WINE POLYPHENOL RESVERATROL INHIBITS CONTRACTIONS OF ISOLATED RAT UTERUS BY ACTIVATION OF SMOOTH MUSCLE INWARDLY RECTIFYING POTASSIUM CHANNELS

* Rad primljen za štampu 30.07.2016. ** Radmila Novaković, Institute of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Serbia; Nebojša Radunović, Institute of Gynecology and Obstetrics, Clinical Center of Serbia, Belgrade, Serbia; Jovana Rajković, Vladimir Đokić, Institute of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Serbia; Aleksandar Petrović, Institute of Food Technolgy and Biochemistry, Faculty of Agriculture, University of Belgrade, Serbia; Branka Ivković, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Serbia; Vitomir Ćupić, Institute of Pharmacologyand Toxicology, Faculty of Veterinary Medicine, University of Belgrade, Serbia; Vladimir Kanjuh, Serbian Academy of Sciences and Arts, Belgrade, Serbia; Helmut Heinle, Institute of Physiology, University of Tuebingen, Germany; Ljiljana Gojko vićBukarica, Institute of Pharmacology ORIGINALNI RAD / ORIGINAL PAPER

Resveratrol is a phytoalexin produced in a number of plant species including grapes.The benefit of resveratrol to health is widely reported.Resveratrol has been found to promote relaxation of non-pregnant and pregnant uterus, but its mechanism of action is unclear.The aims of our study were to investigate the involvement of inwardly rectifying potassium channels (Kir) in inhibitory effects of resveratrol on three models of contractions of non-pregnant rat uterus: the spontaneous rhythmic contractions (SRC), oxytocin-elicited phasic contractions and tonic oxytocin-elicited contractions.Uterine strips were obtained from virgin female Wistar rats in oestrus.Strips were mounted into organ bath for recording isometric tension in Krebs-Ringer solution.Experiments followed a multiple curve design.In order to test the involvement of Kirchannels in a mechanism of action of resveratrol(1-100 µM),BaCl 2 (1 mM),a antagonist of inwardly rectifying potassium channels was used.Resveratrol induced a concentration-dependent relaxation of all models of contractions.BaCl 2 antagonized the response to resveratrolon SRC and oxytocin-elicited phasic contractions.Relaxation achieved by resveratrolon tonic oxytocin-elicited concentrations was insensitive to BaCl 2 .The antagonism of resveratrol effects by inwardly rectifying potassium channels antagonist suggests Materijal i metode rada / Material and methods tes quiescence during gestation (McCloskey i sar., 2014).The roles of the different channels are diverse and depend on the physiological environment.The contraction of myometrium is a complex event and a number of potassium channels play a role in shaping the action potential and modulating myometrial relaxation (Brainard i sar., 2007).To date, several types of K + channels have been identified in the myometrium (Du i sar., 2004; Novaković i sar., 2013; McCloskey i sar., 2014; Novaković i sar., 2015).Kir channels proteins have been identified in pregnant mouse and human myometrium by pharmacological and molecular methods (McCloskey i sar., 2014).
The abnormal contractility might underlie common and important disorders such as infertility, implantation failure, dysmenorrhea, endometriosis, spontaneous miscarriage or preterm birth (Olafsdottir i sar., 2012).The ideal agent for prevention and treatment of uterine abnormal contractility has not yet been found.The mechanism that triggers uterine spasm is currently unclear, although different factors or events seem to be involved.Understanding the processes that lead to relaxation of smooth muscle of the uterus and development of safe and effective tocolytic agents is an important research topic.
The aim of this study is to investigate the participation of Kir channels in inhibitory action of resveratrol on three models of uterus contractility: spontaneous rhythmic contractions (SRC), oxytocin -elicited phasic and oxytocin -elicited tonic contractions of non-pregnant rat uterus.

Animals
The experiments were performed on isolated uterus of the virgin female Wistar rats (200 -250 g), obtained from the animal facilities of the Faculty of Medicine, University of Belgrade.All experiments were conducted in compliance with the Animal Welfare Act of the Republic of Serbia ("Službeni glasnik RS", No. 41/09), the respective regulations ("Službeni glasnik RS", No. 39/10), as well as international legislation (Directive 2010/63/EU and the Guide for the Care and Use of laboratory Animals, 8th ed) with respect to the guidelines of 'Good laboratory practice'.The study was approved by the Ethical Committee of Faculty of Medicine, University of Belgrade, license number 4211/2.
The estrous cycle phase was daily determined before each series of experiments.Vaginal smear was sampled and transmitted to the microscope slide where the cell staining was performed using haemotoxylin and eosin in the general method described by Marcondes i sar.(2002).Based on the appearance of cells during microscopic examination the estrous cycle phase was determined.Only animals which were in estrous phase (45 animals were used) were taken for the experiments and they were sacrificed by cervical dislocation.Then the abdomen was opened in V-shaped form and the both horns of the uterus were isolated.After separating, the tissue sample was held in the isolated organ bath (TSZ-04 / 1.2, Experimetria, Budapest, Hungary) at a temperature of 37° C in Krebs-Ringer solution through which was bubbled into a mixture of 95% oxygen and 5% carbon dioxide, pH 7.4.Segment of the horn of the uterus 1 cm long was placed in the bath by fixing one end of the sample to a holder at the bottom of bath (10 ml), and the other end was attached to the transducer (TSZ-04 / 1.2, Experimetria, Budapest, Hungary) that registers the change of isometric muscle tension.Before the beginning of experiments, samples were incubated for 45 minutes and then they were gradually stretched to an optimal point of tension of 1 g.The contractions were recording by ISOLAB software (Elunit, Belgrade, Serbia) which was connected to the transducer.

Experimental procedure
The inhibitory effects of resveratrol on the contraction of smooth muscles of the rat uterus and the involvement of Kir subtype of K-channel were examined in a separate series of experiments.After the incubation period, 37% of the samples showed a spontaneous rhythmic activity.In these cases, the samples were left for about 30 minutes to stabilize contractions prior to the administration of substances.Phasic contractions were induced by oxytocin (0.2 nM) in the different series of experiments.Tonic contractions were induced by 20 nM of oxytocin.After stabilization of the contractions (~30 minutes) resveratrol was directly cumulatively administered (1 -100 µM) in the bath.In order to investigate the involvement of Kir channels in the mechanism of action of resveratrol, BaCl 2 was used as a non-selective antagonist of Kir channels.After the experiments, concentration -dependent curves with and without the presence of antagonist were designed.

Drugs and solutions
Resveratrol was dissolved in 70% (v/v) ethanol, and the further dilutions were made with distilled water immediately prior to use.In that case working concentrations of ethanol were not greater than 0.01% (v / v) in the bath.The Krebs -Ringer solution had the following composition (expressed in mmol/l): NaCl 120, KCl 5, CaCl 2 2.5, MgSO 4 1.2, NaHCO 3 25, KH 2 PO 4 1.2, and glucose 11.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.Due to the photosensitivity of resveratrol all the experiments were carried out in the dark.A producer of the substances was Sigma-Aldrich Inc., USA.

Treatment of data and statistics
The amplitude of phasic contractions was measured from the baseline to the top of the spike, while the amplitude of tonic contractions was measured from ba-seline to the plateau.The medium values of the amplitude during the control period were taken as 100%.
The concentration of resveratrol, which produces 50% of the maximum effect was considered for the mean effective concentration (EC 50 ) and was calculated for each experiment by using linear interpolation of the graphs.The results were expressed as mean value ± standard error (S.E.M); n indicates the number of experiments.Student's t-test was used for comparison.The probability of P <0.05 was considered statistically significant.Data analysis was carried out by the computer program GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA.
Longitudinal muscle strips of the rat non-pregnant uterus exhibited the SRC of constant amplitude 2.4 ± 0.6 g and frequency 9.5 ± 1.3 contractions per 10 minutes.

Rezultat / Results
Application of low nanomolar concentration of oxytocin (0.2 nM) to the samples produced phasic contractions of constant amplitude 3.1 ± 0.5 g and frequency 12.0 ± 0.8 contractions per 10 minutes.
Used Kir channel antagonist, BaCl 2 did not alter the resting tone of the uterus and did not modify the SRC or contractions evoked by oxytocin (n = 12 all, data not shown).Ba 2+ is often used to examine the physiological roles of Kir channels in cells and tissues.To analyze the contribution of Kir channels to the inhibitory mechanism of resveratrol on contractions of the rat non-pregnant uterus, we used agent that is known to possess a Kir channel-blocking activity -BaCl 2 .Externally applied Ba 2+ suppresses Kir currents in avoltage-dependent manner (Oliver i sar., 1998).Although high concentrations of Ba 2+ can also block Kv channelsat micromolar (< 5 mM) concentrationsthis cation is specific to Kir channels (Inagaki i sar., 1996; Du i sar., 2004; Hibino i sar., 2010).In the present study, BaCl 2 used in concentration of 1 mM significantly antagonized SRC and phasic oxytocin -induced contractions.However, the relaxation of tonic component achieved by resveratrol was not reduced in the presence of BaCl 2 .Accordingly, it seems that Kir channels are not involved in the pathway by which resveratrol induces a relaxation of the tonic contractions.Wang i sar.have published that phasic contractions were more sensitive to resveratrol than basal tension in guinea pig gall bladder (Wang i sar., 2008).The differences between effects of resveratrol on contractions induced by low and high concentrations of oxytocin could come from different sources and routes for initiation and maintenance of phasic and tonic contractions.The phasic contraction of the non-pregnant ratuterus is physiological process with involved Ca 2+ influx via L-types Ca 2+ channels.However, oxytocin, at high concentrations produces tonic contraction resulting from additional mechanisms (Novaković i sar., 2013).It has been shown that Kir7.1 is important to maintain a hyperpolarized membrane potential during uterine quiescence in pregnancy.Kir7.1 channels modulate the action potential, modifying the excitation-contraction cycle by participating in key stages of repolarization (McCloskey i sar., 2014).Pharmacological effects of resveratrolon Kir channels could bean explanation for the inhibition of resveratrol's action on phasic contractions.Previous studies conducted on different tissue models documented variable results of influence of different type of K-channels on resveratrol mechanism of action.Studies conducted on human and rat vascular blood vessels show that BaCl 2 (Novaković i sar., 2006a; Novaković i sar., 2006b) and tetraethylammonium (Buluc i Demirel-Yilmaz, 2006; Gojković-Bukarica i sar., 2008) did not affect the relaxation induced by resveratrol, but 4-AP (Novaković i sar., 2006a; Novaković i sar., 2006b; Gojković-Bukarica i sar., 2008) and glibenclamide (Buluc i Demirel-Yilmaz, 2006) did affect it.The voltage clamp study on the mouse beta cell line revealed that resveratrol (100 μM) blocked K ATP and Kv, but activated BK Ca channels (Chen i sar., 2007).As opposed, the experiments on the rat hearts suggest that resveratrol inhibits L-type Ca 2+ channels and enhances activity of the KATP (Chen i sar., 2008).There are obvious differences in sensitivity to resveratrol among various types of contractions (phasic vs. tonic), species and tissues.
According to our observations, it seems that resveratrol may produce relaxation of SRC and phasic oxytocin-elicited contractions of non-pregnant rat uterus by activation of Kir channel.According to the relative resistance of resvera-

Diskusija / Discussion
Vet. glasnik 70 (3)(4) 121 -129 (2016) Radmila Novaković i sar.: Wine polyphenol resveratrol inhibits contractions of isolated rat uterus by activation of smooth muscle... trol's effects to BaCl 2 on tonic oxytocin-elicited contractions model indicated that resveratrol exerts inhibition by acting on multiple sites.We need further investigation to elucidate the mechanism of action of resveratrol on tonic contractions induced by oxytocin.

Figure 1 .
Figure 1.Antagonism of the inhibitory effect of resveratrol by BaCl 2 (1 mM) on the spontaneous rhythmic contractions (SRC) on the non-pregnant rat uterus.Concentration -response curves for resveratrol in the absence (circle) and presence of BaCl 2 (1 mM, square).The points are the means and the vertical lines show the S.E.M. (n = 5-8).

Figure 2 .
Figure 2. Antagonism of the inhibitory effect of resveratrol by BaCl 2 (1 mM) on phasic contractions provoked by oxytocin (0.2 nM) on the non-pregnant rat uterus.Concentration -response curves for resveratrol in the absence (circle) and presence of BaCl 2 (1 mM, triangle).The points are the means and the vertical lines show the S.E.M. (n = 5-8).

Figure 3 .
Figure 3. Antagonism of the inhibitory effect of resveratrol by BaCl 2 (1 mM) on tonic contractions provoked by oxytocin (20 nM) on the non-pregnant rat ute rus.Concentration -response curves for resveratrol in the absence (circle) and presence of BaCl 2 (1 mM, diamond).The points are the means and the vertical lines show the S.E.M. (n = 5-8)