Regulation of cGMP-induced relaxation and cGMP-dependent
protein kinase in rat myometrium during pregnancy
R. Ann
Word1 and
Trudy L.
Cornwell2
1 Department of Obstetrics and
Gynecology, University of Texas Southwestern Medical Center, Dallas,
Texas 75235-9032; and 2 Division
of Molecular and Cellular Pathology, Department of Pathology,
University of Alabama at Birmingham, Birmingham, Alabama 35294-0019
 |
ABSTRACT |
Increases in
guanosine 3',5'-cyclic monophosphate (cGMP) induced by
nitric oxide (NO), nitrovasodilators, and atrial peptides correlate
with relaxation of vascular smooth muscle. Relaxation of myometrial
smooth muscle by increases in cGMP, however, has required unusually
high concentrations of the cyclic nucleotide. We tested the hypothesis
that the sensitivity of myometrium to relaxation by cGMP is increased
during pregnancy. Aortic smooth muscle was more sensitive to relaxation
by cGMP than myometrial tissues, and, contrary to our hypothesis,
myometrium from pregnant rats was least sensitive. Although levels of
cGMP were elevated after treatment with the NO donor,
S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels of
cGMP-dependent protein kinase (PKG) were significantly decreased in
myometrium from pregnant rats compared with myometrium from nonpregnant
cycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, and
progesterone antagonized this response. We conclude that
1) myometrial tissues from pregnant
rats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP is
accompanied by progesterone-mediated decreases in the level of PKG
expression.
nitric oxide; progesterone; guanosine 3',5'-cyclic
monophosphate phosphodiesterase; oxytocin; atrial natriuretic peptide
 |
INTRODUCTION |
THE BIOCHEMICAL PROCESSES that initiate labor in women
are not known. In addition, the precise mechanisms that serve to
maintain uterine quiescence during pregnancy are not well understood.
Because the incidence of preterm labor has remained constant for
decades, and preterm labor remains a major cause of perinatal mortality and infant morbidity, modulators of myometrial function during pregnancy have been eagerly sought. Recently, it has been proposed that
nitric oxide (NO) may be an endogenous uterine relaxant that contributes to uterine quiescence during pregnancy before term (18,
31). In addition, it has been suggested that relief of NO action may
lead to retreat from uterine quiescence with the onset of spontaneous
uterine contractions of labor (24).
In general, the smooth muscle relaxant effects of NO, atrial
peptides, and drugs that liberate NO are mediated by guanosine 3',5'-cyclic monophosphate (cGMP). The downstream target
for cGMP is believed to be the cGMP-dependent protein kinase (PKG),
which mediates the vasorelaxant effects of cGMP (5, 10, 15). Whereas
increases in cGMP correlate with relaxation in a time- and
concentration-dependent manner in vascular smooth muscle (15), the role
of cGMP as a uterine relaxant is not as well established. For example,
there is no correlation between increases in cGMP and relaxation in
myometrial smooth muscle (7), and increases in cGMP that effect almost
complete relaxation of tonic smooth muscles have relatively little
effect in uterine smooth muscle (7, 29). To resolve this apparent
conundrum, several laboratories have investigated the possibility that
uterine smooth muscle responds to cGMP-mobilizing agents as a function
of the hormonal milieu. Bek et al. (3) reported that estradiol-primed
rat myometrium relaxed in response to atrial natriuretic peptide (ANP).
Potvin and Varma (22) found that myometrium from pregnant or
progesterone-treated rats was refractory to ANP (21, 23). In other
studies, however, it has been suggested that responses to
cGMP-mobilizing agents may be enhanced during pregnancy (31). Thus,
although experimental evidence supports a hormone dependence to the
relaxant properties of cGMP, it remains uncertain whether pregnancy
enhances or diminishes cGMP action in uterine smooth muscle.
In this investigation, we compared the effects of NO donors and cGMP on
contractile properties of aortic smooth muscle and on myometrial smooth
muscle from nonpregnant and pregnant rats. We initially reasoned that,
during pregnancy, myometrial sensitivity to cGMP may be similar to that
of vascular smooth muscle, thereby providing a potential mechanism of
uterine quiescence. The results of these studies, however, indicate
that myometrial tissues obtained from pregnant rats are insensitive to
relaxation by cGMP. Furthermore, the levels of PKG in uterine smooth
muscle are significantly decreased during pregnancy, and this
downregulation of PKG during pregnancy is mediated by progesterone.
These latter findings provide a mechanism, at least in part, for the
diminished sensitivity of uterine smooth muscle to cGMP during
pregnancy.
 |
MATERIALS AND METHODS |
Source of smooth muscle tissues.
Ovariectomized and late pregnant (days
17-21) Sprague-Dawley rats (180-250 g) were
obtained from Sasco Laboratories. Daily subcutaneous injection of
hormones was initiated at 3-wk postovariectomy and continued for a
total of 3 days: 50 µg/kg 17
-estradiol, 2 mg/kg progesterone,
17
-estradiol plus progesterone or vehicle (corn oil, 200 µl). Rats
were killed the day after completion of hormone treatment, and uteri
were dissected. For the late pregnant group, gestational dating was
performed by vaginal smears. The first day of pregnancy was estimated
by estrus pattern with sperm present. All studies were conducted in
accordance with the standards of humane animal care as described in the
National Institutes of Health Guide for the Care and
Use of Laboratory Animals and approved by the
Institutional Review Board for Animal Research. Uterine and aortic
tissues were placed in ice-cold physiological saline solution (PSS; in
mM: 120.5 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4,
20.4 NaHCO3, 2.5 CaCl2, 10.0 D-glucose, and 1.0 pyruvate) and
gassed with 95% O2 and 5%
CO2. The tissues were then
prepared immediately for experimental studies.
Evaluation of smooth muscle contractile properties.
Each uterine horn was opened longitudinally along the mesenteric
border. The endometrium was removed with a cotton-tip applicator and by
sharp dissection using a dissecting microscope. Aortic tissues were
removed, cleaned of adjacent tissues, and opened longitudinally.
Myometrial strips (1 × 3 × 0.5 mm) were cut
parallel to the longitudinal muscle fibers, and aortic strips (0.5 × 1 × 0.2 mm) were cut transversely. Myometrial strips were
hung from Grass FT 0.036 transducers using 6-0 braided silk suture, and aortic strips were mounted with vascular clamps. Clamps or sutures were
attached to steel rods mounted on calibrated mechanical drives. Tissues
were incubated in PSS (37°C), and contractions were recorded on a
Grass model 7 polygraph. Optimal length for maximal force development
(Lo) was
determined by length-force relationships. Strips at
Lo were
conditioned by one contraction in PSS that contained KCl (65 mM)
substituted isotonically for NaCl. Aortic smooth muscle strips were
conditioned by two phenylephrine (0.1 µM)-induced contractions. In
myometrial tissues, active force was quantified by digitizing the
physiograph tracing of isometric force developed during 10 min and
computing the area of active force (30). In each strip, the area of
active force after treatment with test agents was compared with changes
in force development at the corresponding time intervals in separate
control aortic and myometrial strips.
Radioimmunoassay of cGMP.
Muscle strips were mounted for quantification of force generation
(described above) and treated with vehicle or test agents for 5 min.
Tissues were snap frozen with tongs precooled to the temperature of
liquid nitrogen and immediately homogenized in ice-cold extract buffer
that contained HCl (0.1 N) and methanol (50%, vol/vol). Samples were
centrifuged at 10,000 g for 10 min, and supernatants were lyophilized, acetylated, and assayed for cyclic
nucleotide content by radioimmunoassay as described (12). Precipitated
protein was determined using the bichinchoninic acid protein assay
(Pierce, Rockford, IL).
Immunoblot analysis of PKG.
Proteins in tissue extracts were separated by electrophoresis on sodium
dodecyl sulfate (SDS)-polyacrylamide gels (7%). Proteins were
transferred to nitrocellulose at 90 mA for 14-16 h in the presence
of methanol (20%) and SDS (0.1%). Blots were treated with TBST-milk
buffer [10 mM tris(hydroxymethyl)aminomethane, 150 mM NaCl,
0.05% Tween 20, and 0.3% powdered milk] for 1 h and then
incubated overnight with TBST-milk that contained a polyclonal antibody
against bovine lung PKG (1:1,000) at 4°C. Thereafter, the blots
were washed three times with TBST (5 min each) and incubated with goat
anti-rabbit immunoglobulin G conjugated with horseradish peroxidase
(1:10,000). After an extensive washing with TBST, the blots were
developed with a chemiluminescent detection system (enhanced
chemiluminescence Western blotting detection system). Relative amounts
of PKG were quantified using an LKB 2202 Ultroscan laser densitometer.
Quantification was obtained from blots in which densitometric units
were linear with respect to amounts of purified PKG standards and
protein loaded on the gel.
Assay for PKG.
PKG activity was determined from crude soluble extracts. Incorporation
of 32P from
[
-32P]ATP into
histone F2b was monitored using a filter paper assay (16).
Materials.
S-nitroso-N-acetyl-penicillamine
(SNAP) was purchased from Research Biochemicals International (Natick,
MA). The monophosphorothioate activator of PKG,
8-(4-chlorophenylthio)guanosine 3',5'cyclic monophosphate
(8-CPT-cGMP) was obtained from Biolog (La Jolla, CA). M2056 was
obtained from LC Laboratories (Woburn, MA). Radiolabeled compounds
([
-32P]ATP and
[125I]-labeled cGMP)
were from DuPont NEN (Boston, MA), and the bichinchoninic acid protein
assay reagents were from Pierce. The enhanced chemiluminescence Western
blotting detection system was obtained from Amersham (Arlington Heights, IL). All other reagents were from Sigma (St. Louis, MO) or
Fisher Biochemicals (Pittsburgh, PA).
Statistics.
Data are expressed as means ± SE. For multiple comparisons, data
were analyzed using a nonparametric Kruskal-Wallis analysis followed by
2 approximation. Differences
between aorta and myometrium were determined with Mann-Whitney's
U test for two independent variables.
 |
RESULTS |
Effect of activators of guanylate cyclases and cGMP
phosphodiesterase inhibitors on smooth muscle contractility.
To compare the effects of cGMP in vascular and uterine smooth muscles,
we treated aortic and myometrial tissues from pregnant rats with sodium
nitroprusside (SNP), a prototype of NO donors known to increase
cellular levels of cGMP (Fig.
1). Strips were precontracted
with submaximal concentrations of phenylephrine (0.1 µM) or oxytocin
(1 nM) to effect similar degrees of agonist-induced contractions.
Thereafter, the tissues were treated with cumulative concentrations of
SNP (10
8 to
10
4 M). Treatment with SNP
(0.1 µM) resulted in almost complete relaxation of precontracted
aortic smooth muscle (98 ± 0.7%,
n = 5, Fig. 1B) but virtually no detectable
decreases in the frequency or amplitude of oxytocin-induced
contractions in myometrial tissues (Figs.
1D and 2).
This refractoriness to SNP in myometrial tissues was not unique to
oxytocin. Myometrial tissues precontracted with 5-hydroxytryptamine
were also insensitive to relaxation by SNP (data not shown). The
effects of SNP on contractile force of both tissues are summarized in
Fig. 2. Maximal relaxation of myometrial tissues (70%) was effected by
1 × 10
4 M and
half-maximal effects at 3 × 10
5 M. Although high
concentrations of SNP (
100 µM) abolished contractile activity in
oxytocin-contracted myometrial tissues, these effects were irreversible
and spontaneous activity did not resume after several rinses with PSS
or after depolarization with KCl (40 mM). Thus, compared with aortic
smooth muscle, SNP was less effective in relaxing myometrium from
pregnant rats. Similar results were obtained with the NO donor SNAP
(Fig. 2, right). The concentration of SNAP required to effect 50% inhibition of contractile force in
aortic smooth muscle was significantly less than that required for
myometrium (4.2 ± 0.2 × 10
8 M, aorta compared with
2.2 ± 0.4 × 10
4 M
in myometrium, P < 0.01).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of sodium nitroprusside (SNP) on force of contraction in aortic
and myometrial smooth muscle from pregnant rats. Representative
tracings of SNP-induced relaxation of agonist-induced contractions in
aorta (A and
B) and myometrium
(C and
D) from pregnant rats
(day 17). Both tissues were treated
with submaximal concentrations of contractile agents. Aortic smooth
muscle was treated with phenylephrine (PE; 0.1 µM), and myometrial
tissues were treated with oxytocin (OX; 1 nM). Thereafter, the muscle
was treated with vehicle (control strip,
A and
C) or varying concentrations of SNP
(B and
D). Treatment of myometrial tissues
with high concentrations of SNP (0.1 mM) resulted in irreversible
cessation of contractile activity.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of SNP and
S-nitroso-N-acetylpenicillamine
(SNAP) on force of contraction in aortic and myometrial (MYO) smooth
muscle from pregnant rats. Cumulative dose-response curves were
obtained for SNP (left) or SNAP
(right) in aortic ( ) and
myometrial ( ) tissues from pregnant rats. In myometrial tissues,
extent of inhibition of contractile force in response to oxytocin was
determined by computing the area under the curve of isometric force
development after treatment with test agent. Relaxing effect of test
agents was corrected for changes in force development observed at the
corresponding time intervals in control strips. Data represent means ± SE of 4-9 strips from 8 rats.
|
|
The effect of SNP on spontaneous contractions in myometrium from
nonpregnant and pregnant rats was also determined (Fig.
3). Although treatment with SNP resulted in
very small decreases in the amplitude of force development in both
tissues, the predominant effect of SNP was to decrease the frequency of
spontaneous contractions (Fig. 3,
top). Myometrium from nonpregnant
animals in estrus was more sensitive to relaxation by SNP than that
from pregnant animals (Fig. 3,
bottom). A comparison of these data,
along with those presented in Figs. 1 and 2, clearly indicates that
myometrial tissues are less responsive to SNP than aortic smooth
muscle, and myometrium from pregnant animals is even less sensitive
than myometrium from nonpregnant animals. The apparent increased
relaxation of oxytocin-induced contractions (70%, Fig. 2) compared
with spontaneous contractions (10%, Fig. 3) is due to the irreversible
abolishment of contractions in some oxytocin-contracted tissues treated
with high concentrations of SNP.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of SNP on spontaneous contractions in myometrial tissues from
nonpregnant and pregnant rats. Top:
effect of SNP (1 mM) on frequency and amplitude of spontaneous
contractions in myometrium from nonpregnant (solid bar) and pregnant
(open bar) rats. Bottom:
dose-dependent inhibition of spontaneous contractions in myometrium
from nonpregnant ( ) and pregnant ( ) rats. Extent of inhibition
represents the decline in area of isometric force development during
spontaneous contractions 20 min before and after treatment with SNP.
Data represent means ± SE of 3-9 strips for 2 pregnant
(day 17) rats and 3 nonpregnant rats
in estrus. Apparent single points represent data where SE is very
low.
|
|
An alternative mechanism known to elevate cGMP levels was also
tested. Natriuretic peptides elevate cGMP by binding to extracellular receptors that are transmembrane guanylate cyclases. Stimulation of
particulate guanylate cyclase by ANP (0.1 µM) resulted in significant relaxation (85%) in phenylephrine-induced contractions in aorta (Fig.
4). Precontracted myometrial tissues from
pregnant rats, however, were insensitive to relaxation by ANP (Fig. 4).
Thus myometrium from pregnant rats was not sensitive to activators of
either particulate or soluble guanylate cyclases. Insensitivity to
agents that synthesize cGMP may be due to increased cellular levels of
cGMP phosphodiesterase activity. To test this hypothesis, we treated
myometrial and aortic smooth muscles with a selective inhibitor of cGMP
phosphodiesterase (M2056, 10 µM). Inhibition of cGMP
phosphodiesterase resulted in 47 ± 10% relaxation of
aortic smooth muscle within 10 min
(n = 3, Fig.
5A). The
same concentration of M2056 had no effect on oxytocin-induced
myometrial contractions (Fig. 5B).
In addition, preincubation of myometrial tissues with M2056 for up to
20 min did not alter the relaxation response to SNAP
(10
6 M).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of atrial natriuretic peptide (ANP) on contractions in aortic
and uterine smooth muscle from pregnant rats. Representative tracings
of ANP-induced relaxation of precontracted aortic
(A) and myometrial
(B) tissues from pregnant rats
(day 17). Arrows indicate the
addition of ANP (0.1 µM). Tracings are representative of 4 separate
experiments with similar results.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of M2056 on force of contraction in smooth muscle obtained from
the aorta and myometrium of pregnant rats. Aortic
(A) and myometrial
(B) tissues were precontracted with
phenylephrine (0.1 µM) and oxytocin (1 nM), respectively. Thereafter,
tissues were treated with a guanosine 3',5'-cyclic
monophosphate (cGMP) phosphodiesterase inhibitor (M2056, 10 µM).
|
|
cGMP production in myometrial and aortic tissues.
Production of cGMP may be impaired in myometrial tissues, thereby
providing an explanation for the apparent refractoriness of this tissue
to activators of guanylate cyclases. To ensure that cellular levels of
cGMP were increased in myometrial tissues by NO donors and cGMP
phosphodiesterase inhibitors, myometrial and aortic tissues were
treated with SNAP (1 µM), ANP (0.1 µM), M2056 (10 µM), or SNAP + M2056 for 5 min and homogenized, and the tissue content of cGMP was
determined by radioimmunoassay. In both aortic and myometrial tissues,
levels of cGMP increased significantly after treatment with SNAP,
M2056, or SNAP + M2056 (Table 1). In
contrast, ANP resulted in increased tissue levels of cGMP in aortic but
not myometrial tissues from pregnant animals (P = 0.06).
Effect of cGMP analogs on smooth muscle contractility.
We utilized two poorly hydrolyzed analogs of cGMP to assess the
sensitivity of vascular and myometrial smooth muscles to cGMP, thereby
circumventing any requirement for tissue guanylate cyclases and
avoiding the metabolism of cGMP by tissue phosphodiesterases. We
quantified the effects of 8-bromoguanosine 3',5'-cyclic
monophosphate (8-BrcGMP) on oxytocin-induced contractions in myometrium
and aorta (Fig. 6). Precontracted aortic
(Fig. 6A) and myometrial (Fig.
6B) smooth muscle strips were
treated with increasing concentrations of 8-BrcGMP
(10
6 to
10
4 M), and the decrease in
force generation was quantified over time. Whereas 8-BrcGMP
(10
4 M) resulted in
significant inhibition of phenylephrine-induced contractions in aortic
smooth muscle (86 ± 2.7%, n = 5),
precontracted myometrial tissues from pregnant rats responded to
8-BrcGMP (10
4 M) with only
small decreases in both amplitude (17 ± 6%) and frequency (7 ± 6%) of contraction. Similar results were obtained with 8-CPT-cGMP, an
analog of cGMP that is more resistant to cGMP hydrolysis than 8-BrcGMP
(10). Myometrial tissues from nonpregnant rats in estrus, however, were
relaxed with 8-BrcGMP, thereby demonstrating a sensitivity similar to
aortic smooth muscle (Fig. 7). Although myometrial tissues from nonpregnant rats in estrus were relaxed by
10
5 M 8-BrcGMP (Fig.
7A), myometrial tissues from rats in
diestrus or from rats treated with progesterone were like those of
pregnant rats and insensitive to 8-BrcGMP (Fig.
7B). Even high concentrations of
8-BrcGMP (10
4 M) were not
effective in relaxing progesterone-dominated myometrium.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of 8-bromoguanosine 3',5'-cyclic monophosphate
(8-BrcGMP) on force of contraction in aortic and myometrial smooth
muscle. A and
B: representative tracings of the
effect of 8-BrcGMP on contractions in aortic
(A) and myometrial
(B) tissues from pregnant rats
(day 17). 8-BrcGMP was more
effective in relaxing contractions of aortic smooth muscle.
C: concentration-response curves to
8-BrcGMP in aortic ( ) and in myometrial tissues from pregnant rats
( ) and from nonpregnant rats in estrus ( ). Myometrium from
pregnant rats was less responsive to the effects of 8-BrcGMP than aorta
or myometrium from nonpregnant animals in estrus. Data represent
inhibition of the area of isometric force developed during 10 min as
described in MATERIALS AND METHODS.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of 8-BrcGMP on spontaneous contractions in myometrial tissues
from nonpregnant rats. Representative tracings of spontaneous
contractions in myometrial tissues from nonpregnant, cycling rats in
estrus (A) and nonpregnant rats
treated with progesterone (B).
Downward arrow reflects change of bathing medium with physiological
saline solution. Upward arrows indicate addition of the indicated
concentrations of 8-BrcGMP.
|
|
Levels of PKG in aortic and myometrial smooth muscles.
We next examined the expression of the major receptor protein for cGMP,
PKG, in myometrial and aortic tissues. Because myometrial smooth muscle
from pregnant rats was relatively insensitive to relaxation by cGMP, we
analyzed myometrial extracts from nonpregnant, pregnant, and postpartum
rats for PKG by Western blot analysis (Fig.
8). Utilizing a polyclonal antibody to PKG,
we found a single immunoreactive species of 78 kDa in all tissues
examined. Myometrial extracts prepared from nonpregnant cycling rats
contained the highest level of PKG, whereas enzyme levels were
significantly reduced in extracts prepared from term myometrium (Fig.
8). Compared with nonpregnant myometrium, levels of PKG were three- to
fivefold lower in myometrium obtained from pregnant animals at all
gestational days tested (days 12,
15,
17,
18, and
20). Levels of PKG increased in the
postpartum period and by the third postpartum day were similar to
levels observed in nonpregnant animals (Fig. 8). PKG levels were
similar in uterine horns from unilateral pregnant rats, indicating that
regulation of PKG expression may be due to the hormonal milieu rather
than mechanical factors induced by fetal occupancy (data not shown).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 8.
Levels of cGMP-dependent protein kinase (PKG) in nonpregnant, pregnant,
and postpartum rat myometrium. Relative amounts of PKG were determined
by Western blot analysis of extracts prepared from myometrium from
nonpregnant, pregnant, and postpartum rats (10 µg/lane).
Lane 1, nonpregnant rat in estrus;
lane 2, pregnant animal at term
(day 20); lane
3, postpartum day 1;
lane 4, postpartum
day 2; lane
5, postpartum day 3.
Molecular mass markers are indicated to
left.
|
|
Next, we compared the levels of PKG in aorta and myometrium from
nonpregnant animals during defined phases of the estrus cycle (Fig.
9). Levels of PKG were greatest in aortic
tissues. Myometrial extracts prepared from nonpregnant animals in
proestrus or estrus contained relatively lower levels of the enzyme,
yet these levels were greater than those found during late pregnancy
(35% of aorta). Myometrial tissues from animals in diestrus (high
progesterone-to-estrogen ratio) contained lower levels of PKG than
myometrium from animals in estrus, and progesterone treatment of
nonpregnant animals resulted in further decreases in enzyme
immunoreactivity (30% of aorta). These results suggested that PKG was
regulated by ovarian hormones. To test this hypothesis, we determined
the relative amounts of PKG in aorta or myometrium from pregnant rats
and in myometrium from ovariectomized rats treated with vehicle,
estradiol, progesterone, or estradiol and progesterone (Fig.
10). The average densities of the
immunoreactive bands from Western blots utilizing extracts from four
groups of animals are also presented in Fig. 10. The same aortic
extract was included in all blots to serve as an internal control.
Estradiol administration resulted in marked increases in PKG expression
in myometrial tissues to levels similar to those of aortic tissues.
Although progesterone treatment did not affect the low baseline levels
of PKG in myometrium of ovariectomized animals, progesterone
significantly inhibited the estradiol-induced increase in myometrial
PKG expression. The levels of PKG in myometrium from pregnant animals
were similar to those in myometrium from ovariectomized animals or
animals treated with progesterone.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 9.
Immunoreactive PKG in aortic and myometrial homogenates. Western blot
analysis of tissue proteins (25 µg/lane) from aorta (AO), myometrium
from animals in proestrus (PRO), estrus (E), and diestrus (DI), and
myometrium from a progesterone-treated animal (P) and during late
pregnancy (day 20, Preg). Blot is
representative of data obtained from 3 sets of animals. Molecular mass
markers are indicated to left.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 10.
Regulation of PKG levels by ovarian hormones. Relative amounts of PKG
were determined by Western blot analysis of extracts prepared from
aorta or myometrium from pregnant (PREG) or ovariectomized rats treated
with vehicle (CTL), estradiol (E2), progesterone (P), or estradiol + progesterone (E2+P). PKG bands from 4 blots (4 groups of animals) were
quantified by densitometric scanning. Arbitrary units from densitometry
were expressed as percent, with the level in aorta considered 100%.
Inset: representative immunoblot from
one set of animals (lane 1, aorta;
lane 2, CTL; lane
3, E2; lane 4, P;
lane 5, E2+P; lane
6, PREG).
|
|
Activity of PKG in myometrial extracts from nonpregnant and pregnant
rats.
PKG assays were conducted to determine whether the decrease in
immunoreactive PKG in myometrium from pregnant animals correlated with
a decrease in the levels of active enzyme. Rat myometrial extracts were
prepared and assayed for cGMP-stimulated histone kinase activity.
Myometrial extracts prepared from nonpregnant rats
(n = 3) contained high levels of
histone kinase activity, both in the absence or presence of cGMP
(without cGMP, 225 ± 45 pmol · min
1 · mg
1;
with cGMP, 827 ± 92 pmol · min
1 · mg
1).
cGMP-induced PKG activities in extracts from pregnant rats (n = 3, days
12-20) were significantly lower (without cGMP,
62 ± 14 pmol · min
1 · mg
1;
with cGMP, 137 ± 66 pmol · min
1 · mg
1,
P < 0.05). The meaning of the
decreased basal histone kinase activity is not known because many
cellular kinases are capable of phosphorylating histone F2b. Decreased
cGMP-induced activity in myometrium from pregnant rats, however,
indicates decreased PKG enzymatic activity. Thus the decreased
enzymatic activity in pregnant myometrium parallels the decrease in PKG
immunoreactivity by immunoblot analysis.
 |
DISCUSSION |
It is perhaps ironic that some of the earliest investigations with cGMP
were conducted with uterine smooth muscle; however, more than 20 years
later, we have a better understanding of the role of cGMP in vascular
smooth muscle than in myometrial smooth muscle. In the vasculature,
endothelial NO or atrial peptides signal smooth muscle relaxation by
intracellular increases in cGMP and activation of PKG (5, 15). It is
feasible that NO, produced from nerve plexi, decidua (18, 24), or other
resident cells of the myometrium (e.g., macrophages) could diffuse to
myometrial smooth muscle cells, activate soluble guanylate cyclase, and
increase cellular levels of cGMP. Thus NO-induced cGMP elevation may
effect uterine relaxation in a manner analogous to NO action in the
vasculature. It has been suggested that activation of guanylate cyclase
by NO may effect uterine quiescence during pregnancy (18, 24, 31).
However, other roles of NO or cGMP are clearly possible.
The notion that uterine smooth muscle responses to cGMP are modified by
the hormonal milieu has been explored by several laboratories. Bek et
al. (3) initially reported that ANP induced relaxation of rat
myometrium after estrogen treatment. Subsequently, Potvin and Varma
(22) reported that, during pregnancy, the rat uterus was refractory to
relaxation by ANP. The insensitivity to ANP was associated with a
progesterone-mediated decrease in the synthesis of cGMP by ANP (21)
perhaps due to a decrease in ANP receptors (23). We also found that ANP
does not significantly increase cGMP levels in myometrium from pregnant
rats. Stimulation of the soluble guanylate cyclase, however, resulted
in significant increases in tissue levels of cGMP in both myometrium
and aorta. However, myometrial tissues failed to relax in response to
these elevated levels of cGMP. Rapid degradation of cGMP is not the
sole cause of this uterine insensitivity because inhibition of cGMP
phosphodiesterase activity resulted in relaxation of aortic, but not
uterine, smooth muscle from pregnant rats. Furthermore, bypassing
production of cGMP using cell-permeant, phosphodiesterase-resistant
analogs of cGMP (8-BrcGMP and 8-CPT-cGMP) failed to induce uterine
relaxation. These findings suggest that, although decreased synthesis
of cGMP by ANP may contribute to ANP insensitivity in myometrial
tissues during pregnancy, additional mechanisms must be involved to
explain pregnancy-related insensitivity to intracellular cGMP.
In this investigation, we found that myometrium from estrogen-primed
normal cycling rats was more sensitive to 8-BrcGMP than myometrium from
pregnant animals throughout late gestation (days 17-21). These findings are in agreement with
those of Izumi et al. (14) who also reported that high concentrations
(>100 µM) of 8-BrcGMP were required to relax myometrium from
pregnant, laboring animals. We did not observe the reported relaxation
of uterine smooth muscle from pregnant animals (days
16 and 18) by
low-nanomolar concentrations of 8-BrcGMP (13, 32). The effective
concentrations of 8-BrcGMP known to relax smooth muscle are generally
1-100 µM (10). 8-BrcGMP in the low-nanomolar range is probably
insufficient to bind to PKG (10), cGMP phosphodiesterases (25), or any cGMP-binding channel characterized thus far (9).
The effect of SNP on contraction of myometrium from pregnant animals
has also been evaluated by several investigators (13, 22, 31). Although
Potvin and Varma (22) reported no suppression of contractile activity
in response to SNP, Izumi et al. (14) reported that SNP eliminated
spontaneous contractility but did not relax KCl-induced contractions.
In another report, SNP resulted in complete cessation of spontaneous
uterine contractions in pregnant rats (31). In each instance, however,
very high concentrations of SNP (
1 mM) were required to produce an
effect. In comparison, aortic smooth muscle strips are maximally
relaxed with concentrations 10,000-fold less than that required for
myometrium (i.e., 0.1 µM). The effects of SNP in uterine tissue may
be due to nonspecific or even toxic effects of the drug. In the current
study, we found that SNP-induced "relaxation" was irreversible
and spontaneous activity did not resume after removal of the drug from
the bathing solution. Thus responses to high concentrations of SNP
(>1 mM) are not likely to be mediated by cGMP.
The essential role of PKG in mediating smooth muscle relaxation has
been established for vascular and bronchiolar smooth muscle (5, 10,
15). In the current study, the gestational insensitivity of uterine
smooth muscle to cGMP was also correlated with decreased levels of
immunoreactive PKG and lower levels of PKG enzyme activity compared
with tissues that relaxed in response to increases in cGMP. Thus the
sensitivity of smooth muscle to cGMP (aorta > nonpregnant myometrium > pregnant myometrium) parallels the levels of immunoreactive PKG and
PKG enzyme activity. This is the first report of specific hormonal
regulation of PKG expression in cells.
Very little is known about the mechanisms that regulate expression of
PKG. The finding that myometrial PKG levels vary during pregnancy, the
postpartum period, and the estrus cycle suggests that enzyme expression
is regulated by ovarian hormones. Our studies in ovariectomized animals
indicate that estradiol upregulates myometrial PKG and that this
estradiol-induced increase in PKG expression is suppressed by
progesterone. The low levels of estrogen and progesterone in the
immediate postpartum period in the rat are consistent with low levels
of PKG expression in postpartum animals (days
1 and 2) with
restoration of PKG levels by postpartum day
3. The finding that the low levels of PKG in myometrium
from ovariectomized animals were not downregulated further by
progesterone is not surprising because progesterone receptor expression
is regulated by estradiol. Estradiol has also been reported to increase the levels of cGMP in uterine tissues (27). Increased levels of cGMP in
conjunction with elevated levels of PKG would ensure increased PKG
activity in estrogen-primed uterine tissues.
The physiological relevance of this regulatory pathway is unknown. PKG
may mediate thus far uncharacterized physiological responses in uterine
smooth muscle. For example, it is possible that the massive uterine
smooth muscle cell hypertrophy that accompanies pregnancy may involve
downregulation of cGMP action. Antiproliferative properties have been
assigned to cGMP based on studies in mesangial (11) and arterial (6)
smooth muscle cells. Alternatively, cGMP, perhaps through PKG, may also
play a role in remodeling of the extracellular matrix, as it does in
other tissues (19, 26).
In addition to PKG, there are other proteins in reproductive tissues
that are regulated in opposing directions by estrogen and progesterone.
For example, oxytocin receptors, the major gap junction protein,
connexin 43, and interstitial collagenase are upregulated by estradiol
and downregulated by progesterone (28). In most species at term,
progesterone withdrawal, together with increasing levels of estrogen,
leads to increased expression of oxytocin receptors, gap junctions, and
cervical collagenase, thereby providing effective contractions of labor
and cervical ripening. It has also been suggested that the contractile
phenotype of uterine smooth muscle is increased by estradiol treatment
and decreased by progesterone (2). The concept that estrogen promotes a
"contractile" state whereas progesterone gives rise to
"quiescence" suggests that the tissue functions as one or the
other of these physiological states, depending on the hormonal milieu.
It implies that procontractile mechanisms are expressed and functioning
under estrogen domination and prorelaxant mechanisms are acting under
the influence of progesterone. We speculate that estradiol, through PKG
expression, may serve to increase proteins of the contractile phenotype
in uterine smooth muscle. A similar role for PKG has been described in
vascular smooth muscle (4). Progesterone, through suppression of PKG expression, may decrease the contractile phenotype of the cell, whereas
estrogen facilitates smooth muscle function, i.e., force generation and
relaxation.
Previously, we reported that, compared with smooth muscle from bovine
trachealis, myometrial tissues from nonpregnant women were relatively
insensitive to relaxation by cGMP (29). In the current investigation,
we expanded these studies to demonstrate that, during pregnancy,
myometrium from pregnant rats is even less sensitive to relaxation by
cGMP than is myometrium from nonpregnant animals. The lack of
sensitivity to relaxation is correlated with decreased expression of
PKG in myometrial tissues. These results are consistent with those
reported in pregnant human uterine smooth muscle where neither
L-arginine nor inhibitors of NO
synthase result in any alterations of contractility (14a). These in
vitro studies do not support a role for cGMP in the maintenance of
uterine quiescence during pregnancy and are consistent with results
obtained in vivo. In vivo, NO synthase inhibitors do not affect uterine contractility, and neither NO synthase inhibitors nor NO donors alter
the timing of parturition (1, 8, 17). Together, these data provide
evidence that the NO-cGMP-signaling pathway is reduced and unavailable
to mediate uterine quiescence during pregnancy. Downregulation of PKG
may result in phenotypic alterations of myometrial cells that
facilitate other adaptations of uterine smooth muscle during pregnancy
such as cellular hypertrophy and remodeling of the extracellular
matrix.
 |
ACKNOWLEDGEMENTS |
We acknowledge Elizabeth Arnold, Martha Hayes, Dr. Jie Li, and Sharon
Cook for expert technical assistance and Drs. Jim Stull and Tom Lincoln
for thoughtful discussions. We also thank Drs. Diana Tucker and Ayalla
Barnea for providing rat tissues during the early phase of this study.
 |
FOOTNOTES |
This investigation was supported by National Institute of Child Health
and Human Development Grants HD-26164 and HD-11149 (R. A. Word) and
HD-32622 (T. L. Cornwell).
Address for reprint requests: R. A. Word, Dept. of Obstetrics and
Gynecology, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9032.
Received 2 May 1997; accepted in final form 5 December 1997.
 |
REFERENCES |
1.
Ahokas, R. A.,
B. M. Mercer,
and
B. M. Sibai.
Enhanced endothelium-derived relaxing factor activity in pregnant, spontaneously hypertensive rats.
Am. J. Obstet. Gynecol.
165:
801-807,
1991[Medline].
2.
Batra, S.
Hormonal control of myometrial function.
In: The Uterus, edited by T. Chard,
and J. G. Grudzinskas. Melbourne, Australia: Cambridge University Press, 1994, p. 173-192.
3.
Bek, T.,
B. Ottesen,
and
J. Fahrenkrug.
The effect of galanin, CGRP and ANP on spontaneous smooth muscle activity of rat uterus.
Peptides
9:
497-500,
1988[Medline].
4.
Boerth, N. J.,
N. B. Dey,
T. L. Cornwell,
and
T. M. Lincoln.
Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype.
J. Vasc. Res.
34:
245-259,
1997[Medline].
5.
Cornwell, T. L.,
and
T. M. Lincoln.
Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells. Reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase.
J. Biol. Chem.
264:
1146-1155,
1989[Abstract/Free Full Text].
6.
Cornwell, T. L,
G. A. Soff,
A. E. Traynor,
and
T. M. Lincoln.
Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells.
J. Vasc. Res.
31:
330-337,
1994[Medline].
7.
Diamond, J.
Lack of correlation between cyclic GMP elevation and relaxation of nonvascular smooth muscle by nitroglycerin, nitroprusside, hydroxylamine and sodium azide.
J. Pharmacol. Exp. Ther.
225:
422-426,
1983[Abstract].
8.
Diket, A. L.,
M. R. Peirce,
U. K. Munshi,
C. A. Voelker,
S. Eloby-Childress,
S. S. Greenberg,
X. J. Zhang,
D. A. Clark,
and
M. J. S. Miller.
Nitric oxide inhibition causes intrauterine growth retardation and hind-limb disruptions in rats.
Am. J. Obstet. Gynecol.
171:
1243-1250,
1994[Medline].
9.
Finn, J. T.,
M. E. Grunwald,
and
K. W. Yau.
Cyclic nucleotide-gated ion channels: an extended family with diverse functions.
Annu. Rev. Physiol.
58:
395-426,
1996[Medline].
10.
Francis, S. H.,
B. D. Noblett,
B. W. Todd,
J. N. Wells,
and
J. D. Corbin.
Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase.
Mol. Pharmacol.
34:
505-517,
1988.
11.
Garg, U. C.,
and
A. Hassid.
Inhibition of rat mesangial cell mitogenesis by nitric oxide-generating vasodilators.
Am. J. Physiol.
257 (Renal Fluid Electrolyte Physiol. 26):
F60-F66,
1989[Abstract/Free Full Text].
12.
Harper, J. F.,
and
G. Brooker.
Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution.
J. Cyclic Nucleotide Res.
1:
207-218,
1975[Medline].
13.
Izumi, H.,
and
R. E. Garfield.
Relaxant effects of nitric oxide and cyclic GMP on pregnant rat uterine longitudinal smooth muscle.
Eur. J. Obstet. Gynecol. Reprod. Biol.
60:
171-180,
1995[Medline].
14.
Izumi, H.,
C. Yallampalli,
and
R. E. Garfield.
Gestational changes in L-arginine-induced relaxation of pregnant rat and human myometrial smooth muscle.
Am. J. Obstet. Gynecol.
169:
1327-1337,
1993[Medline].
14a.
Jones, G. D.,
and
L. Poston.
The role of endogenous nitric oxide synthesis in contractility of term or preterm human myometrium.
Br. J. Obstet. Gynaecol.
104:
241-245,
1997[Medline].
15.
Lincoln, T. M.
Cyclic GMP and mechanisms of vasodilation.
Pharmacol. Ther.
41:
479-502,
1989[Medline].
16.
Lincoln, T. M.,
C. L. Hall,
C. R. Park,
and
J. D. Corbin.
Guanosine 3':5'-cyclic monophosphate binding proteins in rat tissues.
Proc. Natl. Acad. Sci. USA
73:
2559-2563,
1976[Abstract].
17.
Molnar, M.,
and
F. Hertelendy.
N
-nitro-L-arginine, an inhibitor of nitric oxide synthesis, increases blood pressure in rats and reverses the pregnancy-induced refractoriness to vasopressor agents.
Am. J. Obstet. Gynecol.
166:
1560-1567,
1992[Medline].
18.
Natuzzi, E. S.,
P. C. Ursell,
M. Harrison,
C. Busher,
and
R. K. Reimer.
Nitric oxide synthase activity in the pregnant uterus decreases at parturition.
Biochem. Biophys. Res. Commun.
194:
1-8,
1993[Medline].
19.
Perr, H. A.,
M. F. Graham,
R. F. Diegelmann,
and
R. W. Downs.
Cyclic nucleotides regulate collagen production by human intestinal smooth muscle cells.
Gastroenterology
96:
1521-1528,
1989[Medline].
21.
Potvin, W.,
S. Mulay,
and
D. R. Varma.
Inhibition of the tocolytic activity of atrial natriuretic factor by progesterone and potentiation by progesterone receptor antagonist RU486 in rats.
Br. J. Pharmacol.
104:
379-84,
1991[Abstract].
22.
Potvin, W.,
and
D. R. Varma.
Refractoriness of the gravid rat uterus to tocolytic and biochemical effects of atrial natriuretic peptide.
Br. J. Pharmacol.
100:
341-347,
1990[Abstract].
23.
Potvin, W.,
and
D. R. Varma.
Down-regulation of myometrial atrial natriuretic factor receptors by progesterone and pregnancy and up-regulation by oestrogen in rats.
J. Endocrinol.
131:
259-266,
1991[Abstract].
24.
Sladek, S. M.,
A. C. Regenstein,
D. Lykins,
and
J. M. Roberts.
Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy.
Am. J. Obstet. Gynecol.
169:
1285-1291,
1993[Medline].
25.
Turko, I. V.,
T. L. Haik,
L. M. McAllister-Lucas,
F. Burns,
S. H. Francis,
and
J. D. Corbin.
Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding phosphodiesterases. A putative NKXnD motif for cGMP binding.
J. Biol. Chem.
271:
22240-22244,
1996[Abstract/Free Full Text].
26.
Vargas, S. J.,
S. N. Holden,
P. M. Fall,
and
L. G. Raisz.
Effects of atrial natriuretic factor on cyclic nucleotides, bone resorption, collagen and deoxyribonucleic acid synthesis, and prostaglandin E2 production in fetal rat bone cultures.
Endocrinology
125:
2527-2531,
1989[Abstract].
27.
Vesely, D. L.,
and
D. E. Hill.
Estrogens and progesterone increase fetal and maternal guanylate cyclase activity.
Endocrinology
107:
2104-2109,
1980[Abstract].
28.
Word, R. A. Parturition. In:
Textbook of Reproductive Medicine,
edited by B. R. Carr and R. E. Blackwell. Norwalk,
CT: Appleton and Lange. In press.
29.
Word, R. A.,
M. L. Casey,
K. E. Kamm,
and
J. T. Stull.
Effects of cGMP on [Ca2+]i, myosin light chain phosphorylation, and contraction in human myometrium.
Am. J. Physiol.
260 (Cell Physiol. 29):
C861-C867,
1991[Abstract/Free Full Text].
30.
Word, R. A.,
K. E. Kamm,
and
M. L. Casey.
Contractile effects of prostaglandins, oxytocin, and endothelin-1 in human myometrium in vitro: refractoriness of myometrial tissue of pregnant women to prostaglandins E2 and F2
.
J. Clin. Endocrinol. Metab.
75:
1027-1032,
1992[Abstract].
31.
Yallampalli, C.,
R. E. Garfield,
and
M. Byam-Smith.
Nitric oxide inhibits uterine contractility during pregnancy but not during delivery.
Endocrinology
133:
1899-1902,
1993[Abstract].
32.
Yallampalli, C.,
H. Izumi,
M. Byam-Smith,
and
R. E. Garfield.
An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy.
Am. J. Obstet. Gynecol.
170:
175-185,
1994[Medline].
AJP Cell Physiol 274(3):C748-C756
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society