www.elsevier.com/locate/vph

Vascular Pharmacology

An ethyl acetate fraction obtained from a Southern Brazilian red wine

relaxes rat mesenteric arterial bed through hyperpolarization and

NO-cGMP pathway

Elke Zuleika Schuldta,*, Angela Cristina Beta, Mariana Appel Horta, Carla Ianssenb,

Marcelo Maraschinb, Karina Cklessc, Rosa Maria Ribeiro-do-Vallea

aDepartamento de Farmacologia, CCB, UFSC, Florianopolis, BrazilbLaboratorio de Fitotecnia e Morfogenese Vegetal, CCA, UFSC, Florianopolis, Brazil

cDepartment of Pathology, University of Vermont, HRSF Building, Room 214, Burlington, VT 05405, USA

Received 29 December 2004; received in revised form 23 March 2005; accepted 22 April 2005

Abstract

A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing

the risk of coronary heart disease (CAD). In this study, we investigated the effect of a crude extract (CE), as well as an ethyl acetate fraction

(EAF) obtained from a Brazilian red wine in the mesenteric arterial bed (MAB) from rats.

Our results showed that after the tonus of MAB was increased with phenylephrine (PE), increasing concentrations of CE induced a

concentration-dependent relaxation; moreover, EAF was more potent in relaxing the MAB when compared with CE. In vessels depolarized

with KCl (80 mM) or treated with the Na+/K+-ATPase pump inhibitor, ouabain (OUA; 100 AM), or with the K+ channel blockers: barium

(BaCl2, 100 AM) and tetraethylammonium (TEA; 500 AM ), the effect of EAF was significantly reduced. However, this effect was not altered

by the ATP-dependent K+ (KATP) channel blocker, glibenclamide (GLI; 100 AM) as well as Charybdotoxin (ChTx 10 nM), a nonselective

inhibitor of KCa channels of large and intermediate conductance plus Apamin (Apamin 100 nM), a specific inhibitor of KCa channels of small

conductance. The residual vasodilator effect of EAF observed in vessels pretreated with L-NOARG (100 AM), 1H-[1,2,4,] oxadiazolo[4,3-

alfa]quinoxalin, ODQ (10 AM) or KCl (80 mM), given separately, was reduced by the administration of KCl (40 mM) plus L-NOARG (100

AM). The present study demonstrates that the vasodilator effect of EAF is partially dependent upon membrane hyperpolarization in

combination with nitric oxide (NO) release.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Wine; Mesenteric arterial bed; NO; Hyperpolarization

1. Introduction

Consumption of polyphenol-rich foods, such as fruits

and vegetables, and beverages derived from plants, such as

cocoa, red wine and tea, may represent a beneficial diet in

terms of cardiovascular protection (Yamakoshi et al., 1999;

1537-1891/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.vph.2005.04.002

* Corresponding author. Universidade Federal de Santa Catarina-UFSC,

Coordenadoria Especial de Farmacologia/BLOCO ‘‘D’’, Campus Universi-

tario-Trindade-Florianopolis-SC, CEP: 88049-900, Caixa Postal: 476,

Brazil. Tel.: +55 48 331 9491, +55 48 331 9764; fax: +55 48 337 5479.

E-mail address: elkeschuldt@pop.com.br (E.Z. Schuldt).

Stoclet et al., 2004). Among the numerous plausible

mechanisms by which polyphenols may confer cardiovas-

cular protection, improvement of the endothelial function

and inhibition of angiogenesis and cell migration and

proliferation in blood vessels have been the focus of recent

studies. (Howard et al., 2002; Pal et al., 2002; Stoclet et al.,

2004).

Recently, it was reported that incubation of endothelial

cells with French red wines up-regulated endothelial-type

nitric oxide synthase (eNOS) expression and eNOS activity,

but in contrast, red wines from Germany showed little or no

effect on eNOS expression (Wallerath et al., 2003). Red

43 (2005) 62 – 68

E.Z. Schuldt et al. / Vascular Pharmacology 43 (2005) 62–68 63

wine contains a range of polyphenols derived from the skin

of the grape and these include flavonols (quercetin and

myricetin), gallic acid, condensed tannins (catechin and

epicatechin polymers) and polymeric anthocyanins (Frankel

et al., 1995). Variations in the concentration of these

constituents among red wines may be responsible for the

range of biological effects and this suggests that not all red

wines might have the same pharmacological properties

(Ritchey and Waterhouse, 1999; Leighton et al., 1999;

Howard et al., 2002; Soares de Moura et al., 2004).

Red wine is particularly rich in polyphenol substances

compared to white wine. Polyphenols are potent in

preventing lower density lipoprotein oxidation, the corner-

stone event in the oxidation theory of atherogenesis and may

play an important role in preventing cardiovascular disease

(Schuldt et al., 2000; Vinson et al., 2001).

Since it has been hypothesized that other mediators than

NO may be involved in vasodilator responses in small

vessels (such as coronary and mesenteric arteries), and

because the mechanisms of these mediators may involve K+

channels (Soares de Moura et al., 2004; Dabisch et al.,

2004; Ndiaye et al., 2004), the current study was undertaken

to investigate the role of NO, as well as K+ channels in

mediating the vasodilator responses to an ethyl acetate

fraction (EAF) obtained from a Southern Brazilian red wine,

in vitro.

2. Materials and methods

2.1. Alcohol-free extract and organosolvent fraction

Red wine (variety Bordeaux-Vitis labrusca L.), thought

to have originated from Southern Brazil (Vale do Rio do

Peixe, Tangara-Santa Catarina state), from the 2000 vintage.

The bottles were kept in the dark and at 20–22 -C prior to

analysis.

Aliquots of 300 mL of Bordeaux red wine were collected

and the alcohol-free extract (CE) was obtained under

reduced pressure (80 -C, ¨50 min), following measurement

of the ethanol content using an alcoholometer apparatus

(Incoterm Co.). The polyphenols in the alcohol-free extract

(ca. 100 mL) were isolated by phase partitioning with ethyl

acetate (EtOAc–300 mL, 24 h incubation at room temper-

ature/in the dark). The organic phase (residue) was collected

and the EtOAc was removed by evaporation using an N2

stream. During the preparation of the alcohol-free extract

and the organosolvent fraction (EAF), the samples were

constantly protected from light to avoid photochemical

isomerization of any polyphenol compound.

Since the pharmacological protocols could not be carried

out in the presence of solvents, it was necessary to

determine the level of dry matter beginning with the

obtained residue. To this end, the residue was transferred

to Petri dishes and maintained in an incubator (;60 -C) untilit had reached constant weight. Through the resuspension of

the dry matter in deionized water, samples of CE and EAF

were obtained, which were used in the subsequent pharma-

cological experiments.

2.2. Pharmacological assays—Mesenteric Arterial Bed

The animal care research protocols were in accordance

with the guidelines of our university and were approved by

our Institutional Ethics Committee.

Male Wistar rats were anesthetized with a mixture of

ketamine (Francotar\) and 2-(2,6-xilidino)-5,6-dihydro-4H-

1,3-tiazine chlorhydrate (Rompum\) and a stainless poly-

ethylene cannula (PE-50) was inserted into the superior

mesenteric artery, then flushed with 10 mL of PBS

containing heparin (10 IU/mL) to remove possible blood

clots. After ligating all branches except the 4 or 5 directed to

the terminal ileum, the whole mesentery was cut along the

ileal border and transferred to a humidifying chamber

(McGregor, 1965), where it was perfused with 5 mL of

warm (37 -C) Krebs solution (composition in mM): NaCl,

113; KCl, 4.7; CaCl2, 2.5; KH2PO4, 0.9; NaHCO3, 25;

MgSO4, 1.1; glucose, 11; pH 7.4, saturated with 5% CO2 in

O2. To investigate the possible influence of endogenous

eicosanoids, concentration–response curves (CRC) to CE

an EAF were conducted in the presence of indomethacin.

According to our experiments, indomethacin does not affect

the vasodilator effect of CE as well as EAF (data not

shown), and for this reason, all subsequent experiments

were conducted in the presence of indomethacin (10 AM),

added to Krebs solution.

The perfusion pressure (PP) was measured by a trans-

ducer connected to a preamplifier and chart recorder (Digi-

Med, model 190, USA). Since the flow rate was constant

throughout the whole experimental period (4.0 mL/min),

any pressure alteration reflected changes in vascular

resistance. Drugs were either dissolved in Krebs solution

and perfused at the desired concentration or were adminis-

tered as bolus injections directly into the perfusion stream

(volume=100 AL). All preparations were allowed to

equilibrate for 30 min before the start of the experiments.

After this period, isolated injections of CE or EAF (30–

3000 Ag; bolus) were administered until consistent

responses were obtained. The basal pressure after the

equilibration period was 19.85T0.30 mm Hg (n =20).

Concentrations of CE or EAF were injected after the PP

had been elevated (70–80 mm Hg) with phenylephrine (PE,

750–1500 Ag; added to the perfusion fluid and adjusted to

maintain the same increase in PP). Acetylcholine (ACh, 200

nmol, bolus) was also injected to test endothelium function-

ality before concentration–response curves to CE and EAF

were obtained. All vasodilator responses to ACh, CE and

EAF were obtained in vessels precontracted with PE. As CE

and EAF induced a long-lasting inhibition of the PE

constrictor effect (Register 1), only one dose–response

curve to CE or EAF was obtained in each arterial bed

preparation.

120,0

90,0

60,0

30,0

,00mmhg

30100

300

30001000

Krebs05minutes

Register 1. Representative perfusion pressure (mm Hg) trace showing the

vasodilator effect of EAF in mesenteric arterial bed (MAB) during

phenylephrine (Phe; 750–1500 Ag) treatment. Arrows indicate injections

of EAF (30–3000 Ag).

30 100 300 1000 3000

0

25

50

75

100

CE (+E)EAF (+E)

*

**

Samples [µg]

Dec

reas

e in

PP

(%

)

Fig. 1. Effect of CE and EAF in the isolated MAB: CRC for CE (h) andEAF (?), in the rat mesenteric arterial bed (MAB) precontracted with

phenylephrine. Values represent the meanTS.E.M. of five to six experi-

ments. *p <0.05; **p <0.01, unpaired t-test.

E.Z. Schuldt et al. / Vascular Pharmacology 43 (2005) 62–6864

The vasodilator effects of EAF (30–3000 Ag; bolus)

were studied after perfusion with NN-nitro-l-arginine (L-

NOARG, 100 AM), an inhibitor of NO-synthase, 1H-

[1,2,3]oxadiazolo[4,3-alfa]quinoxalin (ODQ, 10 AM), in

vessels perfused with high K+ solution (KCl 80 mM), to

inhibit EDHF (endothelium-derived hyperpolarizing factor),

and in vessels perfused with KCl 40 mM plus L-NOARG

(100 AM). In this last protocol, the concentration of KCl was

adjusted to 40 mM, in order to maintain the same PP as in

the other experiments (70–80 mm Hg). When high K+ was

used alone, 80 mM KCl was administered instead of 40 mM

KCl, since the concentration of 40 mM KCl was not

sufficient to produce the same contraction, as well as the

same PP (70–80 mm Hg). Moreover, in experiments with

high K+, phenylephrine was not added to the perfusion

fluid.

The vasodilator effects of EAF were also studied after

perfusion with glibenclamide (100 AM), an inhibitor of the

ATP-dependent K+ (KATP) channels, ouabain (100 AM), an

inhibitor of the Na+/K+-ATPase pump, Ba2+(BaCl2; 100

AM), to inhibit inward-rectifier potassium channels (KIR),

Ba2+(100 AM) plus ouabain (100 AM), tetraethylammonium

(TEA, 500 AM) a non-selective K+ channels inhibitor,

Charybdotoxin (ChTx 10 nM), a nonselective inhibitor of

KCa channels of large and intermediate conductance, and

Apamin (Apamin 100 nM), a specific inhibitor of KCa

channels of small conductance.

Since variations in vascular tone can complicate the

analysis of responses, the level of vascular tone was

maintained at a similar level (70–80 mm Hg) throughout

the study so that responses could be compared directly.

2.3. Drugs

The drugs PE, ACh, L-NOARG, ODQ, glibenclamide,

ouabain, TEA, ChTx and Apamin were purchased from

Sigma (St. Louis, MO), and heparin was purchased from

Roche. PE and ACh were dissolved in PBS (phosphate-

buffered saline) and stored as frozen aliquots until required.

L-NOARG, ODQ, glibenclamide, ouabain, BaCL2, TEA,

ChTx and apamin were dissolved in Krebs solution. All

drugs and solutions, as well as CE and EAF, were freshly

prepared before each experiment.

2.4. Statistical analysis

The vasodilator effects of CE and EAF were expressed as

the percentage reduction in the increase in PP induced by

PE. All results are expressed as meansTS.E.M. for the

number of preparations (or for the number of rats). The

results were also expressed as the geometric mean (IC50)

values, accompanied by their respective 95% confidence

limits. The IC50 represents the concentration which pro-

duced 50% of relaxation obtained from phenylephrine

precontracted preparations. The maximal relaxation (Rmax)

corresponds with the maximal response of the highest

concentration used.

One-way ANOVA plus Tukey and Student’s unpaired t

test were used for statistical analysis. Values of p <0.05

were considered statistically significant.

3. Results

Increasing concentrations of CE and EAF injected

through the Mesenteric Arterial Bed (MAB) caused a

concentration-dependent decrease in the PP response.

Although both CE and EAF induced a sustained and long-

lasting vasodilatory effect (Register 1), the activity exhibited

by EAF was clearly more potent than that of CE. Because of

its greater relaxant potency, EAF was chosen for all

subsequent experiments (Fig. 1, Table 1).

In order to investigate whether the vasodilator effect of

EAF is dependent on NO-cGMP and/or endothelium-

derived hyperpolarizing factor (EDHF) pathways, concen-

tration–response curves (CRC) were obtained for EAF, in

the absence and in the presence of L-NOARG or 80 mM

Table 1

Mean IC50 and maximal relaxation (Rmax) values obtained with CE and

EAF, with and without different solutions and drugs, in MAB of

normotensive Wistar rats

Group IC50 (a) (Ag) Rmax (b) (%)

CE 891.25 (538.10–1475.70)*** 78.40T3.67EAF (control) 390.84 (252.27–605.53) 81.88T4.04

+KCl 40+L-NOARG) 1059.25 (906.06–1238.34)*** 49.68T1.69***

+KCl 80 767.36 (457.35–1287.49)*** 61.11T3.64**

+L-NOARG 736.20 (330.17–1641.53)*** 70.57T4.38+GLI 252.34 (128.77–494.51) 82.01T6.05

+OUA 665.27 (474.14–933.45)*** 64.29T1.80*

+BaCl2 638.26 (275.43–1479.02)*** 86.53T1.96

+BaCl2+OUA 743.02 (312.70–1765.51)*** 71.16T5.64+TEA 851.13 (543.30–1333.80)*** 68.60T4.92

+ChTx+Apamin 421.70 (210.71–843.94) 98.01T6.45

+ODQ 881.04 (370.13–2097.20)*** 83.71T3.62

(a) Mean IC50 with the respective 95% confidence limits. ***p <0.001:

significantly different from the EAF (control) group. ANOVA/Tukey test.

(b) MeanTS.E.M. *p <0.05; **p <0.01; ***p <0.001: significantly different

from the EAF (control) group. ANOVA/Tukey test.

30 100 300 1000 3000

0

25

50

75

100

+ GLI 100 µM+ OUA 100 µM+ TEA 500 µM

Control

EAF [µg]

Dec

reas

e in

PP

(%

)

**

**

(A)

(B)

E.Z. Schuldt et al. / Vascular Pharmacology 43 (2005) 62–68 65

KCl. The pretreatment of the vessels with both inhibitors

(alone) significantly reduced, to the same extent, the

vasodilator effect of EAF. However, the combination of L-

NOARG with 40 mM KCl markedly shifted the CRC for

EAF to the right, when compared with the control (Fig. 2,

Table 1).

For this purpose, in order to investigate if the mecha-

nisms by which the EAF exerts its vasodilatory effect occur

mainly by hyperpolarization, CRC for EAF were obtained

in the MAB, after perfusion of GLI, OUA, TEA, barium,

ChTx and Apamin. The perfusion with GLI produced a

30 100 300 1000 3000

0

25

50

75

100

+ KCl 80

+ L-NOARG 100 µM

+ KCl 40 + L-NOARG 100 µM

Control

***

***** ***

******

*****

**

+ ODQ 10 µM

*

***

EAF [µg]

Dec

reas

e in

PP

(%

)

Fig. 2. Effect of EAF in the presence of L-NOARG, ODQ and high K+:

CRC for EAF in the rat mesenteric arterial bed (MAB), in the absence (?)

and in the presence of L-NOARG (r), ODQ (>), L-NOARG plus 40 mM

KCl (h) and 80 mM KCl (g). Values represent the meanTS.E.M. of four to

six experiments. *p <0.05; **p <0.01; ***p <0.001 significant difference

when compared with the control. ANOVA/Tukey test.

small leftward shift in the CRC for EAF. However,

perfusion with OUA or TEA significantly reduced the

vasodilatory effects of EAF (Fig. 3A). Fig. 3B illustrates the

CRC for EAF, obtained with BaCl2, BaCl2+plus OUA and

ChTx plus apamin. Barium and OUA, perfused alone or

simultaneously, displayed inhibitory effects characterized as

parallel rightward shifts of the EAF control curve. However,

ChTx and apamin were not able to promote an inhibitory

effect in EAF-induced relaxation in the MAB (Fig. 3B).

30 100 300 1000 3000

0

20

40

60

80

100

+ BaCl2 100 µM

Control

+ BaCl2 100 µM + OUA 100 µM

*

**

+ ChTx 10 nM + Apamin 100 nM

#

#

EAF [µg]

Dec

reas

e in

PP

(%

)

Fig. 3. Effect of EAF in the presence of the Na+/K+-ATPase pump inhibitor

and K+ channel blockers: CRC for EAF in the rat mesenteric arterial bed

(MAB) precontracted with phenylephrine, in the absence (?) and in the

presence of (A): OUA (h), GLI (r) and TEA (g). (B): BaCl2 (h), BaCl2plus OUA (g) and ChTx plus Apamin (>). Values represent the

meanTS.E.M. of five to six experiments. *p <0.05; **p <0.01 indicate a

significant difference when compared with the control. #p <0.05 indicate

significant difference between BaCl2 and BaCl2+OUA; ANOVA/Tukey

test.

0

20

40

60

80

100

120 Control+ L-NOARG+ ODQ+ KCl40 + L-NOARG+ KCl 80+ BaCl2+ BaCl2 + OUA + ChTx + Apamin+ GLI+ OUA+ TEA

****

***

***

*

Acethylcholine [200 nmol]

Dec

reas

e in

Per

fusi

on

Pre

ssu

re (

%)

Fig. 4. Effect of ACh-mediated relaxation in MAB: effect of a single injection of ACh (200 nmol) in rat mesenteric arterial bed (MAB), in the absence (control)

and in the presence of different solutions and drugs. Values represent the meanTS.E.M. of five to six experiments. *p <0.05; **p <0.01; ***p <0.001 indicate

significant difference when compared with the control. ANOVA/Tukey test.

E.Z. Schuldt et al. / Vascular Pharmacology 43 (2005) 62–6866

In all experiments, after the consistent precontraction of

the preparations with PE or KCl, a single dose of ACh (200

nmol) was administered in the MAB as a positive control.

According to Fig. 4, a high concentration of K+ as well as

Na+/K+-ATPase pump inhibitor and K+ channel blockers

(except GLI and BaCl2) were more efficient in decreasing

the vasodilatory effect of ACh in the MAB, when compared

to control or L-NOARG groups. ODQ was not able to

decrease the vasodilatory effect of ACh in the MAB.

However, the protocol in which the NO-synthase inhibitor

(L-NOARG) was perfused simultaneously with KCl 40 mM

was the more potent in inhibiting the decrease in PP induced

by ACh.

Table 1 shows the IC50 values with their respective

confidence limits (95%) and maximal relaxation (Rmax).

According to Table 1, in all experiments where the EDHF

mechanism was investigated (in protocols with high K+,

Na+/K+-ATPase pump inhibitor or K+ channel blockers,

except glibenclamide), there was an increase in IC50 values,

when compared with the IC50 control group.

4. Discussion

This study was performed in order to investigate the

mechanisms by which an ethyl acetate fraction (EAF),

obtained from a Brazilian red wine, induces a decrease in

PP, in MAB from normotensive Wistar rats. According to

our results, the EAF, and also to some extent the CE, was

effective in inducing a significant decrease in PP, in isolated

vessels of experimental animals (Fig. 1).

Previous data from our laboratory revealed that by

contrast with CE, EAF possesses a high concentration of

phenolic compounds; these compounds include gallic acid,

m-coumaric acid, chlorogenic acid, quercetin and resvera-

trol. In fact, EAF, an enriched polyphenol fraction obtained

by phytochemical partition of CE with ethyl acetate, showed

interesting results towards the scavenging of reactive

oxygen species (ROS), probably as a result of its higher

concentration of phenolic compounds (data not shown).

Therefore, the concentration-dependent relaxant response

induced by EAF in the MAB may be partly due to a radical

scavenging property, mainly superoxide radical. Superoxide

radical can react with NO at a high rate constant, generating

the oxidant peroxynitrite (Malinski et al., 1993; Demiryurek

et al., 1998), that has been implicated in the pathophysiol-

ogy of a variety of diseases including inflammation,

atherosclerosis and arthritis (White et al., 1994). The

antioxidant property of phenolic compounds could increase

the half-life of NO (Fitzpatrick et al., 1995; Dell’Agli et al.,

2004) and in this way contribute to the relaxing effect

verified in our experiment.

The present study suggests that the vasodilator effect of

the EAF partially depends on the release of NO from the

endothelial cells, since the perfusion of the MAB with L-

NOARG, an inhibitor of NO-synthase and ODQ, an

inhibitor of guanylate cyclase, significantly reduced the

effect of EAF (Fig. 2, Table 1). Similar results were found in

a recent study which showed that another inhibitor of NO-

synthase, NG-nitro-l-arginine methyl ester (L-NAME),

significantly reduced the vasodilator effect of a Brazilian

red wine, in the rat MAB (Soares de Moura et al., 2004).

The vasorelaxation induced by NO is dependent on a

reduction in intracellular calcium in smooth muscle cells,

due not only to the activation of soluble guanylate cyclase

(GC), but also to hyperpolarization induced by the opening

of K+ channels (Waldman and Murad, 1987; Tare et al.,

1990). For this purpose, several pieces of evidence suggest

that NO may be only one of several EDRFs mediating the

vasodilator response in small vessels. When NO and

prostaglandin synthesis are blocked, stimulation of the

vascular endothelium is still capable of eliciting vaso-

dilatation in various vascular preparations (Parkington et al.,

2002; Savage et al., 2003).

E.Z. Schuldt et al. / Vascular Pharmacology 43 (2005) 62–68 67

According to Fig. 2, the vasodilator effect of EAF may

involve hyperpolarization of the cells through K+ channels,

since 80 mM KCl also reduced the decrease in PP induced

by EAF in the MAB (Fig. 2, Table 1). In our experiment, the

inhibitory effect of L-NOARG and high K+ on the EAF-

induced vasodilatory activity was further enhanced when

those inhibitors were perfused simultaneously. According to

Fig. 2 and IC50 values described in Table 1, the decrease in

PP brought about by EAF might be due to a combination of

an activation of GC enzyme plus hyperpolarization by the

opening of K+ channels.

Some authors (Li et al., 1997; Naderali et al., 1997) have

reported that flavonoids such as quercetin and resveratrol

are able to open Ca2+-activated K+ channels (KCa).

Resveratrol, a polyphenolic phytoalexin that is generated

in response to stress in specific plants and also grape skins,

has a stimulatory effect on KCa (large-conductance potas-

sium channel) found in human vascular endothelial cells

(Chen and Pace-Asciak, 1996). It is postulated that

increased K+ efflux following the activation of KCa channels

by resveratrol may increase K+ concentration in the

myoendothelial space followed by hyperpolarization of

smooth muscle cells, leading to the dilation of blood vessels

(Bhat et al., 2001).

Another piece of evidence which indicates that EAF

could be involved in the EDHF pathway is illustrated in Fig.

3A and B, since the vasodilator effect of EAF is inhibited by

the perfusion of the MAB with the Na+/K+-ATPase pump

inhibitor, ouabain (OUA) and the different K+ channel

blockers, including tetraethylammonium (TEA) and barium.

The perfusion of the MAB with OUA or TEA markedly

inhibited the decrease in PP induced by EAF, and to the

same extent as in the presence of 80 mM KCl. According to

Soares de Moura et al. (2004), KATP channels are probably

not involved in wine polyphenols-induced vasodilatation in

the MAB; in our experiment, EAF-induced vasorelaxation

was also not inhibited by glibenclamide (Fig. 3A).

The decrease in PP induced by EAF was also partially

inhibited by the perfusion of ouabain plus barium. Even

though the perfusion of the MAB with barium (alone) did

not significantly shift the CRC for EAF to the right, when

barium and ouabain were perfused in the same solution, this

inhibitory effect was more evident (Fig. 3B).

Our results are in accordance with Edwards et al. (1998),

who have shown that the relaxations stimulated by K+ were

similarly inhibited by barium and ouabain in rat hepatic and

mesenteric arteries. These authors proposed that K+ entering

the subendothelial space after efflux through endothelial Kca

channels stimulates smooth muscle hyperpolarization by

activation of KIR and the Na+/K+-ATPase pump on the

smooth muscle cells. In our experiments, although barium

plus ouabain significantly shifted the EAF control curve to

the right, ChTx plus Apamin were not able to inhibit the

decrease in PP induced by EAF (Fig. 3B; Table 1).

Eventually, the pharmacological similarities of the hyper-

polarization activity and the vasodilator effect induced by

EAF (both inhibited by high K+ and some K+ channel

blockers) suggest that the EAF effect could be partially

mediated by activation of KIR and Na+/K+-ATPase pump.

Since it has been hypothesized that other mediators than

NO may be involved in the vasodilator responses to ACh

and because the mechanisms of these mediators may

involve K+ channels (Dabisch et al., 2004), Fig. 4 was

obtained as our positive control, in order to demonstrate the

ACh-mediated relaxation, in all different protocols. Since

the perfusion of L-NOARG and ODQ do not inhibit the

ACh-mediated relaxation in the MAB, this demonstrates

that the ACh mechanism possesses a significant component

that is independent of NO production. These observations

are consistent with those reported in the literature (Wood-

man et al., 2000; Savage et al., 2003), in which NO makes a

small but significant contribution to ACh-mediated vaso-

dilatation with a more substantial role for EDHF, in small

resistance arteries. However, comparing the results of Fig. 4

with the CRC for EAF obtained in the presence of L-

NOARG and ODQ (Fig. 2), EAF appears to be more NO-

dependent when compared with ACh, since the perfusion

with both drugs significantly shifted the CRC for EAF to the

right.

5. Conclusions

This study showed that NO appears to make an important

contribution to EAF-mediated vasodilatation, either via the

NO-cGMP pathway or through hyperpolarization induced

by the opening of K+ channels. The phenolic compounds

present in EAF (such as quercetin and resveratrol) may exert

their effects by activation of KIR and the Na+/K+ ATPase

pump. The present investigation also demonstrated that EAF

induces a vasodilator effect that does not involve the

opening of KATP channels and potassium conductance

(KCa) channels.

In concert with other systemic beneficial effects of red

wine, this could contribute to the cardiovascular protection

enjoyed by moderate drinkers of red wine.

Acknowledgments

The authors are grateful to CNPq (Brazil) for financial

support.

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