BIOLOGY OF REPRODUCTION 61, 1023–1030 (1999)
Regulation of Calcitonin Gene-Related Peptide Receptors in the Rat Uterus During
Pregnancy and Labor and by Progesterone 1
C. Yallampalli,2,3 P.R.R. Gangula,3 S. Kondapaka,3 Li Fang,3 and S. Wimalawansa4
Departments of Obstetrics and Gynecology3 and Internal Medicine,4 The University of Texas Medical Branch,
Galveston, Texas 77555
ABSTRACT
Calcitonin gene-related peptide (CGRP) is a potent smooth
muscle relaxant in a variety of tissues. We recently demonstrated
that CGRP relaxes uterine tissue during pregnancy but not during labor. In the present study we examined whether uterine 125ICGRP binding and immunoreactive CGRP receptors are regulated by pregnancy and labor and by sex steroid hormones. We
found that 125I-CGRP binding to membrane preparations from
uteri was elevated during pregnancy and decreased during labor
and postpartum. Changes in immunoreactive CGRP receptors
were similar to the changes in 125I-CGRP binding in these tissues,
suggesting pregnancy-dependent regulation of CGRP receptor
protein. CGRP receptors were elevated by Day 7 of gestation,
and a precipitous decrease in these receptors occurred on Day
22 of gestation prior to the onset of labor. Both 125I-CGRP-binding and immunofluorescence studies indicated that CGRP receptors were localized to myometrial cells.
Hormonal control of uterine CGRP receptors was assessed
by the use of antiprogesterone RU-486, progesterone, and estradiol-17b. RU-486 induced a decrease in uterine CGRP receptors during pregnancy (Day 19). On the other hand, progesterone prevented the fall in uterine CGRP receptors at term (Day
22). In addition, progesterone also increased uterine CGRP receptors in nonpregnant, ovariectomized rats, while estradiol had
no effects. These hormone-induced changes in uterine CGRP receptors were demonstrated by 125I-CGRP-binding, Western immunoblotting, and immunolocalization methods.
These results indicate that CGRP receptors and CGRP binding in the rat uterus are increased with pregnancy and decreased
at term. These receptors are localized to the myometrial cells,
and progesterone is required for maintaining CGRP receptors in
the rat uterus. Thus, the inhibitory effects of CGRP on uterine
contractility are mediated through the changes in CGRP receptors and may play a role in uterine quiescence during pregnancy.
quiescence until parturition. Several recent studies, including ours [5], provide evidence that nitric oxide (NO) may
be one of the potent smooth muscle-relaxing factors that
may play a role in maintaining uterine quiescence during
pregnancy.
Calcitonin gene-related peptide (CGRP), a neuropeptide,
is the most potent endogenous vasodilator peptide known
[6, 7]. CGRP has been reported to inhibit smooth muscle
contractility in a variety of tissues, including uterus and
fallopian tubes of women [8]. In addition, CGRP-containing nerve fibers are present in the uterus, and therefore
CGRP may play a role in inhibiting uterine activity during
pregnancy [9]. We recently reported that CGRP inhibits
uterine contractility during pregnancy in the rat and that
this effect was dramatically decreased at term during labor
[10]. Furthermore, the changes in relaxation sensitivity to
CGRP of the rat uterus during pregnancy and labor were
associated with similar changes in uterine CGRP-binding
capacity [10]. However, from these studies it is unclear
whether the changes in the CGRP binding in the uterus are
regulated in a pregnancy-dependent manner and whether
female sex steroid hormones are involved in these changes.
In addition, it is unknown whether the changes in CGRP
binding to the uterus are due to changes in the number of
receptors and the involvement of specific cell types in the
uterus. Therefore, studies were designed to 1) measure
changes in CGRP binding to the uterine tissue during gestation and parturition in the rat, 2) determine whether steroid hormones regulate CGRP receptors in the rat uterus,
3) ascertain whether the changes in CGRP binding are due
to changes in CGRP receptor protein, and 4) identify the
uterine cells expressing CGRP receptor protein.
MATERIALS AND METHODS
INTRODUCTION
Animals and Treatments
Pregnancy is associated with significant changes in the
function and structure of the uterus. A quiescent state of
the uterus is essential for successful completion of pregnancy. The relatively quiescent uterine activity is maintained until term, and strong and well-synchronized contractions occur during parturition at term for expulsion of
the fetus. This increased contractile activity is associated
with corresponding increases in oxytocin receptors [1], gap
junction channels [2], calcium channels [3], and endothelin
receptors [4], which have been shown to be involved in
contractile activity regulation. In contrast, little emphasis
has been placed on the factors that might control uterine
Adult nonpregnant (200–210 g BW) and timed-pregnant
Sprague-Dawley rats were purchased from Harlan SpragueDawley (Houston, TX) and maintained on a 12L:12D
schedule. Animals received an ad libitum supply of rat
chow and water. Virgin female rats were mated, and the
day of observation of a vaginal plug with the presence of
sperm was designated Day 1 of gestation. The time of delivery under these circumstances was on Day 22. Timemated rats were killed on Days 1, 5, 6, 7, 8, 18, and 22
prior to and during labor. In addition, one group of nonpregnant and day-after postpartum animals were killed. A
group of pregnant rats on Day 18 were also injected s.c.
with a single dose of an antagonist of progesterone, RU486 (Biomol, Plymouth Meeting, PA; 10 mg/rat) and killed
at 24 h after injection. Finally, progesterone (Sigma Chemical Co., St. Louis, MO; 2 mg/rat, twice a day) was injected
s.c. from Day 20 to Day 22, and animals were killed on
Day 22. Vehicle for steroid treatments (sesame oil) was
injected (0.2 ml) to control animals.
Accepted May 24, 1999.
Received February 1, 1999.
1
Financial support was provided by NIH through grants HD 30273
and HL 58144.
2
Correspondence: Chandra Yallampalli, University of Texas Medical
Branch, 301 University Boulevard, Route 1062, Galveston, TX 77555–
1062. FAX: 409 747 0475; e-mail: [email protected]
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YALLAMPALLI ET AL.
In another set of experiments, virgin female adult rats
were bilaterally ovariectomized (ovx) under general anesthesia using ketamine (45 mg/kg BW; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg BW; Burns
Veterinary Supply, New York, NY). Seven days later (72 h
prior to necropsy), the ovx rats were exposed to one of four
different treatments: 1) estradiol-17b (E2; 2.5 mg/injection,
twice-daily s.c. injections); 2) progesterone (P4; 2 mg/injection, twice-daily s.c. injections); 3) E21P4 (same doses
and frequency as above); and 4) sesame oil only (control).
The steroid hormone injections were prepared with sesame
oil as vehicle (0.2 ml) and injected s.c.
All animals were killed in a CO2 inhalation chamber;
the uteri were removed immediately and cleaned, and the
full-thickness uterus was fixed in Bouin’s solution for paraffin sections or was quickly frozen in liquid nitrogen and
stored at 2708C until used. In some animals, endometrial
tissue was scraped off, using a blunted scalpel [11], from
one uterine horn, and the myometrium was frozen. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.
CGRP Receptor-Binding Assay
The tissues were homogenized in 50 mM Tris(hydroxymethyl)aminomethane buffer (pH 7.4 containing
0.32 M sucrose, 1 mM dithiothreitol [DTT], 5 mM EDTA,
and aprotinin [200 KIU/L]), and the homogenate was centrifuged at 800 3 g for 10 min. The supernatant was recentrifuged at 30 000 3 g for another 30 min. Final membrane pellet was resuspended in fresh Tris(hydroxy
methyl)aminomethane buffer (no sucrose). Membrane
preparations (100–200 mg protein per tube) were incubated at 48C for 150 min with 8.3 3 10212 M 125I-human
CGRP (20 000 cpm/tube; Amersham, Arlington Heights,
IL; now Amersham Pharmacia Biotech, Piscataway, NJ)
with or without varying concentrations (32 3 10215 M to
13 3 1029 M) of unlabeled CGRP. Specific binding was
calculated from the total amount of labeled CGRP bound
minus the amount bound in the presence of 0.5 mM (e.g.,
0.5 mg/tube) unlabeled CGRP counted in a gamma counter. The data were analyzed with Scatchard’s method, and
the results are expressed as 125I-CGRP bound (in femtomoles per milligram) to the membrane protein.
Western Immunoblotting Analysis
For preparation of tissue extracts, 100 mg of uterine tissue was homogenized in 500 ml of lysis buffer (50 mM
Tris[hydroxymethyl]aminomethane-HCl, pH 7.5; 120 mM
NaCl; 0.4% Nonidet P-40; 100 mM NaF; 200 mM NaVO5;
1 mM PMSF; 10 mg/ml leupeptin; 10 mg/ml aprotinin). The
homogenates were incubated on ice for 20 min. After removal of cell debris by centrifugation (14 000 3 g, 30
min), protein was estimated (Pierce kit, Rockford, IL) in
the supernatant. Equal amounts of protein from each preparation were resolved on a 10% SDS-polyacrylamide gel,
transferred to nitrocellulose, probed with primary CGRP
receptor monoclonal antibody (raised against the affinitypurified CGRP receptors from porcine cerebellum, by Dr.
Sunil Wimalawansa [12–14]) for 1 h, washed three times
with TTBS (20 mM Tris[hydroxymethyl]aminomethaneHCl, pH 7.6, 0.05% Tween 20, 100 mM NaCl), and incubated with a secondary antibody coupled to horseradish
peroxidase. After three washes the membrane was developed using the enhanced chemiluminescence system (ECL,
Amersham). Densitometric analysis was performed in the
linear range using SigmaGel software (Sigma).
Immunofluorescent Localization of CGRP Receptors
Immunofluorescent staining procedures were based on a
previously reported [15] method with slight modifications. Rat
uteri were removed and fixed in Bouin’s fixative. After routine
tissue processing procedures of dehydration in ascending
grades of alcohol, clearing in xylene, and infiltration with paraffin, the tissues were embedded in paraffin. Sections (5 mm
thick) were rinsed with normal horse serum, and avidin-biotin
blocking buffer was applied to slides to reduce nonspecific
staining. The primary monoclonal antibody (raised by Dr.
Sunil Wimalawansa) for CGRP receptor in PBS was applied
at room temperature for 90 min. Slides were then incubated
with biotinylated horse anti-mouse IgG (Vector Labs., Burlingame, CA) for 45 min. The detection step was performed
using the fluorescein avidin-D (Vector) for 1 h. Then slides
were washed with PBS buffer, and propidium iodine (Boehringer-Mannheim, Indianapolis, IN) in PBS was applied as
counterstain to visualize the nuclei. Finally, slides were
mounted with Vectashield mounting medium (Vector) and
viewed under a Nikon (Tokyo, Japan) fluorescence microscope. As controls, the sections were incubated with mouse
IgG to substitute for the primary antibody.
Statistics
Results are expressed as mean 6 SEM, and data were
analyzed for statistical differences with Student’s t-test or
one-way ANOVA followed by Bonferroni’s t-test to verify
differences between individual groups. Differences were
considered significant if P , 0.05.
RESULTS
Changes in CGRP Receptors in the Rat Uterus During
Pregnancy and Parturition
I-CGRP peptide binding. Specific binding of 125I-human-a-CGRP to uterine membranes was measured in four
separate uteri each from rats during different stages of pregnancy, during labor, postpartum, and in the nonpregnant
state. Unlabeled human a-CGRP competed for 125I-CGRPbinding sites in a dose-dependent manner. A single class of
binding sites for CGRP was obtained from the uterus [10],
and the dissociation constants were similar among all stages
of pregnancy examined (1.6–2.5 nM). The binding capacity
(Bmax) of the uterus for CGRP (Fig. 1) was 351 6 50 fmol/
mg protein in nonpregnant animals in diestrous stage. These
binding sites in the uterus were increased significantly (ANOVA, P , 0.01) during mid-late pregnancy (Day 18) (685
6 82) compared to those in the nonpregnant state. However, the binding sites for CGRP decreased dramatically on
Day 22. This occurred even in animals that were not in
active labor (vaginal bleeding and delivery of pups), indicating that uterine CGRP-binding capacity decreases at
term regardless of stage of parturition. Furthermore, the reduced CGRP-binding capacity was maintained during the
postpartum period (391 6 68).
Western immunoblotting. To determine whether the changes in 125I-CGRP binding to uterine tissues during pregnancy
and labor are due to variations in the CGRP receptor protein
levels, we analyzed the tissues by Western immunoblotting.
As shown in Figure 2, a single band of CGRP receptor protein
was obtained with a predicted size of 60 kDa. Densitometric
analysis of the CGRP receptor protein from three separate
125
PREGNANCY AND STEROID HORMONES REGULATE UTERINE CGRP RECEPTORS
FIG. 1. CGRP receptors in uteri from nonpregnant rats (NP) and pregnant rats on Day 18 (D18), during nonlabor (D22) and labor at term
(D22), and one day postpartum (PP). Specific binding sites for CGRP in
rat uterus were identified with 125I-human CGRP assay. Results are expressed as 125I-human CGRP bound in femtamoles per milligram of membrane protein (n 5 4). Data were analyzed with Scatchard’s method, and
asterisk indicates that CGRP receptors in rat uteri on Day 18 were statistically different (P , 0.01, ANOVA) when compared with the other 4
groups. The bars represent mean 6 SEM.
FIG. 2. Western immunoblotting analysis of rat uterine homogenates for
CGRP receptors. Top) Representative immunoblot of rat uterus showing
changes in the expression of CGRP receptors in rat uterus from nonpregnant rat (NP), on Day 18 (D18), during spontaneous labor (labor), and
one day postpartum (PP1). Bottom) Densitometric analysis of 60-kDa
band in Western blots (see Materials and Methods for detailed protocol)
of rat uterus. The bars represent mean 6 SEM of triplicate determinations
from 3 separate animals. Groups with different letters at the top of bars
vary significantly (P , 0.05, ANOVA).
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FIG. 3. Expression of CGRP receptors in rat uterus at early stages of
pregnancy. Top) Representative Western analysis of uterus for CGRP receptors from 3 rats each on Day 1 (D01), Day 5 morning (D05a), Day 5
evening (D05p), Day 6 (D06), Day 7 (D07), and Day 8 (D08) of pregnancy. Bottom) Densitometric analysis of 60-kDa band in Western blots
of rat uterus. Bars represent the mean 6 SEM from 3 separate rats. Groups
with asterisks at the top of bars show a significant (P , 0.05, ANOVA)
increase in expression of CGRP receptor as compared to Day 1.
FIG. 4. Changes in CGRP receptor content in various compartments of
rat uterus obtained on Day 18 (D18) and during labor at term (D22).
Specific binding sites for CGRP from whole uteri (1 Endo) and myometrial
compartment (- Endo, following endometrium removal) were identified
with 125I-human CGRP bound in femtomoles per milligram of membrane
protein (n 5 4). Data were analyzed with Scatchard’s method, and values
are means 6 SEM of CGRP receptors in each group. * P , 0.05 compared
with whole uterus (1 Endo) from each stage of gestation (Student’s t-test).
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FIG. 5. Immunofluorescent localization for CGRP receptors in the rat uterus. Sections (3400) from a) nonpregnant animals during diestrus, b) on Day
18, c) during labor, and d) one-day postpartum were used. The green immunofluorescence indicates the presence of CGRP receptors on myometrial
cells. Arrows point to CGRP receptor-positive cells. E, epithelium; M, myometrium. Photomicrographs are representative of results obtained from a
minimum of 3 animals in each group. None of the sections examined showed positive immunostaining when primary antibody was omitted (-antibody)
and was replaced by nonspecific mouse IgG. Red immunofluorescence indicates the cellular nuclei.
PREGNANCY AND STEROID HORMONES REGULATE UTERINE CGRP RECEPTORS
FIG. 6. Effect of RU-486 and P4 on CGRP receptor content during pregnancy on Day 19 (D19) and during labor at term (D22) in rat uterine
membranes. Specific binding sites for CGRP in rat uterus were identified
by 125I-human CGRP assay. Data were analyzed with Scatchard’s method,
and values are mean 6 SEM of 3 separate animals per group. * P , 0.01
compared with control (-) in each group (Student’s t-test).
FIG. 7. Effect of P4 on CGRP receptor expression. Top) Uterine samples
from nonpregnant rats (NP), from pregnant rats on Day 19 (D19) treated
with or without RU-486, and from pregnant rats on Day 22 (D22) treated
with or without P4, were analyzed for the expression of CGRP receptor
protein. Bottom) The relative densitometric analysis of the specific band.
Groups with different letters at the top of the bars vary significantly among
(P , 0.01, ANOVA). The bars represent the mean 6 SEM of triplicate
determinations from 3 different animals.
1027
FIG. 8. Effect of E2 and P4, either alone or in combination, on CGRP
receptors in nonpregnant ovariectomized (ovx) rats. Nonpregnant ovx rats
were treated for 3 days s.c. with either vehicle (oil), E2 (2.5 mg/injection
twice daily), P4 (2 mg/injection twice daily), or the combination of E2 and
P4. Specific binding for CGRP in the rat uterus was measured as indicated
in Figures 1 and 4. Data are means 6 SEM of 3 separate animals. Groups
with asterisks at the top of the bars vary significantly from control (oil)
group (P , 0.05, ANOVA).
FIG. 9. Effects of steroid hormones on the expression of CGRP receptors
in rat uterus. Ovariectomized rat uterine samples were collected after 72h treatment with sesame oil (oil), E2, P4, or E21P4 and (top) analyzed for
the expression of CGRP receptors by Western immunoblotting. Bottom)
The relative densitometric analysis of the bands. Groups with asterisks at
the top of the bars vary significantly from control (oil) (P , 0.01, ANOVA).
The bars represent the mean 6 SEM of triplicate determinations from 3
different animals.
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YALLAMPALLI ET AL.
FIG. 10. Immunofluorescent localization for CGRP receptor in the ovariectomized rat uterus with steroid hormone treatments. Uterine sections (3400)
of ovariectomized rats treated with a) oil, b) E2, c) P4, or d) P41E2 were utilized. E, Epithelium; M, myometrium. Photomicrographs are representative
of results obtained from minimum of 3 animals in each group. None of the sections showed positive immunostaining when primary antibody was
omitted (-antibody) and was replaced by nonspecific mouse IgG. Red immunofluorescence indicates the cellular nuclei.
PREGNANCY AND STEROID HORMONES REGULATE UTERINE CGRP RECEPTORS
uteri from each group showed that this protein was increased
with pregnancy (Day 18) and decreased at term. These changes in protein level mirrored the changes in 125I-CGRP-binding
capacity. We further extended the Western blot analysis of the
CGRP receptor protein to earlier stages of gestation (periimplantation period). Figure 3 shows that the CGRP receptor
protein level in the uterus increased after implantation of the
embryos. The CGRP receptor protein levels were significantly
(P , 0.05) elevated on Days 7 and 8, while these receptor
levels remained unchanged from Day 1 to Day 6 (n 5 3 in
each group).
Localization of CGRP receptors in the uterus. To determine the uterine cell types that contain CGRP receptors,
we used two methods. With the first method, we removed
endometrium from one uterine horn of the uterus, measured
125I-CGRP-binding density of the myometrium, and compared the values with those for the full-thickness contralateral uterine horn from the same group of animals (n 5 4).
Figure 4 shows that the CGRP-binding concentration (fmol/
mg protein) increased with the removal of endometrium in
the uterus from rats during pregnancy as well as during
labor, indicating that the majority of CGRP binding was
located in the myometrial compartment.
To further confirm these observations we utilized immunofluorescent methods and localized the CGRP receptors in the uterus from three separate animals per group. A
representative picture shows that CGRP receptors were localized to the myometrial cells in the uterine sections from
pregnant rats (Fig. 5). Neither epithelial nor stromal cells
contained immunoreactivity to CGRP receptor antibody.
This monoclonal antibody has been shown to be highly
specific for CGRP receptors ([13–15]; Western blotting
data). Control sections without primary antibody showed
no significant staining in any of these cells in pregnant rat
uterus (Fig. 5). Furthermore, the intensity of staining to
myometrial cells was significantly higher in sections from
pregnant (Day 18, Fig. 5b) than from nonpregnant rats (Fig.
5a). Again, the staining intensity decreased with labor (Fig.
5c) and postpartum (Fig. 5d), supporting both the 125ICGRP-binding and Western blotting data presented.
Effects of P4 and RU-486 on Uterine CGRP Receptors
During Pregnancy
Since 125I-CGRP-binding, Western immunoblotting, and
immunofluorescent data all indicated that CGRP receptors
were elevated during pregnancy and decreased at term and
postpartum, we investigated whether these changes were
associated with changes in sex steroid hormone levels that
are known to occur during pregnancy and labor. Figure 6
shows that antiprogesterone, RU-486, inhibited 125I-CGRP
binding to the rat uterus when injected to pregnant rats on
Day 18 (n 5 3). More than 80% of the receptor binding
declined within 24 h after RU-486 injection. Conversely,
injections of P4 from Days 20 to 22 reversed the decline in
uterine 125I-CGRP binding that occurred at term during labor so that the binding capacity in the P4-treated animals
was similar to that during Day 19. Furthermore, the Western blotting of CGRP receptor protein in the uterus from
RU-486- or P4-treated animals (Fig. 7) confirmed the data
obtained with 125I-CGRP binding (Fig. 6) (i.e., antiprogesterone decreased while P4 increased CGRP receptor protein
in the rat uterus during pregnancy).
Steroid Hormone Regulation of Uterus CGRP Receptors
in Nonpregnant Rats
We investigated whether the steroid hormones—E2 and
P4—modulate uterine CGRP receptors in nonpregnant rats.
1029
Ovariectomized adult rats were treated with E2, P4, or
E21P4, and uterine tissues from 3 animals per group were
evaluated for 125I-CGRP binding, Western blotting, and immunolocalization of the receptors. Figure 8 shows that P4,
but not E2, increased 125I-CGRP binding to the uterus. The
binding density was further elevated with the E21P4 treatment. These changes in 125I-CGRP-binding capacity in the
uterus were further supported by the Western blotting data
presented in Figure 9 (i.e., P4 increased CGRP receptors in
the rat uterus). Finally, the CGRP receptor immunostaining
(Fig. 10) to the uterine myometrial cells was also significantly greater in animals treated with P4 (alone or in combination with E2) compared with other groups.
DISCUSSION
Our data demonstrate that an increase in CGRP receptors
in the rat uterus occurs early in pregnancy and that this
elevated level of receptors is maintained until term, when
the receptor level declines. The CGRP receptors are localized to myometrial cells, and the receptor density is upregulated by P4 but not by E2. These data extend our previous report [10] and provide a basis for the observed
changes in the CGRP-induced inhibition of uterine contractility in the rat (i.e., CGRP inhibits uterine activity during pregnancy, but not during labor or postpartum). These
data together with our previous findings [10] provide evidence that CGRP may play a role in the maintenance of
uterine quiescence during pregnancy in the rat and that the
decrease in P4 levels at term could reduce CGRP-induced
uterine relaxation.
Changes in CGRP receptor levels in uterine tissues (i.e.,
increase during pregnancy and the decline at term) parallel
the reported changes in CGRP-mediated inhibitory effects
on uterine contractility [10]. The decline in the CGRP-induced inhibitory effect on contractility at term coincides
with the up-regulation of stimulatory factors of contraction.
Factors influencing contraction of uterine tissues such as
oxytocin receptors, gap junction proteins, and potassium
channels are hormonally regulated. This study indicates that
inhibitory effects of CGRP and its receptor-mediated uterine activity are also well regulated during pregnancy and
by sex steroid hormones. P4, but not E2, increased the number of CGRP receptors, while the antiprogesterone decreased the number of CGRP receptors in the rat uterus.
Conversely, these hormones regulate contractile factors
such as oxytocin receptors and gap junctions in the opposite
direction (i.e., decreased by P4 and increased by E2). Therefore, we suggest that P4-induced increase in the CGRP receptors in the uterus may antagonize the effects of stimulatory factors prior to term. On the other hand, reduction
in uterine CGRP receptors secondary to the decline in P4
levels at term may allow full activation of myometrial contractility by the stimulatory factors.
Understanding the cellular and tissue distribution of
CGRP receptors provides important information regarding
the underlying mechanisms of both regulation of CGRP
receptors and their action within the uterus. Specific localization of CGRP receptors was associated with myometrial
muscle cells of the rat uterus but not with other cell types.
These results provide histologic evidence supporting a biological function for CGRP in the pregnant rat uterus. Here
we demonstrate for the first time the presence of CGRP
receptors histochemically in the rat uterus.
The present study is also the first to investigate the regulation of CGRP receptors in the uterus by E2 and P4. We
demonstrate that both 125I-CGRP binding and immunore-
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YALLAMPALLI ET AL.
active CGRP receptor protein in the uterus are dramatically
elevated by P4, but not by E2. Coadministration of E2 with
P4, however, further elevated the 125I-CGRP binding. These
results suggest that E2 could enhance the ability of P4 to
increase CGRP receptor binding through the induction of
P4 receptors in the uterus [16]. Therefore, it is likely that
uterine CGRP receptors are regulated primarily by P4. A
decline in P4 at term could lead to decreases in uterine
CGRP receptors. This concept is further supported by the
RU-486-induced decreases and P4-induced increases in
uterine CGRP receptors during pregnancy.
The CGRP receptor antibody used for both Western immunoblotting and immunofluorescence is a mouse monoclonal antibody raised against purified CGRP receptors
from cerebellum; it was characterized previously [12–14].
The antibody was previously utilized for ELISA, Western
blotting, and immunohistochemistry [13, 14]. In our study
the antibody reacted specifically with a single band of protein, with the predicted size of 60 kDa, from uterine homogenates. The observation that CGRP receptor protein is
increased during pregnancy and with P4 treatment is novel
and is of considerable significance. P4 may transcriptionally
regulate these receptors and thus the relaxation effects of
CGRP in the uterus, which therefore provides another
mechanism through which P4 may enhance uterine quiescence during pregnancy.
We have demonstrated previously that elevated P4 levels
during pregnancy increase uterine NO synthesis [17–19]
and NO synthase-II (NOS-II) enzyme levels in the rat uterus [15, 18, 20]. Both NOS-II levels and NO synthesis by
the uterus are dramatically decreased with antiprogesterone
[15, 18, 20]. In the present study, P4 administration markedly increased while antiprogesterone inhibited CGRP receptors in the rat uterus. Thus it appears that P4 enhances
the systems that favor uterine relaxation activity during
pregnancy. This important action of P4 may be mediated
via the CGRP system, in addition to the NO system.
The concept that CGRP receptor protein is an essential
component of CGRP binding and signaling mechanism is
further strengthened by the observed correlative changes in
the levels of immunoreactive protein in uterine homogenates, 125I-CGRP-binding capacity, and previously reported
[10] inhibitory effects of CGRP on myometrial contractility. These correlated changes occur not only during pregnancy and labor but also with P4 treatment in ovx rats,
indicating that the primary component for changes in
CGRP-induced effects is the number of CGRP receptors.
Recently, two membrane receptors (RDC1 and CRLR) have
been proposed as putative CGRP receptors [6, 21, 22]. Both
these receptors have been suggested to represent CGRP receptor type 1. The fact that the changes in both CGRP
protein (measured by Western blotting using antibody to
CGRP receptors) and binding (measured by 125I-CGRP
binding) were identical in the uterus suggest that in the rat
uterus these are type 1 CGRP receptors.
In summary, CGRP receptors and CGRP binding in the
rat uterus are increased with pregnancy and decreased with
labor. Uterine CGRP receptors are up-regulated by P4 and
down-regulated by antiprogesterone. Furthermore, CGRP
receptors are localized primarily to the myometrial cells in
the uterus. We propose that the inhibitory effects of CGRP
on uterine contractility are mediated through the changes
in CGRP receptor levels; this may play a role in uterine
quiescence during pregnancy, and a decrease in the CGRP
receptor levels at term could facilitate initiation of labor.
ACKNOWLEDGMENT
We thank Ms. Debbie Servantes for typing the manuscript.
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