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Pregnancy |
Department of Obstetrics and Gynecology,3 University of Texas Medical Branch, Galveston, Texas 77555
Department of Endocrinology,4 Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901
Cardiovascular Disease Research Program,5 North Carolina Central University, Durham, North Carolina 27707
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mechanisms of hormone action, neuropeptides, pregnancy, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is synthesized primarily in the dorsal root ganglia (DRG) [5] that send CGRP-containing nerves peripherally to blood vessels and centrally to the spinal cord [6, 7]. CGRP has been shown to inhibit spontaneous contractions in the uteri of rats and humans [8–10]. Furthermore, previous studies indicate that receptors for CGRP are elevated in pregnant rat and human uteri [10, 11]. Potent vasodilatory effects of CGRP were also noticed in the uterine arteries of sheep and humans. This effect is significantly greater in the arteries of pregnant women [12]. The studies of Grewal et al. [13] found that the vasodilatory effects of CGRP in isolated uterine vascular bed preparations were substantially increased when nonpregnant ovariectomized rats were treated with sex steroid hormones. Collectively, these data suggest that CGRP may play a major role in regulating blood flow to the uterus during pregnancy. Sex steroid hormones may be involved in this process.
Previously, CGRP receptors were classified as CGRP1 or CGRP2 receptors based primarily on their differential affinity for CGRP antagonist, CGRP8-37 [14–16]. However, these differential affinity studies have been inconsistent [17, 18], and a recent report by Rorabaugh et al. [19] questioned the existence of separate CGRP2 receptors. Recent studies from our laboratory showed the existence of a CGRP receptor that is different from a well-characterized CGRP receptor component, calcitonin receptor-like receptor (CRLR) [20, 21]. A monoclonal antibody to the CGRP receptor specifically detects a 66-kDa protein from rat cerebellum and other rat and human tissues but not from SK-N-MC cells, which expresses CRLR [21]. Based on these findings, CGRP receptors are classified as CGRP-A and CGRP-B receptors. CGRP-A receptors consist of a well-characterized 7-transmembrane CRLR; a single transmembrane accessory protein, receptor activity modifying protein 1 (RAMP1); and the CGRP-B receptor that is not related to CRLR [20, 21]. Changes in these receptor complexes and postreceptor signaling may underlie changes in sensitivity to CGRP in uterine vasculature and, therefore, changes in uterine blood flow during pregnancy. In addition, several secondary messenger pathways, including cyclic adenosine monophosphate (cAMP), nitric oxide, and ATP-sensitive K+ channel activation, have been suggested to mediate CGRP-induced vasodilation in a variety of blood vessels.
In the present study, we hypothesized that CGRP-induced relaxation of uterine arteries is greater in the pregnant rat compared to the nonpregnant diestrus rat (NP-DE) and that receptor and postreceptor mechanisms are involved in this process. We further hypothesized that CGRP receptors, CGRP-A and CGRP-B, are elevated during pregnancy. To test these hypotheses, the following experiments were undertaken.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uterine Artery Relaxation Studies
Groups of rats (n = 3–4) that were either pregnant on Day 18 of gestation (250–300 g BW) or nonpregnant (175–200 g BW) at diestrus (NP-DE) were used in the present study. Rats were deeply anesthetized by an intraperitoneal injection of ketamine (45 mg/kg BW; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg BW), and the uterine artery was carefully dissected. Uterine artery rings (2-mm length, 
250–400 µm) 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 segments were mounted in the jaws of a wire myograph (Kent Scientific, Litchfield, CT) for the measurement of isometric tension [22]. The unstretched vessels 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 lumen of each segment was stretched to a length that would give a diameter of 250 (nonpregnant) to 400 µm (pregnant) and were allowed to equilibrate for 15 min. Finally, 5 µM norepinephrine (NE) was added into the chamber to stimulate the contraction 3–4 times. Each time, the uterine artery segment was incubated with NE for 5 min and washed with PSS for 5 more minutes. The relaxation response to CGRP in NP-DE and Day 18 pregnant rats' uterine artery segments was investigated at doses of CGRP between 10-10 and 10-7 M in a cumulative manner. The vessel rings were precontracted with the ED70 concentration of NE that was determined for each vessel.
Mechanisms for the CGRP-Induced Relaxation in Day 18 Pregnant Rat Uterine Artery
The involvement of receptors, various intracellular messengers, and ion channels in CGRP-induced vascular relaxation was investigated using selective inhibitors. In these studies, we first assessed vascular reactivity of uterine arteries of rats on Day 18 of pregnancy to varying doses of CGRP (10-10–10-7 M) with NE preconstriction using a wire myograph. Subsequently, CGRP was washed out with PSS, and the uterine arterial segments were incubated for 30 min in fresh PSS with either CGRP8-37 (10-4 M, a receptor antagonist for CGRP), the inhibitors of adenylate cyclase (SQ 22536, 10-5 M), guanylate cyclase (1H-[1,2,4] oxadizaolo [4,3-a] quinoxalin-1-one ODQ, 10-5 M), adenosine triphosphate-sensitive (glybenclamide, 10-5 M) or calcium-dependent (tetraethylammonium, 10-3 M) potassium (K+) channels, or nitric oxide (NG-nitro-L-arginine methyl ester, L-NAME, 10-4 M). After the incubation period, relaxation responses to cumulative doses of CGRP (10-10–10-7 M) were repeated in NE-precontracted arterial segments. For each segment, the CGRP-induced vasorelaxations were calculated as the percentage of precontracted tension induced by norepinephrine (ED70). All the chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). ODQ was dissolved in 1% DMSO. All the other chemicals were dissolved in cold PSS buffer. The dose(s) we have selected for CGRP8-37, SQ22536, glybenclamide, tetraethylammonium, ODQ, and L-NAME were based on results from other published studies [23] and our preliminary studies (data not shown).
Expression of CRLR and RAMP1 in Rat Uterine Artery Homogenates
Total tissue RNA was isolated from the rat uterine artery by a single-step guanidine thio-cyanate method using the reagent Trizol (Gibco/BRL, Gaithersburg, MD). Complementary DNA (cDNA) was synthesized from 2 µg of total RNA by reverse transcription using the Ready-To-Go RT-PCR beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ) as described by the supplier. For reverse transcription, samples were placed into a thermal cycle for one cycle at 28°C for 15 min, 42°C for 30 min, 99°C for 5 min, and 5°C for 5 min. PCR reactions were initiated by the specific primer sets for CRLR, RAMP1 [24], and 18S rRNA (Ambion Inc., Austin, TX). Briefly, 2.5 µl of the cDNA were mixed with a PCR mixture containing 2.5 mM MgCl2, 1x PCR buffer (Sigma), 200 µM dNTPs (Sigma), 0.2 units of Taq DNA polymerase (Sigma), and 300 nM of forward and reverse primers for CRLR and RAMP1 in a final volume of 50 µl for each reaction. For 18S, 2 ml of primer pair were used as per the supplier's specifications. PCR reactions were carried out on GeneAmp PCR System 9700 (Perkin Elmer, Branchburg, NJ) with the following conditions: An initial denaturation step at 95°C for 5 min was followed by 35 cycles of 30 sec at 95°C, 90 sec at 60°C, and 30 sec at 72°C. Reactions were terminated by a 7-min elongation step at 72°C. The total cycle number was chosen for each from the linear portion of the curve (data not shown). PCR products were visualized on 1.6% agarose gel containing 0.5 µg/ml ethidium bromide, run in 0.5x TBE buffer at 100 V for 1.5 h. Gels were placed on a UV light box, imaged, and analyzed with the Sigma Gel software. The identities of the amplified sequences were verified by direct double-strand sequencing. The sequences showed 100% homology to the published sequences (data not shown). The expressions of CRLR and RAMP1 mRNA were normalized with their respective 18S values. The results were expressed as the ratio of the densitometric readings of CGRP receptor components to 18S readings; therefore, the results indicate relative changes in CRLR and RAMP1 in the rat uterine artery.
Western Immunoblotting Analysis of CGRP-B Receptors
Equal amounts of protein (Pierce Kit, Rockford, IL) from NP-DE and pregnant Day 18 rat uterine artery homogenates were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and primed with primary CGRP receptor monoclonal antibody (raised against the affinity-purified CGRP receptors for porcine cerebellum by Dr. Sunil Wimalawansa) [14] for 1 h. The membrane was washed three times with TTBS (20 mM Tris [hydroxymethyl] aminomethane-HCl, pH 7.6, 0.05% Tween 20, 100 mM NaCl) and then 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 Sigma Gel software (Sigma).
Statistics
The change in initial tension of uterine artery rings in response to CGRP was calculated as a percentage of precontraction induced by norepinephrine. The pD2 (-log EC50), 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 analysis of variance (ANOVA). Unpaired Student t-test or one-way ANOVA was used for comparison of two or more means, respectively (Bonferroni post hoc test). The data are expressed as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CGRP (10-10–10-7 M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in both nonpregnant and pregnant Day 18 uterine arteries (Fig. 1). The overall vasorelaxation sensitivity to CGRP is greater in pregnant uterine arteries (P < 0.05, ANOVA and Bonferronni-Dunn tests) compared to nonpregnant. The pD2 values were significantly lower (P < 0.05) in NP-DE (7.00 ± 0.40) compared to pregnancy (8.00 ± 0.10). Next, we examined whether CGRP-induced uterine artery relaxation in pregnant rats is mediated by its receptors. We used the antagonist of CGRP, CGRP8-37. As shown in Figure 2, blockade of the CGRP receptor by CGRP8-37 abolished CGRP-induced relaxation of the pregnant rat uterine artery (P < 0.05).
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In order to investigate whether nitric oxide and cyclic guanosine monophosphate (cGMP) pathways are involved in CGRP-induced relaxation effects, pregnant Day 18 uterine arteries were incubated for 30 min in the presence of either L-NAME (nitric oxide inhibitor) (10-4 M) or ODQ (inhibitor of guanylate cyclase) (10-5 M) and compared with the relaxant effects of CGRP (control) obtained prior to these drugs being added to the same uterine artery rings. As shown in Figure 3A, L-NAME did not inhibit the vasodilator effects of CGRP in pregnant rat uterine arteries (pD2, 7.52 ± 0.31) compared with control (7.41 ± 0.29). However, the relaxation responses to CGRP were significantly (P < 0.05) attenuated by the inhibitor of guanylate cyclase (ODQ) (pD2, 6.20 ± 0.30) compared with the control group (pD2, 7.36 ± 0.41) (Fig. 3B).
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Next, we examined whether the cyclic adenosine monophosphate (cAMP) generation pathway is involved in CGRP-induced uterine artery relaxation. Day 18 gestation rat uterine artery segments were preincubated with 10-5 M SQ22536, an inhibitor of adenylate cyclase, and dose-response relaxant effects of CGRP were measured. Results showed that the overall relaxation response (P < 0.05, two-way ANOVA) to CGRP was significantly reduced in the presence of the inhibitor of adenylate cyclase (Fig. 4) (pD2; control, 8.00 ± 0.10: SQ 22536, 6.60 ± 0.36). Further, inhibition of adenosine triphosphate-sensitive K+ channels (KATP) with glybenclamide (10-5 M) or calcium-sensitive K+ (KCA) channels with tetraethylammonium (10-3 M) significantly (P < 0.05) altered vasorelaxation to CGRP in pregnant rat uterine arteries (Fig. 5, A and B). The pD2 values for glybenclamide were 6.86 ± 0.24 compared with the control (7.66 ± 0.29). Similarly, pD2 values in the presence of tetraethylammonium were 6.23 ± 0.29 compared with the control (8.00 ± 0.10).
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Pregnancy-Associated Changes in the Expression of mRNA for CRLR and RAMP1 in Rat Uterine Arteries
We measured the mRNA levels for both CRLR and RAMP1 in uterine artery from rats on Day 18 of gestation and in the nonpregnant diestrus (NP-DE) stage. The uterine artery was dissected, cleaned of fat, and subjected to RNA extraction as described in Methods. Figure 6 shows the changes in mRNA for CRLR and RAMP1 in uterine arteries from nonpregnant and Day 18 pregnant rats, expressed relative to 18S from each specimen. The data indicate that both CRLR and RAMP1 mRNA were substantially increased (P < 0.05) in rat uterine arteries obtained from pregnant rats compared with those of nonpregnant rats.
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Western Immunoblotting of CGRP-B Receptors in Rat Uterine Arteries
We next investigated whether CGRP-B receptors are expressed in rat uterine arteries and, if so, whether these receptor levels are altered with pregnancy. As shown in Figure 7, a single band of CGRP-B receptor protein was obtained with a predicted size of 66 kDa. Densitometric analysis of the CGRP-B receptor proteins from the uterine arteries of four animals in each group showed that the levels of this protein were not influenced with pregnancy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have shown that CGRP-induced uterine artery relaxation is greater in pregnant rats compared with those in the nonpregnant state (Fig. 1). Similar observations were reported by Nelson et al. [12] using human uterine arteries, in which the vascular relaxation sensitivity to CGRP is enhanced in pregnant uterine blood vessels compared with nonpregnant individuals. These studies further show that the endogenous content of CGRP-like immunoreactivity was significantly greater in uterine arteries from pregnant patients compared with uterine arteries from nonpregnant patients. We and others reported that CGRP-induced relaxation of uterine smooth muscle is increased during pregnancy in both rats and humans [8–11]. Further, CGRP-induced decreases in blood pressure were significantly greater in pregnant compared with nonpregnant rats [26]. The vasodilatory effects of CGRP in uterine vascular beds appeared to be modulated by female sex steroid hormones [13]. Collectively, these studies suggest that enhancing the uterine artery relaxation sensitivity effects to CGRP may be important to maintaining adequate blood flow to the uteroplacental compartment during pregnancy. Sex steroid hormones may be involved in this process.
Several reports from radioligand and functional studies indicate that CGRP8-37 is a competitive inhibitor of CGRP binding as well as the vasodilatory effects of CGRP [15, 27]. CGRP8-37 inhibits vasodilation of the rat mesenteric arterial bed induced by periarterial nerve stimulation and the hemodynamic actions of CGRP in the conscious rat [28]. Chronic administration of CGRP8-37 to pregnant rats caused an increase in systolic blood pressure and fetal mortality and reduced the weight of fetuses [25]. Acute administration of CGRP8-37 produced a significant increase in mean arterial blood pressure in L-NAME-treated pregnant rats [29]. In the present study, preincubation of uterine artery segments with CGRP8-37 significantly (P < 0.05) inhibited CGRP-induced relaxation (Fig. 2), suggesting that the vasodilatory effect of CGRP in uterine arteries is mediated by its receptors.
Generally, it is accepted that CGRP-induced relaxation in small resistance arteries is due to a direct receptor-mediated effect on the smooth muscle, whereas it is endothelium dependent in the large conduit arteries [30, 31]. The relaxation effects of CGRP are mediated via an increase in the intracellular cAMP concentration in a variety of smooth muscle cells [32–34]. Further, it has been shown that part of the CGRP-induced relaxation in mesenteric arteries is mediated by the opening of ATP-sensitive K+ channels [23]. Moreover, involvement of cAMP pathways in CGRP-induced K+-channel activation in smooth muscle has been reported [35]. Thus, CGRP could increase cAMP or activate K channels in causing uterine artery relaxation.
Next, we investigated whether nitric oxide and cGMP are involved in CGRP-induced vascular effects in pregnant uterine arteries. Results show that ODQ (Fig. 3B) but not L-NAME (Fig. 3A) inhibited the CGRP-induced vascular relaxation effects in pregnant uterine arteries partially but significantly. Studies of Anouar et al. [36] suggested that the relaxant effects of CGRP are abolished by inhibitors of nitric oxide synthase in rat aortas but not in the uterus. Recent studies in mouse uteri demonstrated that the relaxation of the myometrium by CGRP is independent of nitric oxide synthase activity [37]. Moreover, studies by Lu and Fiscus [38] postulated that the relaxation effects of CGRP appeared to be mediated through the cGMP inhibition of type III phosphodiesterase and subsequent accumulation of cAMP in smooth muscles. L-NAME failed to inhibit CGRP-induced uterine artery relaxation, suggesting that the endothelium and the release of nitric oxide in response to CGRP are not major contributors in this setting. However, further studies are required to address the involvement of cGMP on CGRP-induced vasodilation in pregnant rat uterine arteries.
Both ATP-sensitive and Ca2+-activated K+ channels have been implicated in mediating CGRP-induced relaxation for a variety of vascular beds. Glybenclamide, an inhibitor of ATP-sensitive K+ channels, antagonized CGRP effects in mesenteric arteries [39] and pulmonary vascular beds [40] as well as on hypotension in rabbits [41], suggesting the involvement of ATP-sensitive K+ channels. Studies of Nelson et al. [23] demonstrated that CGRP relaxes norepinephrine-contracted human uterine arteries, at least in part, by activation of ATP-sensitive K+ channels but not calcium-activated K+ channels. The present study shows that both glybenclamide and tetraethylammonium reduced the relaxation responses to CGRP in rat pregnant uterine arteries (Fig. 5, A and B). A diversity of opinion exists concerning the types of K+ channels in vascular smooth muscles. Although the ATP-sensitive K+ channel is present, the large conductance Ca2+-activated K+ channel may predominate in a number of vascular smooth muscles, such as the guinea pig mesenteric artery [42] and the human cystic artery [43]. Our findings that both glybenclamide and tetraethylammonium significantly inhibited CGRP-induced uterine artery relaxation suggests the involvement of both ATP- and Ca2+-activated K+ channels in CGRP-induced relaxation of pregnant rat uterine blood vessels.
At the present time, it is unclear whether these signal-transduction pathways involved in CGRP-induced vascular relaxation are independent or interdependent. Data from this study suggest that cAMP, cGMP, and potassium channels appeared to be involved in CGRP-mediated uterine artery relaxation in pregnant rats. Reports indicate that the cAMP pathway is also involved in the CGRP-induced K+ channel activation and in transient decreases in the [Ca2+]I and long-lasting Ca2+ desensitization [44] in vascular smooth muscles [35, 45]. However, a decrease of intracellular Ca2+ in the vascular smooth muscle cells could be the final common pathway of the vascular relaxation effects of CGRP. Additional studies, including assessment of the generation of cAMP and cGMP in uterine arteries in response to CGRP, are required to fully understand the signaling "cross talk" in CGRP-induced uterine artery relaxation.
In this study, we assessed if changes in CGRP receptors in rat uterine arteries could contribute to the increased relaxation sensitivity to CGRP during pregnancy. Both CRLR and RAMP1, components of the CGRP-A receptor, are expressed in the uterine artery, and levels are elevated in pregnant compared with nonpregnant rats (Fig. 6). On the other hand, although CGRP-B receptors are expressed in both NP-DE and pregnant rat uterine arteries, the levels of this protein remained unchanged during pregnancy (Fig. 7). Therefore, we suggest that during pregnancy, elevated levels of CRLR and RAMP1 in uterine artery may contribute to enhance the relaxation sensitivity to CGRP.
Several studies in both animals and humans demonstrated that endogenous vasodilators, such as nitric oxide and prostacyclin, may be essential in maintaining uterine blood flow during pregnancy [1]. Data from the present study, together with our previous findings [25, 46], suggest that CGRP may also play an important role in modulating uteroplacental blood flow during pregnancy and, therefore, maintaining normal fetoplacental growth and survival.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, 301 University Boulevard, Medical Research Building, Room 11.138, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Received: 28 January 2003.
First decision: 21 March 2003.
Accepted: 26 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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