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Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555
ABSTRACT
Cardiovascular adaptation is a hallmark of pregnancy. Here we report on vascular hyperresponsiveness to an endogenous vasodilator, adrenomedullin (ADM), during pregnancy. Intravenous administration of ADM dose dependently decreased the mean arterial pressure, and the decrease was significantly greater in pregnant compared with nonpregnant rats without affecting the heart rate. In endothelium-intact mesenteric artery precontracted by ED70 concentration of norepinephrine, the potency and efficacy of ADM in causing the vasodilation of mesenteric arterial rings from pregnant rats are significantly higher compared with nonpregnant females at diestrus. The magnitude of inhibition of concentration-dependent response to ADM by the inhibition of either soluble guanylate cyclase or adenylate cyclase was greater in pregnant rats. Moreover, ADM-induced cyclic nucleotide generation, both cGMP and cAMP, in the mesenteric artery was elevated during pregnancy and was sensitive to the receptor antagonist, ADM22–52. These findings suggest that during pregnancy the vasodilatory effects of ADM are greater and are associated with increased generation of cyclic nucleotides in resistance vessels, and these changes may be part of the cardiovascular adaptations that occur during pregnancy.
adrenomedullin, blood pressure, cyclic adenosine monophosphate, cyclic guanosine monophosphate, cyclic nucleotides, pregnancy, vascular relaxation
Pregnancy is associated with many cardiovascular adaptations to ensure adequate blood supply to growing fetuses. Despite an increase in blood volume and cardiac output of 40% to 50%, pregnancy is characterized by a decrease in mean arterial blood pressure (MAP) [1]. During pregnancy, the generalized decrease in blood pressure results from reduced vascular resistance, and blood pressure returns to nonpregnancy levels in the postpartum period [2]. The marked vascular functional changes during pregnancy are attributed to several physiologic modifications of a wide range of factors. These include augmented plasma levels of endogenous vasodilators such as adrenomedullin (ADM) [3], calcitonin gene-related peptide (CALCA) [4], modifications of mechanical properties and/or tissue composition leading to increased elasticity of blood vessel walls, and reduced excitation-response coupling for vasoactive substances like angiotensin II, vasopressin, and norepinephrine (NE) [5]. Although there are numerous reports on the pressor agents with respect to vascular reactivity during pregnancy, further investigations are needed on the role of endogenous vasodilators such as ADM on these vascular adaptations during pregnancy.
Adrenomedullin, a 52-amino acid peptide, was discovered in 1993 from a panel of peptides extracted from a pheochromocytoma [6]. Later it was realized that ADM is produced by a wide range of cells, including vascular endothelial and smooth muscle cells. Acute or chronic administration of ADM resulted in a significant decrease in total peripheral resistance accompanied by a fall in blood pressure in both conscious and hypertensive rats [7]. Elevated plasma concentrations of ADM during pregnancy in both humans [8] and rats [3] suggest a potential role of ADM in pregnancy. Moreover, both the maternal and umbilical cord plasma levels of ADM were reported to be elevated in preeclampsia [8]. Because the ADM levels are increased both in normal and preeclamptic pregnancies, we propose that the vascular reactivity to ADM may be increased during pregnancy, and this may be one of the mechanisms of vascular adaptation and may possibly be impaired in preeclampsia. ADM is reported to mediate its effects through both cGMP-dependent [9] and cAMP-dependent [10] pathways. In this study, we hypothesized that ADM-induced vasodilation is enhanced in pregnant rats through increased peripheral arterial vasorelaxation responses to ADM, and the enhanced vasodilation is associated with increased generation of cyclic nucleotides. Thus, in this study we assessed the changes in blood pressure in vivo and the vasorelaxation sensitivity of mesenteric artery in vitro to ADM in pregnant and nonpregnant rats and determined the role of cyclic nucleotides in this process.
All studies were performed using 200–250 g female Sprague Dawley rats obtained from Harlan Sprague Dawley (Houston, TX) that either were timed pregnant or nonpregnant. All rats were maintained in the colony room with a fixed photoperiod of 12L:12D and with access to water and rodent chow ad libitum. Rats were used for in vivo experiments or killed for in vitro experiments either on Day 20 of pregnancy or in nonpregnant state at diestrus stage determined by vaginal smear examination. All procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch and were in accordance with those published by the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996).
Measurement of Mean Arterial Pressure
The animals were anesthetized with ketamine (45 mg/kg–1 body weight; Burns Veterinary Supply, Westbury, NY) and xylazine (5 mg/kg–1 body weight; Burns Veterinary Supply). Catheters (PE-50; Becton Dickinson, Sparks, MD) were inserted into the left carotid artery to continuously measure MAP using a DBP001 direct BP system (Kent Scientific, Litchfield, CT) and into the right jugular vein to administer either vehicle (saline) or ADM. Six hours after surgery, with the animals in a fully awake and free-moving state, MAP and heart rate were continuously measured and recorded using Workbench for Windows data acquisition software, version 3.0 (Strawberry Tree Inc., Sunnyvale, CA). Animals received a bolus injection (200 µl) of either saline or saline containing varying concentrations of ADM (0.1–4 nmol/l). Continuous monitoring of MAP allowed us to assess the peak responses after each dose of ADM administration, and this occurred between 0.5 and 1.5 min, primarily around 1.0 min after injections. Therefore, we calculated the changes in MAP and heart rate after each dose of ADM or saline alone from that immediately before injection.
Preparation of Blood Vessel and Recording of Isometric Tension
The animals were killed by exsanguination under deep anesthesia induced by i.p. injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). The small intestine, including the blood supply, was cut and placed in physiologic salt solution (PSS) and kept on ice. The PSS contained the following composition (mmol/l): NaCl (114), KCl (4.7), KH2PO4 (1.15), Na2HPO4 (1.10), MgSO4.7H2O (1.18), NaHC03 (15), CaCl2 (1.5), and glucose (5.0). Secondary branches of the mesenteric artery then were isolated and cleaned of fat and connective tissue. The arterial segments (length, ~2 mm) were mounted on a wire myograph (Kent Scientific) using tungsten wires and were incubated for 15 min in PSS at 37°C, which was gassed with 95% air and 5% CO2 to maintain pH 7.4. The segment then was stretched to a length that was equivalent to a diameter of 200–225 µm and was incubated for another 15 min. The tissue was activated to contract by the addition of 5 µmol/l NE until reproducible responses were obtained. The relaxation responses were measured at cumulative doses of ADM between 10–9 and 3 x 10–7 mol/l on vessel rings precontracted with an ED70 concentration of NE that was determined for each vessel. Experiments were performed on arteries with intact endothelium. The endothelium was considered intact if acetylcholine (3 µmol/l) was able to cause at least 80% relaxation of arteries precontracted with ED70 concentration of NE. To assess the role of the cGMP or cAMP pathway, concentration-response curves to ADM were constructed before and after incubation of endothelium-intact arterial strips for 30 min in 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µmol/l) or 9-(tetrahydro-2-furany1)-9H-purin-6-amine (SQ22536; 10 µmol/l). In all of these studies, one arterial segment was used per animal, and replicates represent the number of animals in each group.
Measurement of Cyclic Nucleotide Levels
To assess the possibility that the cellular cGMP or cAMP determines the enhanced potency and efficacy of ADM-induced vasodilation during pregnancy, the basal as well as ADM-stimulated intracellular cGMP and cAMP levels were measured by RIA using cGMP [125I] and cAMP [125I] assay systems (Amersham Biosciences, Little Chalfont, England). Briefly, the mesenteric arterial arcade was carefully removed, weighed, and equilibrated for 1 h in 5 ml Krebs buffer containing 100 µmol/l of phosphodiesterase inhibitor, isobutyl-1-methyl-xanthine (IBMX; Sigma Chemical Co.), at 37°C aerated with 95% O2 and 5% CO2. After equilibration, tissues were incubated with 100 nmol/l of ADM for 2 min and were rapidly frozen in liquid nitrogen and homogenized in 1.2 ml of 10% trichloroacetic acid. Tissues used to determine the antagonism by the ADM antagonist, ADM22–52, were preincubated with 100 µmol/l ADM22–52 for 30 min before adding ADM. The cGMP and cAMP standards (2–128 fmol/tube) and samples (cAMP samples, diluted to 1:50) were acetylated by adding triethylamine/acetic anhydride (2:1 [v/v], 25 µl/tube). Labeled cGMP or cAMP bound to its respective antibodies was recovered using magnetic beads coated with goat anti-rabbit immunoglobulin G, and radioactivity was quantified in a gamma counter. Cyclic nucleotide levels are presented as picomoles per milligram tissue weight.
Stock solutions of ADM (100 µmol/l), NE (10 mmol/l), ADM22–52 (100 mmol/l), and SQ22536 (100 mmol/l) were prepared in triple-distilled water, aliquoted, and stored at –80°C. ODQ (10 mmol/l) was dissolved in dimethyl sulfoxide. ADM and ADM22–52 were purchased from American Peptide Co., Inc. (Sunnyvale, CA), whereas NE, SQ 22536, and ODQ were purchased from Sigma.
Data are presented as mean ± SEM. Relaxation to ADM is expressed as 100 minus percentage of the initial pre-contraction to NE. The data were analyzed by SigmaPlot 9.0 and Prism GraphPad Software (San Diego, CA), employing appropriate statistical tools. Means of different groups were analyzed by one-way or two-way ANOVA with Bonferroni posttest. Student paired t-test or two-way repeated measures ANOVA with Bonferroni posttest was used when comparisons were made between control and drug treatments in the same preparation. P 
0.05 was considered statistically significant. Individual concentration-response curves of ADM were subjected to nonlinear regression analysis to determine EC50, which was expressed as pD2 (–log EC50 of the molar concentration of the agonist).
Effect of Pregnancy on ADM-Induced Changes in MAP
To determine the in vivo differences due to pregnancy in ADM-induced vasodilation, the effect of ADM on MAP was recorded in both pregnant and nonpregnant rats. The basal MAP values were 115.58 ± 3.26 mm Hg in nonpregnant and 102.6 ± 3.17 mm Hg in pregnant rats. The decrease in MAP caused by ADM was dose dependent in all animals. In both groups, as illustrated in Figure 1A, the decreases in MAP (
MAP) were significantly (P 0.05) greater in pregnant (maximal response: –14.78 ± 1.52 mm Hg) compared with nonpregnant (maximal response: –8.88 ± 2.76 mm Hg) rats. But no significant change in heart rate was caused by ADM in either group (Fig. 1B).
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Effect of Pregnancy on Vascular Reactivity to ADM In Vitro
The ED70 concentration of NE that was determined for each vessel produced a sustained contraction in endothelium-intact mesenteric artery rings from both pregnant rats and rats that were virgin at diestrus. ADM (10–9 to 3 x 10–7 mol/l), added cumulatively at increments of 0.5 log units, relaxed the vascular rings in a concentration-dependent manner in both pregnant and nonpregnant rats. However, the potency and efficacy of ADM in causing the vasodilation was significantly (P 0.05) higher in pregnant (pD2, 7.49 ± 0.08; Emax 82.52% ± 3.15%; n = 16) compared with nonpregnant females at diestrus (pD2, 7.13 ± 0.15; Emax 68.92% ± 4.01%; n = 12; Fig. 2).
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Effect of Pregnancy on cGMP-Mediated, ADM-Induced Vasodilation and cGMP Levels
The involvement of cGMP in ADM-induced vasodilation was assessed using the guanylate cyclase inhibitor ODQ in vascular reactivity studies. The concentration-response curve to ADM was shifted to the right by 10 µmol/l ODQ in both pregnant and nonpregnant groups. The log shift of ADM response by ODQ was greater in pregnant rats (0.55 ± 0.09; pD2: control, 7.22 ± 0.12; ODQ, 6.53 ± 0.21; Emax: control, 81.66% ± 5.39%; ODQ, 60.62% ± 8.23%; n = 8) compared with nonpregnant rats (0.24 ± 0.05; pD2: control, 6.92 ± 0.12; ODQ, 6.67 ± 0.21; Emax: control, 69.43% ± 4.92%; ODQ, 52.89% ± 6.59%; n = 5; Fig. 3A and Table 1). The ADM-induced cGMP generation was significantly (P 0.05) greater in pregnant (0.28 ± 0.05 pmol/l per mg tissue) compared with nonpregnant (0.14 ± 0.03 pmol/l per mg tissue) rats (Fig. 3B). However, there were no significant differences between arteries from pregnant and nonpregnant rats on their basal cGMP levels. Incubation of arterial strips with ADM22–52 (100 µmol/l), an antagonist to ADM receptors, prevented the ADM-induced cGMP generation in which the cGMP levels were similar to the basal levels (Fig. 3B).
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Effect of Pregnancy on cAMP-Mediated, ADM-Induced Vasodilation and cAMP Levels
The contribution of cAMP in the augmented ADM-induced vasodilation was assessed by inhibiting the adenylate cyclase using SQ22536. The concentration-response curve to ADM was shifted to the right by 10 µmol/l SQ22536 in both pregnant and nonpregnant groups. The log shift of ADM-induced concentration-dependent relaxation curve after inhibition of adenylate cyclase also was significantly (P 0.01) greater in pregnant (0.84 ± 0.10; pD2: control, 7.59 ± 0.05; SQ22536, 6.77 ± 0.08; Emax: control, 87.68% ± 3.03%; SQ22536 63.67% ± 3.45%; n = 9; Fig. 4A and Table 1) compared with nonpregnant (0.28 ± 0.05; pD2: control, 7.23 ± 0.13; SQ 22536, 6.95 ± 0.11; Emax: control, 76.73% ± 4.2%; SQ 22536, 70.98% ± 8.37%; n = 6). Also, the ADM-induced cAMP generation was significantly (P 0.05) higher in pregnant (2.00 ± 0.47 pmol/l per mg tissue) compared with nonpregnant (0.75 ± 0.06 pmol/l per mg tissue) rats (Fig. 4B).
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The primary findings of this study are: 1) the in vivo hypotensive effects of ADM are greater in pregnant rats compared with nonpregnant rats; 2) the heart rate was not affected by ADM; 3) the in vitro vascular reactivity of rat mesenteric artery to ADM is enhanced during pregnancy; 4) the magnitude of inhibition of concentration-dependent response to ADM by the inhibition of soluble guanylate cyclase or adenylate cyclase was greater in pregnant rats compared with nonpregnant rats; 5) the ADM-induced generation of cGMP and cAMP in the mesenteric artery is elevated during pregnancy; and 6) the ADM-induced cyclic nucleotide generation is sensitive to ADM22–52. These findings suggest that during pregnancy the vasodilatory effects of ADM are greater, and this enhanced ADM-induced vasodilation involves increased generation of cyclic nucleotides in resistance vessels such as the mesenteric artery.
Marked cardiovascular adaptations are critical during pregnancy to ensure adequate blood supply to growing fetuses. During pregnancy there is a significant decrease in MAP, despite an increase in blood volume and cardiac output [1]. The vascular functional changes in pregnancy are attributed to several physiologic mechanisms, from augmented plasma levels of endogenous vasodilators like ADM [3] and CALCA [4], to modifications of mechanical properties of blood vessels and reduced excitation-response coupling for vasoconstrictors such as angiotensin II, vasopressin, and NE [5]. In this study we found that the vascular relaxation to the endogenous vasodilatory peptide, ADM, is enhanced during pregnancy, both in vitro, in resistance vessels such as the mesenteric artery, and in vivo, as evidenced by the greater decrease in MAP. The in vivo effects of ADM are consistent with the findings of Makino et al. [11], who have demonstrated the potentiation of the hypotensive effect of ADM in pregnant rats. The increased vascular relaxation reactivity to ADM shown by the mesenteric artery may contribute to the reduced vascular resistance during pregnancy. Yong et al. [2] have suggested that the generalized decrease in blood pressure during pregnancy results from reduced vascular resistance, and the blood pressure returns to nonpregnant levels in the postpartum period. Another reason for potentiated hypotensive effect during late gestation could be due to changes in cardiovascular reflex system. Heesch and Rogers [12] suggested that the baroreflex-mediated increases in efferent renal sympathetic nerve activity may be attenuated in pregnancy. Crandall and Heesch [13] demonstrated that blood pressure decreased to a greater extent in pregnant rats than in nonpregnant rats after captopril treatments. Hence, it is possible that changes in the baroreflex during pregnancy also could be the reason for the potentiated hypotensive action of ADM. But in this study there was no change in heart rate after ADM administration in pregnant or nonpregnant rats, which could rule out the possibility that the larger depressor responses of ADM in pregnant rats are due to less reflex tachycardia, as pregnancy depresses baroreflex function. Yet, it is noted that the heart rate values seem to vary to an extent that the study might be somewhat underpowered to detect any true differences in heart rate, if they exist. However, not all vasodilatory peptides show increased response during pregnancy. For example, the hypotensive effect of atrial natriuretic peptide, which is a potent vasodilator, was blunted in pregnant rats in late gestation compared with nonpregnant rats [14].
ADM mediates its vasodilatory effects through both cGMP-dependent [9] and cAMP-dependent [10] pathways. The metabolic production and plasma level of cGMP, a cellular mediator of vascular smooth muscle relaxation [15] are increased during pregnancy [16]. Hence, we hypothesized that the increased vasodilatory response to ADM during pregnancy corresponds to increased generation of cGMP. Also, Shimekake et al. [10] observed that ADM increased cGMP levels in rat aortic strips. Hayakawa et al. [17] had shown earlier that the vasodilatory effect of ADM was partially endothelium dependent in rat aorta and renal vessels. These authors also have demonstrated a role for cGMP pathway in ADM-induced vasodilation. The inhibition of cGMP-specific type V phosphodiesterase by E-4021 in aorta and renal vessels augmented ADM-induced vasorelaxation. In our study, the ADM-induced vasorelaxation of mesenteric artery was reduced by ODQ, a guanylate cyclase inhibitor, in both pregnant and nonpregnant rats. However, the magnitude of inhibition was higher in pregnant rats, suggesting the involvement of cGMP in the augmented vascular relaxation to ADM seen in pregnant rats. The role of cGMP was further confirmed by the enhanced cGMP generation by ADM in mesenteric artery from pregnant rats. ADM22–52, an antagonist to ADM, prevented the ADM-induced cGMP generation in the mesenteric artery from both pregnant and nonpregnant rats, indicating that ADM-induced cGMP generation is receptor mediated. In vascular smooth muscles, the increased cGMP levels may lead to the opening of various K+ channels [18] or activation of sodium pumps [19], and may cause relaxation. Moreover, Pascoal et al. [20] have demonstrated that pregnancy augmented the endothelium-dependent relaxation of vessels. Hence, it is possible that the increased vascular reactivity to the endogenous vasodilator ADM during pregnancy is due to the activation of the endothelium-dependent cGMP pathway, and this plays a role in vascular adaptations during pregnancy.
We reported recently that ADM generates intracellular cAMP in the mesenteric artery from pregnant rats [21]. Hence, we determined whether there is any difference in the cAMP-mediated, ADM-induced vasorelaxation between nonpregnant and pregnant rats. Surprisingly, the magnitude of reduction of vasorelaxation after inhibition of adenylate cyclase was markedly higher in pregnant rats. Furthermore, the ADM-induced cAMP generation also was significantly higher in the mesenteric artery arcade from pregnant than nonpregnant rats. We have suggested recently that cAMP-dependent protein kinase A plays a role as an intracellular messenger pathway to ADM receptors and activates KCa channels, which in turn reduces intracellular calcium and causes relaxation of the artery [21].
Interestingly, there was no difference between pregnant and nonpregnant groups in the vascular reactivity to ADM after the inhibition of either guanylate cyclase or adenylate cyclase. This suggests that both of these pathways are involved in the vascular hyperresponsiveness to ADM during pregnancy. It may also be due to an interaction between these two pathways, as seen in the rat cerebral artery, where cAMP limits cGMP loss by restricting cGMP efflux [22]. In addition, studies by Lu and Fiscus [23] postulated that relaxation effects of certain vasodilators like CALCA appeared to be mediated through the cGMP inhibition of type II phosphodiesterase and the subsequent accumulation of cAMP in smooth muscles.
The enhanced vasodilatory effect of ADM during pregnancy also may be due to the sensitizing effect of female sex steroids, which are found to be increased during pregnancy. Previously, we have shown that female sex steroids, both estradiol and progesterone, increased ADM-induced vasodilation by increasing the expression of ADM2 receptor components in rat mesenteric artery [24]. Taken together, during pregnancy the elevated female sex steroid levels sensitized the resistance vessels to the endogenous ADM by increasing the expression of its receptor components, which in turn increased the intracellular cyclic nucleotide levels as second messengers leading to increased vasorelaxation.
In conclusion, this study suggests that ADM-induced vasodilation is enhanced both in vitro and in vivo during pregnancy, and the augmented ADM-induced vasodilation does not correspond to altered heart rate. The increased vascular effects of ADM may be due to increased generation of cyclic nucleotides in resistance vessels like mesenteric artery. The increased vascular reactivity of resistance vessels to ADM may contribute to the reduced vascular resistance, and thus for vascular adaptations during pregnancy. Thus, we suggest that ADM may play a role in vascular adaptations during pregnancy.
ACKNOWLEDGMENTS
We thank Mrs. Cheryl R. Welch for administrative support.
FOOTNOTES
1Supported wholly through National Institutes of Health grants HL58144 and HD72650. 
Correspondence: 2 Chandra Yallampalli, Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., MRB, 11.138, Rt. 1062, Galveston, TX 77555-1062. FAX: 409 747 0475; e-mail: chyallam{at}utmb.edu
Received: 10 May 2006.
First decision: 6 June 2006.
Accepted: 11 September 2006.
REFERENCES
This article has been cited by other articles:
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  H.W.F. van Eijndhoven, R. Aardenburg, M.E.A. Spaanderman, J.G.R. De Mey, and L.L.H. Peeters Pregnancy Enhances the Prejunctional Vasodilator Response to Adrenomedullin in Selective Regions of the Arterial Bed of Wistar Rats Reproductive Sciences, December 1, 2007; 14(8): 771 - 779. [Abstract] [PDF]
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