Female Sex Steroids Increase Adrenomedullin-Induced Vasodilation by Increasing the Expression of Adrenomedullin2 Receptor Componen
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《内分泌学杂志》
Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555
Abstract
Based on the favorable effects of female sex steroids in vascular functions and the potent hypotensive effects of adrenomedullin (AM), we hypothesized that AM-induced vasodilation is gender dependent, and female sex steroids enhance this effect. In endothelium-intact rat mesenteric artery, AM (1 nM–0.3 μM)-induced concentration-dependent relaxation was significantly (P < 0.05) higher in females [pD2(–log EC50 of the molar concentration), 7.05 ± 0.10; maximal relaxation response (Emax), 69.2 ± 3.46%] than males (pD2, 6.53 ± 0.08; Emax, 53.28 ± 4.86%). The increased relaxation was lost when the females were ovariectomized (OVX) (pD2, 6.14 ± 0.24; Emax, 39.68 ± 5.68%). The reduced relaxation response in OVX rats was reversed by administration of either progesterone (P4; pD2, 7.18 ± 0.07; Emax, 72.4 ± 2.76%) or 17-estradiol (E2; pD2, 7.00 ± 0.14; Emax, 70.4 ± 4.79%). AM mediates its effects through either AM22–52-sensitive AM1 receptors [composed of calcitonin receptor-like receptors (CLs) and receptor activity-modifying protein (RAMP)2] or AM2 receptors (CL/RAMP3), which can be antagonized more potently by calcitonin gene-related peptide8–37 than AM22–52. Pharmacological characterization suggested the involvement of AM2 receptors in the increased vasodilatory effect of AM in both P4- and E2-treated animals as calcitonin gene-related peptide8–37 (10 μM) was more potent in antagonizing the AM effects (Emax, P4: 25.92 ± 5.32%; E2: 29.11 ± 7.41%) than AM22–52 (100 μM). RT-PCR studies also supported the involvement of AM2 receptors because expression of mRNA levels encoding CL (previously reported) and RAMP3 were increased in P4- or E2-treated OVX rats. In conclusion, AM-induced vasodilation is gender-dependent and increased by female sex steroids by increased expression of AM2 receptor components.
Introduction
GENDER DIFFERENCE IN the incidence of cardiovascular disease is well documented in the literature. Epidemiological evidence suggests that premenopausal women have a reduced incidence of cardiovascular diseases compared with postmenopausal women and men of a comparable age (1). The much lower mortality rate in association with cardiovascular disease in postmenopausal women receiving steroid hormone therapy indicates the cardioprotective role of female sex steroid hormones (2). The cardiovascular protective effects of female sex steroids have also been shown in numerous animal studies (3). Although the underlying mechanisms are not completely understood, the favorable effects of steroid hormones in vascular functions are attributed to several factors such as nitric oxide (4), prostaglandins (5), and calcitonin gene-related peptide (CGRP) (6, 7). However, there are several other factors remaining unexplored, which include adrenomedullin (AM), a structurally related peptide to CGRP.
AM is a 55-amino acid peptide that was discovered in 1993 from a panel of peptides extracted from a pheochromocytoma (8). Later, it was realized that AM was produced by a wide range of cells including vascular endothelial and smooth muscle cells. In rat, cat, sheep, and man, iv infusion of AM results in a potent and sustained hypotension (9, 10, 11). Acute or chronic administration of AM resulted in a significant decrease in total peripheral resistance accompanied by a fall in blood pressure in both conscious and hypertensive rats (12).
The mechanisms via which AM can elicit vascular relaxation are heterogeneous with respect to both species and vascular bed. These are not completely understood but are known to involve both the CGRP and AM receptors (13). Both CGRP and AM are known to act via the same seven-transmembrane G protein-linked receptor [i.e. calcitonin receptor-like receptor (CL)]. The majority of the differences in intracellular signaling pathways activated by CGRP and AM are attributed to the different receptor activity-modifying proteins (RAMPs) that form their receptors. Three RAMPs (RAMP1, RAMP2, and RAMP3) have been identified as a novel family of single-transmembrane domain proteins (14). The association of CL with RAMP1 produces a CGRP-selective CGRP receptor that is antagonized by the CGRP antagonist, CGRP8–37; CL with RAMP2, an AM-selective receptor (AM1) that can be antagonized by the weak AM peptide antagonist AM22–52 and CL with RAMP3 another AM receptor (AM2) that can respond to both CGRP and AM, which can be more potently antagonized by CGRP8–37 than by AM22–52 (13, 15). Thus, RAMPs provide a mechanism whereby a cell could dynamically change its sensitivity from one peptide to another.
Based on the favorable effects of female sex steroids in vascular functions and the potent hypotensive effects of AM, we hypothesized that female sex steroids increase the vasodilation caused by AM in resistant vessels like mesenteric artery. Hence, in the present study, we examined in the rat mesenteric artery: 1) the gender-based differential responsiveness to AM, 2) the role of female sex steroids on AM-induced vasodilation, and 3) the changes in the AM receptor components RAMP2 and RAMP3 based on sex steroid treatments.
Materials and Methods
Animals
Adult male and female nonpregnant (age 9–11 wk) Sprague Dawley rats (Harlan Sprague Dawley, Houston, TX) weighing between 250 and 300 g were used. All rats were maintained in the colony room with fixed photoperiod of 12-h light, 12-h dark cycle and having access to water and rodent chow ad libitum. All procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch (Galveston, TX) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Ovariectomy
Female rats were anesthetized by ip injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). Small incisions were made on each flank, and the ovaries were removed. The muscle layer was suture-closed, and the incisions were closed with wound clips. After 5 d, the ovariectomized (OVX) rats received a small incision in the back of the neck into which either a sc E2 [0.5 mg, 21-d release (OVX-E2 group)] or a progesterone [P4; 5 mg, 21-d release (OVX-P4 group)] or a placebo pellet (OVX group) (Innovative Research of America, Sarasota, FL) was placed. These animals were used after 7 d, and the efficacy of ovariectomy and hormone replacement was confirmed by measuring plasma P4 and E2 levels using DSL RIA kits (Diagnostic System Laboratories, Webster, TX).
Preparation of blood vessel
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 114 mM NaCl, 4.7 mM KCl, 1.15 mM KH2PO4, 1.10 mM Na2HPO4, 1.18 mM MgSO4.7H2O, 15 mM NaHC03, 1.15 mM CaCl2, and 5.0 mM glucose. Secondary branches of the mesenteric artery were then isolated and cleaned off fat and connective tissue. The arterial segments (length, approximately 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 norepinephrine (NE) until reproducible responses were obtained. The relaxation responses were measured at cumulative doses of AM 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. Where indicated, the endothelium was removed by gently rubbing the intimal surface of the vessel with tungsten wire (size, 1 μm). The endothelium was considered intact if acetylcholine (3 μM) caused more than 80% relaxation of arteries precontracted with ED70 concentration of NE and effectively removed (denuded) if acetylcholine failed to relax. The viability of endothelium-denuded arterial rings was confirmed by relaxation induced by sodium nitroprusside (1 μM).
Pharmacological characterization of AM vasodilation
To determine the role of endothelium in the vasodilatory responses caused by AM, concentration-dependent relaxation (1 nM–;0.3 μM) was produced in endothelium-intact and -denuded arterial rings from both OVX-P4 and OVX-E2 rats. To confirm the endothelial contribution or lack of it to AM-induced vasorelaxation, concentration responses to AM (1 nM–;0.3 μM) were performed in the presence (after 30-min incubation) and absence of N-nitro-L-arginine methyl ester (L-NAME) (100 μM), an endothelial nitric oxide synthase (eNOS) inhibitor in endothelium-intact arterial rings. To verify whether the AM-induced vascular relaxation is mediated through AM1 receptors or AM2 receptors, the receptor antagonists AM22–52 and CGRP8–37 were used. Concentration-response curves to AM (1 nM–;0.3 μM) were obtained from endothelium-intact vessel segments precontracted by ED70 concentration of NE. Then, the vessel segment was washed with PSS and was incubated with either AM22–52 (10–5 or 10–4 M) or CGRP8–37 (10–5 M) for 30 min. After the incubation period, relaxation responses to cumulative doses of AM (1 nM–;0.3 μM) were repeated in NE precontracted vessel segments.
Expression of mRNA encoding RAMP2 and RAMP3
Total RNA was isolated from mesenteric artery using Trizol Reagent (Invitrogen, Carlsbad, CA). The quality and quantity of RNA were assessed at A260/280, and all samples showed absorbency ratios ranging between 1.6 and 2.0. The total RNA was treated for genomic DNA contamination using a DNA-free kit (Ambion, Austin, TX). Using total RNA, first strand cDNA was synthesized by RT using the GeneAMP RNA PCR kit (PerkinElmer, Branchburg, NJ) as described by the supplier. For RT, 2 μg total RNA was mixed with 3.0 ml random primer (Invitrogen), 200 μM dNTP solution (Sigma-Aldrich, St. Louis, MO), and 10 U avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) in the presence of 5 U RNAase inhibitor (Invitrogen). Samples were placed into a thermal cycler for one cycle at 42 C for 15 min, 99 C for 5 min, and 5 C for 5 min. The cDNA was stored at –20 C.
PCR using the specific primer sets for RAMP2 and RAMP3 was carried out with cDNA synthesized by RT. Briefly, 2 μl cDNA was mixed with a PCR mixture containing 2.5 mM MgCl2, 1 x 10 x PCR buffer II, 5 U/100 μl Amplitaq DNA polymerase kit (PerkinElmer), 65 μl sterile distilled water, and 0.1 μM of the following set of primers: RAMP2, forward, 5'-GCTGTTACTGCTGCTGTTGC 3', and reverse, 5'-GTCTGCCTCGTACTCCAAGC 3'; and RAMP3, forward, 5'-CTTCTCCCTCTGTTGCTGCT 3, and reverse, 5'-GTCCTGTCCACAGTGCAGTT 3'. Final volume was 100 μl. Primer sequences for rat RAMP2 (GenBank accession no. NM_031646.1) and RAMP3 (GenBank accession no. NM_020100.2) were derived using published sequences from the GenBank database (14). For amplification of 18S, the following primers (Ambion) were used: forward, 5'-AGGAATTGACGGAAGGGCAC-3'; and reverse, 5'-GTGCAGCCCCGGACATCTAAG-3'. The PCRs for RAMP2, RAMP3, and 18S were carried out on a GeneAMP PCR system 9700 (PerkinElmer) with the following conditions. An initial denaturation step at 95 C for 5 min was followed by 35 cycles of 60 sec at 95 C, 90 sec at 63 C, and 30 sec at 72 C, with a final extension cycle of 7 min at 72 C. The total cycle number chosen for each gene was from the linear portion of their respective amplification curve (data not shown).
Drugs used
Stock solutions of AM (100 μM), NE (10 mM), AM22–52 (100 mM), and CGRP8–37 (1 mM) were prepared in triple-distilled water, aliquoted, and stored at –80 C. AM, AM22–52, and CGRP8–37 were purchased from American Peptide Co., Inc. (Sunnyvale, CA), whereas NE was purchased from Sigma-Aldrich.
Statistical analysis
Data are presented as mean ± SE. Relaxation to AM is expressed as 100 – percentage of the initial precontraction to NE. The data were analyzed using Prism GraphPad Software (GraphPad Software Inc., San Diego, CA) employing appropriate statistical tools. Means of different groups were analyzed by one-way ANOVA and subjected to Newman-Keuls multiple comparison test. Student’s paired t test was used when comparisons were made between control and drug treatment in the same preparation. P 0.05 was considered statistically significant. Individual concentration-response curves of AM were subjected to linear regression analysis to determine EC50, which was expressed as pD2 (–log EC50 of the molar concentration of the agonist).
Results
Effect of ovariectomy and female sex steroid pellet placement on plasma sex steroid levels
To confirm the effect of OVX and hormone replacement on the plasma sex hormone levels, P4 and E2 levels were assayed in plasma collected when the animals were used for the experiments. There was significant reduction in the hormone levels after ovariectomy, whereas the hormone levels reached physiological levels after steroid hormone pellet placement. The levels of plasma P4 and E2 were 2.81 ± 0.50 nM (n = 3) and 0.1 ± 0.01 nM (n = 4), respectively, after ovariectomy. In OVX rats receiving P4 or E2 pellets, the plasma P4 levels were 11.43 ± 1.67 nM (n = 7), and the plasma E2 levels were 0.78 ± 0.12 nM (n = 6), respectively.
Effect of gender on the responsiveness of mesenteric artery to AM
The ED70 concentration of NE that was determined for each vessel produced a sustained contraction in endothelium-intact mesenteric artery rings from both male and female at diestrus rats. AM (1 nM–0.3 μM), added cumulatively at increments of 0.5 log units, relaxed the vascular rings in a concentration-dependent manner in both male and female rats. However, the potency and efficacy of AM in causing the vasodilatory response was significantly (P 0.05) lower in males [pD2, 6.53 ± 0.08; maximal relaxation response (Emax), 53.28 ± 4.86%; n = 5; Fig. 1A] compared with females (pD2, 7.05 ± 0.10; Emax, 69.2 ± 3.46%; n = 13; Fig. 1A) at diestrus.
Effect of ovariectomy and female sex steroid treatment on vasorelaxation caused by AM
Because the vascular responsiveness of mesenteric artery to AM was significantly higher in females than males, we hypothesized that female sex steroids were responsible for this increased sensitivity to AM in females. Thus, the AM-induced vasodilatory responses of mesenteric artery segments were compared from female rats upon OVX or OVX with E2 or P4 supplementation. AM (1 nM–0.3 μM) induced a concentration-dependent relaxation of the mesenteric arterial segments from all the groups. The vasodilatory response to AM was markedly reduced in females upon ovariectomy (pD2, 6.14 ± 0.24; Emax, 39.68 ± 5.68%; n = 4, P < 0.05) in comparison with females with intact ovary at diestrus (pD2, 7.05 ± 0.10; Emax, 69.21 ± 3.46; n = 13; Fig. 1A). In addition, the vasodilatory responses to AM in OVX females were similar to the responsiveness to AM in males (Fig. 1A). On the other hand, in female OVX rats, both E2 and P4 significantly (P < 0.05) increased the vasodilatory responses of mesenteric artery to AM. However, as shown in Fig. 1B, there were no significant differences between the E2 (pD2, 7.00 ± 0.14; Emax, 70.4 ± 4.79; n = 11) and P4 (pD2, 7.18 ± 0.07; Emax, 72.4 ± 2.76; n = 8) groups.
Effect of endothelium on the vasodilatory effect of AM
Because female sex steroids alter the responses to vasodilators by influencing the role of endothelium, we determined the contribution of endothelium to the vasodilatory effects of AM by comparing between endothelium-intact and -denuded arterial rings. AM (1 nM–0.3 μM) produced concentration-dependent responses in both endothelium-intact and -denuded tissues (Fig. 2, A and B). However, there were no significant differences between the endothelium-intact and -denuded rings from both OVX-P4 (endothelium-intact, pD2, 7.07 ± 0.11; Emax, 69.9 ± 3.49%; n = 9; endothelium-denuded, pD2, 7.11 ± 0.18; Emax, 71.1 ± 5.57%; n = 3) and OVX-E2 (endothelium-intact, pD2, 7.03 ± 0.13; Emax, 72.8 ± 5.29%; n = 13; endothelium-denuded, pD2, 6.55 ± 0.27; Emax, 63.4 ± 9.27%; n = 6) groups. Because there was a rightward shift, although statistically insignificant, in the OVX-E2 group, we further evaluated the vasodilatory responses in the presence of L-NAME (100 μM), an inhibitor of eNOS. Preincubation with L-NAME for 30 min did not alter the position of the concentration-response curve of AM (Fig. 2C). This dose of L-NAME was sufficient for substantial inhibition of ACh-induced vasorelaxation (data not shown) and, therefore, considered as an effective dose.
Effect of AM22–52 and CGRP8–37 on the vasodilatory effect of AM
Because female sex steroid treatments restored the impaired vasodilatory responses to AM in OVX rats, we next assessed the involvement of receptor subtypes using AM receptor antagonists, AM22–52 and CGRP8–37. Incubation for 30 min with 10 μM AM22–52 failed to shift the concentration-response curve of AM (Fig. 3, A and B), whereas 100 μM AM22–52 shifted the curve to the right in both OVX-P4 and OVX-E2 groups. The Emax values were reduced by 100 μM AM22–52 from 77.55 ± 3.07% to 35.63 ± 6.56%, n = 6 (P 0.05) in OVX-P4 rats and from 77.40 ± 5.24% to 36.00 ± 7.11%, n = 4 (P 0.05) in OVX-E2 rats (Fig. 3, C and D). On the other hand, just 10 μM CGRP8–37 was sufficient to substantially inhibit the vasodilatory responses to AM in mesenteric artery segments from both OVX-P4 (control, pD2, 7.18 ± 0.07; Emax, 72.40 ± 2.76%; n = 4 vs. CGRP8–37, Emax, 25.92 ± 5.35%; n = 4, P 0.01) and OVX-E2 (control, pD2, 6.84 ± 0.10; Emax, 63.39 ± 3.51% vs. CGRP8–37, Emax, 29.16 ± 7.41%; P 0.01; n = 6; Fig. 4, A and B).
Effect of female sex steroid treatments on the expression of mRNA encoding RAMP2 and RAMP3
The above pharmacological investigations suggested the involvement of AM2 receptors (CL/RAMP3) in the enhanced responsiveness to AM in hormone-treated rats. To confirm the role of changes in the AM receptor components, we measured the levels of mRNA encoding RAMP2 and RAMP3 in mesenteric arteries isolated from OVX, OVX-P4, and OVX-E2 rats using RT-PCR. The mRNA levels are expressed relative to those of 18S in each animal. As shown in Fig. 5, mRNA levels for RAMP2 remained unaltered, whereas the mRNA levels for RAMP3 were increased significantly (P < 0.05) in both E2- and P4-treated compared with OVX rats.
Discussion
The major findings from this study are: 1) the AM-induced vasodilatory response in rat mesenteric artery is gender-dependent, i.e. increased in females compared with males; 2) depletion of female sex steroids by ovariectomy abolished the gender differences in the vasodilatory response to AM; 3) administration of either P4 or E2 reversed the reduced vascular reactivity to AM in the OVX animals, confirming the influence of female sex steroids on AM-induced vasodilation; 4) the enhanced vascular responsiveness to AM in the steroid-treated groups is not endothelium-dependent because endothelium denudation or inhibition of eNOS enzyme could not alter the response; and 5) further, both pharmacological and AM receptor component mRNA data suggest the involvement of AM2 receptors in steroid-induced enhanced mesenteric artery relaxation to AM. Therefore, we suggest that steroid hormones regulate AM-induced vasodilatory responses in resistance vessels like mesenteric artery and therefore play a role in the cardiovascular protective effects of female sex steroids.
Gender differences in AM-induced vasodilation
The AM-induced vasodilatory response is significantly greater in female rats compared with males. Gender differences in vascular tone have been described in a multitude of vascular beds in both human and experimental animals (16). For example, -adrenergic agonists such as NE cause less forearm vasoconstriction in women than in men (17). Moreover, age-related alterations in the vasculature enhance vasoconstriction in males relative to females (18). Previously, we have reported that the vasodilatory response to CGRP is lower in males compared with females (19). Several reports in different models of hypertension suggested that the arterial pressure is greater in males than females, and ovariectomy in females accelerated the development of hypertension to a level that is not different from males (20). Gender differences in AM-induced relaxation is also shown by Packer et al. (21) in rat pulmonary artery wherein AM relaxed NE contractions two to four times greater in female than male rats. It is possible that together with the increased vasodilatory responsiveness of resistance vessel like mesenteric artery to endogenous vasodilators such as AM and reduced sensitivity to vasoconstrictors such as NE (22) result in lowered peripheral vascular resistance in females and contribute to gender-related differences in cardiovascular diseases, including hypertension, as reported in epidemiological studies (1).
Effect of ovariectomy and female sex steroid treatment on AM-induced vasodilation
Because AM-induced vasodilation is greater in females, we hypothesized that female sex steroids are responsible for the gender differences in vasorelaxation. The gender differences in the mesenteric arterial relaxation by AM are abolished when the female rats are depleted of steroid sex hormones through bilateral ovariectomy. Reversal of reduced vascular response to AM in the OVX animals by administration of either P4 or E2 confirmed the influence of female sex steroids on the vascular reactivity to AM. P4 is frequently described as opposing the actions of E2; however, this may be an oversimplification, at least with the cardiovascular effects. It is well documented that circulatory P4 levels are increased during pregnancy when there are marked cardiovascular adaptations like volume expansion and vasodilation. Results of studies from our laboratory showed that the vasodilatory effects of CGRP in hypertensive rats were P4-dependent (6). Our previous studies showed that mean arterial blood pressure was significantly higher in adult rats when ovarian hormones are depleted by bilateral ovariectomy (7). Also, hormonal replacement therapy is known to decrease blood pressure in postmenopausal women. Studies in hypertensive experimental animals have supported gender differences in blood pressure and have suggested that female sex hormones may protect against the development of hypertension (23). The data from the current study shows both E2 and P4 increase the AM-induced arterial relaxation to the same magnitude. The enhanced vasodilatory response of resistance vessels like mesenteric artery to AM, which can act in an autocrine or paracrine manner, may be part of the mechanisms involved in the favorable effects of hormonal replacement therapy in postmenopausal women, less incidence of cardiovascular diseases in premenopausal females, and vascular adaptations like reduced peripheral vascular resistance during pregnancy when both E2 and P4 levels are elevated.
Role of endothelium in the augmented AM-induced vasodilation in P4- and E2-treated rats
In the present study, the enhanced vascular relaxation to AM in steroid-treated groups is not endothelium-dependent because endothelium denudation or inhibition of eNOS enzyme could not shift the concentration-response curve of AM in both E2- and P4-treated rats. Because the animals used here were OVX, it is possible that the endothelium-derived factors are affected by ovariectomy because reciprocal changes in endothelium-derived hyperpolarizing factor and nitric oxide system in the mesenteric artery of adult female rats after ovariectomy are reported by Nawate et al. (24).
Pharmacological characterization of AM receptors
We tried to pharmacologically characterize the receptors involved in the enhanced vasodilation of mesenteric artery to AM in steroid-treated rats using the antagonists AM22–52 and CGRP8–37. Receptors for AM are heterodimeric complexes of the CL together with RAMP2 (AM1 receptor) or RAMP3 (AM2 receptor). The three potential consequences of RAMP interaction with the associated receptors are: transport of the receptor to the cell surface, modification of the receptor glycosylation, and direct and indirect modification of the ligand binding site through association with the receptor at the cell surface (25). The AM-selective receptor (AM1) can be antagonized by the weak AM peptide antagonist AM22–52, whereas the AM2 receptor can respond to both CGRP and AM, which can be antagonized more potently by CGRP8–37 compared with AM22–52 (13). In our study, the AM-induced vasodilation was inhibited by 10 μM CGRP8–37, whereas 10 μM AM22–52 could not shift the concentration-response curve in both E2- and P4-treated groups. However, 100 μM AM22–52 could inhibit the AM-induced vasodilation in both E2- and P4-administered groups, indicating CGRP8–37 is more potent than AM22–52 in inhibiting the AM-induced vasodilation, which is the pharmacological character of AM2 receptors. Both AM22–52 and CGRP8–37 are believed to be competitive antagonists, but the Emax of AM is reduced in the presence of these antagonists in our experiments. It is possible that the maximum concentration of the agonist used in these experiments is not sufficient to produce the Emax in the presence of antagonists as that produced in their absence. Hay et al. (26) have demonstrated the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at CL/RAMP2 and CL/RAMP3 receptors transiently expressed in COS 7 cells. AM22–52 (10 μM) antagonized AM at the CL/RAMP2 complex, whereas 10 μM CGRP8–37 was an effective antagonist to AM at the CL/RAMP3 complex. The pA2 values of AM22–52 and CGRP8–37 estimated in functional studies for AM1 receptors were in the order of 7 and around 6, respectively, whereas those for AM2 receptors were less than 5.5 and 6.18, respectively (27). The pharmacological investigations in the current study pointed to the involvement of AM2 receptors in the increased vasodilatory response to AM under the influence of female sex steroids.
Regulation of mRNA levels of AM receptor components by P4 and E2
The role of AM2 receptors is further supported by the increased mRNA levels encoding RAMP3 in mesenteric arteries from E2- and P4-treated OVX rats compared with OVX females. Altered gene expression of AM receptor components is shown (28) in patients with pregnancy-induced hypertension. They showed a significant negative correlation between the RAMP2 mRNA levels in the umbilical artery and uterine muscle and blood pressure, suggesting an important role for the reduced expression of AM receptor components in pregnancy-induced hypertension. Previously, we have demonstrated changes in the gene expression for CL, RAMP1, RAMP2, and RAMP3 in rat uterine tissue during pregnancy, labor, and by steroid hormone treatments (29), suggesting dynamic changes of these receptor components by respective physiological state. In OVX mice exposed to E2 for 6 h, uterine gene expression profiles developed by DNA microarrays showed about 20.4-fold increase in RAMP3 (30), indicating steroid hormone maneuver of RAMP3 expression. We have shown earlier increased expression of CL in rat mesenteric artery by P4 treatment but not E2 (31). RT-PCR data from the current study show increased expression of RAMP3, but not RAMP2, by both P4 and E2 treatments. Increase in expression of both CL and RAMP3 by P4 or RAMP3 alone by E2 may enhance the interaction between CL and RAMP3 and increase the probability of formation of AM2 receptors. However, at this juncture, it is not possible to rule out the involvement of CGRP1 receptors in AM-induced vasodilation, although this is less likely because CGRP1 receptor-mediated effects cannot be blocked by AM22–52 (32). This is further supported by the studies in which CGRP8–37, but not AM22–52, inhibited AM-induced cAMP production in HEK-293 cells cotransfected with hCLR and hRAMP1 (33).
In conclusion, our study suggests that the vasodilatory responses to AM in a resistant vessel-like mesenteric artery are gender-dependent and increased by female sex steroids, both P4 and E2. The enhanced vasodilatory response is not endothelium-dependent. Pharmacological and receptor component mRNA data suggest the involvement of AM2 receptors in steroid-induced enhanced mesenteric artery relaxation to AM. Therefore, we suggest that female sex steroids regulate AM-induced vasodilatory responses in resistance vessels and thereby play a role in steroid-related cardiovascular protection. The gender differences and the influence of female sex steroids on AM-induced vasorelaxation raise interesting questions with regard to our understanding of the gender-related and pre- and postmenopausal differences in cardiovascular diseases reported in epidemiological studies and also the marked cardiovascular adaptations during pregnancy.
Acknowledgments
We thank Cheryl Welch for administrative support in the preparation of this manuscript.
Footnotes
This work was supported by National Institutes of Health Grants HL 58144, HL 72650, and HD 40828.
First Published Online October 6, 2005
Abbreviations: AM, Adrenomedullin; CGRP, calcitonin gene-related peptide; CL, calcitonin receptor-like receptor; E2, estradiol; Emax, maximal relaxation response; eNOS, endothelial nitric oxide synthase; L-NAME, N-nitro-L-arginine methyl ester; NE, norepinephrine; OVX, ovariectomized; P4, progesterone; pD2, –log EC50 of the molar concentration; PSS, physiological salt solution; RAMP, receptor activity-modifying protein.
Accepted for publication September 23, 2005.
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Abstract
Based on the favorable effects of female sex steroids in vascular functions and the potent hypotensive effects of adrenomedullin (AM), we hypothesized that AM-induced vasodilation is gender dependent, and female sex steroids enhance this effect. In endothelium-intact rat mesenteric artery, AM (1 nM–0.3 μM)-induced concentration-dependent relaxation was significantly (P < 0.05) higher in females [pD2(–log EC50 of the molar concentration), 7.05 ± 0.10; maximal relaxation response (Emax), 69.2 ± 3.46%] than males (pD2, 6.53 ± 0.08; Emax, 53.28 ± 4.86%). The increased relaxation was lost when the females were ovariectomized (OVX) (pD2, 6.14 ± 0.24; Emax, 39.68 ± 5.68%). The reduced relaxation response in OVX rats was reversed by administration of either progesterone (P4; pD2, 7.18 ± 0.07; Emax, 72.4 ± 2.76%) or 17-estradiol (E2; pD2, 7.00 ± 0.14; Emax, 70.4 ± 4.79%). AM mediates its effects through either AM22–52-sensitive AM1 receptors [composed of calcitonin receptor-like receptors (CLs) and receptor activity-modifying protein (RAMP)2] or AM2 receptors (CL/RAMP3), which can be antagonized more potently by calcitonin gene-related peptide8–37 than AM22–52. Pharmacological characterization suggested the involvement of AM2 receptors in the increased vasodilatory effect of AM in both P4- and E2-treated animals as calcitonin gene-related peptide8–37 (10 μM) was more potent in antagonizing the AM effects (Emax, P4: 25.92 ± 5.32%; E2: 29.11 ± 7.41%) than AM22–52 (100 μM). RT-PCR studies also supported the involvement of AM2 receptors because expression of mRNA levels encoding CL (previously reported) and RAMP3 were increased in P4- or E2-treated OVX rats. In conclusion, AM-induced vasodilation is gender-dependent and increased by female sex steroids by increased expression of AM2 receptor components.
Introduction
GENDER DIFFERENCE IN the incidence of cardiovascular disease is well documented in the literature. Epidemiological evidence suggests that premenopausal women have a reduced incidence of cardiovascular diseases compared with postmenopausal women and men of a comparable age (1). The much lower mortality rate in association with cardiovascular disease in postmenopausal women receiving steroid hormone therapy indicates the cardioprotective role of female sex steroid hormones (2). The cardiovascular protective effects of female sex steroids have also been shown in numerous animal studies (3). Although the underlying mechanisms are not completely understood, the favorable effects of steroid hormones in vascular functions are attributed to several factors such as nitric oxide (4), prostaglandins (5), and calcitonin gene-related peptide (CGRP) (6, 7). However, there are several other factors remaining unexplored, which include adrenomedullin (AM), a structurally related peptide to CGRP.
AM is a 55-amino acid peptide that was discovered in 1993 from a panel of peptides extracted from a pheochromocytoma (8). Later, it was realized that AM was produced by a wide range of cells including vascular endothelial and smooth muscle cells. In rat, cat, sheep, and man, iv infusion of AM results in a potent and sustained hypotension (9, 10, 11). Acute or chronic administration of AM resulted in a significant decrease in total peripheral resistance accompanied by a fall in blood pressure in both conscious and hypertensive rats (12).
The mechanisms via which AM can elicit vascular relaxation are heterogeneous with respect to both species and vascular bed. These are not completely understood but are known to involve both the CGRP and AM receptors (13). Both CGRP and AM are known to act via the same seven-transmembrane G protein-linked receptor [i.e. calcitonin receptor-like receptor (CL)]. The majority of the differences in intracellular signaling pathways activated by CGRP and AM are attributed to the different receptor activity-modifying proteins (RAMPs) that form their receptors. Three RAMPs (RAMP1, RAMP2, and RAMP3) have been identified as a novel family of single-transmembrane domain proteins (14). The association of CL with RAMP1 produces a CGRP-selective CGRP receptor that is antagonized by the CGRP antagonist, CGRP8–37; CL with RAMP2, an AM-selective receptor (AM1) that can be antagonized by the weak AM peptide antagonist AM22–52 and CL with RAMP3 another AM receptor (AM2) that can respond to both CGRP and AM, which can be more potently antagonized by CGRP8–37 than by AM22–52 (13, 15). Thus, RAMPs provide a mechanism whereby a cell could dynamically change its sensitivity from one peptide to another.
Based on the favorable effects of female sex steroids in vascular functions and the potent hypotensive effects of AM, we hypothesized that female sex steroids increase the vasodilation caused by AM in resistant vessels like mesenteric artery. Hence, in the present study, we examined in the rat mesenteric artery: 1) the gender-based differential responsiveness to AM, 2) the role of female sex steroids on AM-induced vasodilation, and 3) the changes in the AM receptor components RAMP2 and RAMP3 based on sex steroid treatments.
Materials and Methods
Animals
Adult male and female nonpregnant (age 9–11 wk) Sprague Dawley rats (Harlan Sprague Dawley, Houston, TX) weighing between 250 and 300 g were used. All rats were maintained in the colony room with fixed photoperiod of 12-h light, 12-h dark cycle and having access to water and rodent chow ad libitum. All procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch (Galveston, TX) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Ovariectomy
Female rats were anesthetized by ip injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). Small incisions were made on each flank, and the ovaries were removed. The muscle layer was suture-closed, and the incisions were closed with wound clips. After 5 d, the ovariectomized (OVX) rats received a small incision in the back of the neck into which either a sc E2 [0.5 mg, 21-d release (OVX-E2 group)] or a progesterone [P4; 5 mg, 21-d release (OVX-P4 group)] or a placebo pellet (OVX group) (Innovative Research of America, Sarasota, FL) was placed. These animals were used after 7 d, and the efficacy of ovariectomy and hormone replacement was confirmed by measuring plasma P4 and E2 levels using DSL RIA kits (Diagnostic System Laboratories, Webster, TX).
Preparation of blood vessel
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 114 mM NaCl, 4.7 mM KCl, 1.15 mM KH2PO4, 1.10 mM Na2HPO4, 1.18 mM MgSO4.7H2O, 15 mM NaHC03, 1.15 mM CaCl2, and 5.0 mM glucose. Secondary branches of the mesenteric artery were then isolated and cleaned off fat and connective tissue. The arterial segments (length, approximately 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 norepinephrine (NE) until reproducible responses were obtained. The relaxation responses were measured at cumulative doses of AM 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. Where indicated, the endothelium was removed by gently rubbing the intimal surface of the vessel with tungsten wire (size, 1 μm). The endothelium was considered intact if acetylcholine (3 μM) caused more than 80% relaxation of arteries precontracted with ED70 concentration of NE and effectively removed (denuded) if acetylcholine failed to relax. The viability of endothelium-denuded arterial rings was confirmed by relaxation induced by sodium nitroprusside (1 μM).
Pharmacological characterization of AM vasodilation
To determine the role of endothelium in the vasodilatory responses caused by AM, concentration-dependent relaxation (1 nM–;0.3 μM) was produced in endothelium-intact and -denuded arterial rings from both OVX-P4 and OVX-E2 rats. To confirm the endothelial contribution or lack of it to AM-induced vasorelaxation, concentration responses to AM (1 nM–;0.3 μM) were performed in the presence (after 30-min incubation) and absence of N-nitro-L-arginine methyl ester (L-NAME) (100 μM), an endothelial nitric oxide synthase (eNOS) inhibitor in endothelium-intact arterial rings. To verify whether the AM-induced vascular relaxation is mediated through AM1 receptors or AM2 receptors, the receptor antagonists AM22–52 and CGRP8–37 were used. Concentration-response curves to AM (1 nM–;0.3 μM) were obtained from endothelium-intact vessel segments precontracted by ED70 concentration of NE. Then, the vessel segment was washed with PSS and was incubated with either AM22–52 (10–5 or 10–4 M) or CGRP8–37 (10–5 M) for 30 min. After the incubation period, relaxation responses to cumulative doses of AM (1 nM–;0.3 μM) were repeated in NE precontracted vessel segments.
Expression of mRNA encoding RAMP2 and RAMP3
Total RNA was isolated from mesenteric artery using Trizol Reagent (Invitrogen, Carlsbad, CA). The quality and quantity of RNA were assessed at A260/280, and all samples showed absorbency ratios ranging between 1.6 and 2.0. The total RNA was treated for genomic DNA contamination using a DNA-free kit (Ambion, Austin, TX). Using total RNA, first strand cDNA was synthesized by RT using the GeneAMP RNA PCR kit (PerkinElmer, Branchburg, NJ) as described by the supplier. For RT, 2 μg total RNA was mixed with 3.0 ml random primer (Invitrogen), 200 μM dNTP solution (Sigma-Aldrich, St. Louis, MO), and 10 U avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) in the presence of 5 U RNAase inhibitor (Invitrogen). Samples were placed into a thermal cycler for one cycle at 42 C for 15 min, 99 C for 5 min, and 5 C for 5 min. The cDNA was stored at –20 C.
PCR using the specific primer sets for RAMP2 and RAMP3 was carried out with cDNA synthesized by RT. Briefly, 2 μl cDNA was mixed with a PCR mixture containing 2.5 mM MgCl2, 1 x 10 x PCR buffer II, 5 U/100 μl Amplitaq DNA polymerase kit (PerkinElmer), 65 μl sterile distilled water, and 0.1 μM of the following set of primers: RAMP2, forward, 5'-GCTGTTACTGCTGCTGTTGC 3', and reverse, 5'-GTCTGCCTCGTACTCCAAGC 3'; and RAMP3, forward, 5'-CTTCTCCCTCTGTTGCTGCT 3, and reverse, 5'-GTCCTGTCCACAGTGCAGTT 3'. Final volume was 100 μl. Primer sequences for rat RAMP2 (GenBank accession no. NM_031646.1) and RAMP3 (GenBank accession no. NM_020100.2) were derived using published sequences from the GenBank database (14). For amplification of 18S, the following primers (Ambion) were used: forward, 5'-AGGAATTGACGGAAGGGCAC-3'; and reverse, 5'-GTGCAGCCCCGGACATCTAAG-3'. The PCRs for RAMP2, RAMP3, and 18S were carried out on a GeneAMP PCR system 9700 (PerkinElmer) with the following conditions. An initial denaturation step at 95 C for 5 min was followed by 35 cycles of 60 sec at 95 C, 90 sec at 63 C, and 30 sec at 72 C, with a final extension cycle of 7 min at 72 C. The total cycle number chosen for each gene was from the linear portion of their respective amplification curve (data not shown).
Drugs used
Stock solutions of AM (100 μM), NE (10 mM), AM22–52 (100 mM), and CGRP8–37 (1 mM) were prepared in triple-distilled water, aliquoted, and stored at –80 C. AM, AM22–52, and CGRP8–37 were purchased from American Peptide Co., Inc. (Sunnyvale, CA), whereas NE was purchased from Sigma-Aldrich.
Statistical analysis
Data are presented as mean ± SE. Relaxation to AM is expressed as 100 – percentage of the initial precontraction to NE. The data were analyzed using Prism GraphPad Software (GraphPad Software Inc., San Diego, CA) employing appropriate statistical tools. Means of different groups were analyzed by one-way ANOVA and subjected to Newman-Keuls multiple comparison test. Student’s paired t test was used when comparisons were made between control and drug treatment in the same preparation. P 0.05 was considered statistically significant. Individual concentration-response curves of AM were subjected to linear regression analysis to determine EC50, which was expressed as pD2 (–log EC50 of the molar concentration of the agonist).
Results
Effect of ovariectomy and female sex steroid pellet placement on plasma sex steroid levels
To confirm the effect of OVX and hormone replacement on the plasma sex hormone levels, P4 and E2 levels were assayed in plasma collected when the animals were used for the experiments. There was significant reduction in the hormone levels after ovariectomy, whereas the hormone levels reached physiological levels after steroid hormone pellet placement. The levels of plasma P4 and E2 were 2.81 ± 0.50 nM (n = 3) and 0.1 ± 0.01 nM (n = 4), respectively, after ovariectomy. In OVX rats receiving P4 or E2 pellets, the plasma P4 levels were 11.43 ± 1.67 nM (n = 7), and the plasma E2 levels were 0.78 ± 0.12 nM (n = 6), respectively.
Effect of gender on the responsiveness of mesenteric artery to AM
The ED70 concentration of NE that was determined for each vessel produced a sustained contraction in endothelium-intact mesenteric artery rings from both male and female at diestrus rats. AM (1 nM–0.3 μM), added cumulatively at increments of 0.5 log units, relaxed the vascular rings in a concentration-dependent manner in both male and female rats. However, the potency and efficacy of AM in causing the vasodilatory response was significantly (P 0.05) lower in males [pD2, 6.53 ± 0.08; maximal relaxation response (Emax), 53.28 ± 4.86%; n = 5; Fig. 1A] compared with females (pD2, 7.05 ± 0.10; Emax, 69.2 ± 3.46%; n = 13; Fig. 1A) at diestrus.
Effect of ovariectomy and female sex steroid treatment on vasorelaxation caused by AM
Because the vascular responsiveness of mesenteric artery to AM was significantly higher in females than males, we hypothesized that female sex steroids were responsible for this increased sensitivity to AM in females. Thus, the AM-induced vasodilatory responses of mesenteric artery segments were compared from female rats upon OVX or OVX with E2 or P4 supplementation. AM (1 nM–0.3 μM) induced a concentration-dependent relaxation of the mesenteric arterial segments from all the groups. The vasodilatory response to AM was markedly reduced in females upon ovariectomy (pD2, 6.14 ± 0.24; Emax, 39.68 ± 5.68%; n = 4, P < 0.05) in comparison with females with intact ovary at diestrus (pD2, 7.05 ± 0.10; Emax, 69.21 ± 3.46; n = 13; Fig. 1A). In addition, the vasodilatory responses to AM in OVX females were similar to the responsiveness to AM in males (Fig. 1A). On the other hand, in female OVX rats, both E2 and P4 significantly (P < 0.05) increased the vasodilatory responses of mesenteric artery to AM. However, as shown in Fig. 1B, there were no significant differences between the E2 (pD2, 7.00 ± 0.14; Emax, 70.4 ± 4.79; n = 11) and P4 (pD2, 7.18 ± 0.07; Emax, 72.4 ± 2.76; n = 8) groups.
Effect of endothelium on the vasodilatory effect of AM
Because female sex steroids alter the responses to vasodilators by influencing the role of endothelium, we determined the contribution of endothelium to the vasodilatory effects of AM by comparing between endothelium-intact and -denuded arterial rings. AM (1 nM–0.3 μM) produced concentration-dependent responses in both endothelium-intact and -denuded tissues (Fig. 2, A and B). However, there were no significant differences between the endothelium-intact and -denuded rings from both OVX-P4 (endothelium-intact, pD2, 7.07 ± 0.11; Emax, 69.9 ± 3.49%; n = 9; endothelium-denuded, pD2, 7.11 ± 0.18; Emax, 71.1 ± 5.57%; n = 3) and OVX-E2 (endothelium-intact, pD2, 7.03 ± 0.13; Emax, 72.8 ± 5.29%; n = 13; endothelium-denuded, pD2, 6.55 ± 0.27; Emax, 63.4 ± 9.27%; n = 6) groups. Because there was a rightward shift, although statistically insignificant, in the OVX-E2 group, we further evaluated the vasodilatory responses in the presence of L-NAME (100 μM), an inhibitor of eNOS. Preincubation with L-NAME for 30 min did not alter the position of the concentration-response curve of AM (Fig. 2C). This dose of L-NAME was sufficient for substantial inhibition of ACh-induced vasorelaxation (data not shown) and, therefore, considered as an effective dose.
Effect of AM22–52 and CGRP8–37 on the vasodilatory effect of AM
Because female sex steroid treatments restored the impaired vasodilatory responses to AM in OVX rats, we next assessed the involvement of receptor subtypes using AM receptor antagonists, AM22–52 and CGRP8–37. Incubation for 30 min with 10 μM AM22–52 failed to shift the concentration-response curve of AM (Fig. 3, A and B), whereas 100 μM AM22–52 shifted the curve to the right in both OVX-P4 and OVX-E2 groups. The Emax values were reduced by 100 μM AM22–52 from 77.55 ± 3.07% to 35.63 ± 6.56%, n = 6 (P 0.05) in OVX-P4 rats and from 77.40 ± 5.24% to 36.00 ± 7.11%, n = 4 (P 0.05) in OVX-E2 rats (Fig. 3, C and D). On the other hand, just 10 μM CGRP8–37 was sufficient to substantially inhibit the vasodilatory responses to AM in mesenteric artery segments from both OVX-P4 (control, pD2, 7.18 ± 0.07; Emax, 72.40 ± 2.76%; n = 4 vs. CGRP8–37, Emax, 25.92 ± 5.35%; n = 4, P 0.01) and OVX-E2 (control, pD2, 6.84 ± 0.10; Emax, 63.39 ± 3.51% vs. CGRP8–37, Emax, 29.16 ± 7.41%; P 0.01; n = 6; Fig. 4, A and B).
Effect of female sex steroid treatments on the expression of mRNA encoding RAMP2 and RAMP3
The above pharmacological investigations suggested the involvement of AM2 receptors (CL/RAMP3) in the enhanced responsiveness to AM in hormone-treated rats. To confirm the role of changes in the AM receptor components, we measured the levels of mRNA encoding RAMP2 and RAMP3 in mesenteric arteries isolated from OVX, OVX-P4, and OVX-E2 rats using RT-PCR. The mRNA levels are expressed relative to those of 18S in each animal. As shown in Fig. 5, mRNA levels for RAMP2 remained unaltered, whereas the mRNA levels for RAMP3 were increased significantly (P < 0.05) in both E2- and P4-treated compared with OVX rats.
Discussion
The major findings from this study are: 1) the AM-induced vasodilatory response in rat mesenteric artery is gender-dependent, i.e. increased in females compared with males; 2) depletion of female sex steroids by ovariectomy abolished the gender differences in the vasodilatory response to AM; 3) administration of either P4 or E2 reversed the reduced vascular reactivity to AM in the OVX animals, confirming the influence of female sex steroids on AM-induced vasodilation; 4) the enhanced vascular responsiveness to AM in the steroid-treated groups is not endothelium-dependent because endothelium denudation or inhibition of eNOS enzyme could not alter the response; and 5) further, both pharmacological and AM receptor component mRNA data suggest the involvement of AM2 receptors in steroid-induced enhanced mesenteric artery relaxation to AM. Therefore, we suggest that steroid hormones regulate AM-induced vasodilatory responses in resistance vessels like mesenteric artery and therefore play a role in the cardiovascular protective effects of female sex steroids.
Gender differences in AM-induced vasodilation
The AM-induced vasodilatory response is significantly greater in female rats compared with males. Gender differences in vascular tone have been described in a multitude of vascular beds in both human and experimental animals (16). For example, -adrenergic agonists such as NE cause less forearm vasoconstriction in women than in men (17). Moreover, age-related alterations in the vasculature enhance vasoconstriction in males relative to females (18). Previously, we have reported that the vasodilatory response to CGRP is lower in males compared with females (19). Several reports in different models of hypertension suggested that the arterial pressure is greater in males than females, and ovariectomy in females accelerated the development of hypertension to a level that is not different from males (20). Gender differences in AM-induced relaxation is also shown by Packer et al. (21) in rat pulmonary artery wherein AM relaxed NE contractions two to four times greater in female than male rats. It is possible that together with the increased vasodilatory responsiveness of resistance vessel like mesenteric artery to endogenous vasodilators such as AM and reduced sensitivity to vasoconstrictors such as NE (22) result in lowered peripheral vascular resistance in females and contribute to gender-related differences in cardiovascular diseases, including hypertension, as reported in epidemiological studies (1).
Effect of ovariectomy and female sex steroid treatment on AM-induced vasodilation
Because AM-induced vasodilation is greater in females, we hypothesized that female sex steroids are responsible for the gender differences in vasorelaxation. The gender differences in the mesenteric arterial relaxation by AM are abolished when the female rats are depleted of steroid sex hormones through bilateral ovariectomy. Reversal of reduced vascular response to AM in the OVX animals by administration of either P4 or E2 confirmed the influence of female sex steroids on the vascular reactivity to AM. P4 is frequently described as opposing the actions of E2; however, this may be an oversimplification, at least with the cardiovascular effects. It is well documented that circulatory P4 levels are increased during pregnancy when there are marked cardiovascular adaptations like volume expansion and vasodilation. Results of studies from our laboratory showed that the vasodilatory effects of CGRP in hypertensive rats were P4-dependent (6). Our previous studies showed that mean arterial blood pressure was significantly higher in adult rats when ovarian hormones are depleted by bilateral ovariectomy (7). Also, hormonal replacement therapy is known to decrease blood pressure in postmenopausal women. Studies in hypertensive experimental animals have supported gender differences in blood pressure and have suggested that female sex hormones may protect against the development of hypertension (23). The data from the current study shows both E2 and P4 increase the AM-induced arterial relaxation to the same magnitude. The enhanced vasodilatory response of resistance vessels like mesenteric artery to AM, which can act in an autocrine or paracrine manner, may be part of the mechanisms involved in the favorable effects of hormonal replacement therapy in postmenopausal women, less incidence of cardiovascular diseases in premenopausal females, and vascular adaptations like reduced peripheral vascular resistance during pregnancy when both E2 and P4 levels are elevated.
Role of endothelium in the augmented AM-induced vasodilation in P4- and E2-treated rats
In the present study, the enhanced vascular relaxation to AM in steroid-treated groups is not endothelium-dependent because endothelium denudation or inhibition of eNOS enzyme could not shift the concentration-response curve of AM in both E2- and P4-treated rats. Because the animals used here were OVX, it is possible that the endothelium-derived factors are affected by ovariectomy because reciprocal changes in endothelium-derived hyperpolarizing factor and nitric oxide system in the mesenteric artery of adult female rats after ovariectomy are reported by Nawate et al. (24).
Pharmacological characterization of AM receptors
We tried to pharmacologically characterize the receptors involved in the enhanced vasodilation of mesenteric artery to AM in steroid-treated rats using the antagonists AM22–52 and CGRP8–37. Receptors for AM are heterodimeric complexes of the CL together with RAMP2 (AM1 receptor) or RAMP3 (AM2 receptor). The three potential consequences of RAMP interaction with the associated receptors are: transport of the receptor to the cell surface, modification of the receptor glycosylation, and direct and indirect modification of the ligand binding site through association with the receptor at the cell surface (25). The AM-selective receptor (AM1) can be antagonized by the weak AM peptide antagonist AM22–52, whereas the AM2 receptor can respond to both CGRP and AM, which can be antagonized more potently by CGRP8–37 compared with AM22–52 (13). In our study, the AM-induced vasodilation was inhibited by 10 μM CGRP8–37, whereas 10 μM AM22–52 could not shift the concentration-response curve in both E2- and P4-treated groups. However, 100 μM AM22–52 could inhibit the AM-induced vasodilation in both E2- and P4-administered groups, indicating CGRP8–37 is more potent than AM22–52 in inhibiting the AM-induced vasodilation, which is the pharmacological character of AM2 receptors. Both AM22–52 and CGRP8–37 are believed to be competitive antagonists, but the Emax of AM is reduced in the presence of these antagonists in our experiments. It is possible that the maximum concentration of the agonist used in these experiments is not sufficient to produce the Emax in the presence of antagonists as that produced in their absence. Hay et al. (26) have demonstrated the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at CL/RAMP2 and CL/RAMP3 receptors transiently expressed in COS 7 cells. AM22–52 (10 μM) antagonized AM at the CL/RAMP2 complex, whereas 10 μM CGRP8–37 was an effective antagonist to AM at the CL/RAMP3 complex. The pA2 values of AM22–52 and CGRP8–37 estimated in functional studies for AM1 receptors were in the order of 7 and around 6, respectively, whereas those for AM2 receptors were less than 5.5 and 6.18, respectively (27). The pharmacological investigations in the current study pointed to the involvement of AM2 receptors in the increased vasodilatory response to AM under the influence of female sex steroids.
Regulation of mRNA levels of AM receptor components by P4 and E2
The role of AM2 receptors is further supported by the increased mRNA levels encoding RAMP3 in mesenteric arteries from E2- and P4-treated OVX rats compared with OVX females. Altered gene expression of AM receptor components is shown (28) in patients with pregnancy-induced hypertension. They showed a significant negative correlation between the RAMP2 mRNA levels in the umbilical artery and uterine muscle and blood pressure, suggesting an important role for the reduced expression of AM receptor components in pregnancy-induced hypertension. Previously, we have demonstrated changes in the gene expression for CL, RAMP1, RAMP2, and RAMP3 in rat uterine tissue during pregnancy, labor, and by steroid hormone treatments (29), suggesting dynamic changes of these receptor components by respective physiological state. In OVX mice exposed to E2 for 6 h, uterine gene expression profiles developed by DNA microarrays showed about 20.4-fold increase in RAMP3 (30), indicating steroid hormone maneuver of RAMP3 expression. We have shown earlier increased expression of CL in rat mesenteric artery by P4 treatment but not E2 (31). RT-PCR data from the current study show increased expression of RAMP3, but not RAMP2, by both P4 and E2 treatments. Increase in expression of both CL and RAMP3 by P4 or RAMP3 alone by E2 may enhance the interaction between CL and RAMP3 and increase the probability of formation of AM2 receptors. However, at this juncture, it is not possible to rule out the involvement of CGRP1 receptors in AM-induced vasodilation, although this is less likely because CGRP1 receptor-mediated effects cannot be blocked by AM22–52 (32). This is further supported by the studies in which CGRP8–37, but not AM22–52, inhibited AM-induced cAMP production in HEK-293 cells cotransfected with hCLR and hRAMP1 (33).
In conclusion, our study suggests that the vasodilatory responses to AM in a resistant vessel-like mesenteric artery are gender-dependent and increased by female sex steroids, both P4 and E2. The enhanced vasodilatory response is not endothelium-dependent. Pharmacological and receptor component mRNA data suggest the involvement of AM2 receptors in steroid-induced enhanced mesenteric artery relaxation to AM. Therefore, we suggest that female sex steroids regulate AM-induced vasodilatory responses in resistance vessels and thereby play a role in steroid-related cardiovascular protection. The gender differences and the influence of female sex steroids on AM-induced vasorelaxation raise interesting questions with regard to our understanding of the gender-related and pre- and postmenopausal differences in cardiovascular diseases reported in epidemiological studies and also the marked cardiovascular adaptations during pregnancy.
Acknowledgments
We thank Cheryl Welch for administrative support in the preparation of this manuscript.
Footnotes
This work was supported by National Institutes of Health Grants HL 58144, HL 72650, and HD 40828.
First Published Online October 6, 2005
Abbreviations: AM, Adrenomedullin; CGRP, calcitonin gene-related peptide; CL, calcitonin receptor-like receptor; E2, estradiol; Emax, maximal relaxation response; eNOS, endothelial nitric oxide synthase; L-NAME, N-nitro-L-arginine methyl ester; NE, norepinephrine; OVX, ovariectomized; P4, progesterone; pD2, –log EC50 of the molar concentration; PSS, physiological salt solution; RAMP, receptor activity-modifying protein.
Accepted for publication September 23, 2005.
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