| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Departments of Obstetrics/Gynecology and
b Internal Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1062
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
placenta, pregnancy, progesterone, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Pregnancy and female reproductive hormones profoundly influence the vascular system. Several studies suggest that estrogens increase vascular relaxation and maintain blood pressure homeostasis, perhaps through induction of nitric oxide synthesis in endothelial cells [8–10]. Studies by Rylance et al. [11] demonstrated that progesterone (P4) reduces blood pressure. Recent studies from our laboratory demonstrated that vascular relaxation responses to CGRP were enhanced during pregnancy and with estrogen (E2) and P4 treatments in ovariectomized rats [12]. Decreases in CGRP-induced total vascular resistance were substantially greater in pregnant rats and in ovariectomized rats treated with E2 + P4 [13]. Furthermore, both pregnancy and E2 + P4 treatment in ovariectomized rats enhanced the effects of CGRP on vascular resistance in mesenteric, coronary, and renal vasculature [13]. The effects of CGRP on reducing blood pressure in NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide-induced hypertension, were greater during pregnancy and with P4 treatment during postpartum [14]. These data collectively suggest that vasodilator effects of CGRP are increased during pregnancy and that the female sex steroid hormones appear to regulate these effects.
Administration of CGRP reversed hypertension and fetal mortality induced by L-NAME in pregnant rats [15, 16], indicating that CGRP can compensate for a perturbed nitric oxide system. Further, an acute bolus injection of CGRP8-37, a potent and specific CGRP receptor antagonist, produced a significant increase in mean arterial blood pressure in rats treated with L-NAME during pregnancy [14]. The mechanism of this effect appears to be an enhanced vascular responsiveness to CGRP during pregnancy. However, it is unknown whether continuous infusion of CGRP8-37 would elevate blood pressure during pregnancy and adversely affect fetoplacental growth.
Pregnancy is associated with an increase in uteroplacental blood flow and a decrease in uterine vascular resistance [17, 18]. Previous studies demonstrated that the effects of CGRP in isolated uterine vascular bed preparation were substantially increased in ovariectomized rats when they were treated with sex-steroid hormones [19]. Potent vasodilatory effects of CGRP were also reported in the uterine artery of sheep [20] and humans [21, 22], and the effects in women are significantly greater during pregnancy. Firth et al. [23] showed that CGRP relaxes human chorionic plate vasculature. Studies by Mandsager et al. [24] using perfused placental cotyledons from term pregnant women showed that CGRP is a potent vasodilator in placenta. These studies suggest a beneficial role for CGRP in uteroplacental vascular function.
In the present studies, we hypothesized that endogenous CGRP plays an important role in maintaining normal fetoplacental development and vascular adaptations during pregnancy. Further, we assessed whether CGRP-related vascular adaptations are P4-dependent. Studies were, therefore, designed to examine the effects of continuous infusion of CGRP8-37, a CGRP receptor antagonist, to pregnant rats on fetoplacental growth, fetal survival, and blood pressure. Because P4 levels are low in postpartum rats compared with levels during pregnancy [25], and because the role of this hormone in modulating the effect of CGRP8-37 on blood pressure is not known, we investigated whether P4 treatment enhances CGRP8-37 effects on blood pressure during postpartum.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Experimental Protocol
Fetal growth in rats accelerated starting at Day 17 of gestation [26]. Therefore, groups of Day 17 pregnant rats (n = 5) were s.c. infused through osmotic minipumps (Alza, Palto Alto, CA) with a pumping rate of 5 µl/h, with varying doses of CGRP receptor antagonist, CGRP8-37 (0.083–1.33 mg day-1 kg-1 BW) in saline, or with saline only. Dosages of CGRP8-37 were chosen on the basis of their effects on blood pressure in our previous studies [14]. We have previously infused CGRP at Day 17 of gestation to Postpartum Day 6 and found significant effects on blood pressure reduction during pregnancy and postpartum, and improved fetal growth and survival [16, 27]. These studies demonstrate the stability of CGRP peptides in saline in minipumps for an extended period of time. Furthermore, integrity of this antagonist recovered from the minipumps was maintained up to 14 days using mass spectrometry (data not shown).
In the second experiment, groups of five pregnant rats were s.c. infused through osmotic minipumps filled with CGRP8-37 (0.33 mg day-1 kg-1 BW) in saline or saline only beginning at Day 17 of gestation. After spontaneous delivery of pups on Day 22 of pregnancy, the CGRP8-37 group received P4 treatments (2 mg/rat in 0.2 ml of sesame oil twice daily, s.c.) from Postpartum Day 1 to Day 6. Controls received oil only. In both experiments, systolic blood pressures were measured daily by the pneumatic tail cuff method (Narco-Biosystems, Houston, TX). After restraining, the rats were allowed to stabilize. Three consecutive measurements (10-sec intervals) were obtained and these values were averaged for each animal. All measurements were made at the same time of day. In animals that delivered spontaneously, pup weight and mortality were recorded immediately (within 1 h).
To determine the effects of CGRP8-37 on fetal growth and placental development before labor, CGRP8-37 (1.33 mg day-1 kg-1 BW) was continuously infused s.c. from Day 17 of gestation, and animals were killed on Day 20 of gestation. In these animals, placentae and fetuses were isolated, blotted to remove fluids and blood, and immediately weighed.
Human CGRP receptor antagonist, CGRP8-37, was synthesized by the use of solid-phase t-butoxycarbonyl chemistry by one of the authors (S.J.W.), purified, and characterized by mass spectrometry, amino acid analysis, and sequencing. Progesterone was purchased from Sigma Chemical Company (St. Louis, MO).
Statistical Analysis
The weight (in grams) of fetuses, placentae, and pups are expressed as means ± SEM and compared using ANOVA or the Student t-test. Fetal and pup mortality were compared using the chi-square test. Systolic blood pressures (mm Hg) were analyzed using repeated measures ANOVA for comparing changes across all days, with an exchangeable covariance structure for the factors of treatment group, day, and treatment x day interaction. In all analyses, P < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Figure 1 shows longitudinal changes in systolic blood pressure in rats during pregnancy from Day 17 of gestation to Day 6 postpartum. CGRP8-37 (0.083–1.33 mg day-1 kg-1 BW) was continuously infused from Day 17 of pregnancy to postpartum Day 6, and blood pressures were measured daily to assess whether the effects of CGRP8-37 were decreased during the postpartum period, when serum P4 levels were expected to be minimal (Fig. 1). Blood pressure increases following infusion of CGRP8-37 during pregnancy were noted with 0.33 and 1.33 mg day-1 kg-1 BW doses, but not with the 0.083 mg day-1 kg-1 BW dose. However, there were no significant differences in the blood pressure of pregnant rats between 0.33 and 1.33 mg day-1 kg-1 BW. The magnitude of increases in blood pressure during pregnancy persisted for most of the postpartum period, with the 1.33 mg day-1 kg-1 BW dose, but not with the 0.33 mg day-1 kg-1 BW dose.
|
Progesterone Modulation of CGRP8-37 Effects on Blood Pressure in Postpartum Rats
CGRP8-37 at the 0.33 mg day-1 kg-1 BW dose elevated the systolic blood pressure during pregnancy on Days 19 and 20, a period when serum P4 concentrations are still elevated, but not during postpartum, a period of minimal levels of circulatory P4. We examined the modulatory role of P4 on CGRP8-37 effects on blood pressure in postpartum rats (Fig. 2). Small increases in blood pressure were persistent with CGRP8-37 at 0.33 mg day-1 kg-1 BW from Postpartum Day 4 to Postpartum Day 6. However, administration of P4 to rats treated with CGRP8-37 rats substantially increased the blood pressure beginning from Postpartum Day 3 until Postpartum Day 6.
|
Effects of Infusion of CGRP8-37 During Pregnancy on Pup Weight and Mortality
We assessed the role of endogenous CGRP on pregnancy outcome by continuously infusing CGRP8-37 from Day 17 of gestation. The animals were closely monitored throughout the infusion period, and none of the animals delivered prematurely. All animals delivered spontaneously at term. Figure 3 shows the changes in pup weight and pup mortality when CGRP8-37 (0.083–1.33 mg day-1 kg-1 BW) was infused compared with those of saline-treated rats. Mortality rate and the weight of pups were both unchanged in rats receiving 0.083 mg day-1 kg-1 BW. However, the weight of pups in rats receiving 0.33 and 1.33 mg day-1 kg-1 BW were substantially (P < 0.05) lower than saline-treated controls. In these animals, the mortality rate of pups was also significantly (P < 0.05) increased. The differences in pup weights and mortality rate among rats that received CGRP8-37 at 0.33 and 1.33 mg day-1 kg-1 BW were not significant.
|
Effects of CGRP8-37 Infusion During Pregnancy on Fetal and Placental Weights
To further assess whether the effects of CGRP8-37 on fetal growth, fetal mortality, and placental weights could be detected before term on Day 20 of gestation, we infused CGRP8-37 (1.33 mg day-1 kg-1 BW) beginning on Day 17 of gestation. The animals were killed 72 h later and the fetal weights, placental weights, and fetal deaths were recorded. Figure 4 shows that CGRP8-37 substantially decreased both placental (Fig. 4A) and fetal (Fig. 4B) weights compared with those of controls. CGRP8-37 infusion also caused increases in the percentages of dead fetuses compared with age-matched controls (Fig. 4C).
|
DISCUSSION
In this study, we describe the effects of CGRP8-37, a receptor antagonist for CGRP, on systolic blood pressure, fetoplacental growth, and fetal and pup mortality. We also investigated the effects of administration of exogenous P4 to CGRP8-37-induced increases on blood pressure in postpartum rats. Chronic infusion of CGRP8-37 from Day 17 of gestation (the beginning of the rapid fetal growth phase) caused an elevation in systolic blood pressure during pregnancy, reduced fetoplacental growth, and increased pup mortality. These effects of CGRP8-37 on systolic blood pressure, and on pup weight and mortality, were significant with 0.33 and 1.33 mg day-1 kg-1 BW doses but not with the 0.083 mg dose. Elevated blood pressure persisted during postpartum, with the highest dose (1.33 mg) used, and with the 0.33 mg dose, the elevated blood pressure was observed only up to Day 2 of postpartum. Furthermore, when P4 was injected in postpartum animals that received the 0.33 mg day-1 kg-1 BW dose of CGRP8-37, systolic blood pressure was significantly elevated. Infusion of CGRP8-37 decreased the pup weight and increased pup mortality without altering gestational length. Both placental and fetal weights were reduced within 72 h of CGRP8-37 infusion to pregnant rats. These data suggest that endogenous CGRP may play a potent vasodilator role in both reproductive and nonreproductive vascular systems, and in maintaining normal fetoplacental development.
The role of endogenous CGRP in the regulation of blood pressure during pregnancy is not well understood. CGRP is widely distributed in the central and peripheral nervous systems in mammals [3, 4]. Circulatory CGRP levels increase during pregnancy and decrease after delivery in rats and humans [28–30]. Several reports from radio ligand and functional studies indicate that CGRP8-37 is a competitive inhibitor of CGRP binding [31, 32]. Intravenous bolus administration of CGRP8-37 caused inhibition of hypotensive and vasodilator effects of exogenously administered CGRP in rats [33–35]. Studies by Hughes and Brain [36] demonstrated that CGRP8-37 was able to inhibit increases in blood flow in response to capsaicin, an agent that stimulates the release of CGRP from sensory nerve terminals. Furthermore, an increase in skin blood flow by CGRP was blocked by CGRP8-37. However, no studies reported to date have examined the continuous infusion of this receptor antagonist either on blood pressure or on fetal growth. For the first time in the present study, we show that CGRP8-37 elevated systolic blood pressure during pregnancy (Fig. 1), suggesting a role for endogenous CGRP for maintaining normal blood pressure during pregnancy.
Circulatory levels of P4 increase during pregnancy compared to the nonpregnant state. Progesterone levels decline at the end of gestation, with the lowest levels being reached during postpartum [25]. Therefore, the postpartum period serves as a good control for low P4 levels. Because the 0.33 mg CGRP8-37 dose was sufficient to cause increases in systolic blood pressure during pregnancy, we used this dose to assess whether P4 alters the effects of this antagonist on vascular tone during postpartum. Administration of P4 to postpartum rats caused elevated blood pressure when 0.33 mg day-1 kg-1 BW of CGRP8-37 was infused (Fig. 2). The mechanisms involved in the ability of P4 to alter CGRP8-37 effects are not addressed in this study. However, it is possible that P4 may up-regulate CGRP receptors in the blood vessels, and therefore, CGRP8-37 may block the effects of endogenous CGRP on blood pressure in postpartum rats. Moreover, the systolic blood pressure in control animals appear to increase immediately postpartum (Days 1 and 2) compared with Day 17 of pregnancy (Fig. 1), which is consistent with previous findings reported by Liao et al. [37]. These studies suggest that decreases in steroid hormone concentrations (pregnant vs. postpartum) may increase blood pressure in postpartum rats. Several lines of evidence suggest that elevated levels of female sex steroid hormones may play a beneficial role in maintaining vascular adaptations during pregnancy through several locally produced vasoactive agents. The studies by Grewal et al. [19] demonstrated that mean arterial blood pressure was decreased by a bolus injection of CGRP in the presence of estradiol-17ß (E2) and P4 in nonpregnant, ovariectomized rats. We recently reported [12] that female sex steroid hormones significantly elevated vascular relaxation sensitivity to CGRP. Progesterone regulates the blood pressure-lowering effects of CGRP in L-NAME-induced hypertension in postpartum and nonpregnant ovariectomized rats [27]. It has been reported that CGRP receptors are gestationally regulated in rats in both uterus and in resistance blood vessels [38, 39]. These studies, together with our current findings, suggest that P4 appears to regulate endogenous CGRP effects on the vascular system during pregnancy and postpartum, perhaps through the regulation of its receptor levels.
We next examined whether inhibition of endogenous CGRP by CGRP8-37 would inhibit fetoplacental growth and decrease pup survival. These data for the first time show that chronic infusion of CGRP8-37 dose-dependently (Fig. 3) causes fetal growth retardation and increases mortality, similar to the effects we reported with L-NAME treatment [16]. Further, the effects of CGRP8-37 on pup weight and mortality observed in our studies appeared to occur within 72 h after the start of CGRP8-37 infusion. Placental weights were significantly reduced in CGRP8-37-treated rats in parallel with fetal weights, when examined on Day 20 of gestation (Fig. 4). The detailed mechanisms through which CGRP8-37 caused fetoplacental growth retardation are not addressed in this study. However, we suggest that this could occur by decreasing uterine blood flow. Recent reports indicate CGRP decreases uterine vascular resistance in rats [19] and that the uterine artery relaxation sensitivity to CGRP was higher in pregnant women than in nonpregnant women [21, 22] and rats [40]. Potent vasodilatory effects of CGRP were also reported for sheep uterine artery [20]. A reduction in placental weights during CGRP8-37 infusion further suggests that a reduction in uterine blood flow, resulting in placental growth being compromised in these animals. Reduced placental growth could then lead to fetal weight reductions. Moreover, CGRP has been reported to relax chorionic plate vasculature [23] and to decrease perfusion pressure in placental cotyledons [24] in humans. CGRP binding sites in placenta have been previously reported [41]. Therefore, it is possible that CGRP8-37 interfered with endogenous CGRP effects on uterine artery relaxation; therefore, reduced uteroplacental blood flow results in fetal growth retardation.
We and others have previously reported that CGRP is not only a vasodilator, but also a potent uterine smooth muscle relaxant in both rats [42] and humans [43, 44]. In fact, the uterine relaxation sensitivity to CGRP was substantially increased during pregnancy and decreased at term on Day 22 before labor, implicating a role for this peptide in maintaining uterine quiescence during pregnancy [42]. In the current study, infusion of CGRP8-37 from Day 17 of gestation failed to produce preterm labor. Thus, we suggest that inhibition of the uterine relaxant, CGRP alone, may not induce labor and that initiation of labor may require additional events to occur simultaneously, such as higher levels of contractile-associated proteins [45].
In summary, inhibition of endogenous CGRP through continuous infusion of CGRP8-37 during pregnancy caused increases in systolic blood pressure together with fetal growth retardation and increased fetal mortality, and that these effects were dose-dependent. Progesterone modulates CGRP8-37 effects on blood pressure in postpartum rats. The detrimental effects of CGRP8-37 on fetal growth and survival occurred in parallel with the effects on placental weight, indicating that uteroplacental function is compromised in these animals, perhaps through decreases in uterine blood flow. These studies suggest that endogenous CGRP plays an important role in maintaining normal fetoplacental development and vascular adaptations during pregnancy, and that this may be mediated by progesterone. The one or more mechanisms responsible for these adaptations could be due to an increase in CGRP-induced vascular relaxation both in uterine and resistance blood vessels in pregnant rats.
|
FOOTNOTES |
|---|
1 This work was supported in part by the National Institutes of Health through grants HL-58144, HD 30273, and HD-98-004. 
2 Correspondence: Chandrasekhar Yallampalli, 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
Accepted: March 4, 2002.
Received: October 31, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  G. R. Ross and C. Yallampalli Endothelium-Independent Relaxation by Adrenomedullin in Pregnant Rat Mesenteric Artery: Role of cAMP-Dependent Protein Kinase A and Calcium-Activated Potassium Channels J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1269 - 1275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 P.R.R. Gangula, P. Lanlua, R.D. Bukoski, S.J. Wimalawansa, and C. Yallampalli Mesenteric Arterial Relaxation to Calcitonin Gene-Related Peptide Is Increased During Pregnancy and by Sex Steroid Hormones Biol Reprod, November 1, 2004; 71(5): 1739 - 1745. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 H. Yin, L. Chao, and J. Chao Adrenomedullin Protects Against Myocardial Apoptosis After Ischemia/Reperfusion Through Activation of Akt-GSK Signaling Hypertension, January 1, 2004; 43(1): 109 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.R.R. Gangula, C. Thota, S.J. Wimalawansa, R.D. Bukoski, and C. Yallampalli Mechanisms Involved in Calcitonin Gene-Related Peptide-Induced Relaxation in Pregnant Rat Uterine Artery Biol Reprod, November 1, 2003; 69(5): 1635 - 1641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
P < 0.05 for 1.33 mg CGRP8-37
