REVIEW

CURRENT
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 counterbalances many actions of the ACE/AngII/

The renin-angiotensin system (RAS) is an evolution-
ary conserved system, which is present in all
&&
vertebrates [1 ]. RAS has implications in hyperten-
sion 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 angioten-
sinogen, an alpha-2-globulin produced mainly by
the liver, which is cleaved by renin, an enzyme
expressed in the kidney and released in the blood-
stream. This reaction forms angiotensin I, an inac-
tive 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 non-
linear RAS with multifunctional peptides, enzymes
and receptors. The discovery of an ACE2/angioten-
sin-(1–7) [Ang-(1–7)]/Mas protective axis that
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/Fundação Universitária de Cardiologia, Rio Grande
do Sul, Brazil
Correspondence to Robson Augusto Souza dos Santos, MD, PhD,
Department of Physiology and Biophysics, Av. Antonio Carlos, 6627-
ICB-UFMG, 31270-901-Belo Horizonte, MG, Brazil. Tel: +55 31 3409
2956; e-mail: robsonsant@gmail.com
Curr Opin Nephrol Hypertens 2014, 23:130–134
DOI:10.1097/01.mnh.0000441052.44406.92

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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.
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 octa-
peptide 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 adminis-
tration 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 character-
ization of a new peptide from the RAS, the hepta-
peptide, Ala-Arg-Val-Tyr-Ile-His-Pro, alamandine
and its receptor the Mas-related G-coupled receptor
type D (MrgD).
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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 decar-
boxylation (Fig. 1). After Ang-(1–7) perfusion
through the coronary vessels of the rat heart, for-
mation 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, nephro-
pathic patients have an increased alamandine
plasma concentration, suggesting that this peptide
may have, as Ang-(1–7), an important role in path-
ophysiological conditions [8 ].
&&
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 endothelial-
dependent vasorelaxation in aortic rings of mice
and rats. Moreover, the microinjection of alaman-
dine 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 admin-
istration of the inclusion compound alamandine/
HP-b cyclodextrin produced a long-lasting antihy-
pertensive 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
www.co-nephrolhypertens.com 131
 Pathophysiology of hypertension

Angiotensin A Angiotensin (1–7)

A D
H P H P
R F R
V Y I V Y I

Decarboxylation
ACE 2 enzyme

Alamandine

A H P
R
V Y I

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 ].
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
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 ala-
mandine 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: a new member of the angiotensin family Villela et al.

Control
(a) (b) 1 hour of infusion
2.5 3 hours of infusion
4
*

∆ PI/∆ MAP (ms/mmHg)

∆ PI/∆ MAP (ms/mmHg)

2.0
3

1.5
2
1.0

1
0.5

0 0.0
Saline Ala-Ang-(1–7) Saline Ala-Ang-(1–7)

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-GEOD-
24940/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-acti-
vated 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 alaman-
dine to MrgD-transfected cells, no binding was
observed in nontransfected cells or in Mas-trans-
fected cells. Functional studies evaluating nitric
oxide release were also performed in MrgD-trans-
fected cells and showed that alamandine raised
the nitric oxide production in these cells but not
&&
in Mas-transfected cells [8 ]. The actions of alaman-
dine 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-ala-
nine blocked the vasorelaxation induced by alaman-
dine 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 physio-
logical/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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 Pathophysiology of hypertension
REFERENCES AND RECOMMENDED
READING
Papers of particular interest, published within the annual period of review, have
been highlighted as:
& of special interest
&& of outstanding interest
1. Fournier D, Luft FC, Bader M, et al. Emergence and evolution of the renin-
&& angiotensin-aldosterone system. J Mol Med (Berl) 2012; 90:495–508.
A comprehensive review of the emergence and the evolutionary aspect of the RAS.
2. Skeggs LT Jr, Kahn JR, Shumway NP. The preparation and function of
hypertension-converting enzyme. J Exp Med 1956; 103:295–299.
3. Santos RaS, Ferreira AJ, Simões E, Silva AC. Recent advances in the
angiotensin-converting enzyme 2-angiotensin(1-7)-Mas axis. Exp Physiol
2008; 93:519–527.
4. Santos RAS, Simoes AC, Maric C, et al. Angiotensin-(1-7) is an endogenous
ligand for the G protein-coupled receptor Mas. Proc Natl Acad Sci U S A
2003; 100:8258–8263.
5. Jankowski V, Vanholder R, van der Giet M, et al. Mass-spectrometric identi-
fication of a novel angiotensin peptide in human plasma. Arterioscler Thromb
Vasc Biol 2007; 27:297–302.
6. Yang R, Smolders I, Vanderheyden P, et al. Pressor and renal hemodynamic
effects of the novel angiotensin A peptide are angiotensin II type 1A receptor
dependent. Hypertension 2011; 57:956–964.
7. Coutinho DC, Foureaux G, Rodrigues KD, et al. Cardiovascular effects of
angiotensin A: a novel peptide of the renin-angiotensin system. J Renin
Angiotensin Aldosterone Syst 2013. [Epub ahead of print]
8. Lautner RQ, Villela DC, Fraga-silva RA, et al. Discovery and characterization of
&& alamandine, a novel component of the renin-angiotensin system. Circ Res
2013; 112:1104–1111.
This manuscript describes the discovery and identification of alamandine.
134 www.co-nephrolhypertens.com
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
9. Campagnole-Santos MJ, Heringer SB, Batista EN, et al. Differential baror-
eceptor 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 isopro-
terenol-treated rats. Hypertension 2011; 57:477–483.
11. Menon J, Soto-Pantoja DR, Callahan MF, et al. Angiotensin-(1-7) inhibits-
growth 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 recep-
tor 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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