Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4′-Hydroxy-3′-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor

Abstract

Apocynin (aPO, 4′-Hydroxy-3′-methoxyacetophenone) is a cell-permeable, anti-inflammatory phenolic compound that acts as an inhibitor of NADPH-dependent oxidase (NOX). However, the mechanisms through which aPO can interact directly with plasmalemmal ionic channels to perturb the amplitude or gating of ionic currents in excitable cells remain incompletely understood. Herein, we aimed to investigate any modifications of aPO on ionic currents in pituitary GH₃ cells or murine HL-1 cardiomyocytes. In whole-cell current recordings, GH₃-cell exposure to aPO effectively stimulated the peak and late components of voltage-gated Na+ current (I_Na) with different potencies. The EC₅₀ value of aPO required for its differential increase in peak or late I_Na in GH₃ cells was estimated to be 13.2 or 2.8 μM, respectively, whereas the K_D value required for its retardation in the slow component of current inactivation was 3.4 μM. The current–voltage relation of I_Na was shifted slightly to more negative potential during cell exposure to aPO (10 μM); however, the steady-state inactivation curve of the current was shifted in a rightward direction in its presence. Recovery of peak I_Na inactivation was increased in the presence of 10 μM aPO. In continued presence of aPO, further application of rufinamide or ranolazine attenuated aPO-stimulated I_Na. In methylglyoxal- or superoxide dismutase-treated cells, the stimulatory effect of aPO on peak I_Na remained effective. By using upright isosceles-triangular ramp pulse of varying duration, the amplitude of persistent I_Na measured at low or high threshold was enhanced by the aPO presence, along with increased hysteretic strength appearing at low or high threshold. The addition of aPO (10 μM) mildly inhibited the amplitude of erg-mediated K+ current. Likewise, in HL-1 murine cardiomyocytes, the aPO presence increased the peak amplitude of I_Na as well as decreased the inactivation or deactivation rate of the current, and further addition of ranolazine or esaxerenone attenuated aPO-accentuated I_Na. Altogether, this study provides a distinctive yet unidentified finding that, despite its effectiveness in suppressing NOX activity, aPO may directly and concertedly perturb the amplitude, gating and voltage-dependent hysteresis of I_Na in electrically excitable cells. The interaction of aPO with ionic currents may, at least in part, contribute to the underlying mechanisms through which it affects neuroendocrine, endocrine or cardiac function.

1. Introduction

Apocynin (aPO, 4′-Hydroxy-3′-methoxyacetophenone), a polyphenolic compound, is a naturally occurring ortho-methoxy-substitued catechol isolated from a variety of plant sources, including Apocynum cannabinum, Pierorhiza kurroa, and so on [1]. Of note, this compound has been widely used as a selective inhibitor of NADPH-dependent oxidase (NOX) [2,3,4,5]. Alternatively, it has been recognized to be one of the most promising drugs in a variety of pathophysiological disorders, such as inflammatory and neurodegenerative diseases, glioma, and cardiac failure [1,3,5,6,7,8,9,10,11]

aPO has been recently shown to ameliorate cardiac function (e.g., structural remodeling) in heart failure [6,7,11,12,13]. Pituitary cells were previously demonstrated to be expressed in the activity of NOX [14,15]. aPO has been reported to blunt the progression of neuroendocrine alterations induced by social isolation, which were thought to be mainly through its inhibition of NOX activity [16]. However, whether aPO exercises any modifications on ionic currents remains largely unknown.

The voltage-gated Na⁺ (Na_V) channels, nine subtypes of which are denoted Na_V1.1 through Na_V1.9, belong to the larger protein superfamily of voltage-dependent ion channels and their activity plays an essential role in the generation and propagation of action potentials (APs) in electrically excitable cells. The Na_V channels contain four homologous domains (DI-DIV), each of which consists of a six α-helical transmembrane domain (S1–S6) and a reentry P loop between S5 and S6. Na_V1.5 channels primarily underlie AP initiation and propagation in the heart, these channels have also been shown to be critical determinants of AP duration, particularly in the setting of certain arrhythmias (e.g., LQT-3 syndrome) [17,18]. Previous studies have demonstrated the ability of aPO to attenuate angiotensin II-induced activation of epithelial Na⁺ channels in human umbilical vein endothelial cells as well to blunt activation of these channels caused by epidermal growth factor, insulin growth factor-1 or insulin [19,20]. However, the issue of how aPO or other related compounds could perturb the amplitude or kinetic gating of transmembrane ionic currents (e.g., voltage-gated Na⁺ current [I_Na]) still remains unmet.

Therefore, in the present study, the electrophysiological effects of aPO and other related compounds in pituitary GH₃ cells and in HL-1 atrial cardiomyocytes were investigated. We sought to (1) evaluate whether the aPO presence has any effect on the amplitude, gating and voltage-dependent hysteresis (Vhys) of I_Na residing in GH₃ cells; (2) compare the effect of other related compounds on the peak amplitude of I_Na; (3) study the effect of aPO on erg-mediated K⁺ current in GH₃ cells; and (4) investigate the effect of aPO on I_Na in HL-1 cardiomyocytes. Findings from this study, for the first time, provide distinctive evidence to show that, in addition to its effectiveness in suppressing NOX activity, the differential stimulation by aPO of peak and late I_Na may be engaged in varying ionic mechanisms underlying its perturbations on the functional activities of electrically excitable cells (e.g., GH₃ or HL-1 cells).

2. Materials and Methods

2.1. Chemicals, Drugs and Solutions Used in the Present Work

Apocynin (aPO, NSC 2146, NSC 209524, acetovanillone, acetoguaiacone, 4′-Hydroxy-3′-methoxyacetophenone, 1-(4-Hydroxy-3-methoxyphenyl)ethanone, C₉H₁₀O₃, CAS number: 498-02-2, https://pubchem.ncbi.nlm.nih.gov/compound/Acetovanillone (accessed on 16 September 2004)), methylglyoxal (MeG, acetylformaldehyde, pyruvaldehyde, pyruvic aldehyde), norepinephrine, superoxide dismutase (SOD), tefluthrin (Tef), tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were acquired from Sigma-Aldrich (Merck, Taipei, Taiwan), rufinamide (RFM, 1-[(2,6-difluorophenyl]-1H-1,2,3-triazole-4-carboxamide), E-4031 and ranolazine (Ran) were from Tocris (Union Biomed, Taipei, Taiwan), and esaxerenone (ESAX) was from MedChemExpress (Gene-chain, Kaohsiung, Taiwan). Unless noted otherwise, culture media (e.g., F-12 medium), horse serum, fetal bovine or calf serum, L-glutamine, and trypsin/EDTA were purchased from HyCloneTM (Thermo Fisher Scientific, Tainan, Taiwan), while all other chemicals were of laboratory grade and taken from standard sources.

The HEPES-buffered normal Tyrode’s solution used in this work had an ionic composition, comprising (in mM): NaCl 136.5, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.53, glucose 5.5, and HEPES 5.5, and the pH was adjusted with NaOH to 7.4. For measurements of I_Na or I_Na(P), we kept GH₃ or HL-1 cells immersed in Ca²⁺-free, Tyrode’s solution in attempts to avoid the contamination of Ca²⁺-activated K⁺ currents and voltage-gated currents. To record K⁺ currents, we filled up the recording pipette with a solution containing (in mM): K-aspartate 130, KCl 20, KH₂PO₄ 1, MgCl₂ a, Na₂ATP 3, Na₂GTP 0.1, EGTA 0.1, HEPES 5, and the pH was titrated to 7.2 by adding KOH, while to measure I_Na or I_Na(P), we substituted K⁺ ions in internal pipette solution for equimolar Cs⁺ ions and the pH in the solution was adjusted to 7.2 by adding CsOH. All solutions used in this study were prepared using demineralized water from Milli-Q purification system (Merck). On the day of experiments, we filtered the bathing or filling solution and culture medium by using Acrodisc^® syringe filter with a 0.2-μm pore size (Bio-Check, Tainan, Taiwan).

2.2. Cell Preparations

These are provided in the Supplemental Materials mentioned in previous studies [21,22].

2.3. Electrophysiological Measurements

Shortly before experiments, we dispersed cells with 1% trypsin/EDTA solution and an aliquot of cell suspension was quickly placed in a custom-built chamber affixed to the stage of a CKX-41 inverted microscope (Olympus; Taiwan Instrument, Tainan, Taiwan). Ionic currents in GH₃ or HL-1 cells were measured with an RK-400 operational patch-clamp amplifier (Bio-Logic, Claix, France) or an Axopclamp-2B amplifier (Molecular Devices, Sunnyvale, CA, USA), which was equipped with a Digidata 1440A device (Molecular Devices). Ionic currents were recorded in whole-cell or cell-attached configuration of the patch-clamp technique [23,24]. By using a PP-830 vertical puller (Narishige; Taiwan Instrument, Taipei, Taiwan) or a Flaming-Brown P97 horizontal puller (Sutter, Novato, CA, USA), the recording pipettes were pulled from Kimax-51 (#34500) borosilicate glass capillaries (Kimble; Dogger, New Taipei City, Taiwan), and they had tip resistances of 3–5 MΩ in situations when filled with internal pipette solutions stated above. All measurements were undertaken at room temperature (20–25 °C) on the stage of an inverted DM-II fluorescence microscope (Leica; Major Instruments, Kaohsiung, Taiwan). Data acquisition with varying voltage-clamp waveforms (i.e., analog-to-digital and digital-to-analog) was performed using the pClamp 10.7 software suite (Molecular Devices). The liquid junction potentials were zeroed immediately before seal formation was made, and the whole-cell data were corrected.

The signals were monitored and digitally stored on-line at 10 kHz in an ASUS ExpertBook laptop computer (P2451F; Yuan-Dai, Tainan, Taiwan). During the measurements, the Digidata 1440A was operated using pClamp 10.7 software run on Microsoft Windows 7 (Redmond, WA, USA). The laptop computer was placed on the top of an adjustable Cookskin stand (Ningbo, Zhejiang, China) to enable efficient operation during the measurements.

2.4. Whole-Cell Data Analyses

To determine concentration-dependent stimulation of apocynin on the transient (peak) or late I_Na, we kept cells bathed in Ca²⁺-free Tyrode’s solution. During the measurements, we voltage-clamped the examined cell at −80 mV and the brief depolarizing pulse to −10 mV was applied to evoke I_Na. The late I_Na in response to 100 μM aPO was taken as 100% and those (i.e., peak and late I_Na) during exposure to different aPO concentrations (0.3–30 μM) were thereafter compared. The concentration-response data for stimulation of peak or late I_Na in pituitary GH₃ cells were least-squares fitted to the Hill equation. That is,

$$percentage decrease(\%)=\frac{E_{max}\times {\left[aPO\right]}^{n_H}}{E{C}_{50}^{n_H}+{\left[aPO\right]}^{n_H}}$$

(1)

In this equation, [aPO] is the aPO concentration used, ⁿ_H the Hill coefficient, EC₅₀ the concentration needed for a 50% inhibition of peak or late I_Na, and E_max the maximal stimulation of peak or late I_Na caused by the addition of aPO.k.

The stimulatory effect of aPO on I_Na is thought to be explained by a state-dependent activator that binds preferentially to the open state of the Na_V channel. From a simplifying assumption, the first-order binding scheme was given as follows:

(2)

or

$$\frac{dC}{dt}=O\times \beta -C\times \alpha$$

(3)

$$\frac{dO}{dt}=C\times \alpha +O·\left[aPO\right]\times k_{-1}-O\times \beta -O\times k_{+1}^{*}·\left[aPO\right]$$

(4)

$$\frac{d\left(O·\left[aPO\right]\right)}{dt}=O\times k_{+1}^{*}\left[aPO\right]-O·\left[aPO\right]\times k_{-1}$$

(5)

where [aPO] is the aPO concentration applied, and α or β the voltage-gated rate constant for the opening or closing of the Na_v channels, respectively. k*₊₁ or k₋₁ represents the forward (i.e., on or bound) or reverse (i.e., off or un-bound) rate constant of aPO, respectively, while C, O, or O·[aPO] in each term denotes the closed (resting), open, or open-[aPO] state, respectively.

Forward or backward rate constants, k*₊₁ or k₋₁, were respectively determined from the time constants of current decay activated by the brief step depolarization from −80 to −10 mV. The time constants of I_Na inactivation were estimated by fitting the inactivation trajectory of each current trace with a double exponential curve (i.e., fast and slow components of current inactivation). These rate constants would be evaluated using the following equation:

$$\frac{1}{\Delta \mathsf{\tau }}=k_{+1}^{*}\times \left[aPO\right]+k_{-1}$$

(6)

where k*₊₁ or k₋₁, respectively, are ascribed from the slope or from the y-axis intercept at [aPO] = 0 of the linear regression in which the reciprocal time constant (i.e., 1/∆τ) versus varying aPO concentrations was interpolated. ∆τ indicates the difference in the slow component of current inactivation (τ_inact(S)) obtained when the τ_inact(S) value during exposure to each concentration (0.03–30 μM) was subtracted from that in the presence of 100 μM aPO (Figure 1C).

The quasi-steady-state inactivation curve of peak I_Na with or without the aPO addition identified in GH₃ cells was established on the basis of a simple Boltzmann distribution (or the Fermi–Dirac distribution):

$$I=\frac{I_{max}}{1+e^{\frac{\left(V-V_{1/2}\right)qF}{RT}}}$$

(7)

where I_max is the maximal peak I_Na in the absence or presence of 10 μM aPO; V the conditioning potential in mV; V_1/2 the half-maximal inactivation in the relationship of the curve; q the apparent gating charge; F Faraday’s constant; R the universal gas constant; and T the absolute temperature.

2.5. Curve-Fitting Procedures and Statistical Analyses

Curve fitting (linear or non-linear (e.g., exponential or sigmoidal curve)) to various data sets was carried out with the goodness of fit by using various maneuvers, such as the Microsoft “Solver” function embedded in Excel 2019 (Microsoft) and 64-bit OriginPro^® 2016 program (OriginLab; Scientific Formosa, Kaohsiung, Taiwan). The data are presented as the mean ± standard error of the mean (SEM), with sample sizes (n) indicating the number of GH₃ or HL-1 cells from which the data were collected. The Student’s t-test (paired or unpaired) and the analyses of variance (ANOVA-1 or ANOVA-2) with or without repeated measures followed by post-hoc Fisher’s least-significant different test were performed. The analyses were performed using SPSS version 20.0 (Asia Analytics, Taipei, Taiwan). A p value of less than 0.05 was considered to indicate the statistical difference.

3. Results

3.1. Effect of aPO on the Voltage-Gated Na+ Current (I_Na) Recorded from Pituitary GH₃ Cells

In the first stage of measurements, we kept cells immersed in a Ca²⁺-free Tyrode’s solution containing 0.5 mM CdCl₂, the composition of which was stated in Materials and Methods, and we filled up the pipette by using the Cs⁺-containing solution. As the whole-cell configuration was firmly established, we voltage-clamped the tested cell at the level of −80 mV and a brief step depolarization to −10 mV was delivered to activate I_Na with a rapid activation and inactivation [23,25,26]. Of interest, one minute after cells were continually exposed to aPO, the peak amplitude of I_Na was progressively increased, and the concomitant inactivation time course of the current slowed (Figure 1A). In the presence of 10 μM aPO, the peak I_Na amplitude in response to rapid depolarizing pulse from −80 to −10 mV was significantly increased to 445 ± 31 pA (n = 9, p < 0.05) from a control value of 315 ± 22 pA. Additionally, the slow component of the inactivation time constant of I_Na activated by brief membrane depolarization was conceivably prolonged to 65.1 ± 10.2 ms (n = 9, p < 0.05) from a control value of 11.3 ± 2.3 ms (n = 9), although the fast component of the inactivation time constant did not differ significantly between absence and presence of aPO. After washout of aPO, the current amplitude was back to 306 ± 19 pA (n = 8, p < 0.05). Similarly, the deactivation time course of I_Na at −50 mV was prolonged in the presence of aPO.

The relationship between the aPO concentration and the peak or late component of I_Na was further analyzed and constructed in GH₃ cells. Each cell was depolarized from −80 to −10 mV and current amplitudes at different concentrations (0.3–100 μM) of aPO were compared. As can be seen in Figure 1B, the application of aPO resulted in a concentration-dependent increase in peak or late I_Na activated by a short depolarizing pulse. The EC₅₀ value for aPO-stimulated peak or late I_Na was 13.2 or 2.8 μM, respectively, and aPO at a concentration of 100 μM almost fully increased I_Na. The data, therefore, reflect that aPO has a specific stimulatory action on I_Na in GH₃ cells, and that the late component of I_Na was stimulated to a greater extent than the peak component of the current.

3.2. Evaluating aPO’s Time-Dependent Slowing of I_Na Inactivation

It needs to be mentioned that increasing aPO not only resulted in increased amplitude in the peak I_Na but also caused a clear and marked retardation in the magnitude of I_Na inactivation in response to rapid membrane depolarization. According to the first-order reaction scheme (indicated under Materials and Methods), the relationship between 1/∆τ and [aPO] turned out to be linear (Figure 1C). The forward and backward rate constants were estimated to be 0.00898 ms⁻¹μM⁻¹ or 0.0303 ms⁻¹, respectively; thereafter, the apparent dissociation constant (i.e., K_D = k₋₁/k₊₁*) for the binding of aPO to the Na_v channels was consequently yielded to be 3.4 μM, a value which was noticeably close to the estimated EC₅₀ value for aPO-mediated stimulation of late I_Na determined from the concentration-response curve (Figure 1B).

3.3. Effect of aPO on the Current-Voltage (I-V) Relationship or Steady-State Inactivation Curve of I_Na

We continued to examine the stimulatory effect of aPO at different membrane potential, and an I-V relationship of I_Na without or with the aPO addition was constructed. As depicted in Figure 2A, the I-V relationship of I_Na was shifted slightly to more negative potentials during cell exposure to aPO (10 μM). Additionally, the stimulatory effect of aPO on the steady-state inactivation curve of I_Na was further characterized (Figure 2B). In this stage of experiments, a 40-ms conditioning pulse to various membrane potentials (from −120 to +20 mV in 10-mV steps) was delivered to precede the test pulse (40 ms in duration) to −10 mV from a holding potential of −80 mV. Under this experimental protocol, the relationship between the conditioning potentials and the normalized amplitudes of I_Na with or without the addition of aPO (10 μM) was constructed and properly fitted to a Boltzmann type sigmoidal function (indicated under Materials and Methods) by using a non-linear regression analysis. In the absence and presence of 10 μM aPO, the V_1/2 value was noticed to differ significantly (−62.6 ± 1.3 mV (in the control) versus −49.2 ± 1.4 mV (in the presence of aPO); n = 7, p < 0.05); in contrast, the value of q (apparent gating charge) did not differ significantly (2.79 ± 0.12 e (in the control) versus 2.82 ± 0.13 e (in the presence of aPO); n = 7, p > 0.05). Therefore, cell exposure to aPO not only increased the maximal conductance of I_Na, but also shifted the inactivation curve to the rightward direction by approximately 13 mV. However, we found no evident change in the gating charge of the inactivation curve during cell exposure to aPO. As such, it is reasonable to assume that the steady-state I_Na inactivation curve in the presence of this compound was shifted rightward, with no clear adjustment in the gating charge of this curve.

3.4. Effect of aPO on the Recovery from I_Na Inactivation by Using Two-Step Voltage Protocol

We then examined whether the presence of aPO produces any effect on the recovery of I_Na from inactivation. In a two-step voltage protocol, a 50-ms conditioning step to −10 mV inactivated most of the current, and the recovery from current inactivation at the holding potential of −80 mV was examined at different times with a test step (−10 mV, 50 ms), as demonstrated in Figure 3A,B. In the control period (i.e., aPO was not present), the peak amplitude of I_Na nearly completely recovered from inactivation when the interpulse duration was set at 100 ms. The time constant course of recovery from current inactivation in the absence or presence of aPO (10 μM) was least-squares fitted to a single-exponential function with a time constant of 23.3 ± 1.1 or 11.3 ± 0.9 ms (n = 8, p < 0.05), respectively. These experimental observations indicate that cell exposure to aPO produces a significant shortening in the recovery from inactivation of I_Na in GH₃ cells.

3.5. Comparison among Effects of aPO, Tefluthrin (Tef), Tef Plus aPO, aPO Plus Rufinamide (RFM), and aPO Plus Ranolazine (Ran) on the Peak Amplitude of I_Na

Tef, a type-I pyrethroid insecticide, was reported to be an activator of I_Na [23,24,25,27], Ran is recognized as a late I_Na blocker as well as an inhibitor of NOX activity [26,28,29,30], and RFM, known to be an antiepileptic agent, was previously demonstrated to perturb I_Na inactivation [31,32]. For these reasons, we further examined and then compared the effects of these agents on peak I_Na identified in GH₃ cells. As demonstrated in Figure 4, in accordance with previous studies [23], one minute after Tef (10 μM) was applied, it was effective in stimulating peak I_Na. However, in the continued presence of Tef for two minutes, one minute after further addition of 10 μM aPO, peak I_Na was not increased further. In addition, as cells were continually exposed to 10 μM aPO, subsequent application of 10 μM RFM or 10 μM Ran was able to attenuate aPO-induced stimulation of I_Na one minute later. The results imply that aPO and Tef share a similarity to their stimulation of I_Na, and that further addition of RFM or Ran is effective in attenuating aPO-stimulated I_Na in GH₃ cells.

3.6. Stimulatory Action of aPO on I_Na in Methylglyoxal- (MeG-) or Superoxide Dismutase- (SOD-) Treated Cells

One would expect that the effect of aPO on I_Na is engaged in either its inhibition of NOX activity or the reduction in the production of reactive oxygen species. The expression of NOX was previously reported to be distributed in pituitary cells [14,15]. As such, the effect of aPO on I_Na was assessed in cells preincubated with MeG or SOD for 6 h. MeG was previously recognized to be a substrate for NOX activity [33,34,35], while SOD, an antioxidative enzyme, was reported to reduce the production of reactive oxygen species [36]. However, in GH₃ cells preincubated with MeG for 6 h, the I-V relationship of peak I_Na with or without addition of aPO is illustrated in Figure 5. For example, in cells pretreated with MeG (10 μM), aPO (10 μM) could significantly increase the amplitude of I_Na measured at the level of −20 mV from 401 ± 31 to 511 ± 39 pA (n = 7, p < 0.05). Likewise, in SOD-preincubated cells, the addition of aPO (10 μM) increased I_Na amplitude at −20 mV from 409 ± 31 to 515 ± 41 pA (n = 7, p < 0.05). Therefore, these results allowed us to suggest that the stimulatory effect of aPO on I_Na that we obtained in these cells is unlikely to be due to changes in either the production of reactive oxygen species or cytosolic NOX activity.

3.7. Effect of aPO on the Amplitude and Voltage-Dependent Hysteresis (Vhys) of Persistent Na⁺ (I_Na(P))

The Vhys behavior residing in varying types of ion channels (i.e., the difference in current trajectory in response to the upsloping and the downsloping voltages) is currently a subject of extensive research, including Na_V channels [24,37,38]. We next examined whether or how the presence of aPO is able to modify I_Na(P) Vhys activated in response to the upright isosceles-triangular ramp pulse in GH₃ cells. In this stage of our whole-cell current recordings, the tested cell was voltage-clamped at the level of −80 mV and we then applied it with a set of isosceles-triangular ramp pulses ranging between −110 and +50 mV (with a height of 160 mV) of varying ramp duration at a rate of 0.05 Hz through digital-to-analog conversion (Figure 6A). Consistent with previous observations [24,26], the amplitude of I_Na(P) in response to such upright triangular ramp voltage was noticed to display a striking figure-of-eight Vhys (i.e., ∞) in the instantaneous I-V relationship of I_Na(P) with two distinct peaks, i.e., low and high threshold I_Na(P). Alternatively, there is an initial counterclockwise direction, which time goes by, in current trajectory (i.e., high-threshold loop with a peak at −0 mV) activated by the upsloping limb, and following the downsloping limb, a clockwise direction (i.e., low-threshold loop with a peak at −80 mV) ensued (Figure 6B). Of particular interest, one minute after GH₃ cells were exposed to 30 μM aPO alone, the amplitude of I_Na(P) at high or low threshold respectively activated by the upsloping triangular ramp voltage (forward or ascending) or downsloping (backward or descending) limb of upright triangular ramp voltage was increased. The augmentation of low-threshold I_Na(P) produced by 30 μM aPO was observed to be greater than that in the high-threshold one (Figure 6C), for example, as the isosceles-triangular ramp pulse with a duration of 3.2 s (or ramp speed of ±0.1 mV/ms). In the presence of 30 μM aPO, the peak I_Na(P) amplitude measured at the level of −0 mV (i.e., high-threshold I_Na(P)) during the ascending phase of triangular ramp pulse was significantly raised to 175 ± 29 pA (n = 8, p < 0.05) from a control value (measured at the isopotential level) of 151 ± 18 pA (n = 8). Meanwhile, during cell exposure to 30 μM aPO, the peak I_Na(P) amplitude measured at −80 mV during the descending phase of such a ramp concurrently increased from 285 ± 33 to 393 ± 54 pA (n = 8, p < 0.05). Alternatively, the subsequent application of 10 μM Ran, but still in the continued presence of 30 μM aPO, was able to attenuate the aPO-mediated increase of I_Na(P) taken at either high or low threshold amplitude in the Vhys loop. These observations, therefore, enabled us to indicate that the Vhys strength of I_Na(P) activated by isosceles-triangular ramp pulses of varying ramp duration observed in GH₃ cells was enhanced in the presence of aPO (Figure 6B,C).

3.8. Effect of aPO on Erg-Mediated K⁺ Current (I_K(erg)) in GH₃ Cells

Earlier studies have demonstrated that telmisartan, an activator of I_Na, can be effective in inhibiting I_K(erg) [22]. For this reason, we decided to investigate whether aPO exercises any perturbations on I_K(erg). The biophysical and pharmacological properties of I_K(erg) in GH₃ cells have been previously reported [22,39,40,41]. In these whole-cell experiments, we bathed cells in high-K⁺, Ca²⁺-free solution, and the recording pipette was filled up with K⁺-containing solution. The composition of these solutions was detailed under Materials and Methods. The examined cell was voltage-clamped at −10 mV and the linear downsloping ramp pulse from −10 to −100 mV with a duration of 1 s was applied to it. As shown in Figure 7, the addition of 10 μM aPO resulted in a progressive decline in the amplitude of deactivating I_K(erg) in response to such a downsloping hyperpolarizing ramp. However, in the continued presence of aPO, further application of E-4031, an inhibitor of I_K(erg), was able to decease the current amplitude further. Therefore, unlike I_Na induced by aPO, I_K(erg) residing in these cells was subject to being inhibited by its presence.

3.9. Effect of aPO on I_Na Recorded from Murine HL-1 Cardiomyocytes

aPO was previously demonstrated to be a chemo-preventive agent for cardiovascular disorders though the inhibition of NOX activity [35,42,43,44]. In another set of experiments, we tested whether I_Na inherently in heart cells (i.e., HL-1 cardiomyocytes) could still be modified by the presence of aPO. The preparation of these cells was described above under Materials and Methods. Cells were kept bathed in Ca²⁺-free Tyrode’s solution in which 10 mM TEA was included, and the pipette was filled with Cs⁺-enriched solution. Noticeably, as HL-1 cells were continually exposed to aPO at a concentration of 3 or 10 μM, the amplitude of peak I_Na activated by 50-ms depolarizing pulses from −80 to −10 mV was increased; concomitantly, progressive slowing of the inactivation time course of the current was seen (Figure 8A,B). For example, cell exposure to 10 μM aPO resulted in a conceivable increase of peak I_Na from 859 ± 56 to 1381 ± 85 pA (n = 8, p < 0.05); concomitantly, the τ_inact(S) value was significantly raised to 56.3 ± 7.1 ms (n = 8, p < 0.05) from a control value of 7.1 ± 1.4 ms. After washout of aPO (i.e., aPO was removed, but cells were still exposed to Ca²⁺-free Tyrode’s solution containing 10 mM TEA), current amplitude returned 892 ± 58 pA (n = 8, p < 0.05). Alternatively, in the continued presence of aPO (10 μM), further application of either ranolazine (Ran, 10 μM) or esaxerenone (ESAX, 10 μM) was noticed to attenuate aPO-mediated stimulation of I_Na (Figure 8B). Like Ran. ESAX was recently reported to inhibit I_Na [24]. Therefore, consistent to some extent with the observations done in GH₃ cells, the results reflect the effectiveness of aPO in stimulating I_Na in response to the rapid depolarizing step in HL-1 cells.

4. Discussion

The distinctive findings in the present study are that (a) GH₃-cell exposure to aPO could increase I_Na in a concentration, time-, state-, and Vhys-dependent fashion; (b) this agent resulted in the differential stimulation of peak or late amplitude of I_Na activated by abrupt step depolarization with aneffective EC₅₀ value of 13.2 or 2.8 μM, respectively; (c) aPO mildly shifted the I-V curve of I_Na towards the depolarized potentials (i.e., a leftward shift), and it also made a rightward shift in the steady-state inactivation curve of the current towards the right side with no changes in the gating charge of the curve; (d) the recovery of the I_Na block was enhanced in its presence; (e) subsequent addition of rufinamide (RFM) or ranolazine (Ran) counteracted aPO-accentuated I_Na; (f) the stimulatory effect of aPO on I_Na remained unaltered in cells preincubated with MeG or SOD; (g) aPO was capable of increasing the high- or low-threshold amplitude of I_Na(P) elicited by the isosceles-triangular ramp at either upsloping (ascending) or downsloping (descending) limb, respectively; (h) the aPO presence mildly decreased the amplitude of I_K(erg) activated by the downsloping ramp pulse; and (i) the exposure to aPO was effective at increasing the amplitude and inactivation time constant of I_Na in HL-1 atrial cardiomyocytes. Collectively, the present results allow us to reflect that aPO-stimulated changes in the amplitude, gating, and Vhys behavior of I_Na appear to be unlinked to and upstream of its inhibitory action on NOX activity, and that it would participate in the adjustments of varying functional activities in electrically excitable cells (e.g., GH₃ or HL-1 cells), presuming that similar in vivo findings exist.

From the overall I-V relationship of I_Na demonstrated here, there was a slight shift toward more negative potential in the presence of aPO. The steady-state inactivation curve of I_Na in its presence of aPO was also shifted to a rightward direction with no apparent change in the gating charge of the curve. The increased recovery of the I_Na block was demonstrated in its presence. As a result, the window current of I_Na in GH₃ cells was expected to be increased during cell exposure to aPO. Such a small molecule may have higher affinity to the open/inactivated state than to the resting (closed) state residing in the Nav channels, despite the detailed ionic mechanism of its stimulatory action on the channel remaining elusive.

Several lines of clear evidence have been demonstrated to indicate that aPO can inhibit NOX activity and decrease the production of superoxide oxide [2,3,4,16]. Pituitary cells have been previously demonstrated to be expressed in the activity of NOX [14,15,16]. As such, the question arises as to whether the stimulatory effect of aPO on I_Na observed in GH₃ cells may actually result from either the reduction of NOX activity or the decreased level of superoxide anions [15,16]. However, this notion appears to be difficult to reconcile with the present observations disclosing that in GH₃ cells preincubated with MeG or SOD, the stimulatory effect of aPO on I_Na was indeed observed to remain effective. It is also noted that aPO can mildly inhibit the amplitude of I_K(erg). Therefore, under our experimental conditions, the stimulation of I_Na caused by aPO tends to emerge in a manner largely independent of its inhibitory effect on NOX activity; hence, the aPO molecule can exert an interaction at binding site(s) inherently existing on Na_v channels.

Perhaps more important than the issue of the magnitude of the aPO-induced increase in I_Na is that we observed the non-linear Vhys of I_Na(P) in the control period (i.e., aPO was not present) and during cell exposure to aPO or aPO plus Ran, by use of the upright isosceles-triangular ramp voltage command of varying duration through digital-to-analog conversion. In particular, when cells were exposed to aPO, the peak I_Na(p) activated by the forward (ascending or upsloping) end of the triangular ramp of varying duration was observed to be elevated, particularly at the peak level of 0 mV, whereas the I_Na(P) amplitude at the backward (descending or downsloping) end was increased at the peak level of −80 mV. In this respect, the figure-of-eight (i.e., infinity-shaped: ∞) configuration in the Vhys loop activated by the triangular ramp pulse was evidently demonstrated (Figure 6A,B). Additionally, there appeared to be two types of Vhys loops, that is, a low-threshold loop with a peak at −80 mV (i.e., activating at a voltage range near the resting potential) and a high-threshold loop with a peak at 0 mV (i.e., activating at a voltage range near the maximal I_Na elicited by rectangular depolarizing step. The presence of aPO was capable of enhancing the Vhys strength of I_Na(P) and, in its continued presence, further addition of Ran attenuated aPO-increased Vhys loop of the current. In this scenario, findings from the present observations disclosed that the triangular pulse-induced I_Na(P) was detected to undergo striking Vhys change (i.e., initial counterclockwise direction followed by clockwise one) in the voltage-dependence and that such Vhys loops were subject to enhancement by the presence of aPO.

Pertinent to the stimulatory effect of aPO on I_Na is that in this study, due to its effectiveness in increasing the Vhys magnitude of I_Na(P), the voltage-dependent movement of the S4 segment residing in Na_V channels is probably perturbed by this agent; consequently, the coupling of the pore domain to the voltage-sensor domain, which the S1–S4 segments comprise, tended to be facilitated [45,46]. Indeed, the voltage sensor energetically coupled to channel activation, which might be influenced by the aPO molecule, is supposed to be a conformationally flexible region of the Na_V-channel protein. Therefore, these findings can be interpreted to mean either that such I_Na(P), particularly during exposure to aPO, is intrinsically and dynamically endowed with “memory” of previous (or past) events, which is encoded in the conformational (or metastable) states of the Nav-channel protein, or that there is a mode shift of channel kinetics occurring regarding the voltage sensitivity of gating charge movement, which relies on the previous state (or conformation) of the Na_v channel [37,38]. Such a striking type of Vhys natively in Na_V channels would potentially play substantial roles in interfering with electrical behavior, Na⁺ overload, and hormonal sretion in varying types of excitable cells [37]. It is also worth pointing out that the subsequent addition of Ran, still in the continued presence of aPO, did produce a considerable reduction in the aPO-mediated increase in Vhys responding to triangular ramp voltage.

From pharmacokinetic studies in mice [47], following intravenous injection of aPO (5 mg/kg), the peak plasma aPO level was detected at 1 min to reach around 5500 ng/mL (or 33.1 μM). Additionally, aPO was reportedly a selective inhibitor of NOX2 activity with an effective IC₅₀ of 10 μM [48]. According to the data of Figure 1, the IC₅₀ value required for the aPO-stimulated peak or late I_Na was 13.2 or 2.8 μM, respectively, while the K_D value estimated on the basis of minimal reaction scheme was 3.4 μM. It is reasonable to assume, therefore, that aPO-induced changes in the amplitude, gating or Vhys behavior of I_Na presented herein could be highly achievable and of pharmacological relevance.

On the basis of the present experimental observations, despite the inhibitory effect on NOX activity [2,3,4], our results strongly suggest that the stimulatory actions of aPO on transmembrane ionic currents, particularly on Na_V channels, tends to be direct obligate mechanisms. Pyrethroids (e.g., permethrin and cypermethrin), known to activate I_Na, have also been reported to disrupt NOX activity in brain tissue (striatum) [49]. Therefore, through ionic mechanisms shown herein, pyrethroids or other structurally similar compounds are able to adjust the functional activities of varying types of neuroendocrine or endocrine cells, or heart cells, if similar in vivo results exist [6,7,11,12,13,50]. To this end, the overall findings from our study highlight an important alternative aspect that has to be taken into account, inasmuch as there is the beneficial or ameliorating effect of aPO in various pathologic disorders, such as inflammatory or neurodegenerative diseases, and heart failure [1,3,6,7,9,10,11,12,13,16,42].