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a Department of Obstetrics and Gynecology
b Department of Internal Medicine, 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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parturition, placenta, pregnancy, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilator peptide known [3]. CGRP is produced by the tissue-specific alternative splicing of the primary transcript of the calcitonin/CGRP gene [4]. A prominent site of CGRP synthesis is the dorsal root ganglion (DRG) [5]. DRG contain the cell bodies of primary afferent neurons that extend CGRP-containing nerves to peripheral sites such as blood vessels, and CGRP is released into the blood stream via sensory nerve endings. Because CGRP levels in umbilical cord blood are higher than those in the mother's plasma at term [6], it is possible that CGRP in fetal circulation is derived from sensory neurons of the fetus, or even from the placenta itself. CGRP binding sites were demonstrated in many cells and organs throughout the body [3]. Thus the presence and changes in CGRP receptor could therefore regulate CGRP effects in a variety of tissues, including placenta.
CGRP receptors belong to the superfamily of seven-transmembrane (7-TM) receptors, and it has been suggested that they require accessory proteins for their functions. It was recently reported that a family of proteins called receptor activity modifying proteins (RAMPs) modulate the activity of calcitonin receptor-like receptor (CRLR) [7, 8] and calcitonin receptor [9, 10]. Association of RAMP1 protein with CRLR reveals a CGRP receptor [7]. Receptor component protein is believed to work with another 7-TM receptor to form CGRP receptor, which has not been shown to be related to CRLR [11]. A well-characterized [12–14] monoclonal antibody that was raised against ligand affinity-purified CGRP receptor protein from porcine cerebellum does not react with CRLR, and is transiently expressed in human embryonic kidney cells either alone or in combination with RAMP1 (unpublished observations). Further, Western blotting data using this monoclonal antibody to cerebellar CGRP receptor protein demonstrated that this CGRP receptor is expressed in the cerebellum, uterus, lung, and mesenteric artery of rats and in human myometrium, but not in SK-N-MC cells, a cell line that has been demonstrated to express CRLR and RAMPs [7]. In contrast, SK-N-MC cells and rat uterus, but not rat cerebellum, express mRNA for CRLR and RAMP1, further supporting the existence of two separate CGRP receptors: one CRLR-related (CGRP-A receptor) and the other unrelated to CRLR (CGRP-B receptor).
We recently reported that 125I-CGRP binding to membrane preparations from the uterus of rats was elevated during pregnancy and decreased during labor and postpartum [15]. CGRP receptor protein, measured by Western blotting analysis using the CGRP-B receptor monoclonal antibodies in rat uterus were elevated on Day 17 of gestation, with a precipitous decrease in these receptors on Day 22 of gestation before the onset of labor. However, little is known about the existence and regulation of CGRP-B receptor in rat placenta. With the use of monoclonal antibody, our current studies were designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are regulated with the gestational age and by sex-steroid hormones.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Progesterone (P4) and 17ß-estradiol (E2) were purchased from Sigma Chemical Company (St. Louis, MO). RU-486 was obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). ICI 182780 was obtained from Tocris (Ballwin, MO).
Antibodies
Mouse monoclonal anti-CGRP-B receptor antibodies were generated by Dr. S.J. Wimalawansa. These antibodies were prepared against the CGRP affinity-purified proteins from pig cerebellum and characterized as previously described [16]. These antibodies have been screened by ELISA, Western blot analysis, and immunohistochemistry [17], and were previously used to assess CGRP receptors in rat uterus [15], human uterus [18], and cerebellum [17].
Animals and Treatments
Timed, pregnant Sprague-Dawley rats were killed in a carbon dioxide inhalation chamber on Days 17, 18, 19, 20, 21, and 22 of pregnancy before and during labor (six animals for each gestational age). The placentas were removed immediately; cleaned of fat, fetuses, and fetal membranes; and rinsed thoroughly in cold PBS solution. To assess the regulation of placental CGRP-B receptor levels by steroid hormones, we treated pregnant rats with P4, antiprogesterone RU-486, E2, and antiestrogen ICI 182780. Progesterone was given from Day 20 to Day 22 of gestation to maintain adequate P4 levels, because circulatory P4 concentrations decline from Day 20 of pregnancy. Because levels of both estrogen and P4 are steady during Days 15–19, RU-486 and ICI 182780 were given on Day 17 to oppose endogenous steroid hormones. 17ß-Estradiol was given on Day 17 to mimic the increases that occur naturally on Day 22 of gestation. Placentas were also obtained from six pregnant rats injected with P4 (4 mg per rat per day s.c. on Days 20–22), RU-486 (10 mg/rat s.c. on Day 17, at Hours 0 and 6), E2 (5 µg/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 µg/rat, s.c. on Day 17). Placentas were frozen in liquid nitrogen for protein isolation. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch and complied with National Institutes of Health guidelines.
Immunofluoresent Localization of CGRP-B Receptors
Placentas obtained from rats on Day 18 of gestation and during labor were rinsed thoroughly in cold PBS (0.1 M pH 7.4) and fixed in Bouin fixative [19]. After routine tissue processing procedure of dehydration in ascending grades of ethanol, cleaning in xylene, and infiltration with paraffin, the tissues were embedded in paraffin. Sections (5 µm thick) were rinsed with 3% normal horse serum with Triton X-100 for 30 min at room temperature, and then incubated with avidin-biotin blocking buffer to reduce nonspecific staining. The primary monoclonal antibody for CGRP-B receptor in 1% normal horse serum with Triton X-100 was applied to the slides and incubated overnight in a cold room (4°C). After washing three times with PBS on a shaker (300 rotations per minute for 5 min), the slides were incubated with fluorescein-conjugated secondary antibody, Alexa Fluor 488 (1:200, green; Molecular Probes Inc., Eugene, OR), at room temperature for 4 h. The slides were rinsed with PBS for 30 min and then mounted with coverslip using DAPI (Vector Laboratories, Inc., Burlingame, CA). The segments were covered with coverslips and viewed with an Olympus microscope with image-ProPlus software (Olympus Optical Co. Ltd., Tokyo, Japan).
Western Blotting
Lysates of rat placental tissues containing 40 µg of protein were loaded and electrophoresed on SDS-PAGE on a 7.5% Tris-HCl ready gel using a Miniprotein II gel apparatus (Bio-Rad, Richmond, CA) [20]. After electrophoretic transfer to a polyvinylidene difluoride membrane and blocking with 5% nonfat dry milk, the blots were incubated with monoclonal antibody against CGRP-B receptors in a dilution of 1:1000 for 1 h at room temperature. After washing, the blots were incubated with enzyme-conjugated anti-mouse immunoglobulin IgG:horseradish peroxidase for 1 h at room temperature. The blots were then subject to enhanced chemiluminescence using the Western blotting detection system (Amersham, Arlington Heights, IL). Autoradiographs were applied to the blots until satisfactory exposure was achieved. The bands with predicted sizes of 66 kDa were densitometrically scanned and analyzed using a Sigma Gel Analysis System (SPSS Inc., Chicago, IL).
Statistical Analysis
Data are presented as means ± SEM. Differences were tested for significance using ANOVA followed by t-test correction with Bonferroni adjustment. The acceptable value of significance was P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using immunofluorescent methods, we found that immunoreactive CGRP-B receptors were primarily localized in the labyrinthine layer (Lbr) of the placenta from rats on Day 18 of gestation (Fig. 1, A and B) with specific staining in the trophoblasts and villous vessel endothelium and underlying smooth muscle layer. In contrast, the placenta sections from rats during labor showed minimal staining for CGRP-B receptors (Fig. 1, D and E) as evidenced by the lower number of immunopositive cells and reduced intensity of staining in the labyrinth zone. Control sections without primary antibody showed no specific staining in the segments from rats on Day 18 of pregnancy (Fig. 1C) and during labor (Fig. 1F).
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Changes in CGRP-B Receptor Protein in Rat Placenta During Late Pregnancy and Labor
To examine whether CGRP-B receptors are present in rat placenta, we analyzed placental tissues by Western immunoblotting with the monoclonal anti-CGRP-B receptor antibody. As shown in Figure 2, a single band of CGRP-B receptor protein was obtained with a predicted size of 66 kDa in rat placentas. Densitometric analysis of the this protein band of placentas from four to six rats in each group showed that significantly more CGRP receptors were present from Day 17 to Day 22, with maximal expression observed on Day 22 before labor, and this was 10 times higher than that observed on Day 17 (P < 0.01). Further, expression of CGRP receptors in rat placenta decreased precipitously during labor (8% vs. 100% on Day 22 before labor, P < 0.01), suggesting a gestation-dependent regulation of these receptors in rat placenta.
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Effects of Progesterone and RU-486 on Placental CGRP-B Receptors
Because CGRP-B receptors in rat placentas were elevated during late pregnancy, and decreased at term labor, we investigated whether these alterations were associated with changes in sex-steroid hormone levels. As shown in Figure 3, P4 given during Days 20–22 attenuated the fall in placental CGRP-B receptors at term labor, but CGRP-B receptor expression is still lower than that in placentas obtained from rats before labor. Antiprogesterone RU-486 given on Day 17 induced preterm labor in all treated rats and significantly reduced placental CGRP-B receptor protein as shown in Figure 4. The effects were substantial at 6 and 12 h, with only 18% and 41% receptor levels remaining, respectively, compared with 100% before injection. However, placental CGRP-B receptor expression was completely recovered to control levels by 24 h after RU-486 treatment. To further confirm the inhibitory influence of RU-486 on placental CGRP-B receptors, we examined the effect of two sequential injections of RU-486 (10 mg/rat, each injection given at Hours 0 and 6). As shown in Figure 5, the level of CGRP-B receptors remaining in rat placentas was 33% and 54% at 12 and 24 h after two sequential RU-486 injections, compared with 100% in controls (P < 0.05), providing further evidence that P4 is required for maintaining elevated placental CGRP-B receptors in the placenta during gestation.
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Effects of 17ß-Estradiol and ICI 182780 on Placental CGRP-B Receptors
We investigated whether E2 or ICI 182780, an antiestrogen, modulate placental CGRP-B receptors in rats. As shown by the Western blotting data in Figure 6, neither E2 nor ICI 182780 altered placental CGRP-B receptors at 24 h after treatments. However, a significant decrease (34% vs. 100% in control, P < 0.01) in CGRP-B receptor expression was noted 48 h after E2 administration. On the contrary, a profound increase in placental CGRP-B receptors was observed 48 h after ICI 182780 treatments (152% vs. 100% in control, P < 0.01), indicating that E2 inhibited placental CGRP-B receptor expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The definitive rat placenta is formed and differentiated at about Day 12 or Day 13 of gestation [21] within two major regions, the labyrinthine and the trophospongial cell layers. The labyrinth represents the major portion of the placenta and is composed of both maternal blood channels and fetal vessels. Unlike the villous trophoblast in humans, the outermost layer in rat placenta is made up of a fairly thin layer of cytotrophoblast, which is in direct contact with the maternal blood sinus. The second layer is syncytial and sends projections through the discontinuous layer (outermost layer) of cytotrophoblast into the maternal blood spaces. The third layer is the innermost layer and is made of a syncytium that lies just outside the core of vascular fetal mesenchyme [22]. During later stages of pregnancy, this labyrinthine barrier, with its intricate vascularization, is the primary route of maternal-fetal transport [23]. The present study demonstrated the existence of CGRP-B receptors in the labyrinthine barriers, including trophoblast cell and blood vessels, implicating a role for CGRP in the control of blood flow in placenta and the macromolecules passing from the maternal blood to the fetal circulation.
A progressive and significant enhancement in fetal weight occurs during the later third of gestation, commencing at Day 15 of pregnancy [24]. Fetal mass increases from 100 mg to 6500 mg over this time period. Placental weight also increases concurrently, and there is a progressive and significant enhancement in blood flow to uterine tissue [25]. Percentage uterine blood flow directed to placenta is less than 10% at Day 15 of gestation, but it is more than 90% at near term [24]. Therefore, late gestational fetal development is supported by cardiovascular adaptation, including specific control mechanisms such as higher serum levels of CGRP [26], and enhanced expression of CGRP-B receptors in the rat uterus [26] and placentas as shown in this report. Changes in CGRP-B receptors in rat placenta (i.e., an increase during late pregnancy and a decline at term) parallel the alterations in uteroplacental blood supply [27]. Thus, we speculate that the CGRP system may be involved in regulating fetoplacental blood flow to ensure enough oxygen and nutrient supply to the developing fetus.
In present studies, we demonstrate that P4 given during Days 20–22 of pregnancy attenuated the fall in placental CGRP-B receptor at term labor. Further, RU-486 given on Day 17 of pregnancy significantly inhibited placental CGRP-B receptor 6 h after RU-486 administration, suggesting that P4 may be required for maintaining placental CGRP-B receptor expression. However, RU-486-induced inhibition of CGRP-B receptors was not sustained at 24 h after RU-486 administration, suggesting that this phenomenon may be due to a time-related decline of RU-486 effects. The expression of CGRP-B receptors in rat placentas was maintained at lower levels after two sequential injections of RU-486, confirming the inhibitory action of RU-486 on placental CGRP-B receptor expression. The stimulatory effects of P4 on CGRP-B receptor proteins are further supported by our observation that P4 administration during late gestation attenuated the fall in CGRP-B receptors in placenta at term.
We also demonstrate that the CGRP-B receptors in the placenta are substantially lower 48 h after estrogen administration, indicating that E2 inhibits placental CGRP-B receptor expression. This concept is further supported by the ICI 182780-induced increase in placental CGRP-B receptors at 48 h after the treatments. However, the inhibitory action of E2 or stimulatory effects of ICI 182780 were not significant at 24 h after administration, indicating time-related effects of E2 on CGRP-B receptor in placenta. Apparently, further studies will be required to define whether this inhibition in CGRP-B receptors occurs at the transcriptional or translational levels, and whether the protein degradation was enhanced during E2 manipulation. Irrespective of the molecular mechanisms involved, both E2 and P4 appear to regulate CGRP-B receptors in placenta. A fall in P4 during late gestation with a substantial surge in E2 at term [28] may have a profound inhibitory effect on CGRP-B receptors in placenta as observed in our study. Further, these receptors may be more actively reflect the changes in the E2:P4 ratio in rat pregnancy.
During pregnancy, the concentration of CGRP peptide in maternal plasma is elevated both in rats [26] and in humans [29], reaching maximum levels close to term and falling after delivery [30]. Immunoreactive CGRP was first detected in fetal rats at Gestational Day 18, and the incidence considerably increased between Postnatal Days 5 and 14 [31]. The direct correlation of circulating immunoreactive CGRP in the newborn with ascending birth weight may have significance in the development of the fetus.
Circulating immunoreactive CGRP in adults has been shown to come from the release of CGRP from sensory nerve endings. However, the source of CGRP in fetal plasma is not clear. Because CGRP levels in cord blood are higher than those in the mother's plasma at term [6], it is possible that CGRP in fetal circulation is derived from sensory neurons of the fetus, or even from the placenta itself. There is currently no information on whether CGRP can cross the placental barrier in the rats and humans. Therefore, the precise origin of CGRP in the fetoplacental circulation and mechanisms in the regulation of CGRP-B receptor levels in placenta warrant further investigations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, 301 University Boulevard, Route 1062, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Received: 25 March 2002.
First decision: 23 April 2002.
Accepted: 28 May 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  K.E. Green, C. Thota, G.D.V. Hankins, C. Yallampalli, and Y.-L. Dong Calcitonin gene-related peptide stimulates human villous trophoblast cell differentiation in vitro Mol. Hum. Reprod., July 1, 2006; 12(7): 443 - 450. [Abstract] [Full Text] [PDF]
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 Y.-L. Dong, K. E. Green, S. Vegiragu, G. D. V. Hankins, E. Martin, M. Chauhan, C. Thota, and C. Yallampalli Evidence for Decreased Calcitonin Gene-Related Peptide (CGRP) Receptors and Compromised Responsiveness to CGRP of Fetoplacental Vessels in Preeclamptic Pregnancies J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2336 - 2343. [Abstract] [Full Text] [PDF]
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 Y.-L. Dong, S. Vegiraju, M. Chauhan, P. R. R. Gangula, G. D. V. Hankins, L. Goodrum, and C. Yallampalli Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H230 - H239. [Abstract] [Full Text] [PDF]
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Y.L. Dong, S. Vegiraju, M. Chauhan, and C. Yallampalli Expression of calcitonin gene-related peptide receptor components, calcitonin receptor-like receptor and receptor activity modifying protein 1, in the rat placenta during pregnancy and their cellular localization Mol. Hum. Reprod., August 1, 2003; 9(8): 481 - 490. [Abstract] [Full Text] [PDF]
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