REVIEW URRENT C OPINION

Alamandine: a new member of the angiotensin family Daniel C. Villela a, Danielle G. Passos-Silva a, and Robson A.S. Santos a,b

Purpose of review In this article, we review the recent findings regarding a new derivative of angiotensin-(1–7) [Ang-(1–7)], alamandine, and its receptor, the Mas-related G-coupled receptor type D (MrgD) with a special emphasis on its role and how it can be formed. Recent findings Over the last decade, there have been significant conceptual changes regarding the understanding of the renin-angiotensin system (RAS). A cardioprotective axis has been elucidated by the discovery of the Mas receptor for the biologically active Ang-(1–7), and the angiotensin-converting enzyme 2 (ACE2) that coverts Ang II into Ang-(1–7). In addition, several components of the system, such as Ang-(1–12), Angiotensin A (Ang A) and the newly discovered peptide, alamandine, have been identified. Alamandine is generated by catalysis of Ang A via ACE2 or directly from Ang-(1–7). Summary Alamandine is a vasoactive peptide with similar protective actions as Ang-(1–7) that acts through the MrgD and may represent another important counter-regulatory mechanism within the RAS. Keywords alamandine, angiotensin A, angiotensin-(1–7), Mas-related G-coupled receptor type D, renin-angiotensin system

INTRODUCTION The renin-angiotensin system (RAS) is an evolutionary conserved system, which is present in all vertebrates [1 ]. RAS has implications in hypertension and cardiovascular diseases and it was accepted initially as a linear proteolysis pathway with a single active end product, angiotensin II (Ang II). The classical view of this cascade begins with angiotensinogen, an alpha-2-globulin produced mainly by the liver, which is cleaved by renin, an enzyme expressed in the kidney and released in the bloodstream. This reaction forms angiotensin I, an inactive decapeptide, which is cleaved by the angiotensin-converting enzyme (ACE) into Ang II, a biologically active octapeptide [2]. Although Ang II is the main biologically active peptide of the RAS system and has central roles in blood vessels, heart, brain and kidney through its interaction with AT1 receptors and AT2 receptors, several studies have revealed a more complex nonlinear RAS with multifunctional peptides, enzymes and receptors. The discovery of an ACE2/angiotensin-(1–7) [Ang-(1–7)]/Mas protective axis that &&

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counterbalances many actions of the ACE/AngII/ AT1R axis has highlighted the complexity of this system. This counter-regulatory axis has as a central peptide Ang-(1–7), a heptapeptide that is hydrolyzed from Ang II through the action of ACE2 or other peptidases, especially PEP (prolyl-endopeptidase). This peptide can also be formed by another route from the cleavage of Ang I through the enzymes NEP (neutral endopeptidase), prolyl-endopeptidase and TOP (thymet oligopeptidase) or through ACE2,

a National Institute of Science and Technology in Nanobiopharmaceutics, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais and bInstituto de Cardiologia do Rio Grande do Sul/Fundac¸a˜o Universita´ria de Cardiologia, Rio Grande do Sul, Brazil

Correspondence to Robson Augusto Souza dos Santos, MD, PhD, Department of Physiology and Biophysics, Av. Antonio Carlos, 6627ICB-UFMG, 31270-901-Belo Horizonte, MG, Brazil. Tel: +55 31 3409 2956; e-mail: [email protected] Curr Opin Nephrol Hypertens 2014, 23:130–134 DOI:10.1097/01.mnh.0000441052.44406.92 Volume 23  Number 2  March 2014

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Alamandine: a new member of the angiotensin family Villela et al.

KEY POINTS  Alamandine is a new member of the renin-angiotensin system.  Alamandine can be formed from Angiotensin A through hydrolysis by ACE2 or directly from Angiotensin-(1–7).  The effects of alamandine are independent of Mas and can be mediated by a Mas-related receptor, MrgD.  Alamandine produces vasodilation and a long-lasting antihypertensive effect in SHR.

ANGIOTENSIN-CONVERTING ENZYME 2 FORMS ALAMANDINE FROM ANGIOTENSIN A Because Ang A has a high degree of similarity with Ang II, sharing the same C-terminal, the possibility that it could be a substrate for ACE2 was tested. Indeed, this peptide can be hydrolyzed by human ACE2 to form alamandine. The N-terminal amino acid aspartate from Ang II, or probably even from Ang I, is enzymatically decarboxylated into alanine. This peptide can then be cleaved in the C-terminus by ACE2 producing alamandine (Fig. 1) [8 ]. Another route by which alamandine could be produced is directly from Ang-(1–7) through decarboxylation (Fig. 1). After Ang-(1–7) perfusion through the coronary vessels of the rat heart, formation of alamandine was observed, indicating that this peptide can be formed locally as shown for other RAS peptides [8 ]. Alamandine is also an endogenous peptide identified in human blood. Interestingly, nephropathic patients have an increased alamandine plasma concentration, suggesting that this peptide may have, as Ang-(1–7), an important role in pathophysiological conditions [8 ]. &&

which first produce Ang-(1–9) and then through ACE forms Ang-(1–7) [3]. The actions mediated by Ang-(1–7) are done through interaction with Mas, a G-protein coupled receptor [4]. Other angiotensin peptides can also be formed within the RAS and produce biological effects, such as angiotensin (2–8) (Ang III) and angiotensin-(3–8) (Ang IV) [3]. Recently, new components of RAS have been described. Jankowski et al. [5] identified an octapeptide named angiotensin A (Ang A), which is highly similar to Ang II, only differing in the N-terminal in which the Asp1 is decarboxylated into Ala1. This peptide has the same affinity for the AT1 receptor and human AT2 receptor as Ang II, but shows a higher affinity for rat AT2 receptors [5,6]. In addition, Ang A has a smaller vasoconstrictive effect than Ang II in isolated perfused rat kidney, which is not modified in the presence of the AT2 receptor antagonist PD123319, suggesting that Ang A has a lower intrinsic activity at the AT1 receptor [5]. Indeed, intravenous and intrarenal administration of Ang A induced a dose-dependent renal vasoconstrictor response in normotensive rats, and a pressor response in mice [5] and normotensive and hypertensive rats [6], but with less potency than Ang II. This response could be blocked by an AT1 receptor antagonist but was not modified by AT2 receptor ligands [6]. More recently, it has been reported that Ang A administration in rats increased blood pressure at comparable level to Ang II and demonstrated similar cardiac effects (decrease of coronary flow, systolic tension and þdP/dT) which were blocked by the AT1 antagonist losartan [7]. Our group hypothesized that Ang A could be cleaved to form a peptide similar to Ang-(1–7) but with the same N-terminal Asp/Ala modification. The present article reviews the recent discovery and characterization of a new peptide from the RAS, the heptapeptide, Ala-Arg-Val-Tyr-Ile-His-Pro, alamandine and its receptor the Mas-related G-coupled receptor type D (MrgD).

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BIOLOGICAL ACTIONS OF ALAMANDINE Alamandine and Ang-(1–7) differ only in the N-terminal aminoacid Asp/Ala. This may account for the significant similarity of the majority of the biological effects analysed. As previously reported for Ang-(1–7), alamandine produced endothelialdependent vasorelaxation in aortic rings of mice and rats. Moreover, the microinjection of alamandine in medullary areas of the brain, critical for the control of blood pressure, produced cardiovascular effects similar to those produced by Ang-(1–7); increase in blood pressure at the rostral ventrolateral medulla (RVLM) and decrease in blood pressure at caudal ventrolateral medulla (CVLM) [8 ]. Figure 2 shows the effect of intracerebral ventricular (ICV) infusion of Alamandine on baroreflex sensitivity. As previously reported for Ang-(1–7) [9], a selective increase of the bradicardic component of the baroreflex was observed. Another similar effect of Ang-(1–7) and alamandine was observed when an oral formulation of alamandine was tested by inclusion in HP-b cyclodextrin. A single oral administration of the inclusion compound alamandine/ HP-b cyclodextrin produced a long-lasting antihypertensive effect in spontaneously hypertensive rats (SHR) [8 ]. This effect is similar to that produced by oral administration of Ang-(1–7) in SHR (J.N. Braga, R.A.S. Santos, unpublished results). In addition, a significant decrease in collagen I, III and fibronectin

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Pathophysiology of hypertension

Angiotensin (1–7)

Angiotensin A A

H

R V

Y

P

I

D F

H

R V

Y

P

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Decarboxylation enzyme

ACE 2

Alamandine A

H

R V

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MrgD

FIGURE 1. Alamandine formation and receptors. Alamandine can be formed from angiotensin A through the action of ACE2 or from Angiotensin (1–7) through the action of a decarboxylase enzyme. This peptide binds to the Mas-related G-protein coupled receptor, member D (MrgD). The possibility of binding to other unknown receptor cannot be discarded. (This figure is based on illustrations available on http://www.servier.com/Powerpoint-image-bank).

accumulation was observed in isoproterenol-treated rats receiving a dose of alamandine once a day [8 ]. This effect was quite similar to that induced by oral administration of Ang-(1–7) [10]. It should be noted that not all effects of both peptides are the same. For example, no anti-proliferative effect of alamandine, an effect well described for angiotensin-(1–7) [11], could be demonstrated [8 ]. &&

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vasorelaxation effect was fully preserved. These data suggest that alamandine is not a Mas receptor agonist [8 ]. In order to clarify the specificity of alamandine, another Ang-(1–7) antagonist, D-Pro7-Ang-(1–7) was tested. Strikingly, D-Pro7-Ang-(1–7) blocked the vasorelaxation produced by alamandine in aortic rings of mice and the blood pressure effects produced by microinjections of alamandine in the CVLM and RVLM in Fisher rats [8 ]. On the basis of these findings, we formed the hypothesis that alamandine could bind to another receptor related to Mas. Recently, Ang-(1–7) has been shown to be a weak ligand for the Mas-related G-coupled receptor type D (MrgD), suggesting a possible role of this receptor in the Ang-(1–7)/ACE2 axis [12]. As alamandine has a similar structure to that of Ang-(1–7), the hypothesis that alamandine could be a strong ligand for MrgD was tested. This has been proven to be true [8 ]. The MrgD receptor has a single copy gene with orthologs in rats, mice and humans [13]. Initial &&

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ALAMANDINE X ANGIOTENSIN (1–7): SIMILARITIES AND DISSIMILARITIES Although the effects of alamandine resemble those of Ang-(1–7), the receptors for these two peptides differ. It has been established over the last 10 years that Ang-(1–7) is the endogenous ligand for the G-protein coupled receptor Mas. Therefore, an initial hypothesis might be that alamandine could also be a ligand for this receptor. Interestingly, alamandine-induced vasorelaxation, in aortic rings, was not affected by pretreatment with the Mas antagonist, A-779. Furthermore, in aortic rings taken from Mas knockout mice, the alamandine 132

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Alamandine: a new member of the angiotensin family Villela et al.

Control (a)

(b)

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Saline

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FIGURE 2. Effect of central administration of alamandine on baroreflex. Averaged baroreflex sensitivity index [change in pulse interval (PI)/change in mean arterial pressure (MAP), ms/mmHg] produced by (a) phenylephrine (i.v. 5–40 mg/ml) and (b) sodium nitroprusside (i.v. 5–40 mg/ml) before (open bars), 1 h (solid bars) and 3 h (hatched bars) after intracerebral ventricular (ICV) infusion of vehicle (NaCl 0.9%) (n ¼ 5) or alamandine (ICV 4 mg/h) (n ¼ 7). Sprague-Dawley (SD) rats (n ¼ 12, 14–24 weeks of age) were anaesthetized with tribromoethanol (250 mg/kg, i.p.) and instrumented with unilateral stainless steel guide cannulas (22 gauge, 16 mm length), targeted to the lateral ventricle. Drugs were given 1–2 min apart into a femoral vein in 0.1 ml of isotonic NaCl. P  0.05, compared with before injection (Student’s paired t -test).

studies indicated that its expression was restricted to small diameter nociceptive neurons [14], but larger transcriptome studies show its expression in different tissues including muscle, heart and testicle (http://www-test.ebi.ac.uk/gxa/experiment/E-GEOD24940/ENSMUSG00000051207?ef=organism_part) [15]. Agonists and antagonists of MrgD have been found in high throughput screening studies with the goal of finding drugs for pain treatment qthrough calcium mobilization assay in MrgD transfected cells [16,17]. As mentioned above, the amino acid b-alanine has been demonstrated to also be an MrgD agonist, although a high concentration of b-alanine is needed to activate the receptor [18]. This activation leads to elevation of intracellular Ca2þ levels, which triggers the phosphorylation of the mitogen-activated protein kinases, known as extracellular signal regulated kinase (ERK) 1 and ERK2 [19]. Alamandine, however, does not increase Ca2þ in cardiomyocytes (Guatimosin, unpublished results), suggesting that at least part of the action of this peptide would differ from those described for b-alanine. Experiments with FAM-labelled peptide showed that in contrast to the specific binding of alamandine to MrgD-transfected cells, no binding was observed in nontransfected cells or in Mas-transfected cells. Functional studies evaluating nitric oxide release were also performed in MrgD-transfected cells and showed that alamandine raised the nitric oxide production in these cells but not in Mas-transfected cells [8 ]. The actions of alamandine through MrgD were first evaluated in isolated &&

aortic rings by checking whether the effects of b-alanine, considered a putative MrgD ligand, would resemble the vasoactive effects observed by alamandine. Strikingly, b-alanine did not induce a direct relaxing effect as observed with alamandine. However, preincubation of aortic rings with b-alanine blocked the vasorelaxation induced by alamandine in aortic rings of FVB/N mice. In addition, corroborating the central and vasoactive data, the binding of alamandine to the MrgD receptor was blocked by b-alanine and D-Pro7-Ang-(1–7) but not by A-779 [8 ]. Taken together, these observations indicate that alamandine is an endogenous ligand for MrgD. &&

CONCLUSION These novel findings add new understanding of the mechanism by which the RAS influences many critical physiological process. The physiological/pathophysiological conditions that would favour the formation of alamandine, the enzyme(s) responsible for its formation and whether MrgD is the only binding site for this new member of the RAS remain to be clarified. Acknowledgements None. Conflicts of interest There are no conflicts of interest.

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9. Campagnole-Santos MJ, Heringer SB, Batista EN, et al. Differential baroreceptor reflex modulation by centrally infused angiotensin peptides. Am J Physiol 1992; 263:R89–R94. 10. Marques FD, Ferreira AJ, Sinisterra RDM, et al. An oral formulation of angiotensin-(1-7) produces cardioprotective effects in infarcted and isoproterenol-treated rats. Hypertension 2011; 57:477–483. 11. Menon J, Soto-Pantoja DR, Callahan MF, et al. Angiotensin-(1-7) inhibitsgrowth of human lung adenocarcinoma xenografts in nude mice through a reduction in cyclooxygenase-2. Cancer Res 2007; 67:2809–2815. 12. Gembardt F, Grajewski S, Vahl M, et al. Angiotensin metabolites can stimulate receptors of the Mas-related genes family. Mol Cell Biochem 2008; 319: 115–123. 13. Zhang L, Taylor N, Xie Y, et al. Cloning and expression of MRG receptors in macaque, mouse, and human. Brain Res Mol Brain Res 2005; 133:187–197. 14. Liu Y, Yang F-C, Okuda T, et al. Mechanisms of compartmentalized expression of Mrg class G-protein-coupled sensory receptors. J Neurosci 2008; 28: 125–132. 15. Thorrez L, Laudadio I, Van Deun K, et al. Tissue-specific disallowance of housekeeping genes: the other face of cell differentiation. Genome Res 2011; 21:95–105. 16. Ajit SK, Pausch MH, Kennedy JD, Kaftan EJ. Development of a FLIPR assay for the simultaneous identification of MrgD agonists and antagonists from a single screen. J Biomed Biotechnol 2010; 2010: pii: 326020. 17. Zhang R, Yan P-K, Zhou C-H, et al. Development of a homogeneous calcium mobilization assay for high throughput screening of mas-related gene receptor agonists. Acta Pharmacol Sin 2007; 28:125–131. 18. Shinohara T, Harada M, Ogi K, et al. Identification of a G protein-coupled receptor specifically responsive to beta-alanine. J Biol Chem 2004; 279: 23559–23564. 19. Milasta S, Pediani J, Appelbe S, et al. Interactions between the Mas-related receptors MrgD and MrgE alter signalling and trafficking of MrgD. Mol Pharmacol 2006; 69:479–491.

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Alamandine: a new member of the angiotensin family.

In this article, we review the recent findings regarding a new derivative of angiotensin-(1-7) [Ang-(1-7)], alamandine, and its receptor, the Mas-rela...
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