Characterization of angiotensin-(1–7) receptor subtype in mesenteric arteries

Peptides 24 (2003) 455–462

Characterization of angiotensin-(1–7) receptor subtype in mesenteric arteries Liomar A.A. Neves a , David B. Averill a , Carlos M. Ferrario, Mark C. Chappell a , Judy L. Aschner a,b , Michael P. Walkup c , K. Bridget Brosnihan a,∗ a

The Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1932, USA b Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1932, USA c Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1932, USA Received 15 July 2002; accepted 5 December 2002

Abstract Mesenteric arteries from male Sprague-Dawley rats were mounted in a pressurized myograph system. Ang-(1–7) concentration-dependent responses were determined in arteries preconstricted with endothelin-1 (10−7 M). The receptor(s) mediating the Ang-(1–7) evoked dilation were investigated by pretreating the mesenteric arteries with specific antagonists of Ang-(1–7), AT1 or AT2 receptors. The effects of Ang-(3–8) and Ang-(3–7) were also determined. Ang-(1–7) caused a concentration-dependent dilation (EC50 : 0.95 nM) that was blocked by the selective Ang-(1–7) receptor antagonist D-[Ala7 ]-Ang-(1–7). Administration of a specific antagonist to the AT2 receptor (PD123319) had no effect. On the other hand, losartan and CV-11974 attenuated the Ang-(1–7) effect. These results demonstrate that Ang-(1–7) elicits potent dilation of mesenteric resistance vessels mediated by a D-[Ala7 ]-Ang-(1–7) sensitive site that is also sensitive to losartan and CV-11974. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Hypertension; Renin–angiotensin system; Vasodilation; Angiotensin receptors

1. Introduction There is emerging evidence that the biologically active heptapeptide of the renin–angiotensin system, Ang-(1–7), opposes the cardiovascular actions of Ang II. Chronic infusion of Ang-(1–7) in spontaneous hypertensive rats produces a sustained reduction in mean arterial pressure [3,4]. The vasodilator mechanism underlying the depressor action of Ang-(1–7) has been described in a number of vascular regions, including coronary [5,20,36], mesenteric [34,35], cerebral [17,32], aorta [18], and hindlimb [35]. A recent study from our group showed that Ang-(1–7) elicits dilation in canine coronary artery rings by an endothelium dependent mechanism [5]. Recent studies suggest that Ang-(1–7) effects are mediated by an angiotensin receptor distinct from AT1 or AT2 receptors. Autoradiographic studies indicate the presence of specific Ang-(1–7) binding sites in rat mesenteric arteries and aorta [10,28]. Studies using the selective Ang-(1–7) antagonist, D-[Ala7 ]-Ang-(1–7) (D-Ala), provide evidence ∗

Corresponding author. Tel.: +1-336-716-2795; fax: +1-336-716-2456. E-mail address: [email protected] (K.B. Brosnihan).

for the existence of an Ang-(1–7) receptor distinct from the classical AT1 and AT2 receptor subtypes [40,42]. In bovine aortic endothelial cells, saralasin and D-Ala, but neither AT1 nor AT2 receptor antagonists compete for specific binding of Ang-(1–7) [42]. Physiologic studies have shown that D-Ala antagonizes the actions of Ang-(1–7) in the central nervous system [6,33] as well as the hemodynamic and renal effects evoked by the heptapeptide [40,43]. Administration of D-Ala in hypertensive rats [SHR and (mRen2)27 transgenic rats] treated chronically with lisinopril partially reverses the antihypertensive effect of the ACE inhibitor [26,27]. This last finding suggests that endogenous Ang-(1–7) contributes to the blood pressure lowering effect of ACE inhibition. Although previous studies have shown that Ang-(1–7) acts as a vasodilator in several vascular beds, characterization of the receptor(s) by which this peptide promotes vasorelaxation in resistance blood vessels has not been thoroughly determined. Previous studies, using an in situ perfusion system where the vessels were exposed to shear stress, showed a small degree of vasodilation (from 4 to 10%) caused by Ang-(1–7) [15]. Furthermore, these studies were performed without preconstriction, which makes it difficult to

0196-9781/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0196-9781(03)00062-7

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properly assess the full vasodilator capability of Ang-(1–7). Therefore, the aim of the present study was to define the angiotensin receptor subtype(s) at which Ang-(1–7) promotes vasodilation. Vascular responses to the peptide were evaluated in isolated mesenteric resistance arteries under no flow conditions in order to evaluate the pharmacological actions of Ang-(1–7) in the absence of shear stress and determine the capacity of the peptide to act in a paracrine manner.

2. Methods 2.1. Animals Thirty-one 13-week-old male Sprague-Dawley rats were obtained from Charles River Lab (Wilmington, MA, USA) and housed individually for one week under a 12-h light/dark cycle in an AAALAC-approved facility. All protocols were approved by the Animal Care and Use Committee of Wake Forest University School of Medicine and are in compliance with NIH guidelines. 2.2. Isolated mesenteric artery preparation Rats were sacrificed by decapitation on the day of study. A segment of the proximal jejunum with the mesenteric vasculature attached was excised and placed in ice-cold (4 ◦ C) physiological salt solution with the following composition (in mmol/l: KCl 4.8, CaCl2 2.0, KH2 PO4 1.2, MgSO4 1.2, dextrose 11, NaCl 118, and NaHCO3 25). Arteries with an outer diameter of 230–290 ␮m were identified, carefully dissected, and cleaned of adherent adipose tissue. Isolated artery segments with a length of 2–3 mm were transferred to an arteriograph chamber (Living System Instrumentation, Burlington, VT) [22]. The artery segment was cannulated at each end and maintained at an intraluminal pressure of 40 mmHg. Only leak-free preparations that maintained a stable intraluminal pressure were included. Prewarmed buffer (37 ± 0.5 ◦ C) equilibrated with 21% O2 : 5% CO2 : 74% N2 (pH 7.4) was circulated through the vessel chamber at a rate of 38 ml/min; the same gas mixture flowed under the superfusion gas cover. The chamber was set on the stage of an inverted microscope with a video camera attached to the viewing tube. The vessel image was projected on a TV monitor and continuous measurement of the lumen diameter (LD) was made using a Living Systems Instrumentation video dimension analyzer system (Burlington, VT). Signals from a pressure servo system and video dimension analyzer were simultaneously collected by a computer data acquisition system (WinDaq, DATAQ Instruments Inc., Akron, OH), and analyzed by WinDaq Waveform Browser (DATAQ Instruments Inc., Akron, OH). All drugs were added to a buffer reservoir and the buffer was re-circulated. Dosages were expressed as the final cumulative molar concentration in the

buffer solution, assuming zero metabolism. Each mesenteric artery was initially constricted with 50 mmol/l KCl to demonstrate appropriate vascular smooth muscle responses, followed by exposure to 10−5 M acetylcholine to demonstrate intact endothelial-dependent relaxation. Vessels that did not constrict by 50% to KCl and did not dilate by 80%, when acetylcholine was applied, were excluded from further study. After the viability of the vessel had been determined, the vessel was washed and allowed to equilibrate for 30 min prior to the beginning of the experiment. Only one artery was used for each concentration–response curve; however, several arteries were taken from each rat for the different agonist/antagonist combinations studied. At the end of each experiment, 10−5 M acetylcholine was added to each vessel to reaffirm the presence of viable endothelium; vessels that did not dilate by at least 80% were excluded from the analysis. 2.3. Experimental protocol After an equilibration period, the vessels were preconstricted to 34% of their resting diameter with 10−7 M endothelin-1 (ET-1). Time control experiments revealed a gradual and reproducible relaxation of the ET-1 induced maximal constriction over time, necessitating that the measured Ang-(1–7) dilator response be corrected at each time point by the percent spontaneous relaxation. Thus, the luminal diameter of preconstricted vessels in the time control group was recorded every 3 min for 21 min, i.e. the total time of the experiment. The choice of ET-1 as a preconstrictor was made after it was determined that mesenteric resistance arteries underwent complete loss of constrictor tone over 21 min following preconstriction with U46619 or resulted in so potent constriction that the lumen was obliterated producing a vessel incapable of dilation to endothelium dependent or independent dilators. In a separate group of ET-1 preconstricted vessels, progressively increasing cumulative concentrations of Ang-(1–7) (10−10 to 10−5 M) were applied. Vessels were exposed to each peptide concentration for 3 min before the next concentration was added, and the maximal dilation recorded during each 3-min period. A third group of vessels were perfused with 10−7 M D-Ala, the specific Ang-(1–7) antagonist, for 30 min before the vessels were preconstricted with ET-1 and concentration–response curve for Ang-(1–7) was obtained. To determine if AT1 receptors contributed to the Ang-(1–7)-mediated vasodilation, vessels were perfused with losartan (5 × 10−6 M) or CV-11974 (10−8 M) for 30 min before application of progressively increasing concentrations of Ang-(1–7). PD123319 (5 × 10−6 M) was used to test the AT2 receptor contribution. These concentrations of the antagonists were selected based on previous studies demonstrating a maximal effective blocking concentration in mesenteric vessels [31]. Under these conditions, no vasoconstrictor effect of Ang II (10−7 M) was observed up to 60 min after perfusion of losartan (5 × 10−6 M) (data not

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shown). We also examined the vascular reactivity of the mesenteric arteries to Ang-(3–8) and Ang-(3–7) in ET-1 preconstricted vessels to assess whether stimulation of the AT4 receptor could evoke mesenteric dilation. Our experimental condition of preconstricted vessels allows primarily the examination of the vasodilatory effect of Ang-(3–8) and Ang-(3–7). Previous studies have shown that Ang-(3–8) may elicit vasodilation or vasoconstriction via activation of the AT4 receptor [11,30]. In addition, Ang-(3–7) was suggested to be the active form of Ang-(3–8) or may contribute to the functional effects of Ang-(1–7) [23]. For each vessel, dilation at any given peptide concentration was expressed as the difference in lumen diameter from maximum ET-1 constriction divided by the difference between baseline and ET-1 constriction. The time control was subtracted from values in Figs. 3–5. 2.4. Statistical analysis General linear models were used to estimate mean percentage dilation across time while controlling for correlations among measurements and artery segments from the same animal. These models were fit using maximum likelihood; comparisons between mean percent dilation were performed using Wald test [41]. Responses at each concentration of Ang-(1–7) were compared by one-way analysis of variance (ANOVA) followed by Newman-Keuls or Dunn’s multiple comparisons test when significant differences existed between groups. A P-value less than 0.05 was considered statistically significant. Unpaired Student’s t-test was used to compare vessel diameter before and after ET-1. All values are presented as mean ± S.E.M. 2.5. Reagents and chemicals The chemicals used were from Sigma (St. Louis, MO, USA) unless otherwise noted. Ang-(1–7), Ang-(3–7), Ang-(3–8) and D-(Ala)7 -Ang-(1–7) were obtained from Bachem, Inc. (Torrance, CA, USA). These peptides were dissolved in water at an initial concentration of 10−2 M, aliquoted, and stored at −20 ◦ C until use. ET-1 was obtained from Novabiochem (La Jolla, CA) and dissolved in 50:50 ethanol:water solution at an initial concentration of 10−4 M and stored at −20 ◦ C. The AT1 receptor antagonists, losartan and CV-11974, were kindly provided by Merck and Co., Inc. (Wilmington, DE) and Takeda Chemical Industries, Ltd. (Osaka, Japan), respectively, and the AT2 receptor antagonist, PD123319, was provided by Warner-Lambert Parke-Davis (Ann Arbor, MI). Losartan and PD123319 were dissolved in water; CV-11974 was dissolved in 20 mM Na2 CO3 solution at an initial concentration of 10−2 M. On the day of the experiment, the agonists and antagonists were diluted in Krebs-Henseleit solution to achieve the desired final concentration.

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3. Results 3.1. Effect of Ang-(1–7) on vascular reactivity In isolated mesenteric resistance arteries, superfusion of 10−7 M ET-1 significantly reduced the luminal diameter by 65.5±0.5% (from 248.4±3.0 to 85.8±1.8 ␮m, P < 0.001, n = 49). There was no significant difference in baseline diameter among the groups, and neither was there a significant difference in the diameter after ET-1 administration among the groups. Fig. 1A illustrates a typical dilation response to the addition of progressively higher concentrations of Ang-(1–7) (10−10 to 10−5 M); Fig. 1B is a representative time control experiment. The dilator response reached after each dose of Ang-(1–7) is maintained. As shown in Fig. 2, the average dilator response to 12 vessels treated with Ang-(1–7) is significantly greater than the spontaneous dilation observed in the nine control vessels (P = 0.009). Ang-(1–7) displays an EC50 of 47.4 ± 13.1 nM. By removing the influence of the spontaneous dilation of the time control, Fig. 3 shows that the dilation response to Ang-(1–7) plateaus at approximately 10 nM and the corrected EC50 values is 0.95 nM. Pretreatment of the vessels with the specific Ang-(1–7) receptor antagonist, D-Ala, abolished the Ang-(1–7)-induced dilation. Pretreatment with either the AT1 or AT2 blocking agents alone did not alter baseline intraluminal diameter (data not shown). Pretreatment of the mesenteric arteries with the AT2 antagonist, PD123319, did not affect the dilation caused by Ang-(1–7) in mesenteric vessels (19.0 ± 4.9% versus 27.2 ± 6.4% at dose of 10−8 M, P > 0.05) (Fig. 4). Pretreatment with AT1 receptor antagonists, losartan and CV-11974, significantly attenuated the Ang-(1–7) vasodilator effect. The response to Ang-(1–7) at 10−8 M was 8.9 ± 3.4 and 7.4 ± 1.5% with losartan and CV-11974 pretreatment, respectively, values that were significantly different from Ang-(1–7) alone (27.3 ± 3.4%), P < 0.05. To assess the vasodilatory response evoked by stimulation of AT4 receptors on rat mesenteric arteries, complete concentration–response curves to the AT4 receptor agonists, Ang-(3–8) and Ang-(3–7), were determined in ET-1 preconstricted vessels. Neither Ang-(3–8) nor Ang-(3–7) dilated mesenteric resistance arteries (Fig. 5). In addition, there was no evidence of vasoconstriction induced by either peptide.

4. Discussion This study provides a pharmacological characterization of the receptor subtypes mediating the Ang-(1–7) concentration-dependent dilation of mesenteric resistance arteries of Sprague-Dawley rats. The concentrationdependent dilation of ET-1 preconstricted mesenteric resistance vessels by Ang-(1–7) was completely blocked by the Ang-(1–7) selective antagonist, D-Ala. Pretreatment

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Fig. 1. Typical recordings obtained from two mesenteric arteries. Vessels were preconstricted with endothelin-1 (10−7 M) and lumen diameter was monitored after addition of Ang-(1–7) 10−10 to 10−5 M (A) or for 21 min (time control, B). Top chart—typical recording. Bottom chart—moving average.

Fig. 2. Mesenteric arteries were preconstricted with endothelin-1 (10−7 M) and lumen diameter was monitored for 21 min (time control) or after addition of Ang-(1–7) (10−10 to 10−5 M). The average responses are expressed as the percent dilation relative to the reduced vessel diameter induced by endothelin-1. Differences among the means were evaluated using a random and fixed effect general linear model (SAS Institute). ∗ P = 0.009, Ang-(1–7) vs. time control.

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Fig. 3. The spontaneous relaxation of the time control was subtracted in this and the following figures. Mesenteric arteries lumen diameter were assessed after addition of Ang-(1–7) (10−10 to 10−5 M) in the presence or absence of 10−7 M D-[Ala7 ]-Ang-(1–7) pretreatment. Differences among the means were evaluated using one-way analysis of variance (ANOVA) followed by Newman-Keuls or Dunn’s multiple comparisons test. ∗ P < 0.05, compared to Ang-(1–7) alone.

of ET-1 preconstricted vessels with losartan or CV-11974, AT1 receptor antagonists, caused a significant attenuation of the Ang-(1–7)-induced dilation. There was no effect of the AT2 receptor antagonist. Furthermore, in our preparation there did not appear to be an AT4 -mediated relaxation since neither Ang-(3–8) nor Ang-(3–7) caused dilation of ET-1 preconstricted vessels. Thus, the dilatory effect of Ang-(1–7) occurred through a D-Ala-sensitive site that is also recognized by losartan and CV-11974. The results of this study, together with those of Oliveira et. al. [34] and Fernandes et al. [15], reveal that mesen-

teric resistance arteries are substantially more sensitive to the vasodilator actions of Ang-(1–7) than are coronary vessels from dogs or pigs [5,36]. In the current study, the EC50 (0.001 ␮M) for Ang-(1–7) was approximately 2000-fold less than the EC50 (2 ␮M) reported for coronary vessels [5]. One important difference between the studies examining the vascular actions of Ang-(1–7) in mesenteric vessels versus coronary vessels was that the former studies investigated actions of the peptide on resistance level arteries whereas the coronary vessels studied by Brosnihan et al. [5] and Porsti et al. [36] may be viewed as conduit arteries. In fact, for

Fig. 4. The magnitude of relaxation in mesenteric arteries due to addition of Ang-(1–7) (10−10 to 10−5 M) in the presence or absence of losartan (5 × 10−6 M), CV-11974 (10−8 M) or PD123319 (5 × 10−6 M) pretreatment was assessed. ∗ P < 0.05, losartan vs. Ang-(1–7) alone; # P < 0.05, losartan and CV-11974 vs. Ang-(1–7) alone.

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Fig. 5. Ang-(3–8) or Ang-(3–7) was added in a cumulative fashion (10−10 to 10−5 M) after preconstriction of mesenteric arteries of male rats with ET-1.

the mesenteric resistance vessels used in the current experiments, the change in luminal diameter produced by 10−8 M Ang-(1–7) corresponded to a 85% reduction in calculated vascular resistance relative to the ET-1-induced constricted state. Thus, our studies and those of Oliveira et al. [34] and Fernandes et al. [15] have provided evidence that Ang-(1–7) is an important vasodilator near physiological concentrations of the peptide. In the present study, we obtained a complete concentration–response curve to Ang-(1–7) and showed that the Ang-(1–7) dilator response reached a plateau at a concentration of 10−8 M. In comparison to the studies of Oliveira et al. [34] and Fernandes et al. [15] we observed that Ang-(1–7) evoked a greater degree of vasodilation over a larger range of concentrations of the peptide. In part, this may be explained by the differences in vessel size in the two studies (250 ␮m versus 15–25 ␮m), the lack of shear stress in our preparation, and the fact that their preparation was not preconstricted so that the maximal vasodilatory capacity could not be evaluated. These latter differences may account for the greater magnitude of vasodilation in our preparation which would evoke the release of different combinations of endothelial derived relaxing factors that have been previously characterized to be involved in Ang-(1–7)-mediated responses. Additional studies are warranted to determine and quantitate the specific mediators involved in our preparation. Our results, however, agree with those of Oliveira et al. [34] and Fernandes et al. [15] in that D-Ala completely blocked the vasodilator action of Ang-(1–7). The ability of D-Ala to block the vasodilation of Ang-(1–7) agrees with previous studies demonstrating that Ang-(1–7) acted in the vasculature at a unique AT(1–7) receptor [5,15,16,36]. Further support for this receptor was

provided by binding studies using membranes from cultured bovine aortic endothelial cells and supported by the ability of D-Ala to compete for [125 I]-Ang-(1–7) binding [28,42]. In vitro receptor autoradiography of mesenteric vessels revealed that 125 I-[Sar1 Thr8 ]-Ang II binding in the presence of losartan and PD123319 was blocked by Ang-(1–7) and D-Ala [27]. Previous work has shown that D-Ala has a similar affinity as Ang-(1–7) for the vascular endothelial AT1–7 receptor, and that D-Ala is a competitive antagonist at the AT1–7 receptor site [24,42]. Our studies are functional studies and cannot be compared directly to the binding studies of Ang-(1–7) to cultured passage vascular endothelial cells by Tallant et al. [42]. Nevertheless, it was somewhat surprising that Ang-(1–7) at a 100-fold higher concentration did not overcome the blockade of 100 pmol of D-Ala. The most likely explanation for this is that Ang-(1–7) at higher concentrations may compete not only for the D-Ala receptor, but the AT1 receptor as well. The tendency for vasoconstriction in the D-Ala pretreated vessels at Ang-(1–7) concentration of 1 ␮M or greater, as observed in Fig. 3, is consistent with this possibility. Our results differ from previous observations in canine coronary [5], rabbit renal afferent arterioles [38], and rat A2 mesenteric vessels [15] that failed to demonstrate an influence of the AT1 receptor antagonist on the Ang-(1–7) response. However, our studies are not without precedent, since actions of Ang-(1–7) that can be blocked by losartan have been previously described [2,12,37]. In previous studies, it has been demonstrated that Ang-(1–7) has a poor affinity for the AT1 receptor [39,42], thus, it is not likely that Ang-(1–7) at low concentrations is acting at the AT1 receptor. Additional support for this view comes from the finding that D-Ala [8] has no affinity for either AT1 or

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AT2 receptors. Furthermore, it was the smaller, and not the larger, concentrations of Ang-(1–7) that were blocked by losartan and CV-11974, making it unlikely that Ang-(1–7) acted as a competitive antagonist at the AT1 receptor, as proposed by Potts et al. [37]. Our data at the lower concentrations of Ang-(1–7) are consistent with a receptor subtype of Ang-(1–7) that is sensitive to losartan and CV-11974. It has been shown that such receptors exist in the rat kidney [2]. At the higher concentrations of Ang-(1–7), i.e. greater than 1 ␮M, the loss of a significant inhibitory effect of losartan and CV-11974 may reflect a combination of an effect of Ang-(1–7) at an AT1 receptor site that is being blocked by losartan and CV-11974 and an effect of Ang-(1–7) acting at its own receptor. While more investigation is clearly needed, we hypothesize that a positive interaction may exist between AT1 and Ang-(1–7) receptor. Blockade of AT1 receptors has the effect of removing this interaction and, consequently, diminishing the responsiveness of the Ang-(1–7) receptor for its ligand, Ang-(1–7). Pretreatment of mesenteric resistance vessels with an AT2 receptor antagonist PD123319 had no effect on the Ang-(1–7) vasodilator response. Although an AT2 receptor-mediated dilation to Ang II has been postulated for cerebral arteries and renal afferent arterioles [1,21], our results did not support a role for AT2 receptors in the Ang-(1–7) dilator response in the mesenteric vasculature. Moreover, the AT2 receptor has an extremely low affinity for Ang-(1–7) [9,39]. In contrast to Ang-(1–7), Ang II and Ang-(3–8) may act as agonists at AT2 receptors in the mesenteric circulation to promote either vasoconstriction or vasodilation [25,30]. It has been suggested that another metabolite of Ang II, Ang-(3–8) or Ang IV, mediates vasodilation through an AT4 receptor subtype. However, conflicting results have been reported for the vascular actions of Ang-(3–8). Vasodilator effects of Ang-(3–8) have been demonstrated in pulmonary, cerebral, renal cortical, and cochlear circulations [11,13,14,29]. Other reports have demonstrated an AT1 receptor-mediated vasoconstrictor effect of this peptide in the mesenteric and hindlimb vascular beds of the cat and rat [7,19]. To examine the possibility that stimulation of the AT4 receptors in mesenteric resistance vessels might evoke dilation, we investigated the dose response to Ang-(3–8) or Ang-(3–7) in ET-1 preconstricted vessels. Neither peptide produced dilation in the ET-1 preconstricted vessels. We have interpreted these findings as eliminating the AT4 receptor as a site at which Ang-(1–7) mediates dilation in mesenteric resistance vessels. In addition, these data show that Ang-(3–7) is not a mediator of the Ang-(1–7)-induced dilation in mesenteric vessels. In conclusion, these experiments demonstrated that mesenteric resistance arteries have a marked vasodilator response to Ang-(1–7). The dilator responses were blocked by a specific antagonist of Ang-(1–7), D-Ala, and were also attenuated by AT1 receptor antagonists, losartan and CV-11974. Our studies provide no evidence for a role of

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AT2 or AT4 receptors in the mesenteric vasodilator response to Ang-(1–7).

Acknowledgments The authors gratefully acknowledge the technical support of Thuy Smith. This work was supported in part by a grant from the National Institutes of Health, NHLBI-P01 HL58952, and HL62489.

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