CARDIOVASCULAR JOURNAL OF AFRICA • Vol 21, No 3, May/June 2010
AFRICA
133
KwaZulu-Natal and conform to the
Guide to the Care and Use
of Laboratory Animals in Research and Teaching.
28
The pharmacological effects of quercetin were investigated
on isolated, spontaneously contracting portal veins taken from
naïve, normotensive, healthy young adult male and female
Wistar rats (weighing 250–300 g). The animals were kept under
conventional laboratory conditions of temperature, humidity and
light, and allowed free access to food (standard pellet diet) and
tap drinking water
ad libitum
. All the animals used were fasted
for 16 hours, but still allowed free access to water prior to the
commencement of our experiments. Each rat was euthanased by
halothane inhalation, following which the abdomen was opened
and the portal vein (with an
in situ
length of 2–3 cm) was quickly
removed. The harvested venous muscles were cleaned free from
fat and connective tissues and trimmed.
Effect of quercetin on rat isolated portal veins
Each isolated portal vein segment was suspended under an
applied resting tension of 0.5 g in a 30-ml Ugo Basile organ
bath containing Krebs-Henseleit physiological solution (KHS) of
composition, in mM: NaCl, 118; KCl, 4.7; NaHCO
3
, 25.0; MgCl
2
,
1.2; CaCl
2
.2H
2
O, 2.52; NaH
2
PO
4
.2H
2
O, 1.28; and glucose, 5.55;
pH adjusted to 7.4. The bathing KHS was maintained at 35
±
1
o
C
and continuously aerated with carbogen (i.e. 95% O
2
+
5% CO
2
gas mixture). The mounted portal vein preparations were subse-
quently left to equilibrate for 45 to 60 minutes, during which
time the bathing solution was changed every 15 minutes, before
they were challenged with graded concentrations of quercetin
(10
-7
–10
-4
M) and/or reference drugs at different times.
Quercetin and/or the reference drug solutions were added to
the bath fluid sequentially. They were repeated (where neces-
sary) after washing out the previous quercetin or reference drug
concentration four to five times, and allowing each tissue prepa-
ration to rest for five to 10 minutes, or until its tone returned to
the control baseline level. In order to make allowance for chang-
es in tissue sensitivity, two isolated portal veins were always set
up at a time, one used as distilled water-treated control, and the
other as quercetin- or reference drug-treated test preparation. The
control venous muscle strips were only treated with volume/s of
distilled water equivalent to the volume/s of quercetin or refer-
ence drug solutions.
To find out whether the initial brief contractile effect of quer-
cetin on portal vein preparations was mediated through alpha
1
-
adrenoceptor stimulation or L-type voltage-operated calcium
channels, some of the portal vein preparations used were pre-
treated with an alpha
1
-adrenoceptor blocker, prazosin (10
-6
M), or an L-type voltage-dependent calcium channel blocker,
nifedipine (10
-7
M), respectively, 20 minutes before the addi-
tion of quercetin to the bath fluid. The possible involvement of
endothelium-derived relaxing factor (EDRF) and prostacyclin
(PGI
2
) in quercetin-induced vasorelaxations was also investi-
gated by pre-treating the venous tissues with N
G
-nitro-L-arginine
methyl ester (L-NAME, 100
µ
M), a nitric oxide synthase inhibi-
tor; or indomethacin (10
µ
M), a prostanoid synthase inhibitor,
respectively, 20 minutes prior to addition of quercetin to the bath
fluid.
Functional endothelium removal procedure was confirmed
by lack of relaxant effect to a bolus of acetylcholine (10
-6
M)
administration. The possible contribution of cAMP-dependent
protein kinases towards the relaxant effect of quercetin was
examined by pre-incubating the venous tissues with N-p-tosyl-l-
phenylalanine-chloromethyl-ketone (TPCK, 3
µ
M), 20 minutes
prior to addition of quercetin to the bath fluid. Quercetin- and/or
reference drug-induced responses of the smooth muscle prepara-
tions were recorded isometrically by means of Ugo Basile force–
displacement transducers and pen-writing two-channel Gemini
recorders (model 7070).
Drugs
Quercetin dihydrate, L-NAME, acetylcholine chloride, nifed-
ipine hydrochloride, prazosin hydrochloride and indomethacin
were purchased from Sigma-Aldrich Inc. (St Louis, MO, USA).
TPCK was purchased from Bachem (Budendorf, Switzerland).
The salts used to prepare Krebs-Henseleit physiological solution
were purchased from Merck (Germany).
Except for quercetin and indomethacin, all drug solutions
used were prepared by dissolving weighed amounts of the
respective salts in distilled water. Quercetin was dissolved in
dimethylsulfoxide (DMSO). The final concentration of DMSO
was less than 0.08%, which was shown to be devoid of any
observable pharmacological or physiological effect on the
smooth muscle contractile tone. Indomethacin was dissolved in
0.5% w/v sodium bicarbonate immediately before use. Further
dilutions of the drugs were made in KHS. Drug concentrations
quoted in the text refer to final organ bath concentrations.
Statistical analysis
All experimental data obtained are expressed as means (
±
SEM).
Distilled water-induced control means were used as baseline
values. The differences in responses among the different groups
were analysed for statistical significance using the Student’s
t
-test
and two-way analysis of variance (ANOVA, 95% confidence
interval – GraphPad PRISM software, Version 5.00) followed by
Dunnett’s
post-hoc
test. Multiple comparisons of the means were
performed using Bonferroni’s test. In all cases, values of
p
≤
0.05
were taken to imply statistical significance.
Results
The rat isolated portal veins used in this study always exhibited
spontaneous, rhythmic, myogenic contractions with an average
amplitude of 425
±
13 mg (
n
=
8). Quercetin (10
-7
–10
-4
M) raised
the basal tone and caused concentration-dependent and signifi-
cant reductions (
p
<
0.05–0.001) in the contractile amplitudes of
the myogenic contractions. The inhibitory effects of quercetin
on the contractile amplitudes of the veins were always preceded
by initial brief but significant rises (
p
<
0.05) in the basal tones
and, in 75% of the preparations set up, increases in contractile
amplitudes, followed by more pronounced and longer-lasting
secondary relaxations.
Fig. 1 shows a typical trace obtained with quercetin (10
-4
M).
The vasorelaxant effects of quercetin were completely or near
completely reversed by washing out the quercetin concentrations
four to five times, and subsequently allowing the venous tissues
to rest for five to 10 minutes. Pre-incubation of the portal vein
preparations with prazosin (10
-6
M) did not modify the initial
contractile effect of quercetin on the muscles, whereas pre-
incubation of the tissues with nifedipine (10
-7
M) significantly
