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Cardio-Electopharmacology and Vasodilating Mechanisms of Quercetin | OMICS International
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Medicinal Chemistry

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Cardio-Electopharmacology and Vasodilating Mechanisms of Quercetin

Hiroyasu Satoh* and Seiichiro Nishida

Health Life Science, Shitennoji University, Habikino, Osaka 583-8501, Japan

*Corresponding Author:
Dr. Hiroyasu Satoh
Health Life Science Shitennoji University
3-2-1 Gakuenmae, Habikino
Osaka 583-8501, Japan
Tel: +81-72-956- 3181
Fax: +81-72-956-6011
E-mail: [email protected]

Received date: April 29, 2014; Accepted date: June 25, 2014; Published date: June 27, 2014

Citation: Satoh H, Nishida S (2014) Cardio-Electopharmacology and Vasodilating Mechanisms of Quercetin. Med chem 4:523-530. doi:10.4172/2161-0444.1000189

Copyright: © 2014 Satoh H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Quercetin, a kind of flavonoids, exerts the cardiovascular actions. In guinea pig ventricular cardiomyocytes, quercetin depresses the action potential duration (APD) and inhibited the underlying ionic currents ICaL.,IKrec. IK1 in cardiomyocytes. In rat aorta, quercetin (0.1 to 100 μM) relaxed the contraction induced by pretreatment with 5 μM norepinephrine (NE) in aconcentration-dependent manner. NG-monomethyl-L-arginine acetate (L-NMMA) at 100 μM reduced the quercetin (100 μM)-induced vasorelaxation from 97.0 ± 3.7% (n=10, p<0.05) to 78.0 ± 11.6% (n=5, p<0.05). Endothelium removal as well attenuated the vasodilatation. In the presence of both 100 μM L-NMMA and 10 μM indomethacin, the quercetin-induced vasorelaxation was further attenuated by high K (30 mM) or 10 μM tetraethylammonium (TEA). Among KCa channel inhibitors, the quercetin-induced vasodilatation was attenuated by 0.3 μM apamin (sensitive to SK), but not by 30 nM charybdotoxin (sensitive to BK and IK). Under KCl-induced vasoconstriction, the quercetin-induced vasorelaxation was attenuated by PK-C inhibitors; Gö6983 (α-, β-, γ-, δ and ζ-sensitive) produced stronger than Ro-31-8425 (α-, β-, γ- and ε-sensitive). In rat mesenteric artery, the quercetininduced vasodilatation was almost resistant to both 100 μM L-NG-nitro arginine methyl ester (L-NAME) and 100 μM indomethacin. The L-NAME/indomethacin-resistant quercetin-induced   was not modified by TEA (1 mM), but was attenuated by endothelium removal and 100 μM 18α- and 50 μM 18β-glychrrhetinic acids (gap junction inhibitors). Therefore, quercetin dilates the vascular smooth muscle mediated by endothelium-dependent and -independent mechanisms.

Keywords

Quercetin; Vasodilatation; Endothelium; Gap junction; Ionic currents

Introduction

Quercetin, a flavonoid, exists as quercetin glycosides in food, beverages and herbs, and in plasma, as mainly glucuronids or sulfates of quercetin and unconjugated quercetin [1,2]. Quercetin can exhibit significant vasodilatation of rat arteries. In general, flavonoids are vasodilator [3] and scavenger for free radicals [4], and reduce the incidence of cardiovascular diseases and carcinogenesis [5]. Thus, quercetin exerts protective cardiovascular actions through their various pharmacological effects [6,7].

The endothelium dependency of quercetin is predominant in aorta. Most studies (including our previous reports) have shown the endothelium-dependency of the quercetin-induced vasodilatation [7,8]. However, there are several reports that quercetin is less endothelium-dependent or has only a weak endothelium dependency [3,9]. Quercetin can increase the intracellular Ca2+ concentration ([Ca2+] i in the endothelium [10]. The increase of [Ca2+]i leads to endogenous nitric oxide synthase (eNOS) activation and NO production [11], and as a result, may involve with endothelium-derived hyperpolarizing factor (EDHF) for the quercetin-induced vasodilatation [12,13]. Plant polyphenols have been reported to induce EDHF-type relaxation [14]. The cardiovascular pharmacological actions of quercetin we have so far investigated are discussed separately in cardiac and vascular pharmacology.

Cardiac Pharmacology

The cells were prepared from tissue taken from the ventricle muscle of guinea pig hearts, using the methods similar to those described previously [15-17]. Under an anesthesia with sodium pentobarbital (30 mg/kg, i.p), the chest was opened and the aorta was cannulated in situ. The heart was dissected out and perfused with normal Tyrode solution on the Langendorff apparatus. The heart was washed out by high-K+ and low-Cl- solution (KB solution). The temperature of all solutions was maintained at 36.5oC. Effects of quercetin on the action potentials and the ionic currents in the cardiomyocytes were investigated using an Axopatch patch-clamp amplifier (Axon Instruments, Burlingame, CA, U.S.A.) and standard techniques. The composition of the modified Tyrode solution was (in mM): NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.3, glucose 5, and HEPES 5. The pH was adjusted to 7.4 with NaOH. Quercetin (Tocris Biosci., Northpoint, UK) was dissolved with DMSO. The final concentration of DMSO was diluted 100 to 2000 times, and never caused any responses. The pipette solution contained (in mM): K-aspartate 110, KCl 20, MgCl2 1, EGTA 10, Mg- ATP 5, creatine phosphate 5, and HEPES 5 (pH 7.2). All values are given as mean ± S.E.M. The differences of mean values were analyzed by Student’s t-test and ANOVA for paired data, and a p value of less than 0.05 was considered significant. All the experiments were carried out according to the guidelines laid down by the Nara Medical University Animal Welfare Committee, and also under the terms of the Declaration of Helsinki.

Effects on the action potentials

Under the current-clamp experiments, the isolated single cell was stimulated at 1 Hz. The action potential amplitude (APA) and the maximum rate of depolarization (Vmax) significantly decreased by approximately 12% (n=8) at 1-3 μM Table 1. The resting potential (RP) was unaffected. Quercetin at 3 μM prolonged the action potential duration at 75% (APD75) by 11.9 ± 3.4% (n=8, p<0.05) and at 90% repolarizations (APD90) by 17.9 ± 3.0% (n=8, p<0.05), but not at the lower concentrations (0.1-1 μM) [6]. The APD means a period for the membrane repolarization, making T wave on ECG. The APD is clinically reflected directly to QT interval, a period between the depolarization and the repolarization of action potential, mainly responsible for the alteration of the delayed rectifier K+ current IKrec). Thus, the APD prolongation increases the refractory period and simultaneously elevates [Ca2+]i [15,17]. A washout for 15-20 min recovered to approximately 90% of the control value.

  n APA(mV) RP(mV) APD75(ms) APD90(ms) Vmax (V/s)
Control 8 117.4 ± 1.2 -80.9 ± 0.5 77.0 ± 2.2 94.1 ± 2.0 182.8 ± 2.5
Quercetin            
0.1 mM 8 117.6 ± 1.2 -80.6 ± 0.6 78.6 ± 2.4 95.0 ± 2.1 182.1 ± 1.2
0.3 mM 8 117.8 ± 1.1 -80.7 ± 1.0 79.8 ± 3.8 97.3 ± 2.0 182.5 ± 2.3
1 mM 8 114.6 ± 1.4 -80.4 ± 2.3 82.9 ± 2.3 100.3 ± 2.6 173.8 ± 2.21)
3 mM 8 113.7 ± 1.2 -79.7 ± 2.1 86.2 ± 2.01) 110.9 ± 2.31) 161.7 ± 2.42)
Washout 8 110.7 ± 1.0 -80.0 ± 0.6 79.4 ± 2.2 96.8 ± 2.4 172.3 ± 2.2

Table 1: Modulation of the action potential configurations by quercetin in guinea pig ventricular cardiomyocytes.

Effects on the ionic currents

Whole-cell patch voltage-clamp experiments were performed, and test pulses (1 sec duration) were applied to -20 to +60 mV and -40 to -120 mV from a holding potential of -30 mV. The average capacitance was 86.1 ± 2.0 pF (n=44) [17]. Application of quercetin (0.1 to 3 μM) inhibited the L-type Ca2+ current (ICaL) Figure 1. The ICaL at 10 mV decreased by 34.9 ± 3.2% (n=8, p<0.05) at 0.3 μM and by 56.8 ± 3.3% (n=8, p<0.05) at 3 μM. The responses were produced in a concentration-dependent manner. The cells not causing run-down were chose and used for the experiments. Simultaneously, the IKrec at 60 mV increased by 60.4 ± 2.7% (n=8, p<0.001) at 0.3 μM and by 89.7 ± 3.3% (n=8, p<0.001) at 3 μM. In general, the IKrec enhancement may protect a cell due to an APD shortening and a decline of [Ca2+]i, although quercetin prolonged APD. Quercetin decreased the inwardly rectifying k+ current (IK1). Quercetin did not affect the IK1 at lower concentrations, but at 3 μM inhibited it by 12.4 ± 2.1% (n=8, p<0.05). The IK1 is closely related with the RP, and is not yet activated in range of -70 to -90 mV of RP. The INa (as a Vmax) decreased by approximately 18-20% (n=8-9) at higher concentrations (1-3 μM) of quercetin. The effect may produce antiarrhythmic actions. These responses were almost reversible (80-90% of control) after 20 min washout.

medicinal-chemistry-ventricular-cardiomyocytes

Figure 1: Modulation by quercetin of the ionic currents in guinea pig ventricular cardiomyocytes. A: Control. The test pulses were applied 70 mV and -130 mV from a holding potential of -30 mV. B: Quercetin 3 μM. C: Modulation of ICaL in the absence and the presence of quercetin. Current traces at 30 to 70 mV are superimposed. D: Modulation of IK1 by quercetin. Current traces at -130 to -30 mV are presented.

Summary

The experiments in guinea pig ventricular cardiomyocytes showed that (1) quercetin prolonged the APD, (2) other action potential parameters were unaffected, (3) quercetin inhibited ICaL, IK1, and Vmax (INa), but enhanced IKrec, and (4) these responses were almost reversible after a washout. The inhibitions finally lead to decline of [Ca2+]i, resulting in the suppression of the abnormal excitations. Therefore, these electropharmacological effects of quercetin would exert many helpful and protective actions upon cardiac muscle cells under the diseased conditions.

Vascular Pharmacology

Vasodilating effects on rat aorta

Wistar male rats, weighing 150 to 250 g, 7-16 weeks old (n=32), were anesthetized with ether, and euthanized by exsanguination [18,19]. The isolated artery was cut into 1 mm rings in length, suspended between two stainless steel stirrups in a bath filled with 3 ml modified Krebs-Henseleit solution. The modified Krebs-Henseleit solution contained 118 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11.1 mM glucose, 27.2 mM NaHCO3, 0.03 mM ethylene glycol-O,O’-bis (2-aminoethyl)-N,N,N’,N’-tetraacetic acid (EGTA), and 1.8 mM CaCl2. The chamber solution was oxygenated with 95% O2 and 5% CO2 at 36.5?. The removal of endothelial cells was carried out by usual methods, and the almost absence through microscopy was confirmed after the experiments.

Effects on the endothelial cells

On endothelium-derived releasing factor (EDRF): Rat aorta exhibited a strong contraction by 5 μM NE. Quercetin (0.1 to 100 μM) subsequently administrated exhibited the marked endotheliumdependent actions (Table 2). Prior administration of L-NMMA (100 μM) significantly inhibited the quercetin (100 μM)-induced vasodilatation from 97.8 ± 3.7% (n=10) to 78.0 ± 11.6% (n=5, p<0.05). Another NOS inhibitor, L-NAME had the similar effects. This is enforced by the results that removal of endothelium abolished or attenuated the quercetin-induced vasorelaxation. Thus, quercetin decreased the relaxing action by NOS inhibitors. Also, both L-NMMA (100 μM) and indomethacin (10 μM) attenuated the quercetin-induced vasodilatation more than that with L-NMMA alone.

Quercetin
  n 0.1 0.3 1 3 10 30 100 µM
Control 10 1.7±0.5 3.6±0.91) 6.9±0.91) 12.4±1.12) 32.0±5.72) 54.1±8.4 97.8±3.7
Endothelium-denuded 5 1.5±1.3 3.1±1.6 6.9±1.8 11.2±2.4 30.8±4.9 44.1±4.2a) 77.9±2.3a)
L-NAME 100 µM 5 1.9±0.9 3.6±1.1 7.1±2.1 13.2±3.7 21.1±4.6 33.0±5.3a) 69.5±6.1a)
L-NMMA 100 µM 5 1.9±0.6 4.1±1.3 7.3±1.8 12.0±2.4 25.4±4.8 38.7±6.0a) 78.0±11.6a)
L-NMMA 100 µM
+ Indomethacin 10 µM
5 1.9±0.8 4.1±1.3 6.8±1.3 11.0±2.0 18.2±4.7 30.4±6.2a) 65.2±6.6a)

Table 2: Endothelium-dependent vasodilatation induced by quercetin in rat aorta

The endothelium-dependency is important in the quercetininduced vasodilatation. Quercetin causes vasodilatation mainly through NO secretion [19,20], induced by elevating endothelial [Ca2+]i and eNOS phosphorylation [21,22]. The [Ca2+]i elevation in endothelium facilitates other actions such as a production of hydroxyl hydrogen [23], endothelial hyperpolarization [24,25] induced by activation of KCa channels [26], and production of endotheliumderived hyperpolarizing factor (EDHF) [12,27].

On endothelium-derived hyperpolarizing factor (EDHF): Quercetin increases [Ca2+]i in endothelium, stimulating the synthesis of EDHF [13]. Plant polyphenols have been shown to induce the EDHFtype relaxation [14]. The vasodilatation induced by EDHF is considered to be resistant to both inhibitors of NOS and cyclooxygenase [28]. In the presence of L-NMMA (100 μM) and indomethacin (10 μM), quercetin at 100 μM attenuated by 65.2 ± 6.6% (n=5, p<0.001)(Table 3). In aorta, however, the EDHF-related vasorelaxation is told never to observe [29]. So, we examined using rat mesenteric artery as mentioned in Section 4.2.

Quercetin
  n 0.1 0.3 1 3 10 30 100 μM
? Constriction induced by Norepinephrine
A)
Control (Endothelium+) 10 1.7±0.5 3.6±0.91) 6.9±0.92) 12.4±1.12) 32.0±5.73) 54.1±8.43) 97.8±3.73)
Ca2+-free 5 2.1±1.5 3.1±1.4 9.0±2.0 12.8±3.3 22.6±2.5 45.0±3.3a) 67.5±7.7a)
Nicardipine 5 0.0±0.0 1.4±1.4 1.4±1.4 1.4 ±1.4a) 6.8±3.6a) 15.0±7.2a) 61.2±16.6a)
Ro-31-8425 (0.1μM) 5 1.5±1.0 3.0±1.9 6.6±2.5 12.8±4.0 23.0±4.9 42.8±4.2a) 80.8±2.6b)
G?6983 (0.1μM) 5 1.0±0.6 2.0±1.2 2.8±1.7 8.1±2.5 18.8±3.5 40.0±6.2a) 84.6±7.7a)
B)
Endothelium-denuded 5 1.5±1.3 3.1±1.6 6.9±1.8 11.2±2.41) 30.8±4.92) 44.1±4.22) 77.9±2.33)
 +TEA (100μM) 5 1.0±0.75 3.0±1.3 5.5±2.6 9.2±3.6 17.9±4.2a) 31.7±4.9a) 62.5±4.9a)
C)
L-NMMA(100μM )+Indomethacin (10μM) 5 1.9±0.8 4.1±1.3 6.8±1.31) 11.0±2.02) 18.2±4.72) 30.4±6.23) 65.2±6.63)
 +high K+(30mM) 5 0.5±0.3 2.6±1.5 5.1±1.6 9.4±2.3 13.6±1.9 24.4±3.9a) 41.0±5.7a)
 +TEA (100μM) 5 1.2±0.5 1.8±1.5 1.8±1.5 4.0±4.0 8.5±5.5 16.8±7.0a) 43.8±9.5a)
 +Charybdotoxin 5 2.1±1.3 3.8±2.6 4.2±2.0 4.5±2.3 16.8±3.6 28.2 ±2.7 63.4±11.8
 +Apamin 5 0.6±0.6 0.8±0.8 1.6±1.6 3.7±2.7 5.1±1.6 9.4±2.4b) 47.1±11.1b)
? Constriction induced by KCL
A)
Control 10 0.0±0.0 0.8±0.4 0.9±0.4 0.9±0.4 8.8±2.21) 29.9±6.02) 92.8±4.03)
Ro-31-8425 (0.1μM) 5 0.0±0.0 0.0±0.0 0.3±03 1.0±0.6 9.6±3.1 16.0±3.1a) 75.6±7.9a)
G?6983 (0.1μM) 5 0.0±0.0 1.0±1.0 1.1±1.0 1.4±1.2 8.0±1.2 26.8±2.8 49.5±13.7a)

Table 3: The alteration of endothelium-independent vasodilatation induced by quercetin in rat aorta.

On Ca2+-activated K+ (KCa) channel: The elevation of [Ca2+]i induced by quercetin may stimulate the KCa channel. The activation of KCa channel to hyperpolarize the membrane produces the vasodilatation in rat aorta [19,30]. In endothelium-denuded aorta, TEA significantly decreased the quercetin-induced relaxation from 77.9 ± 2.3% to 62.5 ± 4.9% (n=5, p<0.05). The relaxation involved with KCa channel was examined in the presence of indomethacin and L-NMMA. The L-NMMA/indomethacin-resistant relaxation induced by quercetin (100 μM) was significantly reduced by high K+ (30 mM) to 41.0 ± 5.7% (n=5, p<0.05) Table 3. In high K+solution, furthermore, TEA attenuated the L-NMMA/indomethacin-resistant relaxation to 43.8 ± 9.5% (n=5, p<0.05). These results indicate that quercetin modulates the KCa channel.

The KCa channels are classified by their conductances as follows: BK channel (200 pS), IK channel (37 pS), and SK channel (32 pS) [31]. TEA is sensitive to all KCa channels [32,33], apamine to SK channels [34], and carybdotoxin to BK and IK channels [35]. Apamin (0.3 μM), a SK channel inhibitor, strongly decreased the L-NMMA/indomethacinresistant relaxation induced by 30 μM quercetin from 30.4 ± 6.2% to 9.4 ± 2.7% (n=5, p<0.05), and from 65.2 ± 6.6% to 47.1 ±11.4% (n=5, p<0.05) by 100 μM quercetin. But charybdotoxin (30 nM), a BK and IK channel inhibitor, had less or no effect. Therefore, quercetin would possess possibly a selective sensitivity to SK channel, but less to BK and IK channels

On prostaglandin (PG) I2: The pretreatment with both indomethacin and L-NMMA reduced the relaxation to a greater extent than the pretreatment with L-NMMA alone (but not significantly). The PGI2 secretion from endothelium may also partly contribute to the relaxation, as reported previously [20]. Therefore, the endotheliumdependent vasorelaxation induced by quercetin is produced due to EDRF and EDHF, and also partly to PGI2.

Effects on vascular smooth muscle

On L-type Ca2+ channel: L-type Ca2+ channel is regulated by the signal transductions such as cAMP, cGMP and PK-C [15,36,37]. 4-β-phorbol-12,13-dibutyrate (PDB, a PK-C activator) has small stimulation of the ICaL in A7R5 cells [38,39]. Quercetin at 100 μM significantly decreased the vasorelaxation from 97.8 ± 3.7% (n=10) in normal solution to 67.5 ± 3.7% (n=5) in Ca2+-free solution (p<0.01) [7,18]. The quercetin-induced vasorelaxation was also attenuated by nicardipine, and by a switch from normal Krebs’ solution (Ca2+=1.8 mM) to Ca2+-free solution. Also, quercetin dilated the KCl-induced vasoconstriction [3,19]. These findings demonstrate the vasorelxation due to its Ca2+ channel inhibitory action. Satoh [17] has already reported that quercetin is an inhibitor of ICaL channel in cardiomyocytes. These are summarized in Table 3.

On protein kinase C: The Ca2+ mobilization mediated by PK-C stimulation might be dependent on kinds of smooth muscles [40]. PDB caused the vasoconstriction. Quercetin possesses the inhibitory actions of PK-C [3,4]. Quercetin at 100 μM dilated the PDB (300 nM)-induced vasoconstriction by 84.8 ± 6.0% (n=5, p<0.01) Table 3. Under NE-induced vasoconstriction, staurosporine (100 nM), a PK-C inhibitor, decreased the relaxation induced by 100 μM quercetin by approximately 40%. It has been shown that PK-Cα and/or δ are necessary for phorbol ester-mediated constriction of aortic smooth muscle, but are not essential for NE-, vasopressin-, or K+-induced constriction [41]. Furthermore, PK-Cα and δ isozymes are dominant in cultured rat aortic vascular smooth muscle cell, and both isozymes are completely down-regulated by prolonged (16-24 hr) stimulation with the PK-C activator [42]. PK-Cε can modulate phenylephrineinduced contraction in mesenteric artery via calcium-independent pathways [43].

To examine which subtypes of PK-C are related with the quercetininduced constriction, two different types of PK-C inhibitors were chosen; Gö6983 sensitive to α, β, γ, δ and ζ isozymes, and Ro-31- 8425 to α, β, γ and ε isozymes of of PK-C [44]. Ro-31-8425 (0.1 μM) attenuated the quercetin (100 μM)-induced vasorelaxation by 80.8 ± 2.6% (n=5, p<0.001). Gö6983 (0.1 μM), also had similar effects by 84.6 ± 7.6% (n=5, p<0.001). Under the KCl-induced vasoconstriction, both Gö6983 and Ro-31-8425 attenuated the quercetin (100 μM)- induced vasorelaxation by 49.5 ± 7.9% (n=5, p<0.01) and by 75.5 ± 7.9% (n=5, p<0.001), respectively (Table 3). Thus, quercetin might inhibit possibly mediated through PK-Cδ, although it is still difficult to clearly distinguish. In addition, PK-C phosphrylates tyrosin kinase and vasodilator-stimulated phosphoprotein (VASP) as a substrate of cGMP-dependent protein kinase (cGMP-PK) [45]. In this study, however, genistein (tyrosine kinase inhibitor) at 50 μM failed to affect the quercetin-induced constriction (by just 0.7% decrease). The activation of MLCK is abolished by PK-C [40,46]. Furthermore, quercetin inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-jun amino-terminal kinase (JNK) in cultured aortic cells and phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) [47,48].

On Ca2+-activated k+ (KCa) channels: Flavonoids have been shown to produce vasodilatation due to the KCa channel to hyperpolarize the membrane of vascular smooth muscle [30]. The [Ca2+]i in smooth muscle cells initially increases, and then, facilitates the other physiological actions. The vasorelaxation of quercetin is also due to KCa activation involved with EDHF [9]. Recently, quercetin has been demonstrated to activate BK channel in coronary arteries via production of H2O2 [49]. In addition, glibenclamide (a KATP channel inhibitor) have not been reported to affect the quercetin-induced vasodilatation in rat aorta [9]. Anyway, the vasodilatation is responsible for SK channel activation (though not distinguish in endothelium or smooth muscle cells) Table 3.

Summary: The vasodilation mechanisms in rat aorta were due to the inhibitions of (1) NOS by L-NMMA or L-NAME and removal of endothelium, (2) ICaL by Ca2+ antagonist, in Ca2+ free solution and under the KCl constriction, (3) PK-C activation mediated through possibly the PK-Cδ subtype, and (4) KCa channel by apamin but not by charybdotoxin. Furthermore, the vasodilation was produced by (5) both L-NMMA and indomethacin, (6) L-NMMA and indomethacin plus TEA during exposure to high k+ solution, and (7) TEA in endothelium-denuded aorta (Fig. 2). Therefore, quercetin’s effect is due to endothelium-dependent actions mediated through the NO (EDRF), EDHF and partly PGI2 syntheses. As well it is due to endothelium- independent actions mediated through the Ca2+ channel, the KCa channels selective to SK channel, and PK-C [7,18].

Vasodilating Effects on Rat Mesenteric Artery

For this investigation, the similar methods to rat aorta experiments were performed. Mesenteric artery was removed from the same Wistar male rats [18,19]. The isolated mesenteric artery was cut into 1 mm rings in length, suspended between two stainless steel stirrups in a bath filled with modified Krebs-Henseleit solution. The isometric force was recorded using the force-displacement transducer (UL-10GR, Minebea Co., Tokyo, Japan). Rat mesenteric artery also caused the contraction by an application of 1 μM NE. Quercetin (0.1-100 μM) caused the significant vasodilatation in a concentration-dependent manner; at 100μM, the vasodilatation was 99.6 ± 3.0% (n=10, p<0.001). The quercetin-induced vasodilatation was almost consistent with the results in rat aorta [50]. But it was weaker blockade than the results in rat aorta Table 4. Similarly both L-NAME (100 μM) and indomethacin (100 μM) failed to cause a remarkable change in the quercetin-induced vasodilatation, as compared with that with L-NAME alone. Endothelium-denuded arteries also contracted at 1 μM NE. Endothelium removal strongly decreased the quercetin-induced vasodilatation; at 100 μM quercetin by 77.9 ± 2.4% (n=6, p<0.01). Furthermore, endothelium-removal more strongly reduced the quercetin-induced vasodilatation in the presence of L-NAME and indomethacin, indicating that the relaxation is independent of endothelium. Thus, NO may play a minor role for the quercetin-induced vasodilatation in mesenteric artery, different from aorta.

Quercetin
  n 0.1 0.3 1 3 10 30 100 μM
  Control   A   10   1.6 ± 0.94   8.0 ± 1.71)   23.4 ± 3.22) 33.9 ± 2.72) 55.5 ± 3.23)   69.8 ± 3.43)   99.6 ± 3.03)
  Endothelium-denuded   6   1.8 ± 0.55   3.8 ± 1.6b)   8.5 ± 1.9b)   13.0 ± 2.6b)   30.8 ± 4.9)b)   44.2 ± 4.2b)   77.9 ± 2.4b)
  L-NAME 100µM   8   1.3 ± 0.9   3.2 ± 1.8   19.2 ± 3.4   31.0 ± 2.8   51.2 ± 2.0   62.3 ± 4.7   86.0 ± 7.0a)
  B                
  L-NAME+
Indomethacin(100µM)
  8   1.9 ± 0.75   4.1 ± 1.3   19.0 ± 7.3   31.2 ± 6.3   52.5 ± 7.8   63.2 ± 6.9   82.0 ± 2.2
  +TEA(100µM)   6   1.3 ± 0.80   7.5 ± 2.9   20.2 ± 5.7   32.0 ± 3.0   52.5 ± 2.6   67.8 ± 2.4   82.0 ± 2.4
  +glycyrrhetinicacids   9   2.2 ± 1.3   5.5 ± 2.8   11.5 ± 5.0   20.5 ± 5.3   35.0 ± 6.0a)   51.1 ±4.3a)   63.3 ± 5.5b)

Table 4: Modulation of endothelium-dependent relaxation induced by quercetin in rat mesenteric artery.

On KCa channel

The L-NAME/indomethacin-resistant (NO/prostaglandinindependent) relaxation induced by quercetin was never affected by 100 μM TEA (Table 4). Increasing TEA concentration to 1 mM tended to be reduced the resistant vasodilation at 10 to 30 μM quercetin, but not significantly. At 100 μM quercetin, TEA (1 mM) was almost identical to the L-NAME/indomethacin-resistant vasodilatation (82.3 ± 2.6, n=8, p<0.001) [50]. Since quercetin stimulates SK channel in rat aorta [19], quercetin would similarly activate SK channel in mesenteric artery.

On endothelium-derived hyperpolarizing factor (EDHF)

Since the contribution to EDHF-related vasorelaxation is told never to observe in aorta [29], the experiments using rat mesenteric artery were performed. The hyperpolarization induced by EDHF makes the membrane stable, and depresses the ionic channel activity related to the vasoconstriction. The quercetin-induced relaxation in the presence of indomethacin and L-NMMA was reduced by high k+ or TEA. Apamin inhibited the quercetin-induced vasorelaxation. These findings strongly demonstrate that the quercetin-induced vasorelaxation is closely involved with EDHF. Therefore, it is possible that quercetin may increases [Ca2+]i, and then facilitates EDHF synthesis as well as NOS in endothelium.

In rat mesenteric artery, quercetin actually produces NO from endothelium. However, endothelium removal attenuated the quercetin-induced vasodilatation to a greater extent than both L-NAME and indomethacin Table 4. This strongly demonstrates that the endothelium of mesenteric artery plays an important role in the quercetin-induced vasodilatation, mainly dependent on EDHF rather than NO. In resistant vessels than aorta, the EDHF-type relaxation is relatively more predominant [29]. ACh causes the EDHF-type relaxation mediated through KCa channels in fetal aorta [51]. L-NAME attenuated (though be a weak blockade) the quercetin-induced vasodilatation. Thus, NO plays a minor role for the quercetin-induced vasodilatation of mesenteric artery. As well, it may be considered that quercetin is responsible for EDHF-type relaxation, which

EDHF hyperpolarizes to dilate smooth muscle cells. Some candidates for EDHF have already been shown: k+ [52,53], epoxyeicosatrienoic acids (EETs) [52,54], and H2O2 derived from endothelium [23]. In our studies, two mechanisms for the EDHF-induced hyperpolarization have been inferred: (1) EDHF hyperpolarizes smooth muscle cells by stimulating KCa channels [13,28], and (2) the EDHF-mediated response is due to gap junctions, which is confirmed by the electrical communications between endothelium and smooth muscle cells [27,52]. Furthermore, the gap junction for EDHF-type relaxation has already identified by using antibody analysis [55,56].

On gap junction

In our experiments, the quercetin-induced relaxation in the presence of indomethacin and L-NAME was not reduced by high TEA (1 mM). Additional pretreatment with 100 μM 18α- or 50 μM 18β-glychrrhetinic acids (18α- and 18β-GAs) significantly inhibited the L-NAME/indomethacin-resistant vasodilatation from 82.0 ± 2.4% (n=6) to 63.3 ± 5.5% (n=9, p<0.01) (Table 4). 18α- and 18β-GAs are the selective blockers of gap junctions [57]. Endothelium removal also attenuated the quercetin-induced vasodilatation to the same extent as the presence of the inhibitors such as L-NAME, indomethacin, and 18α- and 18β-GAs. So, these findings indicate that the gap junctions contribute to the quercetin-induced vasodilatation. Both 18α- and 18β-GAs reduced the quercetin-induced vasodilatation to a greater extent, as compared with the effect of TEA.

The discrepancy between the results of TEA and 18α- and 18β-GAs might be due to both mechanisms: (1) quercetin elevates endothelial [Ca2+]i and then, activates KCa channels, and (2) quercetin itself has a stimulatory actions on KCa channels. The elevation of [Ca2+]i induced by quercetin activates SK channels [19] to hyperpolarize the endothelium [58,59]. Quercetin would activate SK channel in endothelium via both mechanisms. Thus, TEA failed to inhibit KCa channels both mechanisms, the transduction of the endothelial hyperpolarization to smooth muscle cells is blocked by 18α- and 18β-GAs, and also the endothelial removal. Therefore, the gap junctions involved with EDHF would be responsible for the quercetin-induced vasodilatation [50].

Summary

Rat mesenteric artery possesses almost the similar characteristics to the aorta (Figure 2); (1) quercetin caused a concentrationdependent vasodilatation, (2) the quercetin-induced vasodilation was attenuated by L-NAME, (3) the addition of indomethacin did not cause further modifications, (4) TEA (1 mM) tended to reduce L-NAME/ indomethacin-resistant vasodilatation, (5) at high concentrations (100 μM) of quercetin, TEA failed to produce any effects on L-NAME/ indomethacin-resistant vasodilatation, (6) L-NAME/indomethacinresistant vasodilatation was reduced by 18α- and 18β-GAs, (7) endothelium removal strongly attenuated the quercetin-induced vasodilatation, and (8) 18α- and 18β-GAs reduced the quercetininduced vasodilatation to the same significant extent as the endothelium removal.

medicinal-chemistry-induced-vasorelaxation

Figure 2: Diagram for the mechanism of quecetin-induced vasorelaxation. Quercetin induces [Ca2+]i elevation, leading to NO production and KCa channel activation in endothelium cell. The hyperpolarization induced by the activation of KCa channel inhibits Ca2+ channel in smooth muscle cell. Moreover, modulation of gap junction aggravates the vasodilatation.

The vasodilatation in rat mesenteric artery is also endotheliumdependent. Quercetin produces NO in endothelium, but plays minor role in the quercetin-induced vasodilatation. The vasodilatation induced by quercetin is mainly due to the gap junctions, closely involved with EDHF.

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