JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2013, 64, 6, 795-805
www.jpp.krakow.pl
R. NOVAKOVIC1, B. ILIC2, B. BELESLIN-COKIC2, N. RADUNOVIC3, H. HEINLE4,
R. SCEPANOVIC5, L. GOJKOVIC-BUKARICA1
THE EFFECT OF RESVERATROL ON CONTRACTILITY OF NON-PREGNANT
RAT UTERUS: THE CONTRIBUTION OF K+ CHANNELS
1
Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty, University of Belgrade, Belgrade, Serbia;
2Clinic of Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia, Belgrade, Serbia;
3Institute of Gynecology and Obstetrics, Clinical Center of Serbia, Belgrade, Serbia; 4Institute of Physiology,
University of Tuebingen, Tuebingen, Germany, 5Clinical Center “Dr Dragisa Mišovic”, Belgrade, Serbia
This study was aimed to evaluate resveratrol (1–100 µM) effect on the spontaneous rhythmic contractions (SRC), oxytocininduced (0.2 nM, POxC) phasic and tonic (20 nM, TOxC) contractions of isolated rat uterus. The SRC and POxC were
more sensitive to resveratrol than TOxC (pD2 values: 4.53 and 4.66 versus 4.06). Different blockers of K+ channels
(glibenclamide, tetraethylamonium, iberiotoxin, 4-aminopyridine) antagonized the response to resveratrol on the SRC and
phasic contractions, but did not antagonize the effect of resveratrol on the TOxC. In order to compare the relaxant activities
of resveratrol on the TOxC with that of potassium channel openers, a separate experiments with NS 1619, a highly specific
big Ca2+-sensitive K+ (BKCa) channels opener and pinacidil, a predominant opener of ATP-sensitive K+ (KATP) channels were
done. NS 1619 (10–100 µM) and pinacidil (10–100 µM) produced more potent inhibition of TOxC than resveratrol (pD2
values were 6.00 and 5.29). Iberiotoxin, a highly selective BKCa channels blocker, antagonized the response to NS 1619
and glibenclamide, a highly selective KATP channels blocker, antagonized the response to pinacidil on the TOxC. To test K+and extracellular Ca2+- independent mechanism(s) of resveratrol on TOxC, a K+-rich, Ca2+-free solution was used. Under
this condition, only high concentrations (³30 µM) of resveratrol inhibited TOxC. Western blots analysis confirmed
expression of Kir6.1, Kir6.2, KCa1.1, Kv2.1 and Kv4.2. channel proteins in myometrium. Thus, the effect of resveratrol is
dependent on the types of contractions. The inhibitory response of resveratrol on the SRC and phasic contractions involves
different myometrial K+- channels. When applied in high concentrations, resveratrol has an additional K+- channels
independent mechanism(s) of action. As the effects of NS 1619, pinacidil and resveratrol on the TOxC are different, we can
conclud that resveratrol does not behave as a classical potassium channel opener.
K e y w o r d s : non-pregnant rat uterus, contractility, potassium channels, resveratrol, calcium, oxytocin, calcium channel activator,
potassium channel blocker
INTRODUCTION
A large percentage of women (>50 %) suffer from
dysmenorrhea (1). It is generally accepted that primary
dysmenorrhea is a repercussion of decreased uterine blood flow
caused by the hyper contractility of the uterus smooth muscle and
local contractions of the uterine vessels (2). Up to date, the cause
of dysmenorrhea remains unclear, but the prostaglandins includes
prostaglandin E2 and F2a are important factors of the regulation of
uterine contractility. Also, it has been recently emphasized that
peroxisome proliferator activated receptors are mediators of
prostaglandin E2 synthesis/accumulation in porcine endometrium
(3). Dysmenorrhea is mainly treated with nonsteroidal antiinflamatory drugs (NSAIDs) which suppress prostaglandins
production by inhibition of cyclooxygenase. However, NSAIDs
are not successful in all patients and they have remarkable adverse
effects involving cardiovascular undesirable events, disorders of
kidney and digestive system (1, 2). Accordingly, the ideal agent
for the prevention and treatment of uterine abnormal contractility
has not yet been found. Therefore, development of effective and
safe agents is an important research topic.
Resveratrol is a polyphenol derived from a variety of plant
species, including grapes and red wine (4). During the last decade,
resveratrol has been shown to possess wide spectrum of
pharmacologic properties such as anti-inflammatory, antioxidant,
anticarcinogenic, antiageing, neuroprotective and cardioprotective
effect. Different research indicated many potential targets of
resveratrol, such as cyclooxygenase, antioxidant enzymes, sirtuin
1, estrogen receptor, inflammatory markers and nitric oxide
produced by endothelial NO synthase (5, 6). Our previous reports
indicated that resveratrol inhibits vasocontractile response and
relaxes different arteries and vein by activation of smooth muscle
voltage-gated potassium channels (Kv) and big Ca2+-sensitive K+
channels (BKCa) channels (7-10). But, data from
electrophysiological studies suggest that resveratrol inhibits Ltype Ca2+ channels and enhances activity of the ATP-sensitive K+
channel (KATP) channels in rat hearts (11). As opposed, the
experiments on the mouse beta cell line revealed that resveratrol
796
blocked KATP and Kv, but activated BKCa channels (12). This
discrepancy in the results obtained on the various experimental
models strongly suggests tissue and species selectivity of
resveratrol. However, despite the increasing interest for the effects
of resveratrol on various smooth muscles, the influence of
resveratrol on the contractility of uterus is not entirely defined.
According to our knowledge, there is only one study dealing with
resveratrol effect on the non-pregnant uterus. Hsia et al.
demonstrated that resveratrol inhibited prostaglandin F2aoxytocin-, acetylcholine-, and carbachol-induced uterine
contractions in rats. Also, it inhibited contractions induced by high
K+ concentration and by Ca2+ channel activator (Bay K 8644).
They concluded that resveratrol suppress the increases in
intracellular Ca2+ concentration ([Ca2+]i) induced by different
receptor agonists and blocks influx of external Ca2+ (13).
It is obvious that uterine contractility is a complex and dynamic
physiological process which consists of phasic and prolonged tonic
contractions. In order to determine whether resveratrol influences
myometrium contractility, we investigated three types of uterine
contractions: spontaneous rhythmic contractions (SRC), oxytocininduced phasic contractions and oxytocin-induced tonic
contractions. It is most commonly considered that SRC are the
result of depolarization of membrane and consequent opening Ltype Ca2+ channels. Intracellular Ca2+ transients produced by Ca2+
entry generate phasic spontaneous contractions; however the true
nature of these formations is not fully understood (14). Previously
it has been confirmed that low nanomolar concentration of
oxytocin (£1 nM) induces phasic contractions of rat uterus in
oestrus, but higher concentrations of oxytocin (³20 nM) induce
tonic contractions (15). Therefore, here we used 0.2 nM and 20 nM
of oxytocin for generation of phasic and tonic contractions,
respectively. Phasic contractions are essential for the successful
progression of labor, but they are undesirable for dysmenorrhea.
The prolonged tonic contractions of the uterus may reduce blood
flow in the myometrium and cause hypoxia.
Opening of K+ channel is crucial for achieving the
hyperpolarisation and lowering of the [Ca2+]i, resulting in
relaxation of myometrium. It has been shown that potassium
channels openers inhibited uterus smooth muscle contractility by
activation of different K+ channels (16-18). To date, several types
of K+ channels have been identified by means of pharmacological
and electrophysiological methods in the uterus smooth muscle. The
most studied include the KATP, KV and BKCa channels (19-22).
However, K+ channel family includes more than 100 various
protein subunits (22). The understanding of the most subtypes of
K+ channel and detection of their subunits/proteins in the smooth
muscle of uterus is important. Changes in the expression or activity
of K+ channels can translate into changes in the excitability,
contractility and relaxation of smooth muscles of the uterus.
Therefore, this study was aimed to investigate: 1) the
influence of resveratrol on contractility of rat non-pregnant
uterus and 2) the contribution of different K+ channel subtypes in
the resveratrol action on myometrial contractility.
Western blot study with specific antibodies for Kir6.1 and
Kir6.2 subunit of KATP channels, a- subunits Kv2.1 and Kv4.2
subtype of Kv channels and KCa1.1 subunit of BKCa channels
was performed in order to confirm their presence in the smooth
muscle of the non-pregnant rat uterus in oestrus.
MATERIALS AND METHODS
Animals
Experiments were carried out on the virgin female Wistar
rats (200–250 g) obtained from the animal facilities of the
Faculty of Medicine, University of Belgrade. This study has
been care out in accordance with the Directive 2010/63/EU and
the Guide for the Care and Use of Laboratory Animals (8th ed),
as adopted and promulgated by the United States National
Institute of Health. It has been approved by Ethics Committee of
Faculty of Medicine, University Belgrade (No 4211/2).
The oestrus phase of the oestrous cycle was determined by
microscopic examination of a daily vaginal smear (23). All rats
were killed by cervical dislocation. Uterine horns were cut into
longitudinal segments of approximately 5 mm length and
mounted in 10 mL volume organ bath containing Krebs-Ringer
solution aerated with 95% O2 and 5% CO2 at 37°C. Each strip
was attached with one end via surgical thread to a force
transducer and the other one was held fixed. The preparations
were mounted for isometric recording under 1 g tension and
equilibrated for 45 min. Isometric tension was measured with
TSZ-04/1.2 isolated tissue bath system and force transducer
(Experimetria, Budapest, Hungary). Data were recorded via
computer using IsoLAB software (Elunit, Belgrade, Serbia).
Experimental procedure
After equilibration, some preparations (37%) had
spontaneous rhythmic contractions. The preparations were
allowed to stabilize for at least 30 min before adding drugs. In a
separate series of experiments, uterus strips were stimulated with
oxytocin 0.2 nM or 20 nM to induce contractions and allowed
for 30 min period to assess control contractile performance. In
both types of experiments, concentration-response curves were
constructed by adding resveratrol (1–100 µM) directly to the
bathing solution in a cumulative way, taking the amplitude of the
response immediately before addition of a drug as the control
contraction. The results are expressed as the percentage
inhibition of the control contraction. Increasing concentrations
of resveratrol were added only after the previous concentration
had produced an equilibrium response or after 20 min if no
response was obtained.
In order to compare the effect of resveratrol with the effects
of K+ channels openers, NS 1619 and pinacidil were used. The
uterus strips were stimulated with oxytocin (20 nM) to induce
tonic contractions and allowed for 30 min period to assess
control contractile performance. In both types of experiments,
concentration-response curves were constructed by adding NS
1619 (10–100 µM) or pinacidil (10–100 µM) directly to the
bathing solution in a cumulative way. Increasing concentrations
of K+ channels openers were added only after the previous
concentration had produced an equilibrium response or after 20
min if no response was obtained.
To test the involvement of K+ channels in a mechanism of
action of resveratrol, NS 1619 and pinacidil, the different K+
channel blockers were examined. In separate experiments,
glibenclamide, iberiotoxin, tetraethylammonium chloride
(TEA), 4-aminopyridine (4-AP) were added into the bathing
solution 20 min before exposure to resveratrol, NS 1619 and
pinacidil. The concentration- response curves to resveratrol and
potassium channel openers were obtained in the presence of K+
channel blockers.
To investigate the effect of resveratrol on contractions elicited
only with intracellular source of Ca2+ the protocol was used:
1) Uterus strips were bathed in Krebs solution.
2) The tissue samples were bathed in K+-rich, Ca2+-free
solution in which the concentration of KCl was raised to 60 mM
by isosmotic replacement of NaCl.
3) Strips were stimulated with oxytocin (20 nM) to produce
control contraction.
4) The tissue was returned to Krebs solution for a 30 min.
5) The tissue samples were bathed in K+-rich, Ca2+-free
solution, again.
797
6) A single concentration of resveratrol (1 µM, 3 µM,
10 µM, 30 µM and 100 µM) was added separately to the medium
for 10 minutes and the preparation was stimulated with oxytocin.
The control contraction to oxytocin was taken as 100%. Vehicle
matched control experiments were conducted.
Western blot analysis
Rat uterine tissue samples were homogenized in RIPA buffer
with protease inhibitors (Roche Applied Science, Mannheim,
Germany). Cell lysate was centrifuged for 20 minutes, 11 000
rpm at 4°C and the supernatant was transferred to a new tube.
Protein concentration was measured at 595 nm with
spectrophotometer using the Bio-Rad Protein Assay, based on the
method of Bradford. Prior to SDS PAGE, LDS Sample Buffer
and Reducing Agent were added to the sample, denaturated for 10
minutes at 70°C and loaded into precast 4–12% Bis-Tris gel (Life
Technologies, Carlsbad, CA, USA). After electrophoresis,
separated proteins were transferred to a nitrocellulose membrane.
A membrane was blocked with 5% nonfat dry milk and 0.1%
Tween 20 for one hour at room temperature and probed with
primary antibody for Kir6.1 and Kir6.2 subunit of KATP channels,
a-subunits Kv2.1 and Kv4.2 subtype of Kv channels and KCa1.1
subunit of BKCa channels (Anti-Kir6.1; Anti-Kir6.2; Anti-Kv2.1;
Anti-Kv4.2; Anti-KCa1.1; Alomone Labs, Jerusalem, Israel)
overnight at 4°C. Membranes were probed with HRP-conjugated
antirabbit secondary antibody for 1.5 h at room temperature.
Western blotting kit (Roche, Applied Science, Mannheim,
Germany) was used for the chemiluminescent detections of
proteins. After visualization, the membranes were stripped with
0.2 M NaOH, blocked and reprobed with anti-b-actin antibody
(Abcam, Cambridge, England) and visualized. Protein loading
was normalized to b-actin. ImageQuant software was used for
quantitative analysis (24).
Drugs and solutions
All chemicals were obtained from the Sigma-Aldrich Inc.
St.Louis, MO, USA. Resveratrol and NS 1619 were dissolved in
70% v/v ethanol with further dilution in distilled water before use.
Working concentrations of ethanol in the bath were <0.01% (v/v).
Glibenclamide was dissolved in polyethylene glycol. Stock
solution of pinacidil was dissolved in dilute acid solution (0.1 N
HCl) with further in distilled water before use. Iberiotoxin, TEA, 4AP, and oxytocin were dissolved in distilled water. The KrebsRinger solution had the following composition (mmol/l: NaCl 120,
KCl 5, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose
11). Krebs-Ringer K+- rich, Ca2+- free solution was prepared by
omitting CaCl2 and adding EGTA (1 mM). All drugs were added
directly to the bath in a volume of 50 µL and the concentrations
given are the calculated final concentrations in the bath solution.
Treatment of data and statistics
The amplitude of the tonic contractions was measured from
the baseline to plateau, and the amplitude of the phasic
contractions were measured from the baseline to the end of the
spike. The frequency of SRC and oxytocin-induced contractions
was calculated as a number of cycles in a 10 minutes period of
time. The mean amplitude and frequency during the control
period was taken as 100%.
EC50 value is defined as the concentration of resveratrol
required to produce 50% of the maximum response of elicited
and spontaneous contractions, and it was determined for each
curve by using a non-linear least square fitting procedure of the
individual experimental data, and presented as pD2 (pD2 = –log
EC50). The results are expressed as the means ± standard error of
the mean (S.E.M.); n refers to the number of experiments.
Statistical difference between means was determined by
Fig. 1. Original recordings show the
effects of resveratrol (1–100 µM) on
contractile activity of the nonpregnant rat myometrium: SRC (A),
phasic contractions provoked by low
concentration of oxytocin (0.2 nM,
square) (B), tonic contractions
provoked by high concentration of
oxytocin (20 nM, triangle) (C). SRC,
spontaneous rhythmic contractions;
Oxy, oxytocin.
798
A
Student’s t-test; a value of P<0.05 was considered statistically
significant. Data analysis was performed in Graph Pad Prism
(Graph Pad Software Inc., San Diego, USA).
RESULTS
Effects of resveratrol on spontaneous rhythmic contractions
B
C
Fig. 2. Antagonism of the inhibitory effect of resveratrol by
K+-channel blockers on SRC and contractions provoked by
oxytocin (phasic and tonic) on the isolated non-pregnant rat
uterus. (A) Concentration - response curves for resveratrol on
the SRC, (B) the phasic contractions provoked by oxytocin
(0.2 nM), and (C) the tonic contractions provoked by oxytocin
(20 nM), in the absence (circle) and presence of glibenclamide
(10 µM, square), 4-AP (1 mM, down triangle), TEA (1 mM,
diamond) and iberiotoxin (100 nM, up triangle). The points
are the means and the vertical lines show the S.E.M. (n=5–12).
*P<0.05. SRC, spontaneous rhythmic contractions; GLB,
glibenclamide;
4-AP,
4-aminopyridine;
TEA,
tetraethylammonium; Ibtx, iberiotoxin.
Longitudinal muscle strips of the rat uterus exhibited the
SRC of constant amplitude 2.29±0.9 g and frequency
10.07±1.20 contractions per 10 minutes, as shown in Fig. 1A
(n=12). Resveratrol (1–100 M) inhibited amplitude of SRCs in a
concentration-dependent manner with a pD2 value of 4.53±0.20,
maximal response 93.64±2.53 %, n=12 (Fig. 1A). Used in the
same concentrations, resveratrol significantly inhibited the
frequency of SRC (pD2 value was 4.34±0.66, n=12, Fig. 1A).
Glibenclamide (10 µM), a selective KATP channels blocker
produced a significant rightward shift of the concentrationresponse curve of resveratrol (pD2 values: 4.53±0.20 in the
absence versus 4.33±0.40 in the presence of glibenclamide,
P<0.05, n=7). Glibenclamide failed to produce a suppression of
the maximal response of resveratrol on SRC (93.64±2.53% in
the absence versus 90.50±5.21% in the presence of
glibenclamide, P>0.05, n=7, Fig. 2A).
TEA, a selective inhibitor of calcium-sensitive K+ channels
used in concentration of 1 mM significantly affected resveratrolinduced inhibition (pD2 values: 4.53±0.20 in the absence versus
4.40±0.30 in the presence of TEA, P<0.05, n=6). TEA produced
a significant suppression of the maximal response of resveratrol
on SRC (93.64±2.53% in the absence versus 77.50±3.08% in the
presence of TEA, P<0.05, n=6, Fig. 2A).
Iberiotoxin (100 nM), a highly selective blocker of BKCa
channels, significantly affected resveratrol-induced inhibition
(pD2 values: 4.53±0.20 in the absence versus 4.20±0.51 in the
presence of iberiotoxin, P<0.05, n=5) of amplitude of SRC and
it produced a significant suppression of the maximal response of
resveratrol (93.64±2.53% in the absence versus 76.20±5.66% in
the presence of iberiotoxin, P<0.05, n=5, Fig. 2A).
A predominant blocker of Kv channels, 4-AP (1 mM)
produced a significant rightward shift of the concentrationresponse curve of resveratrol (pD2 values: 4.53±0.20 in the absence
versus 4.41±0.50 in the presence of 4-AP, P<0.05, n=9). However,
4-AP failed to produce a suppression of the maximal response of
resveratrol on SRC (93.64±2.53 % in the absence versus
93.22±6.47 % in the presence of 4-AP, P>0.05, n=9, Fig. 2A).
Iberiotoxin (100 nM) and TEA (1 mM) significantly affected
resveratrol-induced inhibition of frequency of SRC (pD2 values:
4.34±0.66 in the absence versus 3.7 ±0.41 in the presence of
iberiotoxin, n=5; versus 4.01±0.37 in the presence of TEA,
respectively, n=6 P<0.05). Glibenclamide (10 µM, n=7) and 4-AP
(1 mM, n=9) failed to antagonized the reduction of frequency of
SRC by resveratrol (pD2 values were 4.37±0.42 and 4.36±0.30,
respectively).
Effects of resveratrol on phasic contractions provoked by
oxytocin
Application of low nanomolar concentration of oxytocin
(0.2 nM) to bath medium produced phasic contractions of
constant amplitude 2.95±0.70 g and frequency 11.59±1.08
(n=12, Fig. 1B). Resveratrol (1–100 µM) significantly inhibited
amplitude of oxytocin-induced phasic contractions in a
concentration-dependent manner with pD2 value of 4.66±0.28,
maximal responses: 93.67±3.00%, n=12, (Fig. 2B). The
frequency of oxytocin phasic contractions was inhibited by
resveratrol also (pD2 value was 4.69±0.38, n=12, Fig. 2B).
799
A
B
C
Fig. 3. The effects of K+ channels openers NS 1619 and
pinacidil with resveratrol on the tonic contractions induced
by oxytocin (20 nM). (A) Concentration-response curve for
NS 1619, a highly specific BKCa channels opener in the
absence (circle) and presence (up triangle) of iberiotoxin 100
nM; (B) concentration-response curve for pinacidil, a
predominant opener of KATP channels in the absence (down
triangle) and presence (square) of glibenclamide 10 µM; (C)
The order of potency: concentration-response curve for NS
1619 (circle), pinacidil (down triangle) and resveratrol
(diamond). The points are the means and the vertical lines
show the S.E.M. (n=5–12). *P<0.05. GLB, glibenclamide;
Ibtx, iberiotoxin.
Glibenclamide (10 µM,) produced a significant rightward
shift of the concentration-response curve of resveratrol (pD2
values: 4.66±0.28 in the absence versus 4.26±0.73 in the
presence of glibenclamide, P<0.05, n=5) and suppression of the
maximal response (93.67±3.00% in the absence versus
85.40±5.19% in the presence of glibenclamide, P<0.05, n=5,
Fig. 2B).
TEA, used in concentration of 1 mM produced a significant
rightward shift of the concentration-response curve of
resveratrol (pD2 values: 4.66±0.28 in the absence versus
4.19±0.72 in the presence of TEA, P<0.05, n=5). TEA produced
suppression of the maximal response to resveratrol
(93.67±3.00% in the absence versus 74.60±7.44% in the
presence of TEA, P<0.05, n=5, Fig. 2B).
Iberiotoxin (100 nM) produced a significant rightward shift
of the concentration-response curve of resveratrol (pD2 values:
4.66±0.28 in the absence versus 4.30±0.21 in the presence of
iberiotoxin, P<0.05, n=5) without a suppression of the maximal
response (93.67±3.00% in the absence versus 94.56±1.68% in
the presence of iberiotoxin P<0.05, n=5, Fig. 2B).
Application of 4-AP (1 mM,) produced a significant
rightward shift of the concentration-response curve of
resveratrol (pD2 values: 4.66±0.28 in the absence versus
4.34±0.55 in the presence of 4-AP, P<0.05, n=7) with a
significant suppression of the maximal response (93.67±3.00%
in the absence versus 78.00±6.00 % in the presence of 4-AP,
P<0.05, n=7, Fig. 2B).
Iberiotoxin (100 nM) and TEA (1 mM) significantly affected
resveratrol-induced inhibition of frequency (pD2 values:
4.69±0.38 in the absence versus 4.30±0.62 in the presence of
iberiotoxin; versus 4.35±0.71 in the presence of TEA, P<0.05,
n=5, both), too. Glibenclamide (10 µM, n=5) and 4-AP (1 mM,
n=7) failed to produce statistically significant suppression of the
reduction of frequency of phasic contractions provoked by
oxytocin (pD2 values were 4.67±0.83 and 4.60±0.59).
Effects of resveratrol and potassium channel openers on tonic
contractions provoked by oxytocin
Application of high concentration of oxytocin (20 nM) to
bath medium produced tonic contraction with or without phasic
contractions in addition (Fig. 1C). Resveratrol inhibited tonic
contractions induced by oxytocin in the concentration-dependent
manner with pD2 value of 4.06±0.29 and maximal responses of
54.78±4.52%, n=9. The all used K+ channel blockers,
glibenclamide (10 µM, n=5), TEA (1 mM, n=5), iberiotoxin
(100 nM, n=5) and 4-AP (1 mM, n=5) failed to produce a
rightward shift of the concentration-response curve of
resveratrol and suppression of the maximal response on tonic
oxytocin-induced contractions (Fig. 2C).
In order to compare the effect of resveratrol on tonic
contractions induced by oxytocin with the effect of potassium
channel openers, NS 1619, a highly specific BKCa channels
opener and pinacidil, a predominant opener of KATP channels
were used (n=5 both). NS 1619 (10 nM–100 µM) inhibited tonic
contractions induced by oxytocin in the concentration-dependent
manner with pD2 value of 6.00±0.34 and maximal responses of
98.82±1.02%. Iberiotoxin (100 nM), a selective inhibitor of
BKCa channels, produced a significant rightward shift of the
concentration-response curve of NS 1619 (pD2 value in the
presence of iberiotoxin was 4.48±0.64, the maximal response
was 87.00±5.12%, P<0.05 both, n=5, Fig. 3A). Pinacidil
(10 nM–100 µM) inhibited tonic contractions induced by
oxytocin in the concentration-dependent manner with pD2 value
of 5.29±0.20 and maximal responses of 95.25±1.08%.
Glibenclamide (10 µM) produced a significant rightward shift of
the concentration-response curve of pinacidil (pD2 value in the
800
Fig. 4. Original recordings show the
effects of 1 µM and 100 µM of
resveratrol on the responses to
oxytocin (20 nM) in the K+-rich, Ca2+free solution; oxytocin application
induced contraction in K+-rich, Ca2+free Krebs solution. (A) Control; (B)
Oxytocin - induced contraction in K+rich, Ca2+-free Krebs solution after
pretreatment with 1 µM or 100 µM
resveratrol; (C). The effects different
concentrations of resveratrol (1–100
µM) on contractions induced by
oxytocin (20 nM) in the K+-rich, Ca2+free solution. The columns are the
mean values of amplitudes of
contractions ± S.E.M. in the presence
of individual concentrations of
resveratrol (n=5, each). The amplitude
of contractions in absence of
resveratrol was taken as 100% control. * P<0.05 when comparing
effects of individual concentration of
resveratrol.
presence of glibenclamide was 4.61±0.71, suppression of the
maximal response to pinacidil was 74.84±2.96, P<0.05 both,
n=5, Fig. 3B). There are statistically significant difference
between pD2 values of resveratrol, pinacidil and NS 1619
(4.06±0.29 > 5.29±0.20 > 6.00±0.34, respectively, P<0.05 all).
The order of potency was shown in the Fig. 3C.
The all used K+ channel blockers did not alter the resting
tone of the uterus and they did not modify the amplitude of SRC
and contractions evoked by oxytocin (n= 2 all, data not shown).
100% without resveratrol versus 100% with 1 µM, 3 µM or
10 µM of resveratrol, n=15 all, P>0.05, Figs. 4A and 4C).
However, higher concentrations of resveratrol (30 µM and 100
µM) significantly reduced tonic contraction (% of contraction:
100% without resveratrol versus 65±2% with 30 µM of
resveratrol and 47%±2% with 100 µM of resveratrol,
respectively, P<0.05, n=10 both, Figs. 4B and 4C).
Effects of resveratrol on the contraction provoked by oxytocin
in K+-rich, Ca2+-free solutions
Western blotting analysis detected Kir6.1 and Kir6.2 subunit
of KATP channels, a- subunits Kv2.1 and Kv4.2 subtype of Kv
channels and KCa1.1 subunit of BKCa channels in non-pregnant
rat uterus (n=5, each, Fig. 5A). Results were normalized to bactin expression. To quantify the relative abundance of level
expression proteins of Kir6.1, Kir6.2, Kv2.1, Kv4.2 and KCa1.1
densitometry was used. Comparison by densitometry revealed a
significant lower expression level of the Kir6.2 protein than
protein of the others K+ channels subtypes. There were no
In a K+ -rich, Ca2+-free solution, the SRC and the phasic
contraction provoked by low concentration of oxytocin (0.2 nM)
were abolished (n=4 all, data not show), but an application of 20
nM oxytocin induced tonic contraction. Resveratrol in a
concentrations <30 µM did not affect the amplitude of
contractions induced by 20 nM oxytocin (% of contraction:
Western blot analysis
801
Fig. 5. (A) The expression of Kir6.1
and Kir6.2 subunit of KATP channels,
a- subunits Kv2.1 and Kv4.2 subtype
of Kv channels and KCa1.1 subunit of
BKCa channels protein relative to bactin detected by Western blot in nonpregnant rat uterus. (B) Quantitation
of Western blot signals of K+ channels
bands expressed as mean values ±
S.E.M. densitometric units for each
K+ subtypes shown as a histogram
(n=5, each). * P<0.05 when
comparing Kir6.2 subunits protein
expression with Kir6.1, Kv2.1 and
Kv4.2 and KCa1.1 subunit protein
expression.
Table 1. The effects of resveratrol on the frequency of SRC and
phasic contractions provoked by oxytocin (0.2 nM) in the
absence and in the presence of K+ channels blockers.
Type of
contractions
SRC
Oxytocin
provoked
phasic
contractions
pD2
n
Control
GLB
4-AP
TEA
Ibtx
4.34±0.66 a
4.37±0.42
4.36±0.30
4.01±0.37 a
3.47±0.41 a
12
7
9
6
5
Control
GLB
4-AP
TEA
Ibtx
4.69±0.38 a
4.67±0.83
4.60±0.59
4.35±0.71 a
4.30±0.62 a
12
5
7
5
5
Each pD2 value is the mean ± S.E.M. for n experiments. aP<0.05
versus pD2 values in the absence and in the presence of
potassium channels blockers. SRC, spontaneous rhythmic
contractions; GLB = glibenclamide, 4-AP, 4-aminopyridine;
TEA, tetraethylammonium; Ibtx, iberiotoxin.
significant differences in the density of the expression levels
among the others protein subtypes (Fig. 5B).
DISCUSSION
It has been found that resveratrol significantly decreased the
amplitude and frequency of phasic contractions induced by low
concentration of oxytocin (0.2 nM); its sensitivity to resveratrol
was comparable with the sensitivity of SRCs (EC50 were 29 µM
and 23 µM, respectively) to the same agent. Hsia et al. (13) have
shown that 100 µM of resveratrol induced only 40% of
inhibition of oxytocin-induced contractions of rat non-pregnant
uterus. However, in their experiment they used higher
concentrations of oxytocin in order to induced contractions
(1 µM versus 0.2 nM used in our study) (13). The tonic
component of oxytocin contractions had lower sensitivity to
resveratrol (EC50=87 µM) than both types of phasic contractions.
Also, Kawamata et al. (15) have published that phasic
component of oxitocin-induced contractions of non-pregnant
mouse uterus had higher sensitivity to verapamil than its tonic
component. This is in line with previous findings where authors
have shown that phasic contractions of guinea pig gallbladder
were more sensitive to resveratrol than its basal tension (25). On
the other hand, we have shown previously that resveratrol
inhibited tonic contractions of rat aorta (7) and mesenteric artery
(8) with higher potency (EC50 were 10 µM, both) than oxytocininduced tonic contraction investigated here. But, the sensitivity
of tonic contraction of human internal mammary artery
802
(EC50=30 µM) to resveratrol was comparable with results
obtained in this paper (9). It is plausible to conclude that there
are apparent differences in sensitivity to resveratrol among
various types of contractions (phasic versus tonic), species and
tissues.
In order to analyze the contribution of K+ channels to the
inhibition of uterine contractions produced by resveratrol, we
used agents that are known to possess a K+ channel-blocking
activity.
According to the results presented here, it seems that BKCa
channels contributed to the resveratrol inhibition of frequency
of both types of phasic contractions. It has been showed that
iberiotoxin initiated SRC in human myometrium (26) and
enhanced the frequency, whereas Taggart and Wray (27)
showed that same substance had no obvious effect on
frequency. Moreover, neither TEA nor 4-AP affected the
contraction interval of SRC, suggesting that these drugs were
not affecting the frequency (28). However, it has been showed
that the changes in frequency of phasic contractions induced by
oxitocin, are mediated by the sarcoplasmic reticulum Ca2+
release (29). Anwer et al. also showed that when applied to
cultured smooth muscle cell isolated from human myometrium,
iberiotoxin caused depolarization and a rise in [Ca2+]i (26).
Recent data suggest that resveratrol can attenuate intracellular
Ca2+ release from sarcoplasmic reticulum of the rat abdominal
aorta (30). By repolarizing the cell membrane, opening of BKCa
channels may decrease the entry of extracellular Ca2+ thereby,
influence the intracellular Ca2+ transient and contractile
property. Since resveratrol decrease [Ca2+]i, the inactivation
BKCa channels by iberiotoxin and TEA may constitute a
feedback repolarizing system.
Functional studies indicated that the KATP channels have an
important role in regulation of uterus contractility (19).
Stimulation of these channels relaxed the smooth muscle of
uterus (17, 18). Structurally, KATP channels are composed of
pore-forming inward rectifiers channels, Kir6.1 or Kir6.2, and
regulatory subunits, SUR1, SUR2A or SUR2B that assemble to
form an octameric complex (31). The properties of these
channels are different in various tissues due to the combinations
of the subunits forming the channel. It is generally accepted that
glibenclamide is a selective blocker of KATP channels (32). In the
present study, glibenclamide antagonized the effect of
resveratrol on the SRC and of phasic oxytocin-induced
concentration indicating involvement of KATP channels in
resveratrol mechanism of action. Indeed, in the rat heart it was
reported that resveratrol depressed cardiac muscle contraction
and shortened action potential duration due to the activation of
KATP channels (33). But, glibenclamide failed to antagonize the
effect of resveratrol on the tonic oxytocin-induced contractions.
Also, our previous studies have shown that the relaxant effect of
resveratrol on the tonic contraction of rat mesenteric and human
internal mammary artery was not blocked by glibenclamide (7,
8). Accordingly, it seems that KATP channels are not involved in
the pathway by which resveratrol induces a relaxation of the
tonic contractions. To our knowledge, this is the first study
which reveals the protein expression of Kir6.1 and Kir6.2
subunits of KATP channels in rat non-pregnant myometrium. We
obtained that the protein expression level of Kir6.1 subunit was
higher than Kir6.2 subunit when they normalized to b-actin
expression. Earlier papers have showed similar expression ratio
of the two types of Kir channels (Kir6.1/SUR2B and
Kir6.2/SUR1) in the smooth muscle of non-pregnant rat and
human uterus but only at the transcription level (34, 35).
Recently, one study found the protein expression of Kir6.1 and
Kir6.2 subunits in human pregnant myometrium; however the
expression level of Kir6.1 was less compared with Kir6.2 (36).
The expression of KATP channels depends on the gestational stage
and the presence of labor contractions (37). Considering the fact
that Kir6.1/SUR2B is down regulated late in pregnancy (36)
different ratio Kir6.1/Kir6.2 in non-pregnant and pregnant
myometrium is not surprising.
It has previously been shown that potassium channel openers
produce relaxation of different blood vessels via activation of
BKCa channels in the smooth muscle (38). Interestingly,
Malinowsky et al. conclude that perivascular adipose tissue of
human internal mammary artery releases relaxing factor that
seems to act with the involvement of smooth muscle BKCa
channels (39). Previous pharmacological data have shown that
BKCa channels are abundant in myometrium and they are
involved in mediating relaxation of uterine smooth muscle (20).
Their activity are triggered by depolarization and enhanced by
an increase in [Ca2+]i, providing a link between the metabolic
and electrical state of cells. In the different experimental model,
Chen et al. have suggested that resveratrol could activate BKCa
channels in the b cells through an increase of intracellular Ca2+
(12). In contrast, previously it has been found that in endothelial
cells resveratrol stimulates BKCa channels through Ca2+independent mechanism (40). To analyze the possibility that the
inhibitory effect of resveratrol on the myometrium is mediated
via BKCa channels, iberiotoxin and TEA was tested. Iberiotoxin
is highly specific BKCa channels blocker. TEA selectively blocks
the BKCa channels with the IC50 value around 2 mM. But used in
a higher concentrations (up to 10 mM), TEA blocks other types
of K+ channels, too (28). The concentrations of TEA used in our
study were sufficient to inhibit BKCa channels. According to the
results obtained with iberiotoxin, TEA and resveratrol on the
SRC and on the phasic oxytocin-induced contractions, it is
reasonable to conclude that BKCa channels are involved in the
resveratrol mechanism of action on the rat uterus. However, the
effects of resveratrol on the tonic oxytocin-induced contractions
were not sensitive to iberiotoxin and TEA indicating that BKCa
channels are not involved in this resveratrol action. By Western
blot analyses we confirmed the presence of BKCa channels in the
smooth muscle of rat uterus. Basically, BKCa channels consist of
4 a-subunits that form the channel pore and up to 4 regulatory
b-subunits that modify phenotypic and functional diversity (20).
The a-subunits is derived from a single gene, but may exist as
several protein species varying from 83 to 110 kDa,
demonstrating the occurrence of post-translational modification
(41, 42). We obtained the expression of the ~83 kDa protein asubunit for non-pregnant rat myometrium.
We observed that 4-AP (1 mM), a predominant blocker of
Kv channels, antagonized the effect of resveratrol on the SRC
and phasic oxytocin-induced contractions. Previously, in a
model of SRC of non-pregnant rat uterus, it has been shown that
Kv channels were inhibited by 4-AP (1–5 mM) (19). However,
the relaxant effect of resveratrol on the tonic oxytocin-induced
contractions was not blocked by 4-AP. In the same way, 4-AP
did not alter relaxation of tonic contraction of the retinal
arterioles by resveratrol (43). In contrast, a few studies have
confirmed that Kv channels participate in the resveratrol effects
on tonic contractions of the different blood vessels (7-9).
Furthermore, it has been shown that resveratrol inhibits the Kv
channels on the b cells (12). These discrepancies in antagonism
of resveratrol and 4-AP in different tissues may be consequences
of different expression/function of various subtypes of Kv
channels. For example, it has been demonstrated that uterus
smooth muscle cells contain at least three types of Kv current: 4AP insensitive, 4-AP sensitive rapidly inactivating current, 4-AP
sensitive slowly inactivating current (44). Phenotypically, two
main subfamily groups of Kv currents have been observed using
electrophysiological methods which differ in their
pharmacological sensitivity-delayed rectifiers (Kv2.x) and
rapidly inactivating current(Kv4.x) (19). Therefore, we sought to
803
determine whether Kv4.2 and Kv2.1 channels protein are present
in non-pregnant rat uterus. Indeed, the Western blot analysis
confirmed protein expression of both types in similar ratio.
Expression of several Kv1.x and Kv4.x subtypes have been
demonstrated in mouse myometrium cells at the gene expression
and protein level (44). However, the number of expressed
subunits appears to be much larger than the number of apparent
Kv current components. Smith and coworkers (44) suggest that
Kv4.2 subtype could be molecular target for 4-AP in mouse
myometrium. From another side, Kv2.1 is a delayed rectifier
potassium channel ubiquitously expressed in the heart, brain and
neurons (45, 46). But to date there is no evidence for expression
of Kv2.1 channels in myometrial tissue. The data obtained on the
vascular smooth muscle indicates that Kv2.1 current is 4-AP
sensitive (47). According to this, it seems that effect of
resveratrol on phasic contractions appears to be mediated by
Kv4.2 and Kv2.1 subtype of Kv channels present in nonpregnant rat myometrium.
According to our results, it seems that KATP, iberiotoxin,
TEA- and 4-AP-sensitive channels are involved in the inhibition
of SRC and phasic oxytocin-induced contraction of rat uterus
produced by resveratrol. Similar results were obtained for
potassium channel opener, pinacidil on the human pregnant
myometrium and human radial artery (48, 49). The resistance of
resveratrol’s effects achieved by high concentration (³30 µM) to
different K+ channel blockers indicated that resveratrol partly
exerts its inhibition of SRC and phasic oxytocin-induced
contraction by acting on multiple sites. Similarly, K+-channel
independent effects of high concentrations of resveratrol were
obtained in the rat aorta and uterus (7, 13). Moreover, in the
model of oxytocin-induced tonic contractions of rat nonpregnant uterus resveratrol does not behave as a classical
potassium channel openers. The all tested K+ channels blockers
did not antagonize the resveratrol’s effect on this type of
contractions. In contrast, NS 1619, a highly specific BKCa
channels opener and pinacidil, a predominant opener of KATP
channels produced more potent inhibition of tonic oxytocininduced contractions than resveratrol. According to the affinity
of iberiotoxin and glibenclamide, it seems that the effect of NS
1619 and pinacidil on the tonic oxytocin-induced contractions
was mediated by BKCa and KATP channels, respectively.
Thus, apart from different states of the smooth muscle, the
differences between effects of resveratrol on contractions
induced by low and high concentrations of oxytocin might be
explained with different nature and sources of initiation and
maintenance of phasic and tonic contractions, too. Hence,
oxytocin, at low concentrations, produces phasic contraction of
the isolated uterus of the non-pregnant rat which can be
explained by Ca2+ influx via L-types Ca2+ channels. The
oxytocin, at high concentrations produces tonic contraction
resulting from another mechanism which does not appear to
involve Ca2+ influx only (50, 51). Therefore, in order to
investigate whether resveratrol could influence contraction
induces by oxytocin’s intracellular signal pathways, the series of
experiments in the K+ -rich, Ca2+-free Kreb’s solution (the
absence of Ca2+ influx and K+ efflux) were done. Ca2+-free
Kreb’s solution prevent Ca2+ influx in the absence of
extracellular Ca2+ and high K+ abolished the effect of K+channels opening. Under this condition, SRC and phasic
oxytocin-induced contractions were abolished, but an
application of 20 nM oxytocin induced tonic contraction which
was not sensitive to resveratrol used in concentrations lower than
30 µM. These findings provide support for the hypothesis that
³30 µM of resveratrol can inhibit contractions of uterus through
influence on Ca2+- and K+- channels independent intracellular
signaling pathways. According to our observations, it seems that
resveratrol may produce relaxation of non-pregnant uterus by
activation of different smooth muscle K+ channel and/or by
inhibition of the signaling pathways involved in the tonic
contraction of oxytocin. This assumption is in line with the
research on the guinea pig gallbladder smooth muscle (26).
However, the additional experiments are necessary to define
closely the role of the intracellular mechanisms of action of
resveratrol on the tonic oxitocin-induced contractions of rat
uterus. Having in mind that Choi et al. recently suggested that
Ca2+ transport genes may play roles in the uterus for Ca2+
transport and reproductive function, it might be of interest to
investigate whether resveratrol interact with these molecular
structures (52).
The present study clearly demonstrates that resveratrol exert
a potent inhibitory effect on the rat non-pregnant uterine
contractility. The different way of initiation of contractions of
the myometrium influenced differently its response to
resveratrol. Based on K+-channels blockers affinity, it appears
that the inhibitory response of resveratrol on the SRC and phasic
contractions involves different myometrial K+-channels. But
when applied in high concentrations, resveratrol has an
additional K+-channels independent mechanism(s) of action. The
suggestion that effect of resveratrol on the tonic contractions
induced by high concentration of oxytocin is mainly K+ channels
independent, has to be investigated further.
The present study demonstrated, for the first time, the protein
expression ratio of KATP, Kv and BKCa channels in myometrium
of non-pregnant rat. Also, this is the first study which reveals the
protein expression of Kv2.1 and Kir6.1 and Kir6.2 subunits of
KATP channels in rat non-pregnant myometrium.
Based on the results presented here it is plausible to
conclude that resveratrol has potential to be used in prevention
and treatment of uterine abnormal contractility. The
concentration of resveratrol used in our study corresponds to a
reasonable dose for human use (13).
Acknowledgements: This work was supported by the
Scientific Research Grant from Ministry of Education, Science
and Technological Development Government of the Republic of
Serbia (No TR31020 and OI175064).We would like to thank
Mrs. Milena Zabunovic for technical support during this study.
Conflict of interests: None declared.
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play a role in calcium transport in the uterine endometrium?
J Physiol Pharmacol 2011; 62: 499-504.
R e c e i v e d : August 22, 2013
A c c e p t e d : November 13, 2013
Author’s address: Professor Ljiljana Gojkovic-Bukarica,
Institute of Pharmacology, Clinical Pharmacology and Toxicology,
Medical Faculty, University of Belgrade, Belgrade 11129, Serbia.
E-mail: [email protected]