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Abstract

Background The endocannabinoid (eCB) system is strongly involved in the regulation of anxiety and feeding behavior. RVD-hemopressin(α) [RVD-hp(α)], a N-terminally extended form of hemopressin, is a negative allosteric modulator of the cannabinoid (CB) 1 receptor and a positive allosteric modulator of CB2 receptor which has been recently reported to exert anxiolytic/antidepressant and anorexigenic effects after peripheral administration in rats. Pharmacological evidences reported a possible link between brain hypocretin/orexin, monoamine and eCB systems, as regards appetite and emotional behavior control. Considering this, the aim of our work was to investigated the effects of RVD-hp(α) on anxiety like behavior and food intake after central administration and related it to monoamine levels and orexin-A gene expression, in the hypothalamus. Methods We have studied the effects of central RVD-hp(α) (10 nmol) injection on anxiety-like behavior and feeding using different behavioral tests. Hypothalamic levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and gene expression of orexin-A and proopiomelanocortin (POMC) were measured by high performance liquid chromatography (HPLC) and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis, respectively. Results Central RVD-hp(α) administration decreased locomotion activity and stereotypies. Moreover, RVD-hp(α) treatment inhibited anxiogenic-like behavior and food intake, NE levels and orexin-A gene expression, in the hypothalamus. Conclusion Concluding, in the present study we demonstrated that central RVD-hp(α) induced anxiolytic and anorexigenic effects possibly related to reduced NE and orexin-A and POMC signaling, in the hypothalamus. These findings further support the central role of the peptide in rat brain thus representing an innovative pharmacological approach for designing new anorexigenic drugs targeting eCB system.
Accepted Manuscript
Title: Effects of central RVD-hemopressin() administration
on anxiety, feeding behavior and hypothalamic
neuromodulators in the rat
Authors: Lucia Recinella, Annalisa Chiavaroli, Claudio
Ferrante, Adriano Mollica, Giorgia Macedonio, Azzurra
Stefanucci, Marilisa Pia Dimmito, Szabolcs Dvor´
acsk´
o, Csaba
omb¨oly, Luigi Brunetti, Giustino Orlando, Sheila Leone
PII: S1734-1140(17)30801-0
DOI: https://doi.org/10.1016/j.pharep.2018.01.010
Reference: PHAREP 850
To appear in:
Received date: 6-12-2017
Revised date: 26-1-2018
Accepted date: 31-1-2018
Please cite this article as: Lucia Recinella, Annalisa Chiavaroli, Claudio
Ferrante, Adriano Mollica, Giorgia Macedonio, Azzurra Stefanucci, Marilisa
Pia Dimmito, Szabolcs Dvor´
acsk´
o, Csaba T¨omb¨oly, Luigi Brunetti, Giustino
Orlando, Sheila Leone, Effects of central RVD-hemopressin() administration on
anxiety, feeding behavior and hypothalamic neuromodulators in the rat (2010),
https://doi.org/10.1016/j.pharep.2018.01.010
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Title:
Effects of central RVD-hemopressin(α) administration on anxiety, feeding behavior and
hypothalamic neuromodulators in the rat
Short title:
RVD-hemopressin(α) inhibits hypothalamic norepinephrine, orexin-A and POMC
activity
Lucia Recinella, Annalisa Chiavaroli, Claudio Ferrante1, Adriano Mollica1, Giorgia
Macedonio1, Azzurra Stefanucci1, Marilisa Pia Dimmito1, Szabolcs Dvorácskó2, Csaba
Tömböly2, Luigi Brunetti1, Giustino Orlando1*, Sheila Leone1
Affiliations:
1 Department of Pharmacy, G. dAnnunzioUniversity, Chieti, Italy
2 Laboratory of Chemical Biology, Institute of Biochemistry, Biological Research
Centre of the Hungarian Academy of Sciences, Szeged, Hungary
¥ These authors contributed equally to the work.
* Corresponding author:
e-mail: giustino.orlando@unich.it
Tel.: +39 0871 3554755; Fax.: +39 0871 3554762.
Highlights
Central RVD-Hemopressin(α) injection reduced norepinephrine level
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Central RVD-Hemopressin(α) injection reduced orexin-A gene expression
Central RVD-Hemopressin(α) injection reduced POMC gene expression
Abstract
Background: The endocannabinoid (eCB) system is strongly involved in the regulation
of anxiety and feeding behavior. RVD-hemopressin(α) [RVD-hp(α)], a N-terminally
extended form of hemopressin, is a negative allosteric modulator of the cannabinoid
(CB) 1 receptor and a positive allosteric modulator of CB2 receptor which has been
recently reported to exert anxiolytic/antidepressant and anorexigenic effects after
peripheral administration in rats. Pharmacological evidences reported a possible link
between brain hypocretin/orexin, monoamine and eCB systems, as regards appetite and
emotional behavior control. Considering this, the aim of our work was to investigated
the effects of RVD-hp(α) on anxiety like behavior and food intake after central
administration and related it to monoamine levels and orexin-A gene expression, in the
hypothalamus.
Methods: We have studied the effects of central RVD-hp(α) (10 nmol) injection on
anxiety-like behavior and feeding using different behavioral tests. Hypothalamic levels
of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT)
and gene expression of orexin-A and proopiomelanocortin (POMC) were measured by
high performance liquid chromatography (HPLC) and real-time reverse transcription
polymerase chain reaction (RT-PCR) analysis, respectively.
Results: Central RVD-hp(α) administration decreased locomotion activity and
stereotypies. Moreover, RVD-hp(α) treatment inhibited anxiogenic-like behavior and
food intake, NE levels and orexin-A gene expression, in the hypothalamus.
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Conclusion: Concluding, in the present study we demonstrated that central RVD-hp(α)
induced anxiolytic and anorexigenic effects possibly related to reduced NE and orexin-
A and POMC signaling, in the hypothalamus. These findings further support the central
role of the peptide in rat brain thus representing an innovative pharmacological
approach for designing new anorexigenic drugs targeting eCB system.
Abbreviations:
CB1, cannabinoid 1; CBD, cannabidiol; DA, dopamine; eCB, endocannabinoid; Hp,
Hemopressin; 5-HT, serotonin; POMC, proopiomelanocortin; NE, norepinephrine;
RVD-hp(α), RVD-hemopressin(α).
Keywords:
RVD-hemopressin(α)
Anxiety
Orexin-A
Proopiomelanocortin
Food intake
Monoamines
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Introduction
The endocannabinoid (eCB) system plays an important role in mood disorders [1]
Different studies suggested the involvement of cannabinoid receptor activation on
anxiety and depression [2-4]. In addition, the eCB system is involved in the regulation
of food intake, metabolism and calorie storage [5]. However, it is well known that the
first generation of CB1 blockers designed to reduce food intake and body weight, such
as rimonabant, was discontinued due to psychiatric disorders, such as anxiety and
depression [6-9].
The discovery of hemopressin (hp), an hemoglobin α chain-derived peptide and RVD-
hemopressin(α) [RVD-hp(α)], the N-terminally extended peptide of Hp, also known as
PEPCAN-12, revealed a promising research field for the pharmacotherapy of obesity
[10,11]. Hp and RVD-hp(α) were found to bind CB1 receptors, as antagonist/inverse
agonist and negative allosteric modulator, respectively [12-14]. RVD-hp(α) has been
also described as a positive allosteric modulator of CB2 receptor [15]. Recently, we
have also reported the anxiolytic/antidepressent effects of RVD-hp(α), after peripheral
administration in rats [16]. These results are consistent with multiple studies showing
the role of cannabidiol (CBD), an allosteric modulator of CB1/CB2 receptors [17,18], as
potential anxiolytic/antidepressant drug [19,20], possibly acting via the enhancement of
serotonergic and glutammatergic signalling [21,22]. Additionally, CBD inhibited the
hyperphagia induced by CB1 or 5-HT1A receptor agonists [23]. Pharmacological
evidences also reported a cross-talk between orexinergic and eCB systems, as regards
appetite and emotional behavior control [24,25]. Additionally, the orexinergic neurons
activate sympathetic neurons and arousal, playing a crucial role in emotional behavioral
[26]. We have previously suggested that the behavioral activities by peripheral RVD-
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hp(α) could involve modulatory effects on monoaminergic signaling in the prefrontal
cortex [16]. On the other hand, despite the hemopressin peptides have been reported to
cross blood brain barrier [27], a full comprehension of central mechanism of action is
still required. Previously, we observed strong discrepancies between central and
peripheral peptide administration, with particular regards to neuropeptidergic pathways
underlying feeding control [28-30]. Considering these findings, the aim of our work was
to investigate the direct central effects induced by RVD-hp(α), following
intracerebroventricular (icv) administration in the rat, with particular regards to food
intake and anxiety-like behavior. The behavioral data were also related to hypothalamic
NE, DA and 5-HT levels, proopiomelanocortin (POMC) and orexin-A gene expression,
evaluated by high performance liquid chromatography (HPLC) analysis and real time
reverse transcription polymerase chain reaction (RT-PCR), respectively.
Materials and methods
Peptide synthesis and characterization
RVD-hp(α) has been obtained in our laboratory by Fmoc-solid phase peptide synthesis
(Fmoc-SPPS) strategy on 2-CTC (2-chlorotrityl chloride) resin, following the procedure
reported by Mollica and colleagues [31-32]. Fmoc-Lys(Boc)-OH, Fmoc-Hys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH and Fmoc-Ser(tBu)-OH
were used as orthogonally protected building blocks for coupling reactions involving
HATU/TBTU, and basic cocktail solution for Fmoc removal [33].
Chromatographic purification was performed by RP-HPLC semi-preparative C18
column (eluent: ACN/H2O gradient, 5-95% over 32 min) at a flow gradient of 4
mL/min. RVD-hp(α) was characterized by 1H NMR spectra on 300MHz Varian Inova
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spectrometer (Varian Inc., Palo Alto, CA) and mass spectra on Thermo Scientific Q
Exactive (Thermo Fisher Scientific, San Jose, CA), in, the positive mode, capillary
temperature 220°C, spray voltage 2.3 kV, and sheath gas 5 units.
In vivo studies
Male adult Sprague-Dawley rats (200-250 g) were housed in plexiglas cages (2 animals
per cage; 40 cm × 25 cm × 15 cm) and maintained under standard laboratory conditions
(22 ± 1 °C; 60% humidity), on a 12 h/12 h light/dark cycle (light phase: 07:0019:00 h),
with free access to tap water and food, in accordance with the European Community
ethical regulations (EU Directive 2010/63/EU) on the care of animals for scientific
research. RVD-hp (α) was synthesized in our laboratories by using solid phase synthesis
techniques and was diluted in saline at concentrations 10 nmol, as previously reported
[16]. The following experimental groups were designed: Sham-operated (SHAM),
saline-treated (Vehicle) and RVD-hp(α)-treated [RVD-hp(α)]. The initial group size was
n=8 for behavior studies and n=6 for feeding behavior, hypothalamic monoamine levels
and orexin-A gene expression after RVD-hp (α) administration in lateral ventricle.
Stereotaxic surgery
Rats were anesthetized by intraperitoneal injection with ketamine-xilazine (50 and 5
mg/kg, respectively) and placed in a stereotaxic instrument (David Kopf Instruments,
Tujunga, CA). A stainless steel guide cannula (21 G) was inserted stereotaxically to a
depth of 3.5 mm in a position 1.5 mm to the left and 0.8 mm caudal to bregma, as
previously reported [34]. The cannula was secured in position with dental acrylic
(Formatray, Salerno, Italy) and the animal was kept warm during recovery. During
surgery, rats were injected subcutaneously with 1 ml of sterile saline solution and 1 ml
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of 5% glucose solution (Galenica Senese, Siena, Italy) and intraperitoneally with
amoxicillin (20 mg kg1) (Farmalabor, Milano, Italy).
Microinjection
Animals received unilateral microinjections of vehicle (saline) or drugs (1 ul) into
lateral ventricle before being submitted to the behavioral test, by connecting the cannula
with a 5 μl syringe (Hamilton, Switzerland). After behavioral test the rats were
sacrificed, as previously reported [34].
Behavioral test
The animals were brought into the experimental room 30 min prior to the test in order
for them to acclimate to the environment, and were kept in the testing chamber for 5
min prior to each test. All treatments were administered at 09:00 am, and the
experiments performed between 10:00 and 12:00 am. Each test session was recorded by
a video camera connected to a computer; a single video frame was acquired with a
highly accurate, programmable, monochrome frame grabber board (Data
TranslationTM, type DT3153). The intelligent software Smart version 2.5 (Panlab, sl
Bioresearch and Technology, Barcelona, Spain) was used for data processing. The
apparatuses were purchased from 2 Biological Instruments (Besozzo VA, Italy). At the
end of each test, the animals were returned to their home cage, and the apparatus was
cleaned with 75% ethanol and dried before the next procedure.
Locomotor activity was recorded in the home cage over 10 min. The activity monitor
consisted of a black and white video camera, mounted in the top-center of the cage.
Locomotor activity was assessed as horizontal activity, vertical activity, duration of
stereotypic behavior (self-grooming and scratching), time spent in movements and
resting time. Resting time was considered when animal's movements were below a
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threshold set by comparing the score of resting rated manually with the score from the
automated device in preliminary studies [35].
Open field test: To evaluate anxiety-like behavior, each animal was placed in an open
field box (60 × 60 × 60 cm) made of clear Plexiglas with a white laminated sheet of
paper marked into sixteen squares (15 × 15 cm). Immediately after the injection, each
animal was monitored for 10 min. In the open field test, the distance traveled and time
spent into the center were recorded [36].
Elevated plus maze: The apparatus consisted of two open arms (50 × 10 cm) without
side walls, perpendicular to two enclosed arms (50 × 10 × 40 cm) with a central
platform common to all arms (10 × 10 cm). The maze was elevated to a height of 50 cm
above floor level and rats were individually placed in the centre of the maze facing an
open arm. The time spent on open arms, the latency to first exit and the number of
transitions between the arms were recorded during a 10 min test period [34].
Food intake
24 h after RVD-hemopressin(α) (10 nmol) administration, food intake were evaluated,
as previously reported [37]. After completion of feeding and anxiety-like behavioral
test, the animals were sacrificed by CO2 inhalation (100 % CO2 at a flow rate of 20 % of
the chamber volume per minute).
Hypothalamic monoamine extraction and high performance liquid chromatography
(HPLC) determination
Immediately after sacrifice, brains were rapidly removed and individual hypothalami
dissected and subjected to biogenic amine extractive procedures [38]. Briefly, tissues
were homogenized in ice bath for 2 min with Potter-Elvehjem homogenizer in 1 ml of
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0.05 N perchloric acid containing 0.004% sodium EDTA and 0.010% sodium bisulfite.
The homogenate was 5 fold diluted in chromatographic mobile phase and centrifuged at
4,500 x g for 10 min. The supernatant was filtered on 0.45 µm PTFE sterile filters
(Whatman) and directly injected for HPLC. Neurotransmitter recovery was satisfactory
(≥90%) and reproducible, with percentage relative standard deviation ≤10%. The HPLC
apparatus consisting of a Jasco (Tokyo, Japan) PU-2080 chromatographic pump and an
ESA (Chelmsford, MA, USA) Coulochem III coulometric detector, equipped with
micro dialysis cell (ESA-5014b) porous graphite working electrode and solid state
palladium reference electrode. The analytical cell was set at −0.150 V, for detector 1
and at +0.300 V, for detector 2, with a range of 100 nA. The chromatograms were
monitored at the analytical detector 2. Integration was performed by Jasco Borwin
Chromatography software, version 1.5. The chromatographic separation was performed
by isocratic elution on Phenomenex Kinetex reverse phase column (C18, 150 mm×4.6
mm i.d., 2.6 µm). The mobile phase was (10:90, v/v) acetonitrile and 75 mM pH 3.00
phosphate buffer containing octanesulfonic acid 1.8 mM, EDTA 30 µM and
triethylamine 0.015% v/v. Flow rate was 0.6 ml/min and the samples were manually
injected through a 20 µl loop. Neurotransmitter peaks were identified by comparison
with the retention time of pure standard. Neurotransmitter concentrations in the samples
were calculated by linear regression curve (y = bx + m) obtained with standard. Neither
internal nor external standard were necessary for neurotransmitter quantification, in the
hypothalamus homogenate, and all tests performed for method validation yielded results
in accordance to limits indicated in official guidelines for applicability in laboratory
trials. The standard stock solutions of DA, NE and 5-HT at 2 mg/ml were prepared in
bidistilled water containing 0.004% EDTA and 0.010% sodium bisulfite. The stock
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solutions were stored at 4°C. Work solutions (1.25-20.00 ng/ml) were obtained daily
progressively diluting stock solutions in mobile phase.
RNA extraction, reverse transcription and real-time reverse transcription polymerase
chain reaction (real-time RT PCR)
Immediately after sacrifice, hypothalami were rapidly removed, dissected and stored in
RNAlater solution (Ambion, Austin, TX) at −20 ◦C until further processed. Total RNA
was extracted from the hypothalamus using TRI Reagent (Sigma-Aldrich, St. Louis,
MO), as previously reported [37]. Contaminating DNA was removed using 2 units of
RNase-free DNase 1 (DNA-free kit, Ambion, Austin, TX). The RNA solution was
quantified at 260 nm by spectrophotometer reading (BioPhotometer, Eppendorf,
Hamburg, Germany) and its purity was assessed by the ratio at 260 and 280 nm
readings. The quality of the extracted RNA samples was also determined by
electrophoresis through agarose gels and staining with ethidium bromide, under UV
light. One μg of total RNA extracted from each sample in a 20 μl reaction volume was
reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA, USA). Reactions were incubated in a 2720 Thermal
Cycler (Applied Biosystems, Foster City, CA, USA) initially at 25°C for 10 min, then at
37°C for 120 min, and finally at 85°C for 5 s. Gene expression was determined by
quantitative real-time PCR using TaqMan probe-based chemistry (Applied Biosystems,
Foster City, CA, USA). PCR primers and TaqMan probes were obtained from Applied
Biosystems (Assays-on-Demand Gene Expression Products, Rn00565995_m1 for
orexin-A gene; Rn00595020_m1 for POMC gene,) β-actin (Applied Biosystems, Foster
City, CA, USA, Part No. 4352340E) was used as the housekeeping gene. The real-time
PCR was carried out in triplicate for each cDNA sample in relation to each of the
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investigated genes. Data were elaborated with the Sequence Detection System (SDS)
software version 2.3 (Applied Biosystems, Foster City, CA, USA). Gene expression
was relatively quantified by the comparative 2-ΔΔCt method [39].
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 5.01 for Windows
(GraphPad Software, San Diego, CA, USA). All data were collected from each of the
animals used in the experimental procedure and means ± SEM. were determined for
each experimental group and analyzed by unpaired t test (two-tailed P value) and two
way analysis of variance (ANOVA) followed by Bonferroni post-hoc test. As for gene
expression analysis, 1.00 (calibrator sample) was considered the theoretical mean for
the comparison. Statistical significance was accepted at p < 0.05. As regards to the
animals randomized for each experimental group, the number was calculated on the
basis of the “Resource Equation” N=(E+T)/T (10≤E≤20) [40], where E, N and T
represent the numbers of degrees of freedom in an ANOVA, animals and treatment,
respectively.
Results
Exploration behavioral analysis
As shown in Fig. 2, icv RVD-hp(α) (10 nmol) injection induced a significant decrease
of locomotor activity compared to SHAM and vehicle control groups. Two-way
ANOVA showed significant differences in horizontal, vertical activity, time spent in
movements and stereotypic behavior respect to controls (**p<0.005 vs. SHAM and
vehicle)
Anxiety-like behavior
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In the open field test, icv RVD-hp(α) injection decreased the anxiety-related behavior,
as evidenced by a significant increase of travelled distance and time spent in the central
zone respect to controls (Fig. 3A, **p<0.005 vs. SHAM and vehicle). Similarly, in
elevated plus maze test icv RVD-hp(α) injection increased the time spent in the open
arms and number of total transitions, and decreased the latencies to emerge from the
central zone (Fig. 3B, **p<0.005 vs. SHAM and vehicle).
Food intake evaluation
Fig. 4 shows food intake (g) in rats fed a standard diet. Vehicle or peptide were
administered by icv injection, during the light phase, at 9:00 a.m.. Food intake was
evaluated 24 h after treatment in each group of rats. Values represent the means ± SEM.
Compared to SHAM and vehicle control groups, RVD-hemopressin(α) significantly
inhibited food intake (***p<0.001 vs. SHAM and vehicle).
Hypothalamic monoamine levels
Fig. 5 shows decreased NE levels in the hypothalamus after RVD-hp(α) (10 nmol)
injection, compared to SHAM and vehicle control groups (**p<0.005 vs. SHAM and
vehicle). On the other hand, we did not observe any alteration as regards DA and 5-HT
levels, following peptide administration.
POMC and orexin-A gene expression
RVD-hp(α) injection inhibited hypothalamic POMC and orexin-A gene expressions
(Fig. 6), compared to SHAM and vehicle control groups (**p<0.005 vs. SHAM and
vehicle).
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Discussion
The eCB system is well known to modulate anxiety-like and feeding behavior [1,5,41].
In the present study we have shown that RVD-hp(α), a CB1/CB2 allosteric modulator
[13-15], is able to modulate emotional and feeding behavior. In confirming the
anxiolytic and anorexigenic effects, previously described by our research group [11,16],
we also observed a decrease in locomotor activity after central administration. The
contrasting results with our recent published studies could be possibly related to the
different route of administration, thus further supporting the hypothesis of multiple
central and peripheral mechanisms underlying behavioral peptide effects in vivo,
including the possible modulation of adipokines and gut-derived hormones, both
involved in metabolic and behavioral pathways [28,29,38]. Additionally, we observed
that RVD-hp(α) decreased the stereotypic behavior, such as self-grooming and
scratching, which are considered useful index of anxiety behavior, in animal models
[42]. We also observed a decrease in food intake after central RVD-hp(α)
administration, possibly related to reduced hypothalamic NE levels and POMC gene
expression. This is consistent with previous findings suggesting that POMC-derived
peptide β-endorphin and NE are key modulators of appetite and anxiety behavior
induced by CB1 agonists [43,44]. We also found decreased orexin-A gene expression in
the hypothalamus. Multiple studies suggested a possible cross-talk between orexinergic
and eCB systems in rat hypothalamus [24,25,45], which could be involved in appetite,
reward, sleep/wake cycle and nociception [25]. Central administration of orexin-A
stimulates food consumption, while orexin receptor antagonist (SB334867), reduced
feeding [46,47]. On the other hand, orexin-A injection into the brain increased arousal,
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locomotor activity and stereotypies, in rats [48-51]. Moreover, Flores and collaborators
[25] have found orexin and CB1 receptors co-expressed in the hypothalamus.
In the present study, we also found that icv RVD-hp(α) injection decreased NE, and did
not affect DA and 5-HT levels in the hypothalamus. The central noradrenergic system
is involved in the control of arousal [52,53], exploration behavior [54,55] and food
intake [56]. Soya and collaborators suggested that it could mediate orexin-induced
behavioral effects [26]. These studies could clarify our findings of anxiolytic and
anorexigenic effects observed after RVD-hp(α) peripheral and central administration, in
rats. In fact, interference on hypocretinergic transmission might be useful in the control
of appetite and other disorders associated with obesity, such as anxiety, probably
mediated by CB1 receptors.
In conclusion, in the present study we demonstrated that central RVD-hp(α)
administration induced anxiolytic and anorexigenic effects possibly mediated by
reduced NE and orexin-A signaling, in the hypothalamus. These findings further
support the central role of RVD-hp(α) in rat brain, and could represent a perspective in
the pharmaceutical design of new anorexigenic drugs.
Funding
This work was supported by the ÚNKP-16-3 New National Excellence Program of the
Ministry of Human Capacities of Hungary.
Project Authorization
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All the in vivo procedures were approved by local Ethical Committee of “G.
d’Annunzio University” and Italian Ministry of Health (project n. 880 authorized on
24th august 2015).
Conflict of interest statement
The authors declare no conflict interest.
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Legends to figures
Fig. 1. Chemical structure of RVD-hp(α).
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Fig. 2. Locomotor activity in rats treated with a single icv RVD-hp(α) administration
(10 nmol). Compared to SHAM and vehicle, RVD-hp(α) significantly decreased
locomotor and stereotypic activity. Horizontal activity (A), vertical activity (B),
movements (C) and stereotypic movements (D) were recorded for 10 min. Data are
expressed as means ± SEM. (**p<0.005 vs. vehicle).
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Fig.3. Analysis of anxiety-related behavior in rats treated with a single icv RVD-hp(α)
administration (10 nmol). Compared to SHAM and vehicle, RVD-hp(α) decreased
levels of anxiety-like behavior in open field (A) and elevated plus maze test (B). Data
are expressed as means ± SEM. (**p<0.005 vs. SHAM and vehicle).
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Fig. 4. 24 h food intake following icv RVD-hp(α) administration (10 nmol). RVD-hp(α)
significantly inhibited food intake with respect to SHAM and vehicle. Data are
expressed as means ± SEM. (***p <0.001 vs. SHAM and vehicle).
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Fig. 5. Norepinephrine (NE), dopamine (DA) and serotonin (5-hydrohytryptamine, 5-
HT) hypothalamic levels (ng/mg wet tissue), following icv RVD-hp(α) administration
(10 nmol). Data are expressed as means ± SEM. Compared to SHAM and vehicle,
RVD-hp(α) significantly decreased NE levels in the hypothalamus (**p<0.005 vs.
SHAM and vehicle).
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Fig. 6. Relative gene expression orexin-A and POMC in the hypothalamus A after icv RVD-
hp(α) administration (10 nmol), as determined by real-time RT-PCR. Data were calculated
using the 2-Ct method, normalized to -actin mRNA levels, and expressed as relative to
control (calibrator sample, defined as 1.00). Values represent the means ± SEM. (**p<0.005
vs. SHAM and vehicle).
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... RVD-hemopressin(α) is, respectively, a positive and negative allosteric modulator of the cannabinoid 2 and 1 receptors and it is known that the endocannabinoid system is involved in feeding behavior [121]. The central administration of RVD-hemopressin(α) blocked food intake and decreased the hypothalamic expression of orexin A [121] (Table 5). ...
... RVD-hemopressin(α) is, respectively, a positive and negative allosteric modulator of the cannabinoid 2 and 1 receptors and it is known that the endocannabinoid system is involved in feeding behavior [121]. The central administration of RVD-hemopressin(α) blocked food intake and decreased the hypothalamic expression of orexin A [121] (Table 5). Thus, the anorexigenic effect mediated by RVD-hemopressin(α) is associated to a decrease in the hypothalamic orexin A signaling. ...
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To know the processes involved in feeding, the dysregulation of hypothalamic neuropeptides promoting anorexigenic/orexigenic mechanisms must be investigated. Many neuropeptides are involved in this behavior and in overweight/obesity. Current pharmacological strategies for the treatment of obesity are unfortunately not very effective and, hence, new therapeutic strategies must be investigated and developed. Due to the crucial role played by orexins in feeding behavior, the aim of this review is to update the involvement of the orexinergic system in this behavior. The studies performed in experimental animal models and humans and the relationships between the orexinergic system and other substances are mentioned and discussed. Promising research lines on the orexinergic system are highlighted (signaling pathways, heterogeneity of the hypothalamic orexinergic neurons, receptor-receptor interaction, and sex differences). Each of the orexin 1 and 2 receptors plays a unique role in energy metabolism, exerting a differential function in obesity. Additional preclinical/clinical studies must be carried out to demonstrate the beneficial effects mediated by orexin receptor antagonists. Because therapies applied are in general ineffective when they are directed against a single target, the best option for successful anti-obesity treatments is the development of combination therapies as well as the development of new and more specific orexin receptor antagonists.
... Thus, hemopressin may offer behaviorally selective effects on nociception and appetite, without engaging reward pathways (Dodd et al., 2010(Dodd et al., , 2013. Interestingly, intraperitoneal or intracerebral injection of RVD-HP also showed anorexigenic properties in rats, a mechanism that could be partially mediated by lowering of levels of orexin-A, proopiomelanocortin, and agouti-related peptide gene expression Recinella et al., 2018). ...
... Although the presence of hemopressin has not been detected in brain extracts following systemic administration (Fogaça et al., 2015), it induces neuronal activation in specific areas of the mice or rat brain (Dodd et al., 2013;Reckziegel et al., 2017) and produces anxiogenic effects (Fogaça et al., 2015;Leone et al., 2017;Recinella et al., 2018). This could be due to enzymatic cleavage of hemopressin into smaller fragments before entering the central nervous system. ...
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... The light-dark box test and elevated plus maze test assesses brightspace related anxiety. Both these tests were performed as previously reported [21][22][23][24]. ...
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Anxiety and depression have been suggested to increase the risk for post-traumatic stress disorders (PTSD). A link between all these mental illnesses, inflammation and oxidative stress is also well established. Recent behavior studies by our group clearly demonstrate a powerful anxiolytic and antidepressant-like effects of a novel growth hormone releasing hormone (GHRH) antagonist of MIAMI class, MIA-690, probably related to modulatory effects on the inflammatory and oxidative status. In the present work we investigated the potential beneficial effects of MIA-602, another recently developed GHRH antagonist, in mood disorders, as anxiety and depression, and the possible brain pathways involved in its protective activity, in adult mice. MIA-602 exhibited antinflammatory and antioxidant effects in ex vivo and in vivo experimental models, inducing anxiolytic and antidepressant-like behavior in mice subcutaneously treated for 4 weeks. The beneficial effect of MIA-602 on inflammatory and oxidative status and synaptogenesis resulting in anxiolytic and antidepressant-like effects could be related by increases of nuclear factor erythroid 2-related factor 2 (Nrf2) and of brain-derived neurotrophic factor (BDNF) signaling pathways in the hippocampus and prefrontal cortex. These results strongly suggest that GHRH analogs should be tried clinically for the treatment of mood disorders including PTSD.
... They are variously distributed in the central nervous system (CNS), specifically in axons and pre-synaptic terminals, hippocampus, cortex, where they are implicated in the control of memory, sedation, hypothermia, hypotension, and pain sensation [8][9][10] (Fig. 2). The endocannabinoid system is also known to modulate anxiety and feeding behavior interacting with different ligands, as in the case of Hemopressin and RVD-hemopressin [11][12][13]. ...
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Nowadays cardiovascular diseases (CVDs) are the major causes for the reduction of the quality of life. The endocannabinoid system is an attractive therapeutic target for the treatment of cardiovascular disorders due to its involvement in vasomotor control, cardiac contractility, blood pressure and vascular inflammation. Alteration in cannabinoid signalling can be often related to cardiotoxicity, circulatory shock, hypertension, and atherosclerosis. Plants have been the major sources of medicines until modern eras in which researchers are experiencing a rediscovery of natural compounds as novel therapeutics. One of the most versatile plant is Cannabis sativa L., containing phytocannabinoids that may play a role in the treatment of CVDs. The aim of this review is to collect and investigate several less studied plants rich in cannabinoid-like active compounds able to interact with cannabinoid system; these plants may play a pivotal role in the treatment of disorders related to the cardiovascular system.
... The antibody was colocalized with tyrosine hydroxylase and galanin immunolabeling; however, it was not detected in dopaminergic neurons. As described above, there is evidence suggesting the modulatory effects of hemopressin peptides on monoaminergic signaling (Tanaka et al., 2014;Leone et al., 2017;Recinella et al., 2018). ...
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Classically, the endocannabinoid system (ECS) consists of endogenous lipids, of which the best known are anandamide (AEA) and 2 arachidonoylglycerol (2-AG), their enzyme machinery for synthesis and degradation and their specific receptors, cannabinoid receptor one (CB1) and cannabinoid receptor two (CB2). However, endocannabinoids also bind to other groups of receptors. Furthermore, another group of lipids are considered to be endocannabinoids, such as the fatty acid ethanolamides, the fatty acid primary amides and the monoacylglycerol related molecules. Recently, it has been shown that the hemopressin peptide family, derived from α and β chains of hemoglobins, is a new family of cannabinoids. Some studies indicate that hemopressin peptides are expressed in the central nervous system and peripheral tissues and act as ligands of these receptors, thus suggesting that they play a physiological role. In this review, we examine new evidence on lipid endocannabinoids, cannabinoid receptors and the modulation of their signaling pathways. We focus our discussion on the current knowledge of the pharmacological effects, the biosynthesis of the peptide cannabinoids and the new insights on the activation and modulation of cannabinoid receptors by these peptides. The novel peptide compounds derived from hemoglobin chains and their non-classical activation of cannabinoid receptors are only starting to be uncovered. It will be exciting to follow the ensuing discoveries, not only in reference to what is already known of the classical lipid endocannabinoids revealing more complex aspects of endocannabinoid system, but also as to its possibilities as a future therapeutic tool.
... A single i.c.v. administration of RVD-Hpα also produces anxiolytic-like behavior in rats, a result that may correlate with the decrease in norepinephrine levels and orexin-A gene expression in the hypothalamus [94]. However, the α-chain fragment AGH, which is known for its antinociceptive effect following systemic administration, has no influence on locomotor activity in an open field test and the tested parameters of depression models, including forced swimming test and tail suspension test [95]. ...
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Evidence accumulated over the past decades has revealed that red blood cells and hemoglobin (Hb) in the blood play important roles in modulating moods and emotions. The number of red blood cells affects the mood. Hb is the principal content in the red blood cells besides water. Denatured Hb is hydrolyzed to produce bioactive peptides. RVD-hemopressin α (RVD-Hpα), which is a fragment of α-chain (95-103) in Hb, functions as a negative allosteric modulator of cannabinoid receptor 1 and a positive allosteric modulator of cannabinoid receptor 2. Hemorphins, which are fragments of β-chain in Hb, exert their effects on opioid receptors. Two hemorphins, namely, LVV-hemorphin-6 and LVV-hemorphin-7, could induce anxiolytic-like effects. The use of Hb-derived bioactive peptides for the treatment of mood disorders is desirable due to cannabinoid-opioid cross modulation and the critical roles of the two systems in physiological processes, such as memory, mood and emotion.
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Hemopressins ((x)-PVNFKLLSH) or peptide endocannabinoids (pepcans) can bind to cannabinoid receptors. RVD-hemopressin (pepcan-12) was shown to act as endogenous allosteric modulator of cannabinoid receptors, with opposite effects on CB1 and CB2, respectively. Moreover, the N-terminally elongated pepcan-23 was detected in different tissues and was postulated to be the pro-peptide of RVD-hemopressin. Currently, data about the pharmacokinetics, tissue distribution and stability of hemopressin-type peptides are lacking. Here we investigated the secondary structure and physiological role of pepcan-23 as precursor of RVD-hemopressin. We assessed the metabolic stability of these peptides, including hemopressin. Using LC-ESI-MS/MS, pepcan-23 was measured in mouse tissues and human whole blood (∼50 pmol/mL) and in plasma was the most stable endogenous peptide containing the hemopressin sequence. Using peptide spiked human whole blood, mouse adrenal gland and liver homogenates demonstrate that pepcan-23 acts as endogenous pro-peptide of RVD-hemopressin. Furthermore, administered pepcan-23 converted to RVD-hemopressin in mice. In circular dichroism spectroscopy, pepcan-23 showed a helix-unordered-helix structure and efficiently formed complexes with divalent metal ions, in particular Cu(II) and Ni(II). Hemopressin and RVD-hemopressin were not bioavailable to the brain and showed poor stability in plasma, in agreement with their overall poor biodistribution. Acute hemopressin administration (100 mg/kg) did not modulate endogenous RVD-hemopressin/pepcan-23 levels or influence the endocannabinoid lipidome but increased 1-stearoyl-2-arachidonoyl-sn-glycerol. Overall, we show that pepcan-23 is a biological pro-peptide of RVD-hemopressin and divalent metal ions may regulate this process. Given the lack of metabolic stability of hemopressins, administration of pepcan-23 as pro-peptide may be suitable in pharmacological experiments as it is converted to RVD-hemopressin in vivo.
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The endocannabinoid system is activated by the binding of natural arachidonic acid derivatives (endogenous cannabinoids or endocannabinoids) as lipophilic messengers to cannabinoid receptors CB1 and CB2. The endocannabinoid system comprises also many hydrolytic enzymes responsible for the endocannabinoids cleavage, such as FAAH and MAGL. These two enzymes are possible therapeutic targets for the development of new drugs as indirect cannabinoid agonists. Recently a new family of endocannabinoid modulators was discovered; the lead of this family is the nonapeptide hemopressin produced from enzymatic cleavage of the α-chain of hemoglobin and acting as negative allosteric modulator of CB1. Hemopressin shows several physiological effects, e.g. antinociception, hypophagy, and hypotension. It is still matter of debate whether this peptide, isolated from the brain of rats is a real neuromodulator of the endocannabinoid system. Recent evidence indicates that hemopressin could be a by-product formed by chemical degradation of a longer peptide RVD-hemopressin during the extraction from the brain homolysate. Indeed, RVD-hemopressin is more active than hemopressin in certain biological tests and may bind to the same subsite as Rimonabant, which is an inverse agonist for the CB1 receptor and a μ-opioid receptor antagonist. These findings have stimulated several studies to verify this hypothesis and to evaluate possible therapeutic applications of hemopressin, its peptidic derivatives and synthetic analogues, opening new perspectives to the development of novel cannabinoid drugs.
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Background: The endocannabinoid (eCB) system plays an important role in regulating emotional disorders, and is involved, directly or indirectly, in psychiatric diseases, such as anxiety and depression. Hemopressin, a hemoglobin α chain-derived peptide, and RVD-hemopressin(α), a N-terminally extended form of hemopressin, act as antagonist/inverse agonist and negative allosteric modulator of the cannabinoid 1 (CB1) receptor, respectively. Methods: Considering the possible involvement of these peptides on emotional behaviour, the aim of our study was to investigate the behavioural effects of a single intraperitoneal (ip) injection of hemopressin (0.05mg/kg) and RVD-hemopressin(α) (0.05mg/kg), using a series of validated behavioural tests (locomotor activity/open field test, light-dark exploration test, forced swim test) in rats. Prefrontal cortex levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and the gene expression of monoamine oxidase (MAO-B) and catechol-O-methyltransferase (COMT) were measured by high performance liquid chromatography (HPLC) analysis and real-time reverse transcription polymerase chain reaction (RT-PCR), respectively. Results: Hemopressin administration induced anxiogenic and depressive behaviour, decreased monoamine steady state levels in prefrontal cortex, and increased the gene expression of the enzymes involved in their catabolism. By contrast, RVD- hemopressin(α) induced anxiolytic and antidepressive effects, increased monoamines and decreased the enzymes in prefrontal cortex. Conclusion: In conclusion, in the present study we demonstrated behavioral effects induced by peripheral hemopressin and RVD-hemopressin(α) injections, that could involve modulatory effects on monoaminergic signaling, in the prefrontal cortex.
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Background: Hemopressin, VD-hemopressin(α) and RVD-hemopressin(α) are hemoglobin α chain derived-peptides which have been found in mouse brain, and where they modulate cannabinoid (CB) receptor function. The nonapeptide hemopressin has been reported to inhibit feeding after both central and peripheral administration, possibly playing a role of antagonist/inverse agonist of CB1 receptors, and consequently blocking the orexigenic effects of endogenous cannabinoids. VD-hemopressin(α) and RVD- hemopressin(α), are N-terminal extended forms of hemopressin. VD-hemopressin(α) has CB1 agonist activity, and as such it has been shown to stimulate feeding. RVD-hemopressin(α) is reported to play a negative allosteric modulatory function on CB1 receptors, but there are no data on its possible effects on feeding and metabolic control. Methods: We have studied, in rats, the effects of 14 daily intraperitoneal (ip) injections of RVD-hemopressin(α) (10nmol). Results: We found that RVD-hemopressin(α) treatment inhibited food intake while total body weight was not affected. The null effect on body weight despite diminished feeding could be related to decreased uncoupling protein 1 (UCP-1) gene expression in brown adipose tissue (BAT). We also investigated the underlying neuromodulatory effects of RVD-hemopressin(α) and found it to down regulate proopiomelanocortin (POMC) gene expression, together with norepinephrine (NE) levels, in the hypothalamus. Conclusions: In conclusion, RVD-hemopressin(α) administration has an anorectic effect, possibly related to inhibition of POMC and NE levels in the hypothalamus. Despite decreased food intake, body weight is not affected by RVD-hemopressin(α) treatment, possibly due to inhibition of UCP-1 gene expression in BAT.
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Cannabidiol (CBD), the main non-psychotomimetic component of marihuana, exhibits anxiolytic-like properties in many behavioural tests, although its potential for treating major depression has been poorly explored. Moreover, the mechanism of action of CBD remains unclear. Herein, we have evaluated the effects of CBD following acute and chronic administration in the olfactory bulbectomy mouse model of depression (OBX), and investigated the underlying mechanism. For this purpose, we conducted behavioural (open field and sucrose preference tests) and neurochemical (microdialysis and autoradiography of 5-HT1A receptor functionality) studies following treatment with CBD. We also assayed the pharmacological antagonism of the effects of CBD to dissect out the mechanism of action. Our results demonstrate that CBD exerts fast and maintained antidepressant-like effects as evidenced by the reversal of the OBX-induced hyperactivity and anhedonia. In vivo microdialysis revealed that the administration of CBD significantly enhanced serotonin and glutamate levels in vmPFCx in a different manner depending on the emotional state and the duration of the treatment. The potentiating effect upon neurotransmitters levels occurring immediately after the first injection of CBD might underlie the fast antidepressant-like actions in OBX mice. Both antidepressant-like effect and enhanced cortical 5-HT/glutamate neurotransmission induced by CBD were prevented by 5-HT1A receptor blockade. Moreover, adaptive changes in pre- and post-synaptic 5-HT1A receptor functionality were also found after chronic CBD. In conclusion, our findings indicate that CBD could represent a novel fast antidepressant drug, via enhancing both serotonergic and glutamate cortical signalling through a 5-HT1A receptor-dependent mechanism.