Am J Physiol Heart Circ Physiol 293: H1416–H1424, 2007.
First published May 11, 2007; doi:10.1152/ajpheart.00141.2007.
Immunofluorescence localization of the receptor Mas in cardiovascular-related
areas of the rat brain
Lenice K. Becker,1 Gisele M. Etelvino,1 Thomas Walther,2 Robson A. S. Santos,1
and Maria J. Campagnole-Santos1
1
Laboratório de Hipertensão, Departamento de Fisiologia e Biofı́sica, Instituto de Ciências Biológicas, Universidade Federal
de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; and 2Department of Cardiology and Pneumonology,
Charité-Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany
Submitted 3 February 2007; accepted in final form 10 May 2007
angiotensin-(1–7), medulla; hypothalamus
EMERGING EVIDENCE SUGGESTS that several angiotensin (ANG) II
actions can be counterbalanced by its congener, ANG-(1–7),
which may provide a more precise regulation of the physiological functions of the renin-angiotensin system (29, 30). The
physiological role of ANG-(1–7) was more firmly established
by two recent discoveries: 1) the identification of angiotensinconverting enzyme-2, an enzyme that generates ANG-(1–7)
from ANG I or ANG II (11, 12, 36, 37) and 2) the characterization of the G protein-coupled receptor (GPCR) Mas as a
receptor that is associated to several ANG-(1–7) actions (32).
Mas was identified as an ANG-(1–7) receptor in experiments
showing that ANG-(1–7) does not bind to the kidney of
Mas-knockout mice (32). Moreover, Mas-deficient mice completely lack the antidiuretic action of ANG-(1–7) after an acute
water overload, and ANG-(1–7) does not induce a relaxation
response in the aorta of these animals (32). In contrast, Mas-
Address for reprint requests and other correspondence: M. J. CampagnoleSantos, Dept. of Physiology and Biophysics, Federal Univ. of Minas Gerais,
Av. Antonio Carlos, 6627-ICB, 31270-901, Belo Horizonte, MG, Brazil
(e-mail: [email protected]).
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knockout mice preserve the antidiuretic response to vasopressin and the binding of ANG II to ANG II type 1 (AT1) and type
2 (AT2) receptors and of ANG IV to ANG II type 4 (AT4)
receptors in the kidney. In addition, ANG-(1–7) binds to
Mas-transfected Chinese hamster ovary (CHO) cells, and in
Mas-transfected COS or CHO cells, ANG-(1–7) elicit arachidonic acid release that was completely abolished by the Mas
receptor antagonist D-Ala7-ANG-(1–7), A-779, and not affected by AT1 or AT2 receptor antagonists (32). In these and
subsequent studies, Mas was shown to mediate most of the
known peripheral actions of ANG-(1–7), including antidiuresis
(26, 32) vasodilation (22) improvement of endothelial function
(9, 13) and its antifibrotic effect (14).
In the central nervous system, ANG-(1–7) acts as an important neuromodulator, especially in those areas related to tonic
and reflex control of arterial pressure, in the hypothalamus and
in the dorsomedial and ventrolateral medulla (30). At these
sites, the cardiovascular effects induced by ANG-(1–7) are
blocked by A-779 (30, 31), suggesting that in the brain,
ANG-(1–7) actions may be mediated by the interaction with
the GPCR Mas. Previous studies have described high levels of
Mas mRNA in the forebrain areas, including hippocampus,
cortex, and olfactory bulb of the rat and mouse (6, 24). Low
amounts of Mas mRNA were also detected in the medulla
oblongata using RNase protection assay (24). The characterization of Mas as an ANG-(1–7) receptor prompted us to
evaluate whether Mas is present in cardiovascular-related areas
in which biological effects of ANG-(1–7) have been described.
Therefore, we determined the distribution of the GPCR Mas
protein in the rat brain using immunofluorescence.
METHODS
Animals. Wistar rats (300 –350 g) were obtained from the animal
facilities (Centro de Bioterismo) of the Biological Sciences Institute
of the Federal University of Minas Gerais (UFMG, Minas Gerais,
Brazil). All experiments were approved by and performed in accordance with the guidelines established by our Institutional Animal
Welfare Committee (Comite de Ética em Experimentação Animal,
UFMG). The animals were kept in a temperature-controlled room on
a 14-h:10-h light-dark cycle (lights on at 6:00 AM) with free access to
water and food.
Antisera. Polyclonal antisera directed against Mas was produced in
Mas-knockout mice (40) using as antigen, a 12 amino acids peptide
(LAEEKAMNTSSR) corresponding to the NH2-terminal domain of
the mouse Mas protein. This sequence has 100% homology with
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/07 $8.00 Copyright © 2007 the American Physiological Society
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Becker LK, Etelvino GM, Walther T, Santos RAS, Campagnole-Santos MJ. Immunofluorescence localization of the receptor
Mas in cardiovascular-related areas of the rat brain. Am J Physiol
Heart Circ Physiol 293: H1416–H1424, 2007. First published May
11, 2007; doi:10.1152/ajpheart.00141.2007.—The G protein-coupled
receptor Mas was recently described as an angiotensin-(1–7) [ANG(1–7)] receptor. In the present study we evaluated the anatomical
localization of Mas using immunofluorescence in the central nervous
system of adult male Wistar rats. An abundant labeling was found in
the hippocampus, amigdala, anterodorsal thalamic nucleus, cortex,
and hypoglossal nucleus. More importantly, a dense ANG-(1–7)
receptor Mas immunoreactivity was observed in cardiovascular-related areas of the medulla and forebrain, shown in several previous
studies as sites for the action of ANG-(1–7) in the brain. A strong
staining was found in the nucleus of the solitary tract, caudal and
rostral ventrolateral medulla, inferior olive, parvo and magnocellular
portions of the paraventricular hypothalamic nucleus, supraoptic nucleus, and lateral preoptic area. Furthermore, Mas staining was predominantly present in neurons. At the medullary sites, a specific and
high-intensity binding for rhodamine-ANG-(1–7) was also shown.
The specific ANG-(1–7) binding was completely displaced by the
anti-Mas antibody or by the ANG-(1–7) antagonist, A-779. The data
presented provide the first anatomical basis for the physiological role
of ANG-(1–7)/Mas axis in the modulation of different cardiovascular
functions and give new insights for clarifying the role of ANG-(1–7)
in the central nervous system.
Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
Fig. 1. Western immunoblot analysis of Mas receptor expression in the
medulla and hypothalamus. Testis was used as positive control. A protein band
of 33 kDa, equivalent to the Mas protein molecular mass, can be seen.
AJP-Heart Circ Physiol • VOL
brain was then removed, postfixed for 2 h in 10% paraformaldehyde
in PBS (0.02 M, pH 7.4), and then placed in 30% sucrose solution
overnight. Serial coronal sections (30 ␮m) of the brain were then
made in a cryostat. Free-floating sections were hydrated with PBS
(0.02 M, pH 7.4) and permeabilized with 0.2% Tween 20, and the
nonspecific staining was blocked with 5% bovine serum albumin
(BSA), each one for 15 min. Sequentially, the tissues were incubated
with a mouse anti-Mas primary antibody (1:500) at 4°C during 24 h,
for the medulla, and 48 h for the rest of the brain. The sections were
then incubated with an Alexa 594-labeled anti-mouse secondary
antibody during 1 h at room temperature. After incubation, sections
were washed with PBS (0.02 M, pH 7.4) and were mounted onto
chromo-alum-coated slides, air dried, and coverslipped with glycerolPBS mounting media (1:3).
The immunostaining specificity was characterized by preincubating
the antiserum with 30 or 50 ␮g of the immunogenic peptide and
staining the brain tissue as described above.
To identify whether Mas was present in neurons, in additional
experiments brain sections were simultaneously stained with Mas
antibody (1:500); a neuronal marker, anti-Neu-N-Alexa fluor 488
conjugated (1:1,000); and the nuclear marker, Draq-5 (1:200) in the
same conditions as described above.
ANG-(1–7) binding. ANG-(1–7) fluorescent binding in the rat
medulla was obtained using rhodamine (Rho)-ANG-(1–7). Rats (n ⫽
8) were euthanized by decapitation, and the brain was removed and
frozen on dry ice (or liquid nitrogen) and kept in a ⫺80°C freezer.
Serial coronal sections (30 ␮m) of the medulla were then made in a
cryostat, and adjacent sections were mounted onto separated 1%gelatinized slides and dried at 4°C. All the slides were incubated in
phosphate-buffered saline (PBS, 0.02 M, pH 7.4) containing 5% BSA
(5%) and 0.005% bacitracin for 15 min at 4°C. The total binding
sections were next incubated in the same assay buffer described above
with the addition of 0.001% PMSF, 0.005% orthophenantroline, and
10⫺5 M enalaprilat. The nonspecific binding sections were incubated
for 15 min at 4°C in the same assay buffer in the presence of
ANG-(1–7). Other sections were incubated with Mas antibody (1:500)
or the ANG-(1–7) antagonist A-779 at a concentration of 10⫺5 M.
Subsequently, all sections were incubated with Rho-ANG-(1–7)
(6 nM) in assay buffer for 1 h at 4°C. The nonspecific binding sections
were incubated with Rho-ANG-(1–7) in the presence of ANG-(1–7)
(10⫺5 M). The sections were then rinsed (3 times for 1 min in assay
buffer), dried under a stream of air at room temperature, and coverslipped with glycerol-PBS mounting media (1:3).
ANG-(1–7) assay stability. To evaluate the stability of ANG-(1–7)
in the presence of the anti-Mas antibody assay condition, 125I-labeled
ANG-(1–7) (750 nCi) (26) was incubated for 1 h alone or in the
presence of Mas antibody (1:500) and subsequently extracted using
phenyl bond-elut cartridges (Analytichem International). The samples
were dried at 45°C until residue and resuspended in 150 ␮l of aqueous solution containing 0.13% heptafluorobutiric acid (HFBA,
mobile phase A) and 0.13% HFBA-80% acetonitrile in water
(mobile phase B), corresponding to 31% of mobile phase B. The
solution was filtrated (0.45 ␮m, Costar) and injected into a highperformance liquid chromatography system (Shimadzu), equipped
with SLC-6B gradient system and with a Nova Pack C18 column
(3.9 ⫻ 150 mm, particle size 4 ␮m). The experimental condition was
as follows: mobile phase A, 0.13% HFBA; mobile phase B, 0.13%
HFBA-80% acetonitrile; and flow rate, 1.0 ml/min. ANG-(1–7) was
separated with the following gradient: 31% to 41% mobile phase B
during 30 min, 41.1% to 60% mobile phase B during 30.1 to 40 min,
and 60% to 31% mobile phase B during 40.1 to 50 min. Fractions of
1 ml were collected and counted in a gamma counter. Incubation of
125
I-labeled ANG-(1–7) with the anti-Mas mouse serum in the assay
conditions used did not result in any apparent hydrolysis, only one
peak corresponding to tubes 18 –21 was observed before or after
incubation with Mas antibody in the binding assay condition (data not
shown).
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mouse and 91.6% homology with rat Mas, and it is not present in any
other known protein (see Fasta protein database, www.ebi.ac.uk/
fasta33). Mas-knockout mice were submitted to three injections of the
peptide conjugated to keyhole limpet hemocyanin by glutaraldehyde
at 15-day intervals each. Just after the third injection, the antibody
titer was determined by ELISA. The mice were next inoculated
intraperitonealy with Ehrlich tumor cells. After 3–5 days, the peritoneal liquid was removed and centrifuged and the titer of the antibody
was determined in the supernatant by ELISA. This antibody was used
in a recent study in which an anti-human Mas commercially available
antibody was also used for comparison (28a). To further document the
specificity of this antibody, Western blot analyses of brain samples
were performed.
Western blot analysis was performed using a modification of a
previously described protocol (28a). Briefly, four rats were euthanized, the brains were quickly removed, and the medulla and hypothalamus were dissected and homogenized in 2 ml of Tris-acetate
buffer (50 mM, pH 7.4) containing (in ␮g/ml) 1 leupeptin, 1 aprotinin,
and 1 pepstatin and 1 mM phenylmethylsulphonyl fluoride (PMSF).
For a positive control, rat testis, which was previously shown to
express Mas (24), was processed in parallel. The solubilized protein
(50 ␮g for the brain and testis) was separated by electrophoresis in
10% SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding was blocked by the incubation of the membrane with
5% skimmed milk in Tris-base buffer (0.2 M, pH 7.4) containing
0.1% Tween 20. Membranes were incubated with the Mas antibody
(1:500) overnight at 4°C, followed by incubation with horseradish
peroxidase conjugated with secondary antibody (1:2,000) during 1 h
at room temperature. Immunoreactivity bands were visualized by
chemiluminescence. As can be seen in the Fig. 1, brain tissue (medulla
and hypothalamus) and testis gave a single protein band of 33 kDa,
equivalent to the Mas protein molecular mass. Actually, the protein
band appears as a doublet, which may represent different degrees of
glycosylation since Mas possess putative glycosylation sites (see
Fasta protein database).
Immunoflorescence staining procedure. The immunostaining protocol used was modified from Block et al. (4). Briefly, rats (n ⫽ 11)
were euthanized with an overdose of tribromoethanol and were
perfused transcardially with PBS (0.02 M, pH 7.4) for 2 min followed
by 10% paraformaldehyde in PBS (0.02 M, pH 7.4) for 10 min. The
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Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
Fig. 3. Higher magnification (⫻40) images show receptor Mas immunofluorescence in medullary areas: RVLM (left), IO (middle), and CVLM (right). Scale
bar ⫽ 50 ␮m.
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Fig. 2. Illustrative images (⫻10) show receptor Mas immunofluorescence in different areas of the medulla: nucleus tractus solitarius and dorsal motor nucleus
of the vagus complex (NTS/nX; A1), caudal ventrolateral medulla (CVLM; B1), inferior olive (IO; C1), and rostral ventrolateral medulla (RVLM; D1). Staining
disappeared in adjacent coronal sections preincubated with 30 –50 ␮g of the synthetic immmunogen (A2–D2, D3) or incubated without secondary antibody (D4).
Scale bar ⫽ 100 ␮m.
Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
H1419
RESULTS
Fig. 4. Illustrative images (⫻10) show receptor Mas immunofluorescence in
different forebrain areas: paraventricular hypothalamic nucleus (PVN; A1),
supraoptic nucleus (SO; B1), and preoptic area (PO; C1). Staining disappeared
in adjacent coronal sections preincubated with 30 –50 ␮g of the synthetic
immmunogen (A2–C2). Images A and B are a composite of captures of
adjacent regions of 1 section in ⫻10 magnification and were edited with
Microsoft PowerPoint software. Scale bar ⫽ 100 ␮m. 3V, 3rd ventricle.
The immunofluorescence for the Mas receptor was found in
different areas of the rat brain, mainly in those areas of the
medulla and forebrain related to the cardiovascular and hydroelectrolyte controls. In addition, the presence and characteristics of the staining in the different areas of the brain were very
similar in all animals studied. In the medulla, the staining
pattern was documented in relation to the rostrocaudal subdivisions of the nucleus tractus solitarius (NTS). An intense
Mas-positive immunofluorescence was found in the cardiovascular-related areas, such as the caudal ventrolateral medulla
(CVLM), the rostral ventrolateral medulla (RVLM), the NTS,
the dorsal motor nucleus of the vagus (nX), and the inferior
olive (IO), as illustrated in Fig. 2. The images in Fig. 2
represent the more intense staining of each area; however, the
staining pattern was not substantially different throughout the
rostrocaudal axis of all these areas. It is important to emphasize
that the staining was completely eliminated by preabsorption of
the antiserum with 30 –50 ␮g of the synthetic immunogen (Fig.
2, A2–D2 and D3). In addition, no labeling could be seen when
the tissue was incubated only with the primary antibody (antibody autofluorescence) as illustrated in Fig. 2, D4.
Figure 3 presents, in higher magnification (⫻40), the Maspositive cells in the IO and in the RVLM and CVLM. As can
be seen in these higher magnification images, the staining
extends for the whole cell body and, in some cells, also to the
Fig. 5. Higher magnification (⫻40) images showing receptor
Mas immunofluorescence in the paraventricular hypothalamic
nucleus (left) and supraoptic nucleus (right). Scale bar ⫽
50 ␮m.
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Image analyses. Fluorescent images were obtained using confocal
microscopy (Zeiss Confocal LSM 510). Specific laser and channels
for excitation and emission of the florescence were used, as appropriated for each fluorescent compound. The control and stained
images were performed in adjacent slices, and the configuration of the
scan was kept the same for both slices. The images were exported to
Microsoft PowerPoint software (Windows XP platform) and edited.
Some figures were built from 2– 8 superimposed captures of ⫻10
magnification of one section. Adjacent slices were collected for
conventional histology and stained with neutral red (1%) for the
identification of the different brain areas.
Drugs and reagents. ANG-(1–7) and D-Ala7-ANG-(1–7), A779,
were from Bachem Holding; horseradish peroxidase conjugated with
secondary antibody was from Pierce Biotechnology; the fluorescent
secondary antibody, Alexa 594, was from Invitrogen-Molecular
Probes; Rho-ANG-(1–7) was from Phoenix Peptides (USA); neuronal
specific nuclear protein antibody conjugated to Alexa 488, antiNeu-N, was from Chemicon; and the nuclear marker, Draq5, was from
Bio-Status. All other reagents were commercial products of the
highest available grade of purity.
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Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
dendrites. It appears that the cell nucleus remained immunonegative.
In the forebrain, Mas-positive cells were also more strongly
concentrated in the areas related to cardiovascular control and
hydroelectrolite balance control. Figure 4 presents the more
intense labeling for the paraventricular hypothalamic nucleus
(PVN), the supraoptic nucleus (SO), and the preoptic area
(PO). An intense staining for Mas was observed in the SO and
PO in all rostrocaudal axes. Figure 5 presents the Mas-positive
cells in the PVN and in the SO in higher magnification (⫻40).
Mas immunoreactivity in the PVN was observed in both
subdivision, magnocellular, and parvocelullar nucleus. The cell
nucleus in the forebrain areas also appears to remain immunonegative and clear.
Fig. 7. Colocalization of receptor Mas in neuronal cells of
the ventrolateral medulla. The neuron marker, anti-Neu-N,
is shown in green (A), the anti-Mas is shown in red (B),
and the nuclear marker, Draq-5, in blue (C). Image shown
in C represents the overlay, and the image in D is an
enlargement of a neuron shown in C. Scale bar ⫽ 50 ␮m.
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Fig. 6. Illustrative images show receptor Mas
immunofluorescence in areas not related to
cardiovascular control: anterodorsal thalamic
nucleus (Ad; top, left), basomedial and basolateral amygdaloid nucleus (Amg; top, right),
hippocampal nucleus (HC; bottom, left), in
hindlimb area of the frontal cortex (Co;
bottom, middle), and in hypoglossal nucleus
(nXII; bottom, right). Images are a composite
of 2– 6 captures of adjacent regions of 1 section in ⫻10 magnification and were edited
with Microsoft PowerPoint software. Scale
bar ⫽ 100 ␮m.
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Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
(1–7) binding in this region. As shown in Fig. 9, binding of
Rho-ANG-(1–7) was found in different areas of the medulla,
especially in the CVLM, RVLM, and NTS/nX complex. In
addition, the total Rho-ANG-(1–7) binding was greatly displaced by unlabeled ANG-(1–7) and by the ANG-(1–7) receptor antagonist, A-779, and was totally displaced by the antiMas antibody, suggesting that ANG-(1–7) binds to Mas receptor in the medulla.
DISCUSSION
In the present study, using a specific polyclonal antibody
combined with immunofluorescence, we showed, for the
first time, the presence of the GPCR Mas in specific areas of
the brain, including many important cardiovascular-related
sites of the forebrain and medulla. In addition, our data
suggest that the Mas receptor is mainly expressed in neurons. Our results extended previous observations that
showed Mas mRNA, using in situ hybridization with radio-
Fig. 8. Diagrams of frontal sections of the
brain from the atlas of Paxinos and Watson
(Ref. 25) at different levels (number on
diagrams) showing the localization (shaded area) of Mas immunoreactivity into
different areas of the rat brain as observed
in all animals studied. 10, dorsal motor
nucleus of the vagus; 12, hypoglossal nucleus; AD, anterodorsal thalamic nucleus;
Amb, ambiguus nucleus; Amb, amigdala;
AP, area postrema; CVL, caudal ventrolateral reticular nucleus; LPO, lateral PO; 4V;
4th ventricle; LV, lateral ventricle; py, pyramidal tract; RVL, rostro ventrolateral reticular nucleus; LPO, lateral preoptic area.
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Mas immunofluorescence was also found in other areas not
directly related with cardiovascular control or hydroelectrolite
balance (Fig. 6), including the hippocampal nucleus, different
subregions of the frontal cortex, anterodorsal thalamic nucleus,
basomedial and basolateral amygdaloid nucleus, and hypoglossal nucleus (nXII).
To verify in which cellular type the Mas immunostaining
was present, we have incubated medullary sections of the brain
with anti-Neu-N, a neuronal marker, and the fluorescent compound Draq5, a nuclear marker, simultaneously with the Mas
antibody. As illustrated in Fig. 7, the Mas immunoreactivity
was mainly observed in neuronal cells.
Figure 8 summarizes in diagrams, based on the atlas of
Paxinos and Watson (25), the localization of the immunoreactivity for the receptor Mas in different areas of the rat
brain.
Because only low expression of Mas mRNA in the medulla
has been reported in a previous study (24), we next tested
whether the anti-Mas antibody could displace the Rho-ANG-
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Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
labeled Mas cRNA probes, in forebrain areas, such as the
hippocampus and limbic system (6, 24).
The presence of the ANG-(1–7) receptor Mas in cardiovascular-related areas of the rat medulla is in keeping with several
studies describing the biological effects of this heptapeptide in
the same regions (29, 30). The observation that the specific
binding of Rho-labeled ANG-(1–7) to these brain nuclei was
completely displaced by the anti-Mas antibody strongly supports the specificity of the immunoreactive labeling. Mas
mRNA was also detected in a previous study, in the medulla,
although low levels were reported (24).
ANG-(1–7) induces changes in blood pressure when microinjected in many brain areas, including the NTS (hypotension
and bradycardia) (7), RVLM (pressor effect) (15), and the
CVLM (hypotension) (1). The cardiovascular effects elicited
by ANG-(1–7) at these brain regions were abolished by its
selective antagonist, A-779 (1, 15, 31), and not affected by AT1
or AT2 receptor antagonists, which suggested that these effects
were elicited by a selective angiotensin receptor. In addition to
its effect on baseline pressure, it has been shown that, in
opposition to the inhibitory effect of ANG II, ANG-(1–7)
facilitates the baroreflex control of heart rate, either after lateral
ventricle infusion (8, 19) or microinjection into the NTS (10).
The selective effect of ANG-(1–7) on the baroreflex modulation gave a further support for the evidence that ANG-(1–7)
actions also in the brain were mediated by a distinct receptor,
different from AT1 or AT2.. The identification of Mas immunoreactivity in medullary areas involved in cardiovascular
control, in which ANG-(1–7) produces biological effects, provides substantial evidence that the mechanism of the action of
ANG-(1–7), in brain areas involved in cardiovascular regulation, involves its newly described receptor.
It should be pointed out that it has been reported that in some
instances the central and peripheral effects of ANG-(1–7) can
be blocked or attenuated by AT1 or AT2 receptor antagonists
AJP-Heart Circ Physiol • VOL
(2, 16, 17, 27). Whether these results represent interference
with Mas-mediated intracellular mechanisms or a true physical
competition at these binding sites or, yet, are due to a distinct
binding site, as recently proposed by us (35), remains to be
clarified.
Indeed, in the present study we described for the first time
the binding of Rho-ANG-(1–7) to medullary areas, such as the
CVLM, RVLM, and NTS, which correspond to the areas
where immunofluorescent Mas receptor and ANG-(1–7) actions were identified. The blockade of the Rho-ANG-(1–7)
binding by the Mas antibody and A-779 are further evidence
for Mas as a mediator of the actions of ANG-(1–7) in the brain.
Our data showing Mas staining in the hypothalamic areas are
in agreement with those reported by Block et al. (4), which
showed intense immunoreactivity staining for ANG-(1–7) in
the paraventricular, supraoptic, and suprachiasmatic nuclei.
The presence of the peptide and its receptor in close proximity
is consistent with the role of ANG-(1–7) on the modulation of
cardiovascular and neuroendocrine function. ANG-(1–7) was
found to be a potent stimulator of vasopressin release from rat
hypothalamic-neurohypophysial explants (33). In addition, it
has been shown that ANG-(1–7) at the paraventricular hypothalamic nucleus can increase the fire rate of neuronal cells (3)
and increases renal sympathetic nerve activity (34). These
effects are blocked by the selective receptor Mas antagonist,
A-779 (3, 34).
We have also detected the presence of the GPCR Mas in
areas apparently not directly related to cardiovascular function,
such as in the amygdala, hyppocampal nucleus, and different
areas of the cortex. These results are in keeping with previous
studies showing Mas mRNA expression in limbic, thalamic,
and cortical structures (6, 24). It has been shown that the
deletion of Mas leads to an increased durability of long-term
potentiation in the dentate gyrus, without affecting hippocampal morphology, basal synaptic transmission, and presynaptic
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Fig. 9. A–C: total rhodamine (Rho)-angiotensin-(1–7) [ANG-(1–7)] binding in the medullary areas: CVLM (A), RVLM (B), and NTS (C). D: total
Rho-ANG-(1–7) binding in the ventrolateral medulla. E–G: displacement of Rho-ANG-(1–7) binding induced by ANG-(1–7) (10⫺5 M; E), Mas receptor
antagonist (A-779, 10⫺5 M; F), or Mas antibody (Mas Ab, 1:500; G). Scale bar ⫽ 100 ␮m.
Mas IMMUNOFLUORESCENCE IN THE RAT BRAIN
ACKNOWLEDGMENTS
This study was part of the doctoral thesis of L. K. Becker presented to the
Postgraduation Program in Biological Sciences: Physiology and Pharmacology, Instituto de Ciências Biológicas, Federal University of Minas Gerais, and
a recipient of doctoral fellowship from Conselho de Desenvolvimento Cientı́fico e Tecnológico (CNPq).
GRANTS
We thank the following organizations for financial support: CNPq-Fundação de
Amparo a Pesquisa do Estado de Minas Gerais (Programas de Grupos de
Excelência), CNPq (Universal Grant Project), and Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior.
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function (40). In addition, ANG II administration into the
amygdala leads to an AT1 receptor-mediated decrease in the
amplitude of the field potentials in the Mas-knockout mice,
whereas, in normal mice, there was an increase in the amplitude of the field potentials (38). The identification of Mas in
these forebrain areas is consistent with a role of this receptor in
synaptic plasticity and behavior and with the observation that,
centrally, ANG-(1–7) increases learning, memory, and motility
behavior (20). Furthermore, Hellner et al. (18) have recently
reported that ANG-(1–7) enhances long-term potentiation in
the CA1 region of the hippocampus and that this effect was
abolished in Mas-deficient mice, suggesting a role for ANG(1–7)/Mas axis on learning and memory mechanisms.
Although in the present study immunolocalization of ANG(1–7) in the brain was not performed, many studies have shown
the presence of the angiotensin peptides precursor, angiotensinogen (5, 23, 28), in different brain areas where Mas
mRNA (6, 24) or protein (present study) are present. In a recent
and elegant study by Lavoie et al. (21), expressions of renin
and angiotensinogen were observed in adjacent cells of brain
areas where we now report the presence of Mas, including
cardiovascular-related areas, such as NTS and the RVLM, and
noncardiovascular-related areas, such as hypocampus, cortex,
and amigdala. Thus it is very likely that ANG-(1–7) can be
formed and/or released in close proximity to Mas receptor
expression. Future studies will be necessary to evaluate this
possibility.
In summary, the present study shows for the first time the
presence of the ANG-(1–7) receptor Mas in cardiovascular and
hydroelectrolite control areas of the rat brain, providing a
clearer morphological basis for the ANG-(1–7) effects in these
regions. In addition, this study provides new insights for other
possible physiological roles of the ANG-(1–7)/Mas axis in
other brain regions.
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H1424
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