| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Center for Perinatal Biology, Department of Pharmacology and Physiology, Loma Linda University School of Medicine, Loma Linda, California 92350; and 2 Jiangxi Provincial Key Laboratory for Animal Biotechnology, Jiangxi Agricultural University, Nanchang, Jiangxi, China 330045
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
During pregnancy, maternal plasma
cortisol concentrations approximately double. Given that cortisol plays
an important role in the regulation of vascular reactivity, the present
study investigated the potential role of cortisol in potentiation of
uterine artery (UA) contractility and tested the hypothesis that
pregnancy downregulated the cortisol-mediated potentiation. In vitro
cortisol treatment (3, 10, or 30 ng/ml for 24 h) produced a
dose-dependent increase in norepinephrine (NE)-induced contractions in
both nonpregnant and pregnant (138-143 days gestation) sheep UA.
However, this cortisol-mediated response was significantly attenuated
by ~50% in pregnant UA. The 11
-hydroxysteroid dehydrogenase
(11-HSD) inhibitor carbenoxolone did not change the effect of
cortisol in nonpregnant UA but abolished its effect in pregnant UA by
increasing the NE pD2 in control tissues from 6.20 ± 0.05 to 6.59 ± 0.11. The apparent dissociation constant value of
NE 
1-adrenoceptors was not changed by cortisol in
pregnant UA but was decreased in nonpregnant UA. There was no
difference in glucocorticoid receptor density between nonpregnant and
pregnant UA. Cortisol significantly decreased endothelial nitric oxide
(NO) synthase protein levels and NO release in both nonpregnant and
pregnant UA, but the effect of cortisol was attenuated in pregnant UA
by ~50%. Carbenoxolone alone had no effects on NO release in
nonpregnant UA but was decreased in pregnant UA. These results suggest
that cortisol potentiates NE-mediated contractions by decreasing NO
release and increasing NE-binding affinity to
1-adrenoceptors in nonpregnant UA. Pregnancy attenuates
UA sensitivity to cortisol, which may be mediated by increasing type-2
11-HSD activity in UA.
nitric oxide; 1-adrenoceptor; 11-HSD; glucocorticoid receptor
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
DURING PREGNANCY IN SEVERAL SPECIES, including humans and sheep, maternal plasma cortisol concentrations approximately double (19, 32). Whereas the elevation of maternal cortisol may be essential for normal fetal development and homeostasis, either hypercorticism or hypocorticism can result in an increased incidence of fetal growth restriction, prematurity, and fetal or neonatal death. It has been well documented that cortisol plays a key role in the regulation of vascular reactivity and produces a permissive effect in potentiating vasoactive responses to catecholamines through glucocorticoid receptors (GR). Glucocorticoids potentiate vasoconstrictive responses of catecholamines, angiotensin II, vasopressin, and bradykinin, and increased glucocorticoid responsiveness has been associated with an increase in arterial contraction and vascular resistance (5, 10, 18, 22, 41, 42, 48).
The question arises as to whether or to what extent the increased
cortisol levels during pregnancy affect uterine artery contractility and blood flow. Despite the increase in maternal plasma cortisol levels, pregnancy is accompanied by a significant increase in uterine
blood flow. In vivo studies have demonstrated that pregnancy decreases
uterine artery responsiveness to the vasoconstrictor effects of several
agents, including angiotensin II and norepinephrine (NE) (25,
30), although Annibale et al. (1) reported that in
vitro there was increased sensitivity of denuded uterine artery smooth
muscle to -stimulation. It has been demonstrated that pregnancy is
associated with an increase in endothelial nitric oxide (NO) synthase
(eNOS) expression and NO synthesis/release in uterine artery
endothelial cells, which plays a key role in decreased uterine artery
contractility (26, 37, 45, 46, 47). In the present study,
we tested the hypothesis that cortisol potentiated
-adrenoceptor-induced contractions of the uterine artery, which was
attenuated by pregnancy. Specifically, we examined the effect of
cortisol on NE-induced contractions in isolated uterine arteries from
both nonpregnant and pregnant sheep. To determine the potential role of
endothelial NO in the effect of cortisol and its alteration by
pregnancy, we examined the effect of cortisol on NO release and eNOS
protein expression in the endothelium of nonpregnant and pregnant
uterine arteries. The effect of glucocorticoids on vascular reactivity
is regulated by 11-hydroxysteroid dehydrogenase (11-HSD)
(41). Both type 1 and 2 11-HSD have been found in vascular endothelial (7) and smooth muscle cells (6,
42); type 1 11-HSD catalyzes predominantly conversion of
cortisone to active cortisol, whereas type 2 11-HSD converts
cortisol to inactive form cortisone. The present study also examined
the effect of 11-HSD inhibitor carbenoxolone on cortisol-mediated
responses in the uterine artery.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Tissue preparation. Nonpregnant (with intact ovaries) and pregnant (138-143 days gestation) sheep were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The ewes were then intubated, and anesthesia was maintained on 1.5-2.0% halothane in oxygen throughout surgery. An incision in the abdomen was made, and the uterus was exposed. The uterine arteries were isolated and removed without stretching and placed into a modified Krebs solution (pH 7.4) of the following composition (in mM): 115.21 NaCl, 4.7 KCl, 1.80 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 22.14 NaHCO3, and 7.88 dextrose. EDTA (0.03 mM) was added to suppress oxidation of amines. The Krebs solution was oxygenated with a mixture of 95% O2-5% CO2. After removal of the tissues, animals were killed with T-61 (euthanasia solution, Hoechst-Roussel; Somerville, NJ). A total of 35 nonpregnant and 34 pregnant sheep were used. All procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University and followed the guidelines put forward in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The third (nonpregnant) and fourth (pregnant) branches of the main uterine arteries with a similar external diameter (~0.8 mm) were separated from the surrounding tissue, and special care was taken to avoid touching the luminal surface. The arteries were cut into rings of 2 mm in length. For contraction studies, each ring was preincubated in a given culture dish with 2 ml of complete DMEM (Mediatech Cellgro) containing 1% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The tissues were incubated at 37°C in a humidified incubator with 5% CO2-95% air in the absence or presence of cortisol (1, 10, or 30 ng/ml) and/or carbenoxolone (3 µM) for 24 h. To determine the role of endothelium in the effect of cortisol, the endothelium was removed from some arterial rings by gentle rotation of the tissue on an appropriately sized, rough-surfaced blunt hypodermic needle as described previously (17). For NO and eNOS protein measurements, the uterine arteries were cut into segments 20 mm in length. Five segments were placed in a given culture dish with 5 ml DMEM containing 1% fetal bovine serum and penicillin-streptomycin and incubated at 37°C in a humidified incubator with 5% CO2-95% air in the absence or presence of cortisol (10 ng/ml) and/or carbenoxolone (3 µM) for 24 h. After the treatment, a 1-ml sample was taken from the medium for NO measurement, and the endothelium was gently scraped from the vessel lumen of the arterial segments as previously described (26, 46) for determination of eNOS protein levels.Contraction studies.
After cortisol pretreatment, arterial contractions were quantified in
the continuous presence of cortisol in Krebs solution in tissue baths
at 37°C as described previously (17). Isometric tensions
were measured. After 60 min of equilibration in the tissue bath, each
ring was stretched to the optimal resting tension, as determined by the
tension developed in response to potassium chloride (120 mM) added at
each stretch level. Concentration-response curves were obtained by
cumulative addition of NE in approximate one-half log increments.
EC50 values for the agonist in each experiment were taken
as the molar concentration at which the contraction-response curve
intersected 50% of the maximum response and were expressed as
pD2 (
log EC50) values.
-adrenoceptors was determined as previously described
(17). Briefly, the concentration-response curves to NE
were determined before and after the treatment of tissues with
phenoxybenzamine (30 nM for 20 min) to inactivate a fraction of the
receptors and reduce the maximal response to NE by ~50%. The
reciprocal of the concentration of NE before phenoxybenzamine treatment
(1/[A]) was then plotted against the reciprocal of the corresponding
equieffective concentrations after the treatment (1/[A']). The values
for KA and the fraction of active receptors
remaining (q) were calculated as follows (11): 1/[A] = (1 q)/qKA + 1/q[A'], where KA = (slope 1)/intercept and q = 1/slope
(12).
Measurement of NO, nitrite, and nitrate. Cumulated NO release in the culture medium was measured by chemiluminescence method as previously described (50). Because of the instability of NO in oxygenated physiological solution, most of NO is converted to nitrite and further to nitrate. Nitrite and nitrate are relatively stable in the solution and are readily reduced back to NO in vanadium (III)-HCl solution. Samples (0.5 ml) were deproteinized by the addition of 1 ml cold ethanol followed by vortex mixing for 1 min. After incubation on ice for 30 min, samples were centrifuged at 14,000 rpm for 5 min, and the supernatant was collected. A 50-µl aliquot of the supernatant was injected into the gas purge vessel containing 5 ml vanadium (III)-HCl to react for 1 min and reduce nitrate/nitrite in the sample back to NO. To achieve high reducing efficiency, the reduction was performed at 90°C. NO in the sample was then stripped into the head space of the gas purge vessel by bubbling it with helium (12 ml/min) for 60 s. NO in the head space was then drawn into a NO analyzer (model 270B, Sievers Instruments; Boulder, CO) and mixed with ozone (O3) in front of a cooled Hamamastu, red-sensitive photomultiplier tube. The signal from the detector was analyzed by an on-line computer as the area under the peak. The measurement reflected the combined concentrations of nitrite, nitrate, and NO (NOx) of each sample, which were calculated from a standard curve of 10-1,000 pmol nitrate run in each assay.
Western blot analysis of eNOS and GRs.
To determine the effect of cortisol on eNOS protein levels, the
endothelium was gently scraped from the vessel lumen of the arterial
segments after 24-h cortisol treatment as described above. The cells
were then solubilized by sonication in lysis buffer (150 mM NaCl, 50 mM
Tris · HCl, 10 mM EDTA, 0.1% Tween 20, 0.1% -mercaptoetanol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin; pH 7.4). To measure GR protein in
uterine artery endothelium and smooth muscle, the endothelium was
scraped from the vessel lumen of freshly isolated uterine arteries (the
main to third branches) and solubilized. The remaining smooth muscle
was homogenized on ice with a Brinkman Polytron in ice-cold TEGMD
buffer (20 mM Tris, 1 mM EDTA, 10% glycerol, 10 mM sodium molybdate,
and 1 mM dithiothreitol; pH 7.4). The homogenate was centrifuged at
4°C for 45 min at 100,000 g. Protein obtained from both
endothelial and smooth muscle samples was quantified in the supernatant
using a protein assay kit from Bio-Rad. Western blotting of eNOS was
carried out as previously described (45) and that of GR by
the method of O'Donnell et al. (33). Samples were mixed
with an equal volume of 2× sample buffer (0.125 M Tris · HCl,
20% glycerol, 4% SDS, 0.005% bromophenol blue, and 5%
-mercaptoetanol) and heated at 95°C for 5 min. Samples with equal
protein (10 µg for eNOS and 25 µg for GR) were loaded onto a 7.5%
polyacrylamide gel with 0.1% SDS and separated by electrophoresis at
100 V for 2 h. Proteins were then transferred onto an immobilon-P
membrane at 30 V for 60 min at room temperature using a semidry blotter (Bio-Rad). The immobilon-P membrane was probed by mouse monoclonal antiserum for eNOS (1:750, Transduction Laboratories; Lexington, KY)
and rabbit polyclonal antibody for glucocorticoid receptor (1:1,000,
Affinity Bioreagents; Neshanic Station, NJ). Membranes were washed
using Tris-buffered saline and then incubated with horseradish
peroxidase-conjugated goat anti-mouse (1:1,000) and goat anti-rabbit
(1:2,500) antibodies obtained from Amersham (Arlington Heights, IL).
Proteins were visualized with enhanced chemiluminescence (ECL) reagents
(Amersham), and the blots were exposed to hyperfilm. Results were
quantified by a scanning densitometer (model 670, Bio-Rad). Actin was
used to assess equal loading only for within-group analysis.
Materials. NE, cortisol, and carbenoxolone were obtained from Sigma (St. Louis, MO). All electrophoretic and immunoblot reagents were obtained from Bio-Rad Laboratories. All drugs were prepared fresh each day and kept on ice throughout the experiment.
Data analysis. Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad software; San Diego, CA). Results were expressed as means ± SE obtained from the number (n) of experimental animals given. Differences were evaluated for statistical significance (P < 0.05) by one-way ANOVA followed by the Newman-Keuls post hoc test.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Effect of cortisol on NE-induced contractions.
Figure 1 shows the effect of cortisol on
NE-induced contractions of the uterine artery. Cortisol (1, 10, or 30 ng/ml) treatment for 24 h produced a dose-dependent increase, at
the given dose range, in NE-induced contractions of nonpregnant uterine
arteries and increased NE pD2 from a control value of
5.60 ± 0.02 to 5.96 ± 0.04 (P < 0.05),
6.36 ± 0.07 (P < 0.05), and 6.59 ± 0.06 (P < 0.05), respectively (Fig. 1A). In
pregnant uterine arteries, the NE-induced contraction was increased
compared with nonpregnant uterine arteries (NE pD2:
6.21 ± 0.10 vs. 5.60 ± 0.02, P < 0.05). The low dose (1 ng/ml) of cortisol had no effect on NE-induced contractions in pregnant uterine arteries, but the higher doses (10 and
30 ng/ml) increased NE pD2 from a control value of
6.21 ± 0.10 to 6.54 ± 0.05 (P < 0.05) and
6.65 ± 0.09 (P < 0.05), respectively (Fig.
1B). However, the degree of cortisol-mediated potentiation of NE-induced contractions was significantly attenuated in pregnant vs.
nonpregnant uterine arteries (Fig. 2).
Removal of the endothelium diminished the cortisol (10 ng/ml)-mediated
potentiation in both nonpregnant (NE pD2: 6.29 ± 0.10 vs. 6.46 ± 0.17, P > 0.05) and pregnant
(6.93 ± 0.15 vs. 7.14 ± 0.29, P > 0.05)
uterine arteries.
|
|
|
-adrenoceptors in the uterine artery, KA of
NE to -adrenoceptors was determined in intact control and
cortisol-treated tissues using Furchgott's partial irreversible
blockade method. As shown in Fig. 4, the
KA values for NE were higher in nonpregnant (24.6 ± 6.5 µM) than pregnant (5.2 ± 2.0 µM) uterine
arteries (P < 0.05). After cortisol treatment, there
was a significant reduction in NE KA values in
nonpregnant, but not in pregnant, uterine arteries (Fig. 4).
|
Effect of pregnancy on GR protein levels.
Given that effect of cortisol is mediated by the GR, we determined GR
protein levels in nonpregnant and pregnant uterine arteries by Western
blotting. As shown in Fig. 5, the GR was
recognized by the polyclonal antibody at a band of ~97 kDa in both
endothelial scrapings and vascular smooth muscle of the uterine artery,
which is in agreement with the estimated molecular mass previously
reported for rat GR (29). Quantitative analysis of
immunoreactive GR levels indicated that pregnancy did not affect GR
protein levels in either endothelial scrapings or smooth muscle of the
uterine artery (Fig. 5, bottom).
|
Effect of carbenoxolone on cortisol-mediated responses.
To test the hypothesis that pregnancy alters 11-HSD activity in the
uterine artery, the cortisol-mediated potentiation of NE-induced
contractions was examined in the absence and/or presence of the
11-HSD inhibitor carbenoxolone (3 µM for 24 h). As shown in
Fig. 6, carbenoxolone had no effect on
NE-induced contractions in the absence or presence of cortisol in
nonpregnant uterine arteries. In contrast, carbenoxolone potentiated
NE-induced contractions of pregnant uterine arteries by increasing the
NE pD2 from 6.20 ± 0.05 to 6.59 ± 0.11 (P < 0.05) in the absence of cortisol. In the presence
of carbenoxolone, cortisol had no further effect on NE-induced
contractions of pregnant uterine arteries (Fig. 6).
|
Effect of cortisol on eNOS expression and NO release.
Figure 7 shows the effect of cortisol and
carbenoxolone on eNOS protein expression in nonpregnant and pregnant
uterine artery endothelial scrapings. In agreement with a previous
study (45), the representative Western immunoblot showed
that the monoclonal antibody for eNOS detected a single band at the
expected size of 140 kDa in both nonpregnant and pregnant uterine
artery endothelial scrapings. As shown in Fig. 7, there was a decrease
in eNOS protein expression in both pregnant and nonpregnant uterine
artery endothelial scrapings after 24-h pretreatment with cortisol or
carbenoxolone, respectively. Although the carbenoxolone-mediated
decrease in eNOS protein levels were similar in nonpregnant and
pregnant uterine artery endothelial scrapings, the degree of
cortisol-induced reduction in eNOS was significantly less in pregnant
compared with nonpregnant uterine artery endothelial scrapings (Fig.
8). Consistent with its effect on eNOS
protein levels, cortisol decreased NO release 74% in the nonpregnant
uterine artery and 44% in the pregnant uterine artery (Fig.
9). Carbenoxolone alone had no effects on NO release in nonpregnant uterine artery (103.4 ± 15.0 vs.
86.3 ± 3.8 pmol/50 µl, P > 0.05) but
significantly decreased NO release in the pregnant uterine artery
(234.5 ± 39.4 vs. 116.3 ± 27.0 pmol/50 µl,
P < 0.05; Fig. 9).
|
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The present study demonstrated that cortisol plays an important
role in the regulation of uterine artery contractility. There are
several important observations in the present study. First, cortisol
potentiated NE-mediated contractions in both uterine and femoral
arteries from nonpregnant animals. Second, pregnancy selectively
attenuated the cortisol-mediated potentiation in the uterine artery.
Third, cortisol increased NE-binding affinity to -adrenoceptors
selectively in the nonpregnant uterine artery. Fourth, the 11-HSD
inhibitor carbenoxolone increased NE-induced contractions selectively
in the pregnant uterine artery. Fifth, pregnancy did not change GR
density in the uterine artery. Sixth, cortisol downregulated eNOS
protein expression and decreased NO release in uterine artery
endothelium, which was attenuated by pregnancy. Finally, carbenoxolone
inhibited NO release selectively in the pregnant uterine artery.
The results that cortisol potentiated NE-induced contractions of uterine and femoral arteries are in agreement with previous findings demonstrating that corticosteroid hormones play an important role in the regulation of vascular reactivity. It has been demonstrated in vivo that adrenalectomy reduces pressor responsiveness to catecholamines, which can be reversed with glucocorticoid replacement (10, 48). Increased cortisol responsiveness has been associated with an increase in arterial contractile sensitivity to NE and vascular resistance (5, 22, 41, 42). In contrast, although glucocorticoids have been found to have pronounced stimulatory effect on blood pressure in fetal sheep (11, 39, 44), betamethasone treatment for 2 days showed no effect on NE-induced contractions of femoral arteries in fetal sheep (2). This would suggest heterogeneity of glucocorticoid-mediated vascular responses at different developmental stages.
The present study demonstrated, for the first time, that pregnancy attenuated uterine, but not femoral, artery sensitivity to cortisol and decreased cortisol-mediated potentiation of NE-induced contractions of the uterine artery. It has been well documented that the effect of cortisol in potentiating vasoactive responses to catecholamines is mediated by the GR in vascular smooth muscle (13, 22, 28). Given that maternal plasma cortisol concentrations approximately double in sheep (19, 20) and cortisol suppresses the expression of the GR (8), we had expected a decrease in GR density in the pregnant uterine artery. However, immunoreactive GR proteins estimated by Western blotting in the present study were not different in either endothelial scrapings or smooth muscle between nonpregnant and pregnant uterine arteries, suggesting that the pregnancy-associated decrease in uterine artery sensitivity to cortisol was not mediated by decreased GR density. Because immunoreactive GR proteins include both cytosolic and nuclear GR, it is not clear at present whether cytosolic GR availability and binding affinity was altered by pregnancy in the uterine artery. Roesch and Keller-Wood (35) demonstrated differential effects of pregnancy on GR availability and immunoreactivity in the sheep hypothalamus, pituitary, hippocampus, and kidney and suggested significant tissue heterogeneity in the regulation of GR by pregnancy. Because progesterone has antiglucocorticoid effects and binds to GR at a physiological concentration (9), it is speculated that increased progesterone in pregnancy affects uterine artery reactivity locally by decreasing the GR availability in both endothelial scrapings and vascular smooth muscle.
Despite the fundamental importance of cortisol in regulating vascular
reactivity to vasoconstrictors, little is currently known about the
cellular mechanisms of vascular smooth muscle in response to cortisol.
It has been shown that adrenalectomy causes a significant decrease in
1-adrenoceptor density in the rat aorta, which is
restored by dexamethasone replacement (15). We have
demonstrated that NE contracts the uterine artery by acting on
1-adrenoceptors and increasing inositol
1,4,5-trisphosphate (49). The higher binding affinity of
NE to 1-adrenoceptors in pregnant than nonpregnant
uterine arteries may explain in part the increased NE-induced
contractions in pregnant uterine arteries. The present finding that
cortisol significantly decreased dissociation constant of NE to
1-adrenoceptors in nonpregnant uterine arteries suggests
that cortisol-mediated potentiation of NE-induced contractions of
nonpregnant uterine arteries was due, at least in part, to the
increased NE-binding affinity to 1-adrenoceptors. This
is in agreement with previous in vivo studies in the dog and in vitro studies in the rabbit aorta in which cortisol was proposed to increase
the affinity of catecholamine for the adrenergic receptor (3). Nevertheless, the effect of cortisol on catecholamine affinity for the adrenergic receptors has not been previously determined. In the present study, in the absence of exogenous cortisol,
NE-binding affinity to 1-adrenoceptors was greater in
pregnant than nonpregnant uterine arteries. Cortisol increased NE-binding affinity to 1-adrenoceptors in nonpregnant
but not pregnant uterine arteries and eliminated the difference between pregnant and nonpregnant uterine arteries. These studies suggest that
increased 1-adrenoceptor binding affinity in pregnant
compared with nonpregnant uterine arteries may be mediated by an
increase in endogenous cortisol binding to GR in the pregnant uterine
artery due to elevated cortisol levels in pregnancy. Although the
mechanisms underlying cortisol-mediated regulation of agonist-binding
affinity are not clear at present, studies have shown that
glucocorticoids play a crucial role in maintaining coupling of
1-adrenoceptors to G proteins in the rat aorta
(15, 16). This may be an important mechanism by which
cortisol regulates receptor-G protein coupling and hence agonist
binding affinity in vascular smooth muscle.
The finding that the 11-HSD inhibitor carbenoxolone selectively
potentiated NE-induced contractions in the pregnant uterine artery by
increasing NE pD2 in the absence of exogenous cortisol suggests a significant level of endogenous cortisol in the freshly isolated tissues of the uterine arteries. Although we cannot completely rule out any effect of potential cortisol in fetal bovine serum used in
this study, the effect from 1% fetal serum is likely to be minimal,
given that fetal bovine plasma cortisol levels ranged from 3 to 8 ng/ml
(23, 39), which would result in maximal cortisol levels of
0.03-0.08 ng/ml in the medium. Regardless of the source of
cortisol, the finding that carbenoxolone selectively potentiated
NE-induced contractions in the pregnant uterine artery is intriguing
and suggests an increase in type 2 11-HSD activity in the uterine
artery in pregnancy. The effect of glucocorticoids on vascular
reactivity is regulated by 11-HSD (41). The two 11-HSD isozymes catalyze the interconversion of cortisol and cortisone. Type 1 11-HSD has bidirectional activity, whereas type 2 mainly converts cortisol into cortisone, the biologically inactive
form. Both type 1 and 2 11-HSD have been found in vascular endothelial (7) and smooth muscle cells (6,
42). Numerous studies have demonstrated that inhibition of
11-HSD with an enzyme inhibitor such as carbenoxolone increases
cortisol-mediated potentiation of vascular response to NE (5, 22,
41, 42). Although under normal conditions the type 1 isoform
dominates, functioning in the oxo-reductase mode, which converts
cortisone to cortisol in both endothelial and smooth muscle cells, the
two major isoforms are compartmentalized discretely and regulated
differentially by steroids such as estrogen and progesterone
(38). In human pregnancy, placental type 2 11-HSD
activity increases markedly in the third trimester of pregnancy, at a
time when maternal circulating levels of glucocorticoid are rising,
which serves as a protective mechanism for the fetus (36).
The present study suggests an increase in type 2 11-HSD activity in
the uterine artery, which is likely to play an important role in the
local regulation of cortisol concentration by limiting the effect of
cortisol on the uterine artery and protecting it from elevated cortisol
levels during pregnancy.
The present study demonstrated that cortisol significantly decreased NO
release in the uterine artery. In addition, removal of the endothelium
diminished cortisol-mediated potentiation of NE-induced contractions.
These studies suggest that the effect of cortisol on NO production
plays an important role in cortisol-mediated potentiation of uterine
artery contractility. Previous reports concerning the effects of
cortisol on NO synthesis/release are controversial. It has been
suggested that stress-induced increase in plasma NO production is
cortisol independent in the rat (24). Studies (14,
21) in humans showed that mental stress induced transient
endothelial dysfunction and cortisol treatment significantly reduced
plasma nitrate/nitrite concentrations and increased blood pressure. In
addition, studies in adrenalectomized sheep have demonstrated that
withdrawal of glucocorticoid replacement resulted in reduced blood
pressure and pressor responsiveness, which could be restored by the NO
synthase inhibitor
NG-nitro-L-arginine methyl ester
(34). In the present study, the effect of cortisol
on NO production in the uterine artery was examined by directly
measuring NO release. The finding of increased NO release in pregnant
versus nonpregnant uterine arteries is in agreement with our previous
studies (46, 47). Cortisol significantly decreased NO
release in the uterine artery, which was associated with a reduction in
eNOS protein levels in uterine artery endothelial scrapings. A recent
study (43) in cultured human umbilical vein endothelial
cells demonstrated that glucocorticoids decreased eNOS promoter
activity and mRNA stability. We and others have demonstrated that both
eNOS protein and mRNA levels are significantly increased in pregnant
uterine artery endothelial cells (4, 26, 27, 31, 46, 47).
The present finding that the degree of cortisol-mediated inhibition of
eNOS protein expression was decreased in the pregnant uterine artery
may be due in part to the increased eNOS in pregnant uterine artery.
Carbenoxolone decreased eNOS protein levels in both nonpregnant and
pregnant uterine arteries but significantly decreased NO release only
in pregnant uterine arteries. This discrepancy between eNOS protein
levels and NO release is not entirely clear at present but may involve
the regulation of eNOS activity. The finding that carbenoxolone had no
effect on NO release in nonpregnant uterine arteries but significantly decreased NO release in pregnant uterine arteries is consistent with
the contraction studies in which carbenoxolone increased NE-induced
contraction only in pregnant uterine arteries, suggesting that
endothelial effect may play an important role in carbenoxolone-mediated potentiation of NE-induced contraction. This also suggests an increase
in type 2 11-HSD activity in pregnant uterine artery endothelial cells.
In summary, the results indicate that cortisol plays an important role
in the regulation of uterine artery contractility, and its effect is
endothelium dependent. Downregulation of eNOS protein expression and NO
synthesis/release is likely to contribute to the cortisol-mediated
potentiation of uterine artery contraction. More importantly, pregnancy
selectively attenuates uterine artery sensitivity to cortisol. Although
this decreased sensitivity may not be mediated by a decrease in GR
density, the effect of pregnancy on the availability and binding
affinity of GRs in the uterine artery remains to be determined. In
addition, pregnancy may increase type 2 11-HSD activity in uterine
artery endothelial and smooth muscle cells, which is likely to provide
a local protection for the uterine artery to the increased cortisol
levels in pregnancy.
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported in part by National Institutes of Health Grants HL-54094, HL-57787, and HD-31226 and by the Loma Linda University School of Medicine.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: L. Zhang, Center for Perinatal Biology, Dept. of Pharmacology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (E-mail: lzhang{at}som.llu.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.
First published February 28, 2002;10.1152/ajpheart.00842.2001
Received 26 September 2001; accepted in final form 26 February 2002.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Annibale, DJ,
Rosenfeld CR,
and
Kamm KE.
Alterations in vascular smooth muscle contractility during ovine pregnancy.
Am J Physiol Heart Circ Physiol
256:
H1281-H1288,
1989.
2.
Anwar, MA,
Schwab M,
Poston L,
and
Nathanielsz PW.
Betamethasone-mediated vascular dysfunction and changes in hematological profile in the ovine fetus.
Am J Physiol Heart Circ Physiol
276:
H1137-H1143,
1999
3.
Besse, JC,
and
Bass AD.
Potentiation by hydrocortisone of responses to catecholamines in vascular smooth muscle.
J Pharmacol Exp Ther
154:
224-238,
1966
4.
Bird, IM,
Sullivan JA,
Di T,
Cale JM,
Zhang L,
Zheng J,
and
Magness RR.
Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells.
Endocrinology
141:
1107-1117,
2000
5.
Brem, AS,
Bina RB,
Hill N,
Alia C,
and
Morris DJ.
Effects of licorice derivatives on smooth muscle function.
Life Sci
60:
207-214,
1997[ISI][Medline].
6.
Brem, AS,
Bina RB,
King TC,
and
Morris DJ.
Bidirectional activity of 11 -hydroxysteroid dehydrogenase in vascular smooth muscle cells.
Steroids
60:
406-410,
1995[ISI][Medline].
7.
Brem, AS,
Bina RB,
King TC,
and
Morris DJ.
Localization of 2 11-OH steroid dehydrogenase isoforms in aortic endothelial cells.
Hypertension
31:
459-462,
1998
8.
Burnstein, KL,
Bellingham DL,
Jewell CM,
Powell-Oliver FE,
and
Cidlowski JA.
Autoregulation of glucocorticoid receptor gene expression.
Steroids
56:
52-58,
1991[ISI][Medline].
9.
Chou, YC,
and
Luttge WG.
Activated type II receptors in brain cannot rebind glucocorticoids: relationship to progesterone's antiglucocorticoid actions.
Brain Res
440:
67-78,
1988[ISI][Medline].
10.
Darlington, DN,
Kaship K,
Keil LC,
and
Dallman MF.
Vascular responsiveness in adrenalectomized rats with corticosterone replacement.
Am J Physiol Heart Circ Physiol
256:
H1274-H1281,
1989
11.
Dodic, M,
and
Wintour EM.
Effects of prolonged (48 h) infusion of cortisol on blood pressure, renal function and fetal fluids in the immature ovine foetus.
Clin Exp Pharmacol Physiol
21:
971-980,
1994[ISI][Medline].
12.
Furchgott, RF.
The use of -haloalkylamines in the differentiation of receptors and the determination of dissociation constants of receptor agonist complexes.
Adv Drug Res
3:
21-55,
1966.
13.
Gaillard, RC,
Poffet D,
Riondel AM,
and
Saurat JH.
RU486 inhibits peripheral effects of glucocorticoids in humans.
J Clin Endocrinol Metab
61:
1009-1011,
1985[Abstract].
14.
Ghiadoni, L,
Donald AE,
Cropley M,
Mullen MJ,
Oakley G,
Taylor M,
O'Connor G,
Betteridge J,
Klein N,
Steptoe A,
and
Deanfield JE.
Mental stress induces transient endothelial dysfunction in humans.
Circulation
102:
2473-2478,
2000
15.
Haigh, RM,
and
Jones CT.
Effect of glucocorticoids on 1-adrenergic receptor binding in rat vascular smooth muscle.
J Mol Endocrinol
5:
41-48,
1990
16.
Haigh, RM,
Jones CT,
and
Milligan G.
Glucocorticoids regulate the amount of G proteins in rat aorta.
J Mol Endocrinol
5:
185-188,
1990
17.
Hu, XQ,
Longo LD,
Gilbert RD,
and
Zhang L.
Effects of long-term high altitude hypoxemia on 1-adrenergic receptors in the ovine uterine artery: functional and binding studies.
Am J Physiol Heart Circ Physiol
270:
H1001-H1007,
1996
18.
Keller-Wood, M.
Vasopressin response to hyperosmolality and hypotension during ovine pregnancy.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R188-R193,
1994
19.
Keller-Wood, M.
Inhibition of stimulated and basal ACTH by cortisol during ovine pregnancy.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R130-R136,
1996
20.
Keller-Wood, M.
Evidence for reset of regulated cortisol in pregnancy: studies in adrenalectomized ewes.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R145-R151,
1998
21.
Kelly, JJ,
Tam SH,
Williamson PM,
Lawson J,
and
Whitworth JA.
The nitric oxide system and cortisol-induced hypertension in humans.
Clin Exp Pharmacol Physiol
25:
945-946,
1998[ISI][Medline].
22.
Kornel, L.
The role of vascular steroid receptors in the control of vascular contractility and peripheral vascular resistance.
J Steroid Biochem Mol Biol
45:
195-203,
1993[ISI][Medline].
23.
Lay, DC,
Randel RD,
Friend TH,
Carroll JA,
Welsh TH,
Jenkins OC,
Neuendorff DA,
Bushong DM,
and
Kapp GM.
Effects of prenatal stress on the fetal calf.
Domest Anim Endocrinol
14:
73-80,
1997[ISI][Medline].
24.
Leza, JC,
Salas E,
Sawicki G,
Russell JC,
and
Radomski MW.
The effects of stress on homeostasis in JCR-LA-cp rats: the role of nitric oxide.
J Pharmacol Exp Ther
286:
1397-1403,
1998
25.
Magness, RR,
and
Rosenfeld CR.
Systemic and uterine responses to -adrenergic stimulation in pregnant and nonpregnant sheep.
Am J Obstet Gynecol
155:
897-904,
1986[ISI][Medline].
26.
Magness, RR,
Shaw CE,
Phernetton TM,
Zheng J,
and
Bird IM.
Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression.
Am J Physiol Heart Circ Physiol
272:
H1730-H1740,
1997
27.
Magness, RR,
Sullivan JA,
Li Y,
Phernetton TM,
and
Bird IM.
Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NOx.
Am J Physiol Heart Circ Physiol
280:
H1692-H1698,
2001
28.
Marks, R,
Barlow JW,
and
Funder JW.
Steroid-induced vasoconstriction: glucocorticoid antagonist studies.
J Clin Endocrinol Metab
54:
1075-1077,
1982[Abstract].
29.
Miesfeld, R,
Rusconi S,
Godowski PJ,
Maler BA,
Okret S,
Wikstrom AC,
Gustafsson JA,
and
Yamamoto KR.
Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA.
Cell
46:
389-399,
1986[ISI][Medline].
30.
Naden, RP,
and
Rosenfeld CR.
Effect of angiotensin II on uterine and systemic vasculature in pregnant sheep.
J Clin Invest
68:
469-474,
1981.
31.
Nelson, SH,
Steinsland OS,
Wang Y,
Yallampalli C,
Dong YL,
and
Sanchez JM.
Increased nitric oxide synthase activity and expression in the human uterine artery during pregnancy.
Circ Res
87:
406-411,
2000
32.
Nolten, WE,
and
Rueckert PA.
Elevated free cortisol index in pregnancy: possible regulatory mechanisms.
Am J Obstet Gynecol
139:
492-498,
1981[ISI][Medline].
33.
O'Donnel, D,
Francis D,
Weaver S,
and
Meaney MJ.
Effects of adrenalectomy and corticosterone replacement on glucocorticoid receptor levels in rat brain tissue: a comparison between Western blotting and receptor binding assays.
Brain Res
687:
133-142,
1995[ISI][Medline].
34.
Orbach, P,
Wood CE,
and
Keller-Wood M.
Nitric oxide reduces pressor responsiveness during ovine hypoadrenocorticism.
Clin Exp Pharmacol Physiol
28:
459-462,
2001[ISI][Medline].
35.
Roesch, DM,
and
Keller-Wood M.
Differential effects of pregnancy on mineralocorticoid and glucocorticoid receptor availability and immunoreactivity in cortisol feedback sites.
Neuroendocrinology
70:
55-62,
1999[ISI][Medline].
36.
Shams, M,
Kilby MD,
Somerset DA,
Howie AJ,
Gupta A,
Wood PJ,
Afnan M,
and
Stewart PM.
11-Hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in intrauterine growth restriction.
Hum Reprod
13:
799-804,
1998
37.
Sladek, SM,
Magness RR,
and
Conrad KP.
Nitric oxide and pregnancy.
Am J Physiol Regulatory Integrative Comp Physiol
272:
R441-R463,
1997
38.
Sun, K,
Yang K,
and
Challis JR.
Glucocorticoid actions and metabolism in pregnancy: implications for placental function and fetal cardiovascular activity.
Placenta
19:
353-360,
1998[ISI][Medline].
39.
Takeishi, M,
Shibata S,
and
Tsumagari S.
Adrenocorticotropin and cortisol levels in the plasma of bovine fetuses and neonates.
Nippon Juigaku Zasshi
51:
975-980,
1989[Medline].
40.
Unno, N,
Wong CH,
Jenkins SL,
Wentworth RA,
Ding XY,
Li C,
Robertson SS,
Smotherman WP,
and
Nathanielsz PW.
Blood pressure and heart rate in the ovine fetus: ontogenic changes and effects of fetal adrenalectomy.
Am J Physiol Heart Circ Physiol
276:
H248-H256,
1999
41.
Van Uum, SH,
Hermus AR,
Smits P,
Thien T,
and
Lenders JW.
The role of 11-hydroxysteroid dehydrogenase in the pathogenesis of hypertension.
Cardiovasc Res
38:
16-24,
1998
42.
Walker, BR,
Yau JL,
Brett LP,
Seckl JR,
Monder C,
Williams BC,
and
Edwards CR.
11 beta-hydroxysteroid dehydrogenase in vascular smooth muscle and heart: implications for cardiovascular responses to glucocorticoids.
Endocrinology
129:
3305-3312,
1991[Abstract].
43.
Wallerath, T,
Witte K,
Schafer SC,
Schwarz PM,
Prellwitz W,
Wohlfart P,
Kleinert H,
Lehr HA,
Lemmer B,
and
Forstermann U.
Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension.
Proc Natl Acad Sci USA
96:
13357-13362,
1999
44.
Wood, CE,
Cheung CY,
and
Brace RA.
Fetal heart rate, arterial pressure, and blood volume responses to cortisol infusion.
Am J Physiol Regulatory Integrative Comp Physiol
253:
R904-R909,
1987
45.
Xiao, DL,
Bird IM,
Magness RR,
Longo LD,
and
Zhang L.
Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia.
Am J Physiol Heart Circ Physiol
280:
H812-H820,
2001
46.
Xiao, DL,
Liu Y,
Pearce WJ,
and
Zhang L.
Endothelial nitric oxide release in isolated perfused ovine uterine arteries: effect of pregnancy.
Eur J Pharmacol
367:
223-230,
1999[ISI][Medline].
47.
Xiao, DL,
Pearce WJ,
and
Zhang L.
Pregnancy increases endothelium-dependent relaxation of ovine uterine artery: role of nitric oxide and intracellular calcium.
Am J Physiol Heart Circ Physiol
281:
H183-H190,
2001
48.
Yagil, Y,
and
Krakoff LR.
The differential effect of aldosterone and dexamethasone on pressor responses in adrenalectomized rats.
Hypertension
11:
174-178,
1988
49.
Zhang, L,
Pearce WJ,
and
Longo LD.
Noradrenaline-mediated contractions of ovine uterine artery: role of inositol 1,4,5-trisphosphate.
Eur J Pharmacol
289:
375-382,
1995[ISI][Medline].
50.
Zhang, L,
Xiao DL,
and
Bouslough DB.
Long-term high-altitude hypoxia increases plasma nitrate levels in pregnant ewes and their fetuses.
Am J Obstet Gynecol
179:
1594-1598,
1998[ISI][Medline].
This article has been cited by other articles:
|
|
 |
|
  A. E. Michael and A. T. Papageorghiou Potential significance of physiological and pharmacological glucocorticoids in early pregnancy Hum. Reprod. Update, September 1, 2008; 14(5): 497 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Chang and Lubo Zhang Review Article: Steroid Hormones and Uterine Vascular Adaptation to Pregnancy Reproductive Sciences, April 1, 2008; 15(4): 336 - 348. [Abstract] [PDF]
|
|
|
|
 |
|
 F. Li, C. E. Wood, and M. Keller-Wood Adrenalectomy alters regulation of blood pressure and endothelial nitric oxide synthase in sheep: modulation by estradiol Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R257 - R266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhang, D. Xiao, L. D. Longo, and L. Zhang Regulation of {alpha}1-adrenoceptor-mediated contractions of uterine arteries by PKC: effect of pregnancy Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2282 - H2289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Horvath and A. Wanner Inhaled corticosteroids: effects on the airway vasculature in bronchial asthma Eur. Respir. J., January 1, 2006; 27(1): 172 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xiao and L. Zhang Adaptation of uterine artery thick- and thin-filament regulatory pathways to pregnancy Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H142 - H148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xiao, X. Huang, S. Bae, C. A. Ducsay, L. D. Longo, and L. Zhang Cortisol-mediated regulation of uterine artery contractility: effect of chronic hypoxia Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H716 - H722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xiao, X. Huang, W. J. Pearce, L. D. Longo, and L. Zhang Effect of cortisol on norepinephrine-mediated contractions in ovine uterine arteries Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1142 - H1151. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||
