Novel choline analog 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol produces sympathoinhibition, hypotension, and antihypertensive effects

Article abstract of DOI:10.1007/s00210-019-01649-8

The search for new drugs remains an important focus for the safe and effective treatment of cardiovascular diseases. Previous evidence has shown that choline analogs can offer therapeutic benefit for cardiovascular complications. The current study investigates the effects of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) on cardiovascular function and cholinergic-nitric oxide signaling. Synthesized LQFM032 (0.3, 0.6, or 1.2?mg/kg) was administered by intravenous and intracerebroventricular routes to evaluate the potential alteration of mean arterial pressure, heart rate, and renal sympathetic nerve activity of normotensive and hypertensive rats. Vascular function was further evaluated in isolated vessels, while pharmacological antagonists and computational studies of nitric oxide synthase and muscarinic receptors were performed to assess possible mechanisms of LQFM032 activity. The intravenous and intracerebroventricular administration of LQFM032 elicited a temporal reduction in mean arterial pressure, heart rate, and renal sympathetic nerve activity of rats. The cumulative addition of LQFM032 to isolated endothelium-intact aortic rings reduced vascular tension and elicited a concentration-dependent relaxation. Intravenous pretreatment with L-NAME (nitric oxide synthase inhibitor), atropine (nonselective muscarinic receptor antagonist), pirenzepine, and 4-DAMP (muscarinic M1 and M3 subtype receptor antagonist, respectively) attenuated the cardiovascular effects of LQFM032. These changes may be due to a direct regulation of muscarinic signaling as docking data shows an interaction of choline analog with M1 and M3 but not nitric oxide synthase. Together, these findings demonstrate sympathoinhibitory, hypotensive, and antihypertensive effects of LQFM032 and suggest the involvement of muscarinic receptors.

Full text of DOI:10.1007/s00210-019-01649-8

Naunyn-Schmiedeberg's Archives of Pharmacology
https://doi.org/10.1007/s00210-019-01649-8
ORIGINAL ARTICLE
Novel choline analog 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)
piperazin-1-yl)ethan-1-ol produces sympathoinhibition,
hypotension, and antihypertensive effects
1
1
2
3
3
4
Ricardo Menegatti & Flávio S. Carvalho & Luciano M. Lião & Bianca Villavicencio & Hugo Verli & Aline A. Mourão
&
4
4
4
5
6
Carlos H. Xavier & Carlos H. Castro & Gustavo R. Pedrino & Octavio L. Franco & Iransé Oliveira-Silva
&
7
5
8
4,6
Nicole M. Ashpole & Osmar Nascimento Silva
& Elson A. Costa & James O. Fajemiroye
Received: 2 January 2019 /Accepted: 29 March 2019
#
Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
The search for new drugs remains an important focus for the safe and effective treatment of cardiovascular diseases. Previous
evidence has shown that choline analogs can offer therapeutic benefit for cardiovascular complications. The current study
investigates the effects of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) on cardiovascular
function and cholinergic-nitric oxide signaling. Synthesized LQFM032 (0.3, 0.6, or 1.2 mg/kg) was administered by intrave-
nous and intracerebroventricular routes to evaluate the potential alteration of mean arterial pressure, heart rate, and renal
sympathetic nerve activity of normotensive and hypertensive rats. Vascular function was further evaluated in isolated vessels,
while pharmacological antagonists and computational studies of nitric oxide synthase and muscarinic receptors were performed
to assess possible mechanisms of LQFM032 activity. The intravenous and intracerebroventricular administration of LQFM032
elicited a temporal reduction in mean arterial pressure, heart rate, and renal sympathetic nerve activity of rats. The cumulative
addition of LQFM032 to isolated endothelium-intact aortic rings reduced vascular tension and elicited a concentration-
dependent relaxation. Intravenous pretreatment with L-NAME (nitric oxide synthase inhibitor), atropine (nonselective musca-
rinic receptor antagonist), pirenzepine, and 4-DAMP (muscarinic M1 and M3 subtype receptor antagonist, respectively) atten-
uated the cardiovascular effects of LQFM032. These changes may be due to a direct regulation of muscarinic signaling as
docking data shows an interaction of choline analog with M1 and M3 but not nitric oxide synthase. Together, these findings
demonstrate sympathoinhibitory, hypotensive, and antihypertensive effects of LQFM032 and suggest the involvement of
muscarinic receptors.
.
.
.
Keywords Choline analog Muscarinic receptor Nitric oxide synthase Sympathoinhibition
4
*
*
Osmar Nascimento Silva
osmar.silva@catolica.edu.br
Department of Physiology, Universidade Federal de Goiás,
Goiânia, GO 74001-970, Brazil
5
James O. Fajemiroye
olulolo@yahoo.com
S-Inova Biotech, Programa de Pós-graduação em Biotecnologia,
Universidade Católica Dom Bosco, Campo Grande, MS 79117-900,
Brazil
1
2
3
Faculty of Pharmacy, Universidade Federal de Goiás,
Goiânia, GO 74605-170, Brazil
6
7
UniEvangélica, Centro Universitário de Anápolis,
Anápolis, GO 75083-515, Brazil
Department of BioMolecular Sciences, Division of Pharmacology,
School of Pharmacy, University of Mississippi,
University, MS 38677-1848, USA
Chemistry Institute, Universidade Federal de Goiás,
Goiânia, GO 74001-970, Brazil
8
Centro de Biotecnologia, Universidade Federal do Rio Grande do
Sul, Porto Alegre, RS 91500-970, Brazil
Department of Phamacology, Universidade Federal de Goiás,
Goiânia, GO 74001-970, Brazil

Naunyn-Schmiedeberg's Arch Pharmacol
Introduction
Considering these evidences of cholinergic-mediated car-
diovascular changes, researchers are now working to develop
novel compounds targeting mAChR. With the overall goal of
developing a novel cholinergic signaling molecule that could
decrease incidents of cardiovascular dysfunction, the synthe-
sis of a new choline analog 2-(4-((1-phenyl-1H-pyrazol-4-
yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) was pro-
posed and designed from molecular hybridization of
LQFM008 (piperazine derivative) and JWB-1-84-1 (analog
of acetylcholine) (Brito et al. 2017).
Cardiovascular disease is a leading cause of mortality and
is associated with a higher annual death rate than any other
disease category (Stern and Lebowitz 2010). The search
for new drugs that target the cardiovascular system is rec-
ognized as a necessary strategy for effective treatment
(
Fajemiroye et al. 2014). It was suggested that newer
drugs with fewer side effects and with novel mechanisms
of action could provide therapeutic options (Magoon et al.
2
017). Targeting the autonomic nervous system, specifi-
In this study, we investigate 2-(4-(1-phenyl-1H-pyrazol-4-
yl)methyl) piperazin-1-yl)ethan-1-ol (LQFM032)-induced
cardiovascular alterations and the participation of mAChR
mechanisms in regulating cardiovascular responsiveness in
normotensive and hypertensive rats treated with LQFM032.
Pharmacological tools and computational studies were
employed to unravel the molecular mechanism of action and
to investigate non-acetylation of this choline analog by acetyl
coenzyme A and choline acetyltransferase.
cally by reducing sympathetic signaling, is an avenue of
particular interest, as studies have demonstrated signifi-
cantly enhanced sympathetic activity in hypertensive pa-
tients and spontaneously hypertensive rats (SHRs) (Li
et al. 2013; Ye et al. 2013).
The autonomic nervous system controls heart rate,
inotropy, and blood pressure, and regulates the cardiovascular
system. Chronic autonomic sympathetic and parasympathetic
imbalance plays a crucial role in chronic kidney disease, hy-
pertension, renal disease progression, cardiovascular morbid-
ity, and mortality (Penne et al. 2009; Grassi et al. 2011).
Autonomic signals such as epinephrine, norepinephrine, and
acetylcholine contribute to normal physiological responses
within the cardiovascular system, and drive many of the ab-
normal pathophysiological responses (Roy et al. 2015). In
hypertensive rats, renal norepinephrine spillover (an index of
renal sympathetic nerve activity) is increased (Schlaich et al.
Material and methods
Drugs and treatments
Halothane (Cristália, Itapira, SP, Brazil), urethane (Sigma-
G
Aldrich, St. Louis, MO, USA), N -nitro-L-arginine methyl
ester (L-NAME; Sigma-Aldrich, St. Louis, MO, USA), indo-
methacin (Sigma-Aldrich, St. Louis, MO, USA), atropine sul-
phate (Cristália, Itapira, SP, Brazil), 1,1-dimethyl-4-
diphenylacetoxypiperidinium iodide (4-DAMP; Sigma-
Aldrich, St. Louis, MO, USA), phenylephrine (PHE; Sigma-
Aldrich, St. Louis, MO, USA) were used in this study. Saline
(0.3 mL/kg, 0.9% NaCl) was administered as vehicle in con-
trol animals. A 0.3-mL drug solution or vehicle per kilogram
body weight (0.3 mL/kg) was administered intravenously
(IV), while 5 μg of LQFM032 was administered by intra-
cerebroventricular (ICV) injection at a final volume of 5 μL.
All reagents were of analytical purity (≥ 95%).
2
004). The firing frequency of renal sympathetic nerve activ-
ity (RSNA) has been reported to be doubled in SHRs com-
pared to normotensive Wistar–Kyoto rats (Lundin et al. 1984).
Thus, data from human studies and animal models indicate
that the elevation of sympathetic nerve activity plays a causal
role in the hypertensive process.
Although many therapeutic strategies have targeted the ad-
renergic signaling induced by aberrant sympathetic activity,
studies have also suggested the potential benefit of targeting
cholinergic signaling. Choline analogs, including acetylcho-
line, are known to induce hypotensive effects, and several
pharmacological approaches have been used to increase the
responsiveness of blood vessels to acetylcholine-induced va-
sodilation (Tominaga et al. 1994; Hye Khan et al. 2014). A
previous report also demonstrated that pharmacological inhi-
bition of acetylcholinesterase activity led to the attenuation of
hypertension in SHRs (Lataro et al. 2015). The reduction of
heart rate and blood pressure by choline analogs and agonists
of muscarinic acetylcholine receptor (mAChR) suggests that
targeting muscarinic signaling may be beneficial for the treat-
ment of hypertension (Yalcin et al. 2005; Deng et al. 2018). A
better understanding of muscarinic signaling pathways and the
potential roles they play in regulating the heart and vasculature
may provide new therapeutic strategies for treating cardiovas-
cular disease (Harvey 2012).
Synthesis of LQFM032
The procedural details and steps for the synthesis of LQFM032
(molecular weight: 286.18 mg/mmol), as shown in Fig. 1, in-
volve reductive amination of 1-(phenyl)-1Hpyrazole-4-
carbaldehyde and 1-(2-hydroxyethyl)piperazine. This has been
described previously in detail (Brito et al. 2017).
Experimental animals
Adult male normotensive Wistar rats (NWRs) or spontaneous-
ly hypertensive rats (SHRs) with an average weight of 290 ±
25 g and age of 14 ± 2 weeks were provided by the Central

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 1 Synthesis of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032). HCl, hydrochloric acid; MeOH, methanol; Rx
Duff, Duff condition; HTMA, hexamethylenetetramine; CF CO H, trifluoroacetic acid; NaCNBH , sodium cyanoborohydride; ZnCl , zinc chloride
3 2 3 3
Animal House the of Federal University of Goiás. The exper-
imental protocol was approved by the ethics committee of the
Federal University of Goiás (protocol #172/09). Rats with
blood pressure above 150 mmHg were considered SHRs.
The animals were kept under controlled conditions (tempera-
ture 25 ± 1 °C, light/dark cycle of 12 h), and provided free
access to food and water. All procedures were carried out in
strict compliance with the Guiding Principles for Research
Involving Animals and Human Beings (Kilkenny et al. 2010).
used to record alterations in tension baseline. In order to eval-
uate the vascular effects of LQFM032, steady vessel tension
of isolated endothelium-denuded and endothelium-intact aor-
tic rings of NWRs and SHRs was induced by phenylephrine
(PHE) 1 μM prior to the cumulative addition of LQFM032
−12
−11
−10
−9
−8
−7
−6
−5
(10 , 10 , 10 , 10 , 10 , 10 , 10 , or 10 M) and
30 min post-treatment observation. In the experiment, eight
concentration of LQFM032 were added at 8-min intervals in a
cumulative manner. Each concentration was added in a 10-μL
volume (a 10× concentration) after having previously re-
moved 10 μL of the medium in order to maintain the medium
volume constant. LQFM032-induced relaxation in the aortic
rings was calculated as a percentage of the relaxation in re-
sponse to PHE. To test the possibility that the vascular func-
tion of LQFM032 involves NO and prostaglandin release, the
rat aortic rings were pre-incubated with 100 μM L-NAME or
indomethacin 100 μM for 1 h prior to the addition of 1 μM
PHE to induce contraction.
Surgery and intracerebroventricular (ICV)
administration
The skulls of anesthetized rats (with urethane 800 mg/kg i.p.)
were surgically exposed prior to implantation of a stainless-
steel guide cannula (0.6 mm o.d., 23G) 1 mm above the in-
jection site, using a stereotaxic apparatus (Stoelting, Wood
Dale, IL, USA). Stereotaxic coordinates selected for the im-
plantation of the cerebral cannula (1.0 mm posterior to the
bregma, 1.5 mm lateral, and 4.5 mm ventral) were based on
the Paxinos and Watson (1998) rat atlas. Cannulas were fixed
to the skulls with dental cement and metal screws. A tight-
fitting mandrel was kept inside the guide cannula to avoid its
occlusion. After a 7-day recovery period, the femoral artery
and vein were catheterized, and ICV injection was performed
using a 10-μL syringe (Hamilton, Reno, NV, USA).
LQFM032 5 μg was injected at a volume of 5 μL (1 μg/μL)
over a period of 1 min (Kawasaki et al. 1992; Peng et al.
Evaluation of cardiovascular alterations
and mechanism of LQFM032 activity
Cardiovascular effects of peripheral (IV) administration of
saline at 0.3 mL/kg (0.9% NaCl) or LQFM032 at 0.3, 0.6,
and 1.2 mg/kg on RSNA, heart rate (HR) and blood pressure
(
(
BP) were recorded as described in the previous study
Togashi et al. 1990). Baselines of these parameters were re-
corded for 30 min. In separate experiments, animals received
an IV injection of atropine 0.2 mg/kg, L-NAME (infused at
2
009). Incisions were sutured with surgical line following
the intramuscular injection of flunixin (0.02 mL/kg), an anal-
gesic drug at prophylactic dose.
3
μg/kg/min into the femoral vein with a pump; Insight
LTDA, Monte Alto, SP, Brazil), pirenzepine 0.2 mg/kg or 4-
DAMP 0.4 mg/kg prior to the administration of LQFM032 (at
an interval of 60 s) and observed for a duration of 240 s. The
abbreviation Ba.u.^, which means arbitrary units, was adopted
for representative tracings. In the present study, the RSNAwas
recorded and the signal was quantified by integrating the ab-
solute value into 1-s intervals. We used a percentage value of
the RSNA for analysis and graphics confection according to
previous studies (Shi et al. 2007; De Oliveira-Sales et al. 2010;
Pedrino et al. 2016; Pinto et al. 2016; Mourão et al. 2018).
Preparation of aortic rings and studies of vascular
reactivity to LQFM032
Rats were euthanized using a guillotine, and thoracic aortas
were isolated, removed, and cleaned of fat and adherent con-
nective tissues, as described previously (Fajemiroye et al.
2
014). An isometric transducer connected to a data acquisition
system (World Precision Instruments, Sarasota, FL, USA) was

Naunyn-Schmiedeberg's Arch Pharmacol
Bioinformatics analysis
analyzed with two-way ANOVA (Fig. 3b–d) and one-way
ANOVA (Fig. 3e–g) followed by Bonferroni post hoc test.
Crystallographic structures for the nitric oxide synthase
[
NOS1, PDB ID: 1lzx (Li et al. 2002)] and the murine
Effect of intravenous injection of LQFM032 on MAP,
HR, and RSNA of SHRs
muscarinic acetylcholine receptor 3 (mAchR3) complexed
with the inhibitor tiotropium [PDB ID: 4u14 (Thorsen
et al. 2014)] were obtained from the Protein Data Bank
Figure 3a shows representative tracings of the changes in pul-
satile arterial pressure (PAP), heart rate (HR), renal sympathet-
ic nerve activity (RSNA), and integrated RSNA (a.u.) induced
by vehicle (0.3 mL/kg) and LQFM032 (0.3, 0.6 and
(
Berman et al. 2000). We modeled the murine muscarinic
acetylcholine receptor 1 (mAchR1, UniProtKB P08482)
using its human form [PDB ID: 5cxv (Thal et al. 2016)]
as a template with Modeler (Šali and Blundell 1993).
LQFM032 structure was constructed with Avogadro
1.2 mg/kg) in SHRs. Within 30 s of LQFM032 administration,
there were significant changes in MAP, HR, and RSNA of
SHRs (Fig. 3b, c, and d, respectively). The bar chart shows
the peak effect in MAP (−10.0 ± 2.5, −6.0 ± 2.0, and −12.3 ±
(
Hanwell et al. 2012). The protonation of all receptors
was obtained with PROPKA (Dolinsky et al. 2004).
Molecular docking was performed with LQFM032 and
each protein using both DockThor (Custódio et al. 2014;
De Magalhães et al. 2014) and Vina (Trott and Olson
2.2 mmHg; Fig. 3e); HR (−4.6 ± 1.4, −1.7 ± 0.8, or −3.5 ±
.4 bpm; Fig. 3f), and RSNA (−9.8 ± 2.3, −12.7 ± 1.8, or
1
−
21.3 ± 2.9%; Fig. 3g) following intravenous injection of
2
010), using the redocking of co-crystallized ligands as
LQFM032 at 0.3, 0.6, and 1.2 mg/kg, respectively. While a
clear dose-dependent reduction in RSNA was observed in re-
sponse to treatment, HR was significantly reduced only in
animals that received the lowest dose (0.3 mg/kg). Data were
analyzed with two-way ANOVA (Fig. 3b–d) and one-way
ANOVA (Fig. 3e–g) followed by Bonferroni post hoc test.
control in order to validate the method. Intermolecular
interactions were obtained with PoseView (Stierand
et al. 2006), and images were produced with PyMOL
(
DeLano 2002) and Inkscape™.
Data analysis
All values are expressed as mean ± standard error of the mean
Intracerebroventricular (ICV) microinjection
of LQFM032
(
SEM). The average value of MAP, HR, or RSNA was mea-
sured every 30 s after the injection of LQFM032 or vehicle.
Multiple comparisons of groups were carried out through one-
way and two-way analysis of variance (ANOVA) prior to
Bonferroni post hoc test. All analyses were performed using
GraphPad Prism software (San Diego, CA, USA). A value of
p < 0.05 was considered to be significant.
To determine whether the effects of LQFM032 in NWRs and
SHRs were mediated within the central nervous system (CNS),
we next administered LQFM032 (1 μg/μL) directly to the brain
with ICV injection. Figure 4 shows the MAP (a), HR (b), and
RSNA (c) responses to ICV microinjection of LQFM032 (1 μg/
μL). In NWRs and SHRs, the LQFM032 elicited (i) a decrease in
the values of MAP (−10.7 ± 0.3 and −13.8 ± 0.2, respectively)
and RSNA (−11.3 ± 0.3 and −16.6 ± 0.4, respectively), as well
as (ii) a slight increase in HR (1.2 ± 0.09 and 1.8 ± 0.2), when
compared to the vehicle-treated group. Data were analyzed with
one-way ANOVA followed by Bonferroni post hoc test.
Results
Effect of intravenous injection of LQFM032 on MAP,
HR, and RSNA of NWRs
Fig. 2 Cardiovascular and sympathetic effects of LQFM032 acute„
administration in normotensive Wistar rats (NWRs). Representative
tracings showing the changes in pulsatile arterial pressure (PAP), heart
rate (HR), renal sympathetic nerve activity (RSNA), and integrated
RSNA (a.u.) induced by intravenous administration vehicle (0.3 mL/kg)
and LQFM032 (0.3, 0.6, and 1.2 mg/kg) in NWRs (panel a). Panels b, c,
and d show the temporal line graphs of mean arterial pressure (Δ%
MAP), Δ% HR, and Δ% RSNA after intravenous LQFM032 in
NWRs. Panels e, f, and g show the summary data for peak changes in
Δ MAP, Δ HR, and Δ RSNA caused by the dose-dependent effects of
LQFM032 (0.3, 0.6, and 1.2 mg/kg). Data are shown as mean ± SEM.
P < 0.05 [two-way ANOVA (panels b, c, and d) and one-way ANOVA
The line graph in Fig. 2 shows (a) representative tracings of
the changes in pulsatile arterial pressure (PAP), heart rate
(
HR), renal sympathetic nerve activity (RSNA), and integrat-
ed RSNA (a.u.) induced by vehicle (0.3 mL/kg) and
LQFM032 (0.3, 0.6 and 1.2 mg/kg) in NWRs. The temporal
changes in baseline values of (b) MAP, (c) HR, and (d) RSNA
were observed following increasing doses of LQFM032. The
peak effect in MAP (−4.8 ± 0.8, −8.5 ± 2.1, and −16.6 ±
2
1
.8 mmHg; Fig. 2e), HR (−2.0 ± 1.1, −3.6 ± 1.1, or −6.8 ±
.2 bpm; Fig. 2f), and RSNA (−11.1 ± 2.2, −14.1 ± 2.1, or
(
panels e, f and g) followed by Bonferroni post hoc test]. *Different from
−
22.7 ± 2.8%; Fig. 2g) were reduced in a dose-dependent
†
the vehicle; different from the 0.3-mg/kg and 0.6-mg/kg doses of
LQFM032
manner (0.3, 0.6, and 1.2 mg/kg, respectively). Data were

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Naunyn-Schmiedeberg's Arch Pharmacol
Vascular reactivity to LQFM032
LQFM032 docking with the programs DockThor and
Vina. While redocking of NOS and the ligand N-omega-
hydroxy-L-arginine using both docking programs was suc-
cessful, no interactions with LQFM032 were detected, sug-
gesting that it does not interact with NOS. Regarding
mAchR1 and mAchR3 (Fig. 9), LQFM032 was shown to
interact with the key asparagine for inhibition by the in-
verse agonist tiotropium (Asn382). Furthermore,
LQFM032 also interacted with all three tyrosine residues
that form the lid that covers the orthosteric binding site
(Tyr106-Tyr381-Tyr404 and Tyr148-Tyr506-Tyr529 for
mAchR1 and mAchR3, respectively) (Kruse et al. 2012).
Between the two murine acetylcholine receptors,
LQFM032 made contact with key residues in mAchR1
with both docking programs, indicating that it potentially
interacts with the same residues as the inhibitor tiotropium.
The line graph in Fig. 5 depicts concentration–response
curves of cumulative addition of vehicle and LQFM032
to endothelium-intact and endothelium-denuded aortic
rings. LQFM032 caused a concentration-dependent relax-
ation of (a) NWR-isolated aortic rings with endothelium
and (b) SHR-isolated aortic rings with endothelium when
compared with their respective group treated with vehicle
(
control). The maximum relaxant effect of LQFM032 on
PHE-induced contraction was 50.3% ± 6.3% (Fig. 5a) and
5.6% ± 8.4% (Fig. 5b) for NWR- and SHR-endothelium-
intact aortic rings, respectively. Unlike indomethacin
6
1
1
00 μM, the aortic rings pre-incubated with L-NAME
00 μM blocked relaxation induced by the cumulative ad-
−
12
−5
dition of LQFM032 (10 to 10 M) at the plateau phase
of PHE-induced contraction. No significant reduction was
observed in vessel tension of NWR- or SHR-isolated aortic
rings without endothelium. Data were analyzed with two-
way ANOVA followed by the Bonferroni post hoc test.
Discussion
Chemical synthesis and biological screening of compounds
are important strategies for new drug discovery (Fajemiroye
et al. 2017). Recently, a novel choline analog was developed
as a hybrid of piperazine and choline scaffold [Brito et al,
Effect of muscarinic receptors antagonists
on cardiovascular activity of LQFM032
To verify that the effects of LQFM032 1.2 mg/kg were medi-
ated by muscarinic receptors (the more prevalent acetylcho-
line receptors in autonomic effector organs), LQFM032 and
the prototypical muscarinic antagonist atropine were adminis-
tered intravenously. The effect of LQFM032 on (a) MAP, (b)
HR, and (c) RSNA was significantly attenuated by atropine
pretreatment in NWRs (Fig. 6a–c) and SHRs (Fig. 7a–c). One
of the vasomodulating pathways downstream of muscarinic
acetylcholine receptors is nitric oxide synthase (NOS). Thus,
we also assessed whether pretreatment with L-NAME, an in-
hibitor of NOS, could also reduce the effects of LQFM032.
Co-administration of L-NAME with LQFM032 also signifi-
cantly attenuated the effects of this compound on MAP, HR,
and RSNA in NWRs (Fig. 6a–c) and SHRs (Fig. 7a–c).
Because atropine is a nonselective mAChR antagonist, we
assessed the effects of an M1-specific antagonist, pirenzepine,
and an M3-specific antagonist, 4-DAMP. Co-administration
with either the M1 or M3 antagonists significantly attenuated
the hypotensive effects of LQFM032 in SHRs (Fig. 8a–c),
suggesting nonselective agonism of mAChR by LQFM032.
2019]. This compound is of a particular interest, as the six-
membered nitrogen containing a heterocycle is of great signif-
icance for cardioprotection (Rathi et al. 2016). Unlike
LQFM008—one of the parent compounds—the presence of
choline substituents with a piperazine ring greatly enhanced
solubility and increased the possibility of cholinergic-
mediated cardiovascular effects of LQFM032. According to
Rathi et al. (2016), a significant increase in research covering
the activity of the piperazine ring suggests a successful emer-
gence of the pharmacophore. In the present study,
LQFM032—a choline analog—was synthesized in order to
evaluate its effect on cardiovascular parameters.
In order to achieve this objective, the cardiovascular effects
of LQFM032 were determined with respect to the in vivo
physiological readouts in non-hypertensive and
Fig. 3 Cardiovascular and sympathetic effects of LQFM032 acute„
administration in spontaneously hypertensive rats (SHRs).
Representative tracings showing the changes in pulsatile arterial
pressure (PAP), heart rate (HR), renal sympathetic nerve activity
(
RSNA), and integrated RSNA (a.u.) induced by intravenous
administration of vehicle (0.3 mL/kg) and LQFM032 (0.3, 0.6, and
.2 mg/kg) in SHRs (panel a). Panels b, c, and d show the temporal
Data on bioinformatics studies
1
line graphs of mean arterial pressure (Δ% MAP), Δ% HR, and Δ%
RSNA after intravenous LQFM032 in NWRs. Summary data of peak
changes in Δ MAP, Δ HR, and Δ RSNA caused by a dose-dependent
effect of LQFM032 (0.3, 0.6 and 1.2 mg/kg) are shown in panels e, f and
g, respectively. Data are shown as mean ± SEM. P < 0.05 [two-way
ANOVA (panels b, c, and d) and one-way ANOVA (panels e, f, and g)
followed by Bonferroni post hoc test]. *Different from the vehicle;
Having established that LQFM032 modulated muscarinic
and/or downstream nitrous oxide signaling in the intact
cardiovascular system, we next assessed the possible direct
interaction between LQFM032 and NOS or the M1 and M3
cholinergic receptors using molecular docking studies. For
each, a co-crystallized ligand was used as reference for
†
different from the 0.3-mg/kg and 0.6-mg/kg doses of LQFM032

Naunyn-Schmiedeberg's Arch Pharmacol

Naunyn-Schmiedeberg's Arch Pharmacol
by promoting sodium reabsorption and enhancing
preglomerular renal vascular resistance, which in turn
decrease the glomerular filtration rate (DiBona and
Kopp 1997). Thus, the LQFM032-mediated reduction in
RSNA may be beneficial for renal function in hyperten-
sive patients.
Similar to some centrally acting antihypertensive
drugs such as clonidine and moxonidine with nitric oxide
mechanisms (Dobrucki et al. 2001; Moreira et al. 2006),
ICV administration of LQFM032 elicited significant an-
tihypertensive activity in SHRs. Considering this com-
pound as an agonist of muscarinic receptors, our result
seems to be inconsistent with the established role of cen-
trally mediated effects of cholinergic neurons. Blood
pressure regulation has been associated with cholinergic
neuronal activity in numerous brain regions (Buccafusco
1
996). Cholinergic agonists, antagonists, and acetylcho-
linesterase inhibitors enhance or attenuate centrally me-
diated pressor response (Sundaram et al. 1988;
Lazartigues et al. 1999; Kubo 1998).
The lateral parabrachial nucleus comprises cholinergic
neurons that play an important role in the pathogenesis of
Fig. 4 Effects of intracerebroventricular (ICV) administration of
LQFM032 in normotensive Wistar rats (NWRs) and spontaneously hy-
pertensive rats (SHRs). The bar graph shows the effect of ICV microin-
jection of vehicle or LQFM032 (5 μg was injected at a volume of 5 μL
over a period of 1 min) on the (a) mean arterial pressure (Δ MAP), (b)
heart rate (Δ HR), and (c) renal sympathetic nerve activity (RSNA) of
normotensive and hypertensive rats. Data are shown as mean ± SEM. P <
0
.05 (one-way ANOVA followed by Bonferroni post hoc test). *Different
#
†
from the vehicle NWRs; different from the vehicle SHRs; different
from the LQFM032 in NWRs
Fig. 5 Vascular effects of LQFM032. Vasorelaxant effect of LQFM032
−12
−5
(
10 to 10 M) on phenylephrine (PHE 1 μM)-induced pre-contraction
spontaneously hypertensive rats (SHRs). The SHR model is
an important experimental protocol being used for the screen-
ing of antihypertensive drugs (Leong et al. 2015). In summary,
LQFM032 elicited temporal reduction of mean arterial pres-
sure (MAP), heart rate (HR), and renal sympathetic nerve
activity (RSNA) of normotensive rats (NWRs) and SHRs.
An increase in RSNA is known to impair sodium excretion
of endothelium-denuded (E−) or endothelium-intact (E+) aortic rings in
normotensive Wistar rats (NWRs, a) and spontaneously hypertensive rats
−12
−5
(SHRs, b). The vascular function of LQFM032 (10 to 10 M) in-
volves NO and prostaglandin release, and the rat aortic rings were pre-
incubated with 100 μM L-NAME or indomethacin 100 μM for 1 h prior
to the addition of 1 μM PHE to induce contraction (c). Data are shown as
mean ± SEM. P < 0.05 (two-way ANOVA followed by Bonferroni post
†
hoc test). *Different from the vehicle; different from the LQFM032 E−

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 6 Cardiovascular and sympathetic responses evoked by atropine and
L-NAME pretreatments on LQFM032 effects in normotensive Wistar rats
Fig. 8 Pharmacological blockade of the effect of LQFM032 in SHRs
with pirenzepine and 4-DAMP. The effects of intravenous pretreatment
with pirenzepine (PIR 0.2 mg/kg) or 4-DAMP (0.4 mg/kg) on LQFM032
(NWRs). Effects of intravenous pretreatment with atropine (0.2 mg/kg) or
L-NAME (3 μg/kg/min) on LQFM032 1.2 mg/kg-induced changes (Δ) in
MAP (a), HR (b), and RSNA (c). Data are expressed as mean ± SEM
1
.2 mg/kg-induced changes in (a) MAP, (b) HR, and (c) RSNA. Data are
(
*
one-way ANOVA followed by Bonferroni post hoc test). P < 0.05.
Different from the vehicle; different from the LQFM032
expressed as mean ± SEM (one-way ANOVA followed by Bonferroni
#
post hoc test). *p < 0.05 compared to the vehicle-treated group;
#
p < 0.05 compared to the LQFM032-treated group
hypertension (Sundaram et al. 1988). The muscarinic re-
ceptors on the rostral ventrolateral medulla (RVLM; an
essential source of efferent sympathetic activity) are poten-
tial pharmacological targets in a hypertensive state. In ad-
dition, the inhibition of acetylcholinesterase by physostig-
mine and release of acetylcholine by 3,4-diaminopyridine
increased the firing rate of RVLM neurons (Kubo et al.
Hypothetically, the reversible or irreversible competition
for these cellular processes could interfere in the synthesis
and release of endogenous acetylcholine. A decrease in
the level of acetylcholine could trigger positive feedback
to enhance synthesis and release as well as an increase in
the hydrolysis of this neurotransmitter. The previous
unpublished data which showed unaltered activity of
acetylcholinesterase following its incubation with
LQFM032 suggests noninterference of this compound in
the rate of acetylcholine hydrolysis. Together, a disruption
in the synthesis and release of acetylcholine without an
inhibition of its hydrolysis could lead to overall decrease
in its synaptic level in the brain. Additionally, Kubo
1
997). Previous pharmacological and biochemical studies
of the RVLM revealed an enhancement in the release of
acetylcholine and an increase in experimental hypertension
(
Kubo et al. 1997; Kubo 1998).
Being a choline analog, LQFM032 could compete as a
substrate for acetyl coenzyme A and choline acetyltrans-
ferase (i.e. enzymatic acetylating processes), choline high-
affinity transporter, or vesicular acetylcholine transporter.
(
1998) demonstrated the involvement of the hypothalamic
defense area (an area believed to be involved in the hy-
pertension induced by chronic stress) in the release of
acetylcholine in the RVLM and the existence of direct
projections from the hypothalamic structures to the lateral
parabrachial nucleus. This study seems to be consistent
with the demonstration of the anti-anxiety properties of
this cholinergic analog (Brito et al. 2017), and reinforces
the possible link between stress pathway and antihyper-
tensive properties of LQFM032. In the future, additional
studies of specific brain regions will provide a more com-
prehensive understanding of the central mechanisms of
LQFM032-induced cardiovascular alterations and the role
of endogenous acetylcholine.
Despite the central effects of LQFM032, its oral ad-
ministration makes the participation of peripheral
mAChRs in the cardiovascular outcome plausible. An
isolated organ in a physiological solution remains an im-
portant research tool for testing localized or peripheral
effects of drugs. Thus, aortic rings were isolated from
Fig. 7 Cardiovascular and sympathetic responses evoked by atropine and
L-NAME pretreatments on LQFM032 effects in spontaneously hyperten-
sive rats (SHRs). Effects of intravenous pretreatment with atropine
(0.2 mg/kg) or L-NAME (3 μg/kg/min) on LQFM032 1.2 mg/kg-induced
changes (Δ) in MAP (a), HR (b), and RSNA (c). Data are expressed as
mean ± SEM (one-way ANOVA followed by Bonferroni post hoc test).
#
P < 0.05. *Different from the vehicle; different from the LQFM032

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 9 Interaction of LQFM032 and the inhibitor tiotropium with
mAchR1 and mAchR3. a Top: Overview of mAchR1, zooming on
LQFM032 docked by DockThor (purple) and Vina (green) compared to
tiotropium (black). Sticks represent the lid-forming Tyr residues and the
interacting Asn382. Bottom: Predicted interactions between mAchR1 and
LQFM032 according to DockThor (green), Vina (purple), or both (blue).
b Top: Overview of mAchR3 and close-up of LQFM032 docked by
DockThor (purple) and Vina (green) compared to tiotropium (black).
The sticks represent the lid-forming Tyr residues and the interacting
Asn. Bottom: Predicted interactions between mAchR3 and LQFM032
according to DockThor (green), Vina (purple), or both (blue). For both
receptors, interactions with tiotropium are shown in bold, and the lid-
forming Tyr residues and the interacting Asn are shown in italics
normotensive and spontaneously hypertensive rats to test
vascular response to LQFM032. Unlike isolated vessels
without endothelium, this choline analog reduced vascu-
lar tone and elicited a relaxation of isolated aortic rings
with endothelium from normotensive and spontaneously
hypertensive rats. This result suggests the release and
involvement of vasoactives such as nitric oxide (NO),
prostaglandins, and endothelium-derived hyperpolariza-
tion factors. Unlike indomethacin 100 μM (nonsteroid
anti-inflammatory drug/prostaglandin inhibitor), when
the rat aortic rings were pre-incubated with L-NAME
hypothesis and suggest the specific involvement of
muscarinic-nitric oxide pathways.
The attenuation of the LQFM032-induced cardiovascu-
lar activity could be associated with central and/or periph-
eral blockade of muscarinic receptors. According to
Pediani et al. (2016), pirenzepine does not effectively cross
the blood–brain barrier effectively, and as such does not
inhibit M1-mediated cholinergic function in the CNS. In
this manner, the blockade of the effect of intravenously
administered LQFM032 by pirenzepine supports the in-
volvement of peripheral components.
1
00 μM prior to the PHE-induced contraction, cumula-
Although pirenzepine and 4-DAMP have been characterized
pharmacologically by some authors as selective antagonists of
M1 and M3 receptors, respectively (Höglund and Baghdoyan
1997; Pediani et al. 2016; Jaiswal et al. 1989; Borella et al.
2008), the administration of a single dose of pirenzepine
(0.2 mg/kg) or 4-DAMP (0.4 mg/kg) in the present study does
not necessarily confirm a selective blockade of M1 and M3 in the
attenuation of the cardiovascular effects of LQFM032. The close
values of K and K of these antagonists on muscarinic receptors
subtypes seem to weaken the possibility of their selectivity with a
single dose in vivo. The previous study which revealed lack of
correlation in the affinities and abilities of the 4-DAMP to dis-
criminate muscarinic receptor subtypes (Waelbroeck et al. 1992)
supports the need for future evaluation of specific roles of all
muscarinic receptor subtypes in the activity of LQFM032.
The compounds mimicking acetylcholine actions are dila-
tors of vascular beds and are often used as an investigative tool
for the evaluation of endothelial function (Vita et al. 1990;
tive addition of LQFM032 did not elicit relaxation.
Hence, this result supports the participation of NO in
the vascular function of this compound. According to
Stankevicius et al. (2002), these endogenous vasoactives
play important roles in vascular relaxation.
Moreover, the use of pharmacological tools such as
antagonists and computational studies were explored to
unravel the research hypothesis and the potential mecha-
nism of the cardiovascular activity of this choline analog.
The hypothesis of the muscarinic action of this com-
pound and its cardiovascular properties was postulated;
however, it was not tested until this current study. The
attenuation of cardiovascular effects of LQFM032 by L-
NAME (a nonselective NOS inhibitor), atropine (musca-
rinic receptor antagonist), pirenzepine (M1 receptor an-
tagonist), and 4-DAMP (M3 receptor antagonist) pre-
treatments seems to be consistent with our cholinergic
D
B

Naunyn-Schmiedeberg's Arch Pharmacol
Faraci and Sigmund 1999; Klonizakis et al. 2009). The endo-
thelial muscarinic receptors participate in triggering the re-
lease of the vasorelaxing agents, such as NO (Leung et al.
Author’s contribution JOF, NMA and GRP: synthesis and chemical
characterization of LQFM032. BV and HV: in silico assays. JOF, CHX,
CHC, ONS, OLF, IOS: devising the research method/methodology used.
AAM, EAC, CHX, CHC, GRP: conducting research, statistical analysis
and calculations. RM, FSC, LML: devising the concept and assumptions
of the article. All authors wrote the manuscript. All authors read and
approved the manuscript.
2
006). Since L-NAME blocked the effect of LQFM032, it
was assumed that NOS plays an important role in the current
cardiovascular effects.
The NOS has a sympathoinhibitory effect under physi-
ological and disease conditions by acting on the
paraventricular nucleus, the nucleus of the solitary tract,
the rostral ventrolateral medulla, the carotid body, and
nerves in the kidney (Wang and Golledge 2013).
According to Sander et al. (1995), hypertensive responses
to L-NAME in rats with versus without sympathectomy
could be related to inhibition of nitric oxide synthesis.
Since renal nerves transmit signals from the periphery
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All procedures were performed in accordance with the
National Institutes of Health Guidelines for the Care and Use of
Laboratory Animals and were approved by the Ethics Committee of the
Federal University of Goiás (protocol #172/09) as compliant with Brazil
law.
(
kidneys) to the CNS and vice versa (Nishi et al. 2015),
the blockade of the inhibitory effects of LQFM032 on
RSNA by L-NAME pretreatment suggests crosstalk be-
tween the sympathetic nervous system and nitric oxide
signaling.
References
Since NOS catalyzes the synthesis of NO from L-argi-
nine (Ricciardolo et al. 2004), the hypotensive effects of
LQFM032 suggests an increase in (i) the activity of this
enzyme, and (ii) the synthesis and (iii) release of NO. The
overall effect could trigger a reduction in vascular resis-
tance in normotensive Wistar rats. However, these effects
could involve direct activation of endothelial NOS or indi-
rect muscarinic action through the activation of the up-
stream M1 and M3 receptors. Because interactions be-
tween NOS and LQFM032 were not established with our
molecular docking approaches while LQFM032 interacted
with both muscarinic receptors (M1 and M3), our findings
support the aforementioned cholinergic hypothesis.
Berman HM, Westbrook J, Feng Z et al (2000) The Protein Data Bank.
Nucleic Acids Res 28:235–242
Borella TL, De Luca LA, Colombari DSA, Menani JV (2008) Central
muscarinic receptor subtypes involved in pilocarpine-induced sali-
vation, hypertension and water intake. Br J Pharmacol. https://doi.
org/10.1038/bjp.2008.355
Brito AF, Fajemiroye JO, Neri HFS et al (2017) Anxiolytic-like effect of
2
-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol
is mediated through the benzodiazepine and nicotinic pathways.
Chem Biol Drug Des 90:432–442. https://doi.org/10.1111/cbdd.
1
2961
Brito AF, Moreira LKS, Menegatti R, Costa EA (2019) Piperazine deriv-
atives with central pharmacological activity used as therapeutic
tools. Fundam Clin Pharmacol 33:13–24. https://doi.org/10.1111/
fcp.12408
Buccafusco JJ (1996) The role of central cholinergic neurons in the reg-
ulation of blood pressure and in experimental hypertension.
Pharmacol Rev 48:179–211
Custódio FL, Barbosa HJC, Dardenne LE (2014) A multiple minima
genetic algorithm for protein structure prediction. Appl Soft
Comput J 15:88–99. https://doi.org/10.1016/j.asoc.2013.10.029
De Magalhães CS, Almeida DM, Barbosa HJC, Dardenne LE (2014) A
dynamic niching genetic algorithm strategy for docking highly flex-
ible ligands. Inf Sci (NY) 289:206–224. https://doi.org/10.1016/j.
ins.2014.08.002
Conclusions
In summary, experimental data suggest muscarinic-mediated
sympathoinhibitory, hypotensive, and antihypertensive effects
of LQFM032. Future research will be focused on extensive
investigation of the pharmacokinetic and toxicological pro-
files of this new choline analog. Additional modification of
LQFM032 will be carried out in the future to study the struc-
tural activity relationship and to develop analogs that bind a
specific muscarinic receptor subtype.
De Oliveira-Sales EB, Nishi EE, Boim MA et al (2010) Upregulation of
AT1R and iNOS in the rostral ventrolateral medulla (RVLM) is
essential for the sympathetic hyperactivity and hypertension in the
2
K-1C Wistar rat model. Am J Hypertens 23:708–715. https://doi.
org/10.1038/ajh.2010.64
DeLano WL (2002) The PyMOL Molecular Graphics System.
Schrödinger LLC wwwpymolorg Version 1. http://www.pymol.org
Deng AY, deBlois D, Laporte SA et al (2018) Novel pathogenesis of
hypertension and diastolic dysfunction caused by M3R (muscarinic
cholinergic 3 receptor) signaling. Hypertension 72:755–764. https://
doi.org/10.1161/HYPERTENSIONAHA.118.11385
Acknowledgements This work was supported by CAPES, CNPq,
FAPEG and FUNDECT. Osmar N. Silva holds a postdoctoral scholarship
from the National Council of Technological and Scientific Development
(
CNPq) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência
e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) – Brazil
300583/2016-8]. We thank Marcelo Rodrigues Martins (PhD) of the
[
DiBona GF, Kopp UC (1997) Neural control of renal function. Physiol
Rev 77:75–197. https://doi.org/10.1002/cphy.c100043
veterinary pharmacy section Universidade Federal de Goiás for providing
anesthetics freely.

Naunyn-Schmiedeberg's Arch Pharmacol
Dobrucki LW, Cabrera CL, Bohr DF, Malinski T (2001) Central hypo-
tensive action of clonidine requires nitric oxide. Circulation 104:
Leong X-F, Ng C-Y, Jaarin K (2015) Animal models in cardiovascular
research: hypertension and atherosclerosis. Biomed Res Int 2015:1–
11. https://doi.org/10.1155/2015/528757
1
884–1886. https://doi.org/10.1161/hc4101.098281
Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA (2004) PDB2PQR:
an automated pipeline for the setup of Poisson-Boltzmann electro-
statics calculations. Nucleic Acids Res 32:W665–W667. https://doi.
org/10.1093/nar/gkh381
Fajemiroye JO, Amaral NO, da Silva EF et al (2014) Hypotensive and
antihypertensive potential of 4-[(1-phenyl-1H-pyrazol-4-yl) meth-
yl]1-piperazine carboxylic acid ethyl ester: a piperazine derivative.
Life Sci 112:90–96. https://doi.org/10.1016/j.lfs.2014.07.025
Fajemiroye JO, Prabhakar PR, da Cunha LC et al (2017) 22-
azidosalvinorin a exhibits antidepressant-like effect in mice. Eur J
Pharmacol 800:96–106. https://doi.org/10.1016/j.ejphar.2017.02.
Leung HS, Leung FP, Yao X et al (2006) Endothelial mediators of the
acetylcholine-induced relaxation of the rat femoral artery. Vasc
Pharmacol 44:299–308. https://doi.org/10.1016/j.vph.2006.01.010
Li HY, Shimizu H, Flinspach M et al (2002) The novel binding mode of
N-alkyl-N ’-hydroxyguanidine to neuronal nitric oxide synthase
provides mechanistic insights into NO biosynthesis. Biochemistry
41:13868–13875. https://doi.org/10.1021/bi020417c
Li P, Sun HJ, Cui BP et al (2013) Angiotensin-(1-7) in the rostral ventro-
lateral medulla modulates enhanced cardiac sympathetic afferent
reflex and sympathetic activation in renovascular hypertensive rats.
Hypertension 61:820–827. https://doi.org/10.1161/
HYPERTENSIONAHA.111.00191
0
31
Faraci FM, Sigmund CD (1999) Vascular biology in genetically altered
mice: smaller vessels, bigger insight. Circ Res 85:1214–1225
Grassi G, Quarti-Trevano F, Seravalle G et al (2011) Early sympathetic
activation in the initial clinical stages of chronic renal failure.
Hypertension 57:846–851. https://doi.org/10.1161/
HYPERTENSIONAHA.110.164780
Hanwell MD, Curtis DE, Lonie DC et al (2012) Avogadro: an advanced
semantic chemical editor, visualization, and analysis platform. J
Cheminform 4:17. https://doi.org/10.1186/1758-2946-4-17
Harvey RD (2012) Muscarinic receptor agonists and antagonists: effects
on cardiovascular function. Handb Exp Pharmacol 208:299–316
Höglund AU, Baghdoyan HA (1997) M2, M3 and M4, but not M1,
muscarinic receptor subtypes are present in rat spinal cord. J
Pharmacol Exp Ther 281:470–477
Hye Khan MA, Pavlov TS, Christain SVet al (2014) Epoxyeicosatrienoic
acid analogue lowers blood pressure through vasodilation and sodi-
um channel inhibition. Clin Sci (Lond) 127:463–474. https://doi.
org/10.1042/CS20130479
Jaiswal N, Lambrecht G, Mutschler E, Malik KU (1989) Effect of M2
muscarinic receptor antagonist 4-DAMP, on prostaglandin synthesis
and mechanical function in the isolated rabbit heart. Gen Pharmacol
Lundin S, Rickstein S, Thoren P (1984) Renal sympathetic activity in
spontaneously hypertensive rats and normotensive controls, as stud-
ied by three different methods. Acta Physiol Scand 120:265–272.
https://doi.org/10.1111/j.1748-1716.1984.tb00133.x
Magoon R, Choudhury A, Malik Vet al (2017) Pharmacological update:
New drugs in cardiac practice: A critical appraisal. Ann Card
Anaesth 20:S49–S56. https://doi.org/10.4103/0971-9784.197798
Moreira TS, Takakura AC, Sato MA et al (2006) Antihypertensive re-
sponses elicited by central moxonidine in rats: possible role of nitric
oxide. J Cardiovasc Pharmacol 47:780–787. https://doi.org/10.
1
097/01.fjc.0000211794.68152.04
Mourão AA, de Mello ABS, Dos Santos Moreira MC et al (2018) Median
preoptic nucleus excitatory neurotransmitters in the maintenance of
hypertensive state. Brain Res Bull 142:207–215. https://doi.org/10.
1
016/j.brainresbull.2018.06.011
Nishi EE, Bergamaschi CT, Campos RR (2015) The crosstalk between
the kidney and the central nervous system: the role of renal nerves in
blood pressure regulation. Exp Physiol 100:479–484. https://doi.
org/10.1113/expphysiol.2014.079889
Paxinos G, Watson C (1998) The Rat Brain in Stereotaxic Coordinates.
Academic Press, San Diego
Pediani JD, Ward RJ, Godin AG et al (2016) Dynamic regulation of
quaternary organization of the m1 muscarinic receptor by subtype-
selective antagonist drugs. J Biol Chem. https://doi.org/10.1074/jbc.
M115.712562
Pedrino GR, Mourão AA, Moreira MCS et al (2016) Do the carotid body
chemoreceptors mediate cardiovascular and sympathetic adjust-
ments induced by sodium overload in rats? Life Sci 153:9–16.
https://doi.org/10.1016/j.lfs.2016.03.045
Peng J, Wang Y-K, Wang L-G et al (2009) Sympathoinhibitory mecha-
nism of moxonidine: role of the inducible nitric oxide synthase in the
rostral ventrolateral medulla. Cardiovasc Res 84:283–291. https://
doi.org/10.1093/cvr/cvp202
Penne EL, Neumann J, Klein IH et al (2009) Sympathetic hyperactivity
and clinical outcome in chronic kidney disease patients during stan-
dard treatment. J Nephrol 22:208–215
Pinto IS, Mourão AA, da Silva EF et al (2016) Blockade of rostral ven-
trolateral medulla (RVLM) Bombesin receptor type 1 decreases
blood pressure and sympathetic activity in anesthetized spontane-
ously hypertensive rats. Front Physiol 7:205. https://doi.org/10.
2
0:497–502
Kawasaki H, Nakamura S, Takasaki K (1992) Central alpha 2-
adrenoceptor-mediated pressor response to clonidine in conscious,
spontaneously hypertensive rats. Jpn J Pharmacol 59:321–331
Kilkenny C, Browne WJ, Cuthill IC et al (2010) The ARRIVE guidelines
animal research : reporting in vivo experiments. J Pharmacol
Pharmacother 1:94–99. https://doi.org/10.4103/0976-500X.72351
Klonizakis M, Tew G, Michaels J, Saxton J (2009) Impaired microvas-
cular endothelial function is restored by acute lower-limb exercise in
post-surgical varicose vein patients. Microvasc Res 77:158–162.
https://doi.org/10.1016/j.mvr.2008.09.009
Kruse AC, Hu J, Pan AC et al (2012) Structure and dynamics of the M3
muscarinic acetylcholine receptor. Nature 482:552–556. https://doi.
org/10.1038/nature10867
Kubo T (1998) Cholinergic mechanism and blood pressure regulation in
the central nervous system. Brain Res Bull 46:475–481
Kubo T, Taguchi K, Sawai N et al (1997) Cholinergic mechanisms re-
sponsible for blood pressure regulation on Sympathoexcitatory neu-
rons in the rostral ventrolateral medulla of the rat. Brain Res Bull 42:
3
389/fphys.2016.00205
1
99–204. https://doi.org/10.1016/S0361-9230(96)00256-0
Rathi AK, Syed R, Shin HS, Patel RV (2016) Piperazine derivatives for
therapeutic use: a patent review (2010-present). Expert Opin Ther
Pat 26:777–797
Ricciardolo FLM, Sterk PJ, Gaston B, Folkerts G (2004) Nitric oxide in
health and disease of the respiratory system. Physiol Rev 84:731–
Lataro RM, Silva CAA, Tefé-Silva C et al (2015) Acetylcholinesterase
inhibition attenuates the development of hypertension and inflam-
mation in spontaneously hypertensive rats. Am J Hypertens 28:
1
201–1208. https://doi.org/10.1093/ajh/hpv017
Lazartigues E, Brefel-Courbon C, Tran MA et al (1999) Spontaneously
hypertensive rats cholinergic hyper-responsiveness: central and pe-
ripheral pharmacological mechanisms. Br J Pharmacol 127:1657–
7
65. https://doi.org/10.1152/physrev.00034.2003
Roy A, Guatimosim S, Prado VF et al (2015) Cholinergic activity as a
new target in diseases of the heart. Mol Med 20:527–537. https://
doi.org/10.2119/molmed.2014.00125
1
665. https://doi.org/10.1038/sj.bjp.0702678

Naunyn-Schmiedeberg's Arch Pharmacol
Šali A, Blundell TL (1993) Comparative protein modelling by satisfac-
tion of spatial restraints. J Mol Biol 234:779–815. https://doi.org/10.
Togashi H, Yoshioka M, Tochihara M et al (1990) Differential effects of
hemorrhage on adrenal and renal nerve activity in anesthetized rats.
Am J Physiol Circ Physiol 259:H1134–H1141. https://doi.org/10.
1152/ajpheart.1990.259.4.H1134
Tominaga M, Fujii K, Abe I et al (1994) Hypertension and ageing impair
acetylcholine-induced vasodilation in rats. J Hypertens 12:259–268
Trott O, Olson A (2010) AutoDock Vina: improving the speed and accu-
racy of docking with a new scoring function, efficient optimization
and multithreading. J Comput Chem 31:455–461. https://doi.org/10.
1002/jcc.21334.AutoDock
1
006/jmbi.1993.1626
Sander M, Hansen PG, Victor RG (1995) Sympathetically mediated hy-
pertension caused by chronic inhibition of nitric oxide.
Hypertension 26:691–695
Schlaich MP, Lambert E, Kaye DM et al (2004) Sympathetic augmenta-
tion in hypertension: role of nerve firing, norepinephrine reuptake,
and angiotensin neuromodulation. Hypertension 43:169–175.
https://doi.org/10.1161/01.HYP.0000103160.35395.9E
Shi P, Stocker SD, Toney GM (2007) Organum vasculosum laminae
terminalis contributes to increased sympathetic nerve activity induced
by central hyperosmolality. Am J Physiol Integr Comp Physiol 293:
R2279–R2289. https://doi.org/10.1152/ajpregu.00160.2007
Stankevicius E, Martinez AC, Mulvany MJ, Simonsen U (2002) Blunted
acetylcholine relaxation and nitric oxide release in arteries from
renal hypertensive rats. J Hypertens 20:1571–1579
Stern CS, Lebowitz J (2010) Latest drug developments in the field of
cardiovascular disease. Int J Angiol 19:e100–e105
Stierand K, Maaß PC, Rarey M (2006) Molecular complexes at a glance:
automated generation of two-dimensional complex diagrams.
Bioinformatics 22:1710–1716. https://doi.org/10.1093/
bioinformatics/btl150
Vita JA, Treasure CB, Nabel EG et al (1990) Coronary vasomotor re-
sponse to acetylcholine relates to risk factors for coronary artery
disease. Circulation 81:491–497. https://doi.org/10.1161/01.CIR.
81.2.491
Waelbroeck M, Camus J, Tastenoy M, Christophe J (1992) Binding prop-
erties of nine 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP)
analogues to M1, M2, M3 and putative M4 muscarinic receptor
subtypes. Br J Pharmacol 105:97–102
Wang Y, Golledge J (2013) Neuronal nitric oxide synthase and sympa-
thetic nerve activity in neurovascular and metabolic systems. Curr
Neurovasc Res 10:81–89
Yalcin M, Cavun S, Yilmaz MS, Savci V (2005) The involvement of
central cholinergic system in the pressor effect of
intracerebroventricularly injected U-46619, a thromboxane A2 ana-
log, in conscious normotensive rats. Naunyn Schmiedeberg's Arch
Pharmacol 372:31–40. https://doi.org/10.1007/s00210-005-1087-x
Ye ZY, Li DP, Pan HL (2013) Regulation of hypothalamic presympathetic
neurons and sympathetic outflow by group ii metabotropic gluta-
mate receptors in spontaneously hypertensive rats. Hypertension 62:
Sundaram K, Krieger AJ, Sapru H (1988) M2 muscarinic receptors me-
diate pressor responses to cholinergic agonists in the ventrolateral
medullary pressor area. Brain Res. https://doi.org/10.1016/0006-
8
993(88)91032-3
Thal DM, Sun B, Feng D et al (2016) Crystal structures of the M1 and M4
muscarinic acetylcholine receptors. Nature 531:335–340. https://
doi.org/10.1038/nature17188
Thorsen TS, Matt R, Weis WI, Kobilka BK (2014) Modified T4 lyso-
zyme fusion proteins facilitate G protein-coupled receptor
crystallogenesis. Structure 22:1657–1664. https://doi.org/10.1016/
j.str.2014.08.022
2
0
55–262. https://doi.org/10.1161/HYPERTENSIONAHA.113.
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