Clinical Science (2001) 100, 5560 (Printed in Great Britain) 55

The mechanism of resveratrol-induced

vasorelaxation differs in the mesenteric
resistance arteries of lean and obese rats

Ebrahim K. NADERALI*, Sharon L. SMITH*, Patrick J. DOYLE

and Gareth WILLIAMS*
*Diabetes and Endocrinology Research Unit, Department of Medicine, University of Liverpool, UCD, Daulby Street,
Liverpool L69 3GA, U.K., and Novo-Nordisk A/S, Brudelysvej 28, 2880 Bagsvaerd, Denmark

A B S T R A C T

Resveratrol has been shown to induce vasorelaxation. In this study, we investigated the
mechanism(s) of resveratrol-induced vasorelaxation in resistance mesenteric arteries from male
lean and dietary-induced obese rats. Compared with lean rats, arteries from dietary-obese rats
showed significant (P 0.001) endothelial dysfunction, as indicated by a decrease (
20 %) in
maximal acetylcholine-induced vasorelaxation. Resveratrol (535 mol/l) induced concen-
tration-dependent relaxation of mesenteric arteries preconstricted with noradrenaline
(8 mol/l) or KCl (125 mmol/l) from both lean and dietary-obese rats. There were no significant
differences between the two groups, achieving a maximum relaxation of
95 % at a
concentration of 35 mol/l. In noradrenaline-preconstricted arteries from lean rats, NG-nitro-L-
arginine methyl ester (L-NAME ; 100 and 300 mol/l) caused a significant (P 0.01) concen-
tration-dependent rightward shift in reseveratrol activity, with no effect on maximal responses.
However, L-NAME (100 and 300 mol/l) did not alter the effects of reseveratrol on arteries from
dietary-obese rats, giving superimposed concentrationresponses curves. Indomethacin was also
ineffective in altering resveratrol activity in arteries from both lean and dietary-obese rats. In
noradrenaline-precontracted arteries from dietary-obese rats, responses to resveratrol were
not attenuated by endothelial denudation, indicating an action independent of the endothelium.
This study indicates that : (a) the maximal effects of resveratrol on resistance arteries from lean
and dietary-obese rats are not effected by endothelial dysfunction, and (b) the effects of
resveratrol in lean animals (where endothelial function is not impaired), but not in dietary-obese
rats, are mediated via NO.

INTRODUCTION been attributed to resveratrol. The mechanisms by which

Resveratrol is a naturally occurring phenolic trihydroxy-
stilbene, present in a variety of plants [14]. A protective
effect against atherosclerosis and coronary heart disease
through various mechanisms [57], that may include
vasorelaxation [811] and an anti-platelet effect [7], has
resveratrol causes vasodilatation are uncertain, but may
include inhibition of arachidonate metabolism [8,12] and
induction of nitric oxide (NO) synthesis [11,13,14].
The endothelium is vital to various important physio-
logical functions in the arterial wall, including the
regulation of vascular tone [1517]. Endothelial cells

Key words : calcium channels, endothelium, NO synthesis inhibitor, resistance arteries, resveratrol.
Abbreviations : ACh, acetylcholine ; NA, noradrenaline ; L-NAME, NG-nitro-L-arginine methyl ester ; PSS, physiological salt
solution ; KPSS, high-potassium PSS.
Correspondence : Dr E. K. Naderali (e-mail naderali!liverpool.ac.uk).

# 2001 The Biochemical Society and the Medical Research Society

56 E. K. Naderali and others
produce various vasoactive mediators, such as the vaso-
dilator NO and the vasoconstrictor endothelin, both of
which act on the underlying vascular smooth muscle to
modulate arterial contractility [18]. NO production is
regulated by NO synthase, which is stimulated by
mediators and hormones that include acetylcholine
(ACh), bradykinin and insulin [18,19]. A range of deficits
in endothelial function have been identified in several
conditions that are associated with abnormal vascular
reactivity and\or atherogenesis [20,21]. In particular,
impaired NO-mediated vasodilatation has been demon-
strated in obesity, Type II diabetes, hypertension and
hypercholesterolaemia [2225], and in an animal model
of dietary-induced obesity [26], with the most striking
abnormality being blunted vasodilatation in response to
ACh. Therefore in the present study we aimed to
investigate the mechanism(s) of resveratrol-induced vaso-
relaxation in mesenteric resistance arteries from lean and
dietary-obese rats.
METHODS
Experimental procedures
Male Wistar rats (n l 14) were randomly assigned to a
control group (n l 7) or a test group (n l 7). All had free
access to water, and were housed in groups of two
to three under controlled environmental conditions
(1922 mC ; 3040 % humidity) and a 12-h light\dark
cycle (lights on at 07.00 hours). Control animals were fed
a standard laboratory pelleted diet (CRM Biosure,
Cambridge, U.K.), while the test group had free access to
a highly palatable, high-energy diet consisting of 33 %
(by wt.) ground pellet diet, 33 % Nestle! condensed milk,
7 % sucrose and 27 % water, as described previously [27].
The two groups were initially matched for body weight
(Table 1). All animals in the study were maintained on
their respective diet for 16 weeks before being killed by
CO inhalation.
#
Table 1 Physiological and metabolic characteristics of chow-fed and dietary-obese rats
Data are meanspS.E.M. NS, not significant.
Chow-fed
Body weight (g)
Initial 210p4
Final 510.8p10.7
Gain 300.7p16.4
Epididymal fat pad mass (g) 7.9p1.0
Perirenal fat mass (g) 10.2p1.8
Plasma glucose (mM) 10.4p0.8
Plasma triacylglycerols (mM) 0.45p0.02
Plasma non-esterified fatty acids (mM) 0.33p0.04
# 2001 The Biochemical Society and the Medical Research Society
Assessment of metabolic changes
On the day of the experiment, the gonadal and perirenal
fat pads were dissected out and weighed. Blood was
removed by cardiac puncture into cold heparinized tubes.
The plasma was immediately separated by centrifugation
before being frozen for later measurements of blood
analytes [glucose, non-esterified ( free ) fatty acids and
triacylglycerols (triglycerides)]. The plasma glucose con-
centration was determined using a glucose oxidase
method, and non-esterified fatty acid (Boehringer
Mannheim, Milton Keynes, Bucks., U.K.) and triacyl-
glycerol (Sigma Diagnostics, Poole, Dorset, U.K.) con-
centrations were measured using commercial diagnostic
kits.
Assessment of vascular function
Eight third-order mesenteric arteries (180250 m di-
ameter ; 2 mm length) were carefully dissected from each
animal. Each artery was freed of fat and connective tissue
and mounted on two 40 m-diameter stainless-steel wires
in an automated myograph (Cambustion, Cambridge,
U.K.), based on the principle of the Mulvany myograph,
which measures isometric tension generated in response
to various stimuli [28]. The vessels (in duplicate) were
incubated in a 5 ml organ bath containing physiological
salt solution [PSS ; composition (in mM) : NaCl 119, KCl
4.7, CaCl 2.5, MgSO 1.17, NaHCO 25, KH PO 1.18,
# % $ # %
EDTA 0.026 and glucose 5.5] gassed with 95 % O \5 %
#
CO at 37 mC.
#
After a 30 min equilibration period, the lengthtension
characteristics for each vessel were determined as de-
scribed previously [14]. Arteries were allowed a further
30 min to equilibrate before being depolarized twice with
high-potassium PSS (KPSS ; 125 mM) in which NaCl in
normal PSS was replaced by an equimolar concentration
of KCl. Any vessel failing to reach its predetermined
target tension in response to vasoconstriction with KCl
(125 mM) was discarded.
Arteries were then contracted with noradrenaline
(NA ; 8 mol\l). When contractions reached a plateau,
relaxation response curves to either ACh alone or
Dietary-obese Significance
213p5 NS
732.0p15.4 P 0.0001
517.1p14.9 P 0.001
15.9p0.8 P 0.0001
23.4p1.8 P 0.0001
12.2p0.5 NS
0.80p0.05 P 0.001
0.48p0.03 P 0.05

resveratrol (535 mol\l), in the presence or absence of
NG-nitro-L-arginine methyl ester (L-NAME ; 100 and
300 mol\l) or indomethacin (10 mol\l), were carried
out. To evaluate the role of endothelium-derived NO in
NA-induced vasocontriction, arteries were also con-
tracted with NA (8 mol\l) in the presence or absence of
L-NAME (300 mol\l). In another set of experiments,
resveratrol relaxation response curves were carried out
on arteries preconstricted with KCl (125 mmol\l). We
have also examined the effects of resveratrol on arteries
with intact or denuded endothelium precontracted with
NA (8 mol\l).
Endothelium denudation was achieved by gently
rubbing the internal diameter of arteries around 40 m
stainless steel wires. Endothelial function was assessed by
responses to ACh. Failure of arteries to relax to ACh
(100 mol\l) was considered to indicate a state of
endothelial denudation.
Reagents
Noradrenaline, resveratrol, ACh and L-NAME were all
obtained from Sigma. Resveratrol was dissolved in
ethanol. Working concentrations of ethanol in the bath
were 0.01 % (v\v). To exclude possible vascular effects
of ethanol, two arteries from each animal in each
experiment were studied in the presence of the same
volume of ethanol as the test arteries. As reported
previously, there was no significant effect of this low
concentration of ethanol on contractility induced by
either KPSS or NA [29,30].
Statistical analyses
All data are expressed as the meanpS.E.M. for the group.
An average response for all the vessels from a given
animal was determined before group analysis. Stat-
istical significance was tested using repeated-measures
ANOVA or Students t-test, as appropriate. n represents
the number of animals, unless otherwise stated.
RESULTS
Metabolic changes
At the end of the experiment, animals fed on the highly
palatable diet weighed significantly (
40 % ; P 0.001)
more than their lean counterparts. The epididymal and
perirenal fat pads weighed significantly more (
120 % ;
P 0.001) in dietary-induced obese rats than in the
chow-fed controls (Table 1). Moreover, in dietary-
induced obese rats, terminal plasma non-esterified fatty
acid levels were significantly higher (
45 % ; P 0.05)
than in their chow-fed counterparts, as were triacyl-
glycerol levels (
78 % ; P 0.001) (Table 1), indicating
the induction of obesity in animals fed on the highly
palatable diet.
Effects of NO-synthesis inhibition on vasoactivity of resveratrol 57
Figure 1 Effects of ACh on mesenteric arteries from lean
control and dietary-obese rats
Arteries were contracted with NA (8 mol/l). When contractions reached a
plateau, ACh (10 nmol/l100 mol/l) concentrationresponse curves were
performed.
Vascular and contractile responses to NA
There were no significant differences in vessel diameter
between the two groups of arteries used in this study.
Diameters of vessels from lean and dietary-obese animals
were 260p10 m and 250p15 m (n l 7 animals, 40
vessels) respectively.
There were no significant differences in the NA-
induced rise in tension between vessels from chow-fed
and dietary-induced obese rats (6.24p0.61 and
6.66p0.75 mN\mm of artery respectively ; P
0.5). L-
NAME caused a slight increase in NA-induced force in
arteries from both chow-fed (7.20p0.49 mN\mm of
artery) and dietary-induced obese (7.25p0.79 mN\mm
of artery) rats. However, these increases in tension
development were not significant compared with those in
the absence of L-NAME.
Effects of ACh on arteries contracted with
NA
NA (8 mol\l)-precontracted arteries from lean rats
demonstrated progressive relaxation to cumulative ad-
ditions of ACh (10 nmol\l100 mol\l), with a maximal
relaxation of 93p2 % at 100 mol\l ACh. Arteries from
the dietary-obese group that were similarly exposed to
ACh displayed a significant (20p2 % ; P 0.001) de-
crease in maximal relaxation. There was also a significant
rightward shift of the concentrationresponse curves in
arteries from dietary-obese rats compared with those
from lean animals (EC values of 3.14p0.04 and
&!
0.40p0.03 mol\l respectively ; P 0.001) (Figure 1).
Effects of resveratrol on NA- and KCl-
preconstricted arteries
Resveratrol caused a concentration-dependent relax-
ation of arteries precontracted with either NA or KCl,
# 2001 The Biochemical Society and the Medical Research Society
58 E. K. Naderali and others

(a)

Figure 2 Vasorelaxant effects of resveratrol on arteries from (b)

lean control and dietary-obese rats
Arteries were contracted with NA (8 mol/l) or KCl (125 mmol/l). When
contractions reached a plateau, resveratrol (535 mol/l) concentration
response curves were performed.

from both lean control and dietary-obese rats. There was

no significant difference between the vasoactivity of
resveratrol on arteries from lean control and dietary-
obese rats, with a maximal relaxation of up to 99 % being
achieved (Figure 2).

Figure 3 Effects of L-NAME (100 and 300 mol/l) on the

Effects of L-NAME and indomethacin on resveratrol-induced vasorelaxation of mesenteric arteries
resveratrol activity from control (upper panel) and dietary-obese (lower panel)
In arteries from lean animals, L-NAME caused a con- rats
centration-dependent rightward shift of resveratrol- Arteries were contracted with NA (8 mol/l). When contractions reached a
induced vasorelaxation, with no change in maximal plateau, resveratrol (535 mol/l) was added to the arteries in the presence or
relaxation. The EC values for resveratrol absence of L-NAME (100 or 300 mol/l).
&!
were 10.8p0.2 mol\l (control), 15.1p0.3 mol\l
(100 mol\l L-NAME ; P 0.05 compared with control)
and 19.5p0.5 mol\l (300 mol\l L-NAME ; P 0.01
compared with control) (Figure 3, upper panel). In
arteries from dietary-obese rats, L-NAME had no
effect on resveratrol-induced vasorelaxation, with
EC values of 11.2p0.4 mol\l (control), 11.0p0.3
&!
mol\l (100 mol\l L-NAME) and 11.3p0.4 mol\l
(300 mol\l L-NAME) (Figure 3, lower panel).
Indomethacin (10 mol\l) had no significant effect
on the vascular activity of resveratrol on arteries from
lean or dietary-obese rats, giving EC values of
&!
11.1p0.3 mol\l (control) and 11.5p0.5 mol\l (indo-
methacin) for lean rats, and 10.8p0.5 mol\l (control)
and 11.1p0.5 mol\l (indomethacin) for dietary-obese Figure 4 Effect of endothelium denudation on resveratrol
rats. activity on arteries from dietary-obese rats
Arteries were contracted with NA (8 mol/l). When contractions reached a
plateau, resveratrol (535 mol/l) concentrationresponse curves were
Effects of endothelial denudation on performed.
resveratrol activity
In dietary-obese rats, endothelial denudation failed to ence or absence of functional endothelium, giving EC
&!
alter resveratrol activity. Resveratrol caused concen- values of 11.2p0.4 mol\l (endothelium intact) and
tration-dependent vasorelaxation of arteries in the pres- 11.8p0.5 mol\l (endothelium denuded) (Figure 4).

# 2001 The Biochemical Society and the Medical Research Society


DISCUSSION
Several recent studies have indicated vasorelaxant pro-
perties for resveratrol [1114]. Resveratrol has been
shown to inhibit KCl-, noradrenaline- and phenyl-
ephrine-induced contraction of rat aorta [10], guinea-
pig trachea [8] and guinea-pig mesenteric and uterine
arteries [14] in vitro.
The mechanism(s) of action of resveratrol [10,14] are
thought to be both endothelium-dependent and endo-
thelium-independent. The former effect is apparent at
low resveratrol concentrations (1030 mol\l) and is
blocked by inhibitors of NO synthase activity [10,31],
whereas endothelium-independent effects appear at high
resveratrol concentrations (
60 mol\l) and are not
blocked by endothelial denudation or NO synthase
inhibitors [10,11]. In the present study, resveratrol
(535 mol\l) caused concentration-dependent relax-
ation of NA-precontracted arteries from both lean and
dietary-obese rats, indicating the involvement of the
endothelium-dependent component of resveratrol ac-
tivity. Inhibitory effects of L-NAME on the vasoactivity
of resveratrol would have further strengthened such a
hypothesis. Indeed, L-NAME attenuated the responses
to resveratrol of arteries from lean animals, indicating
activation of endothelial NO synthesis by resveratrol.
This finding is in agreement with previous reports in
which resveratrol-induced vasodilatation was attenuated
with inhibitors of NO synthesis [10,31]. However, L-
NAME failed to affect the vasorelaxant property of
resveratrol on arteries from dietary-obese animals, indi-
cating a lack of involvement of the endothelial NO
system in the resveratrol-induced vasorelaxation of ar-
teries from dietary-obese rats. This raises a number of
questions as to the mechanism(s) of resveratrol-induced
vasorelaxation in arteries from animals with dietary-
induced obesity.
The attenuation of ACh-induced vasorelaxation seen
in the present study is in agreement with our previous
report [26] indicating the induction of endothelial dys-
function in dietary-obese animals. Therefore it appears
that the mechanism of resveratrol-induced vasorelaxation
in arteries from obese animals is influenced by the state
of endothelial function. That is, if there is no apparent
endothelial dysfunction, then the mechanism of res-
veratrol-induced vasorelaxation is via the activation and
release of endothelial NO. If, however, the endothelium
is damaged, another component of resveratrols mech-
anism of action becomes more important. This secondary
endothelium-independent effect has been shown to occur
at high concentrations (
60 mol\l) [10,11] ; nonet-
heless, in the present study the endothelium-independent
effect was apparent at all resveratrol concentrations used
(Figures 2 and 3).
In agreement with previous reports [10,14], indo-
methacin did not attenuate responses to resveratrol in the
Effects of NO-synthesis inhibition on vasoactivity of resveratrol 59
present study, indicating that endogenous prostanoids
play little or no part in the vascular activity of resveratrol.
In arteries from dietary-obese rats, resveratrol activity
was similar in the presence or absence of functional
endothelium, suggesting that the endothelium-indepen-
dent component of the effect of resveratrol responses is
not NO-mediated [10,11].
Inhibitors of cyclic nucleotide phosphodiesterases
induce the relaxation of endothelium-denuded aortic
rings [32,33]. Polyphenols have also been shown to
inhibit cyclic nucleotide phosphodiesterases, which
break down the vasorelaxants cAMP and cGMP [34,35].
Such a mechanism might therefore be involved in the
endothelium-independent relaxation induced by resvera-
trol. Resveratrol might also become incorporated into the
smooth muscle membrane, where it could either couple
with a membrane receptor [36] or interact directly with
membrane calcium channels [37], thus inducing endo-
thelium-independent vasorelaxation.
In conclusion, we have shown that resveratrol can in-
duce the relaxation of mesenteric resistance arteries from
both lean and dietary-obese rats. Resveratrol-induced
vasorelaxation may either be endothelium-dependent (at-
tenuated by L-NAME) or endothelium-independent,
in which case endothelium denudation and L-NAME
treatment fail to alter the vasorelaxant effect of res-
veratrol. This vasorelaxant effect of resveratrol may
contribute to reduce blood pressure [38] and, by lowering
myocardial work, may contribute to the beneficial
cardiovascular effects of resveratrol in conditions such as
obesity, Type II diabetes, hypertension and hyper-
cholesterolaemia [2025] where endothelial function is
blunted.
ACKNOWLEDGMENTS
This study was supported by Novo-Nordisk A\S.
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Received 5 May 2000/19 July 2000; accepted 6 October 2000