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a Departments of Obstetrics and Gynecology and
b Internal Medicine, The University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hormonal control of uterine CGRP receptors was assessed by the use of antiprogesterone RU-486, progesterone, and estradiol-17ß. 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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult nonpregnant (200–210 g BW) and timed-pregnant Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (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. Time-mated 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, RU-486 (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.
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-17ß (E2; 2.5 µg/injection, twice-daily s.c. injections); 2) progesterone (P4; 2 mg/injection, twice-daily s.c. injections); 3) E2+P4 (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 -70°C 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 x g for 10 min. The supernatant was recentrifuged at 30 000 x g for another 30 min. Final membrane pellet was resuspended in fresh Tris(hydroxymethyl)aminomethane buffer (no sucrose). Membrane preparations (100–200 µg protein per tube) were incubated at 4°C for 150 min with 8.3 x 10-12 M 125I-human CGRP (20 000 cpm/tube; Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ) with or without varying concentrations (32 x 10-15 M to 13 x 10-9 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 µg/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 µl 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 µg/ml leupeptin; 10 µg/ml aprotinin). The homogenates were incubated on ice for 20 min. After removal of cell debris by centrifugation (14 000 x 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 affinity-purified CGRP receptors from porcine cerebellum, by Dr. Sunil Wimalawansa [12–14]) for 1 h, washed three times with TTBS (20 mM Tris[hydroxymethyl]aminomethane-HCl, 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 µm 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 ± 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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125I-CGRP peptide binding
Specific binding of 125I-human-
-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 -CGRP competed for 125I-CGRP-binding 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 ± 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 ± 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 ± 68).
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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 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 = 3 in each group).
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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 = 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.
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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 125I-CGRP-binding and Western blotting data presented.
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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 = 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).
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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. Ovariectomized adult rats were treated with E2, P4, or E2+P4, 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 E2+P4 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 immunoreactive 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Chandra Yallampalli, University of Texas Medical Branch, 301 University Boulevard, Route 1062, Galveston, TX 77555–1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Accepted: May 24, 1999.
Received: February 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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