Neuropeptides 54 (2015) 59–66

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Neuropeptides

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Role of cerebellar adrenomedullin in blood pressure regulation

Leticia Figueira, Anita Israel ⁎
School of Pharmacy, Laboratory of Neuropeptides, Universidad Central de Venezuela, Caracas Venezuela

a r t i c l e i n f o a b s t r a c t

Article history: Adrenomedullin (AM) and their receptor components, calcitonin-receptor-like receptor (CRLR) and receptor
Received 5 April 2015 activity-modifying protein (RAMP1, RMP2 and RAMP3) are widely expressed in the central nervous system, in-
Received in revised form 27 July 2015 cluding cerebellum. We have shown that AM binding sites are altered in cerebellum during hypertension, sug-
Accepted 29 July 2015
gesting a role for cerebellar adrenomedullinergic system in blood pressure regulation. To further evaluate the
Available online 31 July 2015
role of AM in cerebellum, we assessed the expression of AM, RAMP1, RAMP2, RAMP3 and CRLR in the cerebellar
Keywords:
vermis of 8 and 16 week old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. In
Adrenomedullin addition, the effect of microinjection of AM into rat cerebellar vermis on arterial blood pressure (BP) was deter-
CRLR mined. Animals were sacrificed by decapitation and cerebellar vermis was dissected for quantification of AM,
RAMPs CRLR, RAMP1, RAMP2 and RAMP3 expression using western blot analysis. Another group of male, 16 week old
Cerebellum SHR and WKY rats was anesthetized, and a cannula was implanted in the cerebellar vermis. Following recovery
Vermis AM (0.02 to 200 pmol/5 μL) or vehicle was injected into cerebellar vermis. BP was determined, before and after
AM22–52 treatments, by non-invasive plethysmography. In addition, to establish the receptor subtype involved in AM ac-
Hypertension
tion in vivo, animals received microinjections of AM22–52 (200 pmol/5 μL), an AM1 receptor antagonist, or the
CGRP1 receptor antagonist, CGRP8–37 (200 pmol/5 μL) into the cerebellar vermis, administered simultaneously
with AM or vehicle microinjection. Cannulation was verified post mortem with the in situ injection of a dye solu-
tion. Our findings demonstrated that the expression of CRLR, RAMP1 and RAMP3 was higher in cerebellum of SHR
rats, while AM and RAMP2 expression was lower than those of WKY rats, both in 8 and 16 week old rats. In vivo
microinjection of AM into the cerebellar vermis caused a profound, dose dependent, hypotensive effect in SHR
but not in normotensive WKY rats. Coinjections of a putative AM receptor antagonist, AM22–52 abolished the de-
creases in mean arterial pressure (MAP) evoked by AM, showing that AM acts through its AM1 receptor in the
vermis to reduce MAP. These findings demonstrate a dysregulation of cerebellar AM-system during hyperten-
sion, and suggest that cerebellar AM plays an important role in the regulation of BP. Likewise; they constitute a
novel mechanism of BP control which has not been described so far.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Adrenomedullin (AM) is a 52- (human) or 50-amino acid (rat) pep-
tide which shows some homology with calcitonin gene-related peptide
(CGRP) (Kitamura et al., 1993) and has therefore been added to the cal-
citonin/CGRP/amylin peptide family (Ichiki et al., 1994; Sakata et al.,
1994). AM has two specific receptors, the AM1 and AM2 receptors,
formed from the obligate co-expression of a class-B, G protein coupled
receptor (GPCR), the calcitonin receptor-like receptor (CRLR) and
receptor activity-modifying proteins (RAMP) 2 or 3, respectively
(McLatchie et al., 1998). The calcitonin gene-related peptide 1
(CGRP1) receptor is formed of a complex between CRLR and RAMP1
(McLatchie et al., 1998; Cases and Mora-Macía, 2001). AM1 receptors
are highly selective for AM over CGRP and other peptides while AM2
⁎ Corresponding author at: Apartado Postal 50176, Sabana Grande 1050A, Caracas,
Venezuela.
E-mail address: astern88@gmail.com (A. Israel).
receptors binds both AM and AM2 (intermedin) with high affinities
(Hong et al., 2012). AM also has appreciable affinity for the CGRP1 re-
ceptor (Gibbons et al., 2007).
AM is found in the peripheral circulation, in the cerebrospinal fluid
and in several organs including the central nervous system (CNS)
(Ichiki et al., 1994; Kitamura et al., 1993; Satoh et al., 1995, 1996; Wei
et al., 1998; Wang et al., 2015). In the CNS, AM and its receptors are par-
ticularly localized to the autonomic nuclei, including nucleus tractus
solitarii (NTS), lateral parabrachial nucleus (LPBN) and rostral ventro-
lateral medulla (RVLM) and in cerebral cortex, pituitary gland, thala-
mus, hypothalamus, brainstem, amygdala and cerebellum (Hinson
et al., 2000; Serrano et al., 2000; Juaneda et al., 2003; Macchi et al.,
2006; Owji et al., 1996; Xu and Krukoff, 2004a). Also it was reported
the presence of AM and its receptor components in cerebrospinal
fluid-contacting nucleus (Wu et al., 2015). In cerebellum, AM immuno-
reactivity is located in cerebellar Purkinje cells and mossy terminal
nerve fibers as well as neurons of the cerebellar nuclei (Serrano et al.,
2000).

http://dx.doi.org/10.1016/j.npep.2015.07.003
0143-4179/© 2015 Elsevier Ltd. All rights reserved.
60 L. Figueira, A. Israel / Neuropeptides 54 (2015) 59–66
AM exerts several effects in the CNS. Central AM administration
results in various neuroendocrine responses such as the inhibition of
arginine vasopressin secretion induced by hypovolemic and osmotic
stimuli, and the secretion of oxytocin by activating hypothalamic
oxytocin-producing cells (Serino et al., 1999; Yokoi et al., 1996). In addi-
tion, central administration of AM increases urinary excretion of water,
sodium and potassium, in a dose dependent manner in conscious hy-
drated rats (Israel and Diaz, 2000; Díaz and Israel, 2001), and microin-
jection of AM into the area postrema (AP) and the RVLM causes a
hypertensive effect; while in the paraventricular nucleus (PVN) of the
hypothalamus produces hypotension (Allen et al., 1997; Ji and He,
2002; Xu and Krukoff, 2004b). This evidence indicates that central AM
may play an important role in body fluid homeostasis and central regu-
lation of the cardiovascular system.
Several experimental and epidemiological studies have shown that
AM and its receptor components expression are altered during hyper-
tension (Cases and Mora-Macía, 2001). In effect, it has been shown an
increased expression of AM and its receptor components in rat aorta
and ventricle during hypertension possibly due to a compensatory re-
sponse to the cardiovascular pathophysiological process (Gibbons
et al., 2007; Zhao et al., 2006; Pan et al., 2005).
AM immunoreactivity, AM binding sites and CRLR, RAMP2 and
RAMP3 are expressed in rat cerebellum (Serrano et al., 2000;
Chakravarty et al., 2000; Sone et al., 1997; Uezono et al., 2001), however
little is known about their functional role in this structure. The fact that
AM binding sites are increased in SHR rats' cerebellum when compared
with WKY control (Pastorello et al., 2007) suggest a possible role for cer-
ebellum AM in the blood pressure regulation. Thus, we were prompted
to assess the expression of AM and its receptor components in the cer-
ebellar vermis in Wistar Kyoto (WKY) and spontaneously hypertensive
rats (SHR) of 8 and 16 week old. In addition, the effect on blood pressure
of AM microinjection into the cerebellar vermis in vivo was determined.
2. Materials and methods
2.1. AM and its receptor components expression
Male, 8 and 16 week old WKY and SHR rats, provided from
Venezuelan Institute of Scientific Research (IVIC) (Caracas, Venezuela),
were maintained in single cages under controlled conditions of temper-
ature and photoperiod (lights on 06.00 to 18.00 h) and provided with
free access to tap water and standard laboratory chow.
Cerebellar CRLR, RAMP1, RAMP2, RAMP3 and AM expression was
performed by Western blot analysis by a modification of the method de-
scribed by Urrecheaga et al. (2007). Rats were killed by decapitation, the
skull was rapidly removed, and brain extracted and immediately placed
on ice-cold plate. Cerebellar vermis was dissected, homogenized in ice-
cold lysis buffer for receptor expression assessment (Tris–HCl 200 mM,
CHAPS 160 mM, Na2EDTA 10 mM, PMSF 5 mM and protease inhibitors
cocktail [pepstatin A 5 μM, aprotinin 10 μg/mL, leupeptin 10 μM
pH 7.5]) or in ice-cold lysis buffer for AM expression evaluation (NaCl
50 mM, Tris–HCl 25 mM, Triton X-100 1%, PMSF 1 mM and protease in-
hibitors cocktail [pepstatin A 5 μM, aprotinin 10 μg/mL, leupeptin 10 μM,
pH 8.1]) and centrifuged at 10.000 rpm for 10 min at 4 °C. The superna-
tant was collected and protein concentration was determined by Lowry
et al. (1951) using bovine serum albumin as standard.
For Western blot analysis, samples of rat cerebellar vermis were
subjected to 10% SDS-polyacrylamide gel electrophoresis under
denaturizing and reducing conditions and transferred to a nitrocellulose
membrane. Membranes were incubated for 1 h in blocking buffer (TBS-
Tween 0.1% + 5% non-fat dried milk) at room temperature. Next, mem-
branes were incubated overnight at 4 °C with the following rabbit pri-
mary antibodies: anti-CRLR, anti-RAMP1, anti-RAMP2, anti-RAMP3
and or anti-AM (Santa Cruz, Biotechnology, Inc.), diluted in 3% bovine
serum albumin solution in TBS-Tween 0.1% (1:1000). Then, membranes
were incubated with secondary antibody anti-rabbit IgG conjugated to
horseradish peroxidase (Cell Signaling Technology), for 1 h at room
temperature. After each incubation, membranes were washed 10 min
3 times with TBS-Tween 1×. The immunostaining was visualized by a
chemiluminescent detection system (SuperSignal® West Pico Chemilu-
minescent Substrate) and densitometry analysis was performed using
Quantity One 1-D®-BioRad software. CRLR, RAMP1, RAMP2, RAMP3
and AM expression were normalized with β-actin expression.
2.2. Intracerebellar cannulation
Sixteen-week-old SHR and normotensive WKY male rats were
maintained in single cages under controlled conditions of temperature
and photoperiod (lights on 06.00 to 18.00 h) and provided with free
access to tap water and standard laboratory chow. With the aid of a
stereotaxic instrument and under sodium pentobarbitone anesthesia
(40 mg/kg, intraperitoneally), a cannula was implanted into the cere-
bellar vermis with the following stereotaxic coordinates: antero-
posterior (AP) = −10.3, lateral (L) = 0 and ventral (V) = 2.4 according
to Paxinos and Watson (1986) and Sacchetti et al. (2002). The cannula
was secured to the skull with acrylic cement. A minimum of 3 days
was allowed for recovery. Single intracerebellar injection was made
with a Hamilton syringe fitted with a stop to prevent needle penetration
past the cannula tip. At 09:00 h, half of the rats received AM (0.02 to 200
pmol/5 μL) or vehicle (5 μL) into the cerebellar vermis. To establish the
receptor subtype involved in the hypotensive action of intravermis mi-
croinjection of AM in SHR rats, animals received microinjections of
AM22–52 (200 pmol/5 μL), an AM1 receptor antagonist, or the CGRP1 re-
ceptor antagonist, CGRP8–37 (200 pmol/5 μL) into the cerebellar vermis,
administered simultaneously with AM or vehicle microinjection. To de-
termine whether the effect of AM was site specific, animals received in-
jections of AM (200 pmol/5 μL) or vehicle (5 μL) outside the cerebellar
vermis. And to determine whether the effect of AM was specific, animals
received injections of angiotensin II (ANG II) (200 pmol/5 μL) or vehicle
(5 μL) into cerebellar vermis. Cannula placement was confirmed post-
mortem by examining the distribution of an intravermis injection of
5 μL of fast green dye, given before animal sacrifice. Data were used
only if the dye was distributed in the cerebellar vermis.
2.3. Measurement of cardiovascular responses
Blood pressure (BP) was measured by the tail-cuff method according
to Israel et al. (2000). Systolic and diastolic pressure (SP, DP) and heart
rate were recorded daily using a tail-cuff digital plethysmograph
(Digital Pressure Meter LE 5002 LETICA®, Panlab, S.L. Barcelona-
Spain). To minimize stress, rats were trained daily with the plethysmo-
graph one week prior to the experiment. Cardiovascular parameters
were measured daily at the same time of the day during the training
and experimental periods. Plethysmographic pressure values were val-
idated in early experiments with direct measurements with intra-
arterial catheter recordings (data not shown). Mean arterial pressure
(MAP) was calculated as follows: MAP = DP + 1/3(SP–DP), where
DP: diastolic pressure and SP: systolic pressure.
The procedures used in these experiments were reviewed and ap-
proved by the Animal Care and Use Committee of The Central University
of Venezuela, School of Pharmacy, Caracas, Venezuela. All experiments
were done according to good practice of laboratory animal management
(NIH Guide, 1996).
2.4. Statistics
All data are presented as means ± S.E.M. Statistical differences
between groups were analyzed using Kruskall–Wallis and U Mann–
Whitney analysis. A value of p b 0.05 was considered statistically signif-
icant. Data analysis and graphs were performed using Graph Pad Prism
Program, 5.1 version.
 L. Figueira, A. Israel / Neuropeptides 54 (2015) 59–66 61

Table 1 3.4. Changes of MAP after in situ administration of AM

Characteristics of WKY and SHR, 8 and 16 week old.

Group Weight (g) SP (mm Hg) DP (mm Hg) MAP (mm Hg) In WKY rats, administration of vehicle (V) or AM (200 pmol/5 μL)
WKY 8 weeks 282 ± 2 141 ± 1 89 ± 3 106 ± 2
into cerebellar vermis increased MAP in similar magnitude, either
WKY 16 weeks 415 ± 24 145 ± 3 91 ± 3 108 ± 2 when expressed as increase in MAP or as area under the curve (AUC)
SHR 8 weeks 221 ± 4 167 ± 1⁎ 119 ± 2⁎ 135 ± 1⁎ (V = 134 ± 13 vs. AM = 157 ± 15) (N = 10) (Fig. 5, Panels A and B).
SHR 16 weeks 366 ± 11 174 ± 3#,+ 126 ± 3#,+ 142 ± 3#,+ By contrast, in SHR rats, administration of AM (200 pmol/5 μL) into cer-
Resuts are expressed as mean ± S.E.M. (N = 40). ebellar vermis produced a marked and significant hypotensive action
⁎ p b 0.0001 vs. WKY 8 weeks. when compared with vehicle (reduction of −20 to −40 mm Hg). This
#
p b 0.0001 vs. WKY 16 weeks. effect was also observed when the data where expressed as AUC (V =
+
p b 0.0001 vs. SHR 8 weeks.
264.50 ± 26.0 vs. AM = 99.5 ± 8) (N = 17, p b 0.05) (Fig. 5, Panels C
and D). Microinjections of AM outside of cerebellar vermis did not
3. Results alter MAP in WKY and SHR rats (data not shown). And microinjections
of ANG II or vehicle into cerebellar vermis increased MAP in similar
3.1. Characteristics of SHR and WKY rats of 8 and 16 week old magnitude in both WKY and SHR rats (data not shown). In addition,
AM (0.02 to 200 pmol/5 μL) administration caused a significant and
As shown in Table 1, SP, DP and MAP in 8 and 16 week old SHR rats dose-dependent fall in MAP (Fig. 5, Panels E and F). This effect is also ob-
were significantly higher compared with age matched WKY rats. Like- served when the data are expressed as AUC.
wise, 16 week old SHR showed higher values of SP, DP and MAP when
compared with 8 week old SHR. 3.5. Effect of AM22–52 and CGRP8–37 on the time course of changes in MAP
induced by microinjection of AM in SHR rats

3.2. Effect of age on cerebellar vermis expression of AM and its receptor
components in 8 and 16 week old WKY and SHR rats
In cerebellar vermis of WKY and SHR rats, AM and RAMP2 expres-
sion was unchanged by age (Fig. 1), however age significantly increased
RAMP1, RAMP3 and CRLR expression in both WKY and SHR rats (Fig. 2)
N = 10. p b 0.0001.
3.3. Cerebellar vermis expression of AM, RAMP1, RAMP2, RAMP3 and CRLR
in 8 and 16 week old WKY and SHR rats
SHR rats were microinjected into the vermis with vehicle (5 μL),
AM22–52 (200 pmol/5 μL), AM + AM22–52, CGRP8–37 (200 pmol/5 μL)
or AM + CGRP8–37. As shown in Fig. 6, in SHR rats AM22–52, CGRP8–37
or CGRP8–37 + AM microinjection into cerebellar vermis increased
MAP in similar magnitude as vehicle, either when expressed as increase
in MAP or as AUC. Administration of AM (200 pmol/5 μL) into cerebellar
vermis produced a marked and significant hypotensive action when
compared with vehicle. The AM receptor antagonist, AM22–52 (200
pmol/5 μL), coinjected with AM (200 pmol/5 μL) blocked the AM-
induced decrease in MAP, showing that AM acts through its receptors
in the vermis to reduce MAP (Fig. 6).

It was quantified AM and its receptor components expression in cer- 4. Discussion

ebellar vermis. As is shown in Figs. 3 and 4, there was a reduction in AM
and RAMP2 expression in SHR of 8 and 16 week old, when compared
with age matched WKY rats (N = 10; p b 0.0001); on the contrary,
RAMP1, RAMP3 and CRLR expression was significantly increased in
SHR of 8 and 16 week old when compared with age matched WKY
rats (N = 9, p b 0.001).
AM is an ubiquitous peptide, found widely distributed in the body.
In brain AM and AM specific binding sites are found in areas such
as the cerebral cortex, olfactory bulb, nucleus accumbens, caudate–
putamen, thalamus, hypothalamus, midbrain, medulla oblongata,
circumventricular organs and cerebellum (Hinson et al., 2000; Serrano

Fig. 1. Effect of age on cerebellar vermis AM expression (Panel A) and RAMP2 expression (Panel B) in 8 and 16 week old WKY and SHR rats. Results are expressed as mean ± S.E.M. AM and
RAMP2 expression was normalized with that of β-actin (N = 10).
62 L. Figueira, A. Israel / Neuropeptides 54 (2015) 59–66

Fig. 2. Effect of age on RAMP1 (A), RAMP3 (B) and CRLR (C) expression in cerebellar vermis of 8 and 16 week old WKY and SHR rats. Results are expressed as mean ± S.E.M. RAMP1, RAMP3
and CRLR expression was normalized with that of β-actin (N = 10). *p b 0.0001 vs. WKY 8 week old. #p b 0.0001 vs. SHR 8 week old.

et al., 2000; Juaneda et al., 2003; Macchi et al., 2006). In addition, AM re-
ceptor components are expressed in several brain areas, being found
RAMP1 mRNA in the hippocampus, nucleus accumbens, caudate–puta-
men, cerebral cortex and cerebellum; RAMP2 mRNA is expressed in nu-
merous areas, including autonomic nuclei such as the paraventricular,
supraoptic, arcuate and ventromedial nuclei, as well as the nucleus of
the solitary tract (NTS), area postrema and dorsal motor nucleus of
the vagus, hippocampus, the olfactory bulb, the choroid plexus and cer-
ebellum; and RAMP3 mRNA in the cerebral cortex, thalamus and cere-
bellum (Ueda et al., 2001; Stachniak and Krukoff, 2003). The presence
of AM and its receptor components in the brain suggests the existence
of a central adrenomedullinergic system of physiological relevance.
Our present results demonstrate, for the first time, the presence of
AM and its receptor components in a specific cerebellar area such as
the vermis of WKY and SHR rats, indicating the presence of CGRP1,
AM1 and AM2 receptors in this area (Sakata et al., 1994; Serrano et al.,
2000; Edvinsson et al., 2010; Juaneda et al., 2001). In support to our
findings it has been demonstrated by light and electron microscopy
AM-like immunoreactivity in cerebellar Purkinje cells and mossy
terminal nerve fibers as well as neurons of the cerebellar nuclei
(Serrano et al., 2000). In addition, the presence of RAMP1 and RAMP2
mRNA in Purkinje cells and RAMP 3 mRNA in cerebellar granular cells
has been shown (Ueda et al., 2001). Likewise, CRLR and RAMP1 were
detected on the surface of the Purkinje cell bodies and in their processes
(Edvinsson et al., 2010; Morara et al., 1998). This evidence suggests a
functional role for cerebellar AM and indicates that this peptide could
participate in cerebellar functions as an autocrine/paracrine factor.
The expression and function of adrenomedullergic system compo-
nents could be altered in pathological conditions and during growth.
Our results point to this possibility since they demonstrate that
RAMP1, RAMP3 and CRLR expression in rat cerebellar vermis increases
with age, without changes for AM and RAMP2. Similar findings have
been shown for CGRP1 receptor (CRLR/RAMP1) expressed in cerebellar
cortical neuron and glial, which suffer of marked changes during
cerebellum development. Indeed, autoradiographic and immunofluo-
rescence studies followed by con-focal analysis in rat cerebellum
demonstrated a lower CGRP1 receptor density in Purkinje cells and mo-
lecular cells during development and their increase during maturity,

Fig. 3. Cerebellar vermis expression of AM (Panel A) and RAMP2 (Panel B) in 8 and 16 week old WKY and SHR rats. Results are expressed as mean ± S.E.M. AM and RAMP2 expression was
normalized with that of β-actin (N = 10).*p b 0.0001 vs. WKY 8 weeks. #p b 0.0001 vs. WKY 16 weeks.
 L. Figueira, A. Israel / Neuropeptides 54 (2015) 59–66
Fig. 4. Cerebellar vermis expression of RAMP1 (Panel A), RAMP3 (Panel B) and CRLR (Panel C) in 8 and 16 week old WKY and SHR rats. Results are expressed as mean ± S.E.M. RAMP1,
RAMP3 and CRLR expression was normalized with that of β-actin (N = 10). *p b 0.0001 vs. WKY 8 week old. #p b 0.0001 vs. WKY 16 week old.
suggesting that CGRP1 and its receptor may promote a coordinated de-
velopment of cerebellar glial cells, an effect driven mainly by the CGRP
released by climbing fibers (Morara et al., 2000).
Similar results has been described in the periphery, indeed Hwang
et al. (2007) found that in rats of 3, 12 and 20 months old, there were
age-related increases in lung AM gene expression and peptide levels,
as well as the mRNA levels of CRLR and RAMPs. Likewise, Wong and
Tang (2012) demonstrated that in rats of 1, 7 and 21 days old, preproAM
mRNA levels, CRLR and RAMP2 gene expression increased with age in
lung, kidney, and heart but preproAM mRNA decreased with age in
the adrenal gland. Meanwhile, AM levels increased with age in the
lung but decreased with age in the kidney, the adrenal, and the heart.
Furthermore, as shown in animal models, in midgestation human fetal
lung, AM and its gene and protein expression as well as the AM1 recep-
tor component CRLR and RAMP2 gene expression increased with in-
creasing gestational age. Conversely, the expression of RAMP3,
decreased with increasing gestational age (Ramos et al., 2014). The ev-
idence and our present results suggest that AM and its receptor compo-
nents expression, in different tissues, follow different patterns of
changes during development.
There is functional evidence suggesting that cerebellum may play a
role in the regulation of arterial BP. In effect, it has been shown that
stimulation of several regions of the cerebellum produces changes in ar-
terial BP and heart rate (Nisimaru, 2004; Rector et al., 2006; Tandon
et al., 2006). Electric stimulus of cerebellar cortex posterior to vermis
IX lobe, in anesthetized rabbits induces bradycardia, BP fall and tran-
sient inhibition of sympathetic renal activity (Bradley et al., 1987a;
Rocha et al., 2008). On the contrary, electric stimulation of the rostral re-
gion of fastigial nucleus in anesthetized rabbits produces a pressor re-
sponse (Bradley et al., 1987b).
In support for a role cerebellum in regulation of BP are our previous
results in which we demonstrated autoradiographically differences in
the concentration of AM binding sites in rat cerebellum of normotensive
and genetically hypertensive rats (Pastorello et al., 2007). The increase
in cerebellar AM receptors during hypertension could be interpreted
as an upregulation mechanism of cerebellar binding sites to compensate
the increase in BP in SHR rats; or they could represent a primary alter-
ation which would result in a secondary alteration in the autonomic
regulatory mechanisms in cerebellum with the consequent increase
in BP.
To further get insights in the role of AM in cerebellum, are our pres-
ent results which support the concept of an up-regulation of cerebellar
CGRP1 (CRLR + RAMP1) and AM2 (CRLR + RAMP3) receptors and
a down-regulation of AM1 (CRLR + RAMP2) receptor during
63
hypertension, since they demonstrate that in cerebellar vermis CRLR,
RAMP1 and RAMP3 expression was increased significantly while
RAMP2 was reduced in SHR rats compared with WKY. The reduction
in AM expression in vermis of SHR rats could be responsible for
the up-regulation of AM2 receptors and binding sites observed by auto-
radiography (Pastorello et al., 2007; present results). These findings
support the notion of a dysregulation of AM cerebellar system during
hypertension.
It is known that CRLR/RAMP2 complex constitutes the pathway for
AM biological activity, and it is believed that altered expression of
RAMPs is associated with alterations of AM response (Gibbons et al.,
2007). In effect, during hypertension cerebellar cell phenotype changes
from a high response (high expression of RAMP2) to a low response to
AM (high expression of RAMP3) (Gibbons et al., 2007; present results),
changes which may contribute to the development of hypertension. In
effect, during hypertension there are dynamic changes in the expression
of genes involved in AM signaling, particularly involving a switch from
RAMP2 to RAMP3 expression, which may underlie the marked decrease
in AM expression in cerebellum and/or represent an altered responsive-
ness (Gibbons et al., 2007; present results). Furthermore, upregulation
of RAMP1/RAMP3 expression would promote the interaction of AM
with CGRP1 and AM2 receptors, rather than AM1 receptors, thereby fa-
voring the compensatory mechanism to increased BP. Alternatively,
these changes could be the initial disturbance that would result in dys-
regulation of the mechanisms controlling BP, since these changes are
present from the early stages of life (8 weeks) of hypertensive rats.
Several studies support that hypertension may influence AM and its
receptor component expression. In fact, AM and its receptor compo-
nents expression are altered in cardiovascular system and in CNS during
hypertension. Increases in cardiovascular AM generation and an up-
regulation of the gene expression of ventricles and aortas AM and its re-
ceptor components CRLR, RAMP1 and RAMP3, during L-NAME induced
hypertension has been demonstrated (Pan et al., 2005). Similarly, ex-
pression of AM, RAMP2 and RAMP3, but not CRLR and RAMP1 mRNAs
was increased in left ventricular cardiomiocytes of hypertensive rats
(Bell et al., 2006). Changes of CNS adrenomedullergic system during hy-
pertension have been also shown in brain areas. It was demonstrated
that chronic hypertension induced by one-kidney-one-clip method,
did not alter brain RAMP-1 expression while significantly reduced
RAMP-2 and CRLR, suggesting a stoichiometric shift between CGRP
and AM receptors in favor to the CGRP receptor (Wang et al., 2015). Fur-
thermore, hypertension induced by restraint stress reduced preproAM
mRNA level in the hypothalamic PVN an supraoptic nucleus (SON),
NTS, dorsal motor nucleus of the vagus, AP, and subfornical organ
64 L. Figueira, A. Israel / Neuropeptides 54 (2015) 59–66

Fig. 5. Time course of changes in mean arterial pressure (MAP) induced by microinjection of vehicle, AM (200 pmol/5 μL) or (0.02, 0.2, 2, 20 and 200 pmol/5 μL) into the cerebellar vermis in
WKY (A) and SHR (C and E) rats. Data expressed as area under the curve (AUC) of MAP in WKY (B) and SHR (D and F). Results are expressed as mean ± S.E.M. (N = 10, Panels A and B; N =
17, Panels C, D, E and F). *p b 0.05 vs. vehicle. #p b 0.05 vs. AM 0.02 and 0.2 pmol. ζp b 0.05 vs. AM 2 and 20 pmol/5 μL.

(Shan and Krukoff, 2001). Likewise, increased BP induced by phenyl-
ephrine administration elicited a decrease in RAMP2 mRNA expression
in the PVN and NTS (Stachniak and Krukoff, 2003), suggesting that
changes in RAMP2 mRNA expression may influence brain AM to regu-
late sympathetic activity. Effectively, it has been shown that chronic
stress induced by immobilization for 7 consecutive days produced an
enhanced expression of AM in the cerebrospinal fluid-contacting nucle-
us (Wu et al., 2015) and chronic foot-shock and noise stress for 15 con-
secutive days produced an up-regulation of preproAM mRNA, in the
hypothalamus-pituitary-adrenal (HPA) axis, and down-regulation in
the medulla oblongata and midbrain. This was associated with an in-
creased RAMP3 mRNA expression in the hypothalamus and pituitary
gland and a decrease in the adrenal gland and midbrain (Li et al.,
2004). In this regard, Li et al. (2004) suggested that increased hypotha-
lamic preproAM, RAMP2 and RAMP3 may constitute a protection mech-
anism for resetting hypertension induced by chronic stress, through a
direct or indirect mechanism of action on cardiovascular function.
Meanwhile, the decrease in the expression of preproAM in medulla,
where AM administration causes an increase in BP by increasing the
sympathetic outflow, could provide a protective mechanism of AM to
reset BP. Taken together the evidence suggests that AM may be related
to the development of the stress-induced hypertension.
If cerebellar adrenomedullergic system exerts a physiological role, it
is plausible to expect that the AM administered into the cerebellum may
cause actions in cardiovascular regulation. Our findings point to this
possibility since they demonstrate, for the first time, that microinjection
 L. Figueira, A. Israel / Neuropeptides 54 (2015) 59–66 65

Fig. 6. Effect of AM22–52 (A, B) and CGRP8–37 (C, D) on the time course of changes in mean arterial pressure (MAP) induced by microinjection of AM. SHR rats were microinjected into the
vermis with vehicle (5 μL), AM (200 pmol/5 μL), AM22–52 (200 pmol/5 μL), AM + AM22–52, CGRP8–37 (200 pmol/5 μL) or AM + CGRP8–37. Data are expressed as MAP (A,C) or area under the
curve (AUC) of MAP (B, D). Results are expressed as mean ± S.E.M. (N = 5) *p b 0.05 vs. its own control.

of AM into the cerebellar vermis of hypertensive rats causes a powerful
and significant hypotensive response, which is specific and dose-
dependent. This hypotensive effect is manifested only during hyperten-
sion since AM microinjection into the cerebellar vermis was not able to
reduce BP in normotensive rats. The specificity of the hypotensive ac-
tion of AM administered into the cerebellar vermis is based on the fact
that microinjection of the peptide outside the vermis did not cause
the hypotensive effects in SHR, and in situ administration of a pressor
peptide such as ANG II into cerebellar vermis did not altered BP (results
not shown). In addition, our data show that AM's actions in the cerebel-
lar vermis in SHR rats are mediated by AM1 receptor, as AM22–52, an AM
receptor specific antagonist, blunted AM's hypotensive effect when
both agents were injected together. These results provide the first func-
tional evidence in vivo of a role for AM in the cerebellar vermis in the
control of BP.
The possible cause of the difference in the AM action among normo-
tensive and hypertensive rats may be variable and has been described
for other brain structures, since the infusion of AM reduces BP in both
normotensive and hypertensive rats in a dose dependent manner; how-
ever the fall in BP was higher in hypertensive rats compared to the nor-
motensive (He et al., 1995). Similarly, it was reported that RVLM
neurons of SHR rats are more sensitive and have an increased response
to ANG II with respect to the WKY (Chan et al., 1991). Therefore, the hy-
potensive effect induced by the intracerebellar administration of AM in
SHR could be due to an increase in sensitivity and response in SHR com-
pared with WKY rats. Alternatively, this differential response may be
the manifestation of the cerebellar dysregulation of signaling pathways,
or AM and AM1 receptor expression which are reduced during hyper-
tension (Figueira and Israel, 2013).
In conclusion, our study demonstrated a dysregulation of cerebellar
vermis AM and its receptor components during hypertension. In addi-
tion, they show that under hypertensive conditions AM administered
into cerebellar vermis exerts a profound, dose-dependent hypotensive
effect. These actions are mediated through AM1 receptor. These data
suggest the existence of a cerebellar adrenomedullinergic system of
physiological relevance in BP regulation. Likewise, they constitute a
novel mechanism of BP control which has not been described so far.
Conflicts of interest
Conflicts of interest: none.
Acknowledgments
The authors wish to thank Mr. Banny Caraballo for his technical as-
sistance. This study was supported by Grants from People's Ministry of
Science, Technology and Industry, Science Mission Project, Subproject
7, ECCV No. 2007001585, PEII-20122000760 and CDCH-UCV AIA-
06.8402.2012.
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