Research Article
The Role of Potassium Channels in the Vasodilatation Induced by Resveratrol and Naringenin in Isolated Human Umbilical Vein
DDR
DRUG DEVELOPMENT RESEARCH 76 : 17–23 (2015)
Dragana Protic´,1 Nebojsa Radunovic´,2 Svetlana Spremovic´-Rad-enovic´,2 ´ ,3 Helmut Heinle,4 Aleksandar Petrovic´,5 and Vladimir Zivanovic Ljiljana Gojkovic´-Bukarica1* 1 Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia 2 Faculty of Medicine, University of Belgrade, Clinic for Gynecology and Obstetrics, Clinical Center of Serbia, 11000 Belgrade, Serbia 3 Faculty of Medicine, University of Belgrade, Clinical Center Dr. Dragisa Misovic, 11000, Belgrade, Serbia 4 Institute of Physiology, University of T€ ubingen, T€ ubingen, Germany 5 Department of Food Technology and Biochemistry, Faculty of Agriculture, Nemanjina 6, 11080 Zemun, Serbia
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT Potassium (K1) channels have a key role in the maintenance of smooth muscle tone; a variety of agonists can modify the tone by altering K1-channel activity. The aim of this study was assess the effects of the phenols, resveratrol, and naringenin on K1-channels of the vascular smooth muscle. Segments of human umbilical vein (HUV) without endothelium were precontracted using serotonin (100 lM) or 100 mM K1 to derive cumulative concentration-response curves using increasing concentrations of resveratrol or naringenin. K1-channel inhibitors were added in the bath before resveratrol (1–100 lM) or naringenin (0.01–1 mM) in assess the role of K1-channels in their effects on HUV precontracted by serotonin. 4-Aminopiridine (4-AP; 1 mM), a nonselective blocker of voltage-dependent, tetraethylammonium (TEA; 1 mM) and barium chloride (1 mM), a nonselective blocker of Ca21 -dependent and inward rectifier K1-channels (respectively) induced significant shifts to the right (P < 0.05) of resveratrol. concentrationresponse curves. The effect of naringenin was antagonized by 4-AP (1 mM). 4-AP-, TEA-, and barium chloride-sensitive K1-channels are probably involved in the resveratrol vasodilatatory effect, while naringenin seems to affect 4-AP-sensitive K1-channels. However, other mechanisms of vasodilation induced by C 2015 Wiley Periodicals, Inc. V polyphenols could not be excluded. Drug Dev Res 76 : 17–23, 2015. Key words: polyphenols; resveratrol; naringenin; potassium channels; human umbilical vein
INTRODUCTION
Polyphenols are naturally occurring substances abundant in plants that number more than 8000 compounds that can be classified into several groups. They protect plants from ultraviolet radiation or pathogenic insult. The concentration of polyphenols in fruits like C 2015 Wiley Periodicals, Inc. V
*Correspondence to: Prof. Ljiljana Gojkovic´ Bukarica, Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia. E-mail: [email protected] Received 24 October 2014; Accepted 1 December 2014 Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1111/ddr.21236
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grapes and berries is around 200–300 mg/100 g fresh weight. Also, the products manufactured from these plants contain polyphenols in significant amounts [Pandey and Rizvi, 2009]. Resveratrol and naringenin are polyphenols accessible via the diet and have antioxidant and antiinflammatory properties that can prevent the development of chronic diseases, for example, cardiovascular diseases, cancer, diabetes, infection, and so forth [Scalbert et al., 2005]. Polyphenols also affect vascular tone producing vasorelaxation via mechanisms that directly involve ion channels [Novakovic et al., 2006a, b; Saponara et al., 2006; Gojkovic-Bukarica et al., 2008]. Potassium (K1) channels have an important role in the maintenance of smooth muscle tone particularly in the control of fetoplacental vascular tone [Wareing et al., 2006]. Some blood vessels in the human can maintain vascular tone as the result of K1-channel activity, without adrenergic influences, and are useful to assess the effects of K1-channel modifying substances. The utility of the precontracted human umbilical vein (HUV) to study the effects of vasorelaxant effects of polyphenols has been previously reported with ATP-sensitive K1 (KATP) channels being involved in the resveratrol-induced endothelium-independent HUV vasodilatation [Protic´ et al., 2014]. To better define the mechanism of HUV vasodilatation by resveratrol and naringenin, this study assessed the role(s) of other K1-channels (except KATP) subtypes in the smooth muscle HUV cells in the effects of these polyphenols. MATERIALS AND METHODS
HUV segments were obtained after full-term vaginal delivery from 40 healthy women hospitalized at Clinics for Gynecology and Obstetrics, Clinical Center of Serbia (Belgrade, Serbia). Informed consent was obtained from all patients. The research was approved by the Human Ethics Committee of Medical Faculty, University of Belgrade. After vaginal delivery, medial segments of umbilical cords were immediately placed in Krebs-Ringer solution (mmol/l: NaCl 120, KCl 5, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, Na2EDTA 0.032) and transported to the laboratory at 4 C. Experiments were conducted within 24 h of delivery. Tissue Preparation After umbilical vein rings were cut from umbilical cords, connective tissue was removed and the isolated vein was cut into 5 mm long ring segments. Up to six vascular rings were prepared from each vein. These were randomly allocated for different Drug Dev. Res.
experiments. As the endothelium is dysfunctional after transfer of the umbilicus to the laboratory, it was removed using a stainless-steel wire. Vein rings were mounted between two stainless-steel triangles in an organ bath containing 10 ml Krebs-Ringer bicarbonate and stretched with an initial tension of 3–5 g, at a temperature of 37 C and pH of 7.4. The solution was aerated with 95% O2 and 5% CO2. The HUV rings were equilibrated for 1 h. [Protic´ et al., 2014]. Isometric tension was recorded using a force displacement transducer (K30, Hugo Sachs, Freiburg, Germany) connected to a recording system (R60; Rikadenki, Tokyo, Japan). During equilibration, the organ bath solution was changed every 10 min and tension was adjusted when necessary. Experimental Procedure After equilibration, submaximal contraction in HUV rings was obtained using serotonin (5-HT; 100 lM). Acetylcholine (ACh; 1 lM) was then added to the bath to assess endothelial integrity. The lack of AChinduced vessel relaxation indicated functional removal of the endothelium. After endothelium removal testing, the rings were re-equilibrated for 1 h. Segments of HUV without endothelium were precontracted by 5-HT (100 lM). When a contraction reached a stable plateau, in order to assess the role of K1-channels of the smooth muscle cells in the effect of polyphenols, a K1-channel blocker was added into the organ bath, 20 min before resveratrol or naringenin. Subsequent concentrations of resveratrol or naringenin were added sequentially to the organ bath after previous concentration had produced its equilibrium response. Data and Statistical Analysis Relaxation induced by each concentration of resveratrol or naringenin was measured and expressed as a percentage of the maximum possible relaxation (relaxation back to the baseline tension). The results are expressed as the mean 6 standard error (S. E. M.); n refers to the number of the experiments. The concentrations of polyphenols producing 50% of own maximum response (EC50 value) was determined for each curve using a nonlinear least square fitting procedure of the individual experimental data. All calculations were done using the computer program Graph Pad (Graph Pad Software Inc., San Diego) Statistical difference between means was determined by Student’s t-test and a P value < 0.05 was considered statistically significant. Drugs and Compounds The following were used: 5-HT, ACh, resveratrol, naringenin, 4–aminopyridine (4-AP), tetraethylammonium chloride (TEA), and barium-chloride
POLYPHENOL EFFECTS ON VENOUS POTASSIUM CHANNELS
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Fig. 1. Original recordings of the effects of K1-channel blockers on resveratrol-induced vasodilatation of isolated HUV without endothelium. (A) Vasodilatatory effect of resveratrol on the vein precontracted by serotonin (100 lM). Cumulative concentrations of resveratrol (1– 100 lM, black circles) were added to the organ-bath. Inhibitory effect of (B) 4-AP (1 mM, down-pointing empty triangle); (C) TEA (1 mM, down-pointing black triangle) and (D) BaCl2 (1 mM, up-pointing black triangle) on the resveratrol-induced vasodilatation of HUV without endothelium.
(BaCl2) (Sigma-Aldrich Inc., St. Louis, MO). Resveratrol was dissolved in 70% v/v ethanol and naringenin was dissolved in dimethylsulphoxide (DMSO), and further diluted in distilled water before use. Working concentrations of ethanol and DMSO in the bath were below 0.01%, and had no effect on the responses of the preparations (data not shown). Compounds were added directly into the bath and the concentrations given are the calculated final concentrations in the bath solution. The experiments were performed in a dark room.
concentration–dependent relaxation of HUV precontracted with 100 mM K1 (EC50 values: 17.2 6 2.0 lM vs. 51 6 3 lM in the presence of 100 mM K1,
RESULTS
Control group of experiments: vasorelaxant effect of resveratrol and naringenin to HUV precontracted with serotonin and 100 mM K1. Original recordings of the vasorelaxant effects of resveratrol and naringenin to HUV without endothelium, precontracted with 5-HT are shown in Figures 1A and 2A. Resveratrol (1–100 lM) and naringenin (0.01–1 mM) induced concentration–dependent vasorelaxation of HUV (EC50 value of resveratrol: 17.2 6 2.0 lM and EC50 value of naringenin: 180 6 4 lM, n 5 6, P < 0.01). The Emax for resveratrol and naringenin were 100 6 2%. Resveratrol (1–100 lM) induced
Fig. 2. The effect of 4-AP on naringenin-induced vasodilatation of isolated HUV without endothelium. (A) Original recording of the vasodilatatory effect of naringenin on the vein precontracted by serotonin (100 lM). Cumulative concentrations of naringenin (0.01– 1 mM, white circles) were added to the organ-bath. (B) Original recording of the inhibitory effect of 4-AP (1 mM, down-pointing empty triangle) on the naringenin-induced vasodilatation of HUV without endothelium.
Drug Dev. Res.
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Fig. 3. The effects of K1-channel blockers on resveratrol- or naringenin-induced vasodilatation of isolated HUV without endothelium (panels A and B, respectively). (A) Inhibitory effects of 4-AP (1 mM, down-pointing empty triangle); TEA (1 mM, down-pointing black triangle), and BaCl2 (1 mM, up-pointing black triangle) on the resveratrol-induced vasodilatation of HUV without endothelium. (B) Inhibitory effect of 4-AP (1 mM, down-pointing empty triangle) on the naringenin-induced vasodilatation of HUV without endothelium. Concentration–response curves for resveratrol and naringenin were obtained on the vein precontracted by serotonin (black and white circles, respectively). The effects are expressed as % of vasodilatation of the control contraction. Each point represents the mean 6 S. E. M. (n 5 6 in each group). **P < 0.01 control EC50 values (experiments with resveratrol or naringenin only) vs. curves obtained in presence of K1-channel blockers.
P < 0.01; with maximal responses: 100 6 2% vs. 82 6 2% in presence of 100 mM K1, P < 0.01; n 5 6). Naringenin (0.01–1 mM) induced a concentration– dependent relaxation of HUV precontracted with 100 mM K1 with maximal response 100 6 2% (EC50 values 180 6 4 lM vs. 215 6 3 lM in the presence of 100 mM K1, P < 0.01, n 5 6). The Emax for naringenin in high K1 was 100 6 2%. Effects of K1-channel blockers on resveratroland naringenin-induced vasorelaxation of HUV precontracted with serotonin 4-AP (1 mM), TEA (1, 3 mM), and BaCl2 (1, 3 mM) neither affected the basal tension of HUV without endothelium nor the contraction induced by serotonine (data not shown, n 5 4–6 in each group of experimentals). 4-AP (1 mM, n 5 6), a selective blocker of voltage-sensitive K1- (Kv) channel, inhibited vasorelaxation of HUV induced by resveratrol (EC50 values: Drug Dev. Res.
17.2 6 2.0 lM in control vs. 49 6 3 lM, P < 0.01) with a change in Emax (100 6 2% in control vs. 69 6 2% in the presence of 4-AP, P < 0.01, Figs. 1B and 3A). The vasorelaxant effect of naringenin on the HUV was significantly antagonized by 1 mM of 4-AP (n 5 6; P < 0.01; EC50 values: 180 6 4 lM in control vs. 281.8 6 1 lM in the presence of 4-AP, Figs. 2B and 3B). 4-AP did not modify Emax of naringenin (the maximal response 100% in all experiments). TEA (1 mM, n 5 6), a selective blocker of Ca21-sensitive K1- (KCa) channel, inhibited the effect of resveratrol on HUVs (EC50 values: 17.2 6 2.0 lM in control vs. 28.0 6 3.0 lM, P < 0.05, Figs. 1C and 3A) without any change in Emax (100 6 2% vs. 97 6 2%). However, TEA (1, 3 mM, n 5 6) failed to antagonize the vasorelaxant action of naringenin on HUVs precontracted by serotonin (EC50 values: 180 6 4 lM in control vs. 177.8 6 1 lM in the presence of 1 mM TEA and 176.1 6 2 lM in the presence of 3 mM TEA; data not shown). The maximal responses of naringenin remained unchanged in presence of TEA. BaCl2 (1 mM, n 5 6), an inward rectifier K1(Kir) channel antagonist, inhibited resveratrol– induced relaxation of HUVs (EC50 values: 17.2 6 2.0 lM vs. 50.6 6 2.5 lM, P < 0.01, Figs. 1D and 3A) with a significant change in Emax (100 6 2% vs. 85 6 3%, P < 0.01). In addition, BaCl2 (1, 3 mM, n56) did not antagonize the vasodilator effect of naringenin on HUVs (EC50 values: 180 6 4 lM in control vs. 185.2 6 2 lM in the presence of 1 mM BaCl2 and 183.1 6 2 lM in the presence of 3 mM BaCl2, data not shown). The maximal responses were 100% in all experiments with naringenin in presence of BaCl2. The combination of different K1-channel blockers, 4-AP (1 mM), TEA (1 mM), and BaCl2 (1mM) blocked HUV responses to resveratrol (EC50 values: 51 6 3 lM in series of experiments with 100 mM K1 vs. 68 6 3 lM, P < 0.01, Fig. 4) without any change in Emax values (82 6 2% vs. 80 6 3%, P > 0.05, Fig. 3C). Experiments with the combinations of the same K1-channel blockers with naringenin were not performed since only 4-AP significantly reduced naringenin-induced vasodilation. Higher concentrations of K1-channel blockers were used only when lower concentrations failed to block of polyphenol effects. DISCUSSION
Vascular ion channels represent important targets of polyphenols [Scholz et al., 2010]. The biological significance of the present experimental model as
POLYPHENOL EFFECTS ON VENOUS POTASSIUM CHANNELS
Fig. 4. The effects of the combination of K1-channel blockers and K-reach solution (100 mM of K1) on resveratrol-induced vasodilatation of isolated HUV without endothelium. Concentration–response curves for resveratrol were obtained on the vein precontracted by serotonin (100 lM, white circles) or 100 mM K1 (black circles). The effects are expressed as % of vasodilatation of the control contraction. Each point represents the mean 6 S. E. M. (n 5 6 in each group). **P < 0.01 EC50 value of resveratrol in the presence of 100 mM K1 vs. EC50 value of resveratrol in the presence of the combination of different K1-channel blockers.
well as pharmacokinetics of the polyphenols used have been discussed previously [Gojkovic-Bukarica et al., 2002, Novakovic et al., 2006a, b, 2013; Protic´ et al., 2013, 2014]. In contrast to other veins, the HUV carries oxygenated blood to the target tissue (i.e., from placenta to fetus). In addition, there is no adrenergic innervation of HUVs; rather, its tone depends on the release of vasoactive substances from the surrounding tissue or the bloodstream with K1 channels play a critical role in the control of HUV vascular function [Skulstad et al., 2004; Wareing et al., 2006]. Plasma concentrations of resveratrol and naringenin in vivo appear to be significantly lower than their concentrations used in the present experiments. However, it should be noted that there is a significant intracellular pool of resveratrol in the vascular endothelium [Chen et al., 2013]. High concentrations of resveratrol in the target tissue fit into the concentration range of resveratrol achieved in the present study. Conversely, vascular uptake of naringenin remains to be clarified. In our previously studies, it was established that resveratrol and naringenin induced endotheliumindependent vasorelaxation of HUVs. Resveratrol was a more potent vasorelaxant than naringenin in this experimental model. It was necessary to use an 11-fold higher concentration of naringenin than resveratrol to achieve 50% of maximal vasodilatation of isolated HUVs. In addition, according to functional and Western blot analysis we concluded that smooth muscle KATP channels were involved in the relaxation of HUVs induced by resveratrol, while naringenin
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appears to interact with other K1-channel subtypes or other ion channels. However, glibenclamide did not affect the maximal response to resveratrol and naringenin [Protic´ et al., 2014]. Those results have been a cornerstone of the present investigation. As shown previously, resveratrol and naringenin induced a concentration-dependent relaxation of HUVs precontracted with 100 mM K1. These results indicated the importance of K1-channels in the mechanism of their vasodilatation on isolated HUV [Protic´ et al., 2014]. The present results support this hypothesis. It is well established that high extracellular K1 reduces the chemical gradient for K1 to diffuse out of the cell. Accordingly, the effects of substances affecting K1-channels could be reduced in high K1 [Cairr~ao et al., 2008]. Activation of any type of K1-channel leads to a hyperpolarization of smooth muscle cells and leads to vasodilatation. Vascular smooth muscle cells express a variety of K1-channels including calciumactivated potassium (KCa), KATP-, Kv, and Kir channels [Jackson, 2000; Brayden, 2002]. In our study, selective blockers of KV, KCa, and Kir channels (4-AP, TEA, and BaCl2, respectively) were used to differentiate K1channel mediated vasodilator effects of naringenin and resveratrol. 4-AP (1 mM), was sufficient to block KV channels [Beech and Bolton, 1987]. Similarly, TEA is a selective blocker of BKCa channels in the concentration range used in this study (1 mM; Kd 5 0.29) [Wallner et al., 1999). Finally, barium ions used in the concentration range from 1 to 10 lM inhibit Kir channels in the smooth muscle cells [Quayle et al., 1993]. In the present study, BaCl2 was applied at 1 mM, which was enough to block Kir channels. All of the K1-channel blockers used antagonized the effects of resveratrol on isolated HUVs without endothelium. Accordingly, the K1-channels may play an important role in vasodilatation induced by resveratrol. We have also demonstrated that resveratrol can block vascular KATP channels [Protic´ et al., 2014]. Our results are in agreement with previous findings [Scholz et al., 2010]. For example, endothelium-independent corpus cavernosum relaxation induced by resveratrol appears to largely depend on Kir and KATP channels [Dalaklioglu and Ozbey, 2014]. Also, different blockers of K1-channels (glibenclamide, TEA, iberiotoxin, 4-AP) antagonized the response to resveratrol on the spontaneous rhythmic contractions and phasic contraction of isolated rat uterus [Novakovic et al., 2013). These results are in accordance with our previous findings, which describe the effect of resveratrol on rat mesenteric artery, rat aorta, human internal mammary artery, and so on [Gojkovic-Bukarica et al., 2002; Novakovic et al., 2006a, 2006b]. Drug Dev. Res.
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Simultaneous incubation with 4-AP, TEA, and BaCl2 abolished the effect of resveratrol (1–30 lM), and reduced the effect of resveratrol applied in higher concentration (30–100 lM). These results are consistent with our previous results [Protic´ et al., 2014]. Resveratrol in higher concentrations might produce K1-channel- ndependent vascular effects as well. For example, resveratrol affects [Ca21]i, activates nuclear and extranuclear estrogen receptors [Li and Forstermann, 2009], increases cGMP [el-Mowafy et al., 2002] and reduces the Ca21-sensitivity of muscle fibers [Buluc and Demirel-Yilmaz, 2006]. However, further studies are needed to identify the exact resveratrol targets in the vascular smooth muscle cell at concentrations higher than 30 lM. The effects of naringenin and its mechanisms of action are less well investigated than those of resveratrol. KV channel blockers can antagonize the effects of naringenin. Accordingly, the mechanism of vasodilatation induced by naringenin on HUsV without endothelium probably involve activation of 4AP-sensitive K1-channels. Conversely, naringenin (30 lM) did not modulate KV1.3 channels in human T lymphocytes [Teisseyre et al., 2009]. Also, this polyphenol blocks human ether-a-go-go-related gene channels (KV11.1), which may explain the significant QT prolongation in healthy volunteers induced by grapefruit juice [Lin et al., 2008]. It is obvious that there are apparent differences in sensitivity to naringenin among various experimental models. Also, there are no published results that confirm naringenin as an opener of KATP channels. Conversely, the vasorelaxant effect of naringenin on endothelium-denuded rat vessels was due to the activation of KCa channels in myocytes [Saponara et al., 2006]. In conclusion, functional analysis reveals that 4AP-, TEA-, and BaCl2-sensitive K1-channels play important role in the resveratrol-induced vasodilatation of HUV without endothelium. Conversely, naringenin appears to interact only with 4-AP-sensitive K1-channels. Also, K1- channel-independent vasodilatatory mechanism(s) of these polyphenols cannot be excluded. ACKNOWLEDGMENTS
We would like to thank Mrs Milena Zabunovic´ and Mrs Leposava Bibic´ for technical support during this study. Our work was supported by the Scientific Research Grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia: TR 31020 and OI 175064 (Serbia), and the Alexander von Humboldt Foundation (Germany). Drug Dev. Res.
CONFLICT OF INTEREST
The authors indicated no potential conflicts of interest. REFERENCES Beech DJ, Bolton TB. 1987. The effects of tetraethylammonium ions, 4-aminopyridine or quinidine on K1-currents in single smooth muscle cells of the rabbit portal vein. Biomed Biochim Acta 46:S673–S676. Brayden JE. 2002. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 29:312–316. Buluc M, Demirel-Yilmaz E. 2006. Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium. Vascul Pharmacol 44:231–237. Cairr~ao E, Alvarez E, Santos-Silva AJ, Verde I. 2008. Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical artery. Naunyn Schmiedebergs Arch Pharmacol 376:375–383. Chen ML, Yi L, Jin X, Xie Q, Zhang T, Zhou X, Chang H, Fu YJ, Zhu JD, Zhang QY, et al. 2013. Absorption of resveratrol by vascular endothelial cells through passive diffusion and an SGLT1-mediated pathway. J Nutr Biochem 24:1823–1829. Dalaklioglu S, Ozbey G. 2014. Role of different types of potassium channels in the relaxation of corpus cavernosum induced by resveratrol. Pharmacogn Mag 10:47–52. el-Mowafy AM, Abou-Zeid LA, Edafiogho I. 2002. Recognition of resveratrol by the human estrogen receptor-alpha: a molecular modeling approach to understand its biological actions. Med Princ Pract 11:86–92. Gojkovic-Bukarica L, Hambrock A, L€offler-Walz C, Quast U, Russ U. 2002. Mg21 sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit. Br J Pharmacol 137:429–440. Gojkovic-Bukarica L, Novakovic A, Kanjuh V, Bumbasirevic M, Lesic A, Heinle H. 2008. A role of ion channels in the endothelium-independent relaxation of rat mesenteric artery induced by resveratrol. J Pharmacol Sci 108:124–130. Jackson WF. 2000. Ion channels and vascular tone. Hypertension 35:173–178. Li H, F€orstermann U. 2009. Resveratrol: a multifunctional compound improving endothelial function. Editorial to: “resveratrol supplementation gender independently improves endothelial reactivity and suppresses superoxide production in healthy rats” by S. Soylemez et al. Cardiovasc Drugs Ther 23:425–429. Lin C, Ke X, Ranade V, Somberg J. 2008. The additive effects of the active component of grapefruit juice (naringenin) and antiarrhythmic drugs on HERG inhibition. Cardiology 110:145–152. Novakovic A, Bukarica LG, Kanjuh V, Heinle H. 2006a. Potassium channels-mediated vasorelaxation of rat aorta induced by resveratrol. Basic Clin Pharmacol Toxicol 99:360–364. Novakovic A, Gojkovic-Bukarica L, Peric M, Nezic D, Djukanovic B, Markovic-Lipkovski J, Heinle H. 2006b. The mechanism of endothelium-independent relaxation induced by the wine polyphenol resveratrol in human internal mammary artery. J Pharmacol Sci 101:85–90. Novakovic R, Ilic B, Beleslin-Cokic B, Radunovic N, Heinle H, Scepanovic R, Gojkovic-Bukarica L. 2013. The effect of resveratrol on contractility of non-pregnant rat uterus: the contribution of K(1) channels. J Physiol Pharmacol 64:795–805.
POLYPHENOL EFFECTS ON VENOUS POTASSIUM CHANNELS Pandey KB, Rizvi SI. 2009. Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longev 2: 270–278. ´ B, Novakovic´ R, Kanjuh V, Heinle H, Protic´ D, Beleslin-Cokic Sc´epanovic´ R, Gojkovic´-Bukarica L. 2013. Effect of wine polyphenol resveratrol on the contractions elicited electrically or by norepinephrine in the rat portal vein. Phytother Res 27:1685– 1693. ´ B, Spremovic´-Rad-enovic´ S, Radunovic´ Protic´ D, Beleslin-Cokic N, Heinle H, Sc´epanovic´ R, Gojkovic´ Bukarica L. 2014. The different effects of resveratrol and naringenin on isolated human umbilical vein: the role of ATP-sensitive K1 channels. Phytother Res 28:1412–1418. Quayle JM, McCarron JG, Brayden JE, Nelson MT. 1993. Inward rectifier K1 currents in smooth muscle cells from rat resistance-sized cerebral arteries. Am J Physiol 265:C1363– C1370. Saponara S, Testai L, Iozzi D, Martinotti E, Martelli A, Chericoni S, Sgaragli G, Fusi F, Calderone V. 2006. (1/-)-Naringenin as large conductance Ca(21)-activated K1 (BKCa) channel opener in vascular smooth muscle cells. Br J Pharmacol 149: 1013–1021.
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Scalbert A, Manach C, Morand C, Remesy C, Jimenez L. 2005. Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr 45:287–306. Scholz EP, Zitron E, Katus HA, Karle CA. 2010. Cardiovascular ion channels as a molecular target of flavonoids. Cardiovasc Ther 28:e46–e52. Skulstad SM, Kiserud T, Rasmussen S. 2004. The effect of vascular constriction on umbilical venous pulsation. Ultrasound Obstet Gynecol 23:126–130. Teisseyre A, Duarte N, Ferreira MJ, Michalak K. 2009. Influence of the multidrug transporter inhibitors on the activity of Kv1.3 voltage-gated potassium channels. J Physiol Pharmacol 60:69– 76. Wallner M, Meera P, Toro L. 1999. Molecular basis of fast inactivation in voltage and Ca21-activated K1 channels: a transmembrane beta-subunit homolog. Proc Natl Acad Sci USA 96: 4137–4142. Wareing M, Bai X, Seghier F, Turner CM, Greenwood SL, Baker PN, Taggart MJ, Fyfe GK. 2006. Expression and function of potassium channels in the human placental vasculature. Am J Physiol Regul Integr Comp Physiol 291:R437–R446.
Drug Dev. Res.
The Role of Potassium Channels in the Vasodilatation Induced by Resveratrol and Naringenin in Isolated Human Umbilical Vein
DDR
DRUG DEVELOPMENT RESEARCH 76 : 17–23 (2015)
Dragana Protic´,1 Nebojsa Radunovic´,2 Svetlana Spremovic´-Rad-enovic´,2 ´ ,3 Helmut Heinle,4 Aleksandar Petrovic´,5 and Vladimir Zivanovic Ljiljana Gojkovic´-Bukarica1* 1 Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia 2 Faculty of Medicine, University of Belgrade, Clinic for Gynecology and Obstetrics, Clinical Center of Serbia, 11000 Belgrade, Serbia 3 Faculty of Medicine, University of Belgrade, Clinical Center Dr. Dragisa Misovic, 11000, Belgrade, Serbia 4 Institute of Physiology, University of T€ ubingen, T€ ubingen, Germany 5 Department of Food Technology and Biochemistry, Faculty of Agriculture, Nemanjina 6, 11080 Zemun, Serbia
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT Potassium (K1) channels have a key role in the maintenance of smooth muscle tone; a variety of agonists can modify the tone by altering K1-channel activity. The aim of this study was assess the effects of the phenols, resveratrol, and naringenin on K1-channels of the vascular smooth muscle. Segments of human umbilical vein (HUV) without endothelium were precontracted using serotonin (100 lM) or 100 mM K1 to derive cumulative concentration-response curves using increasing concentrations of resveratrol or naringenin. K1-channel inhibitors were added in the bath before resveratrol (1–100 lM) or naringenin (0.01–1 mM) in assess the role of K1-channels in their effects on HUV precontracted by serotonin. 4-Aminopiridine (4-AP; 1 mM), a nonselective blocker of voltage-dependent, tetraethylammonium (TEA; 1 mM) and barium chloride (1 mM), a nonselective blocker of Ca21 -dependent and inward rectifier K1-channels (respectively) induced significant shifts to the right (P < 0.05) of resveratrol. concentrationresponse curves. The effect of naringenin was antagonized by 4-AP (1 mM). 4-AP-, TEA-, and barium chloride-sensitive K1-channels are probably involved in the resveratrol vasodilatatory effect, while naringenin seems to affect 4-AP-sensitive K1-channels. However, other mechanisms of vasodilation induced by C 2015 Wiley Periodicals, Inc. V polyphenols could not be excluded. Drug Dev Res 76 : 17–23, 2015. Key words: polyphenols; resveratrol; naringenin; potassium channels; human umbilical vein
INTRODUCTION
Polyphenols are naturally occurring substances abundant in plants that number more than 8000 compounds that can be classified into several groups. They protect plants from ultraviolet radiation or pathogenic insult. The concentration of polyphenols in fruits like C 2015 Wiley Periodicals, Inc. V
*Correspondence to: Prof. Ljiljana Gojkovic´ Bukarica, Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia. E-mail: [email protected] Received 24 October 2014; Accepted 1 December 2014 Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1111/ddr.21236
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grapes and berries is around 200–300 mg/100 g fresh weight. Also, the products manufactured from these plants contain polyphenols in significant amounts [Pandey and Rizvi, 2009]. Resveratrol and naringenin are polyphenols accessible via the diet and have antioxidant and antiinflammatory properties that can prevent the development of chronic diseases, for example, cardiovascular diseases, cancer, diabetes, infection, and so forth [Scalbert et al., 2005]. Polyphenols also affect vascular tone producing vasorelaxation via mechanisms that directly involve ion channels [Novakovic et al., 2006a, b; Saponara et al., 2006; Gojkovic-Bukarica et al., 2008]. Potassium (K1) channels have an important role in the maintenance of smooth muscle tone particularly in the control of fetoplacental vascular tone [Wareing et al., 2006]. Some blood vessels in the human can maintain vascular tone as the result of K1-channel activity, without adrenergic influences, and are useful to assess the effects of K1-channel modifying substances. The utility of the precontracted human umbilical vein (HUV) to study the effects of vasorelaxant effects of polyphenols has been previously reported with ATP-sensitive K1 (KATP) channels being involved in the resveratrol-induced endothelium-independent HUV vasodilatation [Protic´ et al., 2014]. To better define the mechanism of HUV vasodilatation by resveratrol and naringenin, this study assessed the role(s) of other K1-channels (except KATP) subtypes in the smooth muscle HUV cells in the effects of these polyphenols. MATERIALS AND METHODS
HUV segments were obtained after full-term vaginal delivery from 40 healthy women hospitalized at Clinics for Gynecology and Obstetrics, Clinical Center of Serbia (Belgrade, Serbia). Informed consent was obtained from all patients. The research was approved by the Human Ethics Committee of Medical Faculty, University of Belgrade. After vaginal delivery, medial segments of umbilical cords were immediately placed in Krebs-Ringer solution (mmol/l: NaCl 120, KCl 5, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, Na2EDTA 0.032) and transported to the laboratory at 4 C. Experiments were conducted within 24 h of delivery. Tissue Preparation After umbilical vein rings were cut from umbilical cords, connective tissue was removed and the isolated vein was cut into 5 mm long ring segments. Up to six vascular rings were prepared from each vein. These were randomly allocated for different Drug Dev. Res.
experiments. As the endothelium is dysfunctional after transfer of the umbilicus to the laboratory, it was removed using a stainless-steel wire. Vein rings were mounted between two stainless-steel triangles in an organ bath containing 10 ml Krebs-Ringer bicarbonate and stretched with an initial tension of 3–5 g, at a temperature of 37 C and pH of 7.4. The solution was aerated with 95% O2 and 5% CO2. The HUV rings were equilibrated for 1 h. [Protic´ et al., 2014]. Isometric tension was recorded using a force displacement transducer (K30, Hugo Sachs, Freiburg, Germany) connected to a recording system (R60; Rikadenki, Tokyo, Japan). During equilibration, the organ bath solution was changed every 10 min and tension was adjusted when necessary. Experimental Procedure After equilibration, submaximal contraction in HUV rings was obtained using serotonin (5-HT; 100 lM). Acetylcholine (ACh; 1 lM) was then added to the bath to assess endothelial integrity. The lack of AChinduced vessel relaxation indicated functional removal of the endothelium. After endothelium removal testing, the rings were re-equilibrated for 1 h. Segments of HUV without endothelium were precontracted by 5-HT (100 lM). When a contraction reached a stable plateau, in order to assess the role of K1-channels of the smooth muscle cells in the effect of polyphenols, a K1-channel blocker was added into the organ bath, 20 min before resveratrol or naringenin. Subsequent concentrations of resveratrol or naringenin were added sequentially to the organ bath after previous concentration had produced its equilibrium response. Data and Statistical Analysis Relaxation induced by each concentration of resveratrol or naringenin was measured and expressed as a percentage of the maximum possible relaxation (relaxation back to the baseline tension). The results are expressed as the mean 6 standard error (S. E. M.); n refers to the number of the experiments. The concentrations of polyphenols producing 50% of own maximum response (EC50 value) was determined for each curve using a nonlinear least square fitting procedure of the individual experimental data. All calculations were done using the computer program Graph Pad (Graph Pad Software Inc., San Diego) Statistical difference between means was determined by Student’s t-test and a P value < 0.05 was considered statistically significant. Drugs and Compounds The following were used: 5-HT, ACh, resveratrol, naringenin, 4–aminopyridine (4-AP), tetraethylammonium chloride (TEA), and barium-chloride
POLYPHENOL EFFECTS ON VENOUS POTASSIUM CHANNELS
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Fig. 1. Original recordings of the effects of K1-channel blockers on resveratrol-induced vasodilatation of isolated HUV without endothelium. (A) Vasodilatatory effect of resveratrol on the vein precontracted by serotonin (100 lM). Cumulative concentrations of resveratrol (1– 100 lM, black circles) were added to the organ-bath. Inhibitory effect of (B) 4-AP (1 mM, down-pointing empty triangle); (C) TEA (1 mM, down-pointing black triangle) and (D) BaCl2 (1 mM, up-pointing black triangle) on the resveratrol-induced vasodilatation of HUV without endothelium.
(BaCl2) (Sigma-Aldrich Inc., St. Louis, MO). Resveratrol was dissolved in 70% v/v ethanol and naringenin was dissolved in dimethylsulphoxide (DMSO), and further diluted in distilled water before use. Working concentrations of ethanol and DMSO in the bath were below 0.01%, and had no effect on the responses of the preparations (data not shown). Compounds were added directly into the bath and the concentrations given are the calculated final concentrations in the bath solution. The experiments were performed in a dark room.
concentration–dependent relaxation of HUV precontracted with 100 mM K1 (EC50 values: 17.2 6 2.0 lM vs. 51 6 3 lM in the presence of 100 mM K1,
RESULTS
Control group of experiments: vasorelaxant effect of resveratrol and naringenin to HUV precontracted with serotonin and 100 mM K1. Original recordings of the vasorelaxant effects of resveratrol and naringenin to HUV without endothelium, precontracted with 5-HT are shown in Figures 1A and 2A. Resveratrol (1–100 lM) and naringenin (0.01–1 mM) induced concentration–dependent vasorelaxation of HUV (EC50 value of resveratrol: 17.2 6 2.0 lM and EC50 value of naringenin: 180 6 4 lM, n 5 6, P < 0.01). The Emax for resveratrol and naringenin were 100 6 2%. Resveratrol (1–100 lM) induced
Fig. 2. The effect of 4-AP on naringenin-induced vasodilatation of isolated HUV without endothelium. (A) Original recording of the vasodilatatory effect of naringenin on the vein precontracted by serotonin (100 lM). Cumulative concentrations of naringenin (0.01– 1 mM, white circles) were added to the organ-bath. (B) Original recording of the inhibitory effect of 4-AP (1 mM, down-pointing empty triangle) on the naringenin-induced vasodilatation of HUV without endothelium.
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Fig. 3. The effects of K1-channel blockers on resveratrol- or naringenin-induced vasodilatation of isolated HUV without endothelium (panels A and B, respectively). (A) Inhibitory effects of 4-AP (1 mM, down-pointing empty triangle); TEA (1 mM, down-pointing black triangle), and BaCl2 (1 mM, up-pointing black triangle) on the resveratrol-induced vasodilatation of HUV without endothelium. (B) Inhibitory effect of 4-AP (1 mM, down-pointing empty triangle) on the naringenin-induced vasodilatation of HUV without endothelium. Concentration–response curves for resveratrol and naringenin were obtained on the vein precontracted by serotonin (black and white circles, respectively). The effects are expressed as % of vasodilatation of the control contraction. Each point represents the mean 6 S. E. M. (n 5 6 in each group). **P < 0.01 control EC50 values (experiments with resveratrol or naringenin only) vs. curves obtained in presence of K1-channel blockers.
P < 0.01; with maximal responses: 100 6 2% vs. 82 6 2% in presence of 100 mM K1, P < 0.01; n 5 6). Naringenin (0.01–1 mM) induced a concentration– dependent relaxation of HUV precontracted with 100 mM K1 with maximal response 100 6 2% (EC50 values 180 6 4 lM vs. 215 6 3 lM in the presence of 100 mM K1, P < 0.01, n 5 6). The Emax for naringenin in high K1 was 100 6 2%. Effects of K1-channel blockers on resveratroland naringenin-induced vasorelaxation of HUV precontracted with serotonin 4-AP (1 mM), TEA (1, 3 mM), and BaCl2 (1, 3 mM) neither affected the basal tension of HUV without endothelium nor the contraction induced by serotonine (data not shown, n 5 4–6 in each group of experimentals). 4-AP (1 mM, n 5 6), a selective blocker of voltage-sensitive K1- (Kv) channel, inhibited vasorelaxation of HUV induced by resveratrol (EC50 values: Drug Dev. Res.
17.2 6 2.0 lM in control vs. 49 6 3 lM, P < 0.01) with a change in Emax (100 6 2% in control vs. 69 6 2% in the presence of 4-AP, P < 0.01, Figs. 1B and 3A). The vasorelaxant effect of naringenin on the HUV was significantly antagonized by 1 mM of 4-AP (n 5 6; P < 0.01; EC50 values: 180 6 4 lM in control vs. 281.8 6 1 lM in the presence of 4-AP, Figs. 2B and 3B). 4-AP did not modify Emax of naringenin (the maximal response 100% in all experiments). TEA (1 mM, n 5 6), a selective blocker of Ca21-sensitive K1- (KCa) channel, inhibited the effect of resveratrol on HUVs (EC50 values: 17.2 6 2.0 lM in control vs. 28.0 6 3.0 lM, P < 0.05, Figs. 1C and 3A) without any change in Emax (100 6 2% vs. 97 6 2%). However, TEA (1, 3 mM, n 5 6) failed to antagonize the vasorelaxant action of naringenin on HUVs precontracted by serotonin (EC50 values: 180 6 4 lM in control vs. 177.8 6 1 lM in the presence of 1 mM TEA and 176.1 6 2 lM in the presence of 3 mM TEA; data not shown). The maximal responses of naringenin remained unchanged in presence of TEA. BaCl2 (1 mM, n 5 6), an inward rectifier K1(Kir) channel antagonist, inhibited resveratrol– induced relaxation of HUVs (EC50 values: 17.2 6 2.0 lM vs. 50.6 6 2.5 lM, P < 0.01, Figs. 1D and 3A) with a significant change in Emax (100 6 2% vs. 85 6 3%, P < 0.01). In addition, BaCl2 (1, 3 mM, n56) did not antagonize the vasodilator effect of naringenin on HUVs (EC50 values: 180 6 4 lM in control vs. 185.2 6 2 lM in the presence of 1 mM BaCl2 and 183.1 6 2 lM in the presence of 3 mM BaCl2, data not shown). The maximal responses were 100% in all experiments with naringenin in presence of BaCl2. The combination of different K1-channel blockers, 4-AP (1 mM), TEA (1 mM), and BaCl2 (1mM) blocked HUV responses to resveratrol (EC50 values: 51 6 3 lM in series of experiments with 100 mM K1 vs. 68 6 3 lM, P < 0.01, Fig. 4) without any change in Emax values (82 6 2% vs. 80 6 3%, P > 0.05, Fig. 3C). Experiments with the combinations of the same K1-channel blockers with naringenin were not performed since only 4-AP significantly reduced naringenin-induced vasodilation. Higher concentrations of K1-channel blockers were used only when lower concentrations failed to block of polyphenol effects. DISCUSSION
Vascular ion channels represent important targets of polyphenols [Scholz et al., 2010]. The biological significance of the present experimental model as
POLYPHENOL EFFECTS ON VENOUS POTASSIUM CHANNELS
Fig. 4. The effects of the combination of K1-channel blockers and K-reach solution (100 mM of K1) on resveratrol-induced vasodilatation of isolated HUV without endothelium. Concentration–response curves for resveratrol were obtained on the vein precontracted by serotonin (100 lM, white circles) or 100 mM K1 (black circles). The effects are expressed as % of vasodilatation of the control contraction. Each point represents the mean 6 S. E. M. (n 5 6 in each group). **P < 0.01 EC50 value of resveratrol in the presence of 100 mM K1 vs. EC50 value of resveratrol in the presence of the combination of different K1-channel blockers.
well as pharmacokinetics of the polyphenols used have been discussed previously [Gojkovic-Bukarica et al., 2002, Novakovic et al., 2006a, b, 2013; Protic´ et al., 2013, 2014]. In contrast to other veins, the HUV carries oxygenated blood to the target tissue (i.e., from placenta to fetus). In addition, there is no adrenergic innervation of HUVs; rather, its tone depends on the release of vasoactive substances from the surrounding tissue or the bloodstream with K1 channels play a critical role in the control of HUV vascular function [Skulstad et al., 2004; Wareing et al., 2006]. Plasma concentrations of resveratrol and naringenin in vivo appear to be significantly lower than their concentrations used in the present experiments. However, it should be noted that there is a significant intracellular pool of resveratrol in the vascular endothelium [Chen et al., 2013]. High concentrations of resveratrol in the target tissue fit into the concentration range of resveratrol achieved in the present study. Conversely, vascular uptake of naringenin remains to be clarified. In our previously studies, it was established that resveratrol and naringenin induced endotheliumindependent vasorelaxation of HUVs. Resveratrol was a more potent vasorelaxant than naringenin in this experimental model. It was necessary to use an 11-fold higher concentration of naringenin than resveratrol to achieve 50% of maximal vasodilatation of isolated HUVs. In addition, according to functional and Western blot analysis we concluded that smooth muscle KATP channels were involved in the relaxation of HUVs induced by resveratrol, while naringenin
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appears to interact with other K1-channel subtypes or other ion channels. However, glibenclamide did not affect the maximal response to resveratrol and naringenin [Protic´ et al., 2014]. Those results have been a cornerstone of the present investigation. As shown previously, resveratrol and naringenin induced a concentration-dependent relaxation of HUVs precontracted with 100 mM K1. These results indicated the importance of K1-channels in the mechanism of their vasodilatation on isolated HUV [Protic´ et al., 2014]. The present results support this hypothesis. It is well established that high extracellular K1 reduces the chemical gradient for K1 to diffuse out of the cell. Accordingly, the effects of substances affecting K1-channels could be reduced in high K1 [Cairr~ao et al., 2008]. Activation of any type of K1-channel leads to a hyperpolarization of smooth muscle cells and leads to vasodilatation. Vascular smooth muscle cells express a variety of K1-channels including calciumactivated potassium (KCa), KATP-, Kv, and Kir channels [Jackson, 2000; Brayden, 2002]. In our study, selective blockers of KV, KCa, and Kir channels (4-AP, TEA, and BaCl2, respectively) were used to differentiate K1channel mediated vasodilator effects of naringenin and resveratrol. 4-AP (1 mM), was sufficient to block KV channels [Beech and Bolton, 1987]. Similarly, TEA is a selective blocker of BKCa channels in the concentration range used in this study (1 mM; Kd 5 0.29) [Wallner et al., 1999). Finally, barium ions used in the concentration range from 1 to 10 lM inhibit Kir channels in the smooth muscle cells [Quayle et al., 1993]. In the present study, BaCl2 was applied at 1 mM, which was enough to block Kir channels. All of the K1-channel blockers used antagonized the effects of resveratrol on isolated HUVs without endothelium. Accordingly, the K1-channels may play an important role in vasodilatation induced by resveratrol. We have also demonstrated that resveratrol can block vascular KATP channels [Protic´ et al., 2014]. Our results are in agreement with previous findings [Scholz et al., 2010]. For example, endothelium-independent corpus cavernosum relaxation induced by resveratrol appears to largely depend on Kir and KATP channels [Dalaklioglu and Ozbey, 2014]. Also, different blockers of K1-channels (glibenclamide, TEA, iberiotoxin, 4-AP) antagonized the response to resveratrol on the spontaneous rhythmic contractions and phasic contraction of isolated rat uterus [Novakovic et al., 2013). These results are in accordance with our previous findings, which describe the effect of resveratrol on rat mesenteric artery, rat aorta, human internal mammary artery, and so on [Gojkovic-Bukarica et al., 2002; Novakovic et al., 2006a, 2006b]. Drug Dev. Res.
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Simultaneous incubation with 4-AP, TEA, and BaCl2 abolished the effect of resveratrol (1–30 lM), and reduced the effect of resveratrol applied in higher concentration (30–100 lM). These results are consistent with our previous results [Protic´ et al., 2014]. Resveratrol in higher concentrations might produce K1-channel- ndependent vascular effects as well. For example, resveratrol affects [Ca21]i, activates nuclear and extranuclear estrogen receptors [Li and Forstermann, 2009], increases cGMP [el-Mowafy et al., 2002] and reduces the Ca21-sensitivity of muscle fibers [Buluc and Demirel-Yilmaz, 2006]. However, further studies are needed to identify the exact resveratrol targets in the vascular smooth muscle cell at concentrations higher than 30 lM. The effects of naringenin and its mechanisms of action are less well investigated than those of resveratrol. KV channel blockers can antagonize the effects of naringenin. Accordingly, the mechanism of vasodilatation induced by naringenin on HUsV without endothelium probably involve activation of 4AP-sensitive K1-channels. Conversely, naringenin (30 lM) did not modulate KV1.3 channels in human T lymphocytes [Teisseyre et al., 2009]. Also, this polyphenol blocks human ether-a-go-go-related gene channels (KV11.1), which may explain the significant QT prolongation in healthy volunteers induced by grapefruit juice [Lin et al., 2008]. It is obvious that there are apparent differences in sensitivity to naringenin among various experimental models. Also, there are no published results that confirm naringenin as an opener of KATP channels. Conversely, the vasorelaxant effect of naringenin on endothelium-denuded rat vessels was due to the activation of KCa channels in myocytes [Saponara et al., 2006]. In conclusion, functional analysis reveals that 4AP-, TEA-, and BaCl2-sensitive K1-channels play important role in the resveratrol-induced vasodilatation of HUV without endothelium. Conversely, naringenin appears to interact only with 4-AP-sensitive K1-channels. Also, K1- channel-independent vasodilatatory mechanism(s) of these polyphenols cannot be excluded. ACKNOWLEDGMENTS
We would like to thank Mrs Milena Zabunovic´ and Mrs Leposava Bibic´ for technical support during this study. Our work was supported by the Scientific Research Grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia: TR 31020 and OI 175064 (Serbia), and the Alexander von Humboldt Foundation (Germany). Drug Dev. Res.
CONFLICT OF INTEREST
The authors indicated no potential conflicts of interest. REFERENCES Beech DJ, Bolton TB. 1987. The effects of tetraethylammonium ions, 4-aminopyridine or quinidine on K1-currents in single smooth muscle cells of the rabbit portal vein. Biomed Biochim Acta 46:S673–S676. Brayden JE. 2002. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 29:312–316. Buluc M, Demirel-Yilmaz E. 2006. Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium. Vascul Pharmacol 44:231–237. Cairr~ao E, Alvarez E, Santos-Silva AJ, Verde I. 2008. Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical artery. Naunyn Schmiedebergs Arch Pharmacol 376:375–383. Chen ML, Yi L, Jin X, Xie Q, Zhang T, Zhou X, Chang H, Fu YJ, Zhu JD, Zhang QY, et al. 2013. Absorption of resveratrol by vascular endothelial cells through passive diffusion and an SGLT1-mediated pathway. J Nutr Biochem 24:1823–1829. Dalaklioglu S, Ozbey G. 2014. Role of different types of potassium channels in the relaxation of corpus cavernosum induced by resveratrol. Pharmacogn Mag 10:47–52. el-Mowafy AM, Abou-Zeid LA, Edafiogho I. 2002. Recognition of resveratrol by the human estrogen receptor-alpha: a molecular modeling approach to understand its biological actions. Med Princ Pract 11:86–92. Gojkovic-Bukarica L, Hambrock A, L€offler-Walz C, Quast U, Russ U. 2002. Mg21 sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit. Br J Pharmacol 137:429–440. Gojkovic-Bukarica L, Novakovic A, Kanjuh V, Bumbasirevic M, Lesic A, Heinle H. 2008. A role of ion channels in the endothelium-independent relaxation of rat mesenteric artery induced by resveratrol. J Pharmacol Sci 108:124–130. Jackson WF. 2000. Ion channels and vascular tone. Hypertension 35:173–178. Li H, F€orstermann U. 2009. Resveratrol: a multifunctional compound improving endothelial function. Editorial to: “resveratrol supplementation gender independently improves endothelial reactivity and suppresses superoxide production in healthy rats” by S. Soylemez et al. Cardiovasc Drugs Ther 23:425–429. Lin C, Ke X, Ranade V, Somberg J. 2008. The additive effects of the active component of grapefruit juice (naringenin) and antiarrhythmic drugs on HERG inhibition. Cardiology 110:145–152. Novakovic A, Bukarica LG, Kanjuh V, Heinle H. 2006a. Potassium channels-mediated vasorelaxation of rat aorta induced by resveratrol. Basic Clin Pharmacol Toxicol 99:360–364. Novakovic A, Gojkovic-Bukarica L, Peric M, Nezic D, Djukanovic B, Markovic-Lipkovski J, Heinle H. 2006b. The mechanism of endothelium-independent relaxation induced by the wine polyphenol resveratrol in human internal mammary artery. J Pharmacol Sci 101:85–90. Novakovic R, Ilic B, Beleslin-Cokic B, Radunovic N, Heinle H, Scepanovic R, Gojkovic-Bukarica L. 2013. The effect of resveratrol on contractility of non-pregnant rat uterus: the contribution of K(1) channels. J Physiol Pharmacol 64:795–805.
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