biomedicines

Article Characterization of Direct Perturbations on Voltage-Gated Sodium Current by Esaxerenone, a Receptor Blocker

Wei-Ting Chang 1,2,3 and Sheng-Nan Wu 4,5,*

1 Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan 71005, Taiwan; [email protected] 2 Division of Cardiovascular Medicine, Chi-Mei Medical Center, Tainan 71004, Taiwan 3 Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan 4 Department of Physiology, National Cheng Kung University Medical College, Tainan 70101, Taiwan 5 Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan 70101, Taiwan * Correspondence: [email protected]; Tel.: +886-6-2353535 (ext. 5334); Fax: +886-6-2362780

Abstract: Esaxerenone (ESAX; CS-3150, Minnebro®) is known to be a newly non-steroidal min- eralocorticoid receptor (MR) antagonist. However, its modulatory actions on different types of ionic currents in electrically excitable cells remain largely unanswered. The present investigations were undertaken to explore the possible perturbations of ESAX on the transient, late and persistent + components of voltage-gated Na current (INa) identified from pituitary GH3 or MMQ cells. GH3-  cell exposure to ESAX depressed the transient and late components of INa with varying potencies.  The IC50 value of ESAX required for its differential reduction in peak or late INa in GH3 cells was Citation: Chang, W.-T.; Wu, S.-N. estimated to be 13.2 or 3.2 µM, respectively. The steady-state activation curve of peak INa remained Characterization of Direct unchanged during exposure to ESAX; however, recovery of peak INa block was prolonged in the Perturbations on Voltage-Gated presence 3 µM ESAX. In continued presence of (10 µM), further addition of 3 µM ESAX Sodium Current by Esaxerenone, a remained effective at inhibiting INa. ESAX (3 µM) potently reversed Tef-induced augmentation of Nonsteroidal Mineralocorticoid INa. By using isosceles-triangular ramp pulse with varying durations, the amplitude of persistent INa Receptor Blocker. Biomedicines 2021, 9, measured at high or low threshold was enhanced by the presence of tefluthrin (Tef), in combination 549. https://doi.org/10.3390/ with the appearance of the figure-of-eight hysteretic loop; moreover, hysteretic strength of the current biomedicines9050549 was attenuated by subsequent addition of ESAX. Likewise, in MMQ lactotrophs, the addition of ESAX also effectively decreased the peak amplitude of I along with the increased current inactivation Academic Editor: Na Chitra Subramanian rate. Taken together, the present results provide a noticeable yet unidentified finding disclosing that, apart from its antagonistic effect on MR receptor, ESAX may directly and concertedly modify the

Received: 7 April 2021 amplitude, gating properties and hysteresis of INa in electrically excitable cells. Accepted: 11 May 2021 + + Published: 13 May 2021 Keywords: esaxerenone; voltage-gated Na current; persistent Na current; current kinetics; hystere- sis; pituitary cell Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Esaxerenone (ESAX; Minnebro®, CS-3150, XL-560, Oklahoma, Japan), known to be a newly oral, non-steroidal selective blocker on the activity of mineralocorticoid receptor (MR), has been growingly used for the management of varying pathologic disorders, Copyright: © 2021 by the authors. such as , refractory hypertension, chronic kidney disease, diabetic Licensee MDPI, Basel, Switzerland. nephropathy, and heart failure [1–10]. Alternatively, the activity of MR has been previously This article is an open access article reported in pituitary cells including GH3 cells or in varying brain regions [11–18]. However, distributed under the terms and whether ESAX exercises any perturbations on electrical activities (e.g., transmembrane conditions of the Creative Commons ionic currents) in pituitary cells is largely unknown. Attribution (CC BY) license (https:// There are nine isoforms (i.e., NaV1.1–1.9 [or SCN1A-SCN5A and SCN8A-SCN11A]) creativecommons.org/licenses/by/ which are expressed in mammalian excitable tissues, including endocrine system [19,20]. 4.0/).

Biomedicines 2021, 9, 549. https://doi.org/10.3390/biomedicines9050549 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 549 2 of 17

Of notice, several inhibitors have been previously described to preferentially block the + late component of voltage-gated Na current (INa), such as ranolazine (Ran) and KMUP- 1 [21,22], while some of NaV-channel activators (e.g., pyrethroids and telmisartan) have been reported in different types of excitable cells [23–26]. As an endocrine-disrupting agent, tefluthrin or cypermethrin is, respectively, type I or II pyrethroid known to activate INa [23,25,27]. The INa can be readily evoked in response to changes in the membrane potential sensed by the channel’s voltage-sensor domains, which are intimately coupled to its pore domain [28–31]. However, at present, whether and how ESAX is capable of interacting directly with NaV channels to perturb the magnitude, gating properties, or hysteretic strength of INa in electrically excitable cells remains little thoroughly reported. In view of the aforesaid considerations, we sought to explore whether there are any possible modifications of ESAX on the amplitude, kinetics, and hysteretic behavior of + voltage-gated Na current (INa) in electrically excitable cells (e.g., pituitary GH3 and MMQ cells). The present study, for the first time, demonstrated that, despite its effectiveness in antagonizing MR activity, the distinguishable inhibition by ESAX of peak and late INa may be caused by one of several ionic mechanisms underlying its marked perturbations on the functional activities of electrically excitable cells, assuming that similar observations exist in vivo.

2. Materials and Methods 2.1. Chemicals, Drugs, and Solutions Used in This Study Esaxerenone (ESAX; Minnebro® CS-3150, XL-550, 1-(2-hydroxyethyl)-4-methyl-N-(4- methylsulfonylphenyl)-5-[2-(trifluoromethyl)phenyl]pyrrole-3-carboxamide, C22H21F3N2O4S, https://pubchem.ncbi.nlm.nih.gov/compound/Esaxerenone, accessed in Oklahoma, Japan, on 26 February 2019) was acquired from MedChemExpress (Genechain, Kaohsiung, Taiwan), aldosterone (Aldo), dexamethasone (Dex), tefluthrin (Tef), tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were from Sigma-Aldrich (Merck Ltd., Taipei, Taiwan), while ranolazine (Ran) was from Tocris (Union Biomed, Taipei, Taiwan). Unless otherwise stated, culture media (e.g., F-12 medium), horse or fetal bovine serum, L-glutamine, and trypsin/EDTA were purchased from HyCloneTM (Thermo Fisher Scientific, Kaohsiung, Taiwan), while all other chemicals, such as aspartic acid, CsOH, CsCl, EGTA, and HEPES, were of laboratory grade and taken from standard sources. The HEPES-buffered normal Tyrode’s solution used in this investigation had an ionic composition, which contained (in mM): NaCl 136.5, CaCl2 1.8, KCl 5.4, MgCl2 0.53, glucose 5.5, and HEPES 5.5, and the solution pH was titrated to 7.4 by adding NaOH. For 2+ measurements of INa, we kept GH3 or MMQ cells bathed in Ca -free, Tyrode’s solution in order to preclude the contamination of Ca2+-activated K+ and voltage-gated Ca2+ currents. To record K+ currents, the solution used to fill up the recording electrode contained (in mM): K-aspartate 130, KCl 20, KH2PO4 1, MgCl2 1, Na2ATP 3, Na2GTP 0.1, EGTA 0.1, HEPES 5, and the solution was adjusted with KOH to pH 7.2. To record INa or INa(P), we substituted K+ ions in the internal solution for equimolar Cs+ ions and the pH value in the solution was then titrated to 7.2 by adding CsOH. All solutions used in this work were prepared using demineralized water from Mill-Q purification system (Merck Ltd., Taipei, Taiwan). On the day of experiments, we filtered the bathing or filling solution and culture medium by using an Acrodisc® syringe filter with a 0.2-µm pore size (Bio-Check, New Taipei City, Taiwan).

2.2. Cell Preparations

The pituitary adenomatous cell line (GH3) was acquired from the Bioresource Col- lection and Research Center (BCRC-60015, http://catalog.bcrc.firdi.org.tw/Bcrc-Content? bid=60015, accessed on 4 April 2012, Hsinchu, Taiwan) [32], while the MMQ cell line (ATCC® CRL-10609, https://www.atcc.org/products/all/CRL-10609.aspx, accessed on 18 April 2019), another established pituitary cell line originally derived from Rattus norvegi- cus pituitary prolactinoma, was from the American Type Cell Collection (Manassas, VA, Biomedicines 2021, 9, 549 3 of 17

USA) through Genechain (Kaohsiung, Taiwan). GH3 cells were maintained in Ham’s F-12 culture medium supplemented with 2.5% fetal bovine serum (v/v), 15% horse serum (v/v), and 2 mM L-glutamine [31], and MMQ cells were cultured in F12 medium supplemented with 10% fetal bovine serum (v/v) and 1.176 g/L NaHCO3 in a humidified incubator at ◦ 30 C with 5% CO2 [33]. The cells were grown as a monolayer culture in a humidified envi- ◦ ronment of 5% CO2/95% air at 37 C. The electrophysiological recordings were commonly made five or six days after the cells had been cultured to 60–80% confluence.

2.3. Electrophysiological Measurements

On the day of the experiments, we gingerly dispersed GH3 or MMQ cells with a 1% trypsin-EDTA solution, and a few drops of cell suspension was thereafter placed in a home-made chamber affixed on the stage of an inverted DM-II microscope (Leica; Major Instruments, Kaohsiung, Taiwan). Cells were kept immersed at room temperature (20–25 ◦C) in normal Tyrode’s solution, the composition of which was provided above, and they were allowed to settle on the chamber’s bottom. The patch electrodes were drawn from 1.8-mm OD round borosilicate glass tubing (Kimax-51 [#34500]; Kimble; Dogger, New Taipei City, Taiwan) using a two-stage pull on a vertical pipet puller (PP-83; Narishige; Major Instruments, Tainan, Taiwan). The recording electrodes used had a tip diameter of ~1 µm and a resistance of 3–5 MΩ. They were mounted in an air-tight holder, having a suction port on the side, and a chloride silver wire was used to make contact with the electrode solution. We recorded different types of ionic currents in the whole-cell mode of standard patch-clamp technique with dynamic adaptive suction (i.e., a decremental change in the suction pressure in response to progressive increase in the seal resistance) with the aid of either an Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA) or an RK-400 amplifier (Bio-Logic, Claix, France) [23,32]. This procedure was noticed to increase the success rate of forming gigaseals and reduced loss of the gigaseal in long-term recordings. For whole-cell current recordings, following seal formation (>1 GΩ) the membrane was ruptured by gentle suction. The liquid junction potentials were zeroed immediately before seal formation was made, and the whole-cell data were corrected.

2.4. Potential and Current Recordings The signals were monitored and stored online at 10 kHz in an ASUS VivoBook Flip 14 laptop computer (TP412FA-0131A10210U; ASUS, Tainan, Taiwan) equipped with a Digidata 1440A interface (Molecular Devices). During the measurements with analog-to-digital and digital-to-analog conversion, the latter device was controlled using pCLAMP v10.7 software (Molecular Devices) run under Microsoft Windows 10 (Redmond, WA, USA). The laptop computer was put on the top of an adjustable Cookskin stand (Ningbo, Zheijiang, China) to allow efficient manipulation during the recordings. Through digital-to-analog conver- sion, the pCLAMP-created voltage-clamped protocols with varying rectangular or ramp waveforms were specifically designed and suited for determining the steady-state or in- stantaneous relationship pf current versus voltage (I–V) and voltage-dependent hysteresis of the current involved.

2.5. Data Analyses

The concentration-response data for inhibition of transient (peak) or late INa in pitu- itary GH3 cells were appropriately fitted to the Hill equation. That is,

[ESAX]nH × E % decrease = max (1) nH nH IC50 + [ESAX]

In this equation, [ESAX] is the ESAX concentration applied, nH the Hill coefficient, Emax the maximal inhibition of transient (peak) or late INa by ESAX, and IC50 the concentration required for a 50% inhibition of transient (peak) or late INa. Biomedicines 2021, 9, 549 4 of 17

The steady-state activation curve of peak INa with or without the ESAX addition observed in GH3 cells was established on the basis of a simple Boltzmann distribution (or the Fermi-Diract distribution) [31]:

GNa(max) GNa =   (2) − V−V1 qF 2 1 + e RT

where GNa(max) is the maximal conductance of peak INa in the absence or presence of 3 µM ESAX; V the membrane potential in mV; V1/2 the half-point of the relationship; q the apparent gating charge; F Faraday’s constant; R the universal gas constant; T the absolute temperature, and RT/F = 25.4 mV.

2.6. Statistical Analyses Curve-fitting (linear or non-linear) to experimental data sets was performed with the goodness of fit by using various maneuvers such as Microsoft Solver function embedded in Excel 2016 (Microsoft) and 64-bit OriginPro®2016 program (OriginLab; Scientific Formosa, Kaohsiung, Taiwan). The averaged results are presented as the mean ± standard error of the mean (SEM), with sample sizes (n) indicative of the number of GH3 or MMQ cells from which the experimental data were collected together. The Student’s t-test (paired or unpaired) and a one-way analysis of variance (ANOVA) followed by post-hoc Fisher’s least-significance difference method, were implemented for the evaluation of differences among means. Statistical significance was determined at a p-value of <0.05, unless otherwise indicated.

3. Results + 3.1. Effect of Esaxerenone (ESAX) on Voltage-Gated Na Current (INa) Measured from GH3 Cells In the first stage of the whole-cell recordings, we measured the effects of ESAX on the amplitude and gating kinetics of INa activated by rapid membrane depolarization. We kept cells immersed in Ca2+-free, Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA), and the recording electrode was filled up with a Cs+-containing solution. As demonstrated in Figure1A, 1 min after GH 3-cells were exposed to ESAX at a concentration of 1 or 3 µM, the amplitude in the peak (i.e., initial) or late (i.e., end-pulse) components of INa by abrupt depolarizing pulse from −80 to −10 mV was progressively depressed. For example, as the rectangular voltage step from −80 to −10 mV with a duration of 40 ms was delivered (indicated in the inset of Figure1A) to activate INa, the addition of 3 µM ESAX was noticed to result in an evident decrease in the peak or late amplitude of INa to 1104 ± 197 or 9 ± 1 pA (n = 8, p < 0.05) from control values of 1498 ± 241 or 19 ± 3 pA (n = 8), respectively. After removal of ESAX, the peak or late amplitude of the current returned to 1452 ± 228 or 18 ± 3 pA (n = 8, p < 0.05), respectively. Moreover, as cells were exposed to 1 µM tetrodotoxin (TTX) alone, peak amplitude of INa evoked by the same voltage protocol was almost abolished, as evidenced by a reduction of current amplitude to 54 ± 6 pA (n = 7, p < 0.01) from a control value of 1486 ± 235 pA (n = 7). Biomedicines 2021, 9, x FOR PEER REVIEW 5 of 17

Biomedicines 2021, 9, 549 5 of 17 natively expressed in GH3 cells, and that this drug tends to be selective for later over peak INa in response to rectangular depolarizing step.

+ Figure 1. Effect of esaxerenone (ESAX) on voltage-gated Na current (INa) measured from pituitary 2+ tumor (GH3) cells. This set of experiments was undertaken in cells bathed in Ca -free, Tyrode’s Figure 1. Effect of esaxerenone (ESAX) on voltage-gated Na+ current (INa) measured from pituitary solution containing 10 mM TEA, and we filled up the electrode by using Cs+-containing solution. tumor (GH3) cells. This set of experiments was undertaken in cells bathed in Ca2+-free, Tyrode’s (solutionA) Representative containing I10Na mMtraces TEA, obtained and we in filled (a) the up control the electrode situation by (i.e.,using ESAX Cs+-containing was not present) solution. and µ µ (duringA) Representative cell exposure INa totraces 1 Mobtained ESAX (b)in (a) or the 3 controlM ESAX situation (c). The (i.e., voltage ESAX pulsewas not protocol present) applied and duringis shown cell in exposure Inset. The to 1 lowerμM ESAX panels (b) inor (3A μ)M show ESAX the (c). expanded The voltage records pulse from protocol each applied dashed is box. (shownB) Concentration-dependent in Inset. The lower panels relationship in (A) show of ESAX the expanded on transient records (or peak) from ( each) and dashed late ( )box. component (B) Concentration-dependentof INa evoked by abrupt membrane relationship depolarization of ESAX on (mean transient± SEM; (or peak)n = 8 ( for○)# and each late point). (█) Eachcomponent smooth ofline INa indicates evoked by the abrupt goodness-of-fit membrane to depolarization the Hill equation, (mean as described ± SEM; n in= 8 Materials for each point). and Methods. Each smooth line indicates the goodness-of-fit to the Hill equation, as described in Materials and Methods. Figure1B shows that the presence of ESAX to the bath can concentration-dependently 3.2.decrease Effect theof ESAX amplitude on the of Steady-State peak or late Current-VoltageINa evoked by (I–V) rapid or depolarizing Conductance-Voltage pulse. According Relationshipthe Hill equation of Peak detailed INa under Materials and Methods, the IC50 value needed for ESAX- mediatedTo characterize block of peak the orinhibitory late INa detectedeffect of inESAX GH 3oncells INa, was we yieldednext examined to be 13.2 whether or 3.2 µthisM, drugrespectively, might exercise the value any of perturbations which was apparently on the steady-state distinguishable I–V relationship between its of suppressive INa in GH3 cells.effects Figure on these 2A twoillustrates components the mean of theI–V current. relationship Our initialof peak experiments; INa with or without therefore, the enable addi- tionus to of reflect 3 μM thatESAX. ESAX It was exercises noticed a that depressant the value action of threshold on the peakor maximal and late voltageINa natively for the I elicitationexpressed of in I GHNa did3 cells, not differ and that between this drug the absence tends to and be selective presence for of later3 μM overESAX. peak TheNa rela-in response to rectangular depolarizing step. tionship of INa-conductance versus membrane potential obtained with or without the ap- plication of 3 μM ESAX was also established and depicted in Figure 2B. Similar experi- 3.2. Effect of ESAX on the Steady-State Current-Voltage (I–V) or Conductance-Voltage mental results were obtained in seven different cells examined. The data; thus, led us to Relationship of Peak INa reflect that no obvious change in the steady-state activation curve of peak INa in GH3 cells To characterize the inhibitory effect of ESAX on I , we next examined whether this was detected during exposure to ESAX. Na drug might exercise any perturbations on the steady-state I–V relationship of INa in GH3 cells. Figure2A illustrates the mean I–V relationship of peak INa with or without the addition of 3 µM ESAX. It was noticed that the value of threshold or maximal voltage for the elicitation of INa did not differ between the absence and presence of 3 µM ESAX. The relationship of INa-conductance versus membrane potential obtained with or without the application of 3 µM ESAX was also established and depicted in Figure2B. Similar experimental results were obtained in seven different cells examined. The data; thus, led us to reflect that no obvious change in the steady-state activation curve of peak INa in GH3 cells was detected during exposure to ESAX. Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 17 Biomedicines 2021, 9, 549 6 of 17

. Figure 2. Effect of ESAX on current versus voltage (I–V) and conductance versus voltage relationships Figure 2. Effect of ESAX on current versus voltage (I–V) and conductance versus voltage relation- of INa identified in GH3 cells. (A) Mean I–V relationship of peak INa taken without () or with ( ) shipsthe addition of INa identified of 3 µM ESAX in GH (mean3 cells.± SEM; (A) nMean= 7 for I–V each relationship point). Inset of shows peak the INa voltage-clamp taken without# (□) or with

(○protocol) the addition delivered. of (B 3) MeanμM ESAX conductance (mean versus ± SEM; voltage n = relationship 7 for each of point). peak INa Insettaken shows in the control the voltage-clamp protocolperiod ( delivered.) and during ( cellB) Mean exposure conductance ( ) to 3 µM ESAX versus (mean voltage± SEM; relationshipn = 7 for each point).of peak Of I notice,Na taken in the con- trolthe period conductance (□) and versus during voltage cell relationship exposure# of(○ the) to current 3 μM betweenESAX (mean the absence ± SEM; and n presence = 7 for each of point). Of notice,ESAX didthe not conductance differ, despite versus the decrease voltage in maximal relationsh conductanceip of the of current peak INa between. the absence and pres- ence of ESAX did not differ, despite the decrease in maximal conductance of peak INa. 3.3. Effect of ESAX on the Recovery of I(Na) Block Recorded from GH3 Cells

In an effort to evaluate ESAX-induced inhibition of peak INa in these cells, we contin- 3.3.ued Effect to investigate of ESAX the on recovery the Recovery from current of I(Na) inactivationBlock Recorded in the from absence GH and3 Cells presence of this drug.In an Ineffort this set to ofevaluate experiments, ESAX-induced a stimulation protocolinhibition consisting of peak of aINa first in (condition- these cells, we contin- ueding) to depolarizing investigate voltage the recovery command whichfrom iscurrent sufficiently inactivation long to reach in athe steady-state absence was and presence of employed, and a second depolarizing (test pulse) was applied at the same potential as the this drug. In this set of experiments, a stimulation protocol consisting of a first (condition- conditioning pulse (Figure3). Then the ratios of the peak I Na in response to the second and ing)first depolarizing pulses were taken voltage as a measure command of recovery which from is sufficiently I(Na) block, and long they to were reach thereafter a steady-state was employed,established and plotteda second versus depolarizing interpulse interval. (test puls Thee)I Nawasrecovery applied from at blockthe same with orpotential as the without the application of 3 µM ESAX is illustrated in Figure3. In the absence or presence conditioning pulse (Figure 3). Then the ratios of the peak INa in response to the second and of 3 µM ESAX, the time course could be described by single exponential with the time firstconstants pulses of were 13.2 ± taken2.1 ms (asn = a8) measure or 21.5 ± of2.7 recovery ms (n = 8), from respectively. I(Na) block, It is; thus, and plausible they were thereafter establishedto anticipate, and from plotted the present versus results, interpulse that the recovery interval. of INa Theblock I wasNa recovery prolonged from by the block with or withoutpresence the of ESAX, application and that of the 3 delayed μM ESAX recovery is illustrated caused by thisin Figure agent may 3. In ascribe the absence largely or presence offrom 3 μ theM openESAX, channel the time block. course could be described by single exponential with the time constants of 13.2 ± 2.1 ms (n = 8) or 21.5 ± 2.7 ms (n = 8), respectively. It is; thus, plausible to anticipate, from the present results, that the recovery of INa block was prolonged by the presence of ESAX, and that the delayed recovery caused by this agent may ascribe largely from the open channel block. Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 17 Biomedicines 2021, 9, 549 7 of 17

Figure 3. ESAX-induced prolongation in the recovery of I block in GH cells. Cells were bathed in Na 3 Ca2+-free, Tyrode’s solution, the electrode was filled with Cs+-containing solution, and a set of two- pulseFigure voltage 3. ESAX-induced protocol was applied prolongation to the examined in the recovery cells. (A )of Representative INa block in GH current3 cells. traces Cells showing were bathed 2+ + currentin Ca recovery-free, Tyrode’s from block solu bytion, use the of two-step electrode protocol was filled with with varying Cs durations-containing (as indicatedsolution, inand the a set of uppermosttwo-pulse part). voltage Current protocol traces was in the applied upper partto the were examined obtained cells. in the ( controlA) Representative period, while current those in traces theshowing lower part current were recovery taken during from cell block exposure by use to 3 ofµM tw ESAX.o-step (B protocol) Relationship with ofvarying relative durations amplitude (as indi- cated in the uppermost part). Current traces in the upper part were obtained in the control period, versus interpulse interval in the absence () and presence ( ) of 3 µM ESAX. The relative amplitude while those in the lower part were taken during cell exposure to 3 μM ESAX. (B) Relationship of of peak INa was taken by the ratio of current amplitudes elicited# by the second depolarizing voltage steprelative and thoseamplitude by the versus first pulse. interpulse Each point interval represents in the the absence mean ±(□)SEM and( npresence= 8). The (○ smooth) of 3 μ lineM ESAX. indicatesThe relative a best amplitude fit to single of exponential. peak INa was taken by the ratio of current amplitudes elicited by the sec- ond depolarizing voltage step and those by the first pulse. Each point represents the mean ± SEM 3.4.(n =Comparisons 8). The smooth among line Effect indicates of ESAX, a best ESAX fit to plussingle Ranolazine exponential. (Ran), ESAX plus Dexamethasone (Dex), Aldosterone (Aldo), and Aldosterone plus ESAX on Peak INa Recorded from GH Cells 3.4.3 Comparisons among Effect of ESAX, ESAX plus Ranolazine (Ran), ESAX plus In another series of whole-cell experiments, we tested the possible effects of ESAX, Dexamethasone (Dex), Aldosterone (Aldo), and Aldosterone plus ESAX on Peak INa Recorded ESAX plus Ran, ESAX plus Dex, Aldo, and Aldo plus ESAX on the peak amplitude of INa activatedfrom GH in3 Cells response to abrupt membrane depolarization from −80 to −10 mV. Ranolazine has beenIn another demonstrated series to of inhibit whole-cellINa potently experiments, [21,32], while we tested Dex could the perturbpossible membrane effects of ESAX, ionESAX currents plus inRan, pituitary ESAX cellsplus orDex, bronchial Aldo, smoothand Aldo myocytes plus ESAX [34,35 on]. Thethe peak expression amplitude of of INa mineralocorticoidactivated in response receptor to abrupt (MR) hasmembrane been shown depolarization previously from to be − expressed80 to −10 mV. in GH Ranolazine3 cells [11,12,18,36,37]. As shown in Figure4A, Aldo (10 µM) alone did not modify peak has been demonstrated to inhibit INa potently [21,32], while Dex could perturb membrane I ; however, further addition of ESAX (3 µM), but still in the presence of Aldo, decreased ionNa currents in pituitary cells or bronchial smooth myocytes [34,35]. The expression of current amplitude. As summarized in Figure4B, ESAX at a concentration of 3 µM produced mineralocorticoid receptor (MR) has been shown previously to be expressed in GH3 cells an inhibitory effect on peak INa in GH3 cells; moreover, the subsequent addition of 10 µM [11,12,18,36,37]. As shown in Figure 4A, Aldo (10 μM) alone did not modify peak INa; how- ranolazine, still in the presence of 3 µM ESAX, was effective at decreasing INa amplitude further.ever, further However, addition as cells of were ESAX continually (3 μM), exposedbut still toin 3theµM presence ESAX, further of Aldo, application decreased of current 10amplitude.µM Dex failed As summarized to modify its decreasein Figure in 4B, the peakESAXINa at. Ona concentration the other hand, of the 3 μ presenceM produced an ofinhibitory 10 µM Aldo effect alone on did peak not I produceNa in GH any3 cells; appreciable moreover, changes the insubsequent the amplitude addition of peak of 10 μM µ Iranolazine,Na; however, still subsequent in the presence addition ofof 3 3 MμM ESAX ESAX, could was reduce effective current at amplitudedecreasing further. INa amplitude Hence,further. its However, seems that as Dex cells or Aldowere did continually not exert anyexposed effect onto 3 ESAX-mediated μM ESAX, further decrease application of of peak INa. 10 μM Dex failed to modify its decrease in the peak INa. On the other hand, the presence of 10 μM Aldo alone did not produce any appreciable changes in the amplitude of peak INa; however, subsequent addition of 3 μM ESAX could reduce current amplitude further. Hence, its seems that Dex or Aldo did not exert any effect on ESAX-mediated decrease of peak INa. Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 17 Biomedicines 2021, 9, 549 8 of 17

FigureFigure 4. Comparison 4. Comparison among among effects effects of ESAX,of ESAX, ESAX ESAX plus plus ranolazine ranolazine (Ran), (Ran), aldosterone aldosterone (Aldo), (Aldo), and and aldosterone aldosterone plus plus ESAX ESAX 2+ on theon the amplitude amplitude of peak of peakINa IdetectedNa detected in GHin GH3 cells.3 cells. Cells Cells were were kept kept bathed bathed in Cain Ca-free,2+-free, Tyrode’s Tyrode’s solution, solution, and and the the recording recording + + electrodeelectrode was was filled filled up withup with Cs Cs-containing-containing solution. solution. The TheINa IwasNa was elicited elicited by by 40 40 ms ms depolarizing depolarizing voltage voltage command command from from −80−80 to to−10 −10 mV, mV, and and current current amplitude amplitude at theat the start star oft theof the voltage voltage pulse pulse was was measured. measured. (A )(A Representative) Representative current current traces traces obtainedobtained in thein the control control (a) (a) and and during during cell cell exposure exposure to 10to µ10M μ aldosteroneM aldosterone (Aldo) (Aldo) (b) (b) or 10or 10µM μ AldoM Aldo plus plus 3 µ 3M μ ESAXM ESAX (c). (c). Inset shows the voltage pulse protocol. (B) Summary bar graph showing effects of ESAX, ESAX plus Ran, ESAX plus Dex, Inset shows the voltage pulse protocol. (B) Summary bar graph showing effects of ESAX, ESAX plus Ran, ESAX plus Dex, Aldo, and Aldo plus ESAX. Each bar represents the mean ± SEM (n = 7–8). * Significantly different from control (i.e., none Aldo, and Aldo plus ESAX. Each bar represents the mean ± SEM (n = 7–8). * Significantly different from control (i.e., none of the agents were present) (p < 0.05), + significantly different from ESAX (3 μM) alone group (p < 0.05), and ** significantly p µ p of thedifferent agents from were aldosterone present) ( < (10 0.05), μM) + alone significantly group ( differentp < 0.05). from ESAX (3 M) alone group ( < 0.05), and ** significantly different from aldosterone (10 µM) alone group (p < 0.05).

3.5. Enhanced Amplitude by Tef of INa Attenuated by ESAX 3.5. Enhanced Amplitude by Tef of I Attenuated by ESAX As an endocrine disruptingNa action, pyrethroids such as cypermethrin have been re- portedAs an to endocrineincrease the disrupting plasma level action, of hormon pyrethroidses including such as Aldo cypermethrin [27]. We, hence, have beennext in- reportedvestigated to increase whether the the plasma presence level of of ESAX hormones could includingmodify Tef-enhanced Aldo [27]. We, INa hence,evoked next in re- I investigatedsponse to rapid whether depolarizing the presence pulse of ESAXin GH3 could cells. modifyIn these Tef-enhanced experiments, whenNa evoked whole-cell in responsecurrent to recordings rapid depolarizing were established, pulse in we GH main3 cells.tained In these the examined experiments, cell whenin voltage whole-cell clamp at current−80 mV recordings and the 40 were ms depolarizing established, wepulse maintained to −10 mV thewas examined applied to cell it. Of in notice, voltage in clamp keeping − − at with80 mVprevious and the observations 40 ms depolarizing [23,25,32], pulse 1 min to of exposing10 mV was cells applied to 10 μM to Tef it. Ofalone, notice, the inpeak keeping with previous observations [23,25,32], 1 min of exposing cells to 10 µM Tef alone, amplitude of INa measured at the start of depolarizing pulse was enhanced, in combination thewith peak a amplitudemeasurable of slowingINa measured in the inactivation at the start ofor depolarizingdeactivation rate pulse of was the current. enhanced, Moreo- in combinationver, subsequent with a measurableaddition of slowingEASX at in a the concentration inactivation of or deactivation3 or 10 μM could rate of attenuate the current. Tef- Moreover, subsequent addition of EASX at a concentration of 3 or 10 µM could attenuate mediated augmentation on depolarization-activated INa (Figure 5). For example, as the Tef-mediated augmentation on depolarization-activated INa (Figure5). For example, as cells were depolarized from −80 to −10 mV to evoke INa, the exposure to 10 μM Tef resulted the cells were depolarized from −80 to −10 mV to evoke INa, the exposure to 10 µM in a significant raise in peak INa amplitude from 557 ± 56 to 1221 ± 187 pA (n = 7, p < 0.05). Tef resulted in a significant raise in peak I amplitude from 557 ± 56 to 1221 ± 187 pA The further application of 3 μM ESAX, Nastill in the continued presence of Tef, greatly re- (n = 7, p < 0.05). The further application of 3 µM ESAX, still in the continued presence of duced to 984 ± 123 pA (n = 7, p < 0.05). Again, the presence of 10 μM Tef evidently increased Tef, greatly reduced to 984 ± 123 pA (n = 7, p < 0.05). Again, the presence of 10 µM Tef the time constant (τinact(S)) in the slow component of INa inactivation from 1.5 ± 0.3 to 21.1 ± evidently increased the time constant (τ ) in the slow component of I inactivation 3.2 ms (n = 7, p < 0.01), while subsequentinact(S) addition of 3 μM ESAX resultedNa in a significant from 1.5 ± 0.3 to 21.1 ± 3.2 ms (n = 7, p < 0.01), while subsequent addition of 3 µM ESAX decline in the τinact(S) value to 12.2 ± 2.1 ms (n = 7, p < 0.05). In this regard, the data reflected resulted in a significant decline in the τ value to 12.2 ± 2.1 ms (n = 7, p < 0.05). In this that the presence of ESAX was capableinact(S) of reversing Tef-mediated enhancement in both regard, the data reflected that the presence of ESAX was capable of reversing Tef-mediated amplitude and τinact(S) of peak INa observed in GH3 cells. enhancement in both amplitude and τinact(S) of peak INa observed in GH3 cells. BiomedicinesBiomedicines2021 2021, 9, ,9 549, x FOR PEER REVIEW 99 of of 17 17

Na 3 FigureFigure 5. 5.Effect Effect of of ESAX ESAX on on tefluthrin tefluthrin (Tef)-mediated (Tef)-mediated augmentation augmentation of ofI NaI recorded recorded from from GH GH3 cells.cells. (A(A)) Representative Representative current current traces traces activated activated by by rapid rapid membrane membrane depolarization depolarization (indicated (indicated in the in upperthe upper part). Current trace labeled a is control, that labeled b was taken during exposure to 10 μM part). Current trace labeled a is control, that labeled b was taken during exposure to 10 µM Tef, Tef, and that labeled c was obtained in the presence of 10 μM Tef plus 10 μM ESAX. The upper and that labeled c was obtained in the presence of 10 µM Tef plus 10 µM ESAX. The upper part in part in (A) shows the voltage-clamp protocol applied. Summary bar graphs shown in (B,C) respec- (A) shows the voltage-clamp protocol applied. Summary bar graphs shown in (B,C) respectively tively demonstrate effects of Tef or Tef plus ESAX on peak INa and the time constant (τinact(S)) in the τ demonstrateslow component effects of ofINa Tef inactivation or Tef plus (mean ESAX ± on SEM; peak n I=Na 7 forand each the timebar). constant* Significantly ( inact(S) different) in the from slow componentcontrols (p < of 0.05),INa inactivation and + significantly (mean different± SEM; nfrom= 7 Tef for (10 each μM) bar). alone * Significantly group (p < 0.05). different from controls (p < 0.05), and + significantly different from Tef (10 µM) alone group (p < 0.05). + 3.6. Augmented Amplitude and Hysteresis by Tef of Persistent Na+ Current (INa(P)) Attenuated 3.6. Augmented Amplitude and Hysteresis by Tef of Persistent Na Current (INa(P)) Attenuated byby ESAX ESAX WeWe next next continued continued to to investigate investigate whether whether cell cell exposure exposure to ESAX to ESAX could could modify modify the Tef- the Tef-mediated enhancement of INa(P) activated in response to the isosceles-triangular ramp mediated enhancement of INa(P) activated in response to the isosceles-triangular ramp pulse pulse in GH3 cells. In these whole-cell current recordings, the examined cell was voltage- in GH3 cells. In these whole-cell current recordings, the examined cell was voltage-clamped atclamped−50 mV at and −50 a mV set ofand upright a set of isosceles-triangular upright isosceles-triangular ramp pulses ramp ranging pulses between ranging− 100between and +50−100 mV and with +50 varying mV with durations varying at adurations rate of 0.05 at Hza rate was of gingerly 0.05 Hz applied was gingerly to it through applied digital- to it to-analogthrough digital-to-analog conversion (Figure conversion6A). In accordance (Figure 6A). with In previous accordance observations with previous [ 25,38 observa-], when tions [25,38], when GH3 cells were exposed to 10 μM Tef alone, the amplitude of INa(P) at GH3 cells were exposed to 10 µM Tef alone, the amplitude of INa(P) at high or low threshold respectivelyhigh or low activated threshold by respectively the upsloping activated (forward, by ascending)the upsloping or downsloping (forward, ascending) (backward, or descending)downsloping end (backward, of upright descending) triangular rampend of voltage upright was triangular raised andramp a strikingvoltage was figure-of- raised eightand a hysteresis striking figure-of-eight (i.e., ∞) in the instantaneoushysteresis (i.e.,I–V ∞) inrelationship the instantaneous of INa(P) I–Vwas relationship concurrently of revealedINa(P) was (Figure concurrently6B,C). For revealed example, (Figure as the isosceles-triangular6B,C). For example, ramp as the pulse isosceles-triangular with a duration oframp 0.8 s pulse (or ramp with speed a duration of ±0.38 of 0.8 mV/ms) s (or ramp was speed applied, of in±0.38 the mV/ms) presence was of 10 applied,µM Tef, in the the peakpresenceINa(P) ofamplitude 10 μM Tef, measured the peak atINa(P) the amplitude level of − measured30 mV (i.e., at high-threshold the level of −30I mVNa(P) (i.e.,) during high- thethreshold ascending INa(P) phase) during of triangular the ascending ramp pulsephase wasof triangular conceivably ramp raised pulse to 62was± 9conceivably pA (n = 7, praised< 0.05) to from62 ± 9 a pA control (n = value7, p < (measured0.05) from a at control the same value level (measured of membrane at the potential)same level of of 21membrane± 4 pA (n potential)= 7). Meanwhile, of 21 ± 4 duringpA (n = exposure7). Meanwhile, to 10 µ duringM Tef, exposure the peak ItoNa(P) 10 μamplitudeM Tef, the measuredpeak INa(P)at amplitude−80 mV duringmeasured the at descending −80 mV during phase the of suchdescending pule was phase concurrently of such pule raised was fromconcurrently 38 ± 7 to raised 58 ± from9 pA 38 (n ±= 7 7, top 58< ± 0.05). 9 pA In(n particular,= 7, p < 0.05). as In depicted particular, in Figure as depicted6B, the in subsequentFigure 6B, the addition subsequent of 3 µ additionM ESAX, of but 3 μ stillM ESAX, in the but presence still in ofthe 10 presenceµM Tef, of was 10 ableμM Tef, to producewas able a to progressive produce a reductionprogressive in reduction the amplitude in the of amplitude high- or low-threshold of high- or low-threshold peak INa(P) measuredpeak INa(P) atmeasured−30 or − at80 − mV30 or to −80 48 mV± 7 to or 48 58 ± 79 or pA 58 ( n± =9 pA 7, p (

inducedinduced increase increase in in the the respective respective high- high- and and low-threshold low-threshold amplitude amplitude of of INa(P)INa(P) activatedactivated by by thethe ascending ascending (rising) (rising) and and descending descending (declining) (declining) phases phases of ofthe the isosceles-triangular isosceles-triangular ramp ramp pulsepulse detected detected in inthese these cells cells (Figure (Figure 6D).6D). It It is is conceivable; conceivable; therefore, therefore, that that the the addition addition of of ESAX,ESAX, but but still still in inthe the presence presence of ofTef, Tef, result resulteded in ina marked a marked reduction reduction in involtage-dependent voltage-dependent hysteretichysteretic strength strength of of the the instantaneous instantaneous I–VI–V relationshiprelationship of of INa(P)INa(P) in inresponse response to toisosceles- isosceles- triangulartriangular ramp ramp voltage voltage in inGH GH3 cells.3 cells.

0.8 sec A 1.6 sec 0 3.2 sec mV -100 0 1000200030004000 msec 100 control

0 pA

-100 0 1000 2000 3000 4000 100 Tef (10 4M)

0 pA

-100 * * 0 1000 2000 3000 4000 100 Tef (10 ΤM)+ESAX (10 πM) 0 pA

-100 0 1000 2000 3000 4000 msec

B 80 control 40

0 pA 0.8 sec -40 3.2 sec 0 -80 -80 -40 0 40 mV 80 Tef (10 ( M) -20 40

0 pA * 0.8 sec pA -40 * 3.2 sec -40 -80 -80 -40 0 40 mV 80 Tef (10 M) 0.8 sec +ESAX (10 +M) 40 -60 3.2 sec 0 pA 0.8 sec 3.2 sec -40 -80 -40 0 -80 mV -80 -40 0 40 mV

Figure 6. Cont. Biomedicines 2021, 9, x FOR PEER REVIEW 11 of 17 Biomedicines 2021, 9, 549 11 of 17

FigureFigure 6. Inhibitory 6. Inhibitory effect effect of ESAX of ESAX on Tef-mediated on Tef-mediated increase increase in persistent in persistent INa (INa(P)INa)( IactivatedNa(P)) activated by by isosceles-triangularisosceles-triangular ramp ramp pulse pulse in GH in3 GHcells.3 cells.In these In thesewhole-cell whole-cell current current recordings, recordings, the potential the potential appliedapplied to the to examined the examined cell was cell held was heldat −50 at mV−50 and mV the and isosceles-tria the isosceles-triangularngular ramp voltage ramp voltage with with

varyingvarying durations durations of 0.4 of to 0.4 3.2 to s (i.e., 3.2 s ramp (i.e., rampspeed speedof ± 0.094 of ± to0.094 0.75 to mV/ms) 0.75 to mV/ms) to activate to I activateNa(P) in INa(P) responsein response to the forward to the forward (from −100 (from to +50−100 mV) to and +50 backward mV) and backward(from +50 to (from −100 +50 mV) to ramp−100 volt- mV) ramp age-clampvoltage-clamp command command was delivered was delivered to it. (A to) Representative it. (A) Representative current current traces tracesobtained obtained in the in control the control period (upper), and during cell exposure to Tef (10 μM) (middle), or to Tef (10 μM) plus ESAX (10 period (upper), and during cell exposure to Tef (10 µM) (middle), or to Tef (10 µM) plus ESAX (10 µM) μM) (lower). The black, red, or blue color indicated in the right upper side respectively denote the (lower). The black, red, or blue color indicated in the right upper side respectively denote the duration duration of isosceles-triangular ramp pulse applied (i.e., 0.8, 1.6 or 3.2 s, (i.e., the ramp speed of ±0.38,of ±0.19 isosceles-triangular or ±0.094 mV/ms)). ramp Purple pulse appliedasterisks (i.e., in the 0.8, middle 1.6 or 3.2part s, of (i.e., (A the) shows ramp Tef-induced speed of ±0.38, aug-±0.19 mentationor ±0.094 in the mV/ms)). amplitude Purple of INa(P) asterisks elicited inby the the middle upsloping part and of (downslopingA) shows Tef-induced ends of the augmentation triangu- lar ramp.in the ( amplitudeB) Representative of INa(P) instantaneouselicited by the I–V upsloping relation of and INa(P) downsloping in response endsto isosceles-triangular of the triangular ramp. ramp(B pulse) Representative with a duration instantaneous of 0.8 s (blackI–V relation color) or of 3.2INa(P) s (bluein response color). The to isosceles-triangular current traces in the ramp up- pulse per partwith are a duration controls, of while 0.8 s those (black in color) the middle or 3.2 or s (blue lower color). part were The respectively current traces acquired in the upper from the part are presencecontrols, of Tef while (10 μ thoseM) alone in the or middle Tef (10 orμM) lower plus part ESAX were (10 respectivelyμM). The dashed acquired orange from arrows the presence in the of middleTef (10partµ andM) alone the right or Tef side (10 ofµ (M)B) show plus ESAX the direction (10 µM). of The INa(P) dashed trajectories orange in arrowswhich time in the passes middle part during the elicitation by the upright isosceles-triangular ramp pulse with a duration of 0.8 s (black and the right side of (B) show the direction of I trajectories in which time passes during the color) or 3.2 s (blue color). The graph in the right sideNa(P) shows an expanded record of the dashed elicitation by the upright isosceles-triangular ramp pulse with a duration of 0.8 s (black color) or box in the left middle part. The asterisks in the middle part of (B) indicate the figure-of-eight (or infinity-shaped:3.2 s (blue color). ∞) loop The (as graph demonstrated in the right in side the showsright side) an expanded of voltage-dependent record of the dashedhysteresis box re- in the left spondingmiddle to part. the triangular The asterisks ramp. in the(C) middleFigure-of-eight part of ( Bpattern) indicate in vo theltage-dependent figure-of-eight (orhysteresis infinity-shaped: of INa(P) activated by isosceles-triangular ramp voltage with a ramp duration of 3.2 s (or a ramp speed Biomedicines 2021, 9, 549 12 of 17

∞) loop (as demonstrated in the right side) of voltage-dependent hysteresis responding to the

triangular ramp. (C) Figure-of-eight pattern in voltage-dependent hysteresis of INa(P) activated by isosceles-triangular ramp voltage with a ramp duration of 3.2 s (or a ramp speed of ±0.094 mV/ms) in the presence of 10 µM Tef. The ascending limb is indicated in blue color, while the descending one is in the orange color. The dashed arrow indicates the direction of current trajectory by which Biomedicines 2021, 9, x FOR PEER REVIEW 12 of 17 time goes. (D) Summary bar graph showing the effects of Tef (10 µM) and Tef (10 µM) plus ESAX (10 µM) on INa(P) amplitude activated by the upsloping and downsloping limb of 0.8-s triangular ramp pulse (mean ± SEM; n = 7 for each bar). Current amplitude in the left side was taken at the of ±0.094 mV/ms) in the presence of 10 μM Tef. The ascending limb is indicated in blue color, levelwhile the of −descending30 mV in one situations is in the orange where color. the The forward dashed (upsloping)arrow indicates limb the direction of triangular of cur- pulse was applied to rent trajectory by which time goes. (D) Summary bar graph showing the effects of Tef (10 μM) and evoke INa(P) (i.e., high-threshold INa(P)), while that in the right side (i.e., low-threshold INa(P)) was at Tef (10 μM) plus ESAX (10 μM) on INa(P) amplitude activated by the upsloping and downsloping −limb80 of mV 0.8-s during triangular the ramp backward pulse (mean (downsloping) ± SEM; n = 7 for end each of bar). the Current pulse. amplitude * Significantly in the left different from control (sidep < was 0.05) taken and at +the significantly level of −30 mV different in situations from where Tef the (10 foµrwardM) alone (upsloping) group limb (p of< triangu- 0.05). lar pulse was applied to evoke INa(P) (i.e., high-threshold INa(P)), while that in the right side (i.e., low- 3.7.threshold Effect INa(P) of) was ESAX at −80 on mV INa duringIdentified the backward in Pituitary (downsloping) MMQ end Cellsof the pulse. * Signifi- cantly different from control (p < 0.05) and + significantly different from Tef (10 μM) alone group (p < 0.05).In another set of experiments, we tested whether INa present in another type of pituitary lactotrophs (i.e., MMQ cells) could still be modified by the presence of ESAX. The3.7. Effect preparation of ESAX on onINa Identified these cellsin Pituitary was MMQ detailed Cells under Materials and Methods. Cells were bathedIn another in Ca set2+ -free,of experiments, Tyrode’s we solution tested whether containing INa present 10 in mM another TEA type and of pitu- the pipet was filled upitary with lactotrophs Cs+-containing (i.e., MMQ cells) solution. could still Of be notice, modified as by MMQ the presence cells wereof ESAX. continually The exposed to preparation on these cells was detailed under Materials and Methods. Cells were bathed ESAXin Ca2+-free, at a concentrationTyrode’s solution of containing 1 or 3 µ M,10 mM the TEA amplitude and the pipet of peak was IfilledNa activated up with by abrupt 40 ms depolarizationCs+-containing solution. from Of− notice,80 to as− 10MMQ mV cells was were appreciably continually exposed diminished, to ESAX inat a combination with theconcentration raised inactivation of 1 or 3 μM, the rate amplitude of the of current peak INa (Figure activated7 ).by Forabrupt example, 40 ms depolari- cell exposure to 3 µM zation from −80 to −10 mV was appreciably diminished, in combination with the raised ESAX gradually decreased peak INa from 267 ± 26 to 187 ± 15 pA (n = 8, p < 0.05). After inactivation rate of the current (Figure 7). For example, cell exposure to 3 μM ESAX grad- washout of the drug (i.e., ESAX was removed, but cells were still exposed to Ca2+-free, ually decreased peak INa from 267 ± 26 to 187 ± 15 pA (n = 8, p < 0.05). After washout of the Tyrode’sdrug (i.e., ESAX solution was removed, containing but cells 10 were mM still TEA), exposed current to Ca2+ amplitude-free, Tyrode’s returned solution to 259 ± 22 pA (containingn = 8, p < 10 0.05). mM TEA), Meanwhile, current amp thelitudeτinact(S) returnedvalue to 259 of ± 22 the pA current (n = 8, p < was 0.05). significantly Mean- shortened fromwhile, the 2.7 τ±inact(S)0.8 value to of 1.7 the± current0.7 ms was ( nsignificantly= 8, p < shortened 0.05). Therefore, from 2.7 ± 0.8 indistinguishable to 1.7 ± 0.7 from the ms (n = 8, p < 0.05). Therefore, indistinguishable from the observations made in GH3 cells, observations made in GH3 cells, the results that we have obtained showed the ability of the results that we have obtained showed the ability of ESAX to inhibit INa in response to ESAXthe rapid to depolarizing inhibit INa stepin responsein these cells. to the rapid depolarizing step in these cells.

Figure 7. Effect if ESAX on INa is present in MMQ pituitary lactotrophs. In this series of experi- Figure 7. Effect if ESAX on INa is present in MMQ pituitary lactotrophs. In this series of experiments, we ments, we kept cells bathed in Ca2+-free Tyrode’s solution and the electrodes were backfilled with 2+ + keptCs+-containing cells bathed solution. in Ca As whole-cell-free Tyrode’s configuratio solutionn was andfirmly the establishe electrodesd, we werevoltage-clamped backfilled with Cs -containing solution.the cell at − As80 mV whole-cell and the depolarizing configuration voltage was step firmly to −10 mV established, followed by we return voltage-clamped to −50 mV was the cell at −80 mV delivered to it. (A) Representative INa traces in response− to depolarizing command pulses− (indi- andcated thein the depolarizing upper part). (a): voltage control; (b): step 1 μ toM ESAX;10 mV(c): 3 followedμM ESAX. Inset by returnin the right to side50 indi- mV was delivered to it. (catesA) Representative an expanded recordINa fromtraces dashed in response box. (B) Summary to depolarizing of the data command showing effect pulses of ESAX (indicated (1 or in the upper part). (a):3 μM) control; on peak (b): amplitude 1 µM ESAX;of INa in MMQ (c): 3 cellsµM (mean ESAX. ± SEM; Inset n in = 8 the for righteach bar). side Current indicates amplitude an expanded record from was measured at the start of 40 ms depolarizing pulse from −80 to −10 mV. * Significantly different dashedfrom control box. (p (

4. Discussion

The principal findings in this study are that (a) the presence of ESAX could depress INa in a concentration, time-, state- and hysteresis-dependent manner identified in GH3 cells; (b) this drug resulted in the differential inhibition of peak or late amplitudes of INa activated by abrupt membrane depolarization with effective IC50 value of 13.2 or 3.2 µM, respectively; (c) ESAX did not modify the steady-state activation curve of peak INa; however, the recovery of INa block was robustly prolonged in its presence; (d) the presence of Tef enhanced both the amplitude and τinact(S) of peak INa, while, further addition of ESAX could effectively counteract Tef-induced modification of INa; (e) subsequent application of ESAX was capable of depressing Tef-induced increase in the high- or low-threshold amplitude of INa(P) elicited by the isosceles-triangular ramp at either upsloping or downsloping limb, respectively; and (f) the presence of ESAX was effective at decreasing the amplitude and gating of INa in pituitary MMQ cells. Taken together with the present observations, the results allow us to reflect that ESAX-perturbed change in the amplitude, gating properties, and hysteretic behavior of INa tends to be independent and upstream of its action on the MR activity, and that it would hence conceivably participate in the adjustments on varying functional activities in electrically excitable cells (e.g., GH3 or MMQ cells) occurring in vivo. In this study, the presence of neither Dex nor Aldo had minimal change on INa. The addition of ESAX, but still in the continued presence of Aldo, was able to decrease the amplitude of peak INa in GH3 cells. Therefore, it seems unlikely that ESAX-mediated inhibi- tion of the amplitude and gating kinetics of INa was largely associated with its blockade of MR. It is also important to be emphasized that the maximal plasma concentration of ESAX was reported previously to reach around 1416 ng/mL (or 3 µM) after 10-day administration with the dose of 100 mg/day [39]. As such, the inhibitory effects of ESAX on peak and late INa that we have obtained here could noticeably be of clinical or therapeutic relevance. In the present investigations, the non-linear voltage-dependent hysteresis of INa(P) in the control period and during cell exposure to Tef or Tef plus ESAX was observed by the upright isosceles-triangular ramp voltage command with varying durations. In particular, as cells were exposed to 10 µM Tef, the peak INa(P) activated at the forward (upsloping) limb of the triangular ramp pulse with varying durations was noticed to be enhanced, particu- larly at the level of −30 mV, whereas the INa(P) amplitude at the backward (downsloping) end was increased at the level of −80 mV. Consequently, in the presence of 10 µM Tef, the figure-of-eight configuration in the hysteretic loop elicited by the triangular ramp pulse was evidently demonstrated (Figure6). That is, there appeared to be the two types of INa(P), that is, low-threshold (i.e., activating at a voltage range near the resting potential) and high-threshold (i.e., activating at a voltage range near the maximal INa achieved) INa(P), observed during cell exposure to 10 µM Tef. The low-threshold INa(P) was noticeably activated (at the voltage range near the resting potential) upon the downsloping end of the triangular ramp pulse; however, the high-threshold (at the voltage range where peak INa was maximally activated) was by the upsloping end of such pulse. As the ramp speed decreased, the area of such hysteretic loop progressively reduced. Therefore, findings from the present observations revealed that the triangular pulse-induced INa(P) was observed to undergo hysteretic change in the voltage dependence found in GH3 cells [29,30,38]. In the present study, as GH3 cells were exposed to Tef, the voltage-dependent move- ment of S4 segment residing in NaV channels could be perturbed; consequently, the cou- pling of the pore domain to the voltage-sensor domain was enhanced [25,27,29]. The voltage sensor energetically coupled to channel activation is supposed to be a conformationally flexible region of the protein. It is; therefore, tempting to speculate that such INa(P), partic- ularly during exposure to Tef, could intrinsically and dynamically possess “memory” of previous or past events, or that mode shift occurs with respect to the voltage sensitivity of gating charge movement, which depends on the previous state (or conformation) of the NaV channel [27,29,38]. Such unique type of hysteretic behavior inherently in NaV channels would potentially play substantial role in influencing electrical behavior, Na+ overload due to an excessive Na+ influx, or hormonal secretion in different types of excitable cells Biomedicines 2021, 9, 549 14 of 17

during exposure to pyrethroid insecticides (e.g., tefluthrin or cypermethrin) [23,25,37,40]. Moreover, it needs to be noticed that the subsequent addition of 3 µM ESAX, still in the continued presence of 10 µM Tef, resulted in a considerable reduction of hysteretic strength responding to triangular ramp voltage. In concert with its antagonistic action on MR, ESAX might exert an additional ameliorat- ing action on salt-induced elevation of blood pressure or on kidney injuries [1,6,8,10,41,42]. ESAX has indeed reported the ability of alleviating effects in hypertensive patients with normal kidney function and in diabetic patients with albuminuria [5]. The activity of NaV channels has been noticeably disclosed to be functionally expressed in varying types of vascular smooth muscle cells [19,32,43–45]. It was also shown that Ran, an inhibitor of late INa [21], could exercise certain beneficial action in the management of pulmonary hypertension [46]. The mRNA transcripts for the α-subunit of NaV1.1, NaV1.2, NaV1.3, and NaV1.6, as well as β1- and β3-subunits of NaV channels, have been previously demon- strated in GH3 cell line [47]. Therefore, it is worth pursuing to a further extent as to which, despite its blockade on MR, ESAX-induced antihypertensive action is linked to its additional inhibitory action on INa (i.e., NaV1.7-encoded current) inherently in vascular smooth myocytes. , another structurally similar agent of ESAX, has been reported previ- ously to perturb Aldo-dependent nuclear import of the MR [48]. The Aldo binding site or MR expression was demonstrated to be present in pituitary cells including GH3 cells [11,12,16,18,36]. However, the effects of ESAX on the amplitude, kinetics, and hys- teretic strength of INa demonstrated in this study were found to be rapid and robust in onset; hence, such hastened action is expected to be independent of its action on the cytosolic activity of MR and it tends to occur in a non-genomic mechanism. On the basis of the present observations, despite the antagonistic effect on MR [2,10], our results strongly imply that the inhibitory action of ESAX on transmembrane ionic chan- nels, particularly on NaV channels, tends to be direct obligate mechanisms. Through ionic mechanisms demonstrated herein, it or other structurally similar non-steroidal compounds (e.g., apararenone and finerenone), which can be preferentially used for oral intake, is able to adjust the functional activities of different types of endocrine or neuroendocrine cells, if similar in vivo findings exist. To this end, even during its perturbing action on the renin- angiotensin-Aldo system [5,6,42,49], findings from this study also highlight an important alternative aspect that needs to be taken into consideration, inasmuch as the beneficial effects of ESAX in different pathologic disorders, such as primary hyperaldosteronism, chronic kidney disease, refractory hypertension, and heart failure, have been currently evaluated [1,3–6,9,39,49–51].

Author Contributions: Conceptualization, S.-N.W.; methodology, S.-N.W.; software, W.-T.C. and S.-N.W.; validation, W.-T.C. and S.-N.W.; formal analysis, S.-N.W.; investigation, W.-T.C. and S.-N.W.; resources, W.-T.C. and S.-N.W.; data curation, S.-N.W.; writing—original draft preparation, S.-N.W.; writing—review and editing, W.-T.C. and S.-N.W.; visualization, W.-T.C. and S.-N.W.; supervision, W.- T.C. and S.-N.W.; project administration, S.-N.W.; funding acquisition, W.-T.C. and S.-N.W. All authors have read and agreed to the published version of the manuscript. Funding: This work was partly supported by a grant from the Ministry of Science and Technology (MOST-108-23140B-006-094), 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 corre- sponding author. Acknowledgments: The authors are grateful to Shin-Yen Chao and Tzu-Hsien Chuang for their as- sistances. Conflicts of Interest: The authors declare that they have no conflict of interest, financial or otherwise. The authors are responsible for the content of writing of the paper. Biomedicines 2021, 9, 549 15 of 17

Abbreviations Aldo, aldosterone; Dex, dexamethasone; ESAX, esaxerenone (1-(2-hydroxyethyl)-4-methyl-N- (4-methylsulfonylphenyl)-5-[2-(trifluoromethyl)phenyl]pyrrole-3-carboxamide); I–V, current versus + voltage; IC50, concentration required for 50% inhibition; INa, voltage-gated Na current; INa(P), per- + + sistent Na current; MR, mineralocorticoid receptor; NaV channel, voltage-gated Na channel; Ran, ranolazine; SEM, standard error of mean; TEA, tetraethylammonium chloride; Tef, tefluthrin; TTX,

tetrodotoxin; τinact(S), time constant in the slow component of current inactivation.

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