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Pregnancy |
Department of Obstetrics and Gynecology,3 University of Texas Medical Branch, Galveston, Texas 77555-1062
Department of Endocrinology,4 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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neuropeptides, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and CGRPß), and adrenomedullin. CGRP and CGRPß differ from each other by three amino acids, and CGRP is the predominant form present in circulation [1–5]. Both of these CGRP peptides are indistinguishable in their biological activities, which include regulation of vascular tone, neurotransmission, and relaxation of smooth muscles of numerous tissues including uterus [6–9]. CGRP induces relaxation of uterus during pregnancy but not during labor, implicating a role in maintaining uterine quiescence during pregnancy [8–10]. In addition, CGRP has been shown to reverse hypertension and fetal growth restriction in nitro-l- arginine methyl ester-treated pregnant rats [11]. Despite the clinical implication of CGRP, therapeutic strategies targeting CGRP receptors have been hindered by the lack of knowledge on functional CGRP receptors.
Although several pharmacological and biochemical studies provide evidence for CGRP receptor heterogeneity, the identity and structure/function relationship of the receptor(s) that bind CGRP have been unclear. Several putative receptors from different mammalian sources have been reported to bind CGRP and activate CGRP-induced secondary messengers. Among these receptors, calcitonin receptor-like receptor (CRLR), which requires an accessory protein, receptor activity modifying protein (RAMP1) is perhaps the most studied CGRP receptor [12]. However, based on the pharmacological studies using affinity of an antagonist to the receptor CGRP8–37 and a linear analog of CGRP, [acetamido-methylcysteine2,7] -CGRP, CGRP receptors were previously classified as CGRP1 and CGRP2 receptors [2, 13, 14]. However, these differential affinity studies have been inconsistent, and several recent reports questioned the existence of separate CGRP2 receptors [15– 17]. Recently we reported existence of a CGRP receptor that is different from CRLR [18, 19]. We have previously shown that a monoclonal antibody to the ligand affinity- purified CGRP receptor specifically detects a 66-kDa protein in rat and human tissues [10, 20–22], which is not CRLR. Based on these findings, CGRP receptors are now classified as CGRP-A and CGRP-B receptors [19]. CGRP-A receptors consist of CRLR, a seven-transmembrane domain class B G-protein coupled receptor (GPCR), and require a single transmembrane domain accessory protein, RAMP1, to become a functional CGRP receptor and CGRP-B receptor, which is not related to CRLR [18, 19].
CRLR contains a large N-terminal (Nt) domain and six highly conserved cysteine residues that form disulfide bonds [23]. Secondary structure prediction of RAMP1 also shows a large Nt domain. There is a growing body of data supporting the involvement of Nt domain of class B GPCRs in ligand receptor activation [24–26]. We hypothesize that Nt domain of CRLR and RAMP1 may be actively involved in determining CGRP binding and specificity and that CGRP-induced relaxation effects in the uterine artery are mediated through Nt-CRLR. Current studies report generation of polyclonal antibodies to the Nt domain of CRLR and RAMP1 and their characterization. We used these antibodies to determine the role of Nt domain of CGRP-A receptor components, CRLR and RAMP1, in CGRP-induced vasorelaxation in rat uterine artery. We also assessed the effects of monoclonal antibodies to CGRP-B receptor to determine the CGRP receptor type involved in CGRP- induced uterine artery relaxation in pregnant rats. We show that Nt domain of CRLR is the major determinant of CGRP-induced relaxation of rat uterine arteries.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Timed-pregnant Sprague-Dawley rats on Day 14 of gestation (300– 320 g BW) 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. The Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, Texas approved all procedures.
Generation of Polyclonal Antibodies
Full length N-terminal domains of CRLR and RAMP1 were cloned, expressed and purified from Escherichia coli (data not shown). Purified recombinant Nt-CRLR and Nt-RAMP1 proteins were used to raise polyclonal antibodies in rabbits. Briefly, 100 µg of Nt-CRLR and Nt-RAMP1 proteins were subjected to 12% SDS-PAGE electrophoresis and the acrylamide gel containing the electrophoresed protein was finely minced and injected into rabbits after collecting the pre-immune serum. Booster injections, with the same amount of protein were given to rabbits at four, eight, and sixteen weeks and serum was collected at seventeen weeks. The antisera for both Nt-CRLR and Nt-RAMP1 were tested for its titer by ELISA.
Characterization of the Polyclonal Antibodies
Polyclonal antibodies were characterized for specificity and linearity by Western immunoblotting. Increasing amounts of Nt-CRLR (5, 10, 20, 40, 60, 80, 100, and 120 ng) and Nt-RAMP1 protein (1, 2, 3, 4, 5, and 6 µg), were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were then incubated with the respective antibodies (1:5000) followed by three washes with 1x TTBS buffer (10mM Tris, 0.05% Tween 20 and 0.9% NaCl) for 10 min each. The membranes were then incubated with 1:5000 dilution of anti-rabbit antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc., Santa Cruz, CA) in 1x TTBS containing 10% non-fat dairy milk for 1 hour. The membranes were then washed with 1x TTBS and developed using the ECL system (Amersham Biosciences, Little Chalfont, UK). The blot was exposed to X-ray film and developed for visualization of protein bands. The linearity of the antibodies was assessed by correlating the immunoreactive band densities with the increasing amounts of Nt-CRLR or Nt-RAMP1 protein used.
The specificity of the antibodies was investigated by using antibody that was preadsorbed with the respective antigen. Briefly 32 µg of Nt- CRLR or Nt-RAMP1 antibody was incubated overnight with 320 µg of the respective antigen at 4°C in 100 µl of phosphate buffered saline, pH 7.4. The reaction mixture was then centrifuged for 10 min at 15800 x g and the supernatant was used as primary antibody after making the required dilution with 1 x TTBS (1:5000). The primary antibodies to Nt- CRLR and Nt-RAMP1, with or without preadsorption were used in the western blotting. The protocol for western immunoblot was similar to that mentioned above. Briefly, 1 µg of the purified Nt-CRLR or Nt-RAMP1 protein and 20 µg of membrane protein from rat uterine tissue homogenate were subjected to 12% SDS-PAGE followed by transfer onto a nitrocellulose membrane. Nitrocellulose membrane was either immunoblotted with the respective antibody or with antisera preadsorbed with the respective antigen.
Preparation of Membrane Protein from Pregnant Rat Uterus
Tissue from pregnant rat uterus (100 mg) was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5; 120 mM NaCl; 0.4% NP-40; 100 mM NaF; 200 mM NaVO5; 1 mM PMSF; 10 µg/ml leupeptin; 10 µg/ml aprotinin). The homogenate was incubated on ice for 20 minutes and tissue debris was removed by centrifugation (14000 x g, 30 minutes). The supernatant containing the membrane protein was used for assessing the specificity of Nt-CRLR and Nt-RAMP1 antibodies.
Cell Culture and Construction of Stable Cell Lines Expressing CRLR and RAMP1
Human embryonic kidney (HEK-293) (ATCC, Manassas, VA) cells were cultured in Dulbecco modified Eagle medium (ATCC) supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Cells were incubated at 37°C under the atmosphere of 95% air:5% CO2. For stable expression, cells were plated in 60 mm plates to a density of 1.5 x 105 cells/plate and transfected with plasmid pcDNA 3 encoding CRLR and RAMP1 (1 µg/well). Transfection was carried out using Lipofectamine according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). HEK-293 cells stably expressing CRLR and RAMP1 were generated by antibiotic G-418 selection of transfected cells.
Visualization of Cell Surface Expression of CRLR and RAMP1
The antibodies to Nt-CRLR and Nt-RAMP1 were further tested for specificity in HEK293 cells stably transfected with pcDNA3 expressing CGRP receptor components, CRLR and RAMP1. HEK293 cells transfected with pcDNA3 containing CRLR and RAMP1 were grown for 24 hours on Lab-Tek tissue culture slides. The cells were fixed with 70% acetone for ten minutes at room temperature followed by washing with PBS. The cells were then incubated either with primary antibodies to Nt-CRLR or Nt-RAMP1, with and without primary antibodies preadsorbed with respective antigens or without any antisera in blocking buffer for one hour. The cells were washed in blocking buffer and were further incubated in the same buffer with Alexa Flour 594 dye conjugated goat anti-rabbit IgG (Vector Laboratories Inc. Burlingame, CA) for one hour at room temperature. The cells were then washed three times with PBS followed by the addition of mounting medium. The cell surface expression of CRLR and RAMP1 were observed under UV fluorescent microscope (OLYMPUS BX60, Tokyo, Japan).
Effects of CGRP Receptor Antibodies on CGRP-Induced Uterine Artery Relaxation
Pregnant rats on day 18 of gestation (250–300 g body weight [BW]) were used in the present study. Rats were deeply anesthetized by an i.p. injection of ketamine (45 mg/kg BW; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg BW; Phoenix Scientific, Inc., St. Louis, MO), and the uterine artery was carefully dissected. Uterine artery rings (2 mm length, 400 µm in diameter) were obtained from the main uterine artery between the middle and lower third of the vascular arcade and placed in physiological salt solution (PSS) kept on ice (pH 7.4). Uterine artery rings were mounted in the jaws of a wire myograph (Kent Scientific, Litchfield, CT) for the measurement of isometric tension [27]. The unstretched vessel rings were allowed to equilibrate for 15 min in PSS that was gassed in 95% air:5% CO2 to maintain pH 7.4 at 37°C. The vessel rings were stretched to a predetermined length that was equivalent to an internal diameter of approximately 400 µm and allowed to equilibrate for another 15 min. The vessels were contracted with 5 µM norepinephrine (NE) until reproducible contractile responses were obtained (three to four times). Each time the artery rings were incubated with NE for 5 min and washed with PSS for another 5 min. Vascular contraction to cumulative doses of NE (10–7 to 10–4 M) were measured to obtain maximal contraction responses for each vessel. The relaxation response to cumulative additions of CGRP (10–10 to 10–7 M) was assessed in uterine artery rings that were precontracted with ED70 concentration of NE. Similar to previous reports [27] we found that ED70 dose of NE maintains contraction response over a longer period of time, and therefore we used this dose for the measurement of vasorelaxation effects of CGRP. The artery rings were incubated with each dose of CGRP for 2 min or until relaxation was stabilized. The relaxation responses of each CGRP dose were calculated as a percentage of tension generated by NE at ED70 dose. In all of these studies, the amplitude of the contraction to NE or relaxation response to CGRP was measured.
The antibodies to Nt-CRLR or Nt-RAMP1 generated and characterized in this study and a previously characterized monoclonal antibody to CGRP-B receptor [13] were utilized to assess the receptor type involved in CGRP signaling in the rat uterine artery. Polyclonal antibodies to Nt- CRLR and Nt-RAMP1 or monoclonal antibody to CGRP-B were added to the muscle bath containing uterine artery segments for 20 min prior to the addition of cumulative doses of CGRP. Preimmune serum from the rabbits was used as control for polyclonal antibodies. In addition, effects of Nt-CRLR antisera preadsorbed with the Nt-CRLR protein prior to adding to the muscle bath was also studied. Varying doses (10–10 to 10–7 M) of CGRP were then added in a cumulative manner and relaxation responses were measured.
Statistics
The change in initial tension of uterine artery rings in response to CGRP was calculated as a percentage of precontraction induced by NE. The PD2 (–logEC50), or concentration of the agent that inhibited 50% of the maximal contraction, was calculated using a nonlinear regression curve (Prism GraphPad Software, Inc, San Diego, CA) from the individual concentration-response relationships. The concentration-response curves were also compared by two-way repeated-measures ANOVA. The data are expressed as means ± SEM for three to five animals per group. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nt-CRLR and Nt-RAMP1 polyclonal antibodies were characterized by studying the linear reaction of the antibodies with their respective antigen and neutralizing the antibody with the antigen. As shown in Figure 1, A and B, both Nt-CRLR and Nt-RAMP1 antibodies showed a linear reaction with increasing amounts of the respective antigen until a plateau was reached. Anti–Nt-CRLR detects a 58- kDa protein in rat uterine membrane and anti–Nt-RAMP1 detects a RAMP1 (16-kDa) protein in the rat uterus. Figure 1, C and D, demonstrate that the detection of these bands by the antibodies was blocked when the antibodies were preadsorbed with their respective antigen prior to use in Western immunoblotting. These data indicate that both Nt- CRLR and Nt-RAMP1 antibodies are specific to the N-terminal domain of CRLR and RAMP1.
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Visualization of Cell Surface Expression of CRLR and RAMP1
It is well established that CRLR and RAMP1, when expressed together, are transported to the cell membrane [12, 28–30]. Nt-CRLR and Nt-RAMP1 polyclonal antibodies were further tested by immunofluorescent localization of these receptors on HEK-293 cells stably transfected with CRLR and RAMP1. Specific immunoflorescent staining of both these receptor components on the plasma membrane was observed as shown in Figure 2. Specificity of these antibodies was further demonstrated by the absence of staining in HEK-293 cells, when antibodies were either eliminated (Fig. 2A) or preadsorbed with respective antigens (Fig. 2B).
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Vasorelaxation Studies Using Nt-CRLR and Nt-RAMP1 Polyclonal Antibodies and CGRP-B Monoclonal Antibody
CGRP (10–10 to 10–7 M) produced a dose-dependent relaxation in rat uterine artery rings precontracted with NE. Incubation of uterine artery segments with Nt-CRLR and Nt-RAMP1 polyclonal antibodies in combination (1:20 000 dilutions each) or with Nt-CRLR antibody alone showed a significant inhibitory effect on the CGRP-induced relaxation of the vessel as compared with the preimmune serum (Fig. 3). In these studies, 1:20 000 dilution of antibodies was found to be optimal based on our pilot observations utilizing 1:40 000 to 1:1000 dilutions (data not shown). The effects of the anti–Nt-CRLR were more substantial as compared with the anti–Nt-RAMP1 on the CGRP-induced vasorelaxation. CGRP-induced uterine artery relaxation was completely abolished when the uterine artery segments were preincubated with the combination of both antibodies (anti–Nt-CRLR + anti–Nt-RAMP1) at the same dilution. The PD2 values for CGRP-induced relaxation in the presence of Nt-CRLR and Nt-CRLR + Nt-RAMP1 antibody were 6.75 ± 0.20 and 6.14 ± 0.35, respectively (P < 0.05), compared with that of preimmune serum (8.0 ± 0.10). The inhibitory effects of antibodies to RAMP1 on CGRP-induced vasorelaxation showed limited dose response (PD2: 7.9 ± 0.14). Because the inhibitory effects of antibodies to Nt-CRLR alone were substantial, we further assessed the specificity of this effect by preadsorbing the antisera with Nt-CRLR protein. The inhibitory effects of Nt-CRLR antibody on CGRP-induced vasorelaxation were completely blocked by the preadsorbed antisera (PD2: –8.03 ± 0.09; Fig. 3). We next investigated the effects of CGRP-B receptor monoclonal antibody on CGRP-induced vasorelaxation in uterine artery segments. As shown in Figure 4, CGRP-B receptor antibody has no effects on CGRP-induced vasorelaxation. Thus, these studies support our hypothesis that CGRP-induced uterine artery relaxation requires CGRP-A receptors and that the Nt-domain of CRLR may be more critical in eliciting CGRP-induced vasorelaxation in rat uterine artery.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study we generated specific polyclonal antibodies to N-terminal domains of CRLR and RAMP1. The specificity of the antibodies was assessed by Western immunoblotting as shown in Figure 1 and immunocytolocalization of CRLR and RAMP1 in HEK-293 cells transfected with CRLR and RAMP1 (Figure 2). As shown in Figure 2, anti– Nt-CRLR and anti–Nt-RAMP1 specifically detect their respective proteins on cell surface in HEK-293 cells transfected with CRLR and RAMP1. In both these methods, the ability of antibody to recognize the antigen was lost with the omission of primary antibody or by neutralization of antibodies with respective antigen protein. Thus, based on these characterization studies, the antibodies are specific to the Nt-domains of CRLR and RAMP1 (CGRP-A receptor components).
In the current study, we used these receptor-specific antibodies to CGRP-A receptor (generated and characterized in this study) and CGRP-B receptor [19] to assess the receptor type involved in mediating CGRP effects in rat uterine artery. CGRP-induced vasorelaxation in pregnant rat uterine artery is blocked when arteries are incubated with the combination of Nt-CRLR and Nt-RAMP1 antibodies (Fig. 3). Nt-CRLR antibody alone is sufficient to substantially block CGRP effects, whereas smaller effects were observed with Nt-RAMP1 antibody. These data suggest the involvement of Nt-CRLR and Nt-RAMP1 in CGRP actions and that CRLR N-terminal domain plays a major role in CGRP-induced vasorelaxation. Present data further support the distinct CGRP receptor pharmacology acquired by CRLR in the presence of RAMP1 and indicate either a direct participation of RAMP1 in forming a selective binding pocket for CGRP or an indirect conformational effect on CRLR to induce optimal CGRP binding [24]. In contrast to the profound effects of Nt-CRLR and Nt-RAMP1 antibodies, the effects of antibodies to CGRP-B receptor were insignificant (Fig. 4). Because the combination of Nt-CRLR and Nt-RAMP1 antibodies could block only 80% of the CGRP-induced relaxation (Fig. 3), the remaining 20% relaxation response may be mediated through other CGRP receptors such as CGRP-B. This is supported by our recent findings that both CGRP-A and CGRP-B receptors are expressed in rat uterine artery [31]. On the other hand, lack of effects of CGRP-B receptor antibodies (Fig. 4) could also result from the possibility that the antigenic epitope on the receptor may not be exposed and thus may not be accessible to the antibody. However, lack of involvement of CGRP-B receptors in CGRP action in uterine artery are further supported by our recent finding that CGRP-B receptor protein levels remain constant at all stages of gestation in rat uterine artery [31], whereas expression of CRLR and RAMP1 protein show an increase with pregnancy as compared with nonpregnant rats (unpublished data). Furthermore, this study, together with our recent report in which we have shown that mRNA levels of CRLR and RAMP1 increased with pregnancy, supports our hypothesis that CGRP effects in pregnant rat uterine artery are primarily mediated through CRLR and RAMP1 [31].
This study shows that Nt-CRLR and Nt-RAMP1 are involved in direct CGRP binding and action. Inhibitory effects of antibodies to Nt-CRLR are more substantial, compared with Nt-RAMP1, suggesting that the antibody binding to the CRLR N-terminal domain may be critical for ligand- induced signaling. A recent study reported that the extracellular domain of RAMP1 is sufficient for normal association with CRLR and that the transmembrane domain contributes to the CGRP affinity and potency [33]. This supports our data, shown in Figure 3, demonstrating inhibitory effects of anti–Nt-RAMP1, although to a lesser extent than Nt-CRLR or Nt-CRLR + Nt- RAMP1 antibody, and that Nt-RAMP1 interacts with either CRLR or CGRP or with both. Defining the precise nature of interaction among CRLR, CGRP, and RAMP1 is important and requires detailed structural and functional studies. However, the present study implies that the major signaling events of CGRP action in the rat uterine artery are mediated through CGRP- A receptor and that polyclonal antibodies for the Nt-domains of CRLR and RAMP1 generated in this study could be useful tools in differentiating functional CGRP receptor type in different tissues.
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FOOTNOTES |
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2 Correspondence: Chandrasekhar Yallampalli, D.V.M., Ph.D., Department of Obstetrics and Gynecology, 301 University Blvd., Medical Research Building, Room 11.138, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Received: 3 October 2003.
First decision: 30 October 2003.
Accepted: 30 January 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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