Mechanisms underlying the vasorelaxant effect of trans-4-methoxy-β-nitrostyrene in the rat mesenteric resistance arteries

Article abstract of DOI:10.1016/j.ejphar.2019.01.058

Mechanisms underlying the vasorelaxant effects of the synthetic nitro compound, trans-4-methoxy-β-nitrostyrene (T4MN) were studied in isolated small resistance arteries from spontaneously hypertensive rats (SHRs). T4MN caused vasorelaxation in endothelium

Full text of DOI:10.1016/j.ejphar.2019.01.058

Author’s Accepted Manuscript
Mechanisms underlying the vasorelaxant effect of
trans-4-methoxy-β-nitrostyrene in the rat mesenteric
resistance arteries
Thayane Rebeca Alves-Santos, Fabiano Elias
Xavier, Gloria Pinto Duarte, Rosivaldo dos Santos
Borges, Pedro Jorge Caldas Magalhães, Saad
Lahlou
www.elsevier.com/locate/ejphar
PII:
S0014-2999(19)30083-4
https://doi.org/10.1016/j.ejphar.2019.01.058
EJP72199
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 25 October 2018
Revised date: 29 January 2019
Accepted date: 31 January 2019
Cite this article as: Thayane Rebeca Alves-Santos, Fabiano Elias Xavier, Gloria
Pinto Duarte, Rosivaldo dos Santos Borges, Pedro Jorge Caldas Magalhães and
Saad Lahlou, Mechanisms underlying the vasorelaxant effect of trans-4-methoxy-
β-nitrostyrene in the rat mesenteric resistance arteries, European Journal of
Pharmacology, https://doi.org/10.1016/j.ejphar.2019.01.058
This is a PDF file of an unedited manuscript that has been accepted for
publication. As a service to our customers we are providing this early version of
the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting galley proof before it is published in its final citable form.
Please note that during the production process errors may be discovered which
could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Mechanisms underlying the vasorelaxant effect of trans-4-methoxy-β-
nitrostyrene in the rat mesenteric resistance arteries
Thayane Rebeca Alves-Santosa, Fabiano Elias Xaviera, Gloria Pinto Duartea, Rosivaldo
dos Santos Borgesb, Pedro Jorge Caldas Magalhãesc, Saad Lahlouc,
*
a
Department of Physiology and Pharmacology, Federal University of Pernambuco,
Recife, PE, Brazil
b
Department of Pharmacy, Federal University of Pará, Belém, PA, Brazil
c
Department of Physiology and Pharmacology, School of Medicine, Federal University
of Ceará, CE, Brazil
*Corresponding author: Prof. Saad Lahlou, Laboratory of Cardiovascular
Pharmacology, Department of Physiology and Pharmacology, School of Medicine,
Federal University of Ceará, Rua Cel. Nunes de Melo 1315, Rodolfo Teófilo, Fortaleza,
CE, 60430-270 Brazil. Tel: +55.85.3366.8334; fax: +55.85.3366.8333. Email addresses:
saad.lahlou@ufc.br; lahlou562@gmail.com

2
Abstract
Mechanisms underlying the vasorelaxant effects of the synthetic nitro compound, trans-
4-methoxy-β-nitrostyrene (T4MN) were studied in isolated small resistance arteries
from spontaneously hypertensive rats (SHRs). T4MN caused vasorelaxation in
endothelium-intact third-order branches of the mesenteric artery pre-contracted with
noradrenaline (NA). This effect was unchanged by indomethacin and atropine but was
significantly reduced by endothelium removal, L-NAME, LY294002, glybenclamide,
TEA, apamin, TRAM 34, or by the association of apamin and TRAM 34. Pretreatment
with the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced
the T4MN-induced relaxation in endothelium-intact, but not in denuded preparations.
Incubation of small resistance arteries with T4MN increased nitric oxide (NO)
production, an effect that was blocked by L-NAME. In Ca2+-free medium, T4MN
inhibits the contractions induced by (i) NA, (ii) exogenous calcium through receptor- or
voltage-operated Ca2+ channels and (iii) those evoked by Ca2+ influx through stores-
operated Ca2+ channels activated by thapsigargin-induced Ca2+ store depletion. In
contrast, T4MN was inert against the transient contraction induced by caffeine in Ca2+-
free medium. In conclusion, T4MN induced effective vasorelaxant effects in isolated
small resistance arteries from SHRs. This vasorelaxation seems to be mediated partly
by an endothelium-dependent mechanism involving activation of Akt/eNOS/NO
pathway and partly by an endothelium-independent mechanism through activation of
sGC/cGMP/PKG pathway in vascular smooth muscle, leading to inhibition of Ca2+
influx from the extracellular milieu and IP3-sensitive intracellular Ca2+ release as well as
activation of potassium channels.
Keywords: NO/cGMP pathway; small resistance arteries; spontaneously hypertensive
rats; trans-4-methoxy-β-nitrostyrene; vasorelaxation.

3
1. Introduction
Hypertension is a common, progressive disorder that constitutes a major risk for
diabetes, stroke, cardiovascular, and renal diseases (GBD, 2015). Systemic mean
arterial pressure is primarily regulated by the total peripheral vascular resistance which
is mainly dependent on small resistance vessels and arterioles that present the greatest
resistance. The endothelium plays an important role for the maintenance of vascular
tone by releasing several endothelium-derived relaxing factors (EDRFs), including
nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor
(EDHF) (Furchgott and Vanhoutte, 1989). Reduced liberation and/or production of
EDRFs cause endothelial dysfunction which is generally observed in conduit and
resistance vessels in human patients and experimental animal models of hypertension
(Sim and Singh, 1987; Bennett et al., 1993; Lüscher, 1994). Accumulated evidences
suggest that in the conduit artery, the endothelium-dependent vasodilatation is mediated
mainly by NO under physiological conditions (Wu et al., 1993; Clark and Fuchs, 1997).
In contrast, EDHF became a more predominant mediator as vessel size became smaller
(Tomioka et al., 1999; Félétou and Vanhoutte, 1999; Chauhan et al., 2003). In
hypertensive conditions, endothelium-dependent vasorelaxation in both small resistance
and conduit arteries was markedly impaired (Jiang et al., 2016). However, Kang et al.
suggested that NO-dependent pathways in the vasorelaxation of small arteries from
hypertensive rats may be increased (Kang et al., 2007).
In a previous study performed in isolated aorta from normotensive rats, we showed
that 1-nitro-2-phenylethane (NPa), a nitro compound isolated from plants (Gottlieb and
Magalhães, 1960), induced vasorelaxant effects through stimulation of the soluble
guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) independently of
endothelial nitric oxide (NO) release, leading to increasing intracellular cGMP levels

4
(Brito et al., 2013). 1-Nitro-2-phenylethene, a structural derivative of NPa, was served
as a lead compound for electronic structural modifications that yielded several new
nitroderivatives such as trans-4-methoxy-β-nitrostyrene (T4MN, Fig. 1). Like NPa,
T4MN displayed an interesting potential to relax rat thoracic aorta through a mechanism
also related to stimulation of sGC/cGMP pathway independently of the presence of
endothelial NO (Arruda-Barbosa et al., 2017). Interestingly, T4MN was nearly 4 times
more potent as a relaxing agent in rat aortic preparations than NPa (Arruda-Barbosa et
al., 2017).
New NO-independent sGC stimulators could be promising candidates not only as
antihypertensive drugs, but also for treatment of pulmonary arterial hypertension, heart
failure, thrombosis, and erectile dysfunction among other diseases (Evgenov et al.,
2006). The present study was undertaken to give further insight into the putative
antihypertensive properties of T4MN. For this purpose, we studied the mechanisms
underlying the vasorelaxant effects of T4MN in isolated small mesenteric arteries from
spontaneously hypertensive rats (SHRs), which are resistance vessels known to play a
relevant role in the regulation of total peripheral resistance and thus systemic blood
pressure (Christensen and Mulvany, 1993).
2. Materials and Methods
2.1. Synthesis of trans-4-methoxy--nitrostyrene
1-((E)-2-nitro-vinyl)-(4-methoxy)-benzene or T4MN was synthesized by the Claisen-
Schmitd procedure (Vogel, 1989; Ford et al., 1994) with p-anisaldehyde and
nitromethane as substrates (0.1 and 0.12 eq., respectively). Full details of the synthesis
of T4MN including its characterization can be found in our previous study performed in
normotensive rats (Arruda-Barbosa et al., 2014).
2.2. Animals

5
Adult male SHRs (age: 16-18 weeks) were obtained from our local colonies maintained
at the Department of Physiology and Pharmacology, Federal University of Pernambuco,
Recife, Brazil. They were kept under conditions of constant temperature (22 ± 2 °C) with
a 12 h light/12 h dark cycle and free access to food and water. All animals were cared for
in compliance with the Guide for the Care and Use of Laboratory Animals of the
Brazilian National Council for Animal Experimentation. All procedures described here
were reviewed and approved the institutional animal ethics committee
(23076.050472/2012-77).
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 118; KCl 5; NaHCO3 25; CaCl2.2H2O
2.5; NaH2PO4 1.18; MgSO4.7H2O 1.18; glucose 11. Calcium-free solutions were
prepared by omitting CaCl2 from normal KHS. Salts were purchased from Merck
(Darmstradt, Germany) and Vetec (Rio de Janeiro, Brazil). Pentobarbital sodium was
purchased from Robifarma Pharmaceutical Industry Ltd. (Hortolândia, São Paulo,
Brazil). Noradrenaline (NA) hydrochloride, 4,5-diaminofluorescein-2 diacetate (DAF-
2DA), acetylcholine (ACh) chloride, atropine sulfate, ethylene glycol-bis(b-aminoethyl
ether)N,N,N´,N´-tetraaceticacid (EGTA), indomethacin, 1H-[1,2,4] oxadiazolo [4,3-a]
quinoxaline-1-one (ODQ), TRAM 34, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-
4-one hydrochloride (LY294002), tetraethylammonium chloride (TEA), glybenclamide,
thapsigargin, L-NG nitroarginine methyl ester (L-NAME), caffeine, and nifedipine were
purchased from Sigma-Aldrich (St Louis, MO, USA). They were first dissolved in
distilled water, ethanol or DMSO then with KHS to achieve desired concentration in the
bath chamber (except EGTA which was added directly to Ca2+-free KHS). Atropine
(muscarinic receptor antagonist), indomethacin (cyclooxygenase inhibitor), ODQ

6
(guanylate cyclase inhibitor), L-NAME (inhibitor of nitric oxide synthase), LY294002
(PI3K inhibitor), apamin (selective blocker of small-conductance Ca2+-activated K+
channels), TRAM 34 (selective blocker of intermediate-conductance Ca2+-activated K+
channels), TEA (blocker of large-conductance Ca2+-activated K+ channels) and
glybenclamide (blocker of ATP-sensitive K+ channels) were applied to the bath 30 min
prior to pre-contraction of endothelium-intact preparations with NA (10 M). T4MN
was dissolved in ethanol, brought up to the desired concentrations using KHS, and
sonicated just before use. It was added in increasing concentrations during steady-state
contractions to investigate its vasorelaxant effects.
2.4. Tissue preparation and experimental protocols
Rats were anesthetized with intraperitoneal injection of pentobarbital (50 mg/kg) and
killed by exsanguination. The entire mesenteric vascular bed was carefully removed and
placed in KHS at temperature of 4°C. The mesenteric artery was carefully dissected
from the surrounding fat and connective tissues. Third-order branches of the superior
mesenteric artery were cut into rings (approximately 2 mm in length) and mounted
using two 40 μm tungsten wires in an isometric small-vessel myograph (Danish
MyoTechnology A/S, Aarhus, Denmark) according to a previously described method
(Mulvany and Halpern, 1997). This devise was connected to an acquisition system
(PowerLab 8/35, ADInstruments, Australia). The rings were maintained in an organ
bath containing warmed (37°C) KHS continuously bubbled with carbogen (5% CO2 in
O2). In one series of experiments, endothelium of some vessels was removed by rubbing
the intimal surface by a human hair. The absence of ACh (1 M)-induced relaxation in
rings pre-contracted by 10 µM NA was taken as an indicator of successful endothelium
denudation. Control rings were exposed only to the vehicle used to dissolve T4MN.
Concentrations of T4MN (0.56-558.1 M) were chosen based on our previous study

7
(Arruda-Barbosa et al., 2017). At the end of each experiment, all resistance arteries
were extensively washed and contracted with KCl (75 mM) to test the viability of the
vessels. All preparations showed functional responses to KCl which confirms their
viability and thereby eliminates putative toxicity of T4MN.
2.5. Determination of nitric oxide production
Endothelial NO levels were visualized and quantified in small arteries using DAF-2DA,
a membrane-permeable fluorescent indicator for NO (Uruno et al., 2005), according to a
previously described method (Gamez-Mendez et al., 2015; Yu et al., 2016). For this
purpose, third-order branches of the superior mesenteric artery were removed of
connective tissue and fat, as described above, and then cut into segments. After 30 min
equilibration in KHS gasified with carbogenic mixture at 37°C, mesenteric segments
were incubated without (control) or with ACh (10 μM) or T4MN (5.6 μM). Thirty min
later, mesenteric segments were placed in a freezing medium (OCT, Electron
Microscopy Science, USA) and frozen in the freezer at -80°C. On the day of
experiment, frozen arterial transverse sections (10 µm) were incubated (30 min) in
phosphate buffer (0.1 M, pH 7.4, 37 °C) containing 10 μM DAF-2DA in a light-
protected humidified chamber. Digital images were collected on a microscope (Eclipse
80i, Nikon, Japan) equipped with a rhodamine/fluorescein filter and camera (DS-U3,
Nikon, Japan) using a 20× objective and the same imaging settings in each case. NO
availability was evaluated by DAF-2 DA mean optical density of the fluorescence. The
images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) by
measurement of the mean optical density of the fluorescence observed in the vessel in
relationship to background staining. Six different areas of each section were analyzed
for the presence of NO examination of the regions marked with green fluorescence.
Some experiments were performed in the presence of L-NAME (100 µM) to evaluate

8
the effect of endothelial NO synthase (eNOS) inhibition on the ACh- or T4MN-induced
NO production. Results were expressed as DAF-2T fluorescence normalized to the
scanned surface.
2.6. Statistical analysis
Results are expressed as the mean ± S.E.M.. IC50 values, defined as the concentration
(µM) of T4MN 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]. The significance (P < 0.05) of results was
assessed using the paired Student’s t-test, the Mann-Whitney U-test, and one-
(concentrations) or two-way (concentrations x treatment) analysis of variance
(ANOVA), followed by Dunnett´s multiple comparison test as appropriate.
3. Results
3.1. Role of the endothelium in the vascular effects of T4MN on sustained
contractions induced by NA
In endothelium-intact preparations, increasing concentrations of T4MN (0.56-558.1
M) fully relaxed the sustained contractions evoked by NA (10 M) in a concentration-
dependent manner (Fig. 2A). These reversible vasorelaxant effects of T4MN were
significantly reduced in endothelium-denuded preparations (Fig. 2A) as evidenced by
the significant increase (8.3 fold) in the IC50 value (Table 1). Similar addition of the
vehicle ethanol alone did not affect the NA-induced contractions (data not shown).
Incubation of endothelium-intact mesenteric preparations with T4MN (0.56-558.1
M), but not its vehicle, also fully relaxed the sustained contractions induced by 60 mM
KCl in a concentration-dependent manner (Fig. 2B), with an IC50 value (10.52 [8.81-
28.67] M) that was significantly higher than that obtained for relaxing NA-induced
contractions (~4 M) in the same preparations (Table 1).

9
3.2. Investigation of the involvement of muscarinic receptor or prostanoid
signaling in T4MN-induced vasorelaxation
Concentration-dependent vasorelaxant effects of T4MN in endothelium-intact resistance
vessels pre-contracted with NA (10 M) remained unaffected by either atropine (1 M)
or indomethacin (10 M) pre-incubation (Fig. 2C), as evidenced by the unaltered IC50
values (Table 1).
3.3. Role of NO/GMPc signaling pathway in T4MN-induced vasorelaxation
In endothelium-intact preparations pre-contracted with NA (10 M), vasorelaxant
potency of T4MN was significantly attenuated by pretreatment with 100 M L-NAME
(Fig. 2C), as evidenced by the significant increase in the IC50 value compared to that
obtained in the absence of the eNOS blocker (Table 1). It is noteworthy that the residual
vasodilator effect in the presence of L-NAME was similar to that observed after
endothelial removal. Vasorelaxation induced by T4MN was also reduced (4.2-fold
increase in IC50 value) in preparations incubated in the concomitant presence of apamin
(1 M) and TRAM 34 (1 M) (Fig. 3 and Table 1). Altogether, these results showed
that eNOS activation and to a lesser extent, EDHF release, contribute to the mediation
of T4MN-induced vasorelaxant effects.
3.3.1. Measurement of nitric oxide production
To further confirm the involvement of released NO from endothelial cells in the
vasorelaxation caused by T4MN, DAF-2T fluorescence studies were performed to
quantify NO production. Incubation of third-order branches of the superior mesenteric
artery for 30 min with T4MN (5.6 μM) significantly increased NO production (Figs. 4A
and 4B), an effect that was of the same order of magnitude as that evoked by the
positive control ACh (10 μM) (Figs. 4A and 4B). Both the stimulatory effects of ACh

10
and T4MN on NO production were prevented in the presence of the NOS inhibitor, L-
NAME (100 μM) (Figs. 4A and 4B).
3.3.2. Involvement of Akt-eNOS signaling
To further define whether Akt-eNOS signaling is involved in the activation of eNOS by
T4MN, resistance vessels were incubated with the PI3K inhibitor, LY294002 (10 M).
We found that blockade of the Akt signaling with LY294002 significantly attenuated
the T4MN-induced vasorelaxation (Fig. 5), as shown by the significant increase in the
IC50 value compared to that obtained in the absence of the PI3K inhibitor (Table 1).
3.3.3. Involvement of sGC activation
As a downstream signaling molecule of NO (Lucas et al., 2000), the role of NO/cGMP
pathway in the vasorelaxation of T4MN was investigated. We found that the selective
inhibitor of sGC ODQ (10 M) significantly shifted to the right the concentration-effect
curve for the relaxant effect of T4MN in endothelium-intact resistance vessels pre-
contracted with 10 M NA (Fig. 6). The IC50 value was significantly higher than that
obtained in the absence of ODQ (Table 1). By contrast, inhibitory effect of ODQ was
not evident in endothelium-denuded preparations (Fig. 6) since no statistical difference
was observed between the IC50 values in the presence and the absence of ODQ (Table
1). It is noteworthy that the IC50 value for the vasorelaxation caused by T4MN in the
presence of L-NAME did not differ statistically from that obtained in either
endothelium-intact or endothelium-denuded preparations pre-incubated with ODQ
(Table 1).
3.4. Role of potassium channels in T4MN-induced vasorelaxation
Role of potassium channels in T4MN-induced relaxation was investigated in
endothelium-intact resistance arteries pre-contracted with NA (10 M). In these vessels,
pre-treatment with TEA (5 mM), glybenclamide (10 M), TRAM 34 (1 M) and

11
apamin (1 M) significantly (Fig. 7) attenuated the vasodilatory effects of T4MN with
IC50 values that were significantly (Table 1) different from that obtained in the absence
of these K+ channel blockers.
3.5. Investigation of the involvement of calcium channels
Under Ca2+-free conditions, we investigated whether T4MN inhibits contractions
induced by a transmembrane influx of exogenous Ca2+ small resistance arteries
depolarized by either KCl (60 mM) in the presence EGTA (50 µM) to activate voltage-
operated calcium channels (VOCCs) or by NA (10 M) in the presence of nifedipine (1
M) to preferentially activate receptor-operated calcium channels (ROCCs). We found
that concentration-dependent contractions evoked by increasing extracellular
concentrations of CaCl2 (0.1-20 mM) in both KCl- (Fig. 8A) and NA-stimulated (Fig.
8B) endothelium-intact mesenteric preparations were abolished by T4MN at 16.7 M
(Figs. 8A and 8B) while they were significantly reduced by T4MN at 5.6 M (Figs. 8A
and 8B). The inhibitory effect of T4MN (5.6 M) against contraction to exogenous Ca2+
in KCl-stimulated preparations was fully prevented by a previous pretreatment with 10
M ODQ (Fig. 8A).
In a separate series of experiments conducted under Ca2+-free conditions, we
investigated whether T4MN interferes with contractions evoked by Ca2+ influx through
store-operated Ca2+ channels (SOCCs) activated by Ca2+ store depletion (Putney et al.,
1997). For this purpose, endothelium-intact mesenteric preparations were initially
bathed in Ca2+-free KHS (containing EGTA 100 µM) and depletion of intracellular Ca2+
stores was achieved through at least three successive exposure to NA (10 M) until no
detectable contraction was recorded. After removing NA from the extracellular medium,
a control cumulative concentration-response curve for Ca2+ (0.1-20 mM) was performed
10 min after the addition to the bath of thapsigargin (0.1 µM), an inhibitor of the sarco-

12
endoplasmic reticulum Ca2+-ATPase (SERCA) (Thastrup et al., 1990). Under these
conditions, contractions due to exogenous addition of Ca2+ were significantly reduced
by 5.6 M T4MN, but were abolished by 16.7 M T4MN (Fig. 8C). The inhibitory
effect of T4MN (5.6 M) was blunted by 10 M ODQ (Fig. 8C).
3.6. Investigation of the involvement of intracellular Ca2+ signaling
To assess whether T4MN affects contractions evoked by sarcoplasmic reticulum Ca2+
channels activated by inositol triphosphate (IP3), endothelium-intact mesenteric rings
were incubated with Ca2+-free KHS (containing 1 mM EGTA) and a transient
contraction was elicited by NA (10 M). This contraction corresponded to 77.24 ±
1.42% (Fig. 9A) of a reference contraction induced by 60 mM K+ in Ca2+-containing
medium. After a subsequent contractile stimulus with 60 mM KCl in normal KHS to
refill Ca2+ internal stores, mesenteric resistance preparations were treated for 5 min with
T4MN bathed in Ca2+-free KHS (containing 1 mM EGTA). Pre-exposure of small
resistance artery preparations to T4MN (5.6 and 16.7 M) significantly reduced the
NA-induced phasic contraction (Fig. 9A). The inhibitory effect of T4MN at 5.6 M was
fully reversed in preparations pretreated with 10 M ODQ (Fig. 9A).
Still under Ca2+-free conditions, we used caffeine as a pharmacological tool to assess
whether T4MN interferes with contractions that involve Ca2+ release from the
sarcoplasmic reticulum via ryanodine-sensitive Ca2+ channels. Caffeine (20 mM)
induced a transient contraction with a magnitude that corresponds to 29.47 ± 2.72% of a
reference contraction induced by 60 mM K+ in Ca2+-containing medium. The magnitude
of this contraction remained unchanged by T4MN either at 5.6 or 16.7 M T4MN (Fig.
9B).
4. Discussion

13
In the current study, T4MN induced effective vasorelaxant effects in small resistance
arteries from SHRs through both endothelium-dependent and endothelium-independent
mechanisms.
The finding that vasorelaxant effects of T4MN were reduced by mechanical removal
of the vascular endothelium suggests that they are partially dependent of the integrity of
the endothelial layer. These effects could be related to endothelium-derived factors,
such as prostaglandins (prostacyclin or PGI2), EDHF or NO (Dusting et al., 1978;
Rubanyi et al., 1990; Vanhoutte, 2001; Hilgers et al., 2006). Released prostaglandins
from endothelial cells is not involved in the vasodilator effect of T4MN, as it remained
unaltered by the non-specific cyclooxygenase inhibitor indomethacin. EDHF plays an
important role mainly in small resistance vessels which are known to determine
peripheral vascular resistance and therefore blood pressure (Shimokawa and Godo,
2016; Tang et al., 2013). In order to evaluate the putative involvement of EDHF in the
T4MN-induced vasorelaxation, mesenteric rings were pre-incubated in the concomitant
presence of apamin and TRAM 34, two blockers of small- and intermediate-
conductance Ca2+-activated K+ channels, respectively (Triggle et al., 2012; Bełtowski,
J., Jamroz-Wiśniewska, 2014) which are known to inhibit the membrane
hyperpolarization and the subsequent vasorelaxation response to EDHF (Furchgott and
Zawadzki, 1980; Wu et al., 1993). Our data showed that, in this condition,
vasorelaxation induced by T4MN was significantly attenuated, suggesting that it could
be partially mediated by hyperpolarization of the cell membrane caused by released
EDHF.
Pretreatment with the eNOS inhibitor L-NAME reduced the T4MN-induced
vasorelaxation without altering the maximal effect (Emax), suggesting that activation of
eNOS leading to released endothelium-derived NO is involved in this effect. Such an

14
involvement of released NO from endothelial cells in the vasorelaxation caused by
T4MN was supported by DAF-2DA fluorescence studies showing that incubation of
third-order branches of the superior mesenteric with T4MN (5.6 μM) resulted in a
significant increase in NO production. Interestingly, this effect caused by T4MN was of
the same order of magnitude as that evoked by ACh (10 μM), used herein as positive
control. Stimulatory effects of both T4MN and ACh on NO production were fully
prevented in mesenteric resistance rings in which eNOS activation is blocked by L-
NAME. It is well known that ACh causes generalized vasodilatation, which is an
indirect effect mediated by released NO from vascular endothelial cells (Vanhoutte et
al., 1995; Arnal et al., 1999; Dimmeler et al., 1999). Pretreatment of endothelium-intact
mesenteric resistance rings with atropine did not inhibit the vasorelaxant effects of
T4MN, a finding that allows us to discard the possibility that T4MN act directly on
vascular smooth muscle (VSM) to induce relaxation.
It is established that phosphatidylinositol-3-kinase (PI3K/Akt) signaling pathway
increases, in a Ca2+-independent manner, the eNOS activity by phosphorylating serine
at 1177, leading to NO production, and consequently vasodilation (Fulton et al., 1999;
Denninger et al., 1999; Triggle et al., 2012). The current study shows that pretreatment
of endothelium-intact resistance mesenteric preparations with LY294002, an inhibitor
of PI3K/Akt pathway, slightly but significantly reduced the T4MN-induced
vasorelaxation. This suggests that Akt-mediated eNOS phosphorylation is involved in
mediating the vasorelaxant responses to T4MN.
The finding that the T4MN-induced vasorelaxation was still observed following
vascular endothelium removal or pretreatment with L-NAME suggests that T4MN has a
direct effect on VSM cells. Following its generation from endothelial cells, NO diffuses
from the endothelial layer into VSM cells to activate sGC which catalyzes the

15
conversion of GTP to the second messenger cGMP (Garthwaite et al., 1995; Lucas et
al., 2000). In the current study, pretreatment with the sGC inhibitor ODQ (Garthwaite et
al., 1995) reduces the T4MN-induced vasorelaxation in endothelium-intact resistance
mesenteric arteries, indicating the involvement of NO/GMPc pathway in this effect.
Since pretreatment with ODQ was inert against the vasorelaxant effects of T4MN in
endothelium-denuded resistance vessels, we discarded the possibility that T4MN was
acting as a direct sGC stimulator in resistance vessels from SHRs. Increased
intracellular concentrations of cGMP activate protein kinase G (PKG), a
serine/threonine kinase composed of an NH2-terminal domain, a regulatory domain, and
a catalytic domain (Hofmann et al., 2006). In turn, PKG, activated by cGMP, can
modulate different target effectors in VSM cells such as potassium channels, VOCCs,
SOCCs and IP3-sensitive Ca2+ channels (Francis et al., 2010).
Potassium channels, one of the downtream signaling target of activated PKG by
cGMP, play an important role in the regulation of vascular tone as they determine
plasma membrane potential (Jackson, 2000; Sobey et al., 2001). Indeed, the current
finding that T4MN exhibit higher potency in inhibiting pharmacomechanical coupling
than electromechanically mediated KCl-induced contractions suggests that opening of
potassium channels in the plasmalemma contributes to the vasodilatation evoked by
T4MN. This was confirmed by our data that T4MN-induced vasorelaxation was
decreased by TEA (blocker of large-conductance Ca2+-activated K+ channels) (Langton
et al., 1991), apamin (selective blocker of small-conductance Ca2+-activated K+
channels) (Murphy and Brayden, 1995), glybenclamide (blocker of ATP-sensitive K+
channels) (Sobey et al., 2001) and TRAM 34 (selective blocker of intermediate-
conductance Ca2+-activated K+ channels) (Agarwal et al., 2013).

16
Calcium influx from the extracellular space through calcium channels is known to play
a crucial role in VSM contraction and is also modulated by PKG activation. It is known
that contraction evoked by high KCl in VSM is due to membrane depolarization,
leading to Ca2+ influx through VOCCs (Somlyo and Somlyo, 1968). On the other hand,
activation of α1-adrenoceptors by NA lead to a biphasic response that is characterized
by an initial phase of contraction elicited by IP3-induced intracellular Ca2+ release from
sarcoplasmic reticulum, followed by a second tonic phase that results from Ca2+ influx
through ROCCs (Ehrlich and Watras, 1988). In our study, T4MN reduced, KCl-induced
contractions in Ca2+-containing medium and, in resistance vessels preparations
depolarized with high KCl in Ca2+-free medium, it also reduced CaCl2-induced
contractions that are due to an increase in Ca2+ influx through VOCCs. The latter effect
was impaired by the selective sGC inhibitor ODQ. Indeed, T4MN also interfered with
pharmacomechanical coupling, as it reduced contractions evoked by NA in Ca2+-
containing medium and those elicited by the cumulative addition of CaCl2 in resistance
vessels stimulated with NA under Ca2+-free conditions in the presence of nifedipine to
remove the indirect influence of VOCC-mediated Ca2+ influx. It is known that SOCCs
are activated by depletion of Ca2+ stores within the sarcoplasmic reticulum, allowing a
capacitative Ca2+ influx into the cytosol and a sustained contraction (Clapham et al.,
2001). Our results revealed that when small resistance rings preparations were
submitted to simultaneous depletion of Ca2+ stores by NA and inhibition of SERCA by
thapsigargin, the sustained contraction induced by the addition of Ca2+ was significantly
reduced by T4MN, an effect that was prevented by pretreatment with ODQ. Taken
together, these findings suggest that T4MN interferes with contractile events elicited by
Ca2+ entry through VOCCs, ROCCs and SOCCs.

17
Finally, experiments were performed under Ca2 +-free conditions to investigate
whether T4MN could affect contractions induced by intracellular events, such as Ca2 +
release from internal stores located in the sarcoplasmic reticulum. Under these
conditions, T4MN did not change the transient contraction evoked by caffeine whereas
it reduced the phasic contraction induced by NA in an ODQ-preventable manner. These
findings suggest that T4MN effectively inhibits Ca2+ mobilization from intracellular
stores mediated by IP3 while it was inert against contractions induced by Ca2+-induced
Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (Karaki and Weis,
1997). A similar result was obtained with other nitroderivatives, such as NPa (Brito et
al., 2013), 1-nitro-2-phenylethene (Arruda-Barbosa et al., 2014) as well as trans-4-
methyl-β-nitrostyrene (Teófilo et al., 2017) in rat aortic preparations. It is noteworthy
that the inability of T4MN to alter the caffeine-induced transient contraction at
concentrations that it prevented the transient contraction induced by NA discards the
putative detrimental effects on cell viability.
In aortic preparations from normotensive rats, vasodilation caused by T4MN occurred
independently upon the integrity of vascular endothelium through stimulation of the
sGC/cGMP pathway (Arruda-Barbosa et al., 2017). The discrepancy that exists
between these findings and those obtained in the current study could be explained by
methodological difference such as the use different species (normotensive vs. SHR,
respectively), stain (Wistar Kyoto vs. Wistar), type of vessels (conduit arteries vs. small
resistance, respectively) and/or contractile agents (phenylephrine vs. NA, respectively).
Unpublished data from our laboratory showed that in a conduit artery (the superior
mesentery artery) from SHRs pre-contracted with the 1-adrenergic receptor agonist
phenylephrine, the IC50 value for the vasorelaxant effect of T4MN was about ~ 22 M
(Alves-Santos et al., 2013). In the current study, the corresponding IC50 value was about

18
~ 4 M. Despite that different contractile agents (phenylephrine vs. NA) were used in
both studies, T4MN displays almost 6-fold greater potency in relaxing small resistance
mesenteric arterial vessels than conduit arteries. This feature may suggest a preferential
action of T4MN in tissues that are more involved in hemodynamic control and
ultimately determine peripheral vascular resistance and in turn blood pressure.
Conclusions
T4MN induced effective vasorelaxant effects in small resistance arteries from SHRs.
This vasorelaxation seems to be mediated partly by an endothelium-dependent
mechanism involving activation of Akt/eNOS/NO signaling pathway leading to cell
endothelial release of NO, and partly by an endothelium-independent mechanism
through activation of sGC/cGMP/PKG pathway in VSM, leading to inhibition of Ca2+
influx from the extracellular milieu and IP3-sensitive intracellular Ca2+ release as well as
to membrane hyperpolarization due to activation of Ca2+-dependent potassium channels.
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this
article.
Funding
This work was supported by the "Conselho Nacional de Pesquisa" [CNPq, Edital
Universal 14/2012, grant number 484656/2012-0] and the "Fundação Amazônica de
Amparo a Estudos e Pesquisas” [FAPESPA, ICAAF, grant number 177/2014]. This
study is part of a Doctoral Thesis developed by Thayane Rebeca Alves dos Santos in
order to obtain his Ph.D. degree in Biochemistry and Physiology at the Federal
University of Pernambuco, Recife, Brazil.

19
References
Agarwal, J.J., Zhu, Y., Zhang, Q.Y., Mongin, A.A., Hough, L.B., 2013. TRAM-34, a
putatively selective blocker of intermediate-conductance, calcium-activated
potassium channels, inhibits cytochrome P450 activity. PLoS One. 7, e63028.
Alves-Santos, T.R., 2013. Estudo dos efeitos cardiovasculares do um derivado do 1-nitro-2-
fenileteno, o 1-nitro-2-[(4'-metoxi)-fenil]-eteno, em ratos espontaneamente hipertensos.
Master Thesis. Federal University of Pernambuco.
Arnal, J.F., Dinh-Xuan, A.T., Pueyo, M., Darblade, B., Rami, J., 1999. Endothelium-
derived nitric oxide and vascular physiology and pathology. Cell. Mol. Life Sci.,
55, 1078-1087.
Arruda-Barbosa, L., Rodrigues, K.M., Souza-Neto, F.D., Duarte, G.P., Borges, R.S.,
Magalhães, P.J.C., Lahlou, S., 2014. Vasorelaxant effects of 1-nitro-2-
phenylethene in rat isolated aortic rings. Vascul. Pharmacol. 63, 55-62.
Arruda-Barbosa, L., Teófilo, T.M., Souza-Neto, F.C.V., Duarte, G.P., Vale, J.K.L.,
Borges, R.S., Magalhães, P.J.C., Lahlou, S., 2017. Trans-4-methoxy-β-
nitrostyrene relaxes rat thoracic aorta through a sGC-dependent pathway. Eur. J.
Pharmacol. 807, 182-189.
Bełtowski, J., Jamroz-Wiśniewska, A., 2014. Hydrogen sulfide and endothelium
dependent vasorelaxation. Molecules 19, 21183-21199.
Bennett, M.A., Watt, P.A., Thurston, H., 1993. Impaired endothelium-dependent
relaxation in two-kidney, one clip Goldblatt hypertension: effect of
vasoconstrictor prostanoids. J. Hypertens. Suppl. 11, S134-135.
Brito, T.S., Lima, F.J., Aragão, K.S., de Siqueira, R.J., Sousa, P.J., Maia, J.G., Filho,
J.D., Lahlou, S., Magalhães, P.J.C., 2013. The vasorelaxant effects of 1-nitro-2-

20
phenylethane involve stimulation of the soluble guanylate cyclase-cGMP
pathway. Biochem. Pharmacol. 85, 780-788.
Chauhan, S., Rahman, A., Nilsson, H., Clapp, L., Macallister, R., Ahluwalia, A., 2003.
NO contributes to EDHF-like responses in rat small arteries: a role for NO stores.
Cardiovasc. Res. 57, 207-216.
Christensen, K.L., Mulvany, M.J., 1993. Mesenteric arcade arteries contribute
substantially to vascular resistance in conscious rats. J. Vasc. Res. 30, 73-79.
Clapham, D.E., Runnels, L.W., Strübing, C., 2001. The TRP ion channel family. Nat.
Ver. Neurosci. 2, 387-396.
Clark, S.G., Fuchs, L.C. 1997. Role of nitric oxide and Ca++-dependent K+ channels in
mediating heterogeneous microvascular responses to acetylcholine in different
vascular beds. J. Pharmacol. Exp. Ther. 282, 1473-1479.
Denninger, J.W., Marletta, M.A., 1999. Guanylate cyclase and the NO/cGMP signaling
pathway. Biochim. Biophys. Acta. 1411, 334-350.
Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., Zeiher, A.M., 1999.
Activation of nitric oxide synthase in endothelial cells by Akt-dependent
phosphorylation. Nature (Lond) 399, 601-605.
Dusting, G.J., Chapple, D.J., Hughes, R., Moncada, S., Vane, J.R., 1978. Prostacyclin
(PGI2) induces coronary vasodilatation in anaesthetised dogs. Cardiovasc. Res.
12, 720-730.
Ehrlich, B.E., Watras, J., 1988. Inositol 1,4,5-trisphosphate activates a channel from
smooth muscle sarcoplasmic reticulum. Nature 336, 583-586.
Evgenov, O.V., Pacher, P., Schmidt, P.M., Haskó G., Schmidt H.H., Stasch, J.P., 2006.
NO-independent stimulators and activators of soluble guanylate cyclase:
discovery and therapeutic potential. Nat. Rev. Drug. Discov. 5, 755-768.

21
Félétou, M., Vanhoutte, P.M., 1999. The alternative: EDHF. 31, 15-22.
Ford, P.W., Narbut, M.R., Belli, J., Davidson, B.S., 1994. Synthesis and structural
properties of the benzopentathiepins varacin and isolissoclinotoxin A. J. Org.
Chem. 59, 5955-5960.
Francis, S.H., Busch, J.L., Corbin, J.D., Sibley, D., 2010. cGMP-dependent protein
kinases and cGMP phosphodiesterases in nitric oxide and cGMP action.
Pharmacol. Rev. 62, 525-563.
Fulton, D., Gratton, J.P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., Franke, T.F.,
Papapetropoulos A., Sessa W.C., 1999. Regulation of endothelium-derived nitric
oxide production by the protein kinase Akt. Nature. 399, 597-601.
Furchgott, R.F., Vanhoutte, P.M., 1989. Endothelium derived relaxing and contracting
factors. FASEB J. 3, 2007-2018.
Furchgott, R.F., Zawadzki, J.V, 1980.. The obligatory role of endothelial-cells in the
relaxation of arterial smooth muscle by acetylcholine. Nature. 288, 373-376.
Gamez-Mendez, A.M., Vargas-Robles, H., Ríos, A., Escalante, B., 2015. Oxidative
stress-dependent coronary endothelial dysfunction in obese mice. PLoS One. 10,
e0138609.
Garthwaite, J., Southam, E., Boulton, C.L., Nielsen, E.B., Schmidt, K., Mayer, B.,
1995. Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol. Pharmacol. 48, 184-188.
GBD 2015., 2016. Risk Factors Collaborators. Global, regional, and national
comparative risk assessment of 79 behavioural, environmental and
occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic
analysis for the Global Burden of Disease Study 2015. Lancet 388, 1659-1724

22
Gottlieb, O.R., Magalhães, M.T., 1960. Essential oil of bark and wood of Aniba
canelilla. Perf. Essen. Oil Res. 50, 69-70.
Hilgers, R.H., Todd, J.Jr, Webb, R.C., 2006. Regional heterogeneity in acetylcholine
induced relaxation in rat vascular bed: role of calcium-activated K+ channels. Am.
J. Physiol Heart Circ. Physiol. 291, H216-H222.
Hofmann, F., Feil, R., Kleppisch, T., Schlossmann, J., 2006. Function of cGMP-
dependent protein kinases as revealed by gene
deletion. Physiol. Rev. 86, 1-23.
Jackson, W.F., 2000. Ion channels and vascular tone. Hypertension 35, 173-178.
Jiang, J., Zheng, J.P., Li, Y., Gan, Z., Jiang, Y., Huang, D., Li, H., Liu, Z., Ke, Y., 2016.
Differential contribution of endothelium-derived relaxing factors to vascular
reactivity in conduit and resistance arteries from normotensive and hypertensive
rats. Clin. Exp. Hypertens. 38, 393-398.
Kang, K.T., Sullivan, J.C., Sasser, J.M., Imig, J.D., Pollock, J.S., 2007. Novel nitric
oxide synthase-dependent mechanism of vasorelaxation in small arteries from
hypertensive rats. Hypertension. 49, 893-901.
Karaki, H., Weis, G.B., 1997. Calcium release in smooth muscle. Life Sci. 44, 111-112,
1977.
Langton, P.D., Nelson, M.T., Huang, Y., Standen, N.B., 1991. Block of calcium-
activated potassium channels in mammalian arterial myocytes by
tetraethylammonium ions. Am. J. Physiol. 260, H927-H934.
Lucas, K.A., Pitari, G.M., Kazerounian, S., Ruiz-Stewart, I., Park, J., Schulz, S.,
Chepenik, K.P., Waldman, S.A., 2000. Guanylyl cyclases and signaling by cyclic
GMP. Pharmacol. Rev. 52, 375-414.

23
Lüscher, T.F., 1994. The endothelium in hypertension: bystander, target or mediator? J.
Hypertens. Suppl. 12, S105-116.
Mulvany M.J., Halpern, W., 1977. Contractile properties of small arterial resistance
vessels in spontaneously hypertensive and normotensive rats. Circ. Res. 41, 19-
26.
Murphy, M.E., Brayden, J.E., 1995. Apamin-sensitive K+ channels mediate an
endothelium-dependent hyperpolarization in rabbit mesenteric arteries. J. Physiol.
489, 723-734.
Putney, J.W.Jr., 1997. Type 3 inositol 1,4,5-trisphosphate receptor and capacitative
calcium entry. Cell. Calcium 21, 257-261.
Rubanyi, G.M., Freay, A.D., Kauser, K., Johns, A., Harder, D.R., 1990.
Mechanoreception by the endothelium: mediators and mechanisms of pressure-
and flow-induced vascular responses. Blood Vessels 27, 246-257.
Shimokawa, H., Godo, S., 2016 . Diverse functions of endothelial NO synthases
system: NO and EDH. J. Cardiovasc. Pharmacol. 67, 361-366.
Sim, M.K., Singh, M., 1987. Decreased responsiveness of the aortae of hypertensive
rats to acetylcholine, histamine and noradrenaline. Br. J. Pharmacol. 90, V147-
150.
Sobey, C.G., 2001. Potassium channel function in vascular disease. Arterioscler.
Thromb Vasc. Biol. 21, 28-38.
Somlyo, A.V., Somlyo. A.P., 1968. Electromechanical and pharmacomechanical
coupling in vascular smooth muscle. J. Pharmacol. Exp. Ther. 159, 129-145.
Tang, G., Yang, G., Jiang, B., Ju, Y., Wu, L. Wang, R., 2013. H₂S is an endothelium
derived hyperpolarizing factor. Antioxid. Redox Signal 19, 1634-1646.

24
Teófilo, T.M., Arruda-Barbosa, L., Rodrigues-Silva, J.M., Vale, J.K.L., Borges, R.S.,
Duarte, G.P., Magalhães, P.J.C., Lahlou, S., 2017. Mechanism of the
vasorelaxant effect induced by trans-4-methyl-β-nitrostyrene, a synthetic
nitroderivative, in rat thoracic aorta. Clin. Exp. Pharmacol. Physiol. 44, 787-794.
Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, M.R., Dawson, A.P., 1990.
Thapsigargin, a tumor promotor, discharges intracellular Ca2+ stores by specific
inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA
87, 2466-2470.
Tomioka, H., Hattori, Y., Fukao, M., Sato, A., Liu, M., Sakuma, I., Kitabatake, A.,
Kanno, M., 1999. Relaxation in different-sized rat blood vessels mediated by
endothelium derived hyperpolarizing factor: importance of processes mediating
precontractions. J. Vasc. Res. 36, 311-320.
Triggle, C.R., Samuel, S.M., Ravishankar, S., Marei, I., Arunachalam, G., Ding, H.,
2012. The endothelium: influencing vascular smooth muscle in many ways. Can.
J. Physiol. Pharmacol. 90, 713-738.
Uruno, A., Sugawara, A., Kanatsuka, H., Kagechika, H., Saito, A., Sato, K., Kudo, M.,
Takeuchi, K., Ito., S., 2005. Upregulation of nitric oxide production in vascular
endothelial cells by all-trans retinoic acid through the phosphoinositide 3-
kinase/Akt pathway. Circulation 112, 727-736.
Vanhoutte, P.M., 2001. Endothelial adrenoceptors. J. Cardiovasc. Pharmacol. 38, 796-
808.
Vanhoutte, P.M., Boulanger, C.M., Mombouli, J.V., 1995. Endothelium-derived
relaxing factors and converting enzyme inhibition. Am. J. Cardiol.76, E3-12.

25
Vogel, A.I., 1989. Vogel's textbook of practical organic chemistry [revised by Furniss,
B.S., Hannaford, A.J., Smith, P.W.G., Tatchell, A.R.] fifth ed. Longman Scientific
and Technical, New York,.
Wu, C.C., Chen, S.J., Yen¸ M.H., 1993. Different responses to acetylcholine in the
presence of nitric oxide inhibitor in rat aortae and mesenteric arteries. Clin. Exp.
Pharmacol. Physiol. 20, 405-412.
Yu, Y., Xiong, Y., Montani, J.P., Yang, Z., Ming, X.F., 2016. En face detection of nitric
oxide and superoxide in endothelial layer of intact arteries. J. Vis. Exp. 2108,
53718.

26
Figure Legends
Fig. 1. Chemical structure of trans-4-methoxy--nitrostyrene.
Fig. 2. Typical trace recordings of the effect of trans-4-methoxy-β-nitrostryrene
(T4MN, 0.56-558.1 μM) on sustained contractions induced by 60 mM KCl in
endothelium-intact resistance mesenteric rings, (A) Concentration-effect curves for
relaxant effects of T4MN (0.56-558.1 μM) in endothelium-intact (E+) and endothelium-
denuded (E-) resistance mesenteric rings pre-contracted with noradrenaline (NA, 10
M), (B) In C, note that vasorelaxant effects of T4MN in endothelium-intact resistance
mesenteric pre-contracted with NA was reduced in the presence of L-NAME (100 M),
but not in the presence of indomethacin (10 M) or atropine (1 M). In panel B, vehicle
added cumulatively at the same concentration (% v/v) range used to dissolve T4MN
was inert against the NA-induced contractions, in preparations with intact endothelium.
Data are expressed as the mean ± S.E.M. (n = 5-6 per group). #P < 0.01 by two-way
ANOVA. Some of error bars are obscured by the size of symbol.
Fig. 3. Vasorelaxant effects of trans-4-methoxy--nitrostryrene (T4MN, 0.56-558.1
M) in resistance mesenteric rings pre-contracted with noradrenaline (NA, 10 M) in
the absence or in the concomitant presence of apamin (1 M) and TRAM 34 (1 M) .
Note that these potassium channel blockers evoked a significant shift to the right of the
relaxant curve of T4MN (#P < 0.01, two-way ANOVA). Data are expressed as the mean
± S.E.M. (n = 5-6 per group).
Fig. 4. In A, Representative fluorographs (20X objective) of 4,5-diaminofluorescein
diacetate (DAF-2DA) signal obtained in transverse sections of vessel walls from
mesenteric resistance tissues without stimulation (Basal, panel A), stimulated with
acetylcholine (ACh, 10 M) alone (panel B) or in the presence of L-NAME (100 M,
panel C), or stimulated with trans-4-methoxy--nitrostryrene (T4MN, 5.6 M) alone

27
(panel D) or in the presence of L-NAME (100 M, panel E). In B, Quantification of
NO production by measuring DAF-2DA fluorescence in mesenteric tissues stimulated
with ACh and T4MN in the absence or the presence of L-NAME. Data are expressed as
the mean ± S.E.M. (n = 5 per group). *P < 0.01, Mann-Whitney U-test with respect to
control (basal). #P < 0.05, Mann-Whitney U-test with respect to the corresponding NO
quantification in the absence of L-NAME.
Fig. 5. Vasorelaxant effects of trans-4-methoxy--nitrostryrene (T4MN, 0.56-558.1
M) in resistance mesenteric rings pre-contracted with noradrenaline (NA, 10 M) in
the absence or in the presence of LY294002 (10 M). Note that the PI3K inhibitor
evoked a significant shift to the right of the relaxant curve of T4MN (#P < 0.05, two-
way ANOVA). Data are expressed as the mean ± S.E.M. (n = 5-6 per group).
Fig. 6. Vasorelaxant effects of trans-4-methoxy--nitrostryrene (T4MN, 0.56-558.1
M) in endothelium-intact (E+) or endothelium-denuded (E-) resistance mesenteric
rings pre-contracted with noradrenaline (NA, 10 M) in the absence or in the presence
of ODQ. Note that this selective inhibitor of sGC evoked a significant shift to the right
of the relaxant curve of T4MN (#P < 0.01, two-way ANOVA). Data are expressed as
the mean ± S.E.M. (n = 5-6 per group). Some of error bars are obscured by the size of
symbol.
Fig. 7. Vasorelaxant effects of trans-4-methoxy--nitrostryrene (T4MN, 0.56-558.1
M) in endothelium-intact resistance mesenteric rings pre-contracted with
noradrenaline (NA, 10 M) in the absence or in the presence of TEA (5 mM),
glybenclamide (Glyb, 10 M), TRAM 34 (1 M) or apamin (1 M). Note that all
potassium channel blockers evoked a significant shift to the right of the relaxant curve
of T4MN (#P < 0.01, two-way ANOVA) only in the endothelium-intact mesenteric
preparations. Data are expressed as the mean ± S.E.M. (n = 5-6 per group).

28
Fig. 8. Inhibitory effects of trans-4-methoxy--nitrostryrene (T4MN, 5.6 and 16.7 M)
on the cumulative concentration-effect curve for CaCl2 (0.1-20 mM) in endothelium-
intact resistance mesenteric rings incubated in Ca2+-free medium and stimulated with 60
mM KCl (A), noradrenaline (NA, 10 M) (B), or with NA after simultaneous depletion
of intracellular Ca2+ stores by NA (10 µM) and inhibition of SERCA by 1 µM
thapsigargin (C). Note that in A and C, inhibitory effect of T4MN was blunted by 10
M ODQ. Data are expressed as the mean ± S.E.M. (n = 5-6 per group). *first
significant effect for control curve (P < 0.05, one-way ANOVA followed by Dunnett´s
test); #P < 0.05, ##P < 0.001 by two-way ANOVA, T4MN vs. concentration-contraction
curve of Ca2+ alone (control).
Fig. 9. Inhibitory effects of trans-4-methoxy--nitrostryrene (T4MN, 5.6 and 16.7 M)
on contractions evoked by noradrenaline (NA, 10 M) (A) or caffeine (20 mM) (B) in
resistance mesenteric rings with intact endothelium maintained under Ca2+-free
conditions. In A, note that inhibitory effect of T4MN (5.6 M) was blunted in the
presence of ODQ (10 M). Black bars indicate a reference 60 mM K+-induced
contraction in Ca2+-containing medium. Data are expressed as the mean ± S.E.M. (n =
5-8 per group). *P < 0.01 by one-way ANOVA followed by Dunnett´s test with respect
to NA-induced contraction in the absence of T4MN.

29
Table 1. IC50 values for the vasorelaxant effects of trans-4-trans-β-nitrostyrene in
resistance mesenteric ring preparations pre-contracted with KCl (60 mM) or
noradrenaline (NA, 10 µM) subjected to various pre-treatments. Values are expressed as
geometric mean [95% confidence interval] and number within parentheses indicates the
a
b
number of preparations for each group. P < 0.05, P < 0.01 by Mann-Whitney U-test
vs. Control (E+) in pharmacological coupling (10 µM NA).
Contractile agent/Pretreatments
IC50 (µM)
n
Noradrenaline
Control (E+)
Endothelium removal (E-)
+ L-NAME
3.92 [0.67-5.47]
(6)
(6)
(6)
(5)
(6)
(5)
(6)
(6)
(6)
(6)
(5)
(5)
(5)
32.45 [20.15-66.50]b
31.74 [25.44-57.30] b
5.68 [3.33-8.02]
+ Atropine
+ Indomethacin
+ LY294002
+ ODQ (E+)
+ ODQ (E-)
+ TEA
+ Glybenclamide
+ Apamin
6.10 [1.82-7.19]
7.67 [5.96-18.50]a
29.84 [22.34-60.86]b
38.52 [20.92-93.90]b
42.24 [20.24-95.75]b
33.60 [28.05-39.75]b
24.79 [17.04-26.07]b
12.20 [6.90-27.40]a
17.32 [9.12-28.10]a
+ TRAM 34
+ [Apamin + TRAM 34]
KCl
Control (E+)
10.52 [8.81-12.88]a
(6)
L-NAME, L-NG-nitroarginine methyl ester; ODQ, 1H-[1,2,4] oxadiazolo [4,3-a]
quinoxaline-1-one; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one
hydrochloride; TEA, tetraethylammonium chloride.