Toxicological Sciences

ToxSci Advance Access originally published online on September 8, 2008
Toxicological Sciences 2008 106(2):454-463; doi:10.1093/toxsci/kfn189

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Time-Dependent Block of Ultrarapid-Delayed Rectifier K⁺ Currents by Aconitine, a Potent Cardiotoxin, in Heart-Derived H9c2 Myoblasts and in Neonatal Rat Ventricular Myocytes

Ya-Jean Wang^*, Bing-Shuo Chen^*,, Ming-Wei Lin^*, An-An Lin, Hsung Peng, Ruey J. Sung and Sheng-Nan Wu^*,,1

^* Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan Department of Anesthesiology, Buddhist Dalin Tzu Chi General Hospital, Chiayi County, Taiwan Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan Department of Life Sciences, National Central University, Taoyuan County, Taiwan

¹ To whom correspondence should be addressed at Department of Physiology, National Cheng Kung University Medical College, No. 1, University Road, Tainan 70101, Taiwan. Fax: +886 6 2362780. E-mail: snwu{at}mail.ncku.edu.tw.

Received April 28, 2008; accepted August 22, 2008

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
Aconitine (ACO), a highly toxic diterpenoid alkaloid, is recognized to have effects on cardiac voltage–gated Na⁺ channels. However, it remains unknown whether it has any effects on K⁺ currents. The effects of ACO on ion currents in differentiated clonal cardiac (H9c2) cells and in cultured neonatal rat ventricular myocytes were investigated in this study. In H9c2 cells, ACO suppressed ultrarapid-delayed rectifier K⁺ current (I_Kur) in a time- and concentration-dependent fashion. The IC₅₀ value for ACO-induced inhibition of I_Kur was 1.4µM. ACO could accelerate the inactivation of I_Kur with no change in the activation time constant of this current. Steady-state inactivation curve of I_Kur during exposure to ACO could be demonstrated. Recovery from block by ACO was fitted by a single-exponential function. The inhibition of I_Kur by ACO could still be observed in H9c2 cells preincubated with ruthenium red (30µM). Intracellular dialysis with ACO (30µM) had no effects on I_Kur. I_Kur elicited by simulated action potential (AP) waveforms was sensitive to block by ACO. Single-cell Ca²⁺ imaging revealed that ACO (10µM) alone did not affect intracellular Ca²⁺ in H9c2 cells. In cultured neonatal rat ventricular myocytes, ACO also blocked I_Kur and prolonged AP along with appearance of early afterdepolarizations. Multielectrode recordings on neonatal rat ventricular tissues also suggested that ACO-induced electrocardiographic changes could be associated with inhibition of I_Kur. This study provides the evidence that ACO can produce a depressant action on I_Kur in cardiac myocytes.

Key Words: aconitine; ultrarapid-delayed rectifier K⁺ current; action potential; H9c2 cell; neonatal rat ventricular myocyte; multielectrode array recording.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
Aconitine (ACO) is an intensely poisonous alkaloid derived from plant species Aconitium (Pullela et al., 2008). It is a proarrhythmic agent known to open tetrotodoxin-sensitive Na⁺ channels in the heart (Wang and Wang, 2003; Wright, 2002). The persistent activation of Na⁺ channels caused by ACO arises from an inhibition of channel inactivation, thus leading to sustained Na⁺ influx (Kunze et al., 1985; Wang and Wang, 2003; Wright, 2002). As a result, ACO can induce action potential (AP) prolongation and cardiac arrhythmias through activation of Na⁺ channels (Kunze et al., 1985). However, more recent work has pointed out that this compound may display other actions in a mechanism unlinked to opening of Na⁺ channels. For example, a previous report showed that ACO can increase the motility of prostate cancer cells in a mechanism unlinked to opening of Na⁺ channels (Fraser et al., 2003). ACO-containing herbal extracts have been also reported to exert an inhibition of proliferation in a variety of neoplastic cells (Chodoeva et al., 2005; Dasyukevich and Solyanik, 2007; Solyanik et al., 2004; Yan et al., 2007) and to have serious toxicity in rat embryos (Xiao et al., 2007). ACO was also recently demonstrated to disrupt intracellular Ca²⁺ homeostasis in isolated cardiac myocytes (Fu et al., 2004) and to induce seizure-like events in rat neocortical brain slices (Voss et al., 2008). At our laboratory, we also reported that this compound can block delayed rectifier K⁺ current in differentiated NG108-15 neuronal cells (Lin et al., 2008).

In the heart, delayed-rectifier K⁺ currents play a fundamental role in determining the duration of cardiac APs. In addition to the transient outward K⁺ currents, the ultrarapidly delayed rectifier K⁺ currents (I_Kur) in rat heart represent one of the major repolarizing currents. These currents in mouse heart can be separated into at least two different components, i.e., I_K,slow1 (encoded by Kv1.5) and I_K,slow2 (encoded by Kv2.1) (Zhou et al., 1998). The inhibition of I_Kur would also be expected to result in prolongation of AP duration and electrocardiographic QT interval. However, it remains unknown whether ACO can prolong cardiac AP and induce QT prolongation via an inhibition of I_Kur.

The H9c2 cell line, which has been established from embryonic rat cardiac ventricle, possesses electrical properties similar to neonatal or developing heart cells (Ménard et al., 1999). After undergoing differentiation, these cells can functionally express L-type Ca²⁺ channels of cardiac and skeletal types and ATP-sensitive K⁺ channels (Ménard et al., 1999; Ranki et al., 2002; Wu et al., 2006). As these cells possess a Kv2.1-type I_Kur, the Kv2.1 (or KCNB1)-cloned K⁺ channel, which exhibits unique gating properties and voltage dependency, was thought to be an important contributor to I_Kur described in H9c2 cells (Lo et al., 2005; Suzuki and Takimoto, 2004; Wang et al., 2002). A previous report at our laboratory has demonstrated the ability of chromanol 293B, a blocker of the slowly activating K⁺ current, to inhibit I_Kur in H9c2 cells in a state-dependent fashion (Lo et al., 2005).

In this study, we attempted to investigate whether ACO has any effects on I_Kur in differentiated H9c2 cells and to determine the mechanism through which this compound interacts with this current. The results clearly demonstrate that in H9c2 cells, ACO can produce a depressant action of I_Kur in a concentration- and time-dependent manner. ACO also induced a prolongation of AP in cultured neonatal rat ventricular myocytes. The major action of this compound on I_Kur presented here is thought to be primarily through an open-channel mechanism.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
Culture of heart-derived H9c2 cells.
The H9c2 cell line, originally derived from embryonic rat ventricles, was obtained from the American Type Culture Collection ([CRL-1446], Manassas, VA). Cells were grown in monolayer culture in 50-ml plastic culture flasks in a humidified atmosphere containing 5% CO₂/95% air (vol/vol) at 37°C. Cells were maintained at a density of 10⁶/ml in Dulbecco's modified Eagle's medium supplemented with 10% heat–inactivated fetal bovine serum and 2mM L-glutamine (Lo et al., 2005; Wang et al., 2006; Wu et al., 2006). To prevent cells from undergoing transdifferentiation into skeletal myoblasts, differentiated H9c2 cells were incubated with all-trans retinoic acid (20nM) for 5–7 days (Ménard et al., 1999). A colorimetric method was often used in examining cell densities in microtiter plates with a tetrazolium salt (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrasolio]-1,3-benzene disulfonate; WST) and an ELISA reader (Dynatech, Chantilly, VA). In a separate series of experiments, differentiated H9c2 cells were preincubated with ruthenium red (30µM) for 5 h before experiments were made.

Isolation and culture of neonatal rat ventricular myocytes.
Cells were isolated from 1- to 2-day-old Sprague-Dawley rats by enzymatic digestion with 0.1% trypsin and 0.03% collagenase. After isolation, cells were plated onto laminin-coated 35–mm dishes at a density of 1 x 10³ cells/mm² and cultured for 48 h in the medium of Dulbecco's modified Eagle's medium and Medium 199 (4:1) containing 10% fetal calf serum, 4mM L-glutamine, 100 units/ml penicillin/streptomycin, and 0.1mM 5-bromo-2-deoxyuridine. 5-Bromo-2-deoxyuridine was used to inhibit fibroblast proliferation. The animal experiments were conducted according to protocols that follow the National Institutes of Health standards and the guidelines for the Care and Use of Experimental Animals. The procedure was approved by the Animal Care and Use Committee of the National Cheng Kung University, Taiwan.

Intracellular Ca²⁺ measurement in H9c2 cells.
Cells were loaded with 3-µM fura-2/AM (Molecular Probes, Eugene, OR) for 45 min at room temperature. Changes in intracellular Ca²⁺ were monitored with single-cell imaging using a TillvisION imaging system equipped with a Polychrome II high-speed monochromator (Till-Photonics, Martinried, Germany). Fura-2 was alternately excited by 340 and 380 nm light delivered from a xenon lamp via a x40, 1.3 NA UV fluor oil objective (Olympus, Tokyo, Japan). The fluorescent images were collected at 510 nm scale every 0.5 s by a Peltier-cooled CCD camera. The ratio of fluorescence, R (340/380 nm), from individual cell was obtained with the aid of TILLvisION software 4.0 (Till-Photonics) (Wang et al., 2006).

Whole-cell patch-clamp recordings.
Cells used for experiments were dissociated, and an aliquot of cell suspension was transferred to a recording chamber mounted on the stage of an inverted DM-IL microscope (Leica Microsystems, Wetzlar, Germany). H9c2 cells or neonatal rat ventricular myocytes were bathed at room temperature (20–25°C) in normal Tyrode's solution containing 1.8mM CaCl₂. Patch pipettes were pulled from Kimax-51 glass capillary tubes (Kimble Glass; Vineland, NJ) using a two-stage electrode puller (PP-830; Narishige, Tokyo, Japan), and the tips were fire polished with a microforge (MF-83; Narishige). The pipettes used had resistances of 3–5 M when immersed in normal Tyrode's solution. Ion currents were measured in the whole-cell mode of the patch-clamp recordings with an RK-400 (Biologic, Claix, France) or an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) (Wang et al., 2006; Wu et al., 2006)

The signals were displayed on an HM-507 oscilloscope (Hameg, East Meadow, NY) and on a Dell 2407WFP-HC LCD monitor (Round Rock, TX). The data were stored online in a Slimnote VX₃ computer (Lemel, Taipei, Taiwan) via a universal serial bus port at 10 kHz through a Digidata-1322A interface (Molecular Devices). This device was controlled by pCLAMP 9.0 software (Molecular Devices). Currents were low-pass filtered at 1 or 3 kHz. Ion currents recorded during whole-cell experiments were digitally stored and analyzed subsequently by use of pCLAMP 9.0, Origin 7.5 software (OriginLab, Northampton, MA) or custom-made macros in Microsoft Excel (Redmont, WA). The pCLAMP-generated voltage-step profiles were generally used to measure the current-voltage (I-V) relations for ion currents (e.g., I_Kur) in neonatal rat ventricular myocytes or in H9c2 cells. AP durations were measured at 90% of repolarization (APD₉₀).

The concentration-response data for inhibition of I_Kur in differentiated H9c2 cells were fitted to the Hill equation:

where [C] is the concentration of ACO, IC₅₀ and n are the concentration required for a 50% inhibition and the Hill coefficient, respectively, and E_max is maximal inhibition of I_Kur induced by ACO.

The steady-state inactivation curve of I_Kur in the presence of ACO was plotted against the test potential V and fit to the Boltzman equation:

where V is the conditioning potential in mV, V_1/2 is the membrane potential for half-maximal inactivation, and k is the slope factor of inactivation curve for I_Kur. The solver subroutine built in Excel (Microsoft) was used to fit the data by a least-squares minimization procedure.

Preparation of multielectrode dish recording system.
One- to 2-day-old Sprague-Dawley rats were used for all the experiments. Transverse slices of ventricular tissue (200 µm thickness) were prepared with a vibrating tissue slicer (DTK-1000, Dosaka, Japan). Slices were immediately transferred to a holding chamber for equilibration at room temperature. The electrograms were recorded within 1 h after preparations were made.

The 64-channel multielectrode dish (MED64) system (Alpha MED Sciences, Tokyo, Japan), a two-dimensional cellular electroactivity monitoring technique based on the methods described by Huang et al. (2007), was employed in this study. The MED-P530A probes (Alpha MED Sciences) with 200µM interpolar distance of electrodes, chamber depth of 10 mm and 64 planar microelectrodes in an 8 x 8 array, were used for spatial-temporal determination of spontaneous firing in neonatal rat ventricular tissue.

During multielectrode array recordings, ventricular tissue, on which a fine mesh net was put with adequate anchorage, was positioned on planar multielectrode array probe. The probe was composed of transparent liquid crystal materials except for the electrodes, thereby allowing the localization of the electrodes in the slice under a microscope. The tissue was superfused with normal Tyrode's solution at room temperature at a flow rate of 2 ml/min. Measurements include R-R interval, R amplitude, and Q-T interval. The end of the T wave is measured as time when the repolarization process returns to the isoelectric point. The Q-Tc interval was calculated as the interval (seconds). The analysis was performed automatically by using custom-made macros in Excel (Microsoft).

Values obtained in this study were provided as means ± SEM with sample sizes (n) indicating the number of cells from which the data were taken, unless noted in multielectrode recordings. The paired or unpaired Student's t-test and ANOVA with the least significance difference method for multiple comparisons were used for the statistical evaluation of differences among means. Statistical significance was determined at a P value of <0.05.

Drugs and solutions.
ACO (acetylbenzoylaconine, C₃₄H₄₇NO₁₁), collagenase, all-trans retinoic acid, and tetraethylammonium chloride were purchased from Sigma Chemical Co. (St Louis, MO), and ruthenium red and pinacidil were from Sigma/RBI (Natick, MA). Dendrotoxin was obtained from Calbiochem (La Jolla, CA), fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes (Eugene, OR), and tetrodotoxin was from Alomone Labs (Jerusalem, Israel). Tissue culture media, trypsin/EDTA, L-glutamine, penicillin-streptomycin, and fungizone were purchased from Life Technologies (Grand Island, NY).

The composition of normal Tyrode's solution was 136.5mM NaCl, 5.4mM KCl, 1.8mM CaCl₂, 0.53mM MgCl₂, 5.5mM glucose, and 5.5mM HEPES-NaOH buffer, pH 7.4. To record I_Kur or membrane potential, the patch pipette was filled with a solution consisting of 140mM KCl, 1mM MgCl₂, 3mM Na₂ATP, 0.1mM Na₂GTP, 0.1mM EGTA, and 5mM HEPES-KOH buffer, pH 7.2. To measure Na⁺ current (I_Na), K⁺ ions inside the pipette solution were replaced with equimolar Cs⁺ ions, and the pH was adjusted to 7.2 with CsOH. In some experiments on intracellular perfusion with ACO, the recording pipettes were filled with 30µM ACO.

Mathematical modeling.
To investigate the effect of ACO on I_Kur in response to simulated AP waveform of rat ventricular myocytes, a basic cell model which mimics electrical behavior of rat ventricular myocytes (Bondarenko et al., 2004; Wu et al., 2006) was implemented. Observations made with this model have been demonstrated to correlate with results of cellular electrophysiology (Bondarenko et al., 2004). Source file related to this modeled cell is available online at http://senselab.med.yale.edu/senselab/modeldb. Simulations were implemented using the program xpp with the aid of the X-Win32 version of XPPAUT on a Hewlett Packard (HP xw9300) Workstation (Palo Alto, CA) (Wu and Chang, 2006). Simulated APs used as voltage templates were replayed to H9c2 cells through a digital-analogue converter built in Digidata-1322A interface (Lo et al., 2001; Wu et al., 2007).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
Effect of ACO on Ultrarapid Delayed Rectifier K⁺ Current (I_Kur)) in Differentiated H9c2 Cells
The whole-cell configuration of the patch-clamp technique was used to evaluate the effect of ACO on ion currents in H9c2 cells preincubated with retinoic acid (20nM). To record K⁺ outward currents and to remove voltage-gated inward currents, cells were bathed in Ca²⁺-free Tyrode's solution containing tetrodotoxin (1µM) and CdCl₂ (0.5mM). When the cell was held at – 50 mV and depolarizing voltage pulses from – 50 to + 50 mV in 10-mV increments were applied with a duration of 1 s, a family of rapidly activating outward currents with a slow inactivation was elicited (Figure 1). This population of K⁺ outward currents, which resembles the Kv2.1-encoded current, has been previously identified as I_Kur (Lo et al., 2005; Suzuki and Takimoto, 2004). Notably, 2 min after exposure to ACO (10µM), the amplitude of I_Kur measured at the end of the voltage pulses was greatly reduced at the potentials ranging from 0 to + 60 mV. For example, when depolarizing pulses from – 50 to + 30 mV were applied, ACO (10µM) significantly decreased the amplitude of I_Kur measured at the end of the voltage pulses from 493 ± 67 to 25 ± 55 pA (n = 9, P < 0.05). After washout of ACO, the amplitude of I_Kur at + 30 mV was partially recovered to 78 ± 58 pA (n = 6).

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 1. Inhibitory effect of ACO on IKur in H9c2 cells. Cells were bathed in Ca2+-free Tyrode's solution containing tetrodotoxin (1µM) and CdCl2 (0.5mM). (A) Superimposed current traces obtained in the absence and presence of ACO (10µM). The cell was depolarized from – 50 mV to various potentials ranging from – 50 to + 60 mV in 10-mV increments. Current traces shown on the upper part are control and those on the lower part were obtained 2 min after addition of ACO (10µM). The uppermost part indicates the voltage protocol used. (B) Averaged I-V relations for initial (Ba) and steady-state (Bb) components of IKur in the absence (filled symbols) and presence (open symbols) of 10µM ACO. Mean ± SEM (n = 9–12). (C) Concentration-response curve for ACO-induced inhibition of IKur. The amplitude of IKur during exposure to ACO was compared with the control value, i.e., in the absence of ACO (mean ± SEM, n = 5–9 for each point). The smooth line represents the best fit to a Hill function. The values for IC50, maximally inhibited percentage of IKur, and the Hill coefficient were 1.4µM, 100%, and 1.2, respectively.

 
The relationship between the concentration of ACO and the percentage inhibition of I_Kur was determined. In these experiments, each cell was depolarized from – 50 to + 50 mV with a duration of 1 s. Current amplitudes were measured at the end of depolarizing pulses. As illustrated in Figure 1C, ACO (0.1–10µM) suppressed the steady-state component of I_Kur in a concentration-dependent manner. With the use of a nonlinear least-squares fit to the data, the half-maximal concentration (i.e., IC₅₀) required for the inhibitory effect of ACO on I_Kur was calculated to be 1.4µM, and at a concentration of 10µM, it almost completely suppressed the steady-state component of I_Kur. Thus, it is clear from our results that ACO can exert a significant action on the inhibition of I_Kur in differentiated H9c2 cells.

Effects of ACO on Activation and Inactivation Time Constants of I_Kur in H9c2 Cells
During exposure to ACO, in addition to the decreased amplitude of I_Kur, the inactivation of I_Kur in response to long-lasting depolarization tended to be accelerated. The time constants of I_Kur activation or inactivation in these cells were further analyzed. As depicted in Figure 2, the time courses of current inactivation in the absence and presence of different concentrations of ACO can be well fitted by a single-exponential process. For example, when depolarizing pulse from – 50 to + 50 mV was evoked with a duration of 1 s, application of ACO (3µM) significantly decreased the mean time constants of I_Kur inactivation from 1017 ± 134 to 132 ± 45 ms (n = 6, P < 0.05). Conversely, no significant change in activation time constant of I_Kur during the exposure to ACO can be demonstrated (data not shown). Therefore, it appears that the exposure to ACO preferentially affects the inactivation time course of I_Kur in H9c2 cells.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 2. Effects of ACO on inactivation time constant of IKur in H9c2 cells. In each cell, depolarizing pulses from – 50 to + 50 mV with a duration of 1 s were applied. (A) Inactivation time courses of IKur at the level of + 50 mV in the absence (a) and presence (b) of 3µM ACO were fitted by a single exponential with a value of 1017 and 126 ms, respectively. Summary of the data showing effects of ACO (1, 3, and 10µM) on inactivation time constants of IK(DR) is illustrated in panel B. tinact is the time constant for IKur inactivation. Each bar represents the mean ± SEM (n = 5–9). *Significantly different from control group. Notably, ACO can decrease the value of inactivation time constant for IKur in a concentration-dependent fashion.

 
Steady-State Inactivation of I_Kur during the Exposure to ACO
To characterize the inhibitory effect of ACO on I_Kur, we next investigated voltage dependence of the effect of ACO on I_Kur in H9c2 cells. Figure 3 shows the steady-state inactivation curve of I_Kur in the presence of ACO (3µM). In these experiments, a 300-ms conditioning pulse to different potentials preceded the test pulse to + 50 mV from a hold potential of – 50 mV. The relationships between the conditioning potentials and the normalized amplitudes of I_Kur were plotted and fitted by the Boltzmann equation. In the presence of ACO (3µM), voltage for half-maximal inactivation (V_1/2) and corresponding slope factor (k) are – 9.8 ± 0.3 and 6.1 ± 0.2 mV (n = 8), respectively. Notably, in the presence of ACO (3µM), the membrane potential at which half-maximal inactivation occurred (V_1/2) was in the range of – 10 mV, a value that corresponds to the plateau level of cardiac AP.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 3. Steady-state inactivation curve of IKur in the presence of ACO in H9c2 cells. In these experiments, the conditioning voltage pulses with a duration of 300 ms to different potentials between – 80 and + 60 mV were applied from a holding potential of – 50 mV. Following each conditioning pulse, a test pulse to + 50 mV was applied to evoke IKur. An example of current traces obtained by two-pulse protocol in the presence of 3µM ACO is illustrated in (A). Upper part indicates the voltage protocol used. (B) Steady-state inactivation curve of IKur obtained in the presence of 3µM ACO. The normalized amplitude of IKur (I/Imax) was constructed against the conditioning potential, and the smooth curve was fitted by the Boltzmann equation (see text for details). Each point represents the mean ± SEM (n = 6–10).

 
Recovery from Block Induced by ACO in H9c2 Cells
Recovery from block was further determined by a two-step pulse protocol consisting of a first (conditioning) depolarizing pulse sufficiently long to allow block to reach a steady state. During exposure to ACO, the membrane potential was then stepped to + 50 mV from – 50 mV for a variable time, after which a second depolarizing pulse (test pulse) was applied at the same potential as the conditioning pulse. The ratios of the peak current amplitudes of I_Kur evoked in response to the test and the conditioning pulse were taken as a measure of recovery from block and then plotted versus interpulse interval (Figure 4). The time course in the presence of 3 and 10µM ACO can be described by a single exponential with a time constant of 2.57 ± 0.22 s (n = 5) and 4.13 ± 0.35 s (n = 6), respectively. Thus, the results indicate that cell exposure to ACO can produce a prolongation of the recovery from inactivation of I_Kur in H9c2 cells.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 4. Time course of the recovery of IKur in the presence of ACO. H9c2 cells, bathed in Ca2+-free Tyrode's solution, were depolarized from – 50 to + 50 mV with a duration of 740 ms, and different interpulse durations were applied. (A) Superimposed current traces obtained in the presence of 10µM ACO. Voltage protocol is shown in the upper part of panel (A). (B) Time course of recovery from inactivation of IKur caused by 3 and 10µM ACO. The time course in the presence of 3 and 10µM ACO was well fitted to a single exponential with a time constant of 2.56 and 4.12 s, respectively. Each point represents mean ± SEM (n = 4–8).

 
Effect of ACO on Averaged I-V Relations of I_Kur in H9c2 Cells Treated with Ruthenium Red
ACO was recently reported to disrupt intracellular Ca²⁺ handling in heart cells (Fu et al., 2004). We also evaluated whether decreased Ca²⁺ release from internal stores can influence ACO-induced inhibition of I_Kur in H9c2 cells. In these experiments, differentiated H9c2 cells were preincubated with ruthenium red (30µM) for 5 h. Ruthenium red, an inhibitor of ryanodine receptors, was previously shown to reverse ACO-induced changes in intracellular Ca²⁺. Our experimental results showed that in ruthenium red–treated cells, the inhibitory effect of ACO on I-V relation of I_Kur was unaltered (Figure 5). There was lack of significant difference in the magnitude of ACO-induced inhibition of I_Kur between control cells and cells treated with ruthenium red. The results could be thus interpreted to mean that the inhibitory effect of ACO on this current in these cells is unrelated to its effects on Ca²⁺ release from internal stores.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 5. Effect of ACO on the averaged I-V relations of IKur in H9c2 cells treated with ruthenium red. H9c2 cells were preincubated with ruthenium red (30µM) for 5 h. In each cell examined, IKur was elicited from – 50 to different potentials ranging from – 50 to + 60 mV with 10-mV increments. Each point represents the mean ± SEM (n = 5–7). Filled squares, control; open squares, in the presence of ACO (10µM).

 
Inhibitory Effect of ACO on I_Kur in Response to Simulated APs
Because the size and time course of I_Kur in response to change in waveforms of cardiac APs were different from those during a rectangular voltage clamp pulse, the effects of ACO on I_Kur elicited by simulated APs generated from a modeled rat ventricular myocyte (Bondarenko et al., 2004) were further investigated. In these experiments, simulated waveforms of cardiac APs were digitally generated as voltage templates and then replayed to evoke I_Kur in H9c2 cells (Lo et al., 2001; Wu et al., 2007). Because ion currents in response to simulated AP waveforms were sensitive to block by 1µM dendrotoxin, the results suggest that I_Kur may be K⁺ flux through the K⁺ channel (Lo et al., 2005). As shown in Figure 6, whole-cell currents elicited by the AP waveforms were recorded from H9c2 cells. Capacitative and leakage currents were estimated using a full-amplitude negative-going AP waveform (Figure 6) and subtracted from total current to yield the AP-evoked I_Kur. Raw, leak, and capacitative currents in response to an AP waveform are illustrated in Figure 6B, and Figure 6C shows the subtracted I_Kur trace. The data shown in Figure 6 are representative of results obtained in seven different other cells. Notably, the component of I_Kur was coincident with the early repolarization of cardiac AP.

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 6. Isolation of IKur in response to a simulated AP of rat ventricular myocyte. The modified AP waveform was originally derived from the model made by Bondarenko et al., 2004. (A) Membrane current evoked by an AP waveform generated from a modeled cell. (B) IKur in response to positive (upper) or negative (lower) AP waveforms. Inset indicates positive and negative AP waveforms. (C) AP waveform (upper) and IKur (lower) determined by the addition of the two membrane currents shown in (B).

 
As shown in Figure 7, the amplitude of I_Kur in response to simulated AP waveforms was significantly reduced when cells were exposed to ACO. For example, after application of 1µM ACO, the peak amplitude of outwards current elicited by simulated waveforms of cardiac AP was decreased by about 35%. Thus, in H9c2 cells, I_Kur evoked during AP waveforms remains sensitive to block by ACO. The results led us to propose that ACO-induced inhibition of I_Kur can influence AP configuration of rat cardiac myocytes, assuming that this current is functionally expressed in vivo.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 7. Effect of ACO on AP waveform–induced IKur in H9c2 cells. (A) Current traces obtained in the absence (a) and presence (b) of 3µM ACO. The upper part shows simulated AP waveform from a modeled rat ventricular myocyte. (B) Summary of the data showing inhibitory effect of ACO (1 and 3µM) on IKur in response to simulated AP generated from a modeled cell. The peak amplitude of IKur elicited by AP waveform was measured. Each bar represents the mean ± SEM (n = 5–8). *Significantly different from control group.

 
Effect of ACO on APs of Cultured Neonatal Rat Ventricular Myocytes
The properties of I_Kur in H9c2 cells may also be different from those functionally expressed in cardiac myocytes. Therefore, the effect of ACO in neonatal rat ventricular myocytes was further evaluated. In the first set of experiments, cells were bathed in normal Tyrode's solution containing 1.8mM CaCl₂, and current-clamp configuration of the patch-clamp technique was made. The typical effect of ACO on membrane potentials is illustrated in Figure 8A. The effect of ACO at a concentration of 3 or 10µM on cardiac APs was noted to consist of a significant prolongation of ventricular AP, along with appearance of early afterdepolarizations, as evidenced previously (Sawanobori et al., 1987). For example, after 2 min of exposing the cells to ACO (10µM), APD₉₀ was greatly prolonged to 785 ± 121 ms from a control of 112 ± 12 ms (n = 6, P < 0.01). However, in continued presence of ACO, further application of tetrodotoxin (1µM) was found to have no significant effects on ACO-induced increase of APD₉₀ (Figure 8B).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 8. Effects of ACO on AP and IKur in cultured neonatal rat ventricular myocytes. Cells were bathed in a normal Tyrode's solution containing 1.8mM CaCl2. (A) Original potential traces showing effect of ACO on APs in neonatal rat ventricular myocytes. Current-clamp experiments were made. Potential trace labeled ‘a’ is control, and those labeled ‘b,’ ‘c,’ and ‘d’ are records obtained 2 min after exposure to 1, 3 and 10µM ACO. (B) Summary of the data showing the effects of ACO (1, 3, and 10µM) on APD90. Each bar represents the mean ± SEM (n = 4–6). *Significantly different from control group. TTX, tetrodotoxin (1µM). (C) Original current traces showing effect of ACO on IKur in neonatal rat ventricular myocytes. Cells were bathed in Ca2+-free Tyrode's solution containing 1µM tetrodotoxin. Whole-cell currents were evoked by 300-ms depolarizing pulses from – 50 to + 50 mV. The upper part indicates the voltage protocol used. a, control; b, 1µM ACO; c, 3µM ACO.

 
Whole-cell voltage clamp experiments were also applied in an attempt to investigate the properties of I_Kur in these cells because ACO-induced prolongation of cardiac APs was insensitive to tetrodotoxin. Consistent with the results made in H9c2 cells, I_Kur could be rapidly activated at depolarized potentials. The exposure to ACO (1 or 3µM) was found to block I_Kur along with increased acceleration of current inactivation (Figure 8C). Similar results were obtained in five different cells.

Voltage-gated I_Na in neonatal rat ventricular myocytes was further investigated. The results showed that tetrodotoxin (1µM) could significantly suppress the peak amplitude of I_Na. However, ACO (3µM) also had an inhibitory effect on I_Na (see Supplementary Information). Therefore, ACO-induced changes of APs seen in neonatal rat ventricular myocytes could not be associated with activation of I_Na as reported previously (Kunze et al., 1985; Wright, 2002).

Multielectrode Array Recordings from the Slices of Neonatal Rat Ventricular Tissues
In a final series of experiments, we also examined if there are any changes in ventricular electrograms when neonatal ventricular tissue was exposed to ACO. Multielectrode recordings were performed to measure electrographic waveforms in neonatal rat ventricular slices (Huang et al., 2007). The mean conduction velocity in spontaneously firing preparations is 31 ± 6 cm/s (n = 5 slices). A representative trace regarding the effect of ACO on ventricular electrograms measured from multielectrode array recordings is illustrated in Figure 9. ACO was found to increase firing frequency and prolong QTc interval significantly. About 5 min of exposing the tissue to ACO (10µM), the amplitude of R wave appeared to vary, along with increased firing of APs. Table 1 shows that exposure to ACO (10µM) resulted in a significant change in the parameters of electrograhic waveforms measured by multielectrode array recordings. However, subsequent application of tetrodotoxin (1µM) did not have any effects on ACO-induced changes in electrogram patterns (data not shown). Taken together, the results suggest that ACO-induced propensity to induce electrophysiological changes in neonatal rat ventricular tissue could be associated with its inhibition of I_Kur.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 9. Multielectrode array recordings in slices of neonatal rat ventricular tissue. Individual recordings shown here were taken from electrode No. 61 of the array. The trace labeled ‘a’ is control and those in ‘b,’ ‘c,’ ‘d,’ and e represent 1, 2, 3, and 4 min after exposure to 10µM ACO, respectively. Notably, 4 min after application of ACO, electrographic waveform mimics the appearance of ventricular tachycardia.

 

View this table: [in this window] [in a new window]   TABLE 1 Summary of the Data Showing Effect of ACO on the Parameters in Electrographic Pattern Recorded from Slices of Neonatal Rat Ventricular Tissues

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
In this study, cell exposure to ACO displayed concentration-, state-, and time-dependent interactions I_Kur. Previous findings at our laboratory have shown that chromanol 293B, a block of the slowly activating K⁺ current, could alter activation kinetics of I_Kur in H9c2 cells (Lo et al., 2005). However, no significant changes in the initial time course of activation in the presence of ACO could be demonstrated, suggesting that ACO-induced block of I_Kur mainly occurred after channel opening. Prior to channel activation, the ACO-binding site is likely to be either in a low-affinity state or inaccessible to the compound.

Application of ACO tends to produce an inactivation-like behavior as it decreases the inactivation time constant of I_Kur in a concentration-dependent manner. In other words, currents that display only a weak and very slow interaction show a peak and rapid decrease to a new steady-state level in the presence of ACO. Based on our experimental data, a block of K⁺ channels in both open and open-inactivated states thus appears to be likely mechanism of ACO-induced inhibition of I_Kur. However, intracellular dialysis with 30µM ACO did not affect amplitude and time course of I_Kur, block by this compound of I_Kur observed in H9c2 cells, or neonatal rat ventricular myocytes may primarily occur via an extracellular site.

In our study, recovery from I_Kur inactivation in the presence of ACO was slow, as more than 10 s was required for the channel to recover completely. Consequently, ACO-induced block of I_Kur is more likely to occur in association with high adrenergic tone because of the fact that under these conditions the availability of K⁺ channels is decreased as a function of firing frequency. In light of steady-state inactivation curve of I_Kur obtained in the presence of ACO, the voltage for half-maximal inactivation was noted to be in the range of the plateau level of cardiac AP. As a determinant of the AP plateau level, changes in I_Kur (or I_Kslow2 in mouse heart) tend to trigger significant alterations in ion flux through Na⁺- or L-type Ca²⁺ channels. It thus remains to be further explored whether block of I_Kur, along with increased current inactivation, can prolong AP duration and induce early afterdepolarizations, thereby leading to facilitation of I_Na or Ca²⁺ currents in cardiac myocytes. Future work is also necessary to assess to what extent ACO can prolong cardiac AP and induce QT prolongation via an inhibition of I_Kur.

Previous work demonstrated that there appears to be a functional coupling between transient outward K⁺ current and L-type Ca²⁺ current in cardiac myocytes (Wang et al., 2006). Under our experimental conditions, major Ca²⁺ channels in differentiated H9c2 myoblasts were blocked by 0.5mM CdCl₂. As extracellular Ca²⁺ was also removed for measurement of I_Kur, there are little or no Ca²⁺ ions permeating through Ca²⁺ channels. The inhibitory effect of ACO on I_Kur presented here is thus unlikely due to actions on Ca²⁺ currents. In our studies, because differentiated H9c2 cells were not found to possess I_Na, there is unlikely to be a functional association between I_Na and I_Kur. In addition, a lack of effect of pinacidil (30µM), a known opener of ATP-sensitive K⁺ channels (Wu et al., 2006), to reverse ACO-induced inhibition of I_Kur in H9c2 cells (data not shown) excludes the possibility that the activity of ATP-sensitive K⁺ channels contributes to its inhibition of I_Kur in these cells.

Our results may help to explain previous findings showing that ACO-containing extracts could increase the motility of prostate cancer cells and suppress the growth of hepatocarcinoma Bel-7402 cells or lung carcinoma cells (Chodoeva et al., 2005; Fraser et al., 2003; Solyanik et al., 2004; Yan et al., 2007), given that those cancer cells did not functionally express the activity of voltage-gated Na⁺ channels. Notably, previous studies have demonstrated that the activity of Kv2 channels might influence proliferation of uterine cancer cells (Suzuki and Takimoto, 2004).

Our results suggest that the ACO-sensitive I_Kur may be carried primarily by Kv2.1 channels. Dysfunction of Kv2.1 channels in mouse ventricular myocytes has been shown to have AP prolongation along with both spontaneous and inducible arrhythmias (Xu et al., 1999). There is also a strong overlap among Drosophila Shab, rat Kv2.1 and human Kv2.1 channels from molecular, pharmacological, and physiological points of view (Ju et al., 2003). Although recent reports demonstrated the expression of Kv2.1 messenger RNA in the human heart (Ördög et al., 2006), no functional Kv2.1 currents have ever been detected. However, ACO may also affect the magnitude of Kv3 current as described in neuroblastoma cells (Lin et al., 2008). Taken together, it is tempting to speculate that ACO actions on I_Kur are the likely cardiac implications in humans. Our experimental results also make a case for examining if the mechanistic direction described here for the effects of ACO has pharmacological and clinical relevance in humans.

The IC₅₀ value of ACO required for block of I_Kur in differentiated H9c2 myoblasts was 1.4µM. This value could be of toxicological relevance as it is similar to that for its known effects on I_Na. Thus, its effective concentration required for the inhibition of I_Kur in H9c2 cells tends to overlap with that for activation of Na⁺ current (Wang and Wang, 2003; Wright, 2002). Similar to tetrodotoxin, ACO exerted a depressant action on I_Na in neonatal rat ventricular myocytes (see Supplementary Information). The exposure to ACO could also be effective in suppressing the amplitude of I_Kur in response to simulated AP from a modeled rat ventricular myocyte. Therefore, to some extent, there appears to be a link between the effect of ACO on I_Kur and its effects on APs or electrographic waveforms in neonatal rat ventricular tissue.

In our study, ACO-induced block of I_Kur in ruthenium red–treated cells is virtually unchanged throughout the depolarizing pulses examined. In intracellular Ca²⁺ measurements, application of ACO (30µM) alone produced no changes in intracellular Ca²⁺ in differentiated H9c2 cells (data not shown). A previous work by Sah et al. (2003) has demonstrated that a decrease in repolarizing K⁺ currents might result in abnormal intracellular Ca²⁺ homeostasis. Therefore, ACO-induced impairment of intracellular Ca²⁺ handling reported in isolated cardiac myocytes tends to be secondarily due to its inhibitory actions on I_Kur. However, we cannot exclude the possibility that ACO or other structurally related compounds are able to interact with accessory Kvb₁ subunits to induce block of I_Kur, as Kvb₁ subunits have been reported to differentially modulate the expression of K⁺ channels in mouse ventricular myocytes (Aimond et al., 2005).

Findings in this study reveal a new molecular mechanism underlying the effects of ACO that may operate in addition to its effects on Na⁺ channel. It remains to be investigated whether channels formed by homotetramers or hetero-oligomers of recombinant Kv2.1 are sensitive to ACO and other structurally related compounds. The nature of the interacting domains between the K⁺ channel and the ACO molecule requires to be further explored. Our data also raise the possibility of its effects on other types of cells that can functionally express Kv2 channels. Indeed, Kv2.1 channels play a role in several tissues, such as pancreas, pulmonary arteries, placental vasculature, and hippocampal and cortical pyramidal neurons (Misonou et al., 2004; Moudqil et al., 2006; Murakoshi and Trimmer, 1999; Park et al., 2006). An effect of ACO on Kv2.1 channels in these tissues could likely have an impact on the functioning of these tissues.

	SUPPLEMENTARY DATA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
Supplementary data are available online at http://toxsci.oxfordjournals.org/.

	FUNDING

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
National Science Council (NSC-97-2320B-006-021), Taiwan; Program for Promoting Academic Excellence and Developing World Class Research Centers; Advanced Biotechnology Education Program, Ministry of Education, Taiwan; Cardiac Electrophysiology and Systems-biology Center, National Cheng Kung University Medical Center, Tainan, Taiwan.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING REFERENCES

 
Aimond F, Kwak SP, Rhodes KJ, Nerbonne JM. Accessory Kvb₁ subunits differentially modulate the functional expression of voltage-gated K⁺ channels in mouse ventricular myocytes. Circ. Res. (2005) 96:451–458.[Abstract/Free Full Text]

Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am. J. Physiol. Heart Circ. Physiol. (2004) 287:H1378–H403.[Abstract/Free Full Text]

Chodoeva A, Bosc J-J, Guillon J, Decendit A, Petraud M, Absalon C, Vitry C, Jarry C, Robert J. 8-O-azeloyl-14-benzoylaconine: A new alkaloid from the roots of Aconitum karacolicum Rapes and its antiproliferative activities. Bioorg. Med. Chem. (2005) 13:6493–6501.[CrossRef][Medline]

Dasyukevich OI, Solyanik GI. Comparative study of anticancer efficacy of aconitine-containing agent BC1 against ascite and solid tumors of Ehrlich's carcinoma. Exp. Oncol. (2007) 29:317–319.[Medline]

Fraser SP, Salvador V, Manning EA, Mizal J, Altum S, Raza M, Berridge RJ, Djamgoz MB. Contribution of functional voltage-gated Na⁺ channel expression to cell behavior involved in the metastatic cascade in rat prostate cancer. I. Lateral motility. J. Cell. Physiol. (2003) 195:479–487.[CrossRef][Web of Science][Medline]

Fu M, Wu M, Wang JF, Qiao YJ, Wang Z. Disruption of the intracellular Ca²⁺ homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocyes. Biochem. Biophys. Res. Commun. (2004) 354:929–936.

Huang CW, Huang CC, Cheng JT, Tsai JJ, Wu SN. Glucose and hippocampal neuronal excitability: Role of ATP-sensitive potassium channels. J. Neurosci. Res. (2007) 85:1468–1477.[CrossRef][Web of Science][Medline]

Ju M, Stevens L, Leadbitter E, Wray D. The roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel. J. Biol. Chem. (2003) 278:12769–12778.[Abstract/Free Full Text]

Kunze DL, Lacerda AE, Wilson DL, Brown AM. Cardiac Na currents and inactivating, reopening, and waiting properties of single cardiac Na channels. J. Gen. Physiol. (1985) 86:691–719.[Abstract/Free Full Text]

Lin MW, Wang YJ, Liu SI, Lin AA, Lo YC, Wu SN. Characterization of aconitine-induced block of delayed rectifier K⁺ current in differentiated NG108-15 neuronal cells. Neuropharmacology (2008) 54:912–923.[CrossRef][Web of Science][Medline]

Lo YC, Yang SR, Huang MH, Liu YC, Wu SN. Characterization of chromanol 293B-induced block of the delayed-rectifier K⁺ current in heart-derived H9c2 cells. Life Sci. (2005) 76:2275–2286.[CrossRef][Web of Science][Medline]

Lo YK, Wu SN, Lee CT, Li HF, Chiang HT. Characterization of action potential waveform-evoked L-type calcium currents in pituitary GH₃ cells. Pflugers Arch. (2001) 442:547–557.[CrossRef][Web of Science][Medline]

Ménard C, Pupier S, Mornet D, Kitzmann M, Nargeot J, Lory P. Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9c2 cardiac cells. J. Biol. Chem. (1999) 274:29063–29070.[Abstract/Free Full Text]

Misonou H, Mohapatra DP, Park EW, Leung V, Zhen D, Misonou K, Anderson AE, Trimmer JS. Regulation of ion channel localization and phosphorylation by neuronal activity. Nat. Neurosci. (2004) 7:711–718.[CrossRef][Web of Science][Medline]

Moudqil R, Michelakis ED, Archer SL. The role of K⁺ channels in determining pulmonary vascular tone, oxygen-sensing, cell proliferation, and apoptosis: Implications in hypoxic pulmonary vasoconstriction and pulmonary arterial hypertension. Microcirculation (2006) 13:615–632.[CrossRef][Web of Science][Medline]

Murakoshi H, Trimmer JS. Identification of the Kv2.1 K⁺ channel as a major component of the delayed rectifier K⁺ current in rat hippocampal neurons. J. Neurosci. (1999) 19:1728–1735.[Abstract/Free Full Text]

Ördög B, Brutyó E, Puskás LG, Papp JG, Varró A, Szabad J, Boldogköi Z. Gene expression profiling of human cardiac potassium and sodium channels. Int. J. Cardiol. (2006) 111:386–393.[CrossRef][Web of Science][Medline]

Park KS, Mohapatra DP, Misonou H, Trimmer JS. Graded regulation of the Kv2.1 potassium channel by variable phosphorylation. Science (2006) 313:976–979.[Abstract/Free Full Text]

Ranki HJ, Budas GR, Crawford RM, Davies AM, Jovanovi A. 17β-Estradiol regulates expression of K_ATP channels in heart-derived H9c2 cells. J. Am. Coll. Cardiol. (2002) 40:367–374.[Abstract/Free Full Text]

Sah R, Ramirez RJ, Oudit GY, Gidrewicz D, Trivieri MG, Zobel C, Backx PH. Regulation of cardiac excitation-contraction coupling by action potential repolarization: Role of the transient outward potassium current (I_to). J. Physiol. (2003) 546:5–18.[Abstract/Free Full Text]

Sawanobori T, Hirano Y, Hiraoka M. Aconitine-induced delayed afterdepolarization in frog atrium and guinea pig papillary muscles in the presence of low concentrations of Ca²⁺. Jpn. J. Physiol. (1987) 37:59–79.[Web of Science][Medline]

Solyanik GI, Fedorchuk AG, Pyaskovskaya ON, Dasyukevitch OI, Khranovskaya NN, Aksenov GN, Sobetsky VV. Anticancer activity of aconitine-containing herbal extract BC1. Exp. Oncol. (2004) 26:307–311.[Web of Science][Medline]

Suzuki T, Takimoto K. Selective expression of HERG and Kv2 channels influences proliferation of uterine cancer cells. Int. J. Oncol. (2004) 25:153–159.[Web of Science][Medline]

Wang SY, Wang GK. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins. Cell. Signal. (2003) 15:151–159.[CrossRef][Web of Science][Medline]

Wang W, Hino N, Yamasaki H, Aoki T, Ochi R. Kv2.1 K⁺ channels underlie major voltage-gated K⁺ outward current in H9c2 myoblasts. Jpn. J. Physiol. (2002) 52:507–514.[CrossRef][Web of Science][Medline]

Wang Y, Cheng J, Tandan S, Jiang M, McCloskey DT, Hill JA. Transient-outward K⁺ channel inhibition facilitates L-type Ca²⁺ current in heart. J. Cardiovasc. Electrophysiol. (2006a) 17:298–304.[CrossRef][Web of Science][Medline]

Wang YJ, Liu YC, Chang HD, Wu SN. Diosgenin, a plant-derived sapogenin, stimulates Ca²⁺-activated K⁺ current in human cortical HCN-1A neuronal cells. Planta Med. (2006b) 72:430–436.[CrossRef][Web of Science][Medline]

Wang YJ, Sung RJ, Lin MW, Wu SN. Contribution of BK_Ca-channel activity in human cardiac fibroblasts to electrical coupling of cardiomyocyte-fibroblasts. J. Membr. Biol. (2006c) 213:175–185.[CrossRef][Web of Science][Medline]

Wright SN. Comparison of aconitine-modified human heart (hH1) and rat skeletal (m1) muscle Na⁺ channels: An important role for external Na⁺ ions. J. Physiol. (2002) 538:759–771.[Abstract/Free Full Text]

Wu SN, Chang HD. Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH₃ cells. Biochem. Pharmacol. (2006) 71:615–623.[CrossRef][Web of Science][Medline]

Wu SN, Chang HD, Sung RJ. Cocaine-induced inhibition of ATP-sensitive K⁺ channels in rat ventricular myocytes and in heart-derived H9c2 cells. Basic Clin. Pharmacol. Toxicol. (2006) 99:510–517.

Wu SN, Wang YJ, Lin MW. Potent stimulation of large-conductance Ca²⁺-activated K⁺ channels by rottlerin, an inhibitor of protein kinase C-d, in pituitary tumor (GH₃) cells and in cortical neuronal (HCN-1A) cells. J. Cell. Physiol. (2007) 210:655–666.[CrossRef][Web of Science][Medline]

Xiao K, Wang L, Liu Y, Peng C, Yan G, Zhang J, Zhuo Y, Li H. Study of aconitine toxicity in rat embryos in vitro. Birth Defects Res. B. Dev. Reprod. Toxicol. (2007) 80:208–212.[CrossRef][Web of Science][Medline]

Xu H, Barry DM, Li H, Brunet S, Guo W, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ. Res. (1999) 85:623–633.[Abstract/Free Full Text]

Yan ZC, Chen D, Wu XZ, Xie GR, Ba Y, Yan Z. Effects of aqueous extracts of Aconitum carmichaeli, Rhizoma bolbostemmatis, Phytolacca acinosa, Panax notoginseng and Gekko swinhonis Guenther on Bel-7402 cells. World J. Gastroenterol. (2007) 13:2743–2746.[Web of Science][Medline]

Zhou J, Jeron A, London B, Han X, Koren G. Characterization of a slowly inactivating outward current in adult mouse ventricular myocytes. Circ. Res. (1998) 83:806–814.[Abstract/Free Full Text]

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