Inhibition of voltage-gated Na+ channels by hinokiol in neuronal cells

Pharmacological Reports 67 (2015) 1049–1054

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Original research article

Inhibition of voltage-gated Na+ channels by hinokiol in neuronal cells Yu-Wen Wang a, Chin-Tsang Yang b, Chi-Li Gong c, Yi-Hung Chen d, Yu-Wen Chen e, King-Chuen Wu f, Tzu-Hurng Cheng g, Yueh-Hsiung Kuo a,h,1, Yuh-Fung Chen i,1, Yuk-Man Leung c,1,* a

Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan School of Chinese Medicine, China Medical University, Taichung, Taiwan Department of Physiology, China Medical University, Taichung, Taiwan d Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan e Department of Physical Therapy, China Medical University, Taichung, Taiwan f Department of Anesthesiology, Eda-Hospital, I-Shou University, Kaohsiung, Taiwan g Department of Biochemistry, China Medical University, Taichung, Taiwan h Department of Biotechnology, Asia University, Taichung, Taiwan i Department of Pharmacology, China Medical University, Taichung, Taiwan b c

A R T I C L E I N F O

Article history: Received 6 November 2014 Received in revised form 27 February 2015 Accepted 31 March 2015 Available online 14 April 2015 Keywords: Hinokiol N2A cells Rat hippocampal CA1 neurons Block Voltage-gated Na+ channels

A B S T R A C T

Background: Hinokiol is a naturally occurring diterpenoid compound isolated from plants such as Taiwania cryptomerioides. Anti-oxidation, anti-cancer, and anti-inflammation effects of this compound have been reported. It is not yet known if hinokiol affects neurons or neuronal ion channel activities. We reported here that hinokiol inhibited voltage-gated Na+ channels (VGSC) in neuronal cells and we characterized the mechanisms of block. Methods: The effects of hinokiol on Na+ channels were examined using the voltage-clamp (whole-cell mode) technique. Results: VGSC was blocked by hinokiol in a concentration-dependent and state-dependent manner in neuroblastoma N2A cells: IC50 are 11.3 and 37.4 mM in holding potentials of 70 and 100 mV, respectively. In the presence of hinokiol there was a 13-mV left shift in steady-state inactivation curves; however, activation gating was not altered. VGSC inhibition by hinokiol did not require channel opening and was thus considered to be closed-channel block. In the presence of hinokiol, since the degree of block did not enhance with stimulation frequency, block by hinokiol thus did not exhibit use-dependence. Recovery from channel inactivation was not significantly affected in the presence of hinokiol. In addition, hinokiol also inhibited VGSC of differentiated neuronal NG108-15 cells and rat hippocampal CA1 neurons. Conclusion: Our results therefore suggest hinokiol inhibited VGSC in a closed-channel block manner and such inhibition involved intensification of channel inactivation. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

Introduction Hinokiol (Fig. 1) is a diterpenoid isolated from the wood of Taiwania cryptomerioides Hayata. This compound has been shown to possess anti-tumor activities against cervical carcinoma (HeLa) and human ovarian carcinoma (HO-8910) cell lines [1] and antioxidant activities [2]. Hinokiol has also been shown to inhibit

* Corresponding author. E-mail address: [email protected] (Y.-M. Leung). 1 These authors contributed equally to this work.

pro-inflammatory enzyme production and generation of TNF-a and nitric oxide from lipopolysaccharide-stimulated RAW macrophages [3,4]. Hepatoprotective activity has also been reported for this compound [5]. Neurons are the cellular basis for fast signal transmission in the nervous system, which regulates many other organ systems; any possible neuromodulatory effects of hinokiol need to be scrutinized before this compound is to be developed as a therapeutic agent. Despite the multitude of pharmacological actions of hinokiol, the effects of hinokiol on neurons in vitro or the nervous system in vivo are unexplored. Voltage-gated Na+ channels (VGSC) permit Na+ influx in neurons during the upstroke phase of action

http://dx.doi.org/10.1016/j.pharep.2015.03.019 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

Y.-W. Wang et al. / Pharmacological Reports 67 (2015) 1049–1054

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In order to yield activation curves, Na+ currents were triggered with increasing depolarization. Conductance (G) was calculated as: G ¼ I=V  V r

Fig. 1. Chemical structure of hinokiol.

where Vr = (RT/zF) ln(Nao/Nai) V is the applied voltage, Vr is the reversal potential of Na+, I is the current, R is the universal gas constant, T is the temperature, z is the ion valency (+1 in this case), and F is the Faraday constant. Nao and Nai are, respectively, extracellular (bath) and intracellular (pipette) Na+ concentrations. The data for voltage-dependence of activation were fitted using the Boltzmann equation: G 1 ¼ Gmax f1 þ exp½ðV 1=2  VÞ=kg

potential and are therefore determinant molecules in excitability. VGSC mutations may lead to epilepsy, and suppression of VGSC activities by phenytoin produces anti-convulsant effects [6]. Inhibition of VGSC also accounts for the anesthetic effects by propofol and lidocaine [7]. In this report we explored the VGSC-blocking action of hinokiol. Voltage-gated Ca2+ channels were not studied since they are not involved in action potential initiation in nonpacemaker neurons. We obtained data showing that hinokiol produced a concentration-dependent and state-dependent block of VGSC in neuroblastoma N2A cells. Detailed mechanisms of blockage were delineated. Hinokiol was also shown to suppress VGSC of differentiated neuronal NG108-15 cells and rat hippocampal CA1 neurons.

Gmax is the maximum conductance, V1/2 is the half-maximal activation potential, and k the slope factor. To yield the steady-state inactivation curve, a two-pulse protocol was deployed: a 10 mV step pulse was preceded by a 2-s pre-pulse of different voltages. The test pulse currents (I) are normalized to the maximal test pulse current (Imax) and then plotted against the pre-pulse voltages. The data for steady-state inactivation were fitted using the Boltzmann equation:

Materials and methods

V1/2 is the half-maximal inactivation potential, and k the slope factor. The Hill equation was used to fit concentration–inhibition curves:

Cell culture and drugs

I 1 ¼ Imax f1 þ exp½ðV  V 1=2 Þ=kg

n

N2A cells and NG108-15 cells were cultured at 37 8C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and penicillin–streptomycin (100 units/ml, 100 mg/ml) (Invitrogen, Carlsbad, CA, USA). For the purpose of differentiating NG108-15 cells into more mature neurons, these cells were incubated in the above-mentioned medium with 0.1% fetal bovine serum, supplemented with 10 mM retinoic acid and 30 mM forskolin for 3 days. Hinokiol was purified from Taiwania cryptomerioides Hayata as described previously, and purity was >99.0% by HPLC analysis [4]. Hinokiol was dissolved in DMSO at 50 mM. Lidocaine was from Sigma (St. Louis, MO, USA) and was dissolved at DMSO at 100 mM. Electrophysiology Voltage-clamp experiments using whole-cell mode were performed as described in previous reports [8,9]. Glass capillary tubes (OD 1.5 mm, ID 1.10 mm, Sutter Instrument, Novato, CA, USA) were pulled using a micropipette puller (P-87, Sutter Instrument), and were subsequently polished using a microforge (Narishige Instruments, Inc., Sarasota, FL, USA). The extracellular bath solution had the following composition (in mM): 140 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 0.02 EGTA (pH 7.4 adjusted with NaOH). The pipette solution had the following composition (in mM): 120 CsCl; 20 TEA-Cl; 8 NaCl; 1 MgCl2; 1 EGTA; 10 HEPES, and 5 MgATP (pH 7.25 adjusted with CsOH). Resistance of the pipettes was between 3 and 6 MV. Currents were recorded with an EPC-10 amplifier with Pulse 8.60 acquisition software. The currents were analyzed by a Pulsefit 8.60 software (HEKA Electronik, Lambrecht, Germany). Data filtering and sampling frequencies were set at, respectively, 2 and 10 kHz. Very soon after a whole-cell mode was established, the holding potential was set at 70 mV or other voltages stated in the text. The cell was stimulated by continuous depolarizing pulses (10 mV, 10-s intervals) to examine drug inhibition. All the experiments were performed at room temperature (25 8C).

Idrug =Icontrol ¼ 1=f1 þ ð½drug=IC50 Þ g Idrug is the current in the presence of hinokiol, Icontrol is the current in the absence of hinokiol, [drug] is the extracellular hinokiol concentration, IC50 is the hinokiol concentration giving rise to 50% inhibition, and n is the Hill coefficient. The effects of hinokiol on Na+ currents were also studied in hippocampal CA1 neurons of 5–10-day-old neonatal Sprague– Dawley rats of both sexes using whole-cell patch clamping. The procedures were approved by the China Medical University Institutional Animal Care and Use Committee and in accordance with the care and use of laboratory animal guidebook from the Chinese Taipei Society of Laboratory Animal Sciences. Soon after the rats were anaesthetized with isoflurane and decapitated, the brains were removed. A block of tissue containing the hippocampus was separated from the brain and glued to the cutting chamber of a tissue slicer with cyanoacrylate glue. Ice-cold Krebs solution used to fill the chambers contained (mM): 127 NaCl, 1.9 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose; this solution was saturated with 95% O2 and 5% CO2. Coronal hippocampal slices (300-mm thick) were prepared by a tissue slicer (DTK-1000, Dosaka, Kyoto, Japan). The slices were then put in an incubation chamber in Krebs solution for 1 h under continuous oxygenation. The slices were then transferred to a recording chamber (volume <0.5 ml). The slice was pressed onto the bottom of the recording chamber with a grid of nylon threads attached to a U-shaped platinum frame and was superfused with Krebs solution for 30 min. Hippocampal neurons were viewed using an upright microscope with a water-immersion objective lens. Pipettes were prepared from thin-walled fiber-filled borosilicate glasses (WPI, OD 2.0 mm, World Precision Instrument, Inc., Sarasota, FL, USA) and filled with pipette solution, which contained (mM): 130 K+ gluconate, 1 MgCl2, 2 CaCl2, 4 ATP, 10 EGTA, and 10 HEPES. These pipettes had resistance from 4 to 6 MV. Currents and membrane potentials were recorded with a Multiclamp 700B amplifier, lowpass filtered at 2 kHz and later analyzed using a pCLAMP software

Y.-W. Wang et al. / Pharmacological Reports 67 (2015) 1049–1054

(version 10.2, Axon Instruments). All experiments were performed at room temperature (25 8C).

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A 1.0 0.8

Results are expressed as means  SEM. Unpaired or paired Student’s t-test was used for two groups. For multiple group comparisons, one-way ANOVA with Tukey’s HSD post hoc test was used. A value of p < 0.05 is considered statistically significant.

I/Imax

Statistical analysis

DMSO Hinokiol

0.6 0.4 0.2 0.0

Results

-140 -120 -100 -80

A 30 μM Hinokiol 500 pA 5 ms

Before Hinokiol

B Idrug/Icontrol

1.2

-70 mV -100 mV

1.0 0.8 0.6 0.4 0.2 0.0 1

10 100 [Hinokiol] μM

1000

Fig. 2. Voltage-triggered Na+ currents in N2A cells were suppressed by hinokiol. (A) Na+ currents were elicited by 10 mV pulses (holding potential was 70 mV) in the absence and presence of 30 mM hinokiol. (B) Concentration–response curves are constructed to show current inhibition by hinokiol at 70 and 100 mV holding potentials. Each data point is the mean  SEM of 3–8 cells.

-40

-20

0

20

B 1.0 0.8

G/Gmax

When hinokiol (30 mM) was applied to the bath, it caused an inhibition of voltage-gated Na+ currents in N2A cells (Fig. 2A). Addition of the solvent DMSO did not affect the Na+ currents (not shown). At a holding potential of 70 mV (a potential at which Na+ channels were partially inactivated; see Fig. 3A), the IC50 value was 11.3 mM and the Hill coefficient was 1.2 (Fig. 2B). At a holding potential of 100 mV (a potential at which Na+ channels were fully available; see Fig. 3A), the IC50 value was 37.4 mM and the Hill coefficient was 0.84. Hence, the inactivated state of the channel affected the block of VGSC by hinokiol (thus state-dependence of block). As the Hill coefficients revealed, there might only be a single binding site in the VGSC for hinokiol. Whether hinokiol modulated Na+ channel gating was investigated. Treatment with 30 mM hinokiol caused a significant left shift in the steady-state inactivation curve (V1/2 = 62.0  2.7 mV and 74.9  3.7 mV in the absence and presence of hinokiol, respectively; p < 0.05) (Fig. 3A). Hinokiol treatment did not alter the slope factors (10.4  2.7 and 8.4  1.8 in the absence and presence of hinokiol, respectively; p > 0.05). As shown in Fig. 3B, Na+ channel voltage-dependence of activation was not modified by hinokiol. We next examined whether hinokiol caused a closed-channel block or open-channel block. In these experiments, a Na+ current

-60

Voltage (mV)

DMSO Hinokiol

0.6 0.4 0.2 0.0 -80

-60

-40

-20

0

20

Voltage (mV) Fig. 3. Steady-state inactivation curves, but not voltage-dependence of activation of VGSC in N2A cells, were affected by hinokiol. (A) A steady-state inactivation protocol was conducted in the absence or presence of 30 mM hinokiol. A 10 mV test pulse was preceded by a pre-pulse of 2 s of different voltages. The test pulse currents were normalized to the maximal test pulse current and plotted against the pre-pulse voltages. The curves were fitted by the Boltzmann equation. Each data point is the mean  SEM of 6–7 cells. (B) Voltage-dependence of activation: increasing depolarizing steps (holding potential of 70 mV and then 10-mV increments) were used to stimulate Na+ currents, and conductance (G) is calculated as described in Materials and methods section. The conductance in the control group and the 30 mM hinokiol-treatment group was then normalized with the respective maximum conductance (Gmax) and then plotted against the applied depolarization voltages. The curves were fitted by the Boltzmann equation. Each data point is the mean  SEM of 4–5 cells.

was firstly triggered and recorded (Fig. 4A left trace) and then stimulation was stopped. The cell was then exposed to hinokiol for 1 min and stimulated the second time to record another current (Fig. 4A right trace). The latter was diminished by 72.3  5.9% (Fig. 4B; n = 4; p < 0.05). These data suggest hinokiol was able to inhibit the Na+ channel while the latter is closed (hence closedchannel block) as during hinokiol exposure (1-min), the Na+ channels had not been stimulated. Lidocaine, a frequently used local anesthetic, inhibits VGSC in a manner that the cumulative block enhances with stimulation frequency (thus use-dependence) [7,9]. Whether hinokiol blocked Na+ currents with frequency-dependence was then studied. Stimulation every 3.0, 1.0, and 0.3 s gave rise to 0.33, 1, and 3.33 Hz, respectively. In the presence of 30 mM lidocaine, the degree of inhibition did not increase at 0.33 Hz, but at 1 and 3.33 Hz, cumulative block enhanced in a frequency-dependent fashion (p < 0.05; Fig. 5). On the contrary, there was no significant enhancement of cumulative block by 10 mM hinokiol at all frequencies. We used 10 mM hinokiol as the latter was equiefficacious to 30 mM lidocaine. Therefore our data suggest the absence of frequency-dependence in hinokiol block. Whether hinokiol affected channel recovery was then examined. A twin-pulse protocol was employed to examine the VGSC recovery, which is fitted with an exponential function (Fig. 6). The

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A Before Hinokiol

After Hinokiol

0.33 Hz

A

I/Iinitial

1.0

500 pA

0.8 0.6

DMSO Hinokiol Lidocaine

0.4

5 ms

0.2 0

B

2

4

6

8

10

8

10

Pulse number

B

80 60 40

*

20

I/Iinitial

Current (% Control)

100

1 Hz

1.0 0.8 0.6

DMSO Hinokiol Lidocaine

0.4

0 Before Hinokiol

After Hinokiol

Fig. 4. Hinokiol caused closed-channel block of Na+ currents in N2A cells. A 10 mV depolarization was used to trigger the first current (current before hinokiol) (A, left trace). The cell received no further triggering and was exposed to 30 mM hinokiol for 1 min. A 10 mV depolarization was used again to trigger the second current (current after hinokiol) (A, right trace). The results are quantified in B. Results are mean  SEM from four cells. *Significantly different (p < 0.05) from the control.

0.2 0

Discussion Although there have been reports describing multiple pharmacological effects of hinokiol such as anti-oxidation, anti-cancer, and anti-inflammation effects, the effects of hinokiol on neuronal physiology or ion channel activities have not been examined. For the first time we reported here the inhibition of VGSC by hinokiol in neuroblastoma N2A cells, differentiated NG108-15 cells and rat hippocampal CA1 neurons. Steady-state (or prolonged) depolarization has been known to cause voltage-gated ion channel inactivation; some ion channel inhibitors bind with higher affinities to partially inactivated channels [7]. For example, dihydropyridines inhibit inactivated L-type voltage-gated Ca2+ channels with much higher potency [10]. Diphenidol (an anti-emetic), lidocaine (a local anesthetic) and osthol (a neuroprotective agent) inhibit partially inactivated VGSC

4

6

Pulse number

C

3.33 Hz

1.0

I/Iinitial

0.8

DMSO control group and hinokiol-treated group recovered with similar time constants of 23.2  3.4 and 30.2  3.3 ms, respectively (n = 6–7; p > 0.05), suggesting channel recovery was not significantly affected in the presence of hinokiol. Whether hinokiol inhibited VGSC in differentiated neuronal NG108-15 cells was investigated. Na+ currents were inhibited by 73.8  15.9% (p < 0.05; n = 4; Fig. 7). We also examined whether hinokiol affected currents in hippocampal CA1 neurons of neonatal rats. Treatment with 30 mM hinokiol suppressed the Na+ current (at 20 mV) by 28.1  4.8% (n = 4; p < 0.05); the effect of hinokiol on outward K+ currents (at +20 mV) was only very mildly inhibited by 6.4  1.9% (n = 4; p < 0.05) (Fig. 8A and B). CA1 neurons exhibited spontaneous generation of action potential. After hinokiol treatment, whilst resting membrane potentials remained unaffected (before and after hinokiol were 57.8  1.2 and 57.9  2.0 mV, respectively), action potential amplitude was significantly reduced by 16.4  0.6% (n = 4; p < 0.05) (Fig. 8C and D).

2

*

0.6 0.4

* * * * * * * * DMSO Hinokiol Lidocaine

0.2 0

2

4

6

Pulse number

8

10

Fig. 5. Frequency-dependence was not observed in hinokiol inhibition of Na+ currents in N2A cells. At a holding potential of 70 mV, cells were triggered by 10 mV pulses at various frequencies (A, 0.33 Hz; B, 1 Hz; C, 3.33 Hz) in the absence or presence of 10 mM hinokiol or 30 mM lidocaine. The maximum current amplitudes of the second to tenth pulses (I) were normalized with the maximum current amplitudes of the first pulse (Iinitial) and then plotted against the number of pulse. Each data point is the mean  SEM of 3 cells. *Significantly different (p < 0.05) from the DMSO control.

with higher affinities [9,11]. Hinokiol joins the above list of drugs as this diterpenoid blocked VGSC more potently when the channel was partially inactivated (Figs. 2B and 3A). Hence, the affinity of the binding site for hinokiol was augmented in inactivated VGSC. In the presence of hinokiol, there was a left-shift of the steady-state inactivation curve (Fig. 3A) but neither activation kinetics nor voltage-dependence of activation was affected (Figs. 1A and 3B). Rather unexpectedly, hinokiol did not significantly affect channel recovery from inactivation (Fig. 6). Taken together, our data suggest hinokiol blocked VGSC not by affecting activation, nor by retarding channel recovery from inactivation, but mainly by intensifying Na+ channel inactivation. It is remarkable that osthol inhibits VGSC in N2A cells in a state-dependent manner, causing both a left-shift of the steady-state inactivation curve and a retardation of channel recovery from inactivation [9]. Hence, there appear to be subtle differences between hinokiol and osthol in their interaction with VGSC.

Y.-W. Wang et al. / Pharmacological Reports 67 (2015) 1049–1054

Fraction recovered

1.0 0.8 0.6 0.4

DMSO Hinokiol

0.2 0.0 0

20

40

60

80

100

Time (ms) Fig. 6. Recovery from channel inactivation was not affected by hinokiol in N2A cells. Channel recovery experiments were performed in cells treated with and without 30 mM hinokiol. A double-pulse protocol was used in which the first and second test pulses (10 mV, 100-ms) were separated by different time intervals. The maximum current amplitude of the second pulse (I2) is normalized with that of the first pulse (I1). The normalized data are plotted against the time intervals. The curves are fitted with a single exponential function. Results are mean  SEM from 6–7 cells of each group.

Before Hinokiol

After Hinokiol

400 pA 3 ms Fig. 7. Voltage-gated Na+ currents in differentiated NG108-15 cells were inhibited by hinokiol. Na+ currents were triggered by 10 mV pulses (holding potential was 70 mV; 10-s intervals) in the absence and presence of 30 mM hinokiol. Similar results were obtained in three other experiments.

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reveal that hinokiol block is a closed-channel block. Lidocaine, by contrast, is a phasic blocker. As shown in Fig. 5, lidocaine block increased progressively as the stimulation frequency was raised to 1 and 3.33 Hz. These data indicate use-dependence or frequencydependence of lidocaine block. Thus, lidocaine gains access to the internal cavity once the channel opens. By contrast, the lack of frequency-dependence of hinokiol block suggests this drug does not bind to the internal cavity. Hinokiol also suppressed VGSC in differentiated NG108-15 cells (Fig. 7), which express a multitude of neuronal VGSC members such as Nav1.1, Nav1.2, Nav1.3, Nav1.6 and Nav1.7 [12]. It is unknown, which VGSC members express in N2A cells, but since these cells were of brain in origin, they presumably express certain neuronal VGSC members. Therefore, hinokiol is expected to cause certain extent of VGSC inhibition in neurons and become neuromodulatory. In fact we did observe hinokiol (at 30 mM) caused a mild inhibition of VGSC and a modest reduction in action potential amplitude in rat hippocampal neurons (Fig. 8). In future development of this drug as a potential therapeutic agent treating cancer and inflammation [1,3,4], such neuromodulatory effects are to be taken into precaution. On the other hand, VGSC inhibition by hinokiol may open other therapeutic opportunities. Inhibition of VGSC is a shared property of many anesthetics such as lidocaine and propofol [7]. VGSC block is believed to be the basic mechanism of the anti-convulsant phenytoin [6]. In addition, anxiolytic drugs such as lamotrigine, carbamazepine, and riluzole are VGSC blockers [13,14]. Whether VGSC block by hinokiol could be translated into anesthetic, anti-convulsant or anti-anxiety applications would await further research into the interaction of hinokiol with different neuronal VGSC members. In conclusion, our results suggest hinokiol inhibited VGSC in a closed-channel block manner and intensified channel inactivation. Conflict of interest

Two modes of channel block, namely, tonic block and phasic block, have been well documented. The former does not depend on channel opening and is thus termed closed-channel block. The latter takes place only after channel opening and is thus termed open-channel block. The degree of open channel block augments with the extent of channel opening (see below). Data in Fig. 4

The authors declare no conflict of interests Funding bodies National Science Council, Taipei, Taiwan; China Medical University, Taichung, Taiwan; Ministry of Education, Taiwan; Department of Health, Taiwan; Eda Hospital, Taiwan. Acknowledgments Y.M.L. would like to thank the Ministry of Science and Technology of Taiwan for providing funding (103-2320-B-039035-). Y.H.K.’s funding has been in part supported by China Medical University, Taiwan under the Aim for Top University Plan of the Ministry of Education, Taiwan, and in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104TDU-B-212-113002). K.C.W. would like to thank Eda Hospital and I-Shou University for support (EDAHP 104004). References

Fig. 8. Effects of hinokiol on Na+ currents and action potentials in rat hippocampal CA1 neurons. At a holding potential of 70 mV, the neuron was depolarized (20 and +20 mV) to elicit voltage-gated Na+ and K+ currents in the absence (A) or presence (B) of 30 mM hinokiol. Spontaneously generated action potentials were recorded before and after the application of 30 mM hinokiol (C and D). Similar results were obtained in three other experiments.

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