was lower at 140 than at 120 days. Aortic
pressure was greater at 140 than at 120 days. There was no difference
in baseline values for pH, PCO2, or
PO2 at 120, 130, or 140 days.
The dose-response study of BQ-788 at 135 ± 3 days revealed that the
doses of BQ-788 studied (1-100 µg) did not change basal pulmonary tone. Baseline values for mean pulmonary arterial pressure, LPA flow, PVR, mean LA pressure, mean aortic pressure, and arterial blood gas tensions did not change. As shown in Fig.
6, the ETB receptor antagonist
BQ-788 at a dose of 1 µg did not block stimulation of the
ETB receptor with SFX-S6c, but higher doses of BQ-788 (10 and 100 µg) blocked ETB receptor stimulation.

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Fig. 6.
Dose-response study of the hemodynamic effect of BQ-788 in the normal
ovine fetal lung at 135 ± 3 days. BQ-788 (1, 10, and 100 µg) did
not change basal fetal pulmonary tone (data not shown). Vasodilation to
SFX-S6c (1 µg over 10 min), a selective ETB receptor
agonist, was blocked by BQ-788 at doses of 10 and 100 µg. PVR,
pulmonary vascular resistance.
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Study of the gestational differences in ETA and
ETB receptor activity revealed that the fall in PVR with
the ETA antagonist BQ-123 was greater at 130 and 140 days
than at 120 days (Fig. 7). Blockade of the
ETB receptor with BQ-788 did not change basal pulmonary
tone at 120, 130, or 140 days.

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Fig. 7.
Maturation-related changes in hemodynamic responses to ET receptor
antagonists in the normal ovine fetal lung at 120, 130, and 140 days
gestation. The percent change in PVR to the selective ETA
receptor antagonist BQ-123 was greater at 130 and 140 days gestation
than at 120 days. Selective ETB receptor blockade with
BQ-788 did not change basal pulmonary tone at 120, 130, or 140 days
gestation.
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 |
DISCUSSION |
Although past studies have demonstrated that ET is present in the fetal
lung and contributes to the regulation of vascular tone during late
gestation, the physiological roles of ET and its regulation in the
normal ovine fetal pulmonary circulation are complex and poorly
understood (4, 5, 17, 22, 38). In this study, we found that
ETA receptor mRNA expression and ETB receptor
mRNA increased from 90 days to 125 and 135 days gestation. These
changes in ETA receptor mRNA expression correlated with findings from physiological studies, demonstrating that ETA
receptor blockade caused pulmonary vasodilation at 130 and 140 days
gestation but not at 120 days. In contrast, ETB receptor
blockade did not change basal PVR. In addition, lung ppET-1 mRNA
expression increased before birth, and ET peptide expression was
greatest at 130 days gestation. We conclude that the ET system is
developmentally regulated and that the increase in ETA
receptor gene expression correlates with the onset of the vasodilator
response to ETA receptor blockade. Although ETB
receptor gene expression increases during late gestation, the balance
of ET receptor activity favors vasoconstriction under basal conditions.
These findings suggest that changes in ET receptor expression and
activities play important roles in regulation of the fetal pulmonary circulation.
Previous studies have shown that ET-1 is present in the perinatal lung
(26) and is vasoactive in the fetus (5, 17, 22, 34). Some investigators
have suggested that the predominant physiological role of endogenous
ET-1 in the normal ovine fetus is vasodilation (4, 40), whereas other
studies have suggested that the predominant role of ET-1 is
vasoconstriction (17, 37, 38). Many of these conclusions are based on
observations based on the effects of infusions of ET-1 in the fetal
lung. However, exogenous infusion of ET-1 may not accurately describe
the hemodynamic effects of endogenous production of ET-1 in the fetal
lung. Circulating plasma concentrations of ET-1 are lower than those
reported to be biologically active (9), and secretion of ET-1 by
endothelial cells is polar and directed in an abluminal direction
toward the interstitial region (36). Brief infusion of ET-1 in the
normal ovine fetal lung causes acute fetal pulmonary vasodilation (5); however, hypertension develops during prolonged infusion (5). Although
ET-1 infusions cause vasodilation in the presence of high pulmonary
vascular tone, similar infusions cause vasoconstriction when the
pulmonary vascular tone is decreased during acute ventilation (4).
Infusion of Big ET-1, the precursor of ET-1, causes only hypertension
without vasodilation (17, 22), suggesting that stimulation of
endogenous ET-1 may have very different effects than brief exogenous
infusions of ET-1. We found that blockade of the ETA
receptor caused vasodilation, whereas blockade of the ETB
receptor did not change basal pulmonary tone. The present study
suggests that under basal conditions the predominant role of endogenous
ET in the ovine fetal circulation from 130 to 140 days is vasoconstriction.
Previous studies have shown that increased pulmonary blood flow and
pulmonary arterial pressure and decreased PVR with advancing gestation
is due to an increase in the total number of arterial vessels.
Increased vasomotor reactivity is related to an increase in the total
amount of smooth muscle, whereas the thickness of muscle in individual
vessels remains constant (23). Prior studies have shown that vascular
reactivity increases during late gestation (24, 29). Similarly, we
found that reactivity to an ETA receptor antagonist
increased during late gestation, suggesting increased ETA
receptor-mediated vasoconstriction under basal conditions.
Little is known about the role of ET during the transition from
intrauterine to newborn life. Studies in humans have revealed increased
circulating ET-1 levels in the fetus and newborn in comparison with
maternal controls (30, 32). Bosentan, a nonselective ETA
and ETB receptor antagonist, did not change the increase in pulmonary blood flow or decrease in PVR with in utero oxygen
ventilation (39). However, more selective antagonists were not studied. Preliminary data from our laboratory suggest that selective blockade of
the ETB receptor attenuates the fall in PVR at birth in the normal late-gestation ovine fetal lamb (20). We speculate that the fall
in ET peptide levels before birth may facilitate the increase in
pulmonary blood flow during the transition to newborn life.
Nonetheless, the precise role of ET-1 at birth requires further investigation.
A potential limitation of our study is that we studied the activity of
the ET receptors under basal conditions but did not evaluate the role
of direct stimulation of the ET receptors. Stimulation of the
ETB receptor causes vasodilation in the ovine fetal lung (17); however, under basal conditions, activity of the ETA
receptor appears greater than that of the ETB receptor.
Because the ET-1 peptide assay used in this study cross-reacts with
ET-2 and ET-3, the role of these peptides requires further study.
However, studies suggest that in the lung, ET-1 activity is greater
than the other ET peptides and that ET-1 is likely more abundant (27,
28)
In summary, expression of ppET-1 mRNA and ET peptide increased during
late gestation in the fetal lamb lung. ETA receptor mRNA
expression and ETB receptor mRNA increased from 90 days to 125 and 135 days gestation. ETA receptor-mediated
vasoconstriction increased from 120 to 140 days, whereas blockade of
the ETB receptor did not change fetal pulmonary tone. We
conclude that the ET system is developmentally regulated and that the
increase in ETA receptor gene expression correlates with
the onset of the vasodilator response to ETA receptor
blockade. Although ETB receptor gene expression increases
during late gestation, the balance of ET receptor activity favors
vasoconstriction under basal conditions.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Thomas Quertermous (Vanderbilt University School of
Medicine, Nashville, TN) and Dr. Scott Magnuson (Abbott Laboratories, Abbott Park, IL) for their kind gifts of cDNA probes. We thank Dr.
Misoo Ellison for assistance in performing the statistical analysis.
 |
FOOTNOTES |
This work was supported by The Children's Hospital Research Institute
Career Development Award (to D. D. Ivy), National Heart, Lung, and
Blood Institute Grant K08-HL-03823-01A1 (to D. D. Ivy), the March of
Dimes Birth Defects Foundation (to D. D. Ivy), the Bugher
Physician-Scientist Training Program (to D. D. Ivy), and an American
Heart Association Established Investigator Award (to S. H. Abman).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. D. Ivy, Dept.
of Cardiology, Box B100, The Children's Hospital, 1056 E. 19th Ave.,
Denver, CO 80218 (E-mail: dunbar.ivy{at}UCHSC.edu).
Received 23 March 1999; accepted in final form 9 November 1999.
 |
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