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1 Perinatal Research
Laboratories, During the follicular phase of the ovarian
cycle, when the local estrogen-to-progesterone ratio is elevated,
uterine blood flow is elevated. This vasodilatory response is
reproduced by exogenous 17
ovary; uterine blood flow; steroids; nitric oxide; mammary; renal
DURING THE FOLLICULAR PHASE of the ovarian cycle,
uterine blood flow (UBF) is elevated just before and during behavioral
estrus (i.e., the proestrous-to-estrous period), a time when the plasma estrogen-to-progesterone ratio is highest. After ovulation and estrus,
during the luteal phase of the estrous cycle, UBF returns to low basal
levels (4-6, 8, 13, 23, 33). Thus changes in UBF
observed during the estrous cycle are temporally and directly associated with the endogenous ratio of estrogen to progesterone in
systemic blood (4, 13, 23, 33) and locally in the uteroovarian lymph
(17, 18) and tissues (30, 38). Markee (25) was the first to demonstrate
that exogenous estrogen administration causes hyperemia
("blushing") of endometrial tissue transplanted into the eye of
the guinea pig or monkey. Since then, many investigators have confirmed
these observations by measuring UBF after estrogen treatment in
numerous species (1, 9, 14, 19, 21, 22, 31, 32, 36). In chronically
instrumented ovariectomized sheep, systemic (1-10 µg/kg iv)
17 We (32) and others (14, 36, 37) have reported that the uterine
vasodilatory response to E2 In the present study, we hypothesized that an increase in the
endogenous ovarian estrogen-to-progesterone ratio or exogenous E2 Synchronization of Follicular and Luteal Phase Ewes
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-estradiol
(E2) administration via a
nitric oxide (NO)-mediated mechanism. We hypothesized that endogenous
ovarian estrogen and exogenous
E2 treatment elevate expression
of endothelial cell-derived NO synthase (eNOS) in uterine, but not in
systemic, arteries. Uterine, mammary, and systemic (renal
and/or omental) arteries were collected from
1) ewes synchronized to the
follicular (day 
1 to
day 0) or luteal
(day 10) phases of the ovarian cycle (n = 4 per phase),
2) ovariectomized ewes 120 min after
systemic vehicle or E2 (5 µg/kg iv) treatment, and 3)
ovariectomized ewes on days 0,
3, 6,
8, and
10 of
E2 (5 µg/kg iv, followed by 6 µg/kg per day) treatment. Expression of eNOS was localized primarily to the endothelium rather than vascular smooth muscle (VSM) in all
arteries examined by immunohistochemistry and Western analysis; inducible NOS was not detected in either endothelium or VSM. Expression of eNOS protein was greater (P < 0.05) in uterine, but not in systemic, artery endothelium-isolated
protein collected from follicular versus luteal phase ewes. Acute
systemic E2 treatment of
ovariectomized ewes increased (P < 0.05) eNOS protein levels in uterine artery endothelium. Prolonged
E2 administration progressively
increased uterine, but not systemic, artery endothelial eNOS protein
expression. Therefore, the increased local estrogen-to-progesterone
ratio during the follicular phase locally elevates eNOS expression, which possibly elevates uterine blood flow. These responses can be
partly reproduced with E2 administration.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-estradiol (E2) causes
maximum uterine vasodilation acutely over a period of 90-120 min
(14, 21, 22). When very low estrogen doses are administered locally, directly into the uterine blood supply (3-4 µg
E2 per uterine horn), maximal
local unilateral UBF vasodilatory responses are induced (14, 21). The
prolonged intravenous infusion of
E2 into ovariectomized sheep
increases cardiac output (systemic flows) and blood volume while
decreasing blood pressure and systemic vascular resistance in a fashion
analogous to postmenopausal estrogen replacement therapy (19, 20, 22).
Recently we (20) compared the acute versus prolonged effects of
E2 administration on blood flow
to both reproductive and nonreproductive vascular beds. Administration of E2 (5 µg/kg iv, followed
by 6 µg/kg per day) induced acute (2 h) and prolonged (3-10
days) increases in uterine and mammary, but not renal or omental, blood flows.
involves the augmentation of nitric oxide synthase (NOS) enzyme
activity and de novo protein synthesis given that
nitro-L-arginine methyl ester
(L-NAME) and cycloheximide,
respectively, decrease
E2-induced increases in UBF.
L-NAME (32) and cyclohexamide
(unpublished observation) treatments also reduce uterine cGMP
production, the second messenger that mediates the physiological
actions of NO in vascular smooth muscle (VSM) (32); moreover, 3 days of
E2 infusion into ovariectomized sheep increases uterine, but not renal, artery NOS-specific activity and nitric oxide (NO)-mediated endothelium-dependent relaxation (37).
These in vivo observations underscore the physiological importance of
NOS activation in this UBF response to estrogen and suggest indirectly
that E2 either acutely (120 min) or chronically (
3 days) increases a protein that regulates
NOS-specific activity or, alternatively, that
E2 elevates the de novo
expression of NOS protein itself.
administered systemically or
locally, at doses and times that cause increases in UBF (14,
19-22), will specifically increase uterine, but not systemic,
artery endothelial cell NOS (eNOS) protein expression. The specific
objectives of this study were to determine
1) whether NOS isoform [eNOS
or inducible NOS (iNOS)] protein levels are increased in
reproductive vascular beds (uterine and mammary) during the follicular
versus the luteal phase of the estrous cycle,
2) whether exogenous systemic or
local E2 administration to
ovariectomized ewes results in an acute rise of eNOS or iNOS protein in
the uterine and mammary artery endothelium, 3) whether the prolonged
administration of intravenous
E2 increases eNOS expression in
reproductive vessels, 4) whether NOS
increases are specific to endothelium versus VSM, and
5) whether systemic (renal
and/or omental) nonreproductive vascular beds, which do not
respond to estrogen with dramatic increases in blood flow (20), have
changes similar to those of reproductive vascular beds (uterine and
mammary arteries) in NOS protein expression.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 to day 0,
n = 4) or luteal (day
10, n = 4). On
day 8 postestrus for each pair, ewes
assigned to the follicular group were given two intramuscular
injections of 5 mg of prostaglandin F2
(PGF2; Lutalyse; Upjohn,
Kalamazoo, MI) 4 h apart, and the ewes assigned to the luteal group
were given two intramuscular saline injections, in volumes equivalent
to that of PGF2, 4 h apart.
This synchronization protocol resulted in ewes showing estrus within
~44-56 h after the first
PGF2 injection (13). We
previously have reported (23) the estrogen and progesterone levels
using this model. Moreover, luteal blood flow (R. R. Magness and T. M. Phernetton unpublished observation) and progesterone fell precipitously
to basal levels within 6 h, and estrogen levels rose progressively
between 18 and 48 h after injection of
PGF2 (5, 23, 33).
Follicular phase ewes were euthanized 44 h after the first injection of
PGF2, and the paired luteal phase ewe was euthanized on the same day, also at ~44-46 h after the first injection of saline. Uterine, mammary, and renal arteries were collected as described in Procurement of Tissues.
Acute Systemic E2
Treatment of Ovariectomized Ewes
for 5 days)
followed by a 2- to 4-day steroid withdrawal period. The
E2 was dissolved in 95%
ethanol and stored at 4°C at a stock concentration of 1 mg/ml for
this and subsequent experiments. This stock solution was diluted in sterile saline (total volume 3 ml) to achieve the appropriate dose
adjusted for the body weight of each animal. It was established in our
previous studies (19, 20, 22) that the maintenance of maximal, normal,
and consistent E2-induced
increases in UBF and cardiac output (systemic flows) were achieved with
this treatment regime. On the day of study, the ewes that had been
randomly paired were given either 5 µg/kg iv
E2
(n = 8) or vehicle (ethanol at the
same volume as E2;
n = 8). At 120-130 min, ewes were
subjected to surgical death, and uterine, renal, and mammary arteries
were collected as described in Procurement of
Tissues. The dose of E2
and time of tissue collection were specifically chosen from previous
dose-response studies and acute (0-150 min) time-course studies
(14, 19, 20, 22) that demonstrated when maximal and steady-state
uterine (8- to 10-fold) and systemic (cardiac output; 25-35%)
vasodilatation occur.
Acute Local E2 Treatment
of Ovariectomized Ewes
vasodilatory responsiveness (14, 22), which was confirmed in at least
three consecutive studies (data not shown). Estrogen replacement was
withheld for 48 h, after which time ewes were injected unilaterally
with a 4-µg E2 bolus into one
randomly selected uterine artery and vehicle (95% ethanol, 3 ml saline
flush) was injected into the contralateral artery (14, 22). Ewes were
subjected to surgical death at 120-130 min, as described in
Procurement of Tissues, while UBF was
elevated maximally only to the uterine horn ipsilateral to the local
unilateral E2 injection, and
the ipsilateral and contralateral uterine arteries were collected.
Prolonged Systemic E2
Infusion Into Ovariectomized Ewes
) for 5 days. After a 4-day
steroid withdrawal period, a 5 µg/kg priming dose of
E2 (or vehicle) was
administered intravenously and
E2 was infused (0.0123 ml/min)
at 6 µg/kg per day for 3 (n = 4), 6 (n = 4), 8 (n = 4), or 10 (n = 4) days. Control sheep (n = 5) were
treated with vehicle (ethanol, saline at the same volume as
E2) for 0, 8, or 10 days. Ewes
were subjected to surgical death as described in
Procurement of Tissues, and uterine,
mammary, renal, and omental arteries were collected for eNOS protein determination.
Procurement of Tissues
Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care and Use Committees of both the Medical School and College of Agriculture and Life Sciences and follow the NIH guide and the "1993 Report of the AVMA Panel on Euthanasia" (1a). After local lidocaine anesthetic was applied to the neck, a 19-gauge polyvinyl catheter was introduced into the jugular vein via a percutaneous needle puncture and advanced to the level of the right ventricle. Ewes were subjected to surgical death with the use of general anesthesia (pentobarbital sodium; Nembutal; 40-50 mg/kg) to maintain tissue perfusion and oxygenation during the time of tissue collection. The uterus, mammary gland, and kidney and/or omentum were removed, and arteries were collected as previously described (24). Briefly, the uterus with the mesometrium, the kidneys with attached hilus, the greater omentum, and the mammary gland were excised rapidly and placed into PBS (8 mM sodium phosphate, 2 mM potassium phosphate, 0.15 M NaCl, pH = 7.4; Sigma Chemical, St. Louis, MO). Arteries were dissected free of connective tissue, fat, and veins and then rinsed free of blood. Intact artery segments were placed directly into lysis buffer (50 mM Tris, 0.15 M NaCl, and 10 mM EDTA, pH 7.4, plus the addition of 0.1% Tween 20, 0.1%-mercaptoethanol,
0.1 M phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml
aprotinin; all from Sigma Chemical). Other portions of
each artery were opened longitudinally, and the endothelium/tunica intima was gently scraped (3-6 times) from the artery and placed directly into lysis buffer using a curved-end spatula. This method results in a relatively pure preparation of endothelial cells devoid of
VSM contamination (24). The remaining "scraped" artery was
"rubbed" with a wet cotton swab, and any remaining adventitia was
extensively removed before the denuded artery (VSM) was placed into
lysis buffer. The endothelium-isolated protein (ENDO), denuded arteries
(VSM), and intact arteries in lysis buffer were frozen in liquid
nitrogen immediately on collection and were stored at 20°C.
Additional intact segments were collected for immunohistochemistry and
fixed in 4% formaldehyde in sodium cacodylate buffer (0.1 M, pH 7.4;
EM Science, Fort Washington, PA) for 24 h and then stored at 4°C in
sodium cacodylate buffer containing 0.01% sodium azide until
dehydration and placement into paraffin blocks (24).
Immunohistochemical Analysis of Arteries
To determine the cellular source of eNOS in arteries, immunohistochemical analysis for eNOS was performed with the use of a Vectastain ABC Elite kit (Vector Labs, Burlingame, CA) and a mouse monoclonal antibody against eNOS from Transduction Laboratories (Lexington, KY) as previously described (24).Preparation of Tissues for Western Analysis
Intact vessels (with endothelium) and denuded vessels (without endothelium) from uterine, mammary, renal, and/or omental arteries were homogenized in lysis buffer and then sonicated. Endothelium-isolated protein (tunica intima) from these same arteries was sonicated. After centrifugation to remove particulate matter from vessel preparations, the protein concentrations of samples were determined using a modified Lowry assay procedure (Bio-Rad, Hercules, CA). With the use of either 10- or 15-well gels, known concentrations of protein (25 or 50 µg for intact and denuded artery homogenates; 8 or 10 µg for endothelial cell lysates) were resolved on 7.5% polyacrylamide gels with 0.1% SDS at 100 V for 2-3 h at room temperature before transfer onto Immobilon-P membrane at 100 V for 2 h at 4°C using the Mini-Protean II system (Bio-Rad). Membranes were blocked with Tris buffer (20 mM Tris basic, 500 mM NaCl, pH 7.5; Sigma Chemical) containing 0.1% Tween 20 and 5% skim milk and were then rinsed briefly in Tris buffer to remove excess milk protein. Primary antibody was diluted in Tris buffer containing 1% BSA and Tween 20, and secondary antibody was dissolved in Tris buffer with Tween 20 and 0.5% skim milk. Primary antibody incubations were for 2 h at room temperature, and secondary antibody incubations were for 1 h, with one 15-min and three 5-min washes with Tris buffer and Tween 20 after each antibody incubation. The primary eNOS monoclonal antibody was from Transduction Laboratories (1:750 dilution), and a positive control, either ovine placental endothelial cell (OPEC) lysates or human umbilical vein endothelial cell (HUVEC) lysates, was included on each blot. The secondary antiserum was a sheep anti-mouse serum (Amersham; 1:3,000 dilution). The primary iNOS antibody was a polyclonal antibody from Affinity Bioreagents (Golden, CO), and stimulated RAW cell [lipopolysaccharide- (LPS) and interferon-
-stimulated mouse
macrophage cell line 264.7] lysates served as a positive control
on each blot. The secondary antiserum for iNOS was a
donkey anti-rabbit serum (Amersham, Arlington Heights, IL; 1:8,000
dilution). The membrane was probed as described by Amersham using the
enhanced chemiluminescence (ECL) kit and exposure to Hyperfilm for 15 min. The relative level of NOS in each sample was determined after ECL
detection by using Bio-Rad scanning transmission densitometer model 670 coupled with Bio-Rad Molecular Analyst software (v. 1.5, Build 468).
Validation of eNOS and iNOS Western Immunoblot Protocol
We first evaluated the specificity of the antibodies to the two NOS isoforms studied. Positive controls used for the two isoforms were as follows: for eNOS, OPEC and HUVEC lysates; for iNOS, liver from a "septic" sheep (see below) and activated mouse macrophages (RAW cells) that were provided with the antibody. The two antibodies only positively recognized their appropriate standards. When these standards and samples were compared with the molecular mass of "rainbow" marker run on the same blot, they were at the expected molecular masses for these isoforms (i.e., eNOS
140-150 kDa and iNOS
130-135 kDa). The ECL signals obtained in this study fell within the linear range of scanning densitometry. Detection of iNOS in
ovine tissues by Western immunoblot, to our knowledge, has not been
reported previously. A rabbit polyclonal antibody (Affinity
Bioreagents) was used to detect iNOS in uterine and systemic artery
preparations. The antibody was initially screened using stimulated
murine RAW cell lysates to determine the optimal dilution of the
primary and secondary antibodies. To determine whether this antibody
recognized sheep iNOS, tissue from a sheep that was given a systemic
dose of LPS (kindly provided by D. Traeber, University of Texas Medical
Branch of Galveston, TX) at a level that causes endotoxemia was
analyzed. Tissue collected from this sheep included lung, liver, and
kidney. Similar tissue was collected from a control animal. Each tissue
was homogenized and sonicated, and the protein concentration was
determined as described in Preparation of Tissues for
Western Analysis. Equal concentrations of
protein (50 µg) from each tissue and animal (LPS-treated or control)
were loaded onto 10-well gels, and proteins were separated by SDS gel electrophoresis [in which a molecular mass marker (Amersham) was included] and transferred onto an Immobilon-P membrane as
described in Preparation of Tissues for Western
Analysis. The membrane was blocked with
milk buffer and then incubated with primary iNOS antibody (1:8,000
dilution; 2 h), followed by incubation with secondary antiserum
horseradish peroxidase-conjugated donkey anti-rabbit (1:8,000, 1 h;
Amersham). Protein levels were examined by ECL detection. A positive
band at an approximate molecular mass of 130 kDa was detected in
stimulated murine RAW cell lysates and in kidney and liver, but not
lung, tissue from a sheep treated with LPS. These three tissues from
untreated animals did not have detectable iNOS, demonstrating that this
antiserum specifically recognizes iNOS in the sheep.
Statistical Analysis
Differences in treatment (follicular vs. luteal phase in intact sheep or acute systemic and local E2
vs. vehicle in ovariectomized sheep) for each tissue type were analyzed
using Student's t-test. Data for
ovariectomized animals treated with prolonged
E2 were analyzed by ANOVA that
measured both treatment and time effects. Means were compared by
Student-Newman-Keuls multiple comparison test.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effects of the Ovarian Cycle on Levels of eNOS and iNOS Protein
Synchronization of the ovarian cycle.
Treatment of sheep with PGF2
decreased (P < 0.01) the size of the
corpus luteum from 10.3 ± 0.3 mm in luteal phase sheep to 6.5 ± 0.5 mm in the follicular phase animals (Table
1). The corpora lutea of the luteal phase
sheep were vascular and reddish in color, whereas the luteal structures
of the PGF2-treated animals
were avascular and blanched. None of the follicular phase ovaries had
ovulated follicles. The size of the large ovarian follicles (>6 mm)
in the follicular phase sheep were also increased compared with that in
luteal phase ewes.
|
Immunohistochemical localization of eNOS during the ovarian cycle. Endothelial cells showed significant positive staining for eNOS in all arteries examined (Fig. 1). Staining in the remaining vessel wall (VSM) was less distinct and patchy, and, therefore, it was unclear whether this was associated with any specific cell type. Renal and mammary artery endothelium showed the highest intensity of endothelial staining. The IgG controls showed little staining of either endothelial cells or VSM. Qualitatively, the uterine artery endothelial eNOS staining was consistently higher during the follicular versus the luteal phase (Fig. 1, top). The overall intensity of the eNOS staining appeared to be similar between follicular and luteal phase ewes in the renal and mammary arteries (Fig. 1, middle and bottom). The immunostaining of eNOS in omental artery cross sections from four additional follicular and luteal phase sheep were similar (data not shown).
|
Quantification of endothelial versus VSM protein expression of NOS by Western immunoblot analysis during the ovarian cycle. A much stronger signal (P < 0.01) was observed in endothelium-isolated proteins than in intact artery homogenates in all arteries examined. In uterine arteries we previously established that the endothelium-isolated protein enriched the eNOS levels 700- to 800-fold (24). Consistent with this, an eNOS band was detected in all of the intact arteries studied, and endothelium removal decreased the level of eNOS protein to nearly nondetectable levels compared with that in intact arteries. The comparison of the relative changes in eNOS protein, expressed as a percentage of mean luteal controls, with the absolute absorbance units per nanogram of protein are shown in Fig. 2 for uterine, renal, and mammary arteries. We observed an average fivefold increase of eNOS concentration in uterine artery endothelium-isolated protein (P = 0.03) collected from follicular versus luteal phase animals; however, endothelium-isolated proteins collected from renal or mammary arteries did not differ significantly (P = 0.79). In intact artery homogenates collected during the follicular versus luteal phase, eNOS concentrations were extremely low and were not significantly different in the uterine (P = 0.42) or renal (P = 0.94) arteries, but they appeared greater in mammary arteries (P = 0.01). Although the intact mammary artery comparison was statistically significant when expressed as a percentage of luteal controls, this difference was not observed in either the ENDO or denuded mammary artery (VSM; data not shown), casting doubt on the biological significance of this mammary artery eNOS response. There also were no major differences in eNOS concentrations observed in VSM homogenates from either uterine or renal arteries collected during the follicular and luteal phases (P > 0.05). Under the current assay conditions, iNOS could not be detected in whole artery homogenate, denuded artery homogenates, or endothelium-isolated protein from any artery collected from the animals in this study, even with maximum protein levels loaded onto the gels. In four additional sheep, eNOS levels for omental artery, ENDO, whole artery, and VSM were similar in the follicular versus luteal phases (data not shown).
|
Effects of Acute Systemic
E2 Treatment on Expression
of eNOS and iNOS
Immunohistochemical localization of eNOS with acute
E2 treatment.
The immunohistochemical localization of eNOS in acute vehicle- versus
E2-treated ovariectomized ewes
is shown in Fig. 3. Greater staining for
eNOS was observed in the endothelium compared with VSM in uterine,
renal, and mammary arteries. Staining in the VSM was faint and patchy
or, in some cases, nonexistent, regardless of treatment. The uterine
artery endothelium, but not VSM, staining appeared to be increased in
the E2- versus vehicle-treated
sheep. Renal and mammary artery endothelium and VSM eNOS were
qualitatively unaltered by acute
E2 treatment. Omental arteries
from three additional acute
E2-treated sheep did not show
alterations in eNOS levels in endothelium or VSM (data not shown).
|
Quantification of effects of acute systemic
E2 on eNOS and iNOS
expression by Western immunoblot analysis.
Western immunoblot studies from acute vehicle- and
E2-treated ovariectomized ewes
are shown in Fig. 4. Levels of eNOS in uterine artery endothelium were significantly elevated by
E2 treatment to 249 ± 70%
of the levels in vehicle-treated controls (P = 0.02). There were no significant
acute E2-mediated changes in
eNOS observed in mammary or renal artery endothelium-isolated protein.
There also were no differences seen in eNOS levels between vehicle and
E2 treatment of ovariectomized
sheep in intact artery homogenates (P = 0.70) or denuded artery homogenates (VSM)
(P = 0.18; data not shown) from any of
the tissues examined, demonstrating the need to isolate the uterine
artery endothelium to be able to quantify these small changes in eNOS
expression by this method. In omental arteries from three additional
acute E2-treated sheep at 120 min, no significant alterations in eNOS protein expression were noted
in ENDO, intact artery, or VSM. In addition, under the present assay
conditions iNOS protein expression could not be detected in the
endothelium or VSM homogenates of all the vessel types we studied (data
not shown).
|
Effects of local acute E2
treatment on expression of eNOS.
To determine the local effects of
E2 on UBF and eNOS expression,
four chronically instrumented ovariectomized nonpregnant sheep were
randomly treated with 4 µg E2
administered into one uterine artery catheter and vehicle into the
contralateral side. Control UBF to the contralateral-treated uterine
horn averaged 10 ± 2 ml/min and was not dramatically altered at 90 (17 ± 5 ml/min) and 120 min (25 ± 9 ml/min). In contrast, in
the uterine horn ipsilateral to the
E2 injection, UBF was observed
to progressively and substantially increase (P < 0.001)
from control values of 14 ± 3 ml/min, plateauing at 90 (147 ± 14 ml/min) and 120 min (168 ± 28 ml/min). During local
E2 responses, the changes in uterine vascular resistance were inverse to UBF, and neither mean arterial pressure nor heart rate was altered significantly
(P > 0.05). The eNOS levels in the
ipsilateral uterine artery endothelium-isolated protein lysates at
120-130 min after unilateral
E2 injection averaged 737 ± 592% of levels in the contralateral vehicle control (data not shown).
We have no explanation for the variability in the local eNOS response,
although it stemmed from the fact that the unilateral increase in eNOS
was observed only in three of the four animals studied. Although this
increase in eNOS expression did not reach statistical significance, it
was statistically equivalent to the 249 ± 70% elevation in uterine
artery eNOS in ENDO with systemically administered
E2, suggesting that there was
indeed a similar effect of local and systemic
E2 on uterine artery eNOS expression.
Effects of Prolonged E2 Infusion on
Expression of eNOS
Immunohistochemical localization of eNOS with prolonged
E2 infusion.
In the ovariectomized sheep, eNOS was immunohistochemically localized
primarily in the endothelium much more than in the VSM, as shown in
Immunohistochemical localization of NOS with acute E2 treatment
and previously (24) for intact animals. Staining in the VSM was faint
and patchy or, in some cases, nonexistent. IgG controls showed little
or no staining of either endothelium or VSM. Figure
5 shows representative micrographs
depicting 6 and 10 days of prolonged
E2 infusion. Compared with
vehicle controls, E2 appeared
to cause a progressive rise in uterine artery endothelial eNOS protein
from 3 to 10 days of infusion. The patchy nature of uterine artery VSM
eNOS staining appeared to be slightly greater in
E2-treated animals on
days 8 and
10, although it is unclear whether
this staining was specific, because these eNOS changes could not be
confirmed on Western immunoblots (see Quantification of effects of prolonged systemic
E2 on eNOS expression by
Western immunoblot analysis). In contrast, in another
reproductive vessel, the mammary artery, we did not observe an increase
in eNOS until 10 days of E2 treatment.
Neither renal nor omental arteries exhibited appreciable changes
in eNOS expression with prolonged
E2 treatment.
|
Quantification of effects of prolonged systemic
E2 on eNOS expression by
Western immunoblot analysis.
The effect of prolonged vehicle versus
E2 infusion in ovariectomized
sheep on ENDO eNOS protein expressions is shown in Fig. 6. Days 3,
6, 8,
and 10 of
E2 infusion are shown, whereas
data for all day 0 and vehicle-treated
animals were not different and therefore were combined. By
day 10 of
E2 infusion, uterine artery ENDO and intact arteries exhibited progressive eNOS increases to
maximums of 576 ± 100 (Fig. 6) and 320 ± 135% (data not
shown), respectively, of control eNOS protein expression,
quantitatively confirming the immunohistochemical observations. Unlike
uterine vasculature, the mammary and omental artery ENDO showed
unaltered eNOS expression until day 10 of E2 treatment, whereas renal
artery ENDO responses were variable and not significantly affected by prolonged E2 treatment. The eNOS
expression in denuded arteries (VSM) in all vessels studied was very
low (nearly nondetectable) and not significantly affected by
E2 treatment.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Although estrogen treatment dramatically increases UBF, and
progesterone administration alone has little vasodilatory effect, when
they are administered simultaneously, progesterone partially attenuates
(25-30%) E2-mediated
elevations in UBF (1, 9, 21, 31). As observed during the ovarian cycle,
the magnitude of this UBF antagonism appears to be related to the
estrogen-progesterone ratio (4, 5, 8, 9, 21, 31). Data from the current study are the first to demonstrate elevations of eNOS protein expression in uterine artery endothelium during the follicular versus
luteal phase of the ovarian cycle when the endogenous
estrogen-progesterone ratio (4, 5, 13, 17, 18, 23, 33) and UBF
(4-6, 8, 25) are elevated. These alterations in endothelial eNOS expression were reproduced with acute or prolonged administration of
E2. These data are consistent
with observations that endothelial-dependent, NO-mediated relaxation
responses of human uterine arteries to acetylcholine are elevated
during phases of the menstrual cycle when estrogen levels are elevated
(3). Because the temporal pattern of cyclical changes in UBF during the
ovarian cycle are associated directly with the endogenous ratio of
estrogen to progesterone in systemic blood (4, 5, 8, 23) and locally in
uterine lymph (17, 18) and uterine tissues (30, 38), the rise in UBF
during the follicular phase is thought to reflect the vasodilator actions of estrogen, whereas the decline in UBF after ovulation results
simply from either the withdrawal of estrogen or from progesterone
blockade of estrogen-induced hyperemia with the development of the
corpus luteum. Our data do not evaluate whether the lower luteal phase
uterine artery endothelial eNOS expression level is associated with
estrogen withdrawal or progesterone inhibition.
Follicular phase increases in uterine artery eNOS levels were specific
to the endothelium, not VSM. Moreover, levels of eNOS in VSM were
dramatically lower (
30 fold) than those observed in
endothelium-isolated protein. Because most of the whole vessel homogenate consists of VSM rather than endothelial cell protein, there
was no difference in eNOS from intact uterine arteries collected during
the ovarian cycle. These data also verify the advantage of evaluating
endothelium-isolated protein for eNOS expression, because the
sensitivity for detecting small changes in expression is greatly
elevated (~700- to 800-fold) over that seen with whole vessel
homogenates (Figs. 2 and 4) (24).
The endothelium also was the primary source of eNOS in mammary
arteries, although the phase of the estrous cycle (follicular vs.
luteal) did not alter eNOS expression in either the endothelium or VSM.
The cellular location, (endothelium >> VSM) of eNOS in ovine
mammary arteries is consistent with that described in goats and cows
(29). Compared with the uterine vascular bed, less is known about the
regulation of blood flow to the mammary gland, although it may be more
dependent on angiogenesis (new vessel growth) rather than vasodilation
(29). Follicular phase increases in eNOS protein expression were
specific to uterine arteries, because there were no effects of the
ovarian cycle on either renal or omental arteries. Blood flows to the
kidney and omentum are not altered in follicular versus luteal phase
sheep (5; R. R. Magness and T. M. Phernetton, unpublished
observations). These data support the theory that follicular
E2 is responsible for the local
uterine artery endothelial eNOS elevation because systemic and local
acute E2 treatment of
ovariectomized ewes resulted in an increase in eNOS protein expression
in uterine, but not renal, omental, or mammary, artery endothelium.
Moreover, in ovariectomized sheep, eNOS protein expression in uterine
artery endothelium-isolated protein was about two- to threefold higher
compared with that in intact arteries. This contrasts the nearly
30-fold difference between endothelium-isolated protein and intact
arteries from "ovary-intact" sheep. These data suggest that
estrogen, progesterone, or other ovarian factors are important in
maintaining the high level of endothelial eNOS protein expression
cells, although this needs further clarification.
The primary NOS isoform in all of the vascular beds studied was eNOS, because iNOS could not be detected under the current assay conditions. In contrast, iNOS protein was detected in the kidney and liver of an LPS-treated sheep but not control sheep, suggesting that eNOS is readily expressed in tissues of healthy sheep and that iNOS is expressed only under pathological conditions. One should be cautious in concluding that there is no iNOS present in VSM, because the sensitivity of the Western assay conditions for the iNOS measurements were based only on LPS-stimulated tissues. These data are consistent with our previous report that 85-90% of the NOS-specific activity in uterine and omental artery endothelium is calcium dependent and that nNOS was not detected by immunohistochemistry and Western analysis (24) as well as with studies demonstrating that ovine fetal pulmonary artery endothelial cells exclusively express eNOS (28).
Systemic E2 causes maximum
uterine vasodilation over a 2-h period (14, 19-22), and when
E2 is administered locally into the uterine arterial blood supply, it causes a maximum local unilateral increase in UBF (14, 21, 22). We have confirmed the unilateral elevation in UBF with local E2
infusion and also demonstrated for the first time that eNOS expression
in uterine artery endothelium is elevated similarly by 2 h of systemic
and locally administered E2.
This uterine vasodilatory response involves the activation of
NOS-specific activity and new protein synthesis, because
L-NAME and cycloheximide,
respectively, will decrease UBF from its
E2-induced maximum (14, 32,
36). Prolonged infusion of E2
was administered at doses previously shown to mimic the beneficial
cardiovascular effects of postmenopausal estrogen replacement therapy
(1, 10, 19, 20). We observed that eNOS protein expression in uterine
artery endothelium was progressively increased by
E2 infusion and that neither
renal nor omental endothelial eNOS was significantly altered until
day 10 of
E2, when the latter was slightly increased. We have recently performed physiological studies to
determine whether, under acute and prolonged
E2 treatment, reproductive and
nonreproductive blood flows are altered (20). Consistent with these
eNOS findings, UBF, but not renal or omental blood flows, were elevated
throughout the 10-day E2
infusion. Although uterine artery endothelial eNOS expression
progressively increased throughout the
E2 infusion (Fig. 6), UBF
peaked at 120 min of infusion but gradually declined thereafter (19,
20). It is unknown why the continued rises in eNOS were not associated with a continued rise in UBF.
In endothelial cell cultures of fetal ovine pulmonary artery (16),
human and bovine aorta (11, 12), and HUVEC (11), E2 increased NO release, eNOS
mRNA, and/or eNOS protein expression via a receptor-mediated
mechanism. However, others have not been able to replicate the finding
that eNOS is upregulated by E2 in aortic endothelium (2, 34). Evidence for the direct effects of
E2 on endothelial cell
expression of eNOS is suggested from a recent study showing that
E2 increases eNOS gene
transcription in fetal pulmonary artery endothelial cells (16) and that
the 5' upstream region of the human eNOS gene contains 11 half-palindromic motifs that may function synergistically to form
estrogen-receptor functional-response elements (26). However, the
observation that acute systemic
E2 treatment increases eNOS
expression in uterine artery endothelium within 2 h suggests possible
posttranscriptional or nongenomic regulation and is not consistent with
time courses in these culture studies in which 24-48 h of
E2 treatment was necessary for
eNOS protein expression to be elevated (11, 12, 16). One explanation is
that estrogen receptor numbers fall with cell passage (11).
Alternatively, in the current in vivo studies, the uterine artery
endothelium is exposed not only to elevated estrogen concentrations but
also to increases in shear stress with the dramatic elevations in UBF
(14, 21, 22). The latter, shear stress, has profound effects to elevate
eNOS expression (35) and greatly increases NO-/endothelial-dependent relaxation responses in myometrial arteries (15). Consistent with in
vitro E2 time courses
(24-48 h) for the induction of eNOS expression (11, 12, 16), the
current findings show that uterine artery eNOS protein is increased 44 h post-PGF2, which we estimate
translates to ~38 h of elevations in endogenous estrogen exposure (5,
13, 33). Furthermore, administration of
E2 to ovariectomized sheep and
to rabbits for 3-4 days increased NO- and endothelium-dependent
relaxation of uterine and carotid arteries, respectively (7, 27, 37).
In the former studies, Veille et al. (37) also showed that intravenous
E2 infusion elevated the
NO-dependent uterine, but not renal, artery vasodilatory responses in
association with increases in calcium-sensitive NOS-specific activity.
This is consistent with the current findings that prolonged systemic
E2 increases uterine, but not
renal, eNOS protein expression. In guinea pigs, 5 days of estrogen
treatment also increased calcium-sensitive NOS activity and eNOS mRNA
expression in skeletal muscle (39).
In conclusion, elevations in the endogenous estrogen-progesterone ratio
increase uterine, but not systemic (renal or omental), artery
endothelial eNOS expression. This uterine arterial endothelial eNOS
response was mimicked by both acute and prolonged
E2 treatment. These studies
support the hypothesis that E2
increases UBF in part by specifically elevating the expression of eNOS
in uterine artery endothelium.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Cindy Goss for help in preparing this manuscript and Dr. Jing Zheng for technical help with the immunohistochemical analysis.
| |
FOOTNOTES |
|---|
This investigation was supported by the United States Department of Agriculture Grant-in-Aid No. 97352044912 and Individual Postdoctoral Grant No. 95372032008 (to K. E. Vagnoni) and by National Institutes of Health Grants HL-49210, HD-33255, HL-57653, and HL-56702.
This study was presented in part at the Tenth World Congress of the International Society for the Study of Hypertension in Pregnancy, Seattle, WA, August 4-8, 1996, and the 13th Rochester Trophoblast Meeting, Banff, Alberta, Canada, September 8-12, 1996.
Present address of K. E. Vagnoni: Dept. of Animal, Dairy, and Veterinary Science, Utah State University, Logan, UT 84322.
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: R. R. Magness, Perinatal Research Laboratories, Dept. of Obstetrics and Gynecology, Univ. of Wisconsin-Madison Medical School, Meriter Hospital/Park-7E, 202 S. Park St., Madison, WI 53715.
Received 10 April 1998; accepted in final form 9 July 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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