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CARDIOVASCULAR

Mechanism of Myricetin Stimulation of Vascular L-type Ca²⁺ Current

Dipartimento di Scienze Biomediche, Università degli Studi di Siena, Siena, Italy

Received November 4, 2004; accepted January 20, 2005.

	Abstract

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An in-depth analysis of the mechanism of the L-type Ca²⁺ current [I_Ca(L)] stimulation induced by myricetin was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Myricetin increased I_Ca(L) in a frequency-, concentration-, and voltage-dependent manner. At holding potentials (V_h) of –50 and –90 mV, the pEC₅₀ values were 4.9 ± 0.1 and 4.2 ± 0.1, respectively; the latter corresponded to the drug-apparent dissociation constant for resting channels, K_R, of 67.6 µM. Myricetin shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction but did not modify the threshold for I_Ca(L) or the T-type Ca²⁺ current. The Ca²⁺ channel blockers nifedipine, verapamil, and diltiazem antagonized I_Ca(L) in the presence of myricetin. Myricetin increased the time to peak of I_Ca(L) in a voltage- and concentration-dependent manner. Washout reverted myricetin effect on both current kinetics and amplitude at V_h of –90 mV while reverting only current kinetics at V_h of –50 mV. At the latter V_h, myricetin shifted the voltage dependence of inactivation and activation curves to more negative potentials by 6.4 and 13.0 mV, respectively, in the mid-potential of the curves. At V_h of –90 mV, myricetin shifted, in a concentration-dependent manner, the voltage dependence of the inactivation curve to more negative potentials with an apparent dissociation constant for inactivated channels (K_I) of 13.8 µM. Myricetin induced a frequency- and V_h-dependent block of I_Ca(L). In conclusion, myricetin behaves as an L-type Ca²⁺ channel agonist that stabilizes the channel in its inactivated state.

Red wine extracts, which are very rich in flavonoids, in bovine aortic endothelial cells, strongly inhibit the synthesis of endothelin-1 (Corder et al., 2001), a vasoactive peptide that is linked to the development of coronary arteriosclerosis. Oral administration of red wine polyphenolic compounds has recently been shown to produce antihypertensive effects in both spontaneously hypertensive (Duarte et al., 2001) and deoxycorticosterone acetate-salt-hypertensive rats (Galisteo et al., 2004a,b), as well as to decrease blood pressure in normotensive rats (Diebolt et al., 2001). In addition, red wine polyphenols increase nitric oxide production in rat aorta (Benito et al., 2002) via the intracellular Ca²⁺ increase in endothelial cells and activation of tyrosine kinases (Martin et al., 2002). These findings help to explain the French paradox (Renaud and de Lorgeril, 1992) and the relationship between the Mediterranean diet based primarily on flavonoid-rich foods (Allium, Petroselinum, and Brassica vegetables and red wine) and the increased longevity (Orgogozo et al., 1997), accompanied by a low incidence of cardiovascular diseases, in Mediterranean populations (Hertog et al., 1995).

The beneficial effect of flavonoids on human health, although highlighted in some prospective epidemiological studies, is not fully established (Hertog et al., 1997). Dietary flavonoids are found in human plasma in sub-micromolar or micromolar concentrations, either as aglycon or as glucosilate/glycosilate metabolites (Paganga and Rice-Evans, 1997; Erlund et al., 2000; Scalbert and Williamson, 2000; Graefe et al., 2001), and some studies have clarified the molecular mechanisms of their effects on vascular tissues. Quercetin (3,3',4',5,7-pentahydroxyflavone), although inducing both endothelium-dependent (Fusi et al., 2003b) and endothelium-independent vasorelaxation in vitro (Duarte et al., 1993; Herrera et al., 1996), has been demonstrated to be an effective stimulator of vascular smooth muscle L-type Ca²⁺ channels (Saponara et al., 2002). This effect, however, is paradoxically accompanied by vasorelaxation, which presumably takes place via pathways [e.g., protein kinase C inhibition (see Duarte et al., 1993), among others] more prominent than L-type channel-mediated Ca²⁺ influx in the hierarchy of functional competences (Fusi et al., 2003b). Myricetin (3,3',4',5,5',7-hexahydroxyflavone) (Fig. 1a) was recently shown to increase I_Ca(L) in rat tail artery myocytes and cause contraction in 20 mM KCl-depolarized rat aorta rings without interacting with the dihydropyridine binding site (Fusi et al., 2003a). The present study provides an in-depth analysis of the L-type Ca²⁺ channel stimulation performed by myricetin on isolated rat tail artery myocytes.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Molecular structure of myricetin (3,3',4',5,5',7-hexahydroxyflavone).

	Materials and Methods

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Cell Isolation Procedure. This study has been carried out in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Male Sprague-Dawley rats (350–450 g; Charles River Italia, Calco, Italy) were anesthetized with a mixture of Ketavet (Gellini, Italy) and Rompum (Bayer, AG, Wuppertal, Germany), decapitated, and bled. Smooth muscle cells, freshly isolated from the tail main artery by means of collagenase treatment in the presence of bovine serum albumin and trypsin inhibitor as previously described (Fusi et al., 2001), exhibited an ellipsoid form (10–15 µm in width, 35–55 µm in length). The cells were continuously superfused with external solution containing 0.1 mM Ca²⁺ (PSS; see below for composition) by using an LKB 2132 peristaltic pump at a flow rate of 500 µl min^–1. At the end of each experiment, both the perfusion system and bath were thoroughly rinsed once with ethanol and several times with water as well as daily with H₂O₂ to avoid contamination of any source. Electrophysiological responses were tested at room temperature (22–24°C) only in those cells that were phase-dense. Cell membrane capacitance averaged 47.9 ± 1.2 pF (n = 71). The time constant of the voltage-clamp averaged 0.7 ± 0.1 ms (n = 71).

Whole-Cell Patch Clamp Recording. Conventional (Hamill et al., 1981) whole-cell patch-clamp method was employed to voltage-clamp smooth muscle cells. Recording electrodes were pulled from borosilicate glass capillaries (WPI, Berlin, Germany) and fire-polished to obtain a pipette resistance of 2 to 5 M when filled with internal solution. A low-noise, high-performance Axopatch 200B (Axon Instruments Inc., Union City, CA) patch-clamp amplifier, driven by an IBM computer in conjunction with an A/D, D/A board (DigiData 1200 A/B series interface; Axon Instruments Inc.), was used to generate and apply voltage pulses to the clamped cells and record the corresponding membrane currents. Current signals, after compensation for whole-cell capacitance, series resistance, and liquid junction potential, were low-pass filtered at 1 kHz and digitized at 3 kHz prior to being stored on the computer hard disk. I_Ca was always recorded in 5 mM Ca²⁺-containing PSS; this concentration was shown to cause maximal peak current in arterial smooth muscle cells (Bolton et al., 1988).

I_Ca was measured over a range of test potentials of 250-ms duration from –50 to 70 mV from a holding potential (V_h) of –50 or –90 mV. Data were collected once the current amplitude had been stabilized (usually 7–10 min after the whole-cell configuration had been obtained). I_Ca did not run down during the following 30 to 40 min under these conditions. To study the effect of some L-type Ca²⁺ channel antagonists (nifedipine, diltiazem, and verapamil) on I_Ca(L) recorded in the presence of myricetin, myocytes were preincubated with the flavonoid, and after a stable response had been attained, cumulative concentrations of the antagonists were added to the bath solution.

A two-pulse protocol was applied to record T-type Ca²⁺ currents [I_Ca(T)], avoiding overlap of I_Ca(L). The cell was first depolarized for a 250-ms period to –40 mV from V_h of –90 mV to elicit I_Ca(T). Following a 6-s return to the initial V_h, a 250-ms clamp pulse to 10 mV was applied to record I_Ca(L), which was taken as 100%.

Steady-state inactivation curves, recorded twice from the same cell (in the absence and presence of the drug, respectively), were obtained using a double-pulse protocol (Rubart et al., 1996). Once various levels of the conditioning potential had been applied for 5 s, followed by a short (5-ms) return to the V_h, a test pulse (250 ms) to 0 mV was delivered to evoke the current. Under control conditions, the 50% inactivation potential, evaluated by fitting a Boltzmann distribution to the first curve, was not significantly different from that of the second curve recorded after 10 min (Fusi et al., 2002).

Activation curves were derived from the current-voltage relationships (see Fig. 2c). Conductance (G) was calculated from the equation G = I_Ca/(E_m–E_Ca), where I_Ca is the peak current elicited by depolarizing test pulses from –50 to 30 mV from V_h of –50 mV, E_m is the membrane potential, and E_Ca is the equilibrium potential for Ca²⁺ (181 mV, as estimated with the Nernst equation). G_max is the maximal Ca²⁺ conductance (calculated at potentials 10 mV). The ratio G/G_max was plotted against the membrane potential and fitted with the Boltzmann equation.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Myricetin stimulation of ICa(L) in rat tail artery myocytes. a, concentration-effect relationship at various Vh. On the ordinate scale, response is reported as percentage of control. The curves show the best fit of the points (pEC50 = 4.9 ± 0.1, nH = 1.1, at Vh of –50 mV, and 4.2 ± 0.1, nH = 1.1, at Vh of –90 mV, respectively). Data points are means ± S.E.M. (n = 3–6). Inset, original recordings of conventional whole-cell ICa(L) elicited with 250-ms clamp pulses to 0 mV from Vh of –90 mV (see schematic diagram), measured in absence (control) or presence of 100 µM myricetin. b, time course of ICa(L) stimulation induced by myricetin at various Vh. Myricetin (20 µM) was applied at the time indicated by the arrow, and peak currents were recorded during a typical depolarization from either –50 or –90 mV to 0 mV, applied every 30 s (0.033 Hz) and subsequently normalized according to the current recorded just prior to myricetin application. Drug washout gives rise either to partial recovery at Vh of –50 mV or to full recovery at Vh of –90 mV from the stimulation. ICa(L) suppression by 300 µMCd2+ is also shown. Data points are mean ± S.E.M. (n = 6).

K⁺ currents were blocked with 30 mM tetraethylammonium chloride in the PSS and Cs⁺ in the internal solution (see below). Current values were corrected for leakage using 300 µM Cd²⁺, which was assumed to completely block I_Ca.

Solutions and Chemicals. PSS contained 130 mM NaCl, 5.6 mM KCl, 10 mM HEPES, 20 mM glucose, 1.2 mM MgCl₂, 5 mM sodium pyruvate, and 0.1 or 5 mM CaCl₂ (pH 7.4). The internal solution for the conventional whole-cell method (pCa 8.4) consisted of 105 mM CsCl, 10 mM HEPES, 11 mM EGTA, 2 mM MgCl₂, 1 mM CaCl₂, 5 mM sodium pyruvate, 5 mM succinic acid, 5 mM oxalacetic acid, 3 mM Na₂ATP, and 5 mM phosphocreatine; pH was adjusted to 7.4 with CsOH. The osmolarity of PSS (320 mOsmol) and that of the internal solution (290 mOsmol) (Stansfeld and Mathie, 1993) were measured with an Osmostat OM 6020 osmometer (Menarini Diagnostics, Florence, Italy).

The chemicals used were collagenase (type XI), tetraethylammonium chloride, bovine serum albumin, trypsin inhibitor, nifedipine, diltiazem, verapamil, CdCl₂, and myricetin (Sigma-Aldrich, Milan, Italy). Myricetin dissolved directly in dimethylsulphoxide and nifedipine dissolved in ethanol were diluted at least 1000 times in PSS prior to use. The resulting concentrations of dimethylsulphoxide and ethanol (below 0.1%) failed to alter the current (data not shown). Final drug concentrations are reported in the text. Unless otherwise stated, 20 µM myricetin was always used. Following control measurements, each cell was exposed to a drug by flushing through the experimental chamber PSS containing that drug.

Statistical Analysis. Acquisition and analysis of data were accomplished by using pClamp 8.2.0.232 [EC] software (Axon Instruments Inc.) and GraphPad Prism version 3.03 (GraphPad Software Inc., San Diego, CA). Data are reported as mean ± S.E.M.; n is the number of cells analyzed (indicated in parentheses), isolated from at least three animals. Statistical analyses and significance as measured by either analysis of variance (followed by Dunnett's post test) or Student's t test for unpaired and paired samples (two-tail) were obtained using GraphPad InStat version 3.05 (GraphPad Software Inc.). In all comparisons, p < 0.05 was considered significant. The current-voltage relationships were calculated on the basis of the peak values (leakage corrected) from the original currents. I_Ca(L) activation was analyzed by measuring the time to peak current or the activation time constant ().

The pharmacological response to myricetin, described in terms of pEC₅₀ (the negative logarithm to base 10 of the molar EC₅₀), was evaluated by global nonlinear regression (Motulsky and Christopoulolos, 2003), assuming that the drug did not affect the maximum of the concentration-response curve in cells clamped at V_h of –90 mV (Fig. 2a). The apparent dissociation constant for resting channels (K_R) was determined by holding the cells at –90 mV and measuring the corresponding concentration-response relationship for myricetin at 0.033 Hz. In fact, the number of channels in the resting state can be assayed by the I_Ca(L) elicited by a large depolarization from hyperpolarized V_h (Bean, 1984), such as –90 mV, where the entire population of L-type Ca²⁺ channels in rat tail artery myocytes is in the resting state (see "Effects of Myricetin on Steady-State Inactivation and Activation Curves for I_Ca(L)"), ready to open in response to depolarization.

	Results

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Effects of Myricetin on I_Ca(L) and I_Ca(T). Figure 2a (inset) shows a typical recording of I_Ca(L) elicited with a clamp pulse to 0 mV from V_h of –90 mV under control conditions and after the addition of 100 µM myricetin. Myricetin enhanced I_Ca(L) in a concentration-dependent manner (Fig. 2a) with an estimated pEC₅₀ of 4.2 ± 0.1 (n = 3–6) that corresponded to a K_R of 67.6 µM. Myricetin enhanced I_Ca(L) with a potency that depended upon V_h. In fact, when V_h was held at –50 mV, the concentration-dependent curve was shifted to the left (pEC₅₀ of 4.9 ± 0.1, n = 3–6; p < 0.001, Student's t test for unpaired samples).

Figure 2b shows the time course of the effects of myricetin on the current recorded at 0.033 Hz from V_h of either –50 or –90 mV to a test potential of 0 mV. After I_Ca(L) had reached steady values, the addition to bath solution of myricetin produced a gradual increase of the current that reached a plateau in about 3 min at all myricetin concentrations used (data not shown). Noticeably, myricetin-induced stimulation of I_Ca(L) was only partially reversible upon drug washout at V_h of –50 mV but completely reversible at V_h of –90 mV. At 2 Hz, under control conditions, the peak amplitude of I_Ca(L) decreased rapidly to a steady-state value of about 70% of that recorded during the first pulse (data not shown). The addition of myricetin just after the first pulse of the second train of pulses caused a similar decrease in peak amplitude of I_Ca(L) followed by a partial recovery to a steady-state value of about 83% of that recorded during the first pulse (data not shown).

The current-voltage relationship in the conventional whole-cell configuration (Fig. 3) shows that myricetin significantly increased the peak inward current in the range between –30 and 10 mV and shifted the apparent maximum by 10 mV in the hyperpolarizing direction without, however, varying the threshold at about –30 mV.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Effect of myricetin on ICa(L)-voltage relationship in rat tail artery myocytes. a and b, original recordings of conventional whole-cell ICa(L) elicited with 250-ms clamp pulses from Vh of –50 mV to test potentials in the range of –50 to 70 mV (see schematic diagram), measured in absence (control) or presence of 20 µM myricetin. c, current-voltage relationships constructed prior to the addition of myricetin (control) and in the presence of myricetin. Data points are mean ± S.E.M. (n = 5). *, p < 0.05; **, p < 0.01; Student's t test for paired samples.

Myocytes used herein express both T- and L-type Ca²⁺ channels (see Petkov et al., 2001). I_Ca recorded at –40 mV from V_h of –90 mV was taken as an indicator of I_Ca(T). This current, in fact, was no longer evident when V_h was –50 mV (data not shown). As shown in Fig. 4, b and c, myricetin still increased I_Ca(L) at 10 mV, although with a lower efficacy compared with V_h of –50 mV (see Fig. 3c). On the contrary, I_Ca(T) was not significantly modified by myricetin (Fig. 4, a and c), even at a concentration of 100 µM (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effect of myricetin on ICa(T) in rat tail artery myocytes. a and b, original recordings of conventional whole-cell ICa elicited with 250-ms clamp pulses to –40 mV (a) and 10 mV (b) from Vh of –90 mV (6-s interpulse; see schematic diagram) measured in the absence (control) or presence of 20 µM myricetin. The two traces in a are superimposed. c, currents measured during the depolarizing pulse to both –40 and 10 mV from Vh of –90 mV were expressed as a percentage of the current evoked by a clamp pulse to 10 mV under control conditions. Each column with the relative bar represents the mean ± S.E.M. (n = 4).

Inhibition by Some L-Type Ca²⁺ Channel Blockers of I_Ca(L) Recorded in the Presence of Myricetin. The antagonistic effects of nifedipine, diltiazem, and verapamil were determined after a stable response to myricetin at V_h of –50 mV was attained. All three Ca²⁺ channel blockers antagonized in a concentration-dependent manner and fully blocked I_Ca(L) recorded in the presence of myricetin (Fig. 5), showing pIC₅₀ values of 8.0 ± 0.2 (nifedipine, n = 5), 5.7 ± 0.1 (diltiazem, n = 5), and 6.0 ± 0.1 (verapamil, n = 5), respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Effects of nifedipine, diltiazem, and verapamil on ICa(L) amplitude recorded in presence of myricetin in rat tail artery myocytes. The amplitude of the current recorded during a typical depolarization from –50 to 0 mV in the presence of myricetin was normalized as 100%. Data points are mean ± S.E.M. (n = 5).

Effects of Myricetin on I_Ca(L) Kinetics. The current evoked at 0 mV from V_h of either –50 or –90 mV activated and then declined with a time course that could be fitted by a monoexponential equation, with a of activation of 3.0 ± 0.3 and 2.02 ± 0.1 ms and a of inactivation of 109.9 ± 9.5 (n = 5) and 85.9 ± 4.2 ms (n = 6), respectively. In the presence of myricetin, activation was delayed, and its time course could be fitted by a biexponential equation at V_h of –50 mV (₁ = 3.4 ± 0.6 and ₂ = 18.9 ± 2.9 ms) and by a monoexponential equation at V_h of –90 mV ( = 3.6 ± 0.5 ms; p < 0.01, Student's t test for paired samples), respectively. This delay in activation, however, markedly reduced the length of the inactivating segment of traces at V_h of –50 mV, thus impeding its fitting; that at V_h of –90 mV increased to 199.3 ± 10.7 ms (p < 0.01). The effect of myricetin on the current kinetics was reversible upon washout, and both of activation and of inactivation returned to control values (3.0 ± 0.4 and 114.2 ± 14.6 ms at V_h of –50 mV, and 2.6 ± 0.2 and 96.4 ± 10.3 ms at V_h of –90 mV, respectively). Myricetin prolonged the time to peak of I_Ca(L) in a voltage-(Fig. 6a) and concentration-dependent manner (Fig. 6b), the latter showing a bell-shaped pattern.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effects of myricetin on time to peak for ICa(L) activation in rat tail artery myocytes. a, time to peak for activation, measured at various membrane potentials, in the absence (control) or presence of myricetin. Columns are mean ± S.E.M. (n = 5). *, p < 0.05, **, p < 0.01, ***, p < 0.001, Student's t test for paired samples. b, time to peak for activation, measured in the absence (control) or presence of different concentrations of myricetin (0.1–100 µM). Columns and relative bars represent mean ± S.E.M. (n = 3–7). **, p < 0.01, Dunnett's post test. Inset, original recordings of conventional whole-cell ICa(L) elicited with 250-ms clamp pulses to 0 mV from Vh of –50 mV, measured in the absence (control) or presence of myricetin.

Effects of Myricetin on Steady-State Inactivation and Activation Curves for I_Ca(L). The voltage dependence of myricetin stimulation was assessed by determining the steady-state inactivation and activation curves for I_Ca(L). At V_h of –50 mV, myricetin significantly shifted the steady-state inactivation curve to more negative potentials (Fig. 7a). The 50% inactivation potentials, evaluated by means of the Boltzmann fitting procedure, were –24.2 ± 1.9 (control) and –30.7 ± 1.5 mV (myricetin; n = 5; p < 0.01, Student's t test for paired samples), respectively. Furthermore, the slope was significantly steeper in the presence of myricetin (slope factor = –8.1 ± 0.7 mV, control and –6.4 ± 0.7 mV, myricetin; p < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of myricetin on both ICa(L) activation and inactivation curves in rat tail artery myocytes. a, steady-state inactivation curves recorded from Vh of –50 mV, obtained in the absence (control) and presence of myricetin, were fitted to the Boltzmann equation. Peak current values were used. Steady-state inactivation curves were obtained using the double-pulse protocol (see Materials and Methods). The current measured during the test pulse is plotted against membrane potential and expressed as relative amplitude. Activation curves were obtained from the current-voltage relationships of Fig. 3c and fitted to the Boltzmann equation (see Materials and Methods). Data point represents the mean ± S.E.M. (n = 5). b, effect of various concentrations of myricetin (3–100 µM) on 50% inactivation potentials obtained from steady-state inactivation curves recorded from Vh of –90 mV, in the absence (control) and presence of myricetin, and fitted to the Boltzmann equation. Columns are mean ± S.E.M. (n = 3–11). **, p < 0.01; Dunnett's post test.

The activation curves calculated from the current-voltage relationships in Fig. 3c were fitted with the Boltzmann equation (Fig. 7a). Myricetin reduced both the 50% activation potential from –2.5 ± 0.7 to –13.6 ± 0.6 mV (n = 5; p < 0.001, Student's t test for paired samples) and the slope factor from 6.6 ± 0.1 mV to 6.1 ± 0.2 mV (p > 0.05).

The apparent dissociation constant of myricetin for inactivated channels (K_I) was determined by the shift of Ca²⁺ channel steady-state availability as a function of myricetin concentration at V_h of –90 mV (Bean et al., 1983). Myricetin shifted the 50% inactivation potential to more hyperpolarizing values in a concentration-dependent manner (Fig. 7b); however, it did not modify the slope factors (–6.7 ± 0.3 mV, control, n = 11; –6.3 ± 0.6 mV, 3 µM myricetin, n = 3; –5.9 ± 0.4 mV, 10 µM myricetin, n = 3; –6.7 ± 0.1 mV, 20 µM myricetin, n = 3; –6.4 ± 0.2 mV, 100 µM myricetin, n = 4). K_I was estimated by plotting the 50% inactivation potentials as a function of myricetin concentration. This relationship fitted the equation V₅₀ = K_control x ln{1/[1 + ([drug]/K_I)]} + V_{50 control}, where V_{50 control} and K_control were the values of the 50% inactivation potential and the slope measured in control conditions, respectively. The value of K_I thus determined was 13.8 ± 2.7 µM.

Frequency-Dependent Block of I_Ca(L) by Myricetin. The frequency dependence of myricetin-induced effects on I_Ca(L) was assessed, as shown in Fig. 8. In the absence of myricetin, 20 depolarizing pulses of 50-ms duration to 0 mV from V_h of –50 mV were applied at different pulse intervals, ranging from 0.5 to 2 s (2, 1, and 0.5 Hz) (Fig. 8a). At the end of the protocol, myricetin was added to the bath solution, and after a 4-min interval without stimulation, the same protocol was repeated. In control conditions, the peak amplitude of I_Ca(L) evoked by the 20th pulse decreased significantly as the frequency of stimulation increased (Fig. 8b, inset), thus suggesting a cumulative channel inactivation. Externally applied myricetin produced a frequency-dependent block of I_Ca(L), the highest frequency causing the greatest inhibition. The frequency-dependent block, calculated by normalizing the current amplitude evoked by the 20th applied stimulus against that induced by the first step-pulse, was significantly lower than that observed under the corresponding control conditions only at 2 Hz (Fig. 8b, inset). Similar results were obtained in parallel experiments wherein Ba²⁺ was substituted for Ca²⁺ as charge carrier (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Tonic stimulation and frequency-dependent block of ICa(L) by myricetin in rat tail artery myocytes. Tonic and frequency dependence of myricetin-induced effects were examined in the range 0.5 to 2 Hz. In the absence of myricetin, 20 depolarizing 50-ms clamp pulses to 0 mV from Vh of either –50 mV (a) or –90 mV (b) were applied at 0.5, 1, or 2 Hz, respectively. Myricetin was added just after the delivery of the first train of pulses, and after a 4-min pause, the same protocol was repeated. The peak amplitude elicited by the first pulse at each frequency in the absence of myricetin was taken as 100%. Inset, peak amplitude elicited by the first pulse of each train (either in the absence or in presence of myricetin) was taken as 100% to better appreciate frequency-dependent block of ICa(L). Data points are mean ± S.E.M. (n = 3). *, p < 0.05 versus the 20th pulse recorded in control condition at 2 Hz; , p < 0.05 versus the 20th pulse recorded in control condition at 0.5 Hz; , p < 0.001 versus the 20th pulse recorded in presence of myricetin at 2 Hz; Student's t test for paired samples or Bonferroni post test, respectively.

The frequency-dependent block of I_Ca(L) by myricetin depended upon V_h (Fig. 8b). In fact, the residual current recorded at the 20th applied stimulus (2 Hz) from V_h of –50 mV was 57.9 ± 3.4% (n = 3), whereas that from V_h of –90 mV was 91.8 ± 1.2% (n = 3, p < 0.001, Student's t test for unpaired samples) (Fig. 8b, inset).

	Discussion

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The present study provides an in-depth analysis of myricetin-induced stimulation of vascular smooth muscle L-type Ca²⁺ channels. This flavonoid acted on L-type, but not T-type Ca²⁺ channels, in a voltage-, concentration-, and frequency-dependent manner. A characteristic of myricetin-induced enhancement of I_Ca(L) was a marked tonic effect, which developed independently of channel activation. In fact, at 0.033 Hz, a frequency allowing full recovery of rat tail artery myocytes Ca²⁺ channels from inactivation (Saponara et al., 2002), the amplitude of I_Ca(L) was increased by myricetin in a way that also depended on V_h. Furthermore, the amplitude of the first I_Ca(L), recorded after a 4-min silent interval subsequent to myricetin addition, increased to a value identical to that obtained gradually in cells stimulated at a frequency of 0.033 Hz for 4 min in the presence of myricetin. This suggests that myricetin does not need an open channel to have access to its putative binding site and display its stimulatory activity (tonic stimulation).

Myricetin induced a peak current enhancement that reached maximum values at weak depolarization conditions, becoming progressively smaller by increasing membrane depolarization, and shifted the maximum of the current-voltage relationship toward more negative potentials without affecting the threshold for I_Ca(L). Thus, the effect of myricetin on the current-voltage relationship might be explained, at least in part, by the activation curve shift toward more hyperpolarizing potentials, which was more pronounced than that observed in the inactivation curve. In addition, myricetin caused a significant change in the slope of the inactivation curve. These results indicate that myricetin may alter the voltage sensitivity of the channel inactivation mechanism. Furthermore, myricetin strongly slowed Ca²⁺ channel activation kinetics in a concentration-dependent manner as well as over a wide range of membrane potentials, although this effect was more pronounced at weak depolarization values. The fact that myricetin slowed the time course of current activation while also shifting the steady-state activation curve to more negative potentials can be explained assuming that the flavonoid, in addition to affecting the voltage dependence of the channels, also interferes with Ca²⁺-channel gating kinetics, slowing (i.e., increasing the rate constants of) the transition from the closed to the open state of the channel. In other ways, it modifies the gating mechanisms. These observations demonstrate that the two effects depend on myricetin occupancy of binding sites at least partly distinct. A similar phenomenon has been recently described for a series of divalent cations (Castelli et al., 2003). Finally, the inward tail currents decayed more slowly in the presence of myricetin (data not shown), possibly as a consequence of the longer-lasting opening of the channel.

Together, all these elements indicate that myricetin shares several basic features with some Ca²⁺ channel agonists such as Bay K 8644 (Wang et al., 1989; Fusi et al., 2003a) and the structurally related flavonoid quercetin (see below; Saponara et al., 2002).

Another feature of myricetin action is the frequency-dependent block of I_Ca(L). Frequency-dependent block of I_Ca(L) and shift of the channel availability toward more hyperpolarizing potentials are generally observed with drugs possessing a greater affinity for the channel in its inactivated state. In fact, myricetin, like verapamil (McDonald et al., 1994), inhibited I_Ca(L) in a frequency-dependent fashion (use-dependent block); i.e., the repetitive depolarization at a relatively high frequency of stimulation potentiated the inhibition of I_Ca(L). Noticeably, this phenomenon was also observed in the presence of Ba²⁺ as charge carrier. Therefore, a direct drug effect, rather than a Ca²⁺-dependent inactivation of the channel subsequent to current stimulation, seems to be implicated. Furthermore, myricetin, like nicardipine (Bean 1984), shifted the voltage dependence of the inactivation curve to more negative potentials. Together, these observations indicate that myricetin stabilizes L-type Ca²⁺ channels in their inactivated state (Bean, 1984) in a voltage-dependent manner. Moreover, its apparent dissociation constant for the inactivated channel (K_I) was about 5 times lower than that for the closed channel (K_R), thus providing an explanation for the shift of the concentration-response curve for I_Ca(L) stimulation, recorded at two different V_h, toward higher drug concentrations. In fact, there was a significant shift of the curve to the right at V_h of –90 mV, i.e., at a greater membrane hyperpolarization, where a smaller number of channels are supposed to be in the inactivated state. Interestingly, the effects of myricetin on both I_Ca(L) kinetics (activation and inactivation) and the slope of steady-state inactivation curve were also affected by V_h, being less pronounced when cell membrane potential was held at –90 mV compared with –50 mV. The reduced drug sensitivity at V_h of –90 mV further supports the hypothesis of a higher affinity of myricetin for the channel in the inactivated state.

Several studies demonstrate how, under certain conditions, drugs affecting L-type Ca²⁺ channels (e.g., dihydropyridines, phenylalkylamines, and benzothiazepines) can have opposite effects on I_Ca(L), i.e., inhibition if activators and activation if blockers (McDonald et al., 1994). Some conditions promote the block of L-type Ca²⁺ channels by dihydropyridine agonists such as Bay K 8644, in particular, high drug concentrations, depolarized V_h, and high pulsing rates. Although the first of these conditions could not be evaluated in the present study because of solubility limits of myricetin, the latter two applied well to the flavonoid. In fact, pulsing rates higher than 1 Hz converted the agonist myricetin into a blocking agent, whereas depolarized V_h favored the progressive accumulation of this block when the interstimulus interval was short. Under these experimental conditions, the blocking activity of the L-type Ca²⁺ channel agonist myricetin clearly emerged.

It might be argued that myricetin effects on I_Ca(L) are not due only to its action on L-type Ca²⁺ channels, but rather the consequence of the recruitment of a third type of Ca²⁺ channel, i.e., N-type Ca²⁺ channel. However, the observed, complete block of the current induced by the three well known L-type Ca²⁺ channel blockers nifedipine, diltiazem, and verapamil in cells exposed to myricetin did not substantiate this hypothesis.

Previous studies have shown how quercetin stimulates I_Ca(L) in cells isolated from rat tail artery (Saponara et al., 2002), in clonal rat pituitary GH₄C₁ (Summanen et al., 2001), as well as GH₃ cells, but not in neuronal NG108 –15 cells (Wu et al., 2003). The electrophysiological characteristics of myricetin Ca²⁺ channel agonism described here (degree of current enhancement, time for maximal effect development, stimulation of current-voltage relationship, stabilization of the inactivated state, lack of effects on I_Ca(T), modulation of voltage dependence, and kinetics of the current) are similar to those already observed with quercetin in the same experimental model (Saponara et al., 2002). Myricetin stimulation of I_Ca(L), however, only partially reverted upon washout of the drug at V_h of –50 mV, at variance with the effect on current kinetics; moreover, myricetin enhancement of I_Ca(L) at V_h of –90 mV, as well as that of I_Ba(L) in the same preparation (Fusi et al., 2003a), were fully reversible. It can be hypothesized that a Ca²⁺ ion occupies a high-affinity Ca²⁺ binding site, thus stabilizing the putative myricetin binding site, as is the case with dihydropyridine Ca²⁺ antagonists (Mitterdorfer et al., 1998). This would take place in the inactivated state of the channel, where a single Ca²⁺ ion is tightly coordinated at the selectivity filter, and the putative myricetin binding domain would assume a high-affinity binding conformation for the drug, thus allowing the formation of a ternary complex. This complex is assumed to stabilize the channel in its inactivated state. Therefore, the replacement of Ca²⁺ with Ba²⁺ as a charge carrier, as well as the decrease in proportion of inactivated channels at more hyperpolarized V_h, should turn the putative myricetin binding domain into a low-affinity binding conformation, thus allowing the complete washout of the drug and the ensuing full reversion of its effects. Of course, this model does not apply to the effects of myricetin on current kinetics that turned to control values even in the presence of Ca²⁺ at V_h of –50 mV, once more supporting the hypothesis that the flavonoid interacts with distinct sites of the channel protein.

In conclusion, the present electrophysiological data point to myricetin as a vascular L-type Ca²⁺ channel agonist characterized by a greater affinity for the channel in the inactivated state. Thus, quercetin and myricetin appear as members of a new class of L-type Ca²⁺ channel modulators of natural source.

	Acknowledgements

 
We thank Dr. Jacopo Magistretti (Università degli Studi di Pavia) for constructive comments on the manuscript. We are also grateful to European Cooperation in the field of Scientific and Technical Research (COST) 926.

	Footnotes

 
This work was supported by grants from the Università degli Studi di Siena (PAR 2002 and 2001 Young Researcher to S.S.) and a grant from the Tuscany Region (Carabiotech 2002).

doi:10.1124/jpet.104.080135.

ABBREVIATIONS: PSS, physiological salt solution; Bay K 8644, (S)-(–)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate.

Address correspondence to: Dr. Fabio Fusi, Dipartimento di Scienze Biomediche, Università degli Studi di Siena, via A. Moro 2, 53100 Siena, Italy. E-mail: fusif{at}unisi.it

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