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CARDIOVASCULAR

Activation of Large-Conductance Calcium-Activated Potassium Channels by Puerarin: The Underlying Mechanism of Puerarin-Mediated Vasodilation

Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, People's Republic of China

Received May 9, 2007; accepted July 24, 2007.

	Abstract

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Puerarin is the main isoflavone found in Pueraria lobata (Willd) Ohwi, which has been used in therapy for various cardiovascular diseases. The present study examined the effects of puerarin on the large-conductance voltage- and Ca²⁺-activated potassium (BK_Ca) channel and on rat thoracic aortas. BK_Ca channels encoded with either (BK-) or / subunits (BK-+1) were heterologously expressed in Xenopus oocytes or human embryonic kidney 293 cells. The activities of BK_Ca channels were measured using excised patch-clamp recordings. Puerarin activated BK-+1 currents with a half-maximal concentration (EC₅₀) of 0.8 nM and a Hill coefficient of 1.11 at 10 µMCa²⁺ and with an EC₅₀ of 12.6 nM and a Hill coefficient of 1.08 at 0 µMCa²⁺. Puerarin (1 nM) induced a 16-mV leftward shift in the conductance-voltage curve for BK-+1 currents at 10 µMCa²⁺ and at 100 nM induced a 26-mV leftward shift at 0 µMCa²⁺. Puerarin mainly increased the BK-+1 channel open probability without changing the unitary conductance. Activation was also detected in the absence of the 1 subunit. A deglycosylated analog of puerarin, daidzein, also activated BK_Ca channels with weaker potency. In addition, puerarin (0.1 to 1000 µM) caused concentration-dependent relaxations of rat thoracic aortic rings contracted with 1 µM noradrenaline bitartrate (EC₅₀ = 1.1 µM). These were significantly inhibited by 50 nM iberiotoxin, a specific blocker of BK_Ca channels. This is the first study demonstrating that puerarin activates BK_Ca channels, especially BK-+1 channels. The activation of the BK_Ca channel probably contributes to the puerarin-mediated vasodilation action.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Activation of mslo+h1 channels coexpressed in Xenopus oocytes by puerarin. A, all traces were recorded in inside-out patches at 10 µM intracellular Ca2+. Top, representative traces under conditions of control, 1 nM puerarin, and washout as indicated. Currents were evoked by voltage steps in the range from–180 to +100 mV for 30 ms in 20-mV steps, after a prepulse to–180 mV as shown at bottom right. Middle, G-V curves from individual patches were normalized and then averaged. The fitted values for the V50 of control and in the presence of 1 nM puerarin are–27 ± 3 and–43 ± 4mV(p < 0.01, n = 6), respectively. Bottom, the voltage dependence of the activation of mslo+h1 currents by 1 nM puerarin at 10 µMCa2+ calculated from the data in the middle panel. *, p < 0.05 versus control, n = 6. B, all traces were recorded in inside-out patches at 0 µM intracellular Ca2+. Top, representative traces are shown under conditions of control, 100 nM puerarin and washout as indicated. Middle, the fitted values for the V50 of control and 100 nM puerarin are 164 ± 6 and 138 ± 11 mV (p < 0.01, n = 5), respectively. The inset shows the chemical structure of puerarin. Bottom, the voltage dependence of the activation of mslo+h1 currents by 100 nM puerarin at 0 µMCa2+ was re-evaluated from the data shown in middle panel. *, p < 0.05 versus control, n = 5.

In this study, we investigated the effects of puerarin on cloned BK_Ca channels and examined the vasodilation effects of puerarin in the presence or absence of the specific BK_Ca channel blocker iberiotoxin (IbTX). These results may help to understand the underlying mechanisms of puerarin-mediated vasodilation.

	Materials and Methods

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Materials. Puerarin and daidzein (7,4'-dihydroxyisoflavone) were purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (purity 99%; Beijing, China). Daidzein was dissolved with dimethyl sulfoxide. The final concentration of dimethyl sulfoxide did not exceed 0.1%, which did not alter the kinetic properties of BK_Ca channels (data not shown). All of the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except where indicated.

Expression of mslo and h1 Channels in Xenopus Oocytes. Methods of expression of mslo and h1 channels in stage V to VI Xenopus oocytes were described previously (Xia et al., 1999; Yao et al., 2005). In brief, oocytes were digested by treatment with 2 mg/ml collagenase I in zero calcium ND-96 solution. Between 2 and 24 h after digestion, 1 to 2 ng (mslo and h1) of cRNA (a gift of Dr. Christopher Lingle, Washington University, St. Louis, MO) was injected into Xenopus oocytes using a Drummond Nanoject II (Drummond Scientific Co., Broomall, PA). After injection, oocytes were then incubated in ND-96 solution at 18°C. Currents were recorded 2 to 7 days after RNA injection. ND-96 solution (pH 7.5) contained the following concentrations: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 2.5 mM sodium pyruvate, and 10 mM H⁺-HEPES. It was supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin for incubation.

Expression of dslo Channels in HEK293 Cells. Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (McCartney et al., 2005). HEK293 cells grew on 24-well plates at a density of 1 x 10⁴/well and were transfected with 0.6 µg of green fluorescent protein/pcDNA3.1 and 0.6 µg of dslo/pcDNA3.1 (a gift of Dr. Christopher Lingle) using Lipofectamine 2000 (Invitrogen). Cells were used for electrophysiological recordings in 1 to 2 days after transfection.

Electrophysiology. Patch pipettes were pulled from borosilicate glass capillaries with a resistance between 2 and 5 megaohms. All experiments in excised patch configurations were performed and recorded using a PC2C patch-clamp amplifier (Inbio Life Science Instrument Co., Ltd., Wuhan City, China) and PClamp software (Molecular Devices, Sunnyvale, CA). Currents were typically digitized at 20 kHz. Macroscopic records were filtered at 10 kHz during digitization. Single-channel records were filtered at 5 kHz and digitized at 10 kHz. For an inside-out patch experiment, the intracellular solution contained 160 mM MeSO₃K, 10 mM H⁺-HEPES, and 2 mM MgCl₂, adjusted to pH 7.0 with methanesulfonic acid (MeSO₃H). The bath solution contained 160 mM MeSO₃K, 10 mM H⁺-HEPES, and 5mM N-hydroxyethylenediaminetriacetic acid with Ca²⁺ added to make 10 µM free Ca²⁺, as defined by the EGTAETC program (E. McCleskey, Vollum Institute, Portland, OR), with the pH adjusted to 7.0.

During recording, drugs and control/wash solutions were puffed locally onto the cell via a puffer pipette containing seven solution channels. The tip (300 µm diameter) of the puffer pipette was located approximately 120 µm from the cell. As determined by conductance tests, the solution around a cell under study was fully controlled by the application solution with a flow rate of 100 µl/min or greater. All experiments were done at room temperature (22–25°C).

Vasoreactivity Measurements. Male Wistar rats (weighing 200 250 g; obtained from the Animal Center, Institute of Health and Epidemic Prevention, Hubei, China) were sacrificed by decapitation under ether anesthesia. The thoracic aortas were excised and cleaned of adherent connective tissue and were cut into 3-mm ring segments. The endothelial layer was removed in some experiments by gently rubbing the intimal surface of the vessels with a hypodermic needle. Conversely, in other cases, the endothelium was maintained. Arterial rings were mounted on two stainless steel hooks in 5-ml organ baths filled with Krebs-Ringer buffer (pH 7.4) of the following composition: 119 mM NaCl, 25 mM NaHCO₃, 11.1 mM glucose, 1.6 mM CaCl₂, 4.7 mM KCl, 1.2 mM KH₂PO₄, and 1.2 mM MgSO₄ and gassed with a mixture of 95% O₂/5% CO₂. The rings were equilibrated for 45 min at +37°C with a resting tension of 1.0 g. During this period, the bathing solution was replaced every 15 min, and if necessary, the basal tone was readjusted to 1.0 g. After the equilibration, the endothelial removal/integrity was confirmed by the administration of acetylcholine (10 µM) to noradrenaline bitartrate (NA; 1 µM)-precontracted vascular rings. A relaxation <10% of the NA-induced contraction was considered the representative of an acceptable lack of the endothelial layer, whereas a relaxation 70% of the NA-induced contraction was considered the representative of an acceptable integrity of the endothelium. Rings were then washed in prewarmed Krebs-Ringer solution until the baseline tone was regained. The rings were then contracted with 1 µM NA to the maximal contraction. Following washout of NA, cumulative concentration-response curves to stepwise cumulative addition of puerarin (0.11000 µM) with or without 50 nM IbTX were established. Each new addition of puerarin was made after the response to the previous addition had attained a steady state. The force of contraction was measured with an isometric force-displacement transducer and registered with a polygraph (RM6240 transducer, RM6240B/C Polygraph; Chengdu Instrument Co., Chengdu, China). Relaxation responses were expressed as a percentage of NA-induced contraction. All animal experiments were approved by the Institutional Animal Care and Use Committee and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication 85-23, revised 1996).

Data Analysis. Data were analyzed with Clampfit (Molecular Devices), SigmaPlot (SPSS Inc., Chicago, IL), and QUB (State University of New York, Buffalo, NY) software. Unless indicated, the data are presented as means ± S.E.M.; statistical significance between two groups and among multiple groups was tested using Student's t test and one-way analysis of variance, respectively. Differences in the mean values were considered significant at a probability of <0.05. Dose-response relationships for puerarin activating BK_Ca channels and relaxing rat thoracic aortic rings were fitted to a Hill equation of the following form: f = f_max/(1 + (EC₅₀ /[puerarin])ⁿ), where f is activation percentage of BK_Ca currents or relaxation percentage of rat thoracic aortic rings, f_max is maximum value of f, and [puerarin] is the concentration of puerarin. EC₅₀ and n denote the puerarin concentration of half-maximal effect and the Hill coefficient, respectively. G-V curves were fitted to a Boltzmann equation of following form: G/G_max = 1/(1 + exp[(V–V₅₀)/k]), where G is the conductance of the channel, G_max is the maximal G, V is the holding potential, V₅₀ is the voltage for half-maximal activation, and k represents the slope factor. G-V curves were generated from steady-state currents. Single-channel analysis was performed using QUB. Single-channel amplitudes were measured by using an all-points histogram of current records. NP_O (the open probability for a multichannel patch) was determined over at least 10 s of recording. NP_O values were calculated from the area under the curve of the Gaussian fit of all-points amplitude histograms. Assuming a Poisson distribution, NP_O = i*X_i, with i = 1... n, where n is the maximum number of simultaneous conducting channels during the observation period, and X_i is the relative area under the curve corresponding to each opening.

	Results

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Puerarin Activating Macroscopic BK-+1 Channel Currents. Macroscopic BK_Ca currents in Xenopus oocytes coexpressing mslo and h1 (mslo+h1) were obtained at different potentials in excised patches. Puerarin does not activate BK_Ca channel currents when applied to the outside of excised cell membrane patches, even at 100 µM concentration (data not shown). However, puerarin potently activated channels when applied to the cytoplasmic side of excised cell membrane in the presence of 0 and 10 µM intracellular Ca²⁺ (Fig. 1). Figure 1A shows the voltage dependence of puerarin-induced enhancement of BK_Ca currents at 10 µMCa²⁺. Application of 1 nM puerarin significantly increased the currents at negative potentials, and the activation was partially reversible. The middle panel summarizes the relationships between conductance and voltage. Puerarin (1 nM) resulted in a 16-mV leftward shift of the V₅₀ of G-V curves. The bottom panel shows the enhancement percentage of BK_Ca currents by puerarin at different potentials. The activation of BK_Ca channel currents by puerarin was markedly inverse voltage-dependent; i.e., there was a larger increase at lower potentials (76.6 ± 13.5 and 27.5 ± 6.1% at–40 and–20 mV, respectively, p < 0.05, versus control, n = 6). Figure 1B summarizes the activation by 100 nM puerarin at 0 µMCa²⁺. Puerarin (100 nM) also stimulated BK_Ca currents and induced a 26-mV leftward shift in the V₅₀ of G-V curves.

Currents of mslo+h1in Xenopus oocytes shown in Fig. 2 were recorded in inside-out patches at–20 and +100 mV with 10 and 0 µM Ca²⁺, respectively. In Fig. 2A, 1 nM puerarin increased BK_Ca currents, and the time courses of the currents, which were activated at various concentrations of puerarin, indicate that the whole process goes in a rapid and partially reversible manner. The dose-response curve is fitted to a Hill equation with an EC₅₀ of 0.8 nM and a Hill coefficient of 1.11. Likewise, puerarin increased the BK_Ca currents at +100 mV and zero Ca²⁺ concentration (Fig. 2B). However, the EC₅₀ and the Hill coefficient derived from the dose-response curve in this case are 12.6 nM and 1.08, respectively, indicating that Ca²⁺ facilitates activation by puerarin.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Concentration dependence of puerarin activation of mslo+h1 currents. A, traces show the activation of BKCa currents coexpressed with mslo and h1 subunits in Xenopus oocytes by 1 nM puerarin in the presence of 10 µM internal Ca2+. Top, example traces show the BKCa currents from an inside-out patch before, during, and after application of 1 nM puerarin at–20 mV. The dashed line represents zero current. Middle, plots show the time course of activation of BKCa currents, which were normalized to control, from four different inside-out patches in the presence of 0.1, 0.5, 1, and 5 nM puerarin at–20 mV. Currents were recorded every 5 s. Bottom, the solid line is a fit to the Hill equation. The EC50 of the dose-response curve of puerarin activation of BKCa currents was 0.8 nM with a Hill coefficient 1.11 (*, p < 0.05 versus control, n = 4). B, the time course and the dose-response curves of puerarin activation of BKCa currents are shown in the middle and bottom panels, at +100 mV and zero intracellular Ca2+. The time course was obtained from an inside-out patch during application of 5, 10, 20, and 100 nM puerarin. Currents were recorded every 15 s. The EC50 value was 12.6 nM with a Hill coefficient 1.08 (*, p < 0.05 versus control, n = 4).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effects of puerarin on single-channel characteristics of BKCa channels coexpressed with mslo and h1 subunits in Xenopus oocytes at 10 µMCa2+. A, single-channel traces were obtained from an inside-out patch before (Control) and after application of 1 nM puerarin. Channels were held at–20 mV in symmetrical 160 KCl solutions. The letters c, o1, and o2 indicate the close, open 1, and open 2 levels, respectively. Records a and b are insets of square section. Each histogram (bottom) illustrates all possible change in open probabilities and unitary conductance after applying puerarin. B, summary of the effect of 1 nM puerarin on NPO (left) and unitary amplitude (right) of single BKCa channels at–20 mV. **, p < 0.01 versus control, n = 6. C, single-channel current-voltage curves are plotted in the absence and presence of 1 nM puerarin (n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Activation by puerarin of BKCa channels in the absence of the 1 subunit at zero Ca2+ concentration. A, top, representative traces of BKCa currents expressed with mslo subunits in Xenopus oocytes were obtained from an inside-out patch before (left) and after (right) application of 1 µM puerarin. The voltage protocol is shown in the inset. Bottom, the V50 of G-V curves for control and 1 µM puerarin are 169 ± 3 and 151 ± 1mV (p < 0.01, n = 5), respectively. B, the concentration-dependent activation curve of mslo obtained from five different patches at + 100 mV. The experimental points were fitted to a Hill function (solid line) with a Hill coefficient of 0.72. The EC50 obtained from the fit was 166.6 nM. *, p < 0.05 versus control, n = 4.

Puerarin Has No Effect on dslo Currents. To estimate the domain of BK_Ca channels that puerarin may bind to, the BK_Ca encoded with dslo subunit expressed in HEK293 cells was used. Puerarin has no effect on the dslo currents at 0 µM Ca²⁺ (data not shown) and 10 µMCa²⁺ (Fig. 5). Therefore, we infer that the possible interaction domain may locate at the S0-S1 and S8-S9 linkers, because the BK_Ca dslo channel contains the above regions significantly different from that of the BK_Ca mslo channel.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Lack of effect of puerarin on BKCa currents expressed with dslo subunits in HEK293 cells. A, example current traces of dslo are shown before (left) and after (right) application of 1 µM puerarin at 10 µM internal Ca2+. Currents were activated at potentials from–180 through +200 mV for 150 ms in 20-mV increments after a prepulse to–180 mV, as shown in inset. B, the V50 of G-V curves are as follows: for control (open symbols), V50 control = 95 ± 6 mV; and for 100 nM puerarin (filled symbols), V50 puerarin = 100 ± 7mV(n = 5). Error bars represent S.E.M.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Activation of mslo currents by daidzein, an analog to puerarin. A, example traces of mslo channels expressed in Xenopus oocytes were obtained from an inside-out patch before (left) and after (right) application of 1 µM daidzein at zero Ca2+. The voltage protocol is shown at the right bottom. B, the V50 of G-V curves are V50 control = 159 ± 9mV (Control, open symbols) and V50 daidzein = 148 ± 5mV(1 µM daidzein, filled symbols) (p < 0.01, n = 5). Error bars represent S.E.M. The chemical structure of daidzein is shown in inset.

Puerarin Relaxing Rat Thoracic Aortic Rings. Puerarin (0.1 to 1000 µM) caused concentration-dependent relaxations in endothelium-intact and endothelium-denuded rat thoracic aortic rings contracted with NA (1 µM) (Fig. 7). The EC₅₀ was 1.1 and 2.2 µM, and the maximal relaxation was 45.9 ± 5.1% (n = 5) and 33.5 ± 4.6% (n = 6) to endothelium-intact and endothelium-denuded aortic rings, respectively, indicating that endothelium was involved in puerarin-induced vasodilation. The specific BK_Ca channel blocker IbTX (50 nM) significantly inhibited puerarin-induced relaxations on both endothelium-intact and endothelium-denuded aortic rings, the fractional inhibition at a puerarin concentration of 1 µM on endothelium-intact and endothelium-denuded aortic rings was 34.0 and 48.0%, respectively, suggesting that the activation of BK_Ca channels also contributes to the puerarin-mediated vasodilation action.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Puerarin-induced relaxation of isolated rat thoracic aortas. Rings were contracted with NA (1 µM) before puerarin (0.1 to 1000 µM) was added cumulatively. Concentration-response curves were obtained on endothelium-intact (circles) or endothelium-denuded (diamonds) aortic rings, in the absence (filled symbols) or in the presence (open symbols) of 50 nM IbTX. *, p < 0.05 versus endothelium-intact group; #, p < 0.05 versus endothelium-denuded group. The inset shows the fractional inhibition of relaxation by 50 nM IbTX at puerarin concentration of 1, 10, and 100 µM on endothelium-intact (filled bars) or endothelium-denuded (open bars) vessels.

	Discussion

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The major findings of this study are as follows. Puerarin potently activated BK-+1 currents in the nanomolar concentration range. It increased the activity of BK-+1 channels with no change in single-channel conductance. Puerarin also stimulated BK- currents with weaker potency. A puerarin analog, daidzein, activated BK- currents with weaker potency than puerarin. Puerarin dilated rat thoracic aortic rings in a concentration-dependent manner, and this relaxation response could be inhibited by IbTX.

Mechanisms of the Effect of Puerarin on BK_Ca Channels. Puerarin potently activated cloned BK-+1 channels, shifting the G-V relationship to the left in cell-free patches. Compared with many other natural BK_Ca channel openers, including BMS-204352 (EC₅₀ 300 nM) (Gribkoff et al., 2001), dehydrosoyasaponin-I (EC₅₀ 100 nM) (Giangiacomo et al., 1998), and mallotoxin (effective concentration 500 nM) (Zakharov et al., 2005), the effective concentration range for puerarin is considerably lower (EC₅₀ values, 0.8 nM at 10 µMCa²⁺ and 12.6 nM at 0 µMCa²⁺). The effect of puerarin on BK_Ca channels is in a voltage- and Ca²⁺-dependent manner. It is very notable that the effect of puerarin is inverse voltage-dependent; i.e., the activation of BK_Ca channels by puerarin is significantly larger at negative potentials, which is similar to another BK_Ca channel opener 12,14-dichlorodehydroabietic acid (Sakamoto et al., 2006). The EC₅₀ of puerarin on BK_Ca channels is a 16-fold augmentation at zero Ca²⁺ compared with at 10 µMCa²⁺, which may imply that Ca²⁺ facilitates the activation of puerarin. On the other side, Ca²⁺ produced a more leftward shift in V₅₀ of BK_Ca channels in the presence of puerarin than that in the absence of puerarin; e.g., the V₅₀ of BK-+1 was–27 ± 3 mV in the absence of 1 nM puerarin but–43 ± 4 mV in the presence of 1 nM puerarin at 10 µMCa²⁺, indicating that puerarin was able to increase the Ca²⁺ sensitivity of BK_Ca channel gating. Single-channel recordings showed that puerarin mainly increased the BK_Ca channel NP_O without changing the unitary conductance. In addition, the Hill coefficients of dose-response curves were approximately unity both at 10 and 0 µMCa²⁺, suggesting only one site for interaction between puerarin and the BK_Ca channel protein.

The Role of the 1 Subunit. Puerarin activating BK_Ca channels was also observed in the absence of the 1 subunit, which indicates that 1 subunit is not required for the activation. However, puerarin has a stronger effect on the BK-+1 channels than the BK- channel alone. In the absence of the 1 subunit, the EC₅₀ of puerarin is greater by 13 times, suggesting that the role of the 1 subunit is probably to facilitate puerarin binding. The 1 subunit is composed of two transmembrane domains, a long extracellular loop, and two short intracellular segments (Knaus et al., 1994), leaving little to construct an intracellular binding site for puerarin. Because puerarin can activate the BK_Ca channels only when applied to the cytoplasmic side of cell membrane, we infer that puerarin-binding sites are located on the intracellular side of the subunits. The N terminus and S0 of the BK- subunit have been supposed to be the possible regions to regulate the gating of BK_Ca channels via interacting with 1 subunits (Wallner et al., 1996). We examined the effect of puerarin on dslo; interestingly, no action was observed. Aligning the sequences of mslo and dslo, we find that there is main difference in two cytoplasmic motifs, i.e., the S0-S1 linker and S8-S9 linker. The sites for puerarin binding to BK_Ca channels may be located in one of these domains. More experiments are needed to determine the precise locations. As we know, both dslo and mslo channels contain the same calcium binding sites in RCK1 (regulator of conductance for K⁺) domain and calcium bowl regions but have very different Ca²⁺ sensitivity (Xia et al., 2002). Furthermore, considering that the S0-S1 and S8-S9 linkers are the major differences in the sequences between dslo and mslo channels, we speculate that they could be the candidates of locations affecting Ca²⁺ sensitivity of BK_Ca channels.

The Function of the Glycosyl Residue of Puerarin. Puerarin is an isoflavone glycoside with a -D-glucopyranoside at 8-position and two hydroxyl groups at 4', 7-positions. The isoflavone nucleus is a rigid and hydrophobic structure, whereas the glycosyl residue and hydroxy group are hydrophilic. Daidzein, an analog of puerarin, has no glycosyl residue and activates BK_Ca channels with weaker potency than puerarin. These results indicate that the -D-glucopyranoside at 8-position plays an important role in puerarin activating BK_Ca channels. Thus, engineering in new BK_Ca channel openers should focus on the modification of the number or location of glycosyl group in the isoflavone nucleus.

Vasodilation and the Activation of BK_Ca Channels Induced by Puerarin. Puerarin caused concentration-dependent relaxations in isolated rat thoracic aortic rings contracted with NA. IbTX, a specific BK_Ca channel blocker, markedly inhibited puerarin-induced relaxations. It is likely, therefore, that the mechanism of this response to puerarin involves opening of BK_Ca channels. In the absence of endothelium, puerarin produced less relaxation than that in the presence of endothelium, indicating that endothelium was also involved in puerarin-mediated vasodilation. However, the inhibition of relaxation by IbTX was not decreased in the absence of endothelium, implying that the contribution of BK_Ca channels to puerarin-mediated vasodilation is independent to the endothelium. It is worth noting that the EC₅₀ of puerarin required to relax aortic rings (1.1 µM) was 1000-fold higher than that required to directly activate BK_Ca channels. This difference is attributed to the fact that puerarin hardly penetrates the cell membrane as mentioned above. However, puerarin could be taken into the cytoplasm through endocytosis. The average diameter of a vesicle is about 140 nm (Zhang et al., 1995), so each vesicle could carry drugs in 3 x 10^–6 of the extracellular concentration into a 10 µm cell in diameter. There are about 200 vesicles taking part in one secretion event (Gillis et al., 1996), and from this we can calculate that the concentration of puerarin in the cytoplasm of vascular smooth muscle cells would be in 6 x 10^–4 of the extracellular concentration, which would be the concentration activating the BK_Ca channels. The plasma concentration of puerarin is in the range of 1 to 1000 µM in humans and animals (Jin et al., 1991; Deng et al., 2004; Wu et al., 2004), so the concentration used in this study is of therapeutic relevance.

In summary, we have provided direct evidences for the activation of cloned BK_Ca channels by puerarin, and shown that BK_Ca channel activation contributes to puerarin-induced vasodilation, which is likely to be a mechanism by which puerarin exerts its action on rat thoracic aortas, in addition to its activation of endothelial nitric-oxide synthase and cAMP pathway, as demonstrated by other investigators (Ma et al., 2003; Yeung et al., 2006). The present findings are of interest for understanding the contribution of BK_Ca openers to lowering blood pressure and improving other cardiovascular symptoms.

	Acknowledgements

 
We thank Dr. Geng Hui, Dr. Tao Yunhai, and Dr. Gao Shangbang for helpful discussions. We thank Prof. Chen Jianguo and Dr. Wang Fang for guidance in vasoreactivity experiments. We thank Prof. John Cram and Prof. He Guangyuan for critical comments on the manuscript.

	Footnotes

 
This work was supported by Grants 30500109 and 30470449 from the Natural Science Foundation of China and the Excellent Doctoral Thesis Research Foundation from Huazhong University of Science and Technology (to S.X.H.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.125567.

ABBREVIATIONS: BK_Ca channel, the large-conductance calcium-activated potassium channel; BK-, the subunit of BK_Ca channels; BK-+1, the and 1 subunit of BK_Ca channels; slo, the subunit of BK_Ca channels; mslo, mouse slo; dslo, drosophila slo; h1, human 1; NA, noradrenaline bitartrate; IbTX, iberiotoxin; HEK, human embryonic kidney; NP_O, the open probability for a multichannel patch; BMS-204352, (3S)-3-(5-chloro-2-methoxyphenyl)-3-fluoro-6-(trifluoromethyl)-1,3-dihydro-2H-indol-2-one.

S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Xiang-Liang Yang, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China. E-mail: yangxl{at}mail.hust.edu.cn

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