gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

The role of potassium channels in the relaxation of rat renal artery induced by resveratrol

Meeting Contribution

  • corresponding author L. Gojkovic-Bukarica - Department of Clinical Pharmacology, Pharmacology and Toxicology, Faculty of Medicine, University Belgrade, Serbia
  • R. Novakovic - Department of Clinical Pharmacology, Pharmacology and Toxicology, Faculty of Medicine, University Belgrade, Serbia
  • J. Cvejic - Department of Pharmacy, Faculty of Medicine, University Novi Sad, Serbia
  • V. Kanjuh - Academy of Sciences and Arts, Belgrade, Serbia
  • M. Atanackovic - Department of Pharmacy, Faculty of Medicine, University Novi Sad, Serbia
  • H. Heinle - Institute of Physiology, University Tbingen, Germany

Deutsche Gesellschaft fr Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft fr Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Dsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf10

doi: 10.3205/10dgaf10, urn:nbn:de:0183-10dgaf100

Published: March 23, 2011

© 2011 Gojkovic-Bukarica et al.
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Abstract

Resveratrol, a polyphenol present in red wine, has been thought to be responsible for cardiovascular benefits associated with moderate wine consumption. The mechanism of cardiovascular benefits probably includes vasorelaxation, antioxidant and anti-platelet effects of resveratrol. The mechanisms by which resveratrol causes vasodilatation are uncertain. The aim of this study was to investigate the mechanism (s) of resveratrol-induced vasorelaxation in rat renal artery (RA) with endothelium. RA rings were precontracted with phenylephrine. Resveratrol induced relaxation of the RA rings. Highly selective blocker of ATP-sensitive K+ channels, glibenclamide as well as selective antagonist of voltage-gated K+ (Kv 1.1.–1.6) channels, margatoxin did not block resveratrol-induced relaxation of RA. A nonselective blocker of Kv channels, 4-aminopyridine and a blocker of big Ca-sensitive K+ (BKCa) channels, charybdotoxin inhibited endothelium-dependent relaxation of RA induced by resveratrol. Resveratrol (100 然) completely relaxed renal artery precontracted with K+-rich solution. In conclusion, we have shown that resveratrol induces relaxation of RA with endothelium. The KV and BKCa channels are involved in this relaxation, but K+ channel- independent mechanism of action also take a part in its vasorelaxant effect.


Introduction

The lower incidence of coronary artery disease in the Southern French and other Mediterranean populations, despite a diet rich in saturated fat and high smoking habits (the so-called French paradox), has been attributed to the prolonged and moderate red wine consumption by these population [1]. It is considered that resveratrol, as a polyphenol, is presented in red wine in significant amounts, and partly responsible for cardiovascular benefits associated with wine consumption. The mechanism of cardiovascular benefits probably includes vasorelaxation, antioxidant and anti-platelet effects of resveratrol [2].

The mechanisms by which resveratrol causes vasodilatation are uncertain. Today is known, that resveratrol-induced vasorelaxation may either be endothelium-dependent or endothelium-independent [3]. Resveratrol might become incorporated into the smooth muscle membrane, where it could either couple with a membrane receptor or interact directly with membrane ion channels, thus inducing endothelium independent vasorelaxation [4], [5]. Recently, it has been shown that some endothelial and peripheral effects of resveratrol are mediated by activation of big Ca2+-activated (BKC) channels and voltage-gated K+ (KV) channels [2], [6].

The resveratrol effect on the rat renal artery was studied before. They have shown that resveratrol induced endothelium-dependent relaxation of different arteries by release of NO [7], [8]. However, the contribution of other mechanism(s) in the resveratrol-induced relaxation of renal artery was not defined. Thus, our study was aimed to define the contribution of different K+ channel subtypes in the resveratrol action on rat renal artery with intact endothelium.


Methods

Animals

Male Wistar rats obtained from the animal facilities of the University of Belgrade of Medicine faculty were used in all experimental procedures. The study was approved by The Ethical Committee of Faculty of Medicine, University of Belgrade. The studies reported in this work have been carried out in accordance with the European regulations on the protection of animals, the Declaration of Helsinki, and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. Vascular rings were prepared from the renal arteries of male Wistar rats, body weight of 250–300 g.

Assessment of vascular function

The renal arteries segments were dissected free from connective tissue. They were cut into rings (3 mm) and mounted between two stainless-steel triangles in an organ bath containing 10 ml Krebs-Ringer-bicarbonate solution (37蚓, pH 7.4), aerated with 95% O2 and 5% CO2. One of the triangles has been attached to a displacement unit allowing fine adjustment of tension and the other was connected to an isometric transducer (K30; Hugo Sachs, Freiburg, Germany). The preparations were allowed to equilibrate for 30 min. During this period, the vessels were washed with a fresh buffer solution every 10 min. We have examined the effects of resveratrol onto the rings with intact endothelium. After the equilibration period, the presence of functional endothelium was assessed. Rings were precontracted with phenylephrine (1 然) and acetylcholine (20 然) was added into the organ bath. The resting tension was 2 g. The vascular rings were allowed a further 30 min to equilibrate before being contracted with phenylephrine (1 然). Concentration–response curves were obtained by the cumulative addition of resveratrol (1–100 然) to ring segments contracted to a stable plateau by adding phenylephrine. 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. Therefore, the following protocol was used: 1) contraction to phenylephrine and concentration-response curve to resveratrol followed by three washes, addition of the different K+ blockers, and a 20-min equilibration period; 2) contraction to phenylephrine and the concentration-response curve to resveratrol was determined. In a separate series of experiments, vascular rings were first contracted with 1 然 phenylephrine to obtain the control contraction. The second contraction was produced by 100 mM K+ (to annul the effect of K+-channel activation) and then the increasing concentration of resveratrol was added. Contractions produced by phenylephrine and 100 mM K+ were of comparable amplitude. K+-rich Krebs-Ringer bicarbonate solution was prepared by equimolar replacement of 100 mM NaCl with 100 mM KCl.

Treatment of data and statistics

Relaxation produced by each concentration of resveratrol was measured and expressed as a percentage of the maximum possible relaxation (i.e., relaxation back to the baseline tension). The concentration of resveratrol producing 50% of its own response (EC50) was determined for each curve by using a non-linear least square fitting procedure of individual experimental data. The results are expressed as the means S.E.M.; n refers to the number of experiments. Statistical difference between means was determined by Student’s t-test, and a P value <0.05 was considered statistically significant. All calculations were done by using the computer program Graph Pad Software (Inc., San Diego, CA USA).

Drugs

The following drugs were used: trans-resveratrol, phenylephrine, acetylcholine, glibenclamide, charybdotoxin, 4-amynopiridine (4-AP), margatoxin, and potassium chloride (KCl) (Sigma-Aldrich, Inc., St. Louis, MO, USA). Resveratrol was 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. Previous experiments showed that the solvents used had no effects on preparations at the concentrations applied. All drugs were added directly to the bath in a volume of 50 無 and the concentrations given are the calculated final concentrations in the bath solution.


Results

Resveratrol (1–100 然) induced a concentration dependent relaxation of rings with endothelium EC50 values of 8.51 0.4 然 (maximal response 100 5%, n=17, Figure 1 [Fig.1]).

Glibenclamide (10 然, n=5), a selective ATP sensitive K+ (KATP) channels inhibitor did not significantly modify the relaxation of the renal artery induced by resveratrol EC50=11.48 3 然, P>0.05; maximal response 100 6% in the presence of glibenclamide, P>0.05 (data not shown).

Charybdotoxin (10 nM, n=9), a potent inhibitor of BKCa channels affect the relaxation of renal artery produced by resveratrol with EC50=21.88 2 然, P<0.05; but did not affect the maximal response 100 5%, P>0.05 (Figure 1 [Fig.1]).

4-AP (1 mM, n=9), a predominant blocker of different Kv channels, inhibited resveratrol-induced relaxation of renal artery with EC50=14.79 0.2 然, P<0.05 but did not affect the maximal response 100 5%, P>0.05 (Figure 1 [Fig.1]).

Margatoxin (30 nM, n=10), a potent inhibitor of KV1.1, KV1.2, KV1.3, and KV1.6 channels KV1 channels, not significantly modify the resveratrol induced relaxation of the renal artery with EC50=11.22 2 然, P>0.05; did not affect the maximal response 99 5%, P>0.05 (Figure 1 [Fig.1]).

In the rat renal artery precontracted with 100 mM K+, relaxant response to resveratrol (1–100 然) was significantly (P<0.05) shifted to the right; maximal response 99 1%, n=10) compared to those obtained in the renal artery precontracted with phenylephrine (maximal response 100 1%, n=10) (Figure 2 [Fig.2]). However, the difference between maximal responses was not significant (P> 0.05).

Glibenclamide (10 然), charybdotoxin (10 nM), 4-AP (1 mM) and margatoxin (30 nM), did not affect the basal tension of renal artery nor the contraction induced by phenylephrine or high K+ (data not shown, n=5–8).


Discussion

Our previous studies suggested vasorelaxant effects of polyphenolic compounds [8], [9]. Resveratrol is thought to be a prime compound of the polyphenols, causing the relaxation of rat aorta, mesenteric and uterine artery of guinea pig, prior contracted by phenylephrine, noradrenaline or KCl [1], [3], [7]. In the present study, we have shown that resveratrol induces concentration-dependent relaxation of rat renal artery with endothelium.

Today is known that endothelium-dependent effect of resveratrol is apparent at low concentrations (10–30 然) and is blocked by inhibitors of NO synthase. The previous reports have shown that resveratrol-induced vasodilatation was attenuated with inhibitors of NO synthesis [7], [8], [10], [11]. In addition, the relaxation induced by resveratrol was blocked by methylene blue, an inhibitor of soluble guanylate cyclase [12]. According to this, it seems that endothelium-dependent vasorelaxation of different blood vessels caused by resveratrol could be mediated by endothelial generation and release of NO. The mechanism underlying the NO-induced vasodilatation has been intensively investigated. Current knowledge suggests a central role for cGMP-dependent activation PKGI which can phosphorylate different membrane proteins. NO can also activate KCa channels and increase the outward potassium current. It has been shown that this action of NO can be both independent and dependent on activation PKGI [13]. Finally, cGMP-dependent inhibition of voltage-gated Ca-channels might also be involved in the mechanism of vasodilatation induced by NO. The relative contribution of each of these PKGI and K+ channel dependent vasodilating mechanism of NO remains to be determined.

To determine whether the K+ channels mediated endothelium-dependent relaxation of rat renal artery induced by resveratrol, we used different potassium channel blockers.

To analyze the contribution of KATP channels to the endothelium-dependent resveratrol-induced relaxation of the renal artery, we used glibenclamide (10 然). Glibenclamide is known as one of the most selective blockers of KATP channels, although when used in a high concentration (>30 然), it may block some other types of K+ channels [14]. In the present study, glibenclamide did not inhibit the relaxation of renal artery induced by resveratrol. Accordingly, it seems that KATP channels are not involved in the pathway by which resveratrol produces a relaxation of the renal artery. This result is in agreement with the view that glibenclamide does not antagonize antinociceptive effect of resveratrol [6].

To analyze the possibility that the endothelium-dependent relaxation of the renal artery, evoked by resveratrol, is mediated via BKCa channels, charybdotoxin was tested. It has been shown that charybdotoxin blocks BKCa channels in vascular smooth muscle [14]. The channel inhibition occurs in the low nanomolar range (Kd ~0.3 and 10 nM). The concentration of charybdotoxin used in our study was sufficient to block BKCa channels. Accordingly, it seems that charybdotoxin-sensitive channels are involved in the mechanism of resveratrol-induced relaxation of the renal artery. This finding is in line to the finding of Wu et al. [1] who demonstrated that resveratrol opens BKCa channels in the vascular endothelial cell.

To analyze the contribution of KV channels to the resveratrol-induced relaxation, we used 4-AP. This compound is the most widely used blocker in the identification of potassium channel types. Using low milimolar concentration, 4-AP achieved some selectivity for KV channels [14]. This feature complies with the results given by our experiments i.e. 4-AP (3 mM) antagonized resveratrol-induced relaxation of renal artery rings with comparable potency. Thus, our finding supports a relevant participation of KV channels in relaxation of renal artery produced by resveratrol. Consistent with this idea is result obtained by Granados-Soto et al. [6] that suggested that activation of KV channels participated in the peripheral nociceptive effect of resveratrol.

We used margatoxin in order to test which subtype of KV channels was included in resveratrol-induced relaxation of HIMA. This peptide is highly selective inhibitor of the KV1 channels, especially 1.1, 1.2, 1.3, and 1.6 subtypes, but display no affinity for the mammalian BKCa channel [15]. KV1.2 and KV1.3 were identified in vascular smooth muscle cell of rat mesenteric artery; KV1.1 and KV1.6 were detected in smooth muscle cell of rat aorta and rat pulmonary artery but not in smooth muscle cell of mesenteric artery [16]. This reflects tissue differences in expression of KV1 channels subtypes. In our study, margatoxin used in concentration (10 nM) sufficient to block KV1.1, KV1.2, KV1.3, and KV1.6 channels, did not influence resveratrol-induced relaxation, suggesting that those channels are not included in the mechanism of resveratrol-induced endothelium-dependent vasodilatation of rat renal artery.

The results of our experiments performed in K+-rich solution also confirm the involvement of K+ channels in resveratrol-induced endothelium-dependent relaxation of the renal artery. However, the ability of resveratrol (100 然) to completely relax renal artery precontracted with K+-rich solution supports the notion that resveratrol acts at least in part through K+ channel-independent mechanism(s). Similar K+ channel-independent effect of resveratrol (over 30 然) in K+-rich solution was obtained in the human mammary artery, rat aorta and mesenteric artery [8], [9], [17]. One possible explanation for this potassium- and endothelium-independent effect is that resveratrol resembles an endogenous signalling molecule [12]. Whether, resveratrol can stimulate endogenous pathways to promote vasodilatation is important issues to address.

In conclusion, we have shown that resveratrol can induce endothelium-dependent relaxation of ret renal artery. It seems that 4-AP-sensitive KV + channels and BKCa channels are included in this relaxation. To define the subtype of KV channels involved in resveratrol-induced relaxation we need further investigations. Its ability to completely relax the rat renal artery precontracted with K+-rich solution suggests that K+ channel-independent mechanism(s) also take a part in its vasorelaxant effect.


Acknowledgements

We thank Mrs. Milena Zabunovic for technical support during this study. Our work was supported by Scientific Research Grants from the Ministry of Science and Technology TP 20027 (Serbia), the Karl & Lore Klein Foundation (Germany) and the Alexander von Humboldt Foundation (Germany).


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