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Endocrinology, doi:10.1210/en.2006-1105
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Endocrinology Vol. 148, No. 4 1727-1735
Copyright © 2007 by The Endocrine Society

Adrenomedullin-2, a Novel Calcitonin/Calcitonin-Gene-Related Peptide Family Peptide, Relaxes Rat Mesenteric Artery: Influence of Pregnancy

Madhu Chauhan, Gracious R. Ross, Uma Yallampalli and Chandra Yallampalli

University of Texas Medical Branch, Galveston, Texas 77555

Address all correspondence and requests for reprints to: Chandra Yallampalli, D.V.M., Ph.D., Distinguished Professor, Department of Obstetrics and Gynecology, 301 University Boulevard, MRB, 11.138, Route 1062, Galveston, Texas 77555-1062. E-mail: chyallam{at}utmb.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adrenomedullin-2 (ADM2), a novel calcitonin/calcitonin-gene-related peptide family peptide, is reported to reduce blood pressure in both normal and hypertensive rats. This study demonstrates gestational regulation of circulatory ADM2 in rat plasma. ADM2 dose-dependently reduces the mean arterial pressure in rats, whereas the hypotensive effect of ADM2 is significantly higher during pregnancy. In addition, immunoreactive ADM2 protein is distributed in perivascular fibers of rat mesenteric artery, and levels of pre-pro-ADM2 are significantly (P < 0.05) elevated in pregnant compared with nonpregnant rat mesenteric artery. Furthermore, incubation of endothelium intact arterial tissue from pregnant rats with ADM217–47, an ADM2 antagonist, shifted the dose-dependent relaxation curve to the right in wire myography. Inhibition of soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 µM) or endothelial nitric oxide synthase with N-nitro-L-arginine methyl ester (100 µM) reduced the relaxation of mesenteric artery induced by ADM2. Inhibition of adenylate cyclase with SQ22536 (10 µM) or protein kinase A with the Rp diastereomer of cyclic adenosine 3',5'-phosphorothioate (10 µM) also reduced the maximal relaxation responses induced by ADM2. Blockade of calcium-activated potassium channels with tetraethylammonium chloride (1 mM) inhibited the ADM2-induced relaxation, whereas blockade of ATP-sensitive potassium channels with glybenclamide (10 µM) did not affect the relaxation response. Hence the mechanism of ADM2-induced vasorelaxation is nitric oxide and receptor mediated and cGMP and cAMP dependent and occurs through activation of calcium-activated potassium channels. In conclusion, rat pregnancy is associated with increased levels of circulatory and vascular tissue ADM2 with concomitant increase in the in vivo hypotensive effect of ADM2 and vascular reactivity of mesenteric artery to ADM2, thus suggesting involvement of ADM2 in vascular adaptations during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MARKED CARDIOVASCULAR ADAPTATIONS are a hallmark of pregnancy to ensure adequate blood supply to the growing fetus. Epidemiological observations show that hypertensive complications of pregnancy such as preeclampsia and fetal growth restriction are preceded by defective development of adaptive vascular response to pregnancy (1). During pregnancy, the generalized decrease in blood pressure results from reduced vascular resistance, and blood pressure returns to nonpregnant levels in the postpartum period (2). The vascular remodeling that occurs during pregnancy is reported to be due to several physiological changes, including augmented plasma levels of endogenous vasodilators such as adrenomedullin (ADM) (3) and calcitonin-gene-related peptide (CGRP) (4), and reduced excitation-response coupling for vasoactive substances like angiotensin II, vasopressin, and norepinephrine (NE) (5). In this report, we demonstrate the association of changes in the functional profile of a novel bioactive peptide, ADM2, to the vascular changes that occur during pregnancy.

Calcitonin (CT), CGRP, ADM, and amylin belong to a unique group of CT/CGRP family peptide hormones important for homeostasis in diverse tissues. CGRP and ADM both are potent vasorelaxants considered to play a role in the adaptive mechanisms in pregnancy through increased vasodilation (4, 6, 7, 8, 9, 10). ADM2 is a 47-amino-acid novel member of the CT/CGRP family peptides discovered simultaneously by two groups in 2004 (11, 12). ADM2 binds to and activates both CGRP and ADM-receptor complexes comprising seven-transmembrane domain class B GPCR, calcitonin-receptor-like receptor (CRLR), and receptor-activity-modifying proteins (RAMPs) (11). CRLR in combination with RAMP1 gives rise to a CGRP receptor, whereas CRLR in combination with RAMP2 or RAMP3 forms ADM receptors (13, 14, 15, 16). ADM2 is shown to be a nonselective agonist for RAMPs but exhibits greater potency with CRLR/RAMP1 and CRLR/RAMP3 (11). Based on phylogenetic analysis, ADM2 and ADM fall into two distinct but closely related groups with 33% structural homology. Roh et al. (11) demonstrated two biologically active forms of ADM2: a 47-amino-acid C-terminally amidated long peptide (IDML) and a 40-amino-acid C-terminally amidated short peptide (IMD8–47) that can be derived from the proteolytic cleavage of the precursor protein.

Immunoreactive ADM2 is present in rat plasma (17). Peripherally administered ADM2 exhibits potent hypotensive effects and has suppressive effects on gastric emptying activities with reduced food intake and water intake (11). Furthermore, ADM2 stimulates vasodilatory activity in the pulmonary vascular bed of rats under elevated vascular tension (18). Thus, ADM2 is a novel peptide, binding to CRLR/RAMP1 and CRLR/RAMP3 receptors and could be important for regulation of diverse physiological processes that have been attributed to CGRP and ADM (11, 13, 17, 19, 20, 21). Recent studies show that ADM2 promoter sequence contains consensus estrogen response elements and that ADM2 is an estrogen-dependent prolactin-releasing factor (20). However, there are no studies on the role of this novel CT/CGRP family peptide in vascular adaptations during pregnancy. Thus, we hypothesized that circulatory ADM2 is gestationally regulated and that ADM2 is involved in vascular adaptations during rat pregnancy. The present study demonstrates the distribution of ADM2 in perivascular nerves in rat mesenteric artery and that the dose-dependent vasorelaxant effects on pregnant rat mesenteric artery are significantly higher when compared with the nonpregnant rat. We also elucidated the mechanisms involved in ADM2-induced relaxation of mesenteric artery. This study suggests a potential role for ADM2 in regulating vascular tone during pregnancy in rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed pregnant and nonpregnant female rats were purchased from Harlan Sprague Dawley (Houston, TX). All animals were given free access to food and water. The institutional animal care and use committee approved the procedures used in the study. Mesenteric arteries from at least four pregnant rats from each group collected were either snap frozen for protein extraction or processed for tissue sectioning for immunohistochemical studies. Segments of mesenteric artery from pregnant or nonpregnant-at-diestrus rats were collected for mounting on the myograph or used for whole-mount assay.

Drugs and solutions
Stock solutions of ADM2 (100 µmol/liter), NE (10 mmol/liter), ADM217–47 (10 mmol/liter), SQ22536 (10 mmol/liter), N-nitro-L-arginine methyl ester (L-NAME) (100 mmol/liter), the Rp diastereomer of cyclic adenosine 3',5'-phosphorothioate (Rp-cAMPS) (10 mmol/liter), tetraethylammonium chloride (TEA) (1 mM) were prepared in triple-distilled water, aliquoted, and stored at –80 C. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 mmol/liter) and glybenclamide (10 mmol/liter) were dissolved in dimethylsulfoxide. ADM2 and ADM217–47 were purchased from Alpha Diagnostics International (Austin, TX), whereas NE, SQ22536. Rp-cAMPS, ODQ, and L-NAME were purchased from Sigma Chemical Co. (St. Louis, MO).

RIA for plasma ADM2 levels
Blood samples were collected in tubes containing aprotinin and spun at 600 x g for 10min at 4 C to obtain plasma and stored at –80 C until the assay. For peptide measurement, 1 ml plasma was acidified with 1% trifluoroacetic acid and centrifuged at 9000 x g for 10 min to pellet large plasma proteins. Remaining proteins were extracted on C-18 Sep-Pak cartridges (Millipore, Bedford, MA), eluted in an acetonitrile-trifluoroacetic acid solution and lyophilized in a Speed-Vac. The ADM2 content in plasma was determined using an ADM2-RIA kit according to the manufacturer’s instruction (Phoenix Pharmaceuticals, Inc., Belmont, CA).

Measurement of mean arterial pressure (MAP)
The animals were anesthetized with ketamine (45 mg/kg body weight) (Burns Veterinary Supply, Westbury, NY) and xylazine (5 mg/kg 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 blood pressure system (Kent Scientific, Litchfield, CT) and into the right jugular vein to administer either vehicle (saline) or ADM2. Six hours after surgery, with the animals in a fully awake and free-moving state, MAP measurements were determined immediately before and for at least 20 min after bolus injection of either the vehicle or varying doses of ADM2 (40, 80, and 160 pmol/kg). Changes in MAP after administration of either saline or different doses of ADM2 from the baseline MAP before the injections were assessed. MAP was continuously monitored in all rats for at least 20 min after each dose of ADM2 or saline. Hypotensive effects were noted immediately after ADM2 injection, and these effects lasted for about 4 min with maximal responses occurring between 0.5 and 2 min. Therefore, we calculated the average MAP measurements between 1 and 2 min after ADM2 injection to compare between different groups. Based on the body weight of animals, the blood volume was calculated using the following formula: blood volume (ml) = 0.06 x body weight (g) + 0.77. Injections of ADM2 at 40, 80, and 160 pmol/kg body weight results in estimated circulating concentrations of 0.65, 1.3, and 2.6 nmol/liter in these rats, respectively.

Preparation of blood vessel for wire myography
The animals were killed by exsanguination under deep anesthesia induced by ip injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). The small intestine, including the blood supply, was cut and placed in physiological salt solution (PSS) and kept on ice. The PSS contained the following composition (mM): 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 were then isolated and cleaned off fat and connective tissue. The arterial segments (length, ~ 2 mm) were mounted on a wire myograph (Kent Scientific, Litchfield, CT) using tungsten wires and 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 was then stretched to a length that was equivalent to a diameter of 200–225 µm and incubated for another 15 min. The tissue was activated to contract by the addition of 5 µM NE until reproducible responses were obtained. The relaxation responses were measured at cumulative doses of ADM2 between 10–9 and 3 x 10–7 M on vessel rings precontracted with 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 µM) caused more than 80% relaxation of arteries precontracted with an ED70 concentration of NE.

Western blotting
Mesenteric artery was homogenized in lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 0.4% Nonidet P-40, 100 mM NaF, 200 mM NaVO5, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Protein homogenate of tissues containing 40 µg protein was loaded and electrophoresed on 4–20% Tris-HCl ready gel using a mini-protein II gel apparatus (Bio-Rad, Richmond, CA) as previously described (22). Separated proteins were electrophoretically transferred onto polyvinylidene difluoride membrane and blocked with 10% nonfat dry milk followed by incubation with the antibodies raised against the C terminal of ADM2 at a dilution of 1:2000 for 2 h at room temperature. After proper washes, the membranes were incubated with antirabbit IgG, horseradish peroxidase-linked secondary antibody (1:5000 dilution) (Amersham, Piscataway, NJ). The blots were then subjected to enhanced chemiluminescence using the Western blotting detection system (Amersham). The membranes were exposed to autoradiography until satisfactory exposure was achieved. The bands were scanned to measure their density and analyzed using a Fluorchem Analysis System (Sigma Gel Software, San Leandro, CA). The protein levels are expressed as the ratio of the housekeeping protein ß-tubulin in the same blot.

Whole-mount staining for ADM2 in mesenteric artery
The mesenteric arteries were immersed in PBS containing 0.5% Triton X-100 overnight, followed by incubation with PBS containing normal goat serum (1:100) for 60 min. The tissues were then incubated in the primary antibodies, rabbit anti-ADM2 1–50 (rat) serum (1:400) for 72 h at 4 C. After incubation, the tissues were washed in PBS, and the site of the antigen-antibody reaction was revealed by incubation in fluorescein-5-isothiocyanate-labeled goat antirabbit IgG (diluted 1:100) (ICN Pharmaceuticals, Inc., Aurora, OH) for 60 min. Thereafter, the tissues were thoroughly washed in PBS, mounted on slides, and coverslipped with glycerol/PBS (2:1 vol/vol) and observed under a confocal laser scanning microscopy (CLSM510; Carl Zeiss, Oberkochen, Germany) equipped with an exciting filter system (458/488 nm for fluorescein-5-isothiocyanate).

Immunolocalization of ADM2 in rat mesenteric artery
Tissue sections of d-18 mesenteric arteries were fixed in 10% formalin for 24 h and embedded in paraffin. Sections of 5 µm thickness were cut with a microtome and placed on coated slides. For immunohistochemical staining, these sections were deparaffinized, rehydrated, and heated for 10 min in sodium citrate buffer (pH 6.0) for antigen retrieval. The sections were then rinsed with PBS (0.1 M, pH 7.4). Endogenous peroxidase was blocked by 3% hydrogen peroxide for 10 min at room temperature. The slides were then washed with PBS (0.1 M, pH 7.4), and blocked with avidin solution followed by biotin, each for 1 h at room temperature. The primary ADM2 antibody, nonimmune serum ADM2 antibody preadsorbed with the antigen (1:90 dilution), was added to the slides and incubated in a cold room (4 C) overnight. The slides were washed three times with PBS containing 1% normal rabbit serum and Triton X-100 for 10 min each on a slow shaker. Secondary antirabbit biotinylated antibody in a 1:1000 dilution was added to the sections followed by incubation for 45 min at room temperature. The slides were washed three times with 1% normal rabbit serum/Triton X-100 and then PBS (0.1 M, pH 7.4) for 10 min each. The AB reagent (Vector Laboratories, Burlingame, CA) solution was added to the slides and was incubated for 1.5 h at room temperature. After the slides were washed with 0.1 M PBS (three times for 10 min each), the diaminobenzidine solution was added and incubated for 3 min or until visible staining was attained. Then the slides were washed with 0.1 M PBS (three times for 3 min each) and then counterstained with hemotoxylin for 1 min and then rinsed with deionized water (three times for 2 min each). The slides were dehydrated and viewed with an Olympus microscope with image ProPlus software.

Measurement of cGMP levels
To assess the possibility that the cellular cGMP determines the enhanced potency and efficacy of ADM2-induced vasodilation during pregnancy, the basal as well as ADM2-stimulated intracellular cGMP levels were measured by RIA using [125I]cGMP assay systems (Amersham Biosciences, Little Chalfont, UK). Briefly, the mesenteric arterial arcade was carefully removed, weighed, and equilibrated for 1 h in 5 ml Krebs buffer containing 100 µmol/liter phosphodiesterase inhibitor, isobutyl-1-methylxanthine (Sigma) at 37 C aerated with 95% O2 and 5% CO2. After equilibration, tissues were incubated with 300 nmol/liter ADM2 for 2 min and rapidly frozen in liquid nitrogen and homogenized in 1.2 ml 10% trichloroacetic acid. According to the manufacturer’s protocol, the cGMP standards (2–128 fmol/tube) and samples were acetylated by adding triethylamine/acetic anhydride (2:1 vol/vol, 25 µl/tube). Labeled cGMP bound to their respective antibodies were recovered by using magnetic beads coated with goat antirabbit IgG, and radioactivity was quantified in a {gamma}-counter. cGMP levels are presented as picomoles per milligram tissue weight.

Statistical analysis
Data are presented as mean ± SE. Relaxation to ADM2 is expressed as 100 minus percentage of the initial precontraction 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 post test or unpaired t test. Student’s paired t test or two-way repeated-measures ANOVA with Bonferroni post test 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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Measurement of immunoreactive ADM2 in rat plasma
ADM2 is reported to be present in the circulation of male rats (17). Plasma RIA for ADM2 was performed in this study to assess whether ADM2 is gestationally regulated in rat pregnancy. Immunoreactive ADM2 was detected in plasma collected from nonpregnant rat and pregnant rats on different days of gestation. Plasma levels of immunoreactive ADM2 in nonpregnant rat were 15 ± 3.4 fmol/ml, which increased with gestational age reaching maximal concentration of 33 ± 4.8 fmol/ml on d 18 of pregnancy (Fig. 1Go). Plasma levels of ADM2 declined on d 22, and the levels were similar to that of nonpregnant rats.


Figure 1
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  		FIG. 1. Plasma levels of ADM2 in nonpregnant (NP) and pregnant rats on different days (D) of pregnancy. RIA was performed with peptide extracted from plasma collected from nonpregnant and pregnant rats on different days of gestation (n = 4 in each group). *, P ≤ 0.05, significant differences when compared with the nonpregnant animals.

 
Influence of pregnancy on the vascular effects of ADM2
To determine the in vivo differences due to pregnancy in ADM2-induced vasodilation, the effect of ADM2 on MAP was recorded in both pregnant and nonpregnant rats. The average basal MAP values were significantly lower in pregnant (98.3 ± 1.7 mm Hg) compared with nonpregnant (115 ± 3.6 mm Hg) rats. The decrease in MAP caused by ADM2 was dose dependent in all animals. In both the groups, as illustrated in Fig. 2AGo, the decreases in MAP ({Delta}MAP) were significantly (P ≤ 0.05) greater in pregnant (maximal response, –14.68 ± 3.0 mm Hg) compared with nonpregnant (maximal response, –5.22 ± 1.4 mm Hg) rats. There were no significant changes in the heart rate after ADM2 infusion in all the groups (Fig. 2BGo). In the isolated endothelium-intact mesenteric artery rings from both pregnant and virgin-at-diestrus rats, the ED70 concentration of NE that was determined for each vessel produced a sustained contraction. ADM2 (10–9 to 10–6 mol/liter), 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 ADM2 in causing the vasodilation was significantly (P ≤ 0.05) higher in pregnant (pD2 = 7.07 ± 0.1; Emax = 84.12 ± 7.05%; n = 10) compared with nonpregnant females at diestrus (pD2 = 6.46 ± 0.2; Emax = 52.9 ± 9.05%; n = 6, Fig. 2BGo). Furthermore, as shown in Fig. 2CGo, the ADM2-induced (0.3 µM) cGMP generation was significantly (P ≤ 0.05) greater in pregnant (0.23 ± 0.04 pmol/liter·mg tissue) compared with nonpregnant (0.11 ± 0.03 pmol/liter·mg tissue) rats. However, there were no significant differences between arteries from pregnant and nonpregnant rats on their basal cGMP levels.


Figure 2
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  		FIG. 2. Influence of pregnancy on vascular effects of ADM2. A, Decrease in MAP in response to systemic administration of varying doses of ADM2 in pregnant and nonpregnant rats; B, no significant changes were observed in the heart rate after ADM2 administration in all groups; C, effect of pregnancy on the concentration-dependent vasodilation induced by ADM2 in endothelium-intact mesenteric arterial strips precontracted by ED70 concentration of NE, isolated from pregnant and nonpregnant-at-diestrus rats; D, ADM2-induced (0.3 µmol/liter for 2 min) generation of cGMP in isolated mesenteric arteries from pregnant and nonpregnant-at-diestrus rats. Data were analyzed by unpaired t test or two-way ANOVA with Bonferroni post test. *, P ≤ 0.05 vs. respective nonpregnant group; bars bearing different letters differ significantly. Numbers of replicates are provided in parentheses.

 
Western blot analysis and immunohistochemical localization of ADM2 in rat mesenteric artery
The antibody used for detecting ADM2 specifically recognizes ADM2 protein and does not cross-react with ADM as shown in Fig. 3AGo. Figure 3BGo demonstrates that a 16-kDa protein band corresponding to pre-pro-ADM2 is expressed in rat mesenteric artery, and its levels are significantly elevated in pregnant compared with the nonpregnant rats (P < 0.05). Using this antibody, we assessed immunolocalization of ADM2 in mesenteric artery tissues. Figure 3CGo shows the immunohistochemical staining of cross-sections of d-18 pregnant rat mesenteric artery, and the ADM2 is localized to the endothelium, vascular smooth muscle, and adventitia.


Figure 3
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  		FIG. 3. Immunolocalization of ADM2 in rat mesenteric arteries. A, Western blot analysis of pre-pro-ADM2 in rat mesenteric artery; dot-blot assay with 5 µg ADM and ADM2 synthetic peptide, demonstrating the specificity of the antibody raised against ADM2: a, incubation of the blot with nonimmune serum; b, incubation of the dot-blot with anti-ADM2 antibody showing specific staining of only ADM2 and no cross-reactivity with its related peptide, ADM. B, In the top panel, 30 µg protein extracted from d 18 mesenteric artery was electrophoresed on 12% SDS-PAGE and probed with anti-ADM2 antibody. As shown, pre-pro-ADM2 protein (16 kDa) is expressed in rat mesenteric artery, and its levels are significantly elevated in pregnancy. Housekeeping protein ß-actin was also analyzed for the respective samples in the same blot for any loading error. Bottom panel shows mean ± SEM of the ratios of densitometric values of target protein and respective ß-actin protein band. *, P ≤ 0.05, significant difference in the level of expression in d-18 pregnant compared with nonpregnant rats. C, Immunohistochemical localization of ADM2: light microscopy micrographs of cross-section of mesenteric artery showing ADM2 immunostaining in the endothelial cells (EC) and smooth muscle (SM) cells and in the adventitia (a). b and c, Incubation of tissues either in absence of the antibody (b) or with the nonimmune serum (c) served as the negative control showing absence of any immunostaining. Magnification, x100. D, Whole-mount staining: confocal laser micrograph of mesenteric artery (whole-mount specimen) showing perivascular nerve fibers containing ADM2 immunoreactivities (c). Incubation of the specimen either in absence of the antibody (a) or with the nonimmune serum (b) served as the negative control. Magnification, x200.

 
Whole-mount staining for ADM2 in rat mesenteric artery
Using whole-mount preparations of rat mesenteric arteries, we show dense immunoreactive ADM2-containing nerve fibers surrounding the artery (Fig. 3DGo). The tissues treated with antibody diluent or preimmune serum demonstrated little or no reactivity.

Effects of ADM2 receptor antagonism or inhibition of nitric oxide synthase/guanylate cyclase on ADM2-induced relaxation of mesenteric artery in pregnant rats
The involvement of ADM2 receptors in ADM2-induced vasodilation during pregnancy was assessed using ADM217–47, an ADM2 antagonist in mesenteric arterial reactivity studies. The concentration-response curve to ADM2 was shifted to the right by preincubation with 10 µmol/liter ADM217–47 for 30 min. The pD2 and Emax values were as follows: control pD2 = 6.9 ± 0.2, and ADM217–47 pD2 = 6.05 ± 0.5; control Emax = 59.34 ± 1.6%, and ADM217–47 Emax = 38.95 ± 8.6%; n = 3 (Fig. 4AGo). Because ADM2-induced relaxation of rat pulmonary vascular bed is NO mediated and ADM2 activates the L-arginine/nitric oxide pathway in rat aorta (18, 23), we assessed whether vascular effects of ADM2 in rat mesenteric artery are altered by nitric oxide synthase inhibitor or guanylate cyclase inhibitor. Preincubation with NO synthase inhibitor L-NAME (100 µmol/liter) or guanylate cyclase inhibitor ODQ (10 µmol/liter) for 20 min reduced the ADM2-induced relaxation response in mesenteric artery (P ≤ 0.05). The respective Emax values before and after incubation were for L-NAME 58.34 ± 1.6 and 45.75 ± 5.83% (n = 6) and for ODQ 58.18 ± 1.23 and 36.94 ± 5.42% (n = 4) (Fig. 4Go, B and C).


Figure 4
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  		FIG. 4. Effect of ADM2 receptor antagonist ADM217–47 (A), nitric oxide synthase inhibitor L-NAME (B), and guanylate cyclase inhibitor ODQ (C) on the ADM2-induced concentration-dependent relaxation of endothelium-intact mesenteric arterial strips precontracted by ED70 concentration of NE from pregnant rats. Data were analyzed by repeated-measures two-way ANOVA followed by Bonferroni post test (n = 5). *, P ≤ 0.05.

 
Effects of inhibition of adenylate cyclase or protein kinase A on ADM2-induced relaxation of mesenteric artery in pregnant rats
The ADM2-induced concentration-dependent relaxation response was developed in mesenteric arterial strips from pregnant rats on d 18 before and after incubation with either SQ22536 (10 µmol/liter), an adenylate cyclase inhibitor, or Rp-cAMPS (10 µmol/liter), a protein kinase A inhibitor. The relaxation response to ADM2 was reduced after inhibition of either adenylate cyclase or protein kinase A. The respective Emax values before and after incubation are for SQ22536 58.55 ± 1.0 and 31.45 ± 6.94% (n = 5) and for Rp-cAMPS 58.18 ± 1.23 and 30.39 ± 5.75% (n = 4) (Fig. 5Go, A and B).


Figure 5
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  		FIG. 5. Effects of inhibition of adenylate cyclase by SQ22536 (A) and inhibition of protein kinase A by Rp-cAMPS (B) on the ADM2-induced concentration-dependent relaxation of endothelium-intact mesenteric arterial strips from pregnant rats precontracted by ED70 concentration of NE. Data were analyzed by repeated-measures two-way ANOVA followed by Bonferroni post test (n = 5). *, P ≤ 0.05.

 
Effect of blockade of KCa or KATP channels on ADM2-induced relaxation of mesenteric artery in pregnant rats
Blockade of KCa channels by TEA (1 mM) inhibited the vasorelaxant effects of ADM2 in pregnant rats (d 18) mesenteric artery, whereas the inhibition of KATP channels did not affect the relaxation response of mesenteric artery to ADM2. The Emax values of ADM2 were reduced from 58.05 ± 0.87 to 30.54 ± 3.78% (n = 6) by TEA (Fig. 6Go, A and B).


Figure 6
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  		FIG. 6. Effect of blockade of calcium-activated potassium channels by TEA (A) and ATP-sensitive potassium channels by glybenclamide (B) on the ADM2-induced concentration-dependent relaxation of endothelium-intact mesenteric arterial strips from pregnant rats precontracted by ED70 concentration of NE. Data were analyzed by repeated-measures two-way ANOVA followed by Bonferroni post test (n = 6). *, P ≤ 0.05; and **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings from this study are that 1) circulatory ADM2 levels increased with gestation and declined at term, 2) systemic administration of ADM2 dose-dependently reduces the mean arterial pressure in rats, whereas the hypotensive effect of ADM2 is significantly higher during pregnancy, 3) the in vitro vascular reactivity of mesenteric artery to ADM2 is also increased during pregnancy, 4) ADM2-induced cGMP generation in mesenteric artery is significantly elevated in pregnant vs. nonpregnant rats, 5) mesenteric artery expressed pre-pro-ADM2, 6) mesenteric artery is innervated by ADM2-containing perivascular fibers, 7) Western blot analysis of mesenteric artery revealed a significant elevation in pre-pro-ADM2 levels with pregnancy, 8) ADM2-induced vasorelaxation involved its receptors and both cGMP and cAMP pathways, and 9) blockade of KCa channels, but not KATP channels, inhibited the ADM2-induced relaxation of mesenteric artery from pregnant rats.

Influence of pregnancy on the vascular effects of ADM2
Pregnancy is associated with large increases of blood flow to meet the increasing requirement of the fetus. The increase in blood flow is associated with remodeling of the resistance artery of mesenteric vasculature, which depends largely on the endothelial cells and circulatory vasoactive agents. ADM and CGRP are potent vasodilators, and their levels are reported to increase with advancing gestation. Sensitivity of the resistance mesenteric arteries to CGRP and ADM is also reported to increase during pregnancy and by female sex steroids (24, 25). ADM2, a novel CT/CGRP family peptide, is present in circulation and is reported to play a role in blood pressure regulation (11, 17). This study shows that the plasma ADM2 levels are significantly (P < 0.05) elevated during pregnancy and declined at term (Fig. 1Go). It is interesting to note that the levels of circulatory CGRP (4) and ADM (3), which also belong to the same family of peptides as ADM2, are also elevated during pregnancy. The hypotensive effects after systemic administration of exogenous ADM2 were significantly higher during pregnancy (Fig. 2AGo). Hence, not only the circulatory levels of ADM2, but also the hypotensive response to ADM2, are elevated during pregnancy. To know whether the augmented hypotensive effect of ADM2 during pregnancy is a reflection of an enhanced relaxation response of peripheral resistance vessels, we studied the vascular reactivity to ADM2 in a resistance vessel like mesenteric artery. Interestingly, the mesenteric arteries from pregnant rats were significantly more sensitive to the ADM2-induced relaxation response than from nonpregnant rats as shown in Fig. 2BGo. Yong et al. (2) have suggested that the generalized decrease in blood pressure during pregnancy results from reduced vascular resistance and that the blood pressure returns to nonpregnant levels in the postpartum period. The increased vascular relaxation reactivity shown by mesenteric artery to ADM2 may be a factor contributing to the reduced vascular resistance during pregnancy. ADM2 is reported to act through CRLR/RAMP receptor complexes (11). Previously, our lab has shown enhanced mRNA expression of CRLR and RAMPs in mesenteric artery and uterus during pregnancy (26, 27). This enhanced expression of CRLR/RAMP receptor complexes in mesenteric artery-like resistance vessels may be a reason for the increased vasorelaxation responses to ADM2 during pregnancy. Furthermore, we looked for any changes in ADM2-induced cGMP generation in mesenteric artery during pregnancy. Previous studies show that the metabolic production and plasma level of cGMP, a cellular mediator of vascular smooth muscle relaxation (28), are increased during pregnancy (29). In vascular smooth muscles, the increased cGMP levels may lead to opening of various K+ channels (30) or activation of sodium pumps (31) and cause relaxation. In this study, we found that pregnancy augmented the ADM2-induced cGMP generation in mesenteric artery (Fig. 2DGo), which could also contribute to the augmented relaxation response during pregnancy.

Expression and localization of ADM2
ADM2 mRNA is expressed largely in pituitary and several other tissues including in rat mesentery (11, 12). The current study shows that pre-pro-ADM2 is expressed in rat mesenteric artery and that its levels in rat mesenteric artery are significantly (P < 0.05) elevated during pregnancy (Fig. 3BGo). Furthermore, using whole-mount sections of mesenteric artery from d-18 pregnant rats, Fig. 3DGo demonstrates that ADM2-like immunoreactivity is distributed in the perivascular nerves surrounding the mesenteric artery. Furthermore, immunohistochemical analysis of the cross-section of mesenteric artery also shows that ADM2 is present in tunica media, tunica intima, and the endothelial cells (Fig. 3CGo). Hobara et al. (32) have demonstrated a similar pattern of expression for ADM in rat mesenteric artery. Our data suggest that rat mesenteric artery is also innervated with ADM2-like immunoreactivity. The current finding that rat mesenteric artery is innervated with ADM2-like immunoreactivity suggests that ADM2 may serve as a neurotransmitter in perivascular nerves. However, additional study is needed to clarify whether ADM2 in mesenteric arteries is synthesized locally and/or transported from dorsal root ganglions similar to that suggested for CGRP (33). The current study suggests that ADM2 may play a role in local control of vascular tone as an autocrine or paracrine regulator.

Mechanisms of ADM2-induced vasorelaxation
The ADM2-induced vasorelaxation appears to be receptor mediated, because ADM217–47, a putative ADM2 C-terminal receptor-binding domain, considered to be an ADM2 antagonist (11), shifted the ADM2-induced concentration-dependent relaxation response to the right (Fig. 4AGo). Although ADM2 can act through any of the CRLR/RAMP receptor complexes (11), the specific receptor complex involved in the vasorelaxation response has to be elucidated. Furthermore, the vasorelaxation response to ADM2 involves the nitric oxide/cGMP pathway because inhibition of nitric oxide synthesis or guanylate cyclase inhibited the relaxation response (Fig. 4Go, B and C). Moreover, the enhanced vasorelaxation response during pregnancy is also associated with the augmented ADM2-induced cGMP generation in mesenteric artery. Inhibition of adenylate cyclase or protein kinase A also inhibited the ADM2-induced vasorelaxation (Fig. 5Go). These data suggest that both cGMP- and cAMP-dependent pathways mediate the vasorelaxation response to ADM2. This could be due to the nonspecific agonist activity of ADM2 on diverse CRLR/RAMP receptor complexes. Furthermore, it may be due to an interaction between these two pathways as reported in rat cerebral artery where cAMP limits cGMP loss by restricting cGMP efflux (34). Also, observations by Lu and Fiscus (35) suggested that relaxation effects of vasodilators such as CGRP appeared to be mediated through the cGMP inhibition of type II phosphodiesterase and subsequent accumulation of cAMP in smooth muscles. Opening of calcium-activated potassium channels is the downstream mechanism of ADM2-induced vasorelaxation, because blockade of KCa channels inhibited the relaxation response, whereas KATP channels are not involved in this process (Fig. 6Go). Hence, ADM2 may activate KCa channels, and the resultant membrane hyperpolarization would inhibit calcium influx via voltage-gated calcium channels in vascular smooth muscle.

In summary, rat pregnancy is associated with increased levels of ADM2 in the circulation and vascular tissues with concomitant increases in the in vivo hypotensive effect of ADM2 and relaxation reactivity of mesenteric artery-like resistance vessels to ADM2. Also, ADM2 is present in various cellular compartments of mesenteric artery as well as perivascular nerves, suggesting a potential paracrine/autocrine role in regulating the vascular tone. Vascular relaxation to ADM2 is mediated through activation of KCa channels. This study provides strong evidence for the role of ADM2 in the vascular adaptations that occur during pregnancy.


   Acknowledgments
 
We acknowledge Cheryl Welch for her administrative support.


   Footnotes
 
The manuscript is supported by Grants HL58144 and HL72620 provided by the National Institutes of Health to C.Y.

First Published Online January 11, 2007

Abbreviations: ADM, Adrenomedullin; CGRP, calcitonin-gene-related peptide; CRLR, calcitonin-receptor-like receptor; CT, calcitonin; L-NAME, N-nitro-L-arginine methyl ester; MAP, mean arterial pressure; NE, norepinephrine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PSS, physiological salt solution; RAMP, receptor-activity-modifying protein; Rp-cAMPS, Rp diastereomer of cyclic adenosine 3',5'-phosphorothioate; TEA, tetraethylammonium chloride.

Received August 14, 2006.

Accepted for publication January 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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