Trans-4-methoxy-β-nitrostyrene relaxes rat thoracic aorta through a sGC-dependent pathway

Article abstract of DOI:10.1016/j.ejphar.2017.05.007

1-Nitro-2-phenylethene (NPe) induces a more potent vasorelaxant effect in rat aorta than its structural analog 1-nitro-2-phenylethane, but mediated through a different mechanism, independent of soluble guanylate cyclase (sGC) stimulation. We hypothesized

Full text of DOI:10.1016/j.ejphar.2017.05.007

European Journal of Pharmacology 807 (2017) 182–189
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Trans-4-methoxy-β-nitrostyrene relaxes rat thoracic aorta through a
sGC-dependent pathway
a
a
Loeste Arruda-Barbosa , Taylena Maria Teófilo , Francisco das Chagas Vasconcelos Souza-
a
b
c
c
Neto , Gloria Pinto Duarte , Joyce Karen Lima Vale , Rosivaldo dos Santos Borges , Pedro Jorge
Caldas Magalhães , Saad Lahlou
a
a,⁎
a
Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, CE, Brazil
Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, PE, Brazil
Department of Pharmacy, Federal University of Pará, Belém, PA, Brazil
b
c
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article:
Trans-4-methoxy-β-nitrostyrene (PubChem
CID: 24864801)
1-Nitro-2-phenylethene (NPe) induces a more potent vasorelaxant effect in rat aorta than its structural analog
1-nitro-2-phenylethane, but mediated through a different mechanism, independent of soluble guanylate cyclase
(sGC) stimulation. We hypothesized that introducing an electron donor into the aromatic moiety might stabilize
Keywords:
Endothelium-independent vasorelaxation
Electronic structure
Trans-4-methoxy-β-nitrostyrene
Guanylate cyclase stimulation
Vascular smooth muscle
NPe, enhancing its potency and/or interaction with sGC. Therefore, trans-4-methoxy-β-nitrostyrene (T4MN)
was synthesized, and mechanisms underlying its vasorelaxant effects were studied in rat aortic ring
preparations. In endothelium-intact preparations, T4MN fully relaxed contractions induced by phenylephrine
(PHE) with a potency similar to that of its parent drug, NPe. This vasorelaxant effect that was unchanged by
endothelium removal, pretreatment with L-NAME, indomethacin, or MDL-12,330 A, but was significantly
2+
reduced by tetraethylammonium, 4-aminopyridine, methyl blue, or ODQ. Under Ca -free conditions, T4MN
did not alter contractions evoked by caffeine, but significantly reduced, in an ODQ-preventable manner, those
2
+
2+
induced by either PHE or extracellular Ca restoration following depletion of intracellular Ca stores in
thapsigargin-treated aortic preparations. Under the same conditions, T4MN also reduced contractions induced
by protein kinase C activator phorbol-12,13-dibutyrate with a potency similar to that evoked by this
nitroderivative against PHE-induced contractions. In conclusion, T4MN induces potent vasorelaxation in rat
aorta by stimulating the sGC-cGMP pathway through a NO-independent mechanism. Introduction of a methoxy
group into the aromatic moiety apparently stabilizes NPe, thereby enhancing its interaction with sGC.
1. Introduction
finding corroborated by the ability of 1-nitro-2-phenylethane to relax
the phenylephrine (PHE)-induced contractions in isolated aortic (de
Nitroderivatives found in higher plants are rare. 1-Nitro-2-pheny-
Siqueira et al., 2010; Brito et al., 2013) or superior mesenteric artery
(Interaminense et al., 2011, 2013) preparations in a concentration-
dependent manner. In rat aortic preparations, vasodilator actions of 1-
nitro-2-phenylethane are mediated through stimulation of the soluble
guanylate cyclase (sGC)-cGMP pathway independently of endothelial
nitric oxide (NO) release, leading to increasing intracellular cGMP
levels (Brito et al., 2013).
lethane was the first nitro compound isolated from plants (Gottlieb and
Magalhães, 1960), and is responsible for the plant's cinnamon-like
scent (Gottlieb, 1972). It is the main constituent of the essential oil
of Aniba canelilla (H.B.K.) Mez [syn. Aniba elliptica A. C. Sm.,
Cryptocarya canelilla Kunth] (Lauraceae), an aromatic plant abundant
in the Amazon region, where it is commonly known as “casca preciosa”
(
precious bark). Previously, we showed that intravenous administra-
Conformational analysis of 1-nitro-2-phenylethane showed that
this molecule can adopt different conformations with varying dihedral
angles between phenyl and nitro groups linked at sp carbon atoms
(Vale et al., 2013). We recently showed that 1-nitro-2-phenylethene
(NPe, Fig. 1A), a structural analog of 1-nitro-2-phenylethane, formed
by substitution of the alkan for the alkene moiety, was nearly 3.5 times
tion of 1-nitro-2-phenylethane induced two periods of hypotension and
bradycardia in either normotensive (de Siqueira et al., 2010) or
hypertensive (Interaminense et al., 2011) rats. The first rapid brady-
cardic and hypotensive phase has a vago-vagal reflex origin while the
delayed hypotension was attributed to a direct vasodilatory effect, a
3
⁎
Correspondence to: Department of Physiology and Pharmacology, Federal University of Ceará, School of Medicine, Rua Cel. Nunes de Melo 1315, Rodolfo Teófilo, Fortaleza, CE
60430-270, Brazil.
http://dx.doi.org/10.1016/j.ejphar.2017.05.007
Received 15 February 2017; Received in revised form 24 April 2017; Accepted 2 May 2017
Available online 03 May 2017
0014-2999/ © 2017 Elsevier B.V. All rights reserved.

L. Arruda-Barbosa et al.
European Journal of Pharmacology 807 (2017) 182–189
free access to food and water. All animals were maintained in
compliance with the Guide for the Care and Use of Laboratory
Animals, published by the US National Institutes of Health (NIH
Publication 85-23, revised 1996). All procedures described here were
reviewed by and approved by local animal ethics committee (126/14).
2.3. Solutions and drugs
The perfusion medium used was a fresh modified Krebs-Henseleit
solution (KHS, pH 7.4) of the following composition (in mM): NaCl
Fig. 1. Chemical structure of 1-nitro-2-phenylethene (or trans-β-nitrostryrene) (A) and
a general procedure for synthesis of its structural analog, trans-4-methoxy-β-nitrostyrene
1
18; KCl 4.7; NaHCO
O 1.18; glucose 11. Calcium-free solutions were prepared by
from normal KHS. Salts were purchased from Merck
Darmstradt, Germany) and Vetec (Rio de Janeiro, Brazil). PHE
3 2 2 2 4 4
25; CaCl ·2H O 2.5; KH PO 1.18; MgSO ·
(B).
7H
2
omitting CaCl
2
more potent at relaxing aortic ring preparations than its parent
molecule (Arruda-Barbosa et al., 2014). However, the mechanism by
which NPe induced vasorelaxation was quite different from that of 1-
nitro-2-phenylethane, as it was independent of sGC stimulation
(
hydrochloride, acetylcholine (ACh) chloride, ethylene glycol-bis(b-
aminoethyl ether)N,N,N´,N´-tetraaceticacid (EGTA), indomethacin,
H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ), tetraethylam-
1
(
Arruda-Barbosa et al., 2014). Given that interactions between drugs
monium chloride (TEA), 4-aminopyridine (4-AP), glybenclamide,
methylene blue (MB), thapsigargin, phorbol-12,13-dibutyrate (PDB),
and receptors (or enzymes) are related not only to hydrophobic
coupling, but also to polar bonds that require a certain electronic
density of specific atoms (Gallé et al., 2013), electronic structure is an
important issue, since it represents energy levels of the molecule that
G
L-N nitroarginine methyl ester (L-NAME), cis-N-(2-phenylcyclopen-
tyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL- 12,330 A), caf-
feine, sodium nitroprusside (SNP) and verapamil were purchased from
Sigma. They were first dissolved in distilled water and then with KHS to
achieve desired concentration in the bath chamber (except EGTA which
can be increased by adding an electron-acceptor (e.g. NO
known that methoxy groups exert two opposite effects: inductive
electron withdrawing) and resonance (electron-donating) effects, but
2
). It is well
(
2+
was added directly to the Ca -free KHS). T4MN and NPe were
dissolved in ethanol, brought up to the desired concentrations using
KHS, and sonicated just before use. Indomethacin (cyclooxygenase
inhibitor), L-NAME (inhibitor of nitric oxide synthase), ODQ (guany-
late cyclase inhibitor), MB (guanylate cyclase inhibitor), MDL-
the latter effect is more powerful than the former, thereby conferring
an electron-donating property upon these groups. Methoxy groups are
strong electron donors due to resonance effects that are comparable to
those of chloro and other halogen substituents, mainly due to their
decreasing oxidation potential (Bessems et al., 1995) and ionization
potential (IP) (Diniz et al., 2004).
In the present study, we hypothesized that methoxy substitution in
the para-position of the aromatic ring would stabilize NPe, further
increasing its interaction with sGC and/or vasorelaxant potency. Thus,
using NPe as a lead compound for electronic structural modifications,
trans-4-methoxy-β-nitrostyrene (T4MN, Fig. 1B) was synthesized, and
mechanisms underlying its vasorelaxant effects were studied in aortic
ring preparations.
1
2,330 A (adenylate cyclase inhibitor), 4-AP (blocker of voltage-
+
+
operated K channels), TEA (nonspecific K channel blocker) or
glybenclamide (blocker of ATP-sensitive K channels) were applied to
+
the bath 10 min prior to pre-contraction with PHE.
2.4. Tissue preparation and experimental protocols
Rats were killed by stunning followed by rapid exsanguination. For
isometric tension recording, the thoracic aorta was removed and placed
in cold oxygenated KHS buffer. Cylindrical ring-like segments of this
artery (1 mm x 5 mm in length), free of fat and connective tissue, were
mounted between two steel hooks in a 5-ml isolated tissue chamber
2
. Materials and methods
.1. Synthesis of trans-4-methoxy-β-nitrostyrene
-((E)−2-nitro-vinyl)-(4-methoxy)-benzene or T4MN was synthe-
2
2 2
containing gassed (95% O and 5% CO ) KHS, at 37 °C, under a resting
tension of 1 g, which was readjusted every 15 min during a 45-min
equilibration period before drug administration. Isometric tension was
recorded using an isometric force displacement transducer (Grass
Model FTO3, Quincy, MA, USA) connected to an acquisition system
1
sized by employing the Claisen-Schmitd procedure (Vogel, 1989; Ford
et al., 1994) with p-anisaldehyde and nitromethane as substrates (0.1
and 0.12 eq., respectively) (Fig. 1B). The aromatic aldehyde was ‘one-
pot’ converted, with a 96% yield, to the corresponding β-nitrostyrene
by treatment with 0.05 eq. of NaOH in methanol and water at 0–10 °C
(
PM-1000; CWE Inc., Akron, OH, USA). Vessels were initially exposed
twice to 60 mM KCl to check their functional integrity. After 30 min,
rings were contracted with a concentration (0.1 μM) of PHE inducing
(
1:2) (Rosini et al., 1991). The resulting precipitate was filtered and
50–70% of the contraction induced by KCl. Then ACh (1 µM) was
dried under vacuum to yield the desired β-nitrostyrene derivative,
T4MN. The trans product is preferable to the cis form due to a
stereoselective reaction that gives a product of low energy. T4MN was
crystallized in ethanol as yellow solid-crystals; m.p. 86.6–88.2 °C (86–
added to assess endothelium integrity. In one series of experiments,
endothelium was removed immediately after dissection by gentle
rubbing of the aortic lumen with a stainless steel wire. The absence
of ACh-induced relaxation was taken as an indicator of successful
endothelium removal. Control rings were exposed only to the vehicle
used to dissolve T4MN. Mechanisms underlying the vasorelaxant
effects of T4MN were studied in aortic preparations contracted with
8
8 °C; Sigma-Aldrich standard). The final product was identified by
1
13
NMR ( H and C NMR) and FT-IR spectroscopy and compared with
properties in the literature (Wang and Wang, 2002). IR νmax (cm
−
1
)
1
8
(
6
1
06.25, 1624.06, 1456.26, 2900, 1600, 1550, 1498, 1375; H NMR
CD SOCD , 300 MHz) δ 7.87 (dd, 2 H), 7.52 (dd, 2 H), 7.47 (d, 1 H),
.87 (d, 1 H), 3.76 (s, 3 H); C NMR (CD
1
the α -adrenergic agonist PHE.
3
3
1
3
3 3
SOCD , 75 MHz) δ 162.89,
2.4.1. Investigation of the role of the vascular endothelium and
39.12, 135.09, 131.33, 122.48, 114.87, 55.50.
potassium channels
Effects of cumulative concentrations of T4MN (0.56–558.1 μM) on
sustained contractions evoked by KCl (60 mM) or PHE (1 μM) were
studied in either endothelium-intact or endothelium-denuded aortic ring
preparations maintained in Ca -containing medium. Vasorelaxant
effects of T4MN were also studied in preparations primed with PHE
2
.2. Animals
2+
Adult male Wistar rats (280–340 g) were kept under conditions of
constant temperature (22 ± 2 °C) with a 12/12 h light/dark cycle and
1
83

L. Arruda-Barbosa et al.
European Journal of Pharmacology 807 (2017) 182–189
2
+
(
1 μM) pre-treated with indomethacin (10 μM) or L-NAME (100 μM).
As a positive control, vascular effects of the NO donor SNP (0.1 nM-
0 μM, n =8) or the vehicle used to dissolve T4MN were studied in PHE-
internal stores. Thereafter, preparations were washed with Ca -free
KHS and a second transient contraction was elicited by caffeine in the
presence of T4MN (167.4 μM), added 5 min prior to caffeine admin-
istration.
1
preconstricted aortic preparations with intact endothelium. In the latter
preparations, vasorelaxant effects of T4MN were studied after pretreat-
ment with TEA (5 mM), glybenclamide (10 μM) or 4-AP (1 mM) to
assess the role of potassium channels.
Finally, inhibitory effects of increasing concentrations (0.56–558.1 μM)
of T4MN on the contraction elicited by the PKC activator PDB (1 μM) were
studied in aortic rings with intact endothelium bathed in Ca -free medium
containing 1 mM EGTA).
2+
(
2
.4.2. Investigation of the involvement of cyclic nucleotides
Aortic preparations with intact endothelium were pretreated with
2.5. Statistical analysis
MDL-12,330 A (3 μM) to assess the involvement of adenylate cyclase
while they were pretreated with ODQ (10 μM) or MB (10 μM) to
investigate the role of guanylate cyclase in vasorelaxant effects of
T4MN.
Results are expressed as mean ± S.E.M.. IC50 values, defined as the
concentration (µM) of T4MN (or NPe) required to produce half-
maximal reduction of the contractile response, were calculated by
interpolation from semi-logarithmic plots, and were expressed as
geometric means [95% confidence interval]. Significance (P < 0.05) of
results was assessed by paired Student's t-test, Mann-Whitney U-test,
and one- or two-way analysis of variance (ANOVA), followed by
Dunnett´s multiple comparison test as appropriate.
2
.4.3. Investigation of the involvement of calcium channels
2+
Under Ca -free conditions, we investigated whether T4MN inhi-
2
+
bits contractions induced by exogenous Ca in aortic ring preparations
with intact endothelium depolarized by either KCl (60 mM) in the
presence of EGTA (50 μM) to activate voltage-operated calcium
channels (VOCCs) or by PHE (1 μM) in the presence of verapamil
3. Results
(
(
1 μM) to preferentially activate receptor-operated calcium channels
ROCCs). Thereafter, a cumulative concentration-response curve for
3.1. Effects of T4MN on sustained contractions induced by KCl or
PHE in aortic rings. Role of the endothelium
2
+
Ca (0.1–20 mM) was developed. After washing the preparation by
changing the bath chamber solution to remove just Ca from the
medium, preparations were incubated for 5 min with T4MN (55.8 or
2
+
In endothelium-containing aortic ring preparations (n =13), T4MN
(0.56–558.1 μM) fully relaxed the sustained contractions induced by
60 mM in a concentration-dependent manner (Figs. 2A, 2C; P < 0.001,
one-way ANOVA), with an IC50 that did not differ significantly from
that obtained in preparations without a functional endothelium (n =10)
(Table 1; P > 0.05, Mann-Whitney U-test). In both preparations,
vasorelaxant effects of T4MN are reversible upon washout. T4MN
(0.56–558.1 μM) also similarly and concentration-dependently relaxed
the sustained contractions evoked by PHE (1 μM) in preparations with
(n =8) or without (n =12) a functional endothelium (Fig. 2D), with IC50
values that were significantly lower than those obtained for relaxing
KCl-induced contractions (Table 1; P < 0.01, Mann-Whitney U-test).
Cumulative addition of vehicle, in the same concentration range used
to dissolve T4MN, has no significant effect on either KCl- (Figs. 2B, 2C; n
=15) or PHE-induced (Fig. 2D; n =15) contractions (P > 0.05, one-way
ANOVA). Furthermore, the positive reference drug, SNP, (0.1 nM-
10 μM) fully relaxed the sustained contractions induced by PHE
(1 μM) in a concentration-dependent manner (P < 0.001, one-way
ANOVA), with an IC50 value of 4.90 (2.96–11.82) nM. Pretreatment of
aortic endothelium-intact rings with L-NAME (100 μM, n =5) (Fig. 2D)
or indomethacin (10 μM, n =5) (Fig. 3C) did alter the vasorelaxant
effects of T4MN against the contractions induced by 1 μM PHE (Table 1;
P > 0.05, Mann-Whitney U-test).
1
67.4 μM). Cumulative concentration-response curve for CaCl
2
(0.1–
2
0 mM) were then repeated and compared with that performed in the
absence of the nitroderivative.
Another series of experiments was conducted under Ca -free
conditions to assess whether T4MN interferes with contractions evoked
by Ca influx through store-operated Ca channels (SOCCs) activated
by Ca store depletion (Putney, 1997). In aortic preparations with
2+
2
+
2+
2
+
2
+
intact endothelium initially bathed in Ca -free KHS (containing EGTA
2
+
1
00 μM), depletion of intracellular Ca stores was achieved through at
least three successive exposures to PHE (10 μM) until no detectable
contraction was recorded. Each stimulus was followed by washing the
preparation with Ca -free KHS to remove PHE from the extracellular
medium. Thereafter, a control cumulative concentration-response
curve for Ca (0.1–20 mM) was performed 10 min after the addition
to the bath of thapsigargin (1 μM), an inhibitor of the sarco-endoplas-
2+
2+
2+
mic reticulum Ca -ATPase (SERCA) (Thastrup et al., 1990). In a
separate set of aortic rings with intact endothelium, T4MN (55.8 or
1
67.4 μM) alone or in the presence of ODQ (10 μM) was added
following thapsigargin treatment and its effects on the cumulative
concentration-response curve for CaCl (0.1–20 mM) were evaluated.
2
2
.4.4. Investigation of the involvement of intracellular calcium
In a first series of experiments, we investigated whether T4MN
2
+
affects contractions evoked by sarcoplasmic reticulum Ca channels
activated by inositol triphosphate (IP ). For this purpose, aortic rings
with intact endothelium were first washed with Ca -free KHS for
min (containing 1 mM EGTA) and a transient contraction was
elicited by PHE (1 μM). After washing the tissues with normal KHS,
3.2. Investigation of the role of potassium channels
3
2
+
Vasorelaxant effects of T4MN (0.56–558.1 μM) against PHE-in-
duced contractions were significantly reduced by TEA (5 mM, n =10) or
4-AP (1 mM, n =8) (Fig. 3A; P < 0.05, two-way ANOVA), as evidenced
by the significant increase in IC50 values (Table 1; P < 0.05, Mann-
Whitney U-test) while they remained unchanged following pre-treat-
ment with glybenclamide (10 μM, n =4) (Fig. 3A; P > 0.05, two-way
ANOVA and Table 1; P > 0.05, Mann-Whitney U-test).
6
2
+
6
0 mM KCl was added to refill internal Ca stores followed by washing
2+
with Ca -free solution and addition of T4MN (55.8 or 167.4 μM),
min later. After 5 min, the second contraction of PHE (1 μM) was
measured in the presence of T4MN alone, or in association with ODQ
10 μM).
In a second series experiments performed under Ca -free condi-
tions, we used caffeine, a pharmacological tool to assess whether T4MN
1
(
2
+
3.3. Investigation of the involvement of cyclic nucleotides
2
+
2+
interferes with contractions induced by Ca -induced Ca release from
the sarcoplasmic reticulum via ryanodine receptors. Aortic rings with
intact endothelium were stimulated with 20 mM caffeine to produce a
transient contraction at 25 °C. After washing with Ca -containing
medium, tissues were stimulated with 60 mM KCl to reload Ca
Concentration-effect curve of T4MN (0.56–558.1 μM) was signifi-
cantly shifted to the right following inhibition of sGC by either ODQ
(10 μM, n =11) or MB (10 μM, n =11) in aortic preparations pre-
contracted with PHE (Fig. 3B, P < 0.05 by two-way ANOVA), with IC50
values significantly higher than the control value (Table 1; P < 0.05,
2
+
2+
1
84

L. Arruda-Barbosa et al.
European Journal of Pharmacology 807 (2017) 182–189
Fig. 2. Typical trace recordings of the effect of trans-4-methoxy-β-nitrostryrene (T4MN, 0.56–558.1 μM) (A) and its vehicle (B) on sustained contractions induced by 60 mM KCl in rat
isolated aortic preparations. Concentration-effect curves for relaxant effects of T4MN (0.56–558.1 μM) in endothelium-intact (E+) and -denuded (E-) aortic rings pre-contracted with
KCl (60 mM) (C) or phenylephrine (PHE, 1 µM) (D). In panel D, note that pretreatment with L-NAME did not alter the vasorelaxant effects of T4MN. In panels C and D, note that
vehicle added cumulatively at the same concentration (% v/v) range used to dissolve T4MN did not change the KCl- and PHE-induced contractions, respectively in preparations with
*
intact endothelium. Results are expressed as means ± S.E.M. (n =10–15 per group). P < 0.05, **P < 0.001 by two-way ANOVA followed by Dunnett´s test with respect to vehicle.
Calibrations: vertical 0.25 g, horizontal 2 min.
2
+
Mann-Whitney U-test). In contract, vasodilating activity of T4MN was
not affected by the adenylate cyclase inhibitor MDL-12,330 A (3 μM, n
A third series of experiments was conducted under Ca -free
conditions to assess whether T4MN interferes with contractions evoked
2+
2+
=
6) (Fig. 3C; p > 0.05, two-way ANOVA).
by Ca influx through SOCCs activated by Ca store depletion. In
aortic preparations in which intracellular Ca stores were depleted
with repeated exposure to PHE followed by thapsigargin, the expected
contractions due to Ca addition (0.1–20 mM, n =12) were unaltered
by 16.7 μM T4MN (n =6, data not shown), but were abolished by
T4MN at 55.8 μM (n =10) (Fig. 5; P < 0.001, two-way ANOVA). Such
an inhibitory effect of T4MN was blunted by 10 μM ODQ (Fig. 5; P <
2+
3
.4. Investigation of the involvement of calcium channels
2+
2+
In endothelium-intact aortic preparations incubated in Ca -free
medium in the presence of a high extracellular KCl concentration
60 mM), increasing extracellular concentrations of CaCl (0.1–
0 mM, n =13) evoked concentration-dependent contractions (P <
.001, one-way ANOVA), an effect that became significant at 0.3 mM
(
2
0.05, two-way ANOVA, n =5).
2
0
(
P < 0.05, one-way ANOVA and Dunnett´s test) and attained a max-
3.5. Investigation of the involvement of intracellular calcium
signaling
imal magnitude at 10 mM (Fig. 4A). These contractions due to VOCC
activation were abolished by T4MN at 167.4 μM (n =5) (Fig. 4A; P <
2
+
0
5
.01, two-way ANOVA) but they remained unaffected by T4MN at
5.8 μM (P > 0.05; two-way ANOVA, n =6) (data not shown).
In aortic rings incubated in Ca -free medium and stimulated with
Under Ca -free conditions, PHE (1 μM) induced a transient contrac-
tion which corresponded to 56.01 ± 6.09% (Fig. 6A, n =14) of a reference
2
+
+
2+
contraction induced by 60 mM K in Ca -containing medium. Pre-
exposure of aortic ring preparations to 167.4 μM (n =6), but not to
55.8 μM (n =5, data not shown) T4MN significantly reduced the PHE-
induced phasic contraction to 2.52 ± 1.31% (Fig. 6A, P < 0.05, one-way
ANOVA followed by Dunnett´s test). This inhibitory effect of T4MN was
reversed in preparations pretreated with 10 μM ODQ (Fig. 6A, n =7).
PHE (1 μM) in the presence of verapamil (1 μM) to preferentially
activate ROCCs (Fig. 4B), T4MN at 167.4 μM (n =6), but not at
5
5.8 μM (n =6, data not shown), also abolished the concentration-
dependent contractions due to CaCl (0.1–20 mM, n =7) (Fig. 4B; P >
.05, two-way ANOVA).
2
0
1
85

L. Arruda-Barbosa et al.
European Journal of Pharmacology 807 (2017) 182–189
Table 1
IC50 values for the vasorelaxant effects of trans-4-methoxy-β-nitrostyrene in aortic ring
preparations pre-contracted with KCl (60 mM) or phenylephrine (1 µM) subjected to
various pre-treatments. Values are expressed as geometric mean [95% confidence
interval] and number within parentheses indicates the number of preparations for each
group.
Contractile agent/Pre-treatments
IC50 (µM)
n
Phenylephrine
Control
Endothelium removal
57.64 [27.85–87.45]
40.09 [32.31–49.50]
372.66 [255.17–490.14]
500.58 [258.35–742.86]
98.23 [51.23–144.74]
55.92 [24.22 −87.62]
325.50 [192.27–453.20]
188.92 [58.32–319.47]
84.59 [65.79–103.35]
66.64 [30.47–102.86]
(08)
(12)
(10)
(08)
(04)
(05)
(11)
(11)
(05)
(06)
a
a
+
+
+
+
+
+
+
+
TEA
4-AP
Glybenclamide
L-NAME
ODQ
Methylene blue
Indomethacin
MDL−12,330 A
a
a
KCl
Control
Endothelium removal
b
120.10 [89.69–150.47]
108.89 [62.56–155.21]
(13)
(10)
b
a
P < 0.05, by Mann-Whitney U-test with respect to value obtained in endothelium-
containing preparations pre-contracted with phenylephrine (control).
b
P < 0.01 by Mann-Whitney U-test with respect to the corresponding values in
preparations pre-contracted with phenylephrine.
Under similar conditions, caffeine (20 mM, n =7) induced a transient
contraction with a magnitude that corresponds to 12.26 ± 3.48% of a
+
2+
reference contraction induced by 60 mM K in Ca -containing medium.
This contraction remained unchanged by 167.4 μM T4MN (Fig. 6B; P >
0
.05, paired Student´s t-test, n =5).
2
+
In aortic rings incubated in Ca -free medium, PDB (1 μM) induced
long-lasting and sustained contraction corresponding to 115.60 ±
+
1
0.30% of the K -induced contraction. When the contraction reached
a steady state, cumulative addition of increasing concentrations (0.56–
58.1 μM) of T4MN (n =9) significantly reduced the PDB-induced
5
contractions in a concentration-dependent manner (P < 0.001, one-way
ANOVA). The IC50 value (75.12 [16.29–133.95] μM) of this effect did
not differ from that of the inhibitory effects of T4MN against PHE-
2+
induced contractions in aortic pre-parations incubated in Ca -con-
taining medium (P > 0.05, Mann-Whitney U-test).
Fig. 3. Effects of pretreatment with tetraethylammonium (TEA, 5 mM), 4-aminopyr-
idine (4-AP, 1 mM), or glybenclamide (Glyb, 10 μM) (A), ODQ (10 µM), methylene blue
(MB, 10 µM) (B), indomethacin (Indo, 10 μM), MDL-12,330 A (3 μM) (C) on the
vasorelaxant effects of trans-4-methoxy-β-nitrostryrene (T4MN, 0.56–558.1 μM) in
endothelium-intact aortic preparations pre-contracted with phenylephrine (1 μM).
Note that only TEA, 4-AP, MB and ODQ pretreatments showed statistical differences
4. Discussion
This study demonstrated that T4MN has interesting vasorelaxant
properties that are independent of endothelium integrity. Although
T4MN was shown to be equipotent with its parent drug NPe, the
mechanism by which it induced vasorelaxation is quite different. This
mechanism seems to involve sGS stimulation, leading to a decrease in
with respect to the control curve (T4MN alone). Results are expressed as means ± S.E.M.
#
(
(
n =4–15 per group). P < 0.01 by two-way ANOVA with respect to the control curve
T4MN alone).
2+
intracellular Ca levels and activation of outward potassium channels
on the plasmalemma, as was reported for 1-nitro-2-phenylethane
turn, PKG, activated by cGMP, can modulate different target effectors in
vascular smooth muscle (VSM) leading to (i) decreased intracellular
calcium levels that can be achieved through inhibition of calcium release
(
Brito et al., 2013).
The vasorelaxant effect of T4MN is independent of the integrity of
2
+
the endothelial layer, as it remained unaltered by removal of the
vascular endothelium. Released NO and prostaglandins from endothe-
lial cells are not involved in the vasodilator effect of T4MN, as it
remained unchanged following pretreatment with L-NAME and in-
domethacin, respectively. Since maximal efficacy for the relaxant effect
of T4MN was unchanged by endothelium removal, it is reasonable to
postulate that its vasodilatory effects have a predominantly myogenic
nature. This hypothesis is reinforced by findings that with the same
from sarcoplasmic reticulum (IP
3
-sensitive Ca
channels) and/or
2
+
decreased influx of extracellular calcium (voltage-operated Ca and
store-operated Ca channels), (ii) hyperpolarization of VSM membrane
potential (outward K channels) and reduction in the sensitivity of the
2+
+
contractile machinery (MLC20). Our data suggest that the vasorelaxant
effects of T4MN involve, at least in part, stimulation of the sGC pathway
independently from endothelial NO release, leading to increased cGMP
levels in aortic rings. This hypothesis is supported by the fact that
vasodilator effects of T4MN in aortic preparations were significantly
inhibited by both ODQ and MB, two guanylate cyclase inhibitors
(Gruetter, 1981; Garthwaite et al., 1995). The present finding contrasts
with a previous study showing that the sGC/cGMP pathway is not
involved in vasodilatation induced by the parent drug, NPe (Arruda-
Barbosa et al., 2014). Cyclic AMP (cAMP), another cyclic nucleotide
potency, T4MN inhibited contractions evoked by the selective α
1
-
agonist, PHE, and those produced by PDB, which activates protein
2+
kinase C under Ca -free conditions.
The actions of cGMP are attributed to activation of protein kinase G
(
PKG), a serine/threonine kinase composed of an NH
2
-terminal domain,
a regulatory domain, and a catalytic domain (Hofmann et al., 2006). In
1
86

L. Arruda-Barbosa et al.
European Journal of Pharmacology 807 (2017) 182–189
Fig. 4. Inhibitory effects of trans-4-methoxy-β-nitrostryrene (T4MN, 167.4 μM) on the
2+
cumulative concentration-effect curve dependent on addition of extracellular Ca (0.1–
0 mM) in endothelium-intact, KCl-stimulated (A) or phenylephrine-stimulated (B)
2
2
+
aortic ring preparations from rats incubated in Ca -free medium. Results are expressed
as means ± S.E.M. (n =5–13 per group); *first significant effect for control curve or for
curves obtained in presence of T4MN (P < 0.05, one-way ANOVA followed by Dunnett´s
Fig. 6. Inhibitory effects of trans-4-methoxy-β-nitrostryrene (T4MN, 167.4 μM) on
contractions evoked by phenylephrine (PHE, 1 μM) (A) or caffeine (20 mM) (B) in
#
2+
test); P < 0.01 by two-way ANOVA, T4MN vs. concentration-contraction curve of Ca
alone (control).
2+
aortic ring preparations with intact endothelium maintained under Ca -free conditions.
Results are expressed as means ± S.E.M. (n =4–14 per group). *P < 0.01 by one-way
ANOVA followed by Dunnett´s test with respect to PHE-induced contraction in the
+
absence of T4MN. K60: reference contraction in response to 60 mM K .
synthesized from intracellular ATP by adenylate cyclase, can also
2
+
activate PKG in VSM, leading to reduction in intracellular Ca and
vasorelaxation (Lincoln et al., 1990). Although activation of PKG by
cAMP requires nearly a 10-fold-higher cAMP concentration than cGMP
concentration, it is unlikely that it contributes to the mediation of
T4MN-induced vasorelaxation since this effect was unaltered by the
adenylate cyclase inhibitor MDL-12,330 A. A similar profile of action
was previously reported for the NO-independent stimulator of sGC, 1-
nitro-2-phenylethane (Brito et al., 2013).
Calcium influx from extracellular space through calcium channels is
known to play a crucial role in VSM contraction. It is known that
contraction evoked by high KCl in VSM is due to membrane depolar-
2+
ization leading to Ca influx through VOCCs (Somlyo and Somlyo,
968). On the other hand, activation of α -adrenoceptors by PHE lead
to a biphasic response that is characterized by an initial phase of
1
1
2+
contraction elicited by IP
3
-induced intracellular Ca release from
sarcoplasmic reticulum followed by a second tonic phase that results
2
+
Fig. 5. Inhibitory effects of trans-4-methoxy-β-nitrostryrene (T4MN, 55.8 μM) on the
from Ca influx through ROCCs (Ehrlich and Watras, 1988). In the
2+
cumulative concentration-effect curve dependent on addition of extracellular Ca (0.1–
0 mM) in endothelium-intact phenylephrine-depolarized aortic ring preparations
present study, T4MN inhibited electromechanically mediated, KCl-
induced contractions and, in aortic preparations depolarized with high
KCl in Ca -free medium, it also reduced or even abolished CaCl -
2
induced contractions that are due to an increase in Ca influx through
VOCCs. T4MN also interfered with pharmacomechanical coupling.
This hypothesis is supported by the ability of T4MN to reduce PHE-
2
2
+
pretreated with thapsigargin, and incubated in Ca -free medium. Results are expressed
2+
as means ± S.E.M. (n =5–12 per group); *first significant effect for control curve (P <
2+
#
0
.05, one-way ANOVA followed by Dunnett´s test); P < 0.001 by two-way ANOVA,
2
+
T4MN vs. concentration-contraction curve of Ca alone (control).
1
87

L. Arruda-Barbosa et al.
European Journal of Pharmacology 807 (2017) 182–189
2
+
induced contractions in Ca -containing medium and to inhibit CaCl
2
-
The HOMO and the lowest unoccupied molecular orbital (LUMO) are
related to nucleophilicity and electrophilicity, respectively. In a recent
molecular modeling study using density functional theory (Oliveira
et al., 2016), Oliveira and coworkers calculated the HOMO-LUMO
energy gap and IP for NPe and T4MN. Compared to NPe, these authors
found that (i) T4MN displayed a lower IP, which is suggestive of a
higher capacity for stabilization of nitrogen-free radicals, and (ii) the
gap energies of frontier orbitals showed that the transition from HOMO
to LUMO is easier for T4MN. Thus, based on these parameters, it can
be concluded that the electronic structure of these two nitroderivatives
must be different. Therefore, it seems that introduction of a methoxy
group into the aromatic moiety stabilizes NPe, increasing its interac-
tion with sGC without changing its vasorelaxant potency.
induced contractions in aortic preparations stimulated with PHE under
Ca -free conditions in the presence of verapamil to remove the
indirect influence of VOCC-mediated Ca influx. Thus, T4MN was
able to interfere with contractile events elicited by Ca entry through
VOCCs and ROCCs. It is known that SOCCs are activated by depletion
of Ca stores within the sarcoplasmic reticulum, allowing a capacita-
tive Ca influx into the cytosol and a sustained contraction (Clapham
et al., 2001). Our results revealed that when aortic preparations were
submitted to simultaneous depletion of Ca
2
+
2
+
2
+
2
+
2+
2
+
stores by PHE and
inhibition of SERCA by thapsigargin, the sustained contraction induced
by the addition of Ca was abolished by T4MN, an effect that was
prevented by pretreatment with ODQ.
2+
In the present study, T4MN-induced relaxation was blunted by
+
pharmacological blockade of K channels with TEA and 4-AP, but not by
5. Conclusions
+
glybenclamide. This suggests that opening of K channels in the
plasmalemma may contribute to vasodilatation evoked by T4MN. Such
a mode of action is consistent with the ability of T4MN to stimulate sGC
and with the finding of higher potency of T4MN on pharmacomechanical
coupling than on electromechanically mediated KCl-induced contrac-
tions. On the other hand, T4MN was unable to change the transient
contraction evoked by caffeine. However, it inhibited the phasic
In conclusion, our findings indicate that the mechanism underlying
the vasorelaxant effects of T4MN is distinctly different from that of its
parent drug, NPe. T4MN-induced vasorelaxation appears mediated by a
sGC pathway leading to increased intracellular cGMP, which in turn
activates PKG. Such an activation mediates the opening of potassium
2+
2+
channels and reduction of intracellular Ca by partial inhibition of Ca
2+
2+
contraction induced by PHE in aortic preparations incubated in Ca
-
influx through either VOCC or intracellular Ca stores. It seems that
introduction of a methoxy group into the aromatic moiety stabilizes NPe,
thereby enhancing its interaction with sGC.
free solution, an effect reversed by pretreatment with ODQ. These
2
+
findings suggest that T4MN effectively inhibits Ca mobilization from
intracellular stores caused by IP while it was inert against contractions
3
2+
2+
induced by Ca -induced Ca release from the sarcoplasmic reticulum
via ryanodine receptors (Karaki and Weiss, 1997). It is noteworthy that
while T4MN was able to relax aortic preparations pre-contracted with
Conflict of interest
The authors declare that there is no conflict of interest regarding
PHE at lower concentrations (IC50 =57.64 µM), its IP
3
signaling
the publication of this article.
blocking effect was evident only at higher concentrations (167.43 µM).
This may suggest that T4MN inhibits contractions of VSM at a signaling
pathway downstream from the receptor activation.
Funding
In concert of all the aforementioned results, T4MN appears to
induce vasorelaxant effects through sGC stimulation independently
from endothelial NO release. Conventional stimulators such YC-1 and
BAY 41–2271 increased cGMP levels in the absence of NO by inducing
a conformational change of sGC probably due to their binding to an
allosteric site in proximity to the heme group (Zhao et al., 1998; Stasch
et al., 2001; Koglin et al., 2003). This stimulatory effect depends on the
presence of a reduced prosthetic heme (i.e., heme iron in the ferrous
state) of sGC since it crucially decreased after heme removal or
oxidation by ODQ (Stasch et al., 2001; Evgenov et al., 2006). By
contrast, heme-independent sGC activators such as BAY 58–2667 bind
preferentially to the NO-insensitive oxidized (i.e., heme iron in the
ferric state) or heme-free forms of sGC. Based upon the present
findings with L-NAME and ODQ, T4MN may likely be characterized
as an sGC stimulator rather than activator, as was previously proposed
for 1-nitro-2-phenylethane (Brito et al., 2013). The sGC-cGMP signal
transduction pathway plays an important role in regulation of pul-
monary vascular tone and resistance in pulmonary arterial hyperten-
sion. Considering that Riociguat (BAY 63–2521), a novel oral NO-
independent stimulator of sGC, is the only treatment currently avail-
able to treat two of the five forms of pulmonary arterial hypertension
This work was supported by the Conselho Nacional de Pesquisa
[CNPq, Edital Universal 14/2012, grant number 484656/2012-0] and
Fundação Amazônica de Amparo a Estudos e Pesquisas” [FAPESPA,
ICAAF, grant number 177/2014]. This study is part of a Doctoral
Thesis developed by Loeste Arruda-Barbosa in order to obtain his
Ph.D. degree in Pharmacological Sciences at the Federal University of
Ceará, Fortaleza, Brazil. Taylena Maria Teófilo is a Doctoral fellow.
Expert reviewing of the manuscript by Dr. Steven D. Aird (Okinawa
Institute of Science and Technology, Okinawa, Japan) is gratefully
acknowledged.
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