Vol. 273, Issue 4, H2009-H2017, October 1997
Regulation of endometrial blood flow in ovariectomized rats:
assessment of the role of nitric oxide
Ren-Sheng
Zhang1,
Paul H.
Guth2,
Oscar U.
Scremin2,
Rajan
Singh1,
Shehla
Pervin1, and
Gautam
Chaudhuri1
1 Departments of
Obstetrics/Gynecology and Molecular and Medical Pharmacology,
School of Medicine, University of California, Los Angeles, and
2 Veterans Affairs Medical
Center, Los Angeles, California 90024
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ABSTRACT |
The purpose of this study was to evaluate the
role of nitric oxide (NO) in the maintenance of basal endometrial blood
flow of ovariectomized rats and in the increase of endometrial blood flow after administration of estradiol 17
(E2). Endometrial blood flow
was repeatedly measured with the
H2 gas clearance technique in
ovariectomized rats.
N
-nitro-L-arginine
methyl ester (L-NAME) dose
dependently reduced basal endometrial blood flow and increased mean
arterial blood pressure and endometrial vascular resistance.
E2 (1 µg/kg iv) increased
endometrial blood flow and reduced endometrial vascular resistance,
which peaked by 2 h after the injection. The vasoconstrictive activity
of L-NAME (an inhibitor for NO
synthesis) was compared with that of phenylephrine (PE, an 
-receptor
agonist acting through an NO-independent mechanism). Doses of
L-NAME (1 and 3 mg/kg iv) were
matched with those of PE (3.2 and 6.4 mg · kg
1 · h1
iv), as they induced an approximately equivalent percent increase in
basal endometrial vascular resistance. The percent increases of
endometrial vascular resistance in
E2-treated animals by the two
agents in matched doses were also of a similar magnitude. When animals
were first treated with L-NAME
or PE, E2 lost the ability to
reduce endometrial vascular resistance. Enzyme activity and gene
expression of NO synthase in the rat uterine tissue were also examined
after E2 treatment, and no
significant changes were observed. These data raise doubts about the
role of NO in the regulation of endometrial blood flow after acute
administration of E2 and
suggest that other mechanisms may be involved.
hydrogen gas clearance; estradiol 17
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INTRODUCTION |
ADMINISTRATION OF estradiol 17
(E2) to ovariectomized animals
increases uterine blood flow (10, 11, 20, 24). This increase in uterine
blood flow occurs after a delay of 30-60 min and peaks ~2 h
after E2 administration (11,
24). Although the time course and the magnitude of this
E2-induced increase in uterine
blood flow have been thoroughly described, the exact mechanism by which
this occurs is not known. Various vasoactive substances, such as
prostanoids and vasoactive polypeptides, have been implicated as the
mediators involved in this phenomenon (4, 5, 11). However, the increase
in uterine blood flow has not been successfully antagonized with
antagonists to the suggested mediators.
The increase in uterine blood flow by
E2 is a localized response,
inasmuch as E2 injection into
one uterine artery of the ewe produced an increase in uterine blood
flow only to the ipsilateral horn (11, 18). Pretreatment with
cycloheximide, an inhibitor of protein synthesis, prevented this
E2-induced increase in uterine blood flow (11), indicating that the production of an enzyme or a
polypeptide is a necessary intermediate for this
E2 response. More recently,
another vasodilator, nitric oxide (NO), has been implicated as a
possible mediator for this E2
response (8, 23, 27). Whereas chronic exposure of tissues to
E2 increases NO synthesis, as
demonstrated by pharmacological (8) and biochemical and molecular
biology techniques (27), the mechanism that underlies the acute effects
of E2 on vascular tone is
controversial. Some investigators reported that the acute
vasorelaxation by E2 is an
endothelium-independent process (13, 28), whereas other investigators
indicated that NO is the mediator (23).
The only evidence that suggests a role of NO in mediating the acute
effects of E2 on the uterine
vascular bed comes from one group of investigators using the sheep
model (23). In support of their hypothesis, these investigators
demonstrated that administration of
N-nitro-L-arginine
methyl ester (L-NAME), an
inhibitor of NO synthase (NOS), was able to reduce, in a dose-dependent
manner, the E2-induced increase
in uterine blood flow. However, the inhibition of NO synthesis leads to
vasoconstriction in all vascular beds, inasmuch as NO is responsible
for maintenance of basal vascular tone (17, 25). It is therefore
possible that the results observed by Van Buren et al. (23) are due to
a physiological antagonism in which the increase of uterine blood flow
by one substance (i.e., E2) is
offset by the decrease in uterine blood flow by another substance (i.e., L-NAME), the net effect
depending on the potencies of the substances involved. In the present
study we reassessed whether NO is the mediator for the acute effects of
E2 on the uterine vascular bed
by using pharmacological, biochemical, and molecular biology
techniques.
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MATERIALS AND METHODS |
Animal Preparation
The experimental procedures have been previously described in detail
(30). Briefly, virgin female Sprague-Dawley rats (200-225 g;
Harlan, Indianapolis, IN) were housed under conditions of controlled temperature and light cycle and were provided free access to food pellets and water. Under pentobarbital sodium anesthesia (40 mg/kg ip),
the animals were ovariectomized bilaterally 3-5 days before the
experiment.
To measure endometrial blood flow using the
H2 gas clearance technique, the
animals were anesthetized with urethan (1.25 g/kg ip). A femoral vein
was cannulated for saline and drug administration, and a carotid artery
was cannulated to monitor the arterial blood pressure on a Gilson
recorder via a pressure transducer (model P23Db, Statham). The body
temperature of the animals was monitored with a rectal probe and was
maintained at 36.5-37.0°C under an incandescent lamp.
For assessments of NOS activity as well as for endothelial NOS (eNOS)
and inducible NOS (iNOS) gene expression in the uterus, an external
jugular vein was cannulated for drug delivery, and the uterine horns
were removed from the animals under anesthesia with urethan (1.25 g/kg
ip). The uterine horns were washed with sterile phosphate-buffered
saline, frozen quickly in liquid nitrogen, and stored in a freezer
until they were homogenized.
H2 Gas Clearance Technique
The H2 gas clearance technique (1)
was used to measure endometrial blood flow of ovariectomized rats.
Details of this technique have been described previously (30). Briefly,
the trachea was intubated with PE-240 tubing to maintain a patent
airway and to facilitate the delivery of 2%
H2 during blood flow measurement. After a midline laparotomy, an Ag-AgCl reference electrode was placed
in the abdominal cavity. A platinum electrode (125 µm diameter, A-M
System, Everett, WA) was then inserted into the endometrium of the
right uterine horn. Care was taken to avoid apparent blood vessels and
to minimize damage to the tissue (30). The uterine horn was then
covered with a thin piece of saline-moist gauze, and the area over the
incision was covered with a piece of Parafilm to prevent evaporation.
To measure endometrial blood flow, the electrodes were connected to a
polarographic and amplifying unit (Val Tech Electronics, Sherman Oaks,
CA) and the electrode current during the inhalation of 2%
H2 and subsequent removal of
H2 was traced on the Gilson
recorder. Before the electrode connection, the output voltage of the
polarographic unit was adjusted to match the electrode voltage between
the platinum electrode and the reference electrode. This was done to
set the electrode current to zero (or close to zero) in the absence of
H2 and to ensure a more reliable recording (30). Through an ADALAB analog-to-digital converter, the
signals were sent to a computer. During the 15 min of
H2 inhalation, the current tracing
gradually rose and reached a plateau as the tissue was saturated with
H2. When
H2 was discontinued for 15-min, a
desaturation curve was generated because of the washout of
H2 from the tissue by blood flow.
The endometrial blood flow was then calculated by analyzing the
H2 desaturation curve using a monoexponential curve-fitting program (12).
NOS Assay
The arginine-to-citrulline conversion assay (2) was used to measure NOS
activity in uterine homogenate. A 20% homogenate of uterine horn was
prepared in 50 mM triethanolamine (TEA)-HCl, pH 7.4, containing 0.1 mM
ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.1 mM EDTA, 0.5 mM dithiothreitol, 1 µM pepstatin A,
and 2 µM leupeptin at 4°C. The homogenate was centrifuged at 20,000 g for 60 min at 4°C, and
the supernatant was used to assay NOS activity. NOS activity was
determined by measuring the formation of
[3H]citrulline from
[3H]arginine (2).
Enzymatic reactions were conducted at 37°C for 10 min in 50 mM
TEA-HCl, pH 7.4, containing 50 µM
L-arginine (77 Ci/mmol, with
~20,000 cpm of
L-[2,3,4,5-3H]arginine-HCl),
100 µM NADPH, 10 µM tetrahydrobiopterin, 10 µM flavin adenine
dinucleotide, 10 µM flavin mononucleotide, 2 mM CaCl2, 50 mM
L-valine, 1 µg of calmodulin,
and 0.2-0.4 mg of supernatant protein in a final incubation volume
of 100 µl. Ca2+-independent NOS
activity was measured in the absence of
Ca2+ and calmodulin and in the
presence of 2 mM EDTA and 2 mM EGTA. Ca2+-dependent NOS activity was
obtained by subtracting the
Ca2+-independent NOS activity from
the total NOS activity. The
L-[2,3,4,5-3H]arginine-HCl
was purified by anionic exchange chromatography on columns of Dowex AG
1-X8, OH form (prepared
from the acetate form), 100-200 mesh, to remove traces of
contaminating
[3H]citrulline.
Enzymatic reactions were terminated by addition of 2 ml of ice-cold
buffer (20 mM sodium acetate, pH 5.5, containing 1 mM
L-citrulline, 2 mM EDTA, and 0.2 mM EGTA), and samples were loaded onto columns (1 cm diameter)
containing 1 ml of Dowex AG 50W-X8,
Na+ form (prepared from
H+ form), that had been
preequilibrated with stop buffer for chromatography. After the 2 ml of
eluate were collected in a test tube, each column was washed again with
2 ml of water and collected in the same test tube. Aquasol-2 (12 ml)
was added to one-half (2 ml) of the final eluate, and the samples were
counted in a liquid scintillation spectrometer (model LS 3801, Beckman).
Reverse Transcriptase Polymerase Chain Reaction
The reverse transcriptase polymerase chain reaction (RT-PCR) technique
was used to assess the eNOS and iNOS gene expression in rat uterus.
Tri-Reagents (Molecular Research Center, Cincinnati, OH) were used to
homogenize rat uterine horns and to extract the total RNA. Reverse
transcription was performed by using 5 µg of total RNA sample, 50 U
of Moloney murine leukemia virus RT, and 100 pmol of oligo(dT) and
running the reaction at 42°C for 30 min. The resulting cDNA samples
were PCR amplified using the Gene Amp RNA PCR kit (Perkin Elmer,
Norwalk, CT) in 100 µl of reaction mixture. The final reaction
mixture was treated by heat denaturation at 94°C for 5 min and PCR
amplification for eNOS or iNOS for 40 cycles, each consisting of
denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and extension at 72°C for 1.5 min. This was followed by a
final extension at 72°C for 5 min. The amplification procedure for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consisted of 30 cycles
each of denaturation at 94°C for 30 s, primer annealing at 55°C
for 30 s, and extension at 72°C for 1 min, using 1 µg of total
RNA sample.
The following primers were used (Custom Primers, GIBCO-BRL,
Gaithersburg, MD): eNOS, 5'-GTGATGGCGAAGCGAGTGAAG-3'
(sense) and 5'-CCGAGCCCGAACACACAGAAC-3' (antisense) (19);
iNOS, 5'-CATGGCTTGCCCCTGGAAGTTTCT-3' (sense) and
5'-CCTCTGATGGTGCCATCGGGCATC-3' (antisense; gene bank accession M8437) (29); GAPDH,
5'-GTGAAGGTCGGTGTCAACCGGATTT-3' (sense) and
5'-CACAGTCTTCTGAGTGGCAGTGAT-3' (antisense) (22). One
hundred nanograms of sense and antisense primers were used in each PCR
in a final volume of 100 µl. The amplified DNA fragments obtained
were of expected base-pair (bp) size: eNOS, 422 bp; iNOS, 747 bp;
GAPDH, 558 bp. Each final PCR product sample (30 µl) was loaded on a
1.5% agarose gel, electrophoresed, and visualized by ethidium bromide
staining under ultraviolet light. The identities of the products were
confirmed by Southern blot hybridization with internal oligonucleotides
that were 32P labeled using
T4-polynucleotide kinase from respective cDNA sequences. We cloned the
PCR products into PCR 2.1 vector using a TA cloning kit (Invitrogen).
Their identity as iNOS, eNOS, and GAPDH was confirmed by DNA sequencing
(data not shown). The quantity of RNA for RT-PCR and the number of
cycles were initially determined to ensure that saturation did not
occur.
Chemicals and Solutions
The stock solution of E2 was
made by dissolving the powder (Sigma Chemical) in 100% ethanol to a
concentration of 250 µg/ml and stored in a freezer. Immediately
before intravenous injection, 10 µl of this stock were mixed with 990 µl of 0.9% NaCl. L-NAME (Sigma Chemical) was dissolved in 0.9% NaCl and stored in a freezer before use. The phenylephrine (PE) solution for intravenous infusion was made by dissolving the PE powder (Sigma Chemical) in 0.9% NaCl to
a concentration of 1 mg/ml and stored in a refrigerator before use.
Lipopolysaccharide (LPS; Sigma Chemical) was dissolved in 0.9% NaCl to
a concentration of 1 mg/ml before intravenous injection.
TEA-HCl, L-arginine-HCl free
base, L-citrulline, pepstatin,
leupeptin, dithiothreitol, sodium acetate, EDTA, EGTA, NADPH, flavin
adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, CaCl2, and calmodulin were
obtained from Sigma Chemical.
L-[2,3,4,5-3H]arginine-HCl
was obtained from Amersham. Dowex AG 1-X8 and Dowex AG 50W-X8 were from
Bio-Rad Laboratories (Richmond, CA).
Experimental Design
After the surgical procedure, a stabilization period of 90-120 min
was allowed before the measurement of endometrial blood flow by the
H2 gas clearance technique (30).
At times indicated, E2 and
L-NAME were administered
intravenously in a volume of 0.1 ml over a 1-min period, and PE was
given as a constant intravenous infusion. Control endometrial blood
flow was measured before administration of these agents. Endometrial
blood flow in response to E2
was measured at 2 h after E2
administration, and the endometrial blood flow in response to
L-NAME or PE was measured
15-30 min after the injection or the start of infusion.
Approximate values of endometrial vascular resistance were obtained by
dividing the mean arterial pressure (MAP) by endometrial blood flow.
Uterine venous pressure was not included in the calculation because of the technical difficulties in measuring it in rats.
For NOS assays and RT-PCR experiments,
E2 or LPS was injected
intravenously in a volume of 0.1 ml over a 1-min period, and the uterus
was removed and frozen in liquid nitrogen at 30 min or 2 h after the
injection. In a separate group of animals, neither E2 nor LPS was given before the
uterus was removed. These animals were in the time
0 group and were used as the negative control for
baseline comparison.
Experimental Protocols
Study I: Cumulative dose response of
L-NAME on MAP, endometrial blood
flow, and endometrial vascular resistance.
After the control endometrial blood flow measurement, cumulative doses
of L-NAME were given at 30-min
intervals to evaluate the role of NO in the regulation of basal
endometrial blood flow and to obtain dose-response effects on MAP,
endometrial blood flow, and endometrial vascular resistance.
Study II: Reversal of the
E2 response by
L-NAME and PE.
Doses of L-NAME (1 and 3 mg/kg)
and PE (3.2 and 6.4 mg · kg1 · h1)
that induced comparable percent increase in endometrial vascular resistance in the absence of E2
were given 2 h after the administration of
E2 to test and compare the
abilities of L-NAME and PE to
reverse the E2-induced changes
in endometrial blood flow and endometrial vascular resistance.
Study III: Blockade of the
E2 response by
L-NAME and PE.
L-NAME injection (1 and 3 mg/kg)
or PE infusion (3.2 and 6.4 mg · kg1 · h1)
was given or started immediately before
E2 injection to compare the
effectiveness of L-NAME and PE
in blocking E2-induced changes in endometrial blood flow and endometrial vascular resistance. In
preliminary experiments we observed that
L-NAME (1 and 3 mg/kg) increased
uterine vascular resistance; this increase was maintained for at least
3 h after its administration.
Study IV: Assessment of NOS activity and eNOS and iNOS gene
expression.
E2, vehicle, or LPS was
administered to the animals to test their effects on uterine NOS
activity as well as on eNOS and iNOS gene expression. LPS was utilized
as the positive control, inasmuch as other investigators (26)
demonstrated an increase in iNOS protein and gene expression after its
administration. The NOS assay was also performed in the presence of
L-NAME to confirm the
specificity of the assay.
Data Analysis
Values are means ± SE. MAP, endometrial blood flow, and endometrial
vascular resistance values were compared using repeated-measures analysis of variance. Values of NOS activity were compared using one-way analysis of variance. Pairwise post hoc comparisons between means were made using Tukey's (least significant difference)
criterion. Differences were considered to be significant at
P < 0.05.
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RESULTS |
Study I: Cumulative Dose Response of
L-NAME on MAP, Endometrial Blood
Flow, and Endometrial Vascular Resistance
Administration of L-NAME dose
dependently produced increases in MAP and endometrial vascular
resistance and a decrease in endometrial blood flow (Fig.
1). The increases in MAP and endometrial vascular resistance and the decrease in endometrial blood flow were
near maximum with 3 mg/kg of
L-NAME.
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Fig. 1.
Response of mean arterial pressure (MAP), endometrial blood flow (EBF),
and endometrial vascular resistance (EVR) to cumulative intravenous
injection of N-nitro-L-arginine
methyl ester (L-NAME).
L-NAME was given at 30-min
intervals as 1, 2, 7, and 20 mg/kg (cumulative doses of 1, 3, 10, and
30 mg/kg). Values are means ± SE from 6 animals.
* Significantly different from control;
@ significantly different
from 1 mg/kg L-NAME group;
# significantly different
from 3 mg/kg L-NAME group
(P < 0.05).
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Study II: Reversal of the
E2 Response by
L-NAME and PE
The E2-induced increase in
endometrial blood flow and decrease in endometrial vascular resistance
were reversed after administration of
L-NAME (Fig.
2) and PE (Fig.
3). The relative changes in endometrial vascular resistance induced by
L-NAME (Fig.
4A) and
PE (Fig. 4B) were also compared
under baseline as well as
E2-treated conditions. Doses of
L-NAME and PE are considered
matched, since the relative changes of endometrial vascular resistance
are similar between the control groups in Fig. 4.
E2-treated animals were
pretreated with 1 µg/kg of E2
2 h before treatment with L-NAME
or PE. No significant difference in relative endometrial vascular
resistance values was found between
L-NAME and PE treatments at
matched doses or between the baseline and the
E2-treated groups.
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Fig. 2.
Reversal of estradiol 17
(E2) response by
L-NAME. MAP, EBF, and EVR were
repeatedly measured in 8 animals under 4 different conditions. Values
are means ± SE. * Significantly different from control;
@ significantly different
from E2 group
(P < 0.05).
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Fig. 3.
Reversal of E2 response by
phenylephrine (PE). MAP, EBF, and EVR were measured at times indicated.
Values are means ± SE from 8 animals. * Significantly
different from control;
@ significantly different
from E2 group
(P < 0.05).
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Fig. 4.
Relative changes of EVR induced by
L-NAME or PE under control or
E2-treated conditions. Data are
from a total of 30 animals. EVR values after
L-NAME administration or during
PE infusion are shown as relative changes normalized to values under
control or E2-treated
conditions and plotted as means ± SE. In
A, data for control group are from
experiments shown in Fig. 1, and data for
E2-treated group are from
experiments shown in Fig. 2. In B,
control group was studied under control conditions, PE was infused at
3.2 or 6.4 mg · kg1 · h1
for 30 min, and data for
E2-treated group are from
experiments shown in Fig. 3.
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Study III: Blockade of the
E2 Response by
L-NAME and PE
A decrease in endometrial vascular resistance induced by
E2 was observed in animals
pretreated with 1 mg/kg of
L-NAME (Fig. 5A), but
not in animals pretreated with 3 mg/kg of
L-NAME (Fig. 5B). The
E2-induced decrease in
endometrial vascular resistance was not observed with infusion of PE at
3.2 mg · kg1 · h1
iv (Fig. 6).
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Fig. 5.
Blockade of E2 response by
L-NAME.
L-NAME [1
mg/kg (A,
n = 7) or 3 mg/kg
(B, n = 8)] was given to animals before injection of 1 µg/kg
E2. MAP, EBF, and EVR were
measured at times indicated and are plotted as means ± SE.
* Significantly different from control
(P < 0.05).
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Fig. 6.
Blockade of E2 response by PE.
A continuous intravenous infusion of PE was started immediately before
injection of 1 µg/kg E2.
Values are means ± SE from 7 animals. * Significantly
different from control;
@ significantly different
from PE group (P < 0.05).
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Study IV: Assessment of NOS Activity and eNOS and iNOS Gene
Expression
There was greater Ca2+-dependent
NOS activity than Ca2+-independent
NOS activity in rat uterus (Fig. 7).
E2 injection did not change the
Ca2+-dependent or the
Ca2+-independent NOS activities
(Fig. 7). By contrast,
Ca2+-independent NOS activity was
increased 2 h after LPS injection (Fig. 8).
This increase of Ca2+-independent
NOS activity was accompanied by an increase in gene expression for iNOS
(Fig. 9,
top), but not for eNOS (Fig. 9,
middle). There was no change in iNOS
(Fig. 9, top) or eNOS (Fig. 9,
middle) gene expression after
E2 administration. Low NOS
activities in the presence of
L-NAME (Figs. 7 and 8) confirmed
the specificity of the assay. Comparable GAPDH gene expressions from
all samples (Fig. 9, bottom)
indicate that equal amounts of RNA were used for each RT-PCR set.
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Fig. 7.
Effect of E2 on NO synthase
(NOS) activity of rat uterus. A total of 15 animals were divided into 3 groups with 5 animals in each group. One group was used for baseline
control. Other 2 groups received 1 µg/kg iv
E2 at 30 min or 2 h before
uterus was removed. Ca2+-dep,
Ca2+ dependent;
Ca2+-indep,
Ca2+ independent;
L-NAME,
104 M
L-NAME. Values are means ± SE.
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Fig. 8.
Effect of vehicle and lipopolysaccharide (LPS) on
Ca2+-independent NOS activity of
rat uterus. At 30 min or 2 h before removal of uterus, animals were
injected intravenously with vehicle for
E2 (0.1 ml of 1% ethanol) or 1 mg/kg LPS. Assay was also performed in presence of
L-NAME in LPS-treated animals to
confirm specificity of assay. Values are means ± SE from 5 animals
in each group. * Significantly different from control at
time 0 (P < 0.05).
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Fig. 9.
Time course of gene expression of inducible NOS (iNOS), endothelial NOS
(eNOS), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat
uterine horn. Lane 1, negative
control, where reverse transcriptase polymerase chain reaction was done
in absence of RNA. Lanes 2-4,
3-5, and
6-8 represent time course after
administration of a single dose of vehicle,
E2, and LPS, respectively. In
each treatment group, 1st point represents time
0 (lanes 2, 5, and
8), 2nd point represents 30 min
(lanes 3, 6, and
9), and 3rd point represents 2 h
(lanes 4, 7, and
10) after administration of each
agent. Lane M,
 X174/Hae III molecular weight
markers. Arrows, position of reverse transcriptase polymerase chain
reaction products for iNOS, eNOS, and GAPDH. Data are from a single
experiment that is representative of 3 separate experiments.
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DISCUSSION |
The identification of endothelium-derived relaxing factor as NO (9, 15)
stimulated numerous investigators to elucidate whether the mechanism by
which E2 modulates vascular
tone is by release of NO (7, 8, 23, 27). The precursor for NO synthesis
is L-arginine (14, 21), and
various analogs of arginine act as competitive inhibitors of NOS (17,
25). These analogs have been utilized to assess the physiological role
of NO in vitro and in vivo (3, 6, 8, 17, 23). Two forms of NOS have been identified: constitutive NOS (cNOS) and iNOS. cNOS is
Ca2+-calmodulin dependent, whereas
iNOS is Ca2+-calmodulin
independent. eNOS, which mediates endothelium-dependent vascular
relaxation through the release of NO, is a type of cNOS.
The relationship between E2 and
NOS is controversial. Gisclard and colleagues (7) obtained a
significant increase in acetylcholine-induced relaxation of the femoral
artery after chronic E2
replacement in castrated rabbits. In these studies, rabbits were
injected daily with 35 µg/kg im
E2, which increased the
circulating E2 concentration to
levels seen during estrus. We (8) also previously demonstrated that the
basal release of NO from the rabbit thoracic aorta was higher in
females than in males, and ovariectomy abolished this difference.
However, some investigators demonstrated that the acute effects of
E2 in modulating vascular tone
of isolated vascular rings are NO independent (13, 28). The primary
objective of this study therefore was to assess whether the
E2-induced acute increase in
endometrial blood flow is mediated by NO. Endometrial, rather than
myometrial, blood flow was estimated, as our previous study indicated
that acute administration of E2
caused more profound changes in the blood flow to the endometrium than
to the myometrium (30).
In study I, administration of
L-NAME to ovariectomized animals
raised the MAP and increased endometrial vascular resistance in a
dose-dependent manner (Fig. 1). This indicated that NO is involved in
the regulation of the basal tone of the uterine vascular bed, even in
the absence of E2, and that a
basal release of NO keeps this vascular bed in a partially dilated
state similar to that suggested for other vascular beds (17). Doses of
1 and 3 mg/kg of L-NAME were
selected for our studies, as MAP and endometrial vascular resistance
were significantly higher than basal values after these doses, and the
responses reached near-maximal values after administration of 3 mg/kg
of L-NAME. In the present study we also observed that when PE was infused at 3.2 and 6.4 mg · kg1 · h1,
the relative increases (normalized to control values) in endometrial vascular resistance under control conditions were comparable to those
produced by 1 and 3 mg/kg of
L-NAME (Fig. 4). Therefore, these doses of PE and L-NAME
were approximately matched in their abilities to raise endometrial
vascular resistance.
The decrease in endometrial vascular resistance after
E2 administration is apparent
by 60 min, and the maximal effect occurs within 2 h (30). In
study II, at 2 h after
E2 injection, administration of
L-NAME (Fig. 2) and PE (Fig. 3)
produced graded increases in endometrial vascular resistance, and the
relative increases in endometrial vascular resistance were comparable
between the two vasoconstrictors (Fig. 4). The magnitudes of these
relative changes in endometrial vascular resistance were not different
from those produced by these agents in the absence of
E2 treatment (Fig. 4). If
E2-induced increase in NO
synthesis was responsible for the decrease in endometrial vascular
resistance, we would expect a much greater increase in endometrial
vascular resistance after L-NAME
administration in E2-treated
than in control animals; we would also expect a greater increase in
endometrial vascular resistance by
L-NAME than by PE in
E2-treated animals, inasmuch as
PE causes vasoconstriction through an NO-independent mechanism. However, our data are not consistent with this notion and, therefore, raise doubt that the increase in endometrial blood flow after acute
E2 treatment is mediated by an
increased synthesis of NO. Our interpretation therefore differs from
that of other investigators (23). This might be due to the fact that
these investigators did not examine the effects of
L-NAME on basal uterine vascular tone and did not use PE as a control vasoconstrictor. The
dose-dependent reduction in
E2-elevated uterine blood flow
by L-NAME in their study could
be simply due to the blockade of NO synthesis that was already present
to the same extent under the baseline conditions.
In a study that first demonstrated that NO synthesis could actually be
induced in vascular tissue (16), active tone was first induced in
isolated aortic rings with PE, then LPS was added to the Krebs solution
in the tissue bath containing the vascular ring. LPS was able to reduce
the vascular tone in these PE-preconstricted vascular rings, whereas
this effect of LPS was not observed in the presence of an inhibitor of
NO synthesis. These findings indicated that the LPS-induced decrease in
vascular tone was mediated by NO. In study
III we utilized a similar protocol to further evaluate whether acute administration of
E2 can increase NO synthesis. As in the isolated aortic ring study (16), the endometrial vascular resistance was initially increased from basal values by infusion of PE
at 3.2 mg · kg1 · h1.
Then, E2 was administered (Fig.
6). A decrease in endometrial vascular resistance was not observed for
up to 3 h after E2
administration, as would be expected if
E2 were to increase NO
synthesis. On the other hand, a slight decrease in endometrial vascular
resistance was observed after
E2 administration only after
the endometrial vascular resistance was increased from basal values by
1 mg/kg, but not by 3 mg/kg, of
L-NAME (Fig. 5). These
observations provide further evidence that the
E2-induced increase in
endometrial blood flow is not mediated by an increase in NO synthesis.
In study IV, administration of
E2 to the animals did not
increase NOS enzyme activity, nor did it enhance NOS gene expression in
the uterus when an increase of endometrial blood flow was expected. On
the other hand, LPS, which we used as our positive control, increased
the Ca2+-calmodulin-independent
NOS enzyme activity and iNOS gene expression in the uterus. These data
further suggest that an
E2-induced increase in
endometrial blood flow is unlikely to be mediated by an increase in NO
synthesis. It is possible that the acute administration of
E2 may be different, in the
ability of modulating NOS activity, from chronic
E2 replacement, where an
increase in the release of NO (8) and an increase in eNOS gene
expression are observed (27). The difference between our studies and
those of other investigators (23) may also be due to differences in experimental designs or animal models. In our studies we assessed the
E2-induced increase in
endometrial blood flow in ovariectomized rats, whereas other
investigators studied the effects of
E2 on total uterine blood flow
in ovariectomized sheep (23).
In conclusion, although the acute effects of
E2 in decreasing endometrial
vascular resistance may be mediated by increased protein synthesis, it
seems unlikely that NOS is increased. Other possible mechanisms need to
be considered.
| |
ACKNOWLEDGEMENTS |
We thank Janis Cuevas for technical expertise in animal
preparations for NOS assay and RT-PCR.
| |
FOOTNOTES |
This work was supported in part by National Heart, Lung, and Blood
Institute Grant HL-46843 and the Laubish Fund for Cardiovascular Research.
Address for reprint requests: G. Chaudhuri, Dept. of OB/GYN, UCLA
School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90024.
Received 11 December 1996; accepted in final form 16 June 1997.
| |
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