Vol. 281, Issue 2, H804-H812, August 2001
Mechanism of uterine vascular refractoriness to endothelin-1
in pregnant sheep
Sherrie
McElvy1,
Suzanne G.
Greenberg1,
John
L.
Mershon1,
Da Seng
Yang1,
Catherine
Magill2, and
Kenneth E.
Clark1
1 Department of Obstetrics and Gynecology, University of
Cincinnati College of Medicine, Cincinnati, Ohio 45267-0526; and
2 Axys Pharmaceuticals, San Francisco, California 94080
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ABSTRACT |
Endothelin-1 (ET-1) is a potent vasoconstrictor
and produces marked pressor responses when given systemically. Studies
in sheep have demonstrated that during pregnancy the uterine
vasculature is refractory to exogenously administered ET-1. We
hypothesize that this pregnancy-dependent refractoriness is due to an
upregulation of local uterine metabolism of ET-1 and/or ETB
receptors and/or downregulation of local uterine ETA
receptors. To investigate these possibilities, 21 nonpregnant and 17 pregnant sheep were used. Dose-response curves to intravenous infusion
of ET-1 and phenylephrine were generated for pregnant and nonpregnant
sheep. ET-1 infused systemically demonstrated vasoconstriction in the systemic and renal vasculature of pregnant and nonpregnant animals and
vasoconstriction in the uterine vasculature of nonpregnant animals. The
pregnant animals showed no uterine vascular response to ET-1. In
contrast, phenylephrine showed vasoconstriction in the systemic, renal,
and uterine circulations in both pregnant and nonpregnant sheep. After
experimentation, the animals were euthanized, and tissues were
harvested for Western blot and activity analysis of neutral
endopeptidase (NEP) or RT-PCR analysis of endothelin-converting enzyme
(ECE) and ETA and ETB receptors. The content
and activity of NEP in the uterine and renal vasculature of pregnant
and nonpregnant animals were similar. RT-PCR demonstrated the presence
of ECE in the uterine vasculature of pregnant and nonpregnant sheep.
ETA receptor mRNA was significantly reduced in pregnant
compared with nonpregnant sheep, whereas ETB receptor mRNA
remained unchanged. We conclude that the uterine vascular refractoriness seen in the pregnant sheep is due to a downregulation of
ETA receptors.
estrogen; placenta; kidney
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INTRODUCTION |
SINCE THE DISCOVERY OF
ENDOTHELIN-1 (ET-1) (10, 30), a potent
vasoconstrictive peptide, there have been many studies investigating the mechanism and effects of this molecule (27).
Our laboratory (31), investigating the effects of ET-1 in
sheep, demonstrated that during pregnancy the ovine uterine vasculature
is specifically refractory to the vasoconstrictive effects of ET-1. The
vasoconstrictive actions of ET-1 occur through its interaction with
ETA receptors. Previous studies from our laboratory
(17) and others (4, 8, 28) have demonstrated
that ET-1 plays a role in maintaining vascular tone. In our previous
study (17), we used a selective ETA receptor
inhibitor to block the interaction of ET-1 with the ETA
receptor in pregnant and nonpregnant sheep. We found that after
ETA receptor blockade, uterine vascular resistance
decreased and uterine blood flow significantly increased. In contrast,
similar blockade of ETA receptors in pregnant sheep was
associated with a slight decrease in uterine blood flow.
ETA receptor blockade reduced systemic arterial pressure in
both pregnant and nonpregnant sheep, suggesting a role for ET-1 in
regulating systemic tone (17). These findings suggest that
the pregnancy-dependent refractoriness seen in our earlier study may be
due to either an upregulation of local uterine metabolism of ET-1 or an
alteration of local uterine ETA and/or ETB
receptors in the uteroplacental vasculature of the pregnant sheep.
With regard to ET-1 metabolism, several enzymes are known to contribute
to the in vivo degradation of ET-1, including carboxypeptidase A,
deamidase, and neutral endopeptidase (NEP) (6, 13, 20); of
these, NEP is thought to be the most predominant (1). NEP is a 90- to 100-kDa glycoprotein widely distributed on mammalian cells
and acts as an endopeptidase on oligopeptides, cleaving peptide bonds
on the amino side of hydrophobic amino acids (16). It has
been determined by molecular cloning to be one of the family of
membrane-anchored ectoenzymes (type II integral membrane protein) with
a short NH2-terminal cytoplasmic domain of 27 amino acids (7). Among its potential substrates are substance P,
bradykinin, atrial natriuretic peptide, angiotensins, and ETs
(16, 29).
Interestingly, it has been reported that, in the rat uterus, NEP mRNA
can be affected by estrogen and progesterone (21); furthermore, NEP activity is upregulated during pregnancy in the rat
uterus, suggesting a role for NEP in regulating uterine smooth muscle
cell contraction in late pregnancy (19). This led us to
speculate that an increase in NEP during pregnancy may increase metabolism of ET-1 in the uterine vasculature of pregnant animals, thus
offering a potential explanation for our previously reported uterine
refractoriness to exogenously administered ET-1 (31).
ET receptors belong to the superfamily of G protein-coupled receptors
and exist in two subtypes, ETA and ETB. In
vascular tissue, ETA receptors are expressed on vascular
smooth muscle and are responsible for vasoconstriction
(23). ETB receptors are expressed on the
vascular endothelium and mediate the transient vasodilator response to
ET-1 through the release of nitric oxide (NO) and/or prostacyclin
(23).
Because it is unknown whether the uterine vascular refractoriness seen
in the pregnant sheep is due to an increased metabolism of ET-1 by NEP
or an alteration of ETA and/or ETB receptors,
we undertook this study to compare the content and activity of NEP and
to determine the presence and quantity of ETA and
ETB receptors in the uterine arteries of pregnant and
nonpregnant sheep. These cellular results were compared with the
hemodynamic responses to ET-1 in the renal and uterine vasculature.
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MATERIALS AND METHODS |
Animals and Surgical Preparation
Twenty-one nonpregnant (50-70 kg) and seventeen pregnant
ewes (110-115 days gestation) were purchased from two commercial vendors (Joy Russell; Williamsburg, OH; and Tom Morris; Reistertown, MD) and used in various portions of this investigation. All studies were conducted under an approved Institutional Animal Care and Use
Committee protocol, and the sheep were housed in a facility approved by
the American Association for Accreditation of Laboratory Animal Care.
Our surgical procedures and instrumentation of pregnant and nonpregnant
sheep with maternal femoral artery and vein catheters, fetal femoral
artery and vein catheters, and maternal uterine and renal artery flow
probes have been previously published (9).
In this study, all nonpregnant animals were ovariectomized to control
for cyclic fluctuations in endogenous estrogen. All but eight of the
nonpregnant sheep were instrumented with chronic indwelling catheters
and flow probes as described above; these instrumented ewes received
estradiol-17
(1 µg/kg iv in ethanol) each evening postsurgery to
prevent uterine atrophy. To determine whether NEP, ECE, or ET receptors
were affected by estradiol-17, the eight nonpregnant sheep not
instrumented were divided into two groups: 1) an
estrogen-depleted group receiving no estrogen and euthanized 7 days
postovariectomy; and 2) an estrogen-treated group receiving
estradiol-17 (1 µg/kg iv) each evening for 7 days and euthanized
after the administration of an estrogen bolus (1 µg/kg iv), which
assured that tissue harvesting was at peak estrogen response (~2 h
after giving estrogen).
All pregnant animals were instrumented as stated above. The pregnant
and nonpregnant animals that were surgically instrumented were given at
least 7 days to recover before participating in experimental protocols.
The pregnant animals were euthanized after experimentation and before
138 days gestation to prevent labor and fetal delivery. In all
animals used for tissue collection, the uterine artery and renal
cortex were rapidly removed after euthanasia, and tissues were
immediately frozen in liquid nitrogen and stored at 
80°C.
Experimental Protocols
ET-1 and phenylephrine dose-response curves.
To confirm the uterine-specific refractoriness to systemically
administered ET-1, dose-response curves were generated in conscious chronically instrumented animals. Six pregnant and six nonpregnant sheep, all instrumented as described above, were allowed to recover, and baseline recordings of mean arterial pressure (MAP), heart rate,
and uterine and renal blood flow were obtained. After baseline measurements, animals received intravenous infusion of either ET-1 in a
series of six doses (0.1, 0.3, 1.0, 3.0, 10, and 30 ng · kg
1 · min1) or
phenylephrine in a series of four doses (0.1, 0.3, 1.0, and 3.0 µg · kg1 · min1). Each
drug was administered for a period of 10 min per dose, with graduated
doses to create cumulative dose-response curves. Arterial pressure,
uterine blood flow, and renal blood flow were recorded continuously
throughout the ET-1 or phenylephrine infusion, and uterine and renal
vascular resistances were calculated as MAP divided by the respective
blood flow. The nonpregnant animals used in this portion of the study
received estrogen each evening (1 µg/kg iv) to prevent uterine
atrophy but were not estrogenized at the time of the dose-response experiments.
To assess whether the specific uterine refractoriness to ET-1 observed
in pregnant animals could be attributed to compensatory vasodilation
via stimulation of NO release, ET-1 dose-response experiments were
repeated before and after administration of
N
-nitro-L-arginine methyl
ester (L-NAME) in a cohort of six pregnant sheep. After baseline measurements, animals received an
intravenous infusion of ET-1 in an abbreviated series of four doses
(1.0, 3.0, 10.0, and 30.0 ng · kg1 · min1) for a
total of 10 min per dose, with data recorded as above. Sheep were
allowed a 3-h reequilibration period, which was more than adequate for
all parameters to return to baseline. L-NAME (10 mg/kg) was
then administered as an intravenous bolus, and new baseline
measurements were recorded. The ET-1 cumulative dose-response curve was
then repeated.
Western blot analysis of NEP.
Twelve pregnant, eight nonpregnant estrogen-treated, and five
nonpregnant estrogen-depleted animals were used for this analysis. Frozen renal cortex and secondary uterine arteries were pulverized on
dry ice, homogenized, and suspended in 50 mM Tris · HCl with 5 mg/ml each of aprotinin and leupeptin and 0.1 M phenylmethylsulfonyl fluoride. The protein concentration of each homogenate was determined using a modification of the Lowry technique (detergent compatible protein assay, BioRad; Hercules, CA).
Samples (10 µg protein/well) were separated by electrophoresis on
8-16% gradient gels. After separation, samples were transferred to nylon membranes (MagnaGragh, MSI) by electrophoresis at 0.750 A for
4 h in a cold room. After transferring, the membranes were incubated in a blocking solution [5% nonfat dry milk, 5% gelatin, and 1% normal goat serum in Tris-buffered saline (TBS)] for 1 h
at room temperature on an orbital shaker. Membranes then received 3- to
15-min washes with TBS containing 0.5% Tween 20. Washed membranes were
incubated in primary antibody (polyclonal NEP antibody B58, 1:50,000;
gift from Axys Pharmaceutical; San Francisco, CA) overnight at room
temperature. Membranes were washed in Tween 20-TBS for 3- to 15-min
washes and incubated in secondary antibody (goat anti-rabbit IgG
conjugated to horseradish peroxidase, 1:10,000; Promega; Madison, WI)
for 2 h at room temperature on an orbital shaker. Membranes were
again washed with Tween 20-TBS for 3- to 15-min washes. Protein bands
on the membranes were developed on X-ray film using a nonradioactive
chemiluminescence reagent (Renaissance, NEN Life Sciences;
Boston, MA). To estimate the NEP content of each sample, the
density of detected bands was compared with that of bands obtained from
a dilutional series of recombinant human NEP.
NEP activity analysis.
For activity determination, tissues from the same sheep as above were
used, and the activity was estimated using a modification of the method
described by Albrightson et al. (2). The tissues were
homogenized in 100 mM MES buffer (pH 6.5, containing 300 mM NaCl) to
yield a protein concentration of 2-4 µg/µl. Assays were
performed in 96-well microtiter plates and initiated by the addition of
20 µl of NEP substrate
(glutaryl-alanine-alanine-phenylalanine-4-methoxy-2-naphthylamide, 1 mM; Sigma; St. Louis, MO). The reaction was allowed to proceed at
37°C for 60 min to generate the product
phenylalanine-4-methoxy-2-naphthylamide. This product was then further
hydrolyzed to 4-methoxy-2-naphthylamide by the addition of 20 µl of
10 µg/ml L-aminopeptidase in the presence of 2.5 µM
phosphoramidon (Sigma), with the reaction again allowed to proceed at
37°C for 60 min. The reaction was terminated by adding 10 µl of
10% trichloroacetic acid, and the final product was visualized by
adding of 150 µl of 0.05% Fast Garnet GBC (Sigma) and incubating for
30 min at room temperature. Absorbance of the developed product was
measured at 570 nm (with a reference of 630 nm to correct for sample
turbidity) using a microplate spectrophotometer (MRX, Dynex
Technologies; Chantilly, VA), and a standard curve using
4-methoxy-2-naphthylamide (0-200 µmol; Sigma) was generated. Aminopeptidase controls (reaction run in the absence of sample) and
NEP-positive controls (60, 6, and 0.6 µg recombinant human NEP; Axys
Pharmaceuticals) were also included on each assay plate.
RT-PCR analysis and quantification of ECE-1 and ETA
and ETB receptors.
Ten pregnant, four estrogen-depleted, and four estrogen-treated sheep
were used in this analysis. The methods for total RNA preparation and
RT-PCR have been previously described (18). In brief,
frozen uterine arteries were homogenized in 4 M guanidine thiocyanate
followed by a phenol chloroform extraction. Total RNA (5 µg) was
subjected to RT-PCR initially using bovine primers for the ET receptors
(3) and ECE-1 (12). These ovine PCR products
were subcloned and sequenced, and the partial ovine cDNA clones are
reported in Genbank (Accession Nos. AF293847, AF349439, and AF294269
for the ETA receptor, ETB receptor, and ECE-1, respectively). On the basis of the ovine sequences obtained, ovine primers were designed and synthesized for the ETA and
ETB receptors as well as for ECE-1. For the ETA
receptor, a 369-bp product was amplified using the following primers:
5'-TGGTCACAGCCATTGAGATTG-3' (sense) and 5'-TGAAGAGGGAACCAGCACAGA-3'
(antisense). For the ETB receptor, a 460-bp product was
amplified using the following primers: 5'-TCTGCTTGCTCCATCCCACT-3'
(sense) and 5'-TGATTGGCACCAGCAGCATA-3' (antisense). For ovine ECE-1, a
307-bp product was amplified using the following primers:
5'-ACATGATCTGGAACCTGGTGC-3' (sense) and 5'-TTCCTTGGCTGATTTCCGAG-3'
(antisense). Depending on the intensity of the amplified product,
35-45 cycles were employed for amplification. To ensure that the
equivalent cDNA template was used in each reaction, amplification of
-actin was used as an internal control. The primer sequences for
-actin were 5'-GACATGGAGAAGATCTGGCACC-3' (sense) and
5'-GAGCTTCTCCTTGATGTCACGC-3' (antisense). The PCR products were
electrophoresed on 1% agarose gels and stained with ethidium bromide.
Quantification of the PCR products was performed by computerized
densitometry (AlphaImager 2200, Alpha Innotech; San Leandro, CA), with
all reactions run on a single gel for comparison. The densities were
normalized to the corresponding actin bands.
Statistical analysis.
Data were analyzed by one-way ANOVA, two-way ANOVA with repeated
measures, or Student's t-test as appropriate. Data are
presented as means ± SE. Results were considered significant at
the P < 0.05 level.
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RESULTS |
Dose-Response Curves
When ET-1 was infused systemically in a cumulative series of six
doses, nonpregnant sheep responded with dose-dependent uterine vasoconstriction, whereas pregnant sheep were almost unresponsive to
the vasoconstrictive effect of ET-1. Figure
1 demonstrates this ET-dependent increase
in uterine vascular resistance, which resulted in a marked and
progressive decrease in uterine blood flow in the nonpregnant animals
and no effect on uterine blood flow or vascular resistance in the
pregnant animals. This pregnancy-associated refractoriness to ET-1
appeared to be specific to the uterine vasculature, because measurement
of renal blood flow and renal vascular resistance in response to ET-1
showed very similar responses between nonpregnant and pregnant sheep
(Fig. 2). In both groups, renal blood
flow fell from baseline in a dose-dependent fashion by up to
40-50% as a result of the increase in renal vascular resistance.
As in the uterine vasculature, the majority of the response
occurred at the highest three doses of ET-1. Both pregnant and
nonpregnant sheep exhibited a dose-dependent increase in MAP in
response to ET-1 (Fig. 3). This response
was slightly, although not significantly, blunted in pregnant animals.
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Fig. 1.
Changes in uterine blood flow (A) and uterine
vascular resistance (B) in response to increasing doses of
endothelin-1 (ET-1) infusion in nonpregnant and pregnant sheep.
*P < 0.05 and ** P < 0.01 vs. control
period. P < 0.0001, pregnant vs. nonpregnant
dose-response curves.
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Fig. 2.
Changes in renal blood flow (A) and renal
vascular resistance (B) in response to increasing doses of
ET-1 infusion in nonpregnant and pregnant sheep. *P < 0.05 and **P < 0.01 vs. control period.
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Fig. 3.
Changes in mean arterial pressure in response to
increasing doses of ET-1 infusion in nonpregnant and pregnant sheep.
**P < 0.01 vs. control period.
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It is possible that the uterine vascular refractoriness to ET-1
observed in the pregnant sheep is due to compensatory release of a
local vasodilator such as NO. Therefore, ET-1 dose-response curves were
repeated in pregnant animals in the presence of L-NAME, an
inhibitor of NO synthase. In this cohort of six sheep, uterine blood
flow again did not change in response to increasing doses of
intravenous ET-1, and this refractoriness was not affected by
administration of L-NAME (Fig.
4). As expected, baseline uterine blood
flow was slightly decreased after NO inhibition (from 540 ± 58 to
403 ± 44 ml/min), and in fact the entire dose-response curve was
shifted to lower values in the presence of L-NAME. This suggests that while endogenous NO does contribute to the regulation of
basal uterine tone in the pregnant sheep, it does not appear to be
involved in the mechanism of pregnancy-associated uterine vascular
refractoriness to ET-1.
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Fig. 4.
Changes in uterine blood flow in pregnant animals in
response to increasing doses of ET-1, either alone or in the presence
of 10 mg/kg
N-nitro-L-arginine methyl
ester (L-NAME). n = 6.
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To determine whether the uterine vascular refractoriness to ET-1
observed in pregnant animals was specific to ET-1, dose-response experiments were repeated using a second vasoconstrictor,
phenylephrine. While the uterine vasculature was slightly less
sensitive to phenylephrine during pregnancy, this was not significant
and was quite different from the complete refractoriness observed in
response to ET-1. By the highest dose of phenylephrine, uterine
vascular resistance more than doubled in pregnant sheep, and this was
associated with an ~50% reduction in uterine blood flow (Fig.
5). In the renal vascular bed, the
response to phenylephrine was also slightly but not significantly
blunted in pregnant animals, similar to what was observed in the uterus
(data not shown). Overall, the renal vasculature was much less
sensitive to phenylephrine than to ET-1, with renal blood flow
falling by only 25% from baseline even at the highest dose of
phenylephrine. MAP responses to phenylephrine were very similar between
pregnant and nonpregnant animals, with a significant increase in
pressure observed only at the highest dose in both groups (data not
shown).
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Fig. 5.
Changes in uterine blood flow (A) and uterine
vascular resistance (B) in response to increasing doses of
phenylephrine infusion in nonpregnant and pregnant sheep.
*P < 0.05 and **P < 0.01 vs. control
period.
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Western Blot Analysis and Enzymatic Activity for NEP
A representative Western blot is shown in Fig.
6, with bands corresponding to NEP
detected in homogenized uterine artery samples shown on the
right. In Fig. 6, left, an example of a standard curve generated from a dilutional series of recombinant human NEP
(1,250-39 pg) is shown; similar dilutional "standard curves" were included on each gel to allow quantitation of NEP content in
tissue samples. Figure 7 demonstrates
that there was no significant difference in the content of NEP in
uterine arteries from all three groups of sheep. Estrogen-depleted
nonpregnant animals were found to have a slightly lower NEP content
than the pregnant sheep (35 ± 8.9 vs. 56 ± 7.6 pg/µg
protein), whereas the estrogen-treated nonpregnant animals were found
to have an insignificant increase in total NEP (69 ± 19.3 pg/µg
protein). Similarly, the NEP content measured in the renal cortex was
not significantly different in all three groups of sheep (pregnant:
100 ± 24.5; estrogen-depleted nonpregnant: 132 ± 67.1;
estrogen-treated nonpregnant: 152 ± 34; Fig. 7).
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Fig. 6.
Right: representative Western blot for measurement of
neutral endopeptidase (NEP) content detected in the homogenized uterine
artery taken from 2 estrogen-depleted nonpregnant (control), 2 estrogen-treated nonpregnant, and 2 pregnant sheep. Left:
example of a standard curve generated from a dilutional series of
recombinant human (rh)NEP; quantitation of NEP content in tissue
samples was estimated by comparing the densities of detected bands
against those in the standard curve.
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Fig. 7.
Top: amount of NEP detected in the homogenized
uterine artery and renal cortex taken from pregnant, estrogen-depleted
nonpregnant (nonpreg control), and estrogen-treated nonpregnant
(nonpreg + Est) sheep. Bottom: activity of NEP detected
in the homogenized uterine artery and renal cortex taken from pregnant,
estrogen-depleted nonpregnant, and estrogen-treated nonpregnant sheep.
Neither NEP content nor activity was significantly different between
groups.
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When the above tissues were further analyzed to determine total NEP
activity, it was found that uterine arteries taken from all three
groups had similar levels of total NEP activity (pregnant: 54 ± 3; estrogen-depleted nonpregnant: 57 ± 4; estrogen-treated nonpregnant: 51 ± 3 nmol
product · h1 · mg protein1;
Fig. 7). Thus it does not appear that the uterine vascular
refractoriness to ET-1 observed in pregnant sheep is mediated by
pregnancy-related differences in the local metabolism of ET-1. In
support of this finding, there was no correlation between NEP activity
and uterine blood flow measured immediately before death in any of the
groups studied (data not shown). In renal cortical tissue, total NEP activity was substantially higher, as expected due to greater content;
but again, levels were similar between all three groups of sheep
(pregnant: 208 ± 21; estrogen-depleted nonpregnant: 184 ± 87; estrogen-treated nonpregnant: 234 ± 9 nmol product
· h1 · mg protein1; Fig.
7).
RT-PCR Analysis and Quantification
With the use of RT-PCR, partial cDNA clones for ovine ECE-1
and ETA and ETB receptors were obtained in this
study. The sequence for ovine ECE-1 (Genbank Accession No. AF294269) is
nearly identical to the bovine cDNA (12) (97% at the
nucleotide level and >99% at the protein level). Compared with the
human ECE-1 sequence (32), this ovine partial cDNA is 91 and 96% identical at the nucleotide and protein level, respectively.
The ovine ETA and ETB receptor partial cDNA
clones (Genbank Accession Nos. AF293847 and AF349439, respectively) are
also highly homologous to the bovine cDNA (3), 98 and
100% identical at the nucleotide and protein levels, respectively. The
ovine ETA receptor shows 90% identity at the nucleotide
level and 98% identity at the protein level to the human
ETA receptor (11). The ovine ETB
receptor shows 90.5% identity at the nucleotide level and 96%
identity at the protein level to the human ETB receptor
(Genbank Accession No. XM007108).
RT-PCR amplification of total RNA from uterine arteries of pregnant and
nonpregnant sheep demonstrated the presence of ECE-1 mRNA in all three
groups. The bands in each of these groups demonstrated some variation
in density. On the other hand, ETA receptor mRNA differed
significantly between the pregnant and nonpregnant sheep. Figure
8 is representative of these RT-PCR
results. This diagram depicts the results from two representative sheep
from each group for ECE-1, ETA receptor, ETB
receptor, and -actin (control). Densitometry analysis of
ETA receptor products (Fig.
9) revealed that this message was
significantly reduced in pregnant compared with nonpregnant control
ewes (from 1.45 ± 0.29 to 0.93 ± 0.07 units).
Interestingly, ETA receptor mRNA was also somewhat
suppressed by estrogen treatment in the nonpregnant animals (from
1.45 ± 0.29 to 0.98 ± 0.09 units), although this did not
reach statistical significance. Densitometry analysis of
ETB receptor products showed that while the message for
this receptor subtype is present in the ovine uterine vasculature, it
does not appear to be affected by either estrogen treatment or
pregnancy (Fig. 9).
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Fig. 8.
RT-PCR products of the ETA receptor, ETB
receptor, endothelin-converting enzyme (ECE), and -actin in uterine
arteries of representative nonpregnant (2 estrogen treated and 2 estrogen depleted) and pregnant sheep.
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Fig. 9.
Densitometry analysis of RT-PCR products of
ETA and ETB receptors detected in uterine
arteries of estrogen-depleted nonpregnant (NP control),
estrogen-treated nonpregnant (NP + Est), and pregnant sheep. *
P < 0.05 vs. nonpregnant control.
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DISCUSSION |
From earlier studies in our laboratory, we know that there is a
local uterine refractoriness to ET-1 in pregnant sheep
(31). Because we hypothesized that the uterine vascular
refractoriness seen in the pregnant sheep is due to increased ET-1
metabolism and/or downregulation of ETA receptors and/or
upregulation of ETB receptors, we undertook this study to
investigate the content and activity of NEP as well as to determine the
presence and quantity of ET receptor subtypes in the uterine
vasculature. Figure 10 depicts the
maturation, degradation, and receptor interaction of ET-1 and should be
referenced as we discuss various points in this pathway as potential
mechanisms for the uterine refractoriness seen in pregnant sheep.
ET-1 is the active metabolite produced by the enzymatic cleavage of Big
ET-1. There are two isoforms of this enzyme, ECE-1 and ECE-2. ECE-1 was
discovered first and thought only to be extracellular (plasma
membrane), but is now known to be intracellular along with ECE-2
(24, 25). A decrease in ECE could lead to
decreased conversion of Big ET-1 to ET-1, which would potentiate
uterine refractoriness. We looked at the expression of ECE-1 mRNA in
uterine arteries and found its expression and quantity to be similar in all groups (pregnant and nonpregnant sheep). These findings allow us to
exclude decreased ECE-1 in the pregnant sheep as the mechanism of
uterine refractoriness. However, because we did not look at ECE-2, it
is uncertain whether a difference in the presence or quantity of ECE-2
exists. Studies by others have shown that the quantity of ECE-2 mRNA is
only 1-2% of that of ECE-1 mRNA in cultured endothelial cells
(29).
Another mechanism that may result in the uterine refractoriness seen in
the pregnant sheep is an upregulation of NEP during pregnancy.
Recently, NEP has been found in the ovine uterus including the
endometrial stroma, myometrial smooth muscle cells, and vasculature in
early pregnancy (22). Our investigation demonstrates a
slight estrogenic upregulation of NEP in the uterine arteries, but this did not reach statistical significance. Furthermore, the measured activity in the uterine arteries and renal cortex showed no difference in pregnant and nonpregnant animals. Therefore, NEP upregulation does
not appear to be the mechanism responsible for pregnant uterine vasculature refractoriness. It is possible that other ET-1 degradation pathways not addressed by the present study may be upregulated during
pregnancy. Clearly, there are a variety of proteases that can
inactivate ET peptides (23), and while NEP appears to be the most predominant, it is certainly not specific for ET. Indeed, a
novel and seemingly highly specific ET-1-inactivating
metalloendopeptidase has been recently described in kidneys from
both rats (15) and humans (14). In addition,
receptor-mediated mechanisms (i.e., ETB receptor) may
significantly contribute to ET-1 clearance (5). Whether or
not any of these pathways is affected by pregnancy remains to be determined.
Another potential mechanism for the observed uterine refractoriness to
ET may involve pregnancy-dependent alterations in vascular receptors
for ET-1. ET-1 typically interacts with two receptor types,
ETA or ETB (Fig. 10). ETB receptors
typically mediate vasodilation via the release of NO; therefore, it is
possible that a pregnancy-dependent upregulation of ETB
receptors in the ovine uterine vasculature would serve to offset any
local vasoconstrictive response to ET-1 during pregnancy. However, the
results of the present study suggest that the ETB receptor
message in the ovine uterine artery is not altered during pregnancy,
nor is it influenced by estrogen in nonpregnant animals. With regard to
ETA receptors, interaction of ET-1 with this receptor
subtype produces vasoconstrictive effects, and these systemic responses
are blocked by ETA receptor-specific inhibitors in pregnant
and nonpregnant animals (17). Whereas ETA
receptor blockade in nonpregnant animals is associated with reductions
in uterine vascular tone, in the pregnant animals there is only minimal
effect of blockade of ETA receptors on uterine hemodynamics. Therefore, our current study examined the presence and
quantity of the ETA receptor message. We found that in
pregnancy there was a significant reduction in the ETA
receptor mRNA in the uterine vasculature. It is possible that the mRNA
may not correspond with protein expression in the ET system; this could not be ruled out in the present study due to the current unavailability of ovine-specific ET receptor antibodies. However, the RT-PCR findings
are consistent with the hemodynamic responses found in this study that
demonstrate little or no response to ET-1 in the uterine vasculature of
pregnant sheep despite normal dose-dependent systemic and uterine
vascular responses in nonpregnant sheep as well as dose-dependent
systemic responses in the pregnant sheep. The results of the present
study therefore suggest that the uterine refractoriness seen in the
pregnant sheep is the result of a downregulation of
ETA receptors.
Activation by ET-1 of a local uterine vasodilator system is another
mechanism that could contribute to the observed uterine refractoriness
to ET-1. In other words, if administration of ET-1 were to stimulate
the local uterine release of a vasodilator, this would offset the
vasoconstrictive effect of ET-1. NO, which is produced by the vascular
endothelium, would be a likely candidate for this, particularly because
stimulation of ETB receptors could mediate this response
(23, 27). We explored this possibility in six pregnant
animals by repeating the ET-1 dose-response curve in the presence of
L-NAME, an inhibitor of NO synthase. In this cohort, we
found that the uterine refractoriness to ET-1 was not altered by NO
blockade; thus it does not appear that NO is involved in the mechanism.
This, however, does not rule out the possible involvement of other
potential vasodilators such as prostacyclin.
ET-1, derived from the vascular endothelium, likely plays an important
role in the normal regulation of local as well as global vascular tone.
Specific uterine vascular refractoriness to ET-1 during normal
pregnancy may serve an important function to protect against sudden
and/or substantial decreases in uteroplacental perfusion at times when
locally generated or circulating ET-1 becomes elevated. If this
refractoriness is the result of a pregnancy-dependent downregulation of
uterine ETA receptors, as suggested by the present study,
then it is possible that disease states associated with decreased
uterine blood flow (such as preeclampsia) may involve an aberration in
this protective mechanism. This could lead to the uterine vasculature
being more sensitive to fluctuations in endogenous ET-1, resulting in
increased uteroplacental vascular tone and ultimately in decreased
uterine blood flow.
| |
ACKNOWLEDGEMENTS |
The authors gratefully acknowledge Jeanne Hirth for performing the
Western blot and activity analysis for NEP. We also thank R. Scott
Baker and Angella Friedman for diligent assistance with the daily
maintenance of animals used in this study.
| |
FOOTNOTES |
This research work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-49901, HL-51051, and HL-56714 as well as by
American Heart Association Fellowship Grant #9920620V (to S. McElvy).
Address for reprint requests and other correspondence: K. E. Clark, Dept. of Obstetrics and Gynecology, PO Box 670526, Univ. of
Cincinnati, Cincinnati, OH 45267-0526 (E-mail:
Kenneth.Clark{at}UC.edu).
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.
Received 21 September 2000; accepted in final form 25 April 2001.
| |
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