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Article

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

1
Department of Physiology, National Cheng Kung University Medical College, No. 1, University Road, Tainan 70101, Taiwan
2
Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan 70101, Taiwan
*
Author to whom correspondence should be addressed.
Submission received: 6 August 2021 / Revised: 23 August 2021 / Accepted: 31 August 2021 / Published: 3 September 2021
(This article belongs to the Special Issue Recent Advances in the Discovery of Novel Drugs on Natural Molecules)

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 (INa) with different potencies. The EC50 value of aPO required for its differential increase in peak or late INa in GH3 cells was estimated to be 13.2 or 2.8 μM, respectively, whereas the KD value required for its retardation in the slow component of current inactivation was 3.4 μM. The current–voltage relation of INa 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 INa inactivation was increased in the presence of 10 μM aPO. In continued presence of aPO, further application of rufinamide or ranolazine attenuated aPO-stimulated INa. In methylglyoxal- or superoxide dismutase-treated cells, the stimulatory effect of aPO on peak INa remained effective. By using upright isosceles-triangular ramp pulse of varying duration, the amplitude of persistent INa 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 INa as well as decreased the inactivation or deactivation rate of the current, and further addition of ranolazine or esaxerenone attenuated aPO-accentuated INa. 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 INa 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+ (NaV) channels, nine subtypes of which are denoted NaV1.1 through NaV1.9, 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 NaV 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. NaV1.5 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 [INa]) 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 INa residing in GH3 cells; (2) compare the effect of other related compounds on the peak amplitude of INa; (3) study the effect of aPO on erg-mediated K+ current in GH3 cells; and (4) investigate the effect of aPO on INa 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 INa 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 INa or INa(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 INa or INa(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 INa, 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 INa. The late INa in response to 100 μM aPO was taken as 100% and those (i.e., peak and late INa) during exposure to different aPO concentrations (0.3–30 μM) were thereafter compared. The concentration-response data for stimulation of peak or late INa in pituitary GH3 cells were least-squares fitted to the Hill equation. That is,
p e r c e n t a g e   d e c r e a s e ( % ) = E m a x × [ a P O ] n H E C 50 n H + [ a P O ] n H
In this equation, [aPO] is the aPO concentration used, nH the Hill coefficient, EC50 the concentration needed for a 50% inhibition of peak or late INa, and Emax the maximal stimulation of peak or late INa caused by the addition of aPO.k.
The stimulatory effect of aPO on INa is thought to be explained by a state-dependent activator that binds preferentially to the open state of the NaV channel. From a simplifying assumption, the first-order binding scheme was given as follows:
Biomedicines 09 01146 i001
or
d C d t = O × β C × α
d O d t = C × α + O · [ a P O ] × k 1 O × β O × k + 1 * · [ a P O ]
d ( O · [ a P O ] ) d t = O × k + 1 * [ a P O ] O · [ a P O ] × k 1
where [aPO] is the aPO concentration applied, and α or β the voltage-gated rate constant for the opening or closing of the Nav 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 INa 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:
1 Δ τ = k + 1 * × [ a P O ] + k 1
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 (τinact(S)) obtained when the τinact(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 INa 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 = I m a x 1 + e ( V V 1 / 2 ) q F R T
where Imax is the maximal peak INa in the absence or presence of 10 μM aPO; V the conditioning potential in mV; V1/2 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 (INa) 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 INa with a rapid activation and inactivation [23,25,26]. Of interest, one minute after cells were continually exposed to aPO, the peak amplitude of INa was progressively increased, and the concomitant inactivation time course of the current slowed (Figure 1A). In the presence of 10 μM aPO, the peak INa 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 INa 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 INa at −50 mV was prolonged in the presence of aPO.
The relationship between the aPO concentration and the peak or late component of INa 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 INa activated by a short depolarizing pulse. The EC50 value for aPO-stimulated peak or late INa was 13.2 or 2.8 μM, respectively, and aPO at a concentration of 100 μM almost fully increased INa. The data, therefore, reflect that aPO has a specific stimulatory action on INa in GH3 cells, and that the late component of INa was stimulated to a greater extent than the peak component of the current.

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

It needs to be mentioned that increasing aPO not only resulted in increased amplitude in the peak INa but also caused a clear and marked retardation in the magnitude of INa 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., KD = k−1/k+1*) for the binding of aPO to the Nav 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 INa 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 INa

We continued to examine the stimulatory effect of aPO at different membrane potential, and an I-V relationship of INa without or with the aPO addition was constructed. As depicted in Figure 2A, the I-V relationship of INa 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 INa 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 INa 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 V1/2 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 INa, 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 INa 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 INa Inactivation by Using Two-Step Voltage Protocol

We then examined whether the presence of aPO produces any effect on the recovery of INa 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 INa 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 INa 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 INa

Tef, a type-I pyrethroid insecticide, was reported to be an activator of INa [23,24,25,27], Ran is recognized as a late INa 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 INa inactivation [31,32]. For these reasons, we further examined and then compared the effects of these agents on peak INa 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 INa. However, in the continued presence of Tef for two minutes, one minute after further addition of 10 μM aPO, peak INa 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 INa one minute later. The results imply that aPO and Tef share a similarity to their stimulation of INa, and that further addition of RFM or Ran is effective in attenuating aPO-stimulated INa in GH3 cells.

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

One would expect that the effect of aPO on INa 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 INa 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 INa 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 INa 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 INa 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 INa 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+ (INa(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 NaV channels [24,37,38]. We next examined whether or how the presence of aPO is able to modify INa(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 INa(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 INa(P) with two distinct peaks, i.e., low and high threshold INa(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 INa(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 INa(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 INa(P) amplitude measured at the level of −0 mV (i.e., high-threshold INa(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 INa(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 INa(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 INa(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 (IK(erg)) in GH3 Cells

Earlier studies have demonstrated that telmisartan, an activator of INa, can be effective in inhibiting IK(erg) [22]. For this reason, we decided to investigate whether aPO exercises any perturbations on IK(erg). The biophysical and pharmacological properties of IK(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 IK(erg) in response to such a downsloping hyperpolarizing ramp. However, in the continued presence of aPO, further application of E-4031, an inhibitor of IK(erg), was able to decease the current amplitude further. Therefore, unlike INa induced by aPO, IK(erg) residing in these cells was subject to being inhibited by its presence.

3.9. Effect of aPO on INa 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 INa 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 INa 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 INa from 859 ± 56 to 1381 ± 85 pA (n = 8, p < 0.05); concomitantly, the τinact(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 INa (Figure 8B). Like Ran. ESAX was recently reported to inhibit INa [24]. Therefore, consistent to some extent with the observations done in GH3 cells, the results reflect the effectiveness of aPO in stimulating INa 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 INa in a concentration, time-, state-, and Vhys-dependent fashion; (b) this agent resulted in the differential stimulation of peak or late amplitude of INa 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 INa 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 INa block was enhanced in its presence; (e) subsequent addition of rufinamide (RFM) or ranolazine (Ran) counteracted aPO-accentuated INa; (f) the stimulatory effect of aPO on INa remained unaltered in cells preincubated with MeG or SOD; (g) aPO was capable of increasing the high- or low-threshold amplitude of INa(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 IK(erg) activated by the downsloping ramp pulse; and (i) the exposure to aPO was effective at increasing the amplitude and inactivation time constant of INa 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 INa 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 INa demonstrated here, there was a slight shift toward more negative potential in the presence of aPO. The steady-state inactivation curve of INa 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 INa block was demonstrated in its presence. As a result, the window current of INa 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 INa 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 INa was indeed observed to remain effective. It is also noted that aPO can mildly inhibit the amplitude of IK(erg). Therefore, under our experimental conditions, the stimulation of INa 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 Nav channels.
Perhaps more important than the issue of the magnitude of the aPO-induced increase in INa is that we observed the non-linear Vhys of INa(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 INa(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 INa(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 INa elicited by rectangular depolarizing step. The presence of aPO was capable of enhancing the Vhys strength of INa(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 INa(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 INa is that in this study, due to its effectiveness in increasing the Vhys magnitude of INa(P), the voltage-dependent movement of the S4 segment residing in NaV 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 NaV-channel protein. Therefore, these findings can be interpreted to mean either that such INa(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 Nav channel [37,38]. Such a striking type of Vhys natively in NaV 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 INa was 13.2 or 2.8 μM, respectively, while the KD 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 INa 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 NaV channels, tends to be direct obligate mechanisms. Pyrethroids (e.g., permethrin and cypermethrin), known to activate INa, 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].

Supplementary Materials

The details of cell preparation in Materials and Methods were mentioned in Supplementary Material which is available online https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/biomedicines9091146/s1.

Author Contributions

Conceptualization, S.-N.W., H.-Y.C. and T.-H.C.; methodology, S.-N.W.; software, S.-N.W.; validation, H.-Y.C., T.-H.C. and S.-N.W.; formal analysis, S.-N.W.; investigation, H.-Y.C., T.-H.C. and S.-N.W.; resources, S.-N.W.; data curation, S.-N.W.; writing—original draft preparation, S.-N.W.; writing—review and editing, T.-H.C., H.-Y.C. and S.-N.W.; visualization, H.-Y.C., T.-H.C., and S.-N.W.; supervision, S.-N.W.; project administration, S.-N.W.; funding acquisition, S.-N.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was in part supported by a grant from the Ministry of Science and Technology (MOST-110-2320-B-006-028), Taiwan.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original data is available upon reasonable request to the corresponding author.

Acknowledgments

H.-Y.C. and T.-H.C. received the student assistantship from the Ministry of Science and Technology, Taiwan, while S.-N.W. received a Talent Award for the Outstanding Researchers from Ministry of Education, Taiwan.

Conflicts of Interest

The authors declare no competing interests that are directly relevant to the present study.

Abbreviations

AP, action potential; aPO (apocynin, 4′-Hydroxy-3′-methoxyacetophenone); EC50, concentration required for 50% stimulation; erg, ether-à-go-go-related gene; ESAX, esaxerenone; I-V, current versus voltage; IK(erg), erg-mediated K+ current; INa, voltage-gated Na+ current; INa(P), persistent Na+ current; KD, dissociation constant; MeG, methylglyoxal; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; Nav channel; voltage-gated Na+ channel; NADPH, nicotinamide adenine dinucleotide phosphate; NOX, NADPH oxidase; Vhys, voltage-dependent hysteresis; Ran, ranolazine; RFM, rufinamide; SEM, standard error of the mean; SOD, superoxide dismutase; τinact(S), slow component of inactivation time constant; TEA, tetraethylammonium chloride; Tef, tefluthrin; TTX, tetrodotoxin.

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Figure 1. Effect of aPO on the peak and late components of voltage-gated Na+ current (INa) identified in pituitary GH3 cells. These experiments were undertaken in cells bathed in Ca2+-free Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA), whereas the recording pipette was filled up with Cs+-enriched solution. (A) Representative INa traces activated by brief depolarizing pulse (indicated in the upper part). a: control (i.e., aPO was not present); b: 3 μM aPO; c: 10 μM aPO. (B) Concentration-dependent stimulation of aPO on peak or late INa (mean ± SEM; n = 8 for each point). The peak (□) or late () amplitude of the current was measured at the beginning or end of a 40-ms depolarizing pulse from −80 to −10 mV. Data analysis was performed by ANOVA-1 (p < 0.05). Each continuous line illustrates the goodness-of-fit to the Hill equation, as elaborated in Materials and Methods. The vertical broken line indicates the EC50 value required for 50% stimulation of the current (peak or late INa). (C) The relationship of the reciprocal to the time constant (i.e., 1/∆τ) versus the aPO concentration was plotted (mean ± SEM; n = 7–11 for each point). From the binding scheme (indicated under Materials and Methods), the forward (k+1*) or backward (k−1) rate constant for aPO-accentuated INa in GH3 cells was computed to be 0.00898 ms−1μM−1 or 0.0303 ms−1, respectively.
Figure 1. Effect of aPO on the peak and late components of voltage-gated Na+ current (INa) identified in pituitary GH3 cells. These experiments were undertaken in cells bathed in Ca2+-free Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA), whereas the recording pipette was filled up with Cs+-enriched solution. (A) Representative INa traces activated by brief depolarizing pulse (indicated in the upper part). a: control (i.e., aPO was not present); b: 3 μM aPO; c: 10 μM aPO. (B) Concentration-dependent stimulation of aPO on peak or late INa (mean ± SEM; n = 8 for each point). The peak (□) or late () amplitude of the current was measured at the beginning or end of a 40-ms depolarizing pulse from −80 to −10 mV. Data analysis was performed by ANOVA-1 (p < 0.05). Each continuous line illustrates the goodness-of-fit to the Hill equation, as elaborated in Materials and Methods. The vertical broken line indicates the EC50 value required for 50% stimulation of the current (peak or late INa). (C) The relationship of the reciprocal to the time constant (i.e., 1/∆τ) versus the aPO concentration was plotted (mean ± SEM; n = 7–11 for each point). From the binding scheme (indicated under Materials and Methods), the forward (k+1*) or backward (k−1) rate constant for aPO-accentuated INa in GH3 cells was computed to be 0.00898 ms−1μM−1 or 0.0303 ms−1, respectively.
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Figure 2. Stimulatory effect of aPO on averaged current–voltage (I-V) relationship (A) and steady-state inactivation curve (B) of INa present in GH3 cells. Cells were kept bathed in Ca2+-free Tyrode’s solution containing 10 mM TEA. (A) Averaged I-V relationships of INa in the absence () and presence () of 10 μM aPO (mean ± SEM; n = 8 for each point). The examined cell was held at −80 mV and the 40-ms voltage pulse ranging from −80 to +40 mV in 10-mV steps was delivered to it. The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different level of voltages) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05). (B) Effect of aPO on the steady-state inactivation curve of INa taken without () or with () the addition of 10 μM aPO. In these experiments, the conditioning voltage pulses with a duration of 40 ms to various membrane potentials between −120 and +20 mV were applied from a holding potential of −80 mV. Following each conditioning potential, a test pulse to −10 mV with a duration of 40 ms was delivered to activate INa. The normalized amplitude of INa (I/Imax) was constructed against the conditioning potential and the sigmoidal curves were optimally fitted by the Boltzmann equation (indicated under Materials and Methods). Each point represents the mean ± SEM (n = 7). The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different level of conditioning potentials) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05).
Figure 2. Stimulatory effect of aPO on averaged current–voltage (I-V) relationship (A) and steady-state inactivation curve (B) of INa present in GH3 cells. Cells were kept bathed in Ca2+-free Tyrode’s solution containing 10 mM TEA. (A) Averaged I-V relationships of INa in the absence () and presence () of 10 μM aPO (mean ± SEM; n = 8 for each point). The examined cell was held at −80 mV and the 40-ms voltage pulse ranging from −80 to +40 mV in 10-mV steps was delivered to it. The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different level of voltages) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05). (B) Effect of aPO on the steady-state inactivation curve of INa taken without () or with () the addition of 10 μM aPO. In these experiments, the conditioning voltage pulses with a duration of 40 ms to various membrane potentials between −120 and +20 mV were applied from a holding potential of −80 mV. Following each conditioning potential, a test pulse to −10 mV with a duration of 40 ms was delivered to activate INa. The normalized amplitude of INa (I/Imax) was constructed against the conditioning potential and the sigmoidal curves were optimally fitted by the Boltzmann equation (indicated under Materials and Methods). Each point represents the mean ± SEM (n = 7). The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different level of conditioning potentials) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05).
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Figure 3. Effect of aPO on the time course of recovery from INa inactivation. The cell tested was depolarized from −80 to −10 mV with a duration of 50 ms, and voltage-clamp commands with varying durations of interpulse interval (i.e., the interval between the first and second pulses) were applied to it. (A) Superimposed INa traces in the presence of 10 μM aPO. The upper part shows the voltage protocol applied. The dashed arrow indicates the trajectory of current inactivation elicited by different durations of interpulse pulse. (B) Effect of aPO on the time course of recovery from current inactivation, as the cells examined were depolarized from −80 to −10 mV. : control; : aPO (10 μM). Each smooth line was optimally fitted by a single-exponential function. The relative amplitude denotes that the peak INa taken at the second pulse is divided by that at the first one. Each point represents the mean ± SEM (n = 8). The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different interpulse intervals) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05).
Figure 3. Effect of aPO on the time course of recovery from INa inactivation. The cell tested was depolarized from −80 to −10 mV with a duration of 50 ms, and voltage-clamp commands with varying durations of interpulse interval (i.e., the interval between the first and second pulses) were applied to it. (A) Superimposed INa traces in the presence of 10 μM aPO. The upper part shows the voltage protocol applied. The dashed arrow indicates the trajectory of current inactivation elicited by different durations of interpulse pulse. (B) Effect of aPO on the time course of recovery from current inactivation, as the cells examined were depolarized from −80 to −10 mV. : control; : aPO (10 μM). Each smooth line was optimally fitted by a single-exponential function. The relative amplitude denotes that the peak INa taken at the second pulse is divided by that at the first one. Each point represents the mean ± SEM (n = 8). The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different interpulse intervals) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05).
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Figure 4. Effect of aPO, tefluthrin (Tef), Tef plus aPO, aPO plus rufinamide (RFM), and aPO plus ranolazine (Ran) on peak amplitude of INa identified in GH3 cells. (A) Representative INa traces activated by depolarizing pulse (as indicated in the upper part). a: control; b: 10 μM aPO; c: 10 μM aPO plus 10 μM RFM. (B) Summary bar graph showing effect of aPO, Tef, Tef plus aPO, aPO plus RFM, and aPO plus Ran on peak INa (mean ± SEM; n = 8–10 for each bar). The number of the control group is 10, while those in other groups are 8. Data analysis was performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05) and ** significantly different from aPO (10 μM) alone group (p < 0.05).
Figure 4. Effect of aPO, tefluthrin (Tef), Tef plus aPO, aPO plus rufinamide (RFM), and aPO plus ranolazine (Ran) on peak amplitude of INa identified in GH3 cells. (A) Representative INa traces activated by depolarizing pulse (as indicated in the upper part). a: control; b: 10 μM aPO; c: 10 μM aPO plus 10 μM RFM. (B) Summary bar graph showing effect of aPO, Tef, Tef plus aPO, aPO plus RFM, and aPO plus Ran on peak INa (mean ± SEM; n = 8–10 for each bar). The number of the control group is 10, while those in other groups are 8. Data analysis was performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05) and ** significantly different from aPO (10 μM) alone group (p < 0.05).
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Figure 5. Stimulatory effect of aPO on averaged I-V relationship of INa in GH3 cells treated with methylglyoxal (MeG) (A) or with superoxide dismutase (SOD) (B). GH3 cells were preincubated with 10 μM MeG for 6 h. Cells were bathed in Ca2+-free Tyrode’s solution and the pipette was filled up with Cs+-containing solution. The cell tested was maintained at −80 mV and the depolarizing pulses ranging between −80 and +40 mV were thereafter delivered to it. Each point represents the mean ± SEM (n = 7). Inset denotes the voltage-clamp protocol used. or □: control; ●or : aPO (10 μM). Noticeably, in MeG- or SOD-treated cells, the stimulatory effect of aPO on the overall I-V relationships of peak INa was altered little. The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data taken at different levels of voltages) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05).
Figure 5. Stimulatory effect of aPO on averaged I-V relationship of INa in GH3 cells treated with methylglyoxal (MeG) (A) or with superoxide dismutase (SOD) (B). GH3 cells were preincubated with 10 μM MeG for 6 h. Cells were bathed in Ca2+-free Tyrode’s solution and the pipette was filled up with Cs+-containing solution. The cell tested was maintained at −80 mV and the depolarizing pulses ranging between −80 and +40 mV were thereafter delivered to it. Each point represents the mean ± SEM (n = 7). Inset denotes the voltage-clamp protocol used. or □: control; ●or : aPO (10 μM). Noticeably, in MeG- or SOD-treated cells, the stimulatory effect of aPO on the overall I-V relationships of peak INa was altered little. The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data taken at different levels of voltages) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05).
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Figure 6. Effect of aPO on voltage-dependent hysteresis (Vhys) of persistent INa (INa(P)) activated by isosceles-triangular ramp pulses with varying ramp duration in GH3 cells. In this series of whole-cell current recordings, we voltage-clamped the tested cell at −80 mV and the isosceles-triangular ramp voltage with varying duration of 0.4 to 3.2 s (i.e., ramp speed of ±0.1 to 0.8 mV/ms) to activate INa(P) in response to the forward (i.e., ascending from −110 to +50 mV) and backward (descending from +50 to −110 mV) that was thereafter applied to it. (A) Representative INa(P) traces obtained in the control period (upper, aPO was not present), and during cell exposure to 10 μM aPO (lower). The uppermost part shows varying durations of isosceles-triangular ramp pulse applied. Of notice, the presence of aPO can augment the INa(P) amplitude elicited by the upsloping and downsloping limbs of the triangular ramp. (B) Representative instantaneous I-V relation of INa(P) in response to isosceles-triangular ramp pulse (the voltage between −100 and +50 mV) with a duration of 3.2 s (as indicated in the left side of panel (B)). Current trace in the left side is control, while that in the right side was acquired from the presence of 10 μM aPO. The dashed arrows in the left side show the direction of INa(P) trajectory in which time passes during the elicitation by the upright isosceles-triangular ramp pulse. Of interest, a striking figure-of-eight (or infinity-shaped: ∞) exists in the Vhys trajectory responding to the triangular ramp. (C) Summary bar graph demonstrating the effect of aPO and aPO plus Ran on INa(P) amplitude activated by the upsloping and downsloping limbs of 3.2-s triangular ramp pulse (mean ± SEM; n = 8 for each bar). Current amplitudes in the left side were taken at the level of 0 mV in situations where the 1.6-s ascending (upsloping) end of the triangular pulse was delivered to elicit INa(P) (i.e., high-threshold INa(P), while those in the right side (i.e., low-threshold INa(P)) was at −80 mV during the descending (downsloping) end of the pulse. Current amplitude measured is illustrated in the absolute value. Data analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from controls (p < 0.05) and ** significantly different from aPO (30 μM) alone groups (p < 0.05).
Figure 6. Effect of aPO on voltage-dependent hysteresis (Vhys) of persistent INa (INa(P)) activated by isosceles-triangular ramp pulses with varying ramp duration in GH3 cells. In this series of whole-cell current recordings, we voltage-clamped the tested cell at −80 mV and the isosceles-triangular ramp voltage with varying duration of 0.4 to 3.2 s (i.e., ramp speed of ±0.1 to 0.8 mV/ms) to activate INa(P) in response to the forward (i.e., ascending from −110 to +50 mV) and backward (descending from +50 to −110 mV) that was thereafter applied to it. (A) Representative INa(P) traces obtained in the control period (upper, aPO was not present), and during cell exposure to 10 μM aPO (lower). The uppermost part shows varying durations of isosceles-triangular ramp pulse applied. Of notice, the presence of aPO can augment the INa(P) amplitude elicited by the upsloping and downsloping limbs of the triangular ramp. (B) Representative instantaneous I-V relation of INa(P) in response to isosceles-triangular ramp pulse (the voltage between −100 and +50 mV) with a duration of 3.2 s (as indicated in the left side of panel (B)). Current trace in the left side is control, while that in the right side was acquired from the presence of 10 μM aPO. The dashed arrows in the left side show the direction of INa(P) trajectory in which time passes during the elicitation by the upright isosceles-triangular ramp pulse. Of interest, a striking figure-of-eight (or infinity-shaped: ∞) exists in the Vhys trajectory responding to the triangular ramp. (C) Summary bar graph demonstrating the effect of aPO and aPO plus Ran on INa(P) amplitude activated by the upsloping and downsloping limbs of 3.2-s triangular ramp pulse (mean ± SEM; n = 8 for each bar). Current amplitudes in the left side were taken at the level of 0 mV in situations where the 1.6-s ascending (upsloping) end of the triangular pulse was delivered to elicit INa(P) (i.e., high-threshold INa(P), while those in the right side (i.e., low-threshold INa(P)) was at −80 mV during the descending (downsloping) end of the pulse. Current amplitude measured is illustrated in the absolute value. Data analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from controls (p < 0.05) and ** significantly different from aPO (30 μM) alone groups (p < 0.05).
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Figure 7. Effect of aPO on erg-mediated K+ current (IK(erg)) in GH3 cells. The experiments were undertaken in cells that were bathed in high-K+, Ca2+-free solution containing 1 μM tetrodotoxin (TTX), and the recording pipette was filled up with K+-containing internal solution. (A) Representative IK(erg) traces obtained in the control (a) and during cell exposure to 10 μM aPO (b). The examined cell was held at −10 mV and a downsloping ramp from −10 to −100 mV with a duration of 1 s (indicated in the inset) was applied to it. The dashed arrow indicates the direction of current trajectory in which time passes, while the asterisk shows the inwardly-rectifying property of IK(erg). (B) Summary bar graph showing effect of aPO and aPO plus E-4031 on the amplitude of IK(erg) (mean ± SEM; n = 8 for each bar). Current amplitude (i.e., peak IK(erg) amplitude) was measured at the level of −70 mV. Data analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05) and ** significantly different from the aPO (10 μM) alone group (p < 0.05).
Figure 7. Effect of aPO on erg-mediated K+ current (IK(erg)) in GH3 cells. The experiments were undertaken in cells that were bathed in high-K+, Ca2+-free solution containing 1 μM tetrodotoxin (TTX), and the recording pipette was filled up with K+-containing internal solution. (A) Representative IK(erg) traces obtained in the control (a) and during cell exposure to 10 μM aPO (b). The examined cell was held at −10 mV and a downsloping ramp from −10 to −100 mV with a duration of 1 s (indicated in the inset) was applied to it. The dashed arrow indicates the direction of current trajectory in which time passes, while the asterisk shows the inwardly-rectifying property of IK(erg). (B) Summary bar graph showing effect of aPO and aPO plus E-4031 on the amplitude of IK(erg) (mean ± SEM; n = 8 for each bar). Current amplitude (i.e., peak IK(erg) amplitude) was measured at the level of −70 mV. Data analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05) and ** significantly different from the aPO (10 μM) alone group (p < 0.05).
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Figure 8. Effect of aPO on depolarization-activated INa present in HL-1 cardiomyocytes. In this set of experiments, we kept cells immersed in Ca2+-free Tyrode’s solution and the electrode was filled with Cs+-enriched solution. When whole-cell configuration was established, we voltage-clamped the cell at −80 mV and the brief depolarization to −10 mV was delivered to it. (A) Representative INa traces activated by depolarizing command pulse (indicated in the upper part). a: control; b: 3 μM aPO; c: 10 μM aPO. (B) Summary bar graph showing effects of aPO, aPO plus ranolazine (Ran), and aPO plus esaxerenone (ESAX) on peak amplitude of INa in HL-1 heart cells (mean ± SEM; n = 8 for each bar). Current amplitude was measured at the beginning of 50-ms depolarizing pulses from −80 to −10 mV. Data analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05) and ** Significantly different aPO (10 μM) alone group (p < 0.05).
Figure 8. Effect of aPO on depolarization-activated INa present in HL-1 cardiomyocytes. In this set of experiments, we kept cells immersed in Ca2+-free Tyrode’s solution and the electrode was filled with Cs+-enriched solution. When whole-cell configuration was established, we voltage-clamped the cell at −80 mV and the brief depolarization to −10 mV was delivered to it. (A) Representative INa traces activated by depolarizing command pulse (indicated in the upper part). a: control; b: 3 μM aPO; c: 10 μM aPO. (B) Summary bar graph showing effects of aPO, aPO plus ranolazine (Ran), and aPO plus esaxerenone (ESAX) on peak amplitude of INa in HL-1 heart cells (mean ± SEM; n = 8 for each bar). Current amplitude was measured at the beginning of 50-ms depolarizing pulses from −80 to −10 mV. Data analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05) and ** Significantly different aPO (10 μM) alone group (p < 0.05).
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Chuang, T.-H.; Cho, H.-Y.; Wu, S.-N. Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4′-Hydroxy-3′-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor. Biomedicines 2021, 9, 1146. https://0-doi-org.brum.beds.ac.uk/10.3390/biomedicines9091146

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Chuang T-H, Cho H-Y, Wu S-N. Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4′-Hydroxy-3′-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor. Biomedicines. 2021; 9(9):1146. https://0-doi-org.brum.beds.ac.uk/10.3390/biomedicines9091146

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Chuang, Tzu-Hsien, Hsin-Yen Cho, and Sheng-Nan Wu. 2021. "Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4′-Hydroxy-3′-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor" Biomedicines 9, no. 9: 1146. https://0-doi-org.brum.beds.ac.uk/10.3390/biomedicines9091146

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