Am J Physiol Regulatory Integrative Comp Physiol
281: R261–R268, 2001.
Effect of in vivo fetal infusion of dexamethasone at 0.75
GA on fetal ovine resistance artery responses to ET-1
CHERYL C. DOCHERTY,1 JUDIT KALMAR-NAGY,1 MARC ENGELEN,1
STEVEN V. KOENEN,1 MARK NIJLAND,1 RHODA E. KUC,2
ANTHONY P. DAVENPORT,2 AND PETER W. NATHANIELSZ1
1
Laboratory for Pregnancy and Newborn Research, Department of Biomedical Sciences, College of
Veterinary Medicine, Cornell University, Ithaca, New York 14853; and 2Clinical Pharmacology Unit,
University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom
Received 18 July 2000; accepted in final form 6 March 2001
GLUCOCORTICOIDS REGULATE IMPORTANT functions that prepare the fetus for metabolic adaptations essential for
extrauterine life, in particular, fetal lung maturation
(4). Treatment of women in preterm labor with ante-
natal glucocorticoids lessens the incidence of neonatal
morbidity (8). The National Institutes of Health recently advised routine administration of antenatal betamethasone or dexamethasone (DM) for 48 h to all
pregnant women at risk of premature delivery before
32 wk of gestation (30a).
Direct infusion of the synthetic glucocorticoids, DM
and betamethasone, to the fetal sheep at 125 days
gestation (⬃0.8 gestation) increases fetal blood pressure (13). A rise in blood pressure could be manifested
via an increase in cardiac output and/or an increase in
total peripheral resistance. The fetal heart operates
near the upper limit of its function curve and therefore
has a limited capacity to increase its cardiac output
(42). Therefore, any change in blood pressure in the
fetus is likely to result from an increase in total peripheral resistance. In support of this view, we previously reported that betamethasone-induced fetal hypertension in sheep is associated with increased fetal
femoral vascular resistance (13). Furthermore, isolated
femoral resistance arteries from fetuses exposed to
betamethasone exhibit altered vascular responsiveness (1).
The mechanisms by which glucocorticoids alter vascular responsiveness in specific fetal vascular beds are
unknown but may involve a disturbance in the balance
of vasodilator- and/or vasoconstrictor-mediated effects.
Endothelin-1 (ET-1), produced by endothelial (44) and
vascular smooth muscle (34) cells, is a potent vasoconstrictor peptide that probably plays an important role
in the maintenance of basal vasomotor tone (16). ET-1
may contribute to fetal hemodynamic changes (19, 31).
Glucocorticoids increased the pre-pro-ET-1 mRNA levels in vascular smooth muscle cells (25) and isolated
rat aorta (33). Cortisol and/or DM have also been
shown to selectively induce ET-1 release (20) and to
potentiate ET-1-induced inositol trisphosphate (IP3)
production (35) in vascular smooth muscle cells.
We hypothesized that antenatal glucocorticoids increase peripheral vascular resistance in the ovine fetus
Address for reprint requests and other correspondence: P. W.
Nathanielsz, Laboratory for Pregnancy and Newborn Research,
Dept. of Biomedical Sciences, College of Veterinary Medicine, Cornell Univ., Ithaca, NY 14853 (E-mail: [email protected]).
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. Section 1734
solely to indicate this fact.
glucocorticoids; blood pressure; fetal sheep
http://www.ajpregu.org
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society
R261
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Docherty, Cheryl C., Judit Kalmar-Nagy, Marc Engelen, Steven V. Koenen, Mark Nijland, Rhoda E. Kuc,
Anthony P. Davenport, and Peter W. Nathanielsz.
Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on
fetal ovine resistance artery responses to ET-1. Am J Physiol
Regulatory Integrative Comp Physiol 281: R261–R268,
2001.—At 110–111 days gestation, instrumented fetal sheep
were administered saline or dexamethasone (2.2 ␮g 䡠 kg⫺1 䡠
h⫺1 iv) for 48 h. Measurement of fetal blood pressure showed
a greater increase in dexamethasone-treated (n ⫽ 6) compared with control (n ⫽ 5) fetuses (7.3 ⫾ 2.3 vs. 0.6 ⫾ 2.3
mmHg, P ⬍ 0.05). Fetuses were delivered by cesarean section, and the femoral muscle and brain were obtained under
halothane anesthesia. Femoral and middle cerebral arteries
(⬃320-␮m internal diameter) were evaluated using wire
myography. Sensitivity to KCl (2.5–125 mM) and the magnitude of the maximal vasoconstriction to 125 mM K⫹ were
similar in femoral and middle cerebral arteries from dexamethasone-treated vs. control fetuses. Acetylcholine-induced
vasorelaxation was similar in femoral arteries from control
and dexamethasone-treated fetuses. Middle cerebral arteries
did not relax to acetylcholine. Sensitivity to endothelin-1
(ET-1; 0.1 pM-0.1 ␮M) and magnitude of the ET-1-induced
vasoconstriction were greater in femoral arteries from dexamethasone-treated vs. control fetuses (P ⬍ 0.05). Autoradiographical studies with receptor-specific ligands demonstrated increased ETA-receptor binding, the principal
receptor subtype, in femoral muscle vessels (P ⬍ 0.001) but
decreased ETA-receptor binding in middle cerebral arteries
(P ⬍ 0.01) from dexamethasone-treated compared with control fetuses. Relatively little ETB-receptor binding was evident in all tissues examined. We conclude that hyperreactivity to ET-1, due to increased ETA-receptor binding, may be
involved in the dexamethasone-induced increase in peripheral vascular resistance in fetal sheep in vivo.
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ET-1-INDUCED VASOCONSTRICTION OF FETAL OVINE ARTERIES
MATERIALS AND METHODS
Animals. Rambouillet-Columbia crossbred ewes (Ovis aries) carrying a single fetus of known gestational age were
studied. Animals had free access to food and water except for
the 24 h before surgery. The experimental protocol was
approved by the Institutional Animal Care and Use Committee at Cornell University. All facilities were approved by the
American Association for the Accreditation of Laboratory
Animal Care.
Surgical preparation. Surgery was performed at 104–105
dGA as previously described (30). The ewes were pretreated
with 1 g of ketamine intramuscularly and 1 mg of glycopyrrolate intramuscularly and induced with 4% halothane for 4
min before intubation. In brief, both ewes and fetuses were
instrumented with polyvinyl catheters inserted into the carotid artery and jugular vein. A catheter was also placed in
the amniotic cavity. All animals were allowed 5 days postoperative recovery. During this time, the ewes received a daily
dose of 1 g of ampicillin sodium intramuscularly (Polycillin-N, Bristol Laboratories, Syracuse, NY) and 500 mg of
ampicillin sodium into the amniotic sac. Phenylbutazone
(EQUAPHEN, Pharma Cerricalas, Shirley, NY) was administered twice daily (0.5 g orally) for 3 days postsurgery to
provide analgesia.
DM treatment and tissue collection. At 110–111 dGA, fetuses received an infusion of either saline (n ⫽ 5) or DM
(AZIUM, Schering, NY) (2.2 ␮g 䡠 kg⫺1 䡠 h⫺1; n ⫽ 6) intravenously for 48 h. After 48 h of infusion, fetuses were removed
during cesarean section (112–113 dGA), and femoral muscle
from the left side and the brain were obtained under halothane general anesthesia and collected in ice-cold physiological salt solution (PSS) for wire myography studies. Another
section of the same tissue was embedded in tissue-freezing
medium (Triangle Biomedical Sciences, Durham, NC), immersed in ice-cold isopentane, snap-frozen in liquid nitrogen,
and stored at ⫺80°C. Fetuses and ewes were then euthanized
by exsanguination under halothane general anesthesia.
Wire myography. Second-order middle cerebral arteries
and femoral arteries were immediately dissected from the
collected tissue. Isolated arteries (⬃2 mm in length) were
mounted on two 40-␮m tungsten wires in a small vessel wire
myograph as previously described (27). It was not always
possible to dissect out enough vessels from each animal to
study all the agonists in each animal. This accounts for the
variation in the number of vessels examined in the different
portions of the study. Vessels were equilibrated in PSS at
37°C and continuously aerated with 95% O2-5% CO2 (pH 7.4)
for 30 min. Vessel dimensions were then normalized as
previously described (27). Briefly, arteries were stretched in
a stepwise manner, and the internal circumference and corresponding wall tension as a result of each stretch were
calculated by a Myodaq program (J. P. Trading) and plotted
to produce a resting wall tension-internal circumference
curve for that particular artery. Intersection of the curve
with a 100-mmHg isobar line determined the point on the
fitted exponential curve corresponding to the internal circumference that the artery would have attained in situ when
relaxed and exposed to a transmural pressure of 13.3 kPa
(100 mmHg), as determined by the Laplace relationship;
termed L100 (27). Preliminary studies conducted by us on
fetal ovine middle cerebral and femoral vessels showed that
active tension development is maximal in ovine fetal resistance arteries when set to 0.9 L100 (normalization value
obtained from interpolation of wall tension-internal circumference curve). Thus experiments described in this paper
were performed at 0.9 L100. There was no significant difference in the calculated internal diameter of arteries between
control and DM-treated groups for femoral arteries [control:
314.1 ⫾ 39.6 ␮m, means ⫾ SE (n ⫽ 4) vs. DM: 232.6 ⫾ 34.2
␮m (n ⫽ 5)]. However, middle cerebral arteries from DMtreated fetuses (355.2 ⫾ 18.6 ␮m, n ⫽ 4) were smaller than
those from age-matched control fetuses (444.2 ⫾ 26.2 ␮m,
n ⫽ 4, P ⬍ 0.05).
After several washes and a further 30-min equilibration,
vessel responsiveness was tested by stimulation with NEK [5
␮M norepinephrine (NE) in 125 mM potassium-substituted
PSS (KPSS)], KPSS alone, 5 ␮M NE alone, and a second
exposure to NEK. Cumulative concentration-response curves
(CRC) were performed to KCl (2–125 mM) and ET-1 (10
pM-0.3 ␮M). The relaxing effects of the cumulative addition
of ACh (1 nM-10 ␮M) on femoral arteries were assessed after
preconstriction with 5 ␮M NE. Because NE-induced vasoconstriction was not maintained in middle cerebral arteries, 50
mM KCl were used to constrict these arteries. Additions were
performed after a plateau had been attained to the preceding
concentration.
Receptor autoradiography. ETA receptors were visualized
in duplicate cryostat sections chosen at random following
incubation with 125I-PD-151242 using quantitative autoradiography, using methods developed for human vessels (23,
32). 125I-PD-151242 has previously been characterized in
vascular smooth muscle having subnanomolar affinity for the
ETA receptor but micromolar affinity at ETB (10, 11). At the
concentration used, 125I-PD-151242 would occupy ⬍0.03% of
the ETB subtype and the ligand would not detect any ETB
receptors present on endothelial cells. At present, there is no
general agreement as to the diameter of a resistance vessel;
the term has been applied to vessels ranging in diameter
from ⬍2 mm (40) to ⬍500 ␮m (26). The diameter of ⬃100
vessels analyzed by autoradiography ranged from 45 to 450
␮m. It is likely that vessels of varying diameter along the
vascular tree would contribute in some degree to vascular
resistance.
Briefly, 10-␮m thick cryostat-cut sections of femoral muscle and frontal cortex were mounted on Poly-Prep slides
(Sigma, St. Louis, MO) and stored at ⫺80°C until they were
processed. Slides were preincubated in HEPES buffer (50
mM HEPES containing 5 mM MgCl2 and 0.3% wt/vol BSA,
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by augmenting vasoconstrictor responses to ET-1. We
therefore examined vasoconstrictor responses to ET-1
in isolated fetal ovine femoral and middle cerebral
resistance arteries, following exposure of the fetal
sheep to a DM infusion for 48 h at 110–111 days
gestational age (dGA; 0.75 gestation). Autoradiographic techniques using receptor subtype-specific ligands were used to quantify the density of ETA and
ETB receptors in the vessels and surrounding tissue.
Because alteration in vascular responsiveness with
DM exposure may reflect changes in the endothelium,
we also examined responses of preconstricted arteries
to the endothelium-dependent relaxatory agent ACh.
We chose these specific fetal vascular beds because the
cerebral circulation is “protected” at times of oxygen
deficiency and undergoes vasodilation and increased
flow (7). In contrast, the femoral bed is unprotected,
acting as a reservoir of peripheral vascular resistance
to maintain perfusion pressure in protected vascular
beds. We chose a stage of gestation to approximate the
period of gestation at which antenatal glucocorticoids
are used therapeutically in human pregnancy.
ET-1-INDUCED VASOCONSTRICTION OF FETAL OVINE ARTERIES
mide were purchased from Sigma. ET-1 was obtained from
Bachem (Torrance, CA).
Data analysis. Tension is expressed as milliNewton per
millimeter artery length or as a percentage of the maximal
response to K⫹. Relaxation is expressed as a percentage of
the preinduced tension. CRC were constructed by fitting data
to the logistic sigmoid equation (log agonist concentration vs.
effect), using the GraphPad Prism program (GraphPad Software, San Diego, CA). Sensitivity (pEC50) to the agonists is
expressed as the negative log of the effective molar concentration of the agonist required to elicit 50% of the maximum
response. Magnitude of responses and pEC50 values were
compared between groups by Student’s unpaired t-test. Data
from receptor autoradiography studies were analyzed by
computer-assisted image analysis (Quantimet 970, Leica).
The autoradiographic image of nonspecific binding was digitally subtracted from the total to calculate specific binding
densities in attomoles per millimeter squared by interpolation from a standard curve. Differences were considered
statistically significant at P ⬍ 0.05. All results are presented
as means ⫾ SE, and n refers to the number of animals
studied.
RESULTS
Blood pressure measurements. Measurement of fetal
blood pressure following the 48-h in vivo protocol
showed a significantly greater increase in the DMtreated compared with the saline-treated control fetuses [7.3 ⫾ 2.3 (n ⫽ 6) vs. 0.6 ⫾ 2.3 mmHg (n ⫽ 5), P ⬍
0.05], compared with pretreatment values for each
group. Data on fetal arterial blood gases and fetal
cardiovascular function before and after 46 h of infusion in both groups are given in Table 1.
K ⫹-induced vasoconstriction. Sensitivity to KCl-induced vasoconstriction was similar in femoral (Fig. 1A)
and middle cerebral (Fig. 1B) arteries from control and
DM-treated fetuses (pEC50 ⬃1.45 and ⬃1.8 for femoral
and middle cerebral arteries, respectively). The magnitude of the maximal vasoconstriction evoked by 125
Table 1. BL fetal blood gases 2 h before
administration of vehicle (n ⫽ 5) or Dex
(n ⫽ 5) and after 46 h of Inf
pH
BL
Inf
PCO2, mmHg
BL
Inf
PO2, mmHg
BL
Inf
FBP, mmHg
BL
Inf
FHR, beats/min
BL
Inf
Vehicle
Dex
7.364 ⫾ 0.007
7.347 ⫾ 0.005
7.330 ⫾ 0.021
7.346 ⫾ 0.013
52.1 ⫾ 0.5
54.3 ⫾ 1.1
50.4 ⫾ 1.4
50.8 ⫾ 1.3
22.5 ⫾ 1.2
21.6 ⫾ 1.6
27.3 ⫾ 1.4
26.5 ⫾ 1.2
32.4 ⫾ 1.7
32.6 ⫾ 2.8
34.7 ⫾ 2.5
42.8 ⫾ 3.0*
172.8 ⫾ 1.5
172.6 ⫾ 2.6
174.4 ⫾ 3.2
178.4 ⫾ 3.3
Values are means ⫾ SE. Baseline (BL) cardiovascular data during
the 12 h before infusion (Inf) and in the final 12 h of the Inf of vehicle
or dexamethasone (Dex) Inf. The only significant change was the rise
in fetal blood pressure (FBP) in Dex-exposed fetuses. FHR, fetal
heart rate. * P ⬍ 0.05.
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pH 7.4) for 30 min at room temperature (23°C). To visualize
the ET-receptor subtypes present in these tissues, the wash
buffer was replaced with HEPES buffer containing 0.1 nM
125
I-ET-1 (to label both receptor subtypes), 0.1 nM 125I-PD151242 (10) (to label ETA receptors), or 0.3 nM 125I-BQ3020
(24) (to identify ETB receptors). Nonspecific binding was
defined by the inclusion of 1 ␮M of the corresponding unlabeled peptide. After a 2-h incubation period, the slides were
washed in ice-cold Tris buffer (50 mM, pH 7.4) and apposed to
radiation-sensitive film (Hyperfilm Bmax, Amersham Pharmacia Biotech, Buckinghamshire, UK) for 5 days. Autoradiograms were analyzed by measuring the diffuse-integrated
optical density using a computer-assisted image-analysis
system (Quantimet 970, Leica, Milton Keynes, UK) equipped
with a shading corrector (with the correction performed in
real time before each scan). The shading corrector compensated for any variation in illumination so that optical densities were measured accurately throughout the autoradiogram. The white level (100% transmission) was set, and the
scanner dark current (the current flowing in the scanner in
the absence of a signal) that would otherwise contribute to
the gray image was eliminated.
The density of ET receptors within the vessels present
throughout the tissue sections was measured by digitizing
each autoradiographic image into an array of 630,000 image
points each with a gray value in the range 0–255. A cursor
was used to draw around each vessel wall displayed on a
high-resolution monitor; the computer isolated each area
defined by these marks and measured the integrated optical
density. When all measurements had been made for a particular section, the threshold for detecting the autoradiogram
was increased to produce a template that was used to align
the autoradiographic image of an adjacent section used to
define the nonspecific binding. The second image was digitally subtracted from the first to measure the amount of
specific binding. The resulting optical densities were converted to the amount of specifically bound radioligand in
attomoles per millimeter squared by interpolation from the
standard curve. Corrections were made for decay of the
radioligand from the time at which these measurements
were made to the midpoint between apposing and developing
the film.
A standard curve was constructed for each film by coexposing calibrated 125I standards with each film and generating an appropriate curve. The autoradiographic image of
each standard was detected, and the cursor was used to draw
around and isolate the image. The integrated optical density
for each standard together with the area was determined.
The radioactivity of each standard, measured by gamma
counting (corrected for the efficiency of the counter), was
divided by the area to calculate radioactivity in dpm per
millimeter squared. For each standard curve, the image
analyzer generated a natural log plot of optical density vs.
radioactivity that gave a linear relationship (see Ref. 9 for a
discussion of the statistical analysis of the data). Tissue
sections were exposed to produce optical density values falling within the linear range (0.1 to 0.8) that is below the
optical density of one, at which point radiation-sensitive
films saturate and cannot record further increases in tissue
radioactivity. The interassay coefficient of variation was
⬍3% (9).
Drugs and solutions. PSS was of the following composition
(in mM): 119 NaCl, 4.7 KCl, 1 KH2PO4⫺, 0.9 MgSO42⫺, 2.5
CaCl2, 11.1 glucose, 2.5 NaHCO3⫺, and 0.021 EDTA. KPSS
was prepared by equimolar substitution of NaCl by KCl in
PSS. Chemicals for PSS, NE, bitartrate salt, and ACh bro-
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ET-1-INDUCED VASOCONSTRICTION OF FETAL OVINE ARTERIES
DISCUSSION
Fig. 1. KCl-induced vasoconstriction (as % of own maximum response) in femoral (A) and middle cerebral (B) resistance arteries
from saline-treated control (E) and dexamethasone (DM)-treated (F)
fetal sheep (means ⫾ SE, n ⫽ 4–6).
mM K⫹ was also similar in femoral (0.49 ⫾ 0.15 vs.
0.83 ⫾ 0.14 mN/mm) and middle cerebral (1.3 ⫾ 0.28
vs. 0.9 ⫾ 0.1 mN/mm) arteries from control and DMtreated fetuses.
ACh-induced response. Sensitivity to ACh was similar in femoral arteries from control (pEC50: 6.99 ⫾
0.14) and DM-treated fetuses (pEC50: 7.3 ⫾ 0.37). Arteries from both groups relaxed to near baseline tension (Fig. 2A). Preconstricted middle cerebral arteries
did not relax to ACh. Indeed, increasing concentrations
of ACh tended to augment the preinduced tone, and
this was particularly notable in arteries from DMtreated fetuses (Fig. 2B).
ET-1-induced vasoconstriction. ET-1 sensitivity was
significantly greater in femoral arteries from DMtreated fetal sheep compared with controls (pEC50:
8.17 ⫾ 0.03 vs. 7.83 ⫾ 0.14, P ⬍ 0.05) (Fig. 3). In middle
cerebral arteries from DM-treated fetuses, a pronounced tachyphylaxis was noted to ET-1 concentrations greater than 10 nM. Because a plateau in the
ET-1-induced vasoconstriction was not obtained in the
middle cerebral arteries in this group, it was not possible to calculate a pEC50 value. pEC50 for ET-1 in
middle cerebral arteries from control fetuses was calculated as 8.34 ⫾ 0.02 (n ⫽ 4). The contraction elicited
by ET-1 (expressed as mN/mm) was augmented in
femoral arteries from DM-treated compared with control fetuses (Fig. 3A). In middle cerebral arteries from
control fetuses, the maximum vasoconstriction was at-
In the present study, we demonstrated that ET-1induced in vitro vasoconstriction of fetal ovine femoral
resistance arteries was increased following in vivo administration of DM directly to the fetus for 48 h at
Fig. 2. Response to acetylcholine (ACh) (as % of preinduced tone) in
femoral (A) and middle cerebral (B) resistance arteries from salinetreated control (E) and DM-treated (F) fetal sheep (means ⫾ SE,
n ⫽ 4–6).
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tained at 0.1 ␮M ET-1 (1.51 ⫾ 0.29 mN/mm). However,
a comparatively smaller maximum response was
evoked by 0.1 nM ET-1 in middle cerebral arteries from
DM-treated fetuses (0.95 ⫾ 0.06 mN/mm); tachyphylaxis occurred with higher concentrations (Fig. 3B).
Tachyphylaxis was not seen in femoral arteries in the
ET-1 concentration range examined.
Distribution of ET-receptor subtypes. 125I-PD-151242
identified a significantly higher density of ETA in small
femoral muscle vessels from DM-treated compared
with control fetuses, but no effect of DM was noted in
the muscle tissue surrounding the femoral arteries
(Figs. 4 and 5, C and D). In contrast, DM treatment
was associated with a decrease in ETA receptors in
middle cerebral artery branches (Figs. 4 and 5, A and
B). In sections from both vascular beds, in marked
difference to the intense binding visualized with 125IPD-151202, relatively little ETB-receptor density could
be detected within the vessel walls using 125I-BQ3020,
and there was no difference between experimental
groups (results not shown).
ET-1-INDUCED VASOCONSTRICTION OF FETAL OVINE ARTERIES
110–111 dGA. As in previous studies, we chose to
administer DM directly to the fetus so that we could
have complete control over fetal exposure without confounds due to maternal and placental metabolism (13).
We have previously demonstrated that a dose of
10 ␮g/h in fetuses weighing ⬃3.32 kg produced concentration of fetal plasma DM of 8 ⫾ 1 ng/ml. These
plasma levels are in the same range as human fetal
plasma levels obtained at cesarean section within 24 h
following maternal glucocorticoid administration (22).
The stimulatory effect of prenatal DM exposure on
femoral vascular reactivity was manifested by a decrease in the concentration of ET-1 required to elicit
50% of the maximal vasoconstriction and an augmented maximal response. In accordance with the
functional results, autoradiography studies demonstrated an increase in ETA receptor-ligand binding
number in small resistance-sized femoral vessels from
DM-treated fetuses. Hence, DM-induced increase in
ET-1 responsiveness appears to be, at least in part,
manifested by an increase in ETA-receptor binding.
To date, relatively few studies have examined the
effect of in vivo glucocorticoid administration on intact
artery ET-1 responsiveness in either fetal or adult
vessels. In contrast to our own findings, reactivity to
ET-1 was not altered in isolated aortic and carotid
conduit arteries from rabbits treated with DM in vivo
(37). Discrepancies in results may be related to differ-
ences in species, developmental age, vessel size, and
vascular preparation.
Middle cerebral arteries from DM-treated sheep
showed a pronounced tachyphylaxis to ET-1 concentrations greater than 10 nM. However, in cerebral arteries
from control fetuses, the maximal vasoconstriction was
not attained until 0.1 ␮M ET-1. Compared with the
femoral vascular bed, a downregulation of ETA-receptor binding was shown in middle cerebral artery
branches from treated fetuses, indicating vascular bed
specificity in DM-induced changes. The greater susceptibility of middle cerebral arteries from DM-exposed
compared with control fetuses to ET-1-induced tachyphylaxis may be related to the comparatively smaller
number of vasoconstrictor ETA receptors in the former;
therefore, tachyphylaxis occurs with relatively lower
concentrations of ET-1 compared with control arteries.
However, the aforementioned explanation is entirely
speculative, and this interesting phenomenon warrants further investigation.
Several studies indicate that glucocorticoids alter
ETA receptors. Glucocorticoids were shown to induce
upregulation of ETA receptor mRNA in human osteoblastic cells (5). However, in A7r5 vascular smooth
muscle cells, although DM increased pre-pro-ET-1
mRNA abundance, it decreased ETA receptor mRNA
abundance and reduced the number of maximal ET-1binding sites without altering their binding affinity.
This suggests that in in vitro culture conditions, DM
downregulates the expression of this receptor subtype,
possibly in part by the autocrine production of ET-1
(21). Similarly, RU-28362, a pure glucocorticoid agonist, caused a decrease in ETA-receptor number but not
affinity in vascular smooth muscle cells in vitro from
spontaneously hypertensive adult rats (33). Likewise,
using a rat vascular smooth muscle cell line (A-10) that
displays high-density and high-affinity ETA receptors,
pretreatment with DM was shown to reduce ETA receptor mRNA and the number of ET receptors by
50–60% without changing their affinity. Furthermore,
this downregulation of ET receptors was accompanied
Fig. 4. Measurements of optical density for specific binding of 125IPD-151242 (amol/mm2) in cerebral arteries, vessels located in femoral muscle, and femoral muscle tissue from saline-treated control
(open bars) and DM-treated (filled bars) fetal sheep (means ⫾ SE,
n ⫽ 4–5). **P ⬍ 0.01; ***P ⬍ 0.001.
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Fig. 3. Endothelin-1 (ET-1)-induced vasoconstriction (mN/mm) in
femoral (A) and middle cerebral (B) resistance arteries from salinetreated control (E) and DM-treated (F) fetal sheep (means ⫾ SE, n ⫽
4–6, *P ⬍ 0.05 control vs. DM).
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ET-1-INDUCED VASOCONSTRICTION OF FETAL OVINE ARTERIES
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Fig. 5. Computer-generated, colorcoded images showing binding of the
ETA-selective ligand 125I-PD-151242 to
sections of frontal cortex from control
vehicle-treated fetus (A) and DMtreated fetus (B) and to femoral muscle
from control vehicle-treated fetus (C)
and DM-treated fetus (D). The autoradiographic image of nonspecific binding
was digitally subtracted from the total
to produce the image of specific binding. Density of binding in amol/mm2
was coded according to the scale bar by
interpolation from a standard curve.
Highest levels of binding are shown in
red, with black below the level of detection.
by an attenuated response to ET-1 in DM-pretreated
cells (29). Similar findings have been reported in endothelial cells. For example, in human cerebromicrovascular endothelial cells that express high-affinity ETA
receptors in culture, pretreatment with DM decreased
the number of ET-1-binding sites without changing the
binding affinity (39).
Importantly, the majority of previous studies examining the interaction of glucocorticoids and ET-1 has
used isolated or cultured vascular-derived cells. However, phenotypic changes in ET-receptor subtypes have
been demonstrated in cultured vascular smooth muscle
cells (36). For example, ETA receptor was shown to be
predominant in early-passage cells and ETB receptors
in late-passage cells, whereas only ETA receptor
mRNA was expressed in intact media of rat aorta (14).
Therefore, attempting to relate the previous findings in
cultured vascular cells to those we have obtained in
isolated intact arteries becomes complex. Furthermore,
of considerable importance is the fact that we examined fetal arteries, which are functionally distinct from
adult arteries. Because antenatal glucocorticoid treat-
ET-1-INDUCED VASOCONSTRICTION OF FETAL OVINE ARTERIES
animal model to elucidate further the action of glucocorticoids on the ET system.
Our findings suggest that the nature of the increased
ET-1-induced responsiveness in arteries from DMtreated fetuses is vascular bed specific. Tissue specificity of glucocorticoid-mediated effects has previously
been reported. For example, DM was shown to increase
ET-1 mRNA before downregulation of ETB receptor
mRNA level at the hypothalamus and cerebellum but
not other areas of the rat brain (38). Thus the influence
of glucocorticoids on ET-1 responsiveness of cerebral
arteries may well be differential depending on their
anatomic location. The tachyphylaxis we noted in DMexposed middle cerebral arteries may represent a tissue-specific protective mechanism to ensure maintained and adequate cerebral blood flow, particularly
at this crucial time of development.
In summary, the findings of our study provide further evidence for the interaction of glucocorticoids and
the potent vasoconstrictor ET-1. Our results indicate
that the DM-induced increase in peripheral vascular
resistance, which we have previously demonstrated in
fetal sheep (13), may involve increased ET-1 responsiveness in femoral arteries. The mechanism of this
increased ET-1-induced vasoconstriction may, at least
in part, be due to an increase in ETA-receptor binding.
Therefore, hyperreactivity to vasoconstrictor agonists,
such as ET-1, may be involved in glucocorticoid-induced hypertension in fetal sheep.
This research was supported by grants from Wellcome Trust, UK
and National Institutes of Health HD-21350. A. P. Davenport acknowledges grant support from the British Heart Foundation and
Royal Society, UK. C. C. Docherty was supported by a Wellcome
Trust Travelling Research Fellowship, UK. S. V. Koenen was supported by the Ter Meulen Funds, Royal Netherlands Academy of
Arts and Sciences.
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