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

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.

The voltage-gated Na + (Na V ) channels, nine subtypes of which are denoted Na V 1.1 through Na V 1.9, 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 V 1.5 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 GH 3 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 GH 3 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 GH 3 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., GH 3 or HL-1 cells).
The HEPES-buffered normal Tyrode's solution used in this work had an ionic composition, comprising (in mM): NaCl 136.5, KCl 5.4, CaCl 2 1.8, MgCl 2 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 GH 3 or HL-1 cells immersed in Ca 2+ -free, Tyrode's solution in attempts to avoid the contamination of Ca 2+ -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, KH 2 PO 4 1, MgCl 2 a, Na 2 ATP 3, Na 2 GTP 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).

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

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 GH 3 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 Ex-pertBook 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.

Whole-Cell Data Analyses
To determine concentration-dependent stimulation of apocynin on the transient (peak) or late I Na , we kept cells bathed in Ca 2+ -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 GH 3 cells were least-squares fitted to the Hill equation. That is, In this equation, [aPO] is the aPO concentration used, n H the Hill coefficient, EC 50 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: 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 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: 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: 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): 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) 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: 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 GH 3 cells was established on the basis of a simple Boltzmann distribution (or the Fermi-Dirac distribution): 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.

Curve-Fitting Procedures and Statistical Analyses
Curve fitting (linear or non-linear (e.g., exponential or sigmoidal curve)) to data sets was carried out with the goodness of fit by using various maneuvers, suc Microsoft "Solver" function embedded in Excel 2019 (Microsoft) and 64-bit Ori 2016 program (OriginLab; Scientific Formosa, Kaohsiung, Taiwan). The data a sented as the mean ± standard error of the mean (SEM), with sample sizes (n) ind the number of GH3 or HL-1 cells from which the data were collected. The Student From the binding scheme (indicated under Materials and Methods), the forward (k +1 *) or backward (k −1 ) rate constant for aPO-accentuated I Na in GH 3 cells was computed to be 0.00898 ms −1 µM −1 or 0.0303 ms −1 , respectively.

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 GH 3 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.

Effect of aPO on the Voltage-Gated Na+ Current (I Na ) Recorded from Pituitary GH 3 Cells
In the first stage of measurements, we kept cells immersed in a Ca 2+ -free Tyrode's solution containing 0.5 mM CdCl 2 , 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 GH 3 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 EC 50 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 GH 3 cells, and that the late component of I Na was stimulated to a greater extent than the peak component of the current.

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 −1 µM −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 EC 50 value for aPO-mediated stimulation of late I Na determined from the concentration-response curve ( Figure 1B).

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. omedicines 2021, 9, x FOR PEER REVIEW or without the addition of aPO (10 μM) was constructed and properly mann type sigmoidal function (indicated under Materials and Methods linear regression analysis. In the absence and presence of 10 μM aPO, t noticed to differ significantly (−62.6 ± 1.3 mV (in the control) versus −49. presence of aPO); n = 7, p < 0.05); in contrast, the value of q (apparent g not differ significantly (2.79 ± 0.12 e (in the control) versus 2.82 ± 0.13 e (i aPO); n = 7, p > 0.05). Therefore, cell exposure to aPO not only increased ductance of INa, but also shifted the inactivation curve to the rightward proximately 13 mV. However, we found no evident change in the gat inactivation curve during cell exposure to aPO. As such, it is reasonab the steady-state INa inactivation curve in the presence of this compound w ward, with no clear adjustment in the gating charge of this curve. In these experiments, the conditioning voltage pulses 40 ms to various membrane potentials between −120 and +20 mV were applied tential of −80 mV. Following each conditioning potential, a test pulse to −10 mV 40 ms was delivered to activate INa. The normalized amplitude of INa (I/Imax) was the conditioning potential and the sigmoidal curves were optimally fitted by th tion (indicated under Materials and Methods). Each point represents the mean statistical analyses were undertaken by ANOVA-2 for repeated measures, p (fact data ken at different level of conditioning potentials) < 0.05, p (factor 2, groups b and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fishe difference test, p < 0.05). 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 I Na 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 I Na. The normalized amplitude of I Na (I/I max ) 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).

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 GH 3 cells.  . Each smooth line was optimally fitted by a single-exponential func plitude denotes that the peak INa taken at the second pulse is divided by that point represents the mean ± SEM (n = 8). The statistical analyses were underta repeated measures, p (factor 1, groups among data ken at different interpul (factor 2, groups between the absence and presence of aPO) < 0.05, p (interac by post-hoc Fisher's least-significant difference test, p < 0.05).

Comparison among Effects of aPO, Tefluthrin (Tef), Tef Plus aPO, aPO (RFM), and aPO Plus Ranolazine (Ran) on the Peak Amplitude of INa
Tef, a type-I pyrethroid insecticide, was reported to be an activat Ran is recognized as a late INa blocker as well as an inhibitor of NOX and RFM, known to be an antiepileptic agent, was previously demons aPO (10 µM). Each smooth line was optimally fitted by a single-exponential function. The relative amplitude denotes that the peak I Na 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). 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 GH 3 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 GH 3 cells.

Comparison among
Biomedicines 2021, 9, x FOR PEER REVIEW

Stimulatory Action of aPO on INa in Methylglyoxal-(MeG-) or Superoxide D (SOD-) Treated Cells
One would expect that the effect of aPO on INa is engaged in either it NOX activity or the reduction in the production of reactive oxygen species. T of NOX was previously reported to be distributed in pituitary cells [14,15 effect of aPO on INa was assessed in cells preincubated with MeG or SOD fo previously recognized to be a substrate for NOX activity [33][34][35], while SO dative enzyme, was reported to reduce the production of reactive oxyge However, in GH3 cells preincubated with MeG for 6 h, the I-V relationship o or without addition of aPO is illustrated in Figure 5. For example, in cells p MeG (10 μM), aPO (10 μM) could significantly increase the amplitude of I the level of −20 mV from 401 ± 31 to 511 ± 39 pA (n = 7, p < 0.05). Likewise, cubated cells, the addition of aPO (10 μM) increased INa amplitude at −20 m 31 to 515 ± 41 pA (n = 7, p < 0.05). Therefore, these results allowed us to su stimulatory effect of aPO on INa that we obtained in these cells is unlikel

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 GH 3 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 SODpreincubated 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. The cell tested was maintained at −80 mV and the depolarizi pulses ranging between −80 and +40 mV were thereafter delivered to it. Each point represents t mean ± SEM (n = 7). Inset denotes the voltage-clamp protocol used. ■ or □: control; •or ○: aPO ( μM). Noticeably, in MeG-or SOD-treated cells, the stimulatory effect of aPO on the overall I-V re tionships of peak INa was altered little. The statistical analyses were undertaken by ANOVA-2 f repeated measures, p (factor 1, groups among data taken at different levels of voltages) < 0.05 (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, follow by post-hoc Fisher's least-significant difference test, p < 0.05).

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 current trajectory in response to the upsloping and the downsloping voltages) is current a subject of extensive research, including NaV channels [24,37,38]. We next examin whether or how the presence of aPO is able to modify INa(P) Vhys activated in response the upright isosceles-triangular ramp pulse in GH3 cells. In this stage of our whole-c current recordings, the tested cell was voltage-clamped at the level of −80 mV and we th applied it with a set of isosceles-triangular ramp pulses ranging between −110 and +50 m (with a height of 160 mV) of varying ramp duration at a rate of 0.05 Hz through digit to-analog conversion ( Figure 6A). Consistent with previous observations [24,26], the am plitude of INa(P) in response to such upright triangular ramp voltage was noticed to displ a striking figure-of-eight Vhys (i.e., ) in the instantaneous I-V relationship of INa(P) wi two distinct peaks, i.e., low and high threshold INa(P). Alternatively, there is an initial cou terclockwise direction, which time goes by, in current trajectory (i.e., high-threshold loo with a peak at −0 mV) activated by the upsloping limb, and following the downslopi limb, a clockwise direction (i.e., low-threshold loop with a peak at −80 mV) ensued (Figu 6B). Of particular interest, one minute after GH3 cells were exposed to 30 μM aPO alon the amplitude of INa(P) at high or low threshold respectively activated by the upslopin triangular ramp voltage (forward or ascending) or downsloping (backward or descen ing) 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 hig threshold one (Figure 6C), for example, as the isosceles-triangular ramp pulse with a d ration of 3.2 s (or ramp speed of ±0.1 mV/ms). In the presence of 30 μM aPO, the peak IN amplitude measured at the level of −0 mV (i.e., high-threshold INa(P)) during the ascendi 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 I Na 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).

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 GH 3 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., highthreshold 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 GH 3 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 GH 3 cells was enhanced in the presence of aPO ( Figure 6B,C).
Biomedicines 2021, 9, x FOR PEER REVIEW the descending phase of such a ramp concurrently increased from 285 ± 33 to 393 (n = 8, p < 0.05). Alternatively, the subsequent application of 10 μM Ran, but sti continued presence of 30 μM aPO, was able to attenuate the aPO-mediated inc INa(P) taken at either high or low threshold amplitude in the Vhys loop. These obser therefore, enabled us to indicate that the Vhys strength of INa(P) activated by isosc angular ramp pulses of varying ramp duration observed in GH3 cells was enhance presence of aPO (Figures 6B,C).  6-s ascending (upsloping) end of the tr pulse was delivered to elicit INa(P) (i.e., high-threshold INa(P), while those in the right side ( threshold INa(P)) was at −80 mV during the descending (downsloping) end of the pulse. Cur plitude measured is illustrated in the absolute value. Data analyses were performed by A (p < 0.05). * Significantly different from controls (p < 0.05) and ** significantly different from μM) alone groups (p < 0.05).

Effect of aPO on Erg-Mediated K + Current (IK(erg)) in GH3 Cells
Earlier studies have demonstrated that telmisartan, an activator of INa, can b tive in inhibiting IK(erg) [22]. For this reason, we decided to investigate whether aP cises any perturbations on IK(erg). The biophysical and pharmacological properties in GH3 cells have been previously reported [22,[39][40][41]. In these whole-cell expe we bathed cells in high-K + , Ca 2+ -free solution, and the recording pipette was filled K + -containing solution. The composition of these solutions was detailed under M and Methods. The examined cell was voltage-clamped at −10 mV and th downsloping ramp pulse from −10 to −100 mV with a duration of 1 s was applied shown in Figure 7, the addition of 10 μM aPO resulted in a progressive declin Figure 6. Effect of aPO on voltage-dependent hysteresis (Vhys) of persistent I Na (I Na(P) ) activated by isosceles-triangular ramp pulses with varying ramp duration in GH 3 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 I Na(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 I Na(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 I Na(P) amplitude elicited by the upsloping and downsloping limbs of the triangular ramp. (B) Representative instantaneous I-V relation of I Na(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 I Na(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 I Na(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 I Na(P) (i.e., high-threshold I Na(P) , while those in the right side (i.e., low-threshold I Na(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).

Effect of aPO on Erg-Mediated K + Current (I K(erg) ) in GH 3 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 GH 3 cells have been previously reported [22,[39][40][41]. In these whole-cell experiments, we bathed cells in high-K + , Ca 2+ -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. . Current amplitude (i.e., peak IK(erg) amplitude) was measured at the level of −70 mV analyses were performed by ANOVA-1 (p < 0.05). * Significantly different from control (p < and ** significantly different from the aPO (10 μM) alone group (p < 0.05).

Effect of aPO on INa Recorded from Murine HL-1 Cardiomyocytes
aPO was previously demonstrated to be a chemo-preventive agent for cardiovas disorders though the inhibition of NOX activity [35,[42][43][44]. In another set of experim we tested whether INa inherently in heart cells (i.e., HL-1 cardiomyocytes) could st modified by the presence of aPO. The preparation of these cells was described abov der Materials and Methods. Cells were kept bathed in Ca 2+ -free Tyrode's solution in w 10 mM TEA was included, and the pipette was filled with Cs + -enriched solution. N ably, as HL-1 cells were continually exposed to aPO at a concentration of 3 or 10 μM amplitude of peak INa activated by 50-ms depolarizing pulses from −80 to −10 mV increased; concomitantly, progressive slowing of the inactivation time course of the rent was seen ( Figure 8A,B). For example, cell exposure to 10 μM aPO resulted in a ceivable increase of peak INa from 859 ± 56 to 1381 ± 85 pA (n = 8, p < 0.05); concomit the τinact(S) value was significantly raised to 56.3 ± 7.1 ms (n = 8, p < 0.05) from a co value of 7.1 ± 1.4 ms. After washout of aPO (i.e., aPO was removed, but cells wer exposed to Ca 2+ -free Tyrode's solution containing 10 mM TEA), current amplitud turned 892 ± 58 pA (n = 8, p < 0.05). Alternatively, in the continued presence of aP μM), further application of either ranolazine (Ran, 10 μM) or esaxerenone (ESAX, 10 was noticed to attenuate aPO-mediated stimulation of INa ( Figure 8B). Like Ran. ESAX recently reported to inhibit INa [24]. Therefore, consistent to some extent with the obs tions done in GH3 cells, the results reflect the effectiveness of aPO in stimulating 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 Ca 2+ -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 Ca 2+ -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 GH 3 cells, the results reflect the effectiveness of aPO in stimulating I Na in response to the rapid depolarizing step in HL-1 cells.

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 isoscelestriangular 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 , and aPO plus esaxerenone (ESAX) on peak amplitude of I Na 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).

Discussion
The distinctive findings in the present study are that (a) GH 3 -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 EC 50 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., GH 3 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 GH 3 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 GH 3 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 GH 3 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 IC 50 of 10 µM [48]. According to the data of Figure 1, the IC 50 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].