Clin Exp Pharmacol Physiol. 2017;44:787–794. wileyonlinelibrary.com/journal/cep © 2017 John Wiley & Sons Australia, Ltd
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787 O R I G I N A L A R T I C L EMechanism of the vasorelaxant effect induced by trans- 4-
methyl-
β
- nitrostyrene, a synthetic nitroderivative, in rat
thoracic aorta
Taylena Maria Teófilo
1|
Loeste Arruda-Barbosa
1|
Jussara Mathyelle
Rodrigues-Silva
1|
Joyce Karen Lima Vale
3|
Rosivaldo Santos Borges
3|
Gloria Pinto Duarte
2|
Pedro Jorge Caldas Magalhães
1|
Saad Lahlou
11School of Medicine, Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, CE, Brazil 2Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, PE, Brazil
3Department of Pharmacy, Federal University
of Pará, Belém, PA, Brazil
Correspondence
Saad Lahlou, Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil. Email: lahlou562@gmail.com
Funding information
CNPq, Edital Universal 14/2012, Grant/Award Number: 484656/2012-0; FAPESPA, ICAAF, Grant/Award Number: 177/2014
Summary
Mechanisms underlying the vasorelaxant effects of trans- 4- methyl- β- nitrostyrene (T4MeN) were studied in rat aortic rings. In endothelium- intact preparations, T4MeN fully and similarly relaxed contractions induced by phenylephrine (PHE) (IC50 = 61.41
[35.40- 87.42] μmol/L) and KCl (IC50 = 83.50 [56.63- 110.50] μmol/L). The vasorelax -ant effect of T4MeN was unchanged by endothelium removal, pretreatment with L- NAME, indomethacin, tetraethylammonium, ODQ or MDL- 12,330A. Under Ca2+- free conditions, T4MeN significantly reduced with a similar potency: (i) phasic contractions induced by PHE, but not by caffeine; (ii) contractions due to CaCl2 in aortic prepara -tions stimulated with PHE (in the presence of verapamil) or high KCl; (iii) contrac-tions evoked by the restoration of external Ca2+ levels after depletion of intracellular Ca2+ stores in the presence of thapsigargin. In contrast, T4MeN was more potent at inhibit -ing contractions evoked by the tyrosine phosphatase inhibitor, sodium orthovanadate, than those induced by the activator of PKC, phorbol- 12,13- dibutyrate. These results suggest that T4MeN induces an endothelium- independent vasorelaxation that ap -pears to occur intracellularly through the inhibition of contractions that are independ -ent of Ca2+ influx from the extracellular milieu but involve phosphorylation of tyrosine residues.
K E Y W O R D S
endothelium-independent vasorelaxation, nitroderivative, trans-4-methyl-β-nitrostyrene,
tyrosine residues phosphorylation, vascular smooth muscle
1
|
INTRODUCTION
β- Nitrostyrenes derivatives display several biological activities: an -tibacterial,1 fungicidal,2 antileishmanial,3 anti- inflammatory,4,5 anti-platelet,6 pro- apoptotic,7,8 and anticancer.9,10 Nitroderivatives occur
rarely in nature. 1- Nitro- 2- phenylethane is the first nitro compound to have been isolated from plants,11 and is responsible for the
cinnamon scent of two Brazilian species of the Family Lauraceae,
Ocotea pretiosa and Aniba canellila.12 Recently, it was reported that
analogue, 1- nitro- 2- phenylethane.13 1- Nitro- 2- phenylethene can therefore be used as a scaffold to synthesize more potent novel de -rivatives with potential as antihypertensive drugs.
In the present study, trans- 4- methyl- β- nitrostyrene (T4MeN) was
synthesized (Figure 1B), and mechanisms underlying its vasorelaxant effects were studied in rat aortic ring preparations. We hypothesized that introducing an electron- releasing group such methyl in the para- position of the aromatic moiety of 1- nitro- 2- phenylethene would con -fer improved vasorelaxant activity in comparison with its parent drug, 1- nitro- 2- phenylethene.
2
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RESULTS
2.1
|
Vasorelaxant effects of T4MeN in aortic rings:
role of the endothelium
Incubation of endothelium- intact preparations (n=6) with increas -ing concentrations of T4MeN (0.6- 612.8 μmol/L) fully relaxed
aortic preparations previously contracted by PHE (1 μmol/L) in a
concentration- dependent manner (Figure 2A, c; P<.001, one- way ANOVA). Vehicle had no such effect (Figure 2B, n=6). The IC50 of the T4MeN effect did not differ significantly from that obtained in endothelium- denuded preparations (n=6) (Table 1, P>.05, Mann- Whitney U- test). Endothelium- intact aortic rings under sustained contractions evoked by eletromechanical coupling (60 mmol/L KCl) were also concentration- dependently relaxed by T4MeN, with an IC50 that was not statistically different from that recorded in PHE- elicited pharmacomechanical coupling (Table 1; P>.05,
Mann- Whitney U- test). As a positive control, sodium nitroprusside (0.1 nmol/L- 10 μmol/L) fully relaxed sustained contractions
in-duced by PHE (1 μmol/L) with an IC50 of 4.90 (2.96- 11.82) nmol/L.
Pretreatment of aortic endothelium- intact rings with L- NAME (100 μmol/L, n=7) (Figure 2D) or indomethacin (10 μmol/L, n=5) F I G U R E 1 The chemical structure of (A) 1- nitro- 2- phenylethene
and (B) a general procedure for synthesis of trans- 4- methyl- β- nitrostyrene
F I G U R E 2 Typical trace recordings of the effect of (A) trans- 4- methyl- β- nitrostryrene (T4MeN, 0.6- 612.8 μmol/L)
and (B) its vehicle on sustained
contractions induced by 60 mmol/L KCl in rat isolated aortic preparations. (C) Vascular effects of T4MeN in endothelium- intact (E+) and endothelium- denuded (E- ) aortic rings pre- contracted with phenylephrine (PHE, 1 μmol/L) and in endothelium- intact
preparations pre- contracted with KCl (60 mmol/L). ( ) Vehicle; ( ) PHE (E+); ( ) PHE (E–); ( ) KCl (E+).
(D) Effects of pretreatment with L- NAME (100 μmol/L) and indomethacin (Indo,
10 μmol/L) on the vasorelaxant effects of
T4MeN in endothelium- intact aortic rings pre- contracted with PHE. ( ) Vehicle; ( ) PHE (E+); ( ) +L- NAME; ( ) +Indomethacin. Data are expressed as mean ± SEM (n=5-7 per group). *P<.05 by
(Figure 2D) did not alter the vasorelaxant effects of T4MeN against the contractions induced by 1 μmol/L PHE (Table 1; P>.05, Mann-
Whitney U- test).
2.2
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Investigation of the role of potassium
channels and cyclic nucleotides
Potassium channels were not involved in the vasorelaxant effects of T4MeN as they were unaltered by pretreatment with TEA (5 mmol/L, n=7) (Figure 3, Table 1; P>.05, Mann- Whitney U- test). Likewise,
pre-treatment with ODQ (10 μmol/L, n=8) or with the adenylyl cyclase in -hibitor MDL- 12,330A (3 μmol/L, n=6) did not affect the vasorelaxant
activity of T4MeN (Figure 3, Table 1; P>.05, Mann- Whitney U- test).
2.3
|
Investigation of the involvement of
calcium channels
Under Ca2+- free conditions, we investigated whether T4MeN inhibits
contractions induced by a transmembrane influx of exogenous Ca2+.
For this purpose, aortic ring preparations were depolarized with KCl (60 mmol/L) in the presence of EGTA (50 μmol/L) to activate voltage-
operated calcium channels. In another set of experiments, aortic rings were pretreated with verapamil (1 μmol/L) to block voltage- operated
calcium channels and stimulated by PHE (1 μmol/L) to preferentially
activate receptor- operated calcium channels. Figure 4 shows that concentration- dependent contractions due to the addition of CaCl2 (0.1-
20 mmol/L) in both KCl- (Figure 4A) and PHE- stimulated (Figure 4B) aortic preparations remained unaffected by 18.5 μmol/L T4MeN (n=6
and 6, respectively), but were significantly reduced by 61.3 μmol/L T4MeN (n=5 and 9, respectively; P<.01, two- way ANOVA).
In a separate series of experiments conducted under Ca2+- free
conditions, the putative inhibitory effects of T4MeN against contrac-tions evoked by Ca2+ influx through store- operated Ca2+ channels
activated by Ca2+ store depletion14 were also investigated. For this
purpose, aortic preparations were initially bathed in Ca2+- free Krebs-
Henseleit solution (KHS) (containing EGTA 100 μmol/L) and depletion
of intracellular Ca2+ stores was achieved through at least three succes
-sive exposures to PHE (10 μmol/L) until no detectable contraction was
recorded. After removing PHE from the extracellular medium, a control cumulative concentration- response curve for Ca2+ (0.1- 20 mmol/L)
was performed 10 minutes after thapsigargin (1 μmol/L), an inhibitor of
the sarco- endoplasmic reticulum Ca2+- ATPase (SERCA) was added to
the bath.15 Such contractions due to exogenous addition of Ca2+ were
unaltered by 18.5 μmol/L T4MeN (n=8), but were almost abolished by
61.3 μmol/L (n=6) T4MeN (Figure 4C; P<.001, two- way ANOVA).
2.4
|
Investigation of the involvement of intracellular
calcium signalling
To investigate whether T4MeN affects contractions evoked by sarcoplasmic reticulum Ca2+ channels activated by inositol triphos
-phate (IP3), aortic preparations were bathed in Ca2+- free KHS
(con-taining EGTA 1 mmol/L) and a transient contraction was recorded after the addition of PHE (1 μmol/L). This contraction
corre-sponded to 38.0 ± 5.1% (Figure 5A, n=6) of a reference contraction induced by 60 mmol/L K+ in Ca2+- containing medium. After a
sub-sequent contractile stimulus with 60 mmol/L KCl in normal KSH to refill Ca2+ internal stores, aortic preparations were treated for
5 minutes with 18.5 or 61.3 μmol/L T4MeN in Ca2+- free KHS (con -taining EGTA 1 mmol/L). Pre- exposure of aortic ring preparations to T4MeN at 18.5 (n=6) or 61.3 (n=6) μmol/L significantly reduced
the magnitude of PHE- induced phasic contractions to 18.0 ± 4.2 and 8.2 ± 3.3% (Figure 5A; P<.05, one- way ANOVA followed by
Dunnett′s test), respectively.
In a second series of experiments performed under Ca2+- free
con-ditions, we used caffeine as a pharmacological tool to assess whether
T A B L E 1 IC50 values for the vasorelaxant effects of trans- 4- methyl- β- nitrostyrene in aortic ring preparations pre- contracted with
KCl (60 mmol/L) or phenylephrine (1 μmol/L) 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/
Pretreatments IC50 (μmol/L) n
Phenylephrine
Control 61.41 [35.40–87.42] (6)
Endothelium removal 73.21 [41.89–104.50] (6)
+TEA 103.30 [34.25–172.40] (7)
+L- NAME 80.80 [30.55–131.10] (7)
+ODQ 79.41 [64.92–93.90] (8)
+Indomethacin 83.39 [14.55–152.20] (5)
+MDL- 12,330A 149.50 [52.80–246.30] (6)
KCl
Control 83.50 [56.63–110.50] (6)
TEA, tetraethylammonium chloride; L- NAME, L- NG- nitroarginine me
-thyl ester; ODQ, 1H- [1,2,4] oxadiazolo [4,3- a] quinoxaline- 1- one; MDL- 12,330A, cis- N- (2- phenylcyclopentyl)- azacyclotridec- 1- en- 2- amine
hydrochloride.
F I G U R E 3 Effects of the pretreatment with tetraethylammonium (TEA, 5 mmol/L), ODQ (10 μmol/L) or MDL- 12,330A (3 μmol/L)
on the vasorelaxant effects of trans- 4- methyl- β- nitrostryrene
(T4MeN, 0.6- 612.8 μmol/L) in endothelium- intact aorta contracted
with phenylephrine (1 μmol/L). ( ) Vehicle; ( ) PHE (E+); ( )
T4MeN interferes with contractions that involve Ca2+ release from
the sarcoplasmic reticulum via ryanodine- sensitive Ca2+ channels.
Stimulation of aortic preparations with caffeine (20 mmol/L, at 25°C) induced a transient contraction with a magnitude that corresponded to 18.2 ± 5.1% (n=7) of a reference contraction induced by 60 mmol/L K+ in Ca2+- containing medium. The magnitude of this contraction
re-mained unchanged by T4MeN, either at 18.5 (n=7) or 61.3 (n=7) μmol/L (Figure 5B; P>.05, one- way ANOVA followed by Dunnett′s test).
2.5
|
Inhibitory effects of T4MeN on
contractions induced by phorbol 12,13- dibutyrate
(PDB) or orthovanadate
In aortic rings incubated in Ca2+- free medium (in the presence of
1 mmol/L EGTA), when contractions induced by the PKC activator,
PDB (1 μmol/L), reached steady- state, cumulative addition of increas -ing concentrations (0.6- 612.8 μmol/L) of T4MN (Figure 6A, n=7)
significantly reduced the PDB- induced contractions in a concentration- dependent manner (P<.001, one- way ANOVA). The IC50 (258.01
[119.80- 397.41] μmol/L) was significantly higher than that for the
inhibitory effects of T4MeN against PHE- induced contractions in aor -tic preparations incubated in Ca2+- containing medium (P<.01, Mann-
Whitney U- test).
Under the same conditions, increasing concentrations of T4MeN (0.6-612.8 μmol/L) also significantly reduced the contractions
evoked by the tyrosine phosphatase inhibitor, sodium orthovanadate (3 mmol/L, n=7) (Figure 6B) in a concentration- dependent manner (P<.001, one- way ANOVA). The IC50 (18.64 [3.24- 34.04] μmol/L) for
this effect was significantly (P<.01, Mann- Whitney U- test) lower than
that evoked by T4MeN against contractions evoked by PHE (normal KHS) or PDB (under Ca2+- free conditions).
3
|
DISCUSSION
The present study shows that T4MeN displays interesting vasorelax -ant actions that in rat aorta are independent of the integrity of the endothelium. This effect seems mediated through a myogenic mecha -nism rather than via inhibition of Ca2+ influx from the extracellular F I G U R E 4 Inhibitory effects of trans- 4- methyl- β- nitrostryrene
(T4MeN, 18.5 and 61.3 μmol/L) on the cumulative concentration-
effect curve for CaCl2 (0.1- 20 mmol/L) in endothelium- intact
aortic rings (maintained in Ca2+- free medium) stimulated with (A)
60 mmol/L KCl, (B) 1 μmol/L phenylephrine (PHE), or (C) with PHE
after simultaneous depletion of intracellular Ca2+ stores by PHE
(10 μmol/L) and inhibition of SERCA by 1 μmol/L thapsigargin. Data
are expressed as mean ± SEM (n=6- 8 per group). #P<.01 by two- way
ANOVA, T4MeN vs concentration- contraction curve of Ca2+ alone. ( ) Control; ( ) T4MeN (18.5 μmol/L); ( ) T4MeN (61.3 μmol/L)
F I G U R E 5 Inhibitory effects of trans- 4- methyl- β- nitrostryrene
(T4MeN, 18.5 and 61.3 μmol/L) on contractions evoked by
(A) phenylephrine (PHE, 1 μmol/L) or (B) caffeine (20 mmol/L)
in endothelium- intact aortic rings maintained under Ca2+- free
milieu, and appears mainly dependent on levels of phospholyrated tyrosine residues. Vascular endothelium plays an important role controlling vascular tone, mainly through synthesis/liberation of endothelial- derived relaxing factors (EDRFs), such as NO and pros -tacyclin (PGI2).16 The vasorelaxant effect of T4MeN is independent
of the integrity of the endothelial layer, as it remained unaltered by vascular endothelium removal. Neither released NO nor prostaglan-dins from endothelial cells are involved in the vasorelaxant effect of T4MeN, as it remained unchanged following pretreatment with L- NAME or the non- selective cyclooxygenase inhibitor, indomethacin, respectively. Because the non- selective KCa channel blocker, TEA, did not change the vasorelaxant effect of T4MeN, it is improbable that this effect resulted from the putative ability of T4MeN to open K+-
channels. Additionally, T4MeN- induced vasorelaxation remained un -changed when aortic preparations were pretreated with the guanylyl cyclase inhibitor, ODQ17 and the adenylate cyclase inhibitor, MDL-
12,330A.18,19 This indicates that neither cGMP- dependent nor cAMP-
dependent process are involved in mediating the relaxant effect of T4MeN in aortic preparations.
Calcium influx from the extracellular medium through different calcium channels plays a crucial role in vascular smooth muscle (VSM) contraction. It is known that store- operated Ca2+ channels are acti
-vated by depletion of Ca2+ stores within the sarcoplasmic reticulum,
allowing a capacitative Ca2+ influx into the cytosol and a sustained
contraction.20 Our results revealed that when aortic preparations were
subjected to simultaneous depletion of Ca2+ stores by PHE and inhibi
-tion of SERCA by thapsigargin, the sustained contrac-tion induced by the addition of Ca2+ was abolished by T4MeN. Contraction evoked by
high KCl in VSM is due to membrane depolarization, leading to Ca2+
in-flux through voltage- operated calcium channels. This increases [Ca2+] i,
and produces sustained contraction following myosin light chain phos-phorylation.21 T4MeN interferes with contractile events mediated by
Ca2+ entry through voltage- operated calcium channels since it inhibited
contractions evoked by KCl in Ca2+- containing medium and those elic
-ited by the cumulative addition of CaCl2 in aortic preparations depolar-ized with high KCl, under Ca2+- free conditions. In pharmacomechanical
coupling, activation of α1- adrenoceptors by PHE leads 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 is due to increased Ca2 + sensiti
-zation of the contractile apparatus and to Ca2+ influx through receptor-
operated calcium channels.22,23 Our results show that T4MeN reduced PHE- induced contractions in Ca2+- containing medium and also it
in-hibited CaCl2- induced contractions in aortic preparations stimulated
with PHE under Ca2+- free conditions. Taken together, these findings
suggest that T4MeN interferes with contractile events elicited by Ca2+
entry through both voltage- operated calcium channels and receptor- operated calcium channels (and also store- operated Ca2+ channels).
T4MeN inhibited both electromechanical and pharmacomechanical couplings with similar potencies, a finding that supports the idea of a myogenic action as the underlying mechanism by which T4MeN in-duces its vasorelaxant effect.
Accordingly, we investigated whether T4MeN interferes with con-tractions induced by intracellular events, such as Ca2+ release from
internal stores in the sarcoplasmic reticulum. In Ca2+- free medium,
T4MeN was ineffective against transient contractions evoked by caf -feine, whereas it inhibited phasic contractions induced by PHE. This indicates that T4MeN effectively inhibits Ca2+ mobilization from intra
-cellular stores mediated by IP3 without interfering with contractions that involve Ca2+ release from the sarcoplasmic reticulum via ryanodine-
sensitive Ca2+ channels.24,25 A similar activity profile has been reported for other nitroderivatives, such as 1- nitro- 2- phenylethane26 and its
structural analogue, 1- nitro- 2- phenylethene.13 It is noteworthy that the IP3 signalling blocking effect of T4MeN was evident at lower con -centrations (18.5 μmol/L) compared to its ability to relax aortic
prepa-rations pre- contracted with PHE (IC50 = 61.41 μmol/L).
PKC is abundant in VSM cells and plays an important role in the regulation of vascular tone as it increases Ca2 + sensitivity to contrac
-tile proteins.27,28 Under physiological conditions, PKC can be activated
by diacylglycerol generated by the binding of PHE to α1- adrenoceptors
and pharmacologically by PDB, a potent- specific PKC activator.29,30 Under Ca2 +- free conditions (in the presence of EGTA), incubation of
endothelium- containing aortic preparations with PDB elicited a sus-tained contraction, an effect that was reversed by T4MeN. This sug -gests that T4MeN may interfere with cellular events that increase PKC- mediated Ca2+ sensitivity of contractile proteins. However, it is
unlikely that T4MeN could act as a PKC inhibitor since its potency to
F I G U R E 6 Effects of cumulative and increasing concentrations of trans- 4- methyl- β- nitrostryrene (T4MeN, 0.6- 612.8 μmol/L) on
sustained contractions induced by (A) phorbol 12,13- dibutyrate (PDB, 1 μmol/L) or (B) by sodium orthovanadate (3 mmol/L) in aortic
ring preparations maintained under Ca2 +- free conditions. Data are
relax contractions induced by PDB was less than that to relax contrac-tions induced by phenylephrine.
Protein tyrosine kinases are involved in many different signalling pathways. Among these, phosphorylation of tyrosyl residues following tyrosine kinase activation is strongly implicated in the mechanism of agonist- induced contraction in VSM.31,32 The level of phosphorylated tyrosine residues is regulated by a balance between the actions of ty-rosine kinases and tyty-rosine phosphatases. Sodium orthovanadate, a potent protein tyrosine phosphatase inhibitor, induces smooth mus-cle contractions through the reduction of dephosphorylated tyro -sine residues, thus indirectly increasing the level of phosphorylated tyrosine.33,34 This vasoconstriction seems partially mediated through calcium released from intracellular stores35 and by Rho- kinase- dependent inactivation of myosin light chain phosphatase via sig -nalling downstream of Src- induced transactivation of the epidermal growth factor receptor.36 Under Ca2 +- free conditions, T4MeN was
able to fully relax contractions evoked by sodium orthovanadate with greater potency than that in preparations pre- contracted with PHE in Ca2+- containing medium. This suggests that vasorelaxant effects of
T4MeN preferentially depend on the level of phosphorylated tyrosine residues. Measurement of tyrosine phosphorylation by direct exper-imental methods may certainly strengthen the argument for such a mechanism of action of T4MeN.
Contrary to expectations, the methyl substitution in the para- position of the aromatic ring did not increase the potency of 1- nitro- 2- phenylethene′s vasorelaxant effect in rat aortic rings (70.74 [31.62- 109.86] μmol/L13). However, it seems that this substitu-tion conferred upon T4MeN greater selectivity against contracsubstitu-tions evoked by enhanced tyrosine phosphorylation than its parent drug, 1- nitro- 2- phenylethene (93.26 [6.32- 249.74] μmol/L,13).
This study shows, for the first time, that T4MeN induces an endothelium- independent relaxation of aortic preparations pre- contracted by phenylephrine, an effect that seems to occur intracel-lularly. This most likely occurs via inhibition of contractions that are independent of extracellular Ca2+ influx, preferentially those evoked
by enhanced tyrosine phosphorylation through an indirect increase of tyrosine kinase activity.
4
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METHODS
4.1
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Synthesis of trans- 4- methyl-
β
- nitrostyrene
1- ((E)- 2- nitro- vinyl)- (4- methyl)- benzene or T4MeN was synthesized
by the Claisen- Schmitd procedure37,38 using p- tolualdehyde and ni -tromethane as substrates (0.1 and 0.12 eq., respectively) (Figure 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 (1:2).39 The resulting precipitate was filtered and dried under vacuum to give the desired β- nitrostyrene
derivative, T4MeN. The trans product is preferential than to the cis
form due to a stereoselective reaction that gives a product of low en-ergy. T4MeN was crystallized in ethanol as yellow solid- crystals; m.p. 101.6- 103.9°C (102- 104°C; Sigma- Aldrich standard, St Louis, MO,
USA). The final product was identified by NMR (1H and 13C NMR) and FT- IR spectroscopy and compared with properties in the literature.40
IR νmax (/cm) 495.71, 810.10, 964.41, 1330.0, 1496.76, 1604.77,
1631.78; 1H NMR (CDCl
3, 300 MHz) δ 8.1 (d, 2H), 7.59 (d, 2H), 7.43 (d, 1H), 7.26 (dd, 1H), 2.41 (s, 3H).
4.2
|
Animals
Adult male Wistar rats (280- 340 g) were kept under conditions of constant temperature (22 ± 2°C) with a 12 hour light/12 hour dark cycle and 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 and approved by the institutional animal ethics committee (127/2014).
4.3
|
Solutions and drugs
The perfusion medium used was a fresh modified KHS, pH 7.4) of the following composition (in mmol/L): NaCl 118; KCl 4.7; NaHCO3 25; CaCl2.2H2O 2.5; KH2PO4 1.18; MgSO4.7H2O 1.18; glucose 11.
Calcium- free solutions were prepared by omitting CaCl2 from nor-mal KHS. Salts were purchased from Merck (Darmstadt, Germany) and Vetec (Rio de Janeiro, Brazil). Phenylephrine (PHE) hydro-chloride, acetylcholine hydro-chloride, 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), tetraethylammonium chloride (TEA), thapsigargin, phorbol- 12,13- dibutyrate (PDB), L- NG- nitroarginine methyl ester (L- NAME), cis- N- (2- phenylcyclopentyl)- azacyclotridec- 1- en- 2- amine hydrochloride (MDL- 12,330A), caffeine, sodium nitroprusside, 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 was added directly to Ca2+- free KHS). T4MeN was dissolved
in ethanol, brought up to the desired concentration using KHS, and sonicated just before use. Indomethacin (cyclooxygenase inhibitor), L- NAME (inhibitor of nitric oxide synthase), ODQ (guanylyl cyclase inhibitor), MDL 12,330A (adenylyl cyclase inhibitor) or TEA (blocker of large- conductance Ca2+- activated K+ channels) were applied to
the bath 10 minutes prior to pre- contraction of endothelium- intact preparations with PHE (1 μmol/L). T4MeN was added in increasing
concentrations during steady- state contraction to investigate its vasorelaxant effects.
resting tension of 1 g, which was readjusted every 15 minutes dur-ing a 45- minutes equilibration period before drug administration. Isometric tension was recorded using an isometric force displace-ment transducer (Grass Model FTO3, Quincy, MA, USA) connected to an acquisition system (PM- 1000; CWE Inc., Akron, OH, USA). Vessels were initially exposed twice to 60 mmol/L KCl to check their functional integrity. After 30 minutes, rings were contracted with a concentration (0.1 μmol/L) of PHE inducing 50- 70% of the con -traction induced by KCl. Then acetylcholine (1 μmol/L) was added
to assess endothelium integrity. In one series of experiments, en -dothelium was removed immediately after dissection by gentle rub-bing of the aortic lumen with a stainless steel wire. The absence of acetylcholine- induced relaxation was taken as an indicator of suc-cessful endothelium removal. Control rings were exposed only to the vehicle used to dissolve T4MeN.
4.5
|
Statistical analysis
Results are expressed as the mean ± SEM. IC50 values, defined as the concentration (μmol/L) of T4MeN required to produce half- maximal
reduction of the contractile response induced by an agonist, were calculated by interpolation from semi- logarithmic plots, and were expressed as geometric mean [95% confidence interval]. The signifi-cance (P<.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.
ACKNOWLEDGEMENTS
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 Taylena Maria Teófilo in order to obtain his Ph.D. degree in Pharmacological Sciences at the Federal University of Ceará, Fortaleza, Brazil. Loeste Arruda- Barbosa 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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How to cite this article: Teófilo TM, Arruda-Barbosa L, Rodrigues-Silva JM, et al. Mechanism of the vasorelaxant effect induced by trans- 4- methyl- β- nitrostyrene, a synthetic
