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Pregnancy |
Department of Obstetrics & Gynecology,3 University of Texas Medical Branch, Galveston, Texas 77555-1062
Division of Cancer Treatment and Diagnosis,4 National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Department of Anatomy,5 Mahidol University, Nakhon Pathom, 73170, Thailand
Department of Endocrinology, Metabolism, and Nutrition,6 Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903-0019
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, polypeptide receptors, pregnancy, progesterone, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CGRP has a potent vasodilator effect in many blood vessels and is produced by the tissue-specific alternative splicing of the primary transcript of the calcitonin/CGRP gene [9]. This peptide is distributed throughout the central and peripheral nervous systems and is located in areas that are involved in cardiovascular functions [10–12]. The neuronal cell bodies in the dorsal root ganglia are a prominent site of CGRP synthesis. The CGRP-containing nerves extend peripherally to blood vessels and centrally to the spinal cord [13, 14].
Earlier pharmacological observations indicate that CGRP receptors have been classified into two types: CGRP1 receptor, which has a high affinity for the CGRP-antagonist CGRP8–37 but is not activated by diacetoamidomethyl cysteine (cysACM)-CGRP, a linear agonist of CGRP; and CGRP2 receptor, which has a lower affinity for CGRP8–37 but is activated by cysACM-CGRP. However, the results of these differential-affinity studies have been inconsistent [15, 16], and a recent report by Rorabaugh et al. [17] questioned the existence of separate CGRP2 receptors. Recent molecular data suggest that CGRP receptors use accessory proteins to form functional receptor complexes and that receptor diversity is increased by the combinatorial use of transmembrane receptors and receptor-associated proteins. Calcitonin receptor-like receptor (CRLR), a member of the superfamily of seven transmembrane receptors, functions as a CGRP receptor in the presence of receptor activity-modifying protein 1 (RAMP1) [18]. In turn, RAMP1 acts as a chaperone protein, is required for correct routing of CRLR to the cell surface, and contributes to the pharmacologic specificity of CRLR.
Previously, a novel CGRP receptor from porcine cerebellum cell membranes was identified and purified to homogeneity [19]. This protein has a very high affinity for CGRP, and a partial sequence of the receptor indicates no homology with CRLR. A mouse monoclonal antibody has been raised against CGRP receptors purified from rat cerebellum [19–22], and it has been used for Western immunoblot analysis of neuronal tissues, blood vessels, cardiac atria, and the uterus in the rat [23, 24]. Studies using transfected human embryonic kidney (HEK) 293 cells (American Type Culture Collection [ATCC], Manassas, VA) from our laboratory indicate that this monoclonal antibody could not detect any immunoreactive protein band in any of the cells transfected with human CRLR plus human RAMP1; however, rat uterus and mesenteric artery homogenates showed a single immunoreactive band at approximately 66 kDa [25]. We also reported that this protein could not be detected in the human neuroblastoma cell line SK-N-MC (ATCC), which expresses CRLR [25]. Additionally, studies by Fluhmann et al. [26] indicate that mRNA for CRLR could not be detected in the rat cerebellum and spinal cord.
Based on mRNA and protein analysis [25], we proposed that CGRP receptors could have two types, one related to CRLR (CGRP-A receptor) and another not related to CRLR (CGRP-B receptor). It is possible that both CGRP-A and CGRP-B receptors are expressed and functional in rat blood vessels, and regulate the relaxation responses to CGRP. The present studies were focused on the expression and regulation of CGRP receptors in mesenteric vessels during pregnancy and with sex steroid hormone treatment in rats. Hence, we hypothesize that increased CGRP receptors in the resistance blood vessels and mesenteric arteries, during pregnancy, and with sex steroid hormone treatment may give rise to the vasodilator effects of CGRP that we observed previously [7]. Therefore, the present studies were aimed at 1) determining if CGRP-A and -B receptors existed in the rat mesenteric arteries; 2) if so, whether the concentrations of receptors for CGRP in these vessels were elevated during pregnancy; and 3) whether the steroid hormones estradiol-17ß (E2) and progesterone (P4) would alter the CGRP receptors in these blood vessels.
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MATERIALS AND METHODS |
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Pregnant rats Adult nonpregnant (body weight, 180–220 g) and timed pregnant (body weight, 300–325 g) rats were purchased from Harlan Sprague Dawley (Houston, TX). All animals were housed in a climate-controlled room with a 12L:12D photoperiod and were fed, ad libitum, standard rat chow with water. Virgin female rats were mated, and the day of observation of a vaginal plug with the presence of sperm was designated as Day 1 of gestation. Timed-pregnant rats (n = 4) were killed on Days 5, 8, 10, 16, and 18 and on Day 22 during labor. On Day 17 of gestation, at 0800 h, pregnant rats were injected subcutaneously with a single dose of a P4 antagonist, RU-486 (10 mg/rat in 0.2 ml of sesame oil; Biomol, Plymouth Meeting, PA). Groups of four rats were killed at 6, 12, or 24 h after treatment. Progesterone (2 mg/rat in 0.2 ml of sesame oil twice a day; Sigma Chemical Co., St. Louis, MO) was injected subcutaneously from Day 20 to Day 22 of gestation, and animals (n = 4) were killed on Day 22. Control animals were treated with vehicle (sesame oil; Sigma). A group of nonpregnant rats at the diestrous stage were also killed as controls for pregnant rats.
Nonpregnant rats Groups of nonpregnant rats, regardless of stage of the estrous cycle, were bilaterally ovariectomized while under ketamine (45 mg/kg body weight; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg body weight; Burns Veterinary Supplies, New York, NY) anesthesia. Seven days after ovariectomy, groups of four rats were treated subcutaneously either with E2 (2.5 µg/injection twice daily for 3 days in 0.2 ml of sesame oil), P4 (2 mg/injection twice daily for 3 days in 0.2 ml of sesame oil), a combination of E2 and P4 (same doses and frequency as above), or vehicle (sesame oil). The doses of E2 and P4 used in the present studies are similar to those we have used previously to mimic the levels achieved during pregnancy, and these levels are substantially higher compared with those of nonpregnant, ovary-intact animals (data not shown).
All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch. All animals were killed in a CO2 inhalation chamber; mesenteric arteries and thoracic aorta were removed immediately, quickly frozen in liquid nitrogen, and stored at -70°C until used. All tissues were thawed in ice before homogenization. Total RNA and protein were isolated from mesenteric arteries or aorta (n = 4 from each group) from nonpregnant and pregnant rats using the reagent Trizol (100 mg of tissue homogenized in 0.7 ml of Trizol at 4°C) according to the manufacturer's protocol (Gibco BRL, Gaithersburg, MD). The RNA was used for determining CGRP-A-receptor components, whereas protein was used for CGRP-B measurements.
Determination of CGRP-A Receptors in Mesenteric Arteries and Aorta
Reverse transcription The quality and quantity of RNA were assessed at A 260/280, and all samples showed absorbency ratios ranging between 1.6 and 2.0. The total RNA was treated for genomic DNA contamination using a DNA-free kit (Ambion, Austin, TX). Using total RNA, first-strand cDNA was synthesized by reverse transcription (RT) using the GeneAmp RNA PCR Kit (Perkin Elmer, Branchburg, NJ) as described by the supplier. For RT, 2 µg of total RNA was mixed with 3.0 ml of random primer (Invitrogen, Carlsbad, CA), 200 µM dNTP solution (Sigma), and 10 U of AMV reverse transcriptase (Promega, Madison, WI) in the presence of 5 U of RNase inhibitor (Invitrogen). Samples were placed into a thermal cycler for one cycle at 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min. The cDNA was stored at -20°C.
Polymerase chain reaction Polymerase chain reaction (PCR) using the specific primer sets for CRLR and RAMP1 was carried out with cDNA synthesized by RT. Briefly, 2 µl of the cDNA was mixed with a PCR mixture containing 2.5 mM MgCl2, 1x of 10x PCR buffer II, 5 U/100 µl of Amplitaq DNA polymerase Kit (Perkin Elmer), 65 µl of sterile distilled water, and 0.1 µM of the following sets of primers: CRLR, 5'-GCTCTGTGAAGGCATTTAC-3' (forward) and 5'-CAGAATTGCTTGAACCTCTC-3' (reverse); RAMP1, 5'-GAGACGCTGTGGTGTGACTG-3' (forward) and 5'-TCGGCTACTCTGGACTCCTG-3' (reverse). Final volume was 100 µl. Primer sequences for rat CRLR (accession no. U 17473) and RAMP1 (accession number AJ 001014) were derived using published sequences from the GenBank database [18]. For amplification of 18S, the following primers (Ambion) were used: forward, 5'-AGGAATTGACGGAAGGGCAC-3'; reverse, 5'-GTGCAGCCCCGGACATCTAAG-3'. The PCRs for CRLR, RAMP1, and 18S were carried out on a GeneAmp PCR system 9700 (Perkin Elmer) with the following conditions: An initial denaturation step at 95°C for 5 min was followed by 35 cycles of 60 sec at 95°C, 90 sec at 63°C, and 30 sec at 72°C, with a final extension cycle of 7 min at 72°C. The total cycle number chosen for each gene was from the linear portion of their respective amplification curve (data not shown).
Electophoresis and Gel Imaging
The PCR products were visualized on 1.6% agarose gels containing 0.5 µg/ml of ethidium bromide and run for 1.5 h at 100 V in 0.5x Tris-borate-EDTA buffer. The DNA signals on the gel were imaged under ultraviolet light using a Polaroid camera (Photodyne, Inc., New Berlin, WI), and density-gradient measurements were performed using the Fluorchem digital imaging system (Alpha Innotech Corp., San Leandro, CA). The levels of expression of CRLR and RAMP1 were calculated as a ratio of their respective 18S values. The identities of amplified sequences were verified by sequencing the gel-extracted PCR product. All receptor components showed 100% homology with their respective published sequences (data not shown). Negative controls were run as PCR using total RNA in place of cDNA, and no signal was detectable when run on agarose gel (data not shown).
Determination of CGRP-B Receptors in Blood Vessels
Western immunoblot analysis Equal amounts of total protein (20 µg each) from each preparation were resolved on a 10% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane by electroelution, and probed with primary CGRP-B-receptor monoclonal antibody (1:2000; raised against the affinity-purified CGRP receptors from porcine cerebellum by Dr. Sunil Wimalawansa [21]) for 1 h, washed three times with TTBS (20 mM Tris-[hydroxymethyl] aminomethane-HCl [pH 7.6], 0.05% Tween 20, 10 mM NaCl), and incubated with an anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibody coupled to horseradish peroxidase. After three washes, the membrane was developed using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol. Densitometric analysis was performed in the linear range using Sigma Gel Software (Sigma). The specificity of this monoclonal antibody has been demonstrated by [125I]CGRA binding to the protein from porcine cerebellum following immunoprecipitation with the antibody (906/id).
[125I]CGRP Binding
In one set of experiments, groups of nonpregnant, ovariectomized rats (n = 20) were treated either with P4 (2 mg/injection twice daily for 3 days) or vehicle (sesame oil). Mesenteric arteries were collected, pooled, and stored at -70°C until used.
The tissues were homogenized in 50 mM Tris-(hydroxymethyl) aminomethane buffer (pH 7.4, containing 0.32 M sucrose, 1 mM dithiothreitol, 5 mM EDTA, and 200 KIU/L of aprotinin), and the homogenate was centrifuged at 800 x g for 10 min. The membrane pellet was prepared as reported previously [23]. Membrane preparations (100 µg protein/tube) were incubated in 300 µl of total reaction volume at 4°C for 150 min with 8.3 x 10-12 M [125I]human CGRP (specific activity, 2000 Ci/mmol; 20 000 cpm/tube; catalog no. IM184; Amersham, Arlington Heights, IL [now Amersham Pharmacia Biotech, Piscataway, NJ]) with or without varying concentrations (32 x 10-15 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 0.4 µM (e.g., 0.5 µg/tube) unlabeled CGRP. The data were analyzed with the Scatchard method and the results expressed as CGRP bound (in fmol/mg) to the membrane protein.
Statistics
Results are expressed as the mean ± SEM. Data were analyzed for statistical differences with the Student t-test or one-way ANOVA followed by the Bonferroni t-test to verify differences between individual groups. Differences were considered to be significant if P < 0.05.
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RESULTS |
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Figure 2 illustrates the changes in CGRP-B-receptor protein concentrations in mesenteric arteries from nonpregnant rats and rats on Days 10, 16, and 18 of gestation and during spontaneous labor on Day 22. As shown in Figure 2, a single band of CGRP-B-receptor protein was obtained with a predicted size of 66 kDa. Densitometric analysis of the CGRP-B-receptor protein from mesenteric arteries from four rats in each group showed that this protein increased progressively with pregnancy from Day 10 until Day 18 and then decreased at term. Occasionally, in some tissues/treatments, a faint, nonspecific band of smaller size was noticeable, which appeared to be nonspecific.
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Because CGRP-receptor concentrations were increased during pregnancy and decreased at term, we investigated whether these changes were associated with changes in sex steroid hormone levels during pregnancy and labor. In this set of experiments, we first examined if CRLR and RAMP1 mRNA expressions were altered with RU-486 treatment on Day 17 of gestation. Next, we investigated whether treatment of pregnant rats with P4 between Days 20 and 22 of gestation maintained the CRLR and RAMP1 mRNA levels in these vessels. The expression of both CRLR and RAMP1 in mesenteric arteries was substantially lower after treatment with RU-486 (Fig. 3). Treatment with P4 from Days 20 to 22 of gestation prevented the term-associated decline in CRLR (Fig. 3A) and RAMP1 (Fig. 3B) mRNA expression in the mesenteric arteries. Figure 4 shows that RU-486 significantly (P < 0.05) inhibited the mesenteric artery concentrations of CGRP-B-receptor proteins compared to Day 17 pregnant rats, with maximal effects occurring within 6 h of treatment. Conversely, treatment with P4 from Days 20 to 22 reversed the decline in CGRP-B-receptor protein concentration in mesenteric arteries that occurred at term during labor (Fig. 4).
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Next, we investigated the effects of the female sex steroid hormones E2 and P4 on the mesenteric artery CGRP-A-receptor components CRLR and RAMP1 and CGRP-B receptors in nonpregnant, ovariectomized rats. Three-day treatments with P4, but not with E2, significantly (P < 0.05) elevated both CRLR (Fig. 5A) and RAMP1 (Fig. 5B) mRNA expression. The expression of CRLR did not change significantly with E2 treatment. However, the responses appeared to be somewhat variable in four replicate animals, and all data were expressed as a ratio of 18S expression. Western immunoblot analysis (Fig. 6) showed that both P4 and combined E2 and P4 treatments increased CGRP-B-receptor protein expression in these blood vessels. On the other hand, E2 treatment significantly (P < 0.05) inhibited the CGRP-B-receptor protein concentrations in mesenteric arteries.
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To evaluate whether increases in CGRP-receptor expression with P4 treatment results in elevated [125I]CGRP-binding sites in this vessel, we performed binding studies using [125I]CGRP in one set of experiments. A single class of binding sites for CGRP was obtained from mesenteric arteries, and the dissociation constants were similar in both vehicle-treated (Kd = 1.14 nM) and P4-treated (Kd = 2.34 nM) rats. However, as shown in Figure 7, the CGRP-binding sites were increased with P4 treatment (870 ± 78 fmol/mg protein) in nonpregnant, ovariectomized rats compared to vehicle-treated rats (384 ± 30 fmol/mg protein).
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We next assessed whether changes in CGRP-A- and CGRP-B-receptor levels are specific to resistance vessels, such as the mesenteric artery. For this, we compared the changes in CGRP-receptor levels in a conduit vessel, the thoracic aorta, to those associated with pregnancy and with steroid hormone treatments in this same tissue. We observed that mRNA levels for both CRLR (Fig. 8A) and RAMP1 (Fig. 8B) were unchanged with pregnancy compared to either term or nonpregnant diestrous stage in these blood vessels. Figure 9 shows the representative Western immunoblot analysis of CGRP-B-receptor proteins from the thoracic aorta of diestrous rats and on Day 18 of pregnancy and during labor. As shown in Figure 9, no significant differences in CGRP-receptor protein expression associated with the stage of pregnancy or labor (P < 0.09) were identified. Similarly, the expression for both CGRP-A and CGRP-B receptors in the thoracic aorta was unchanged with hormone treatments in nonpregnant, ovariectomized rats (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies demonstrated that CGRP induces vasodilation of human coronary and mesenteric arteries [27, 28]. The CGRP receptor-antagonist CGRP8–37 can inhibit vasodilation of rat mesenteric arteries induced by periarterial nerve stimulation, suggesting that endogenous CGRP receptors may participate in regulating vasodilation of the mesenteric vasculature [29, 30]. Results of previous studies from our laboratory demonstrated that CGRP-induced vascular relaxation is increased during pregnancy in the mesenteric artery [31]. In the present study, we have shown, to our knowledge for the first time, that in a resistance blood vessel, mesenteric artery CGRP receptors are elevated during pregnancy and decreased at term (Figs. 1 and 2). Furthermore, treatment with antiprogesterone RU-486 causes decreases in CGRP receptors, whereas P4 treatment during late gestation maintains CGRP receptors in resistance blood vessels at similar levels to those observed during pregnancy (Figs. 3 and 4). It is well documented that circulatory P4 levels are increased during pregnancy and decreased at term. Results of studies from our laboratory suggest that the vasodilatory effects of CGRP in hypertensive rats appear to be P4 dependent [6]. Taken together, these studies suggest that P4 upregulates CGRP receptors in resistance blood vessels and, therefore, maintains vascular adaptations during pregnancy, when P4 levels are elevated.
Because vasodilator effects of CGRP are elevated with sex steroid hormones in nonpregnant rats and with pregnancy, we hypothesized that both P4 and estrogen may increase CGRP-receptor concentrations in resistance blood vessels. Our results show that both CRLR and RAMP1 mRNA expression were elevated only with P4 treatment; E2 was not effective in adult ovariectomized rats (Fig. 5). However, P4 upregulated and E2 downregulated the CGRP-B-receptor protein in mesenteric arteries (Fig. 6). Several studies suggest a role for estrogens in the regulation of vascular tone and blood pressure homeostasis [32, 33] through nitric oxide [34, 35]. Interestingly, in the present study, we observed a significant decline in CGRP-B-receptor proteins in mesenteric arteries with E2 treatment. We have also noticed that both the expression of CGRP-A-receptor (RT-PCR) and CGRP-B-receptor (Western blot analysis) levels were decreased in these blood vessels at term, when estrogen levels were elevated (Figs. 1 and 2). On the other hand, CGRP-B-receptor proteins and CGRP-A-receptor component mRNA levels, as well as binding sites for CGRP in the mesenteric vessels, were significantly elevated by P4 (Figs. 5–7), suggesting that increases in receptor levels with P4 treatment result in parallel increases in binding to [125I]CGRP. Based on these observations, we speculate that increased circulatory levels of estrogen and/or a fall in P4 at term may be responsible for reduced CGRP-A- and CGRP-B-receptor expression in the mesenteric artery. Thus, our studies demonstrated that the increased vasodilator effects of CGRP during pregnancy appear to be mediated through the elevated levels of CGRP receptors in resistance blood vessels. Progesterone may be involved in this process.
The present studies further demonstrated that receptors for CGRP were also expressed in the thoracic aorta, a conduit blood vessel (Figs. 8 and 9). However, the expression of both CGRP-A and CGRP-B receptors in this blood vessel were not regulated either gestationally (Figs. 8 and 9) or by sex steroid hormones (data not shown). It is well known that blood pressure within large arteries is controlled by the resistance of small arteries and arterioles, which thus maintain blood flow. Because CGRP is a smooth muscle relaxant, we speculate that CGRP receptors in smooth muscle cells of resistance blood vessels may play an important role in maintaining vascular tone during pregnancy. At present, we do not know if secondary messenger systems, such as cAMP and cGMP, are involved in the depressor effects of CGRP. Several lines of work suggest that the vasodilator effects of CGRP are mediated through its receptors being coupled to these secondary messenger systems. It has been shown that part of the CGRP-induced relaxation in uterine arteries is mediated by the opening of ATP-sensitive K+ channels [36, 37]. Moreover, involvement of the cAMP pathway in CGRP-induced K-channel activation in vascular smooth muscle has been reported [38]. Additional studies are required to further assess the mechanisms responsible for CGRP-mediated vasodilation during pregnancy and in the presence of P4.
In summary, CGRP receptors (CGRP-A-receptor components as measured by RT-PCR, CGRP-B-receptor proteins as measured by Western blot analysis, and receptor binding as determined by [125I]CGRP binding) are expressed in rat mesenteric vessels and in the thoracic aorta. During pregnancy, a progressive increase occurs in both CGRP-A- and CGRP-B-receptor levels in the mesenteric arteries, but not in the aorta. The P4 maintains and the RU-486 downregulates mesenteric artery CGRP receptors during pregnancy. The P4, but not E2, upregulates CGRP receptors in the mesenteric arteries of adult ovariectomized rats. Therefore, we propose that increased CGRP-receptor expression in resistance vessels plays a role in the increased vasodilator effects of CGRP and, therefore, in the increased vasodilation that occurs during pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics & Gynecology, 301 University Blvd., Medical Research Building, Rm. 11.138, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Received: 20 August 2003.
First decision: 11 September 2003.
Accepted: 25 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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