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. 2021 May 13;9(5):549.
doi: 10.3390/biomedicines9050549.

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

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

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

Wei-Ting Chang et al. Biomedicines. .

Abstract

Esaxerenone (ESAX; CS-3150, Minnebro®) is known to be a newly non-steroidal mineralocorticoid 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 estimated to be 13.2 or 3.2 μM, respectively. The steady-state activation curve of peak INa remained unchanged during exposure to ESAX; however, recovery of peak INa block was prolonged in the presence 3 μM ESAX. In continued presence of aldosterone (10 μM), further addition of 3 μM ESAX remained effective at inhibiting INa. ESAX (3 μM) potently reversed Tef-induced augmentation of INa. By using isosceles-triangular ramp pulse with varying durations, the amplitude of persistent INa measured at high or low threshold was enhanced by the presence of tefluthrin (Tef), in combination with the appearance of the figure-of-eight hysteretic loop; moreover, hysteretic strength of the current was attenuated by subsequent addition of ESAX. Likewise, in MMQ lactotrophs, the addition of ESAX also effectively decreased the peak amplitude of INa along with the increased current inactivation 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 amplitude, gating properties and hysteresis of INa in electrically excitable cells.

Keywords: current kinetics; esaxerenone; hysteresis; persistent Na+ current; pituitary cell; voltage-gated Na+ current.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1
Effect of esaxerenone (ESAX) on voltage-gated Na+ current (INa) measured from pituitary tumor (GH3) cells. This set of experiments was undertaken in cells bathed in Ca2+-free, Tyrode’s solution containing 10 mM TEA, and we filled up the electrode by using Cs+-containing solution. (A) Representative INa traces obtained in (a) the control situation (i.e., ESAX was not present) and during cell exposure to 1 μM ESAX (b) or 3 μM ESAX (c). The voltage pulse protocol applied is shown in Inset. The lower panels in (A) show the expanded records from each dashed box. (B) Concentration-dependent relationship of ESAX on transient (or peak) (○) and late (■) component of INa evoked by abrupt membrane depolarization (mean ± SEM; n = 8 for each point). Each smooth line indicates the goodness-of-fit to the Hill equation, as described in Materials and Methods.
Figure 2
Figure 2
Effect of ESAX on current versus voltage (I–V) and conductance versus voltage relationships of INa identified in GH3 cells. (A) Mean I–V relationship of peak INa taken without (□) or with (○) the addition of 3 μM ESAX (mean ± SEM; n = 7 for each point). Inset shows the voltage-clamp protocol delivered. (B) Mean conductance versus voltage relationship of peak INa taken in the control period (□) and during cell exposure (○) to 3 μM ESAX (mean ± SEM; n = 7 for each point). Of notice, the conductance versus voltage relationship of the current between the absence and presence of ESAX did not differ, despite the decrease in maximal conductance of peak INa.
Figure 3
Figure 3
ESAX-induced prolongation in the recovery of INa block in GH3 cells. Cells were bathed in Ca2+-free, Tyrode’s solution, the electrode was filled with Cs+-containing solution, and a set of two-pulse voltage protocol was applied to the examined cells. (A) Representative current traces showing current recovery from block by use of two-step protocol with varying durations (as indicated in the uppermost part). Current traces in the upper part were obtained in the control period, while those in the lower part were taken during cell exposure to 3 μM ESAX. (B) Relationship of relative amplitude versus interpulse interval in the absence (□) and presence (○) of 3 μM ESAX. The relative amplitude of peak INa was taken by the ratio of current amplitudes elicited by the second depolarizing voltage step and those by the first pulse. Each point represents the mean ± SEM (n = 8). The smooth line indicates a best fit to single exponential.
Figure 4
Figure 4
Comparison among effects of ESAX, ESAX plus ranolazine (Ran), aldosterone (Aldo), and aldosterone plus ESAX on the amplitude of peak INa detected in GH3 cells. Cells were kept bathed in Ca2+-free, Tyrode’s solution, and the recording electrode was filled up with Cs+-containing solution. The INa was elicited by 40 ms depolarizing voltage command from −80 to −10 mV, and current amplitude at the start of the voltage pulse was measured. (A) Representative current traces obtained in the control (a) and during cell exposure to 10 μM aldosterone (Aldo) (b) or 10 μM Aldo plus 3 μM ESAX (c). 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 of the agents were present) (p < 0.05), + significantly different from ESAX (3 μM) alone group (p < 0.05), and ** significantly different from aldosterone (10 μM) alone group (p < 0.05).
Figure 5
Figure 5
Effect of ESAX on tefluthrin (Tef)-mediated augmentation of INa recorded from GH3 cells. (A) Representative current traces activated by rapid membrane depolarization (indicated in the upper part). Current trace labeled a is control, that labeled b was taken during exposure to 10 μM Tef, and that labeled c was obtained in the presence of 10 μM Tef plus 10 μM ESAX. The upper part in (A) shows the voltage-clamp protocol applied. Summary bar graphs shown in (B,C) respectively demonstrate effects of Tef or Tef plus ESAX on peak INa and the time constant (τinact(S)) in the slow component of INa inactivation (mean ± SEM; n = 7 for each bar). * Significantly different from controls (p < 0.05), and + significantly different from Tef (10 μM) alone group (p < 0.05).
Figure 6
Figure 6
Inhibitory effect of ESAX on Tef-mediated increase in persistent INa (INa(P)) activated by isosceles-triangular ramp pulse in GH3 cells. In these whole-cell current recordings, the potential applied to the examined cell was held at −50 mV and the isosceles-triangular ramp voltage with varying durations of 0.4 to 3.2 s (i.e., ramp speed of ± 0.094 to 0.75 to mV/ms) to activate INa(P) in response to the forward (from −100 to +50 mV) and backward (from +50 to −100 mV) ramp voltage-clamp command was delivered to it. (A) Representative current traces obtained in the control period (upper), and during cell exposure to Tef (10 μM) (middle), or to Tef (10 μM) plus ESAX (10 μM) (lower). The black, red, or blue color indicated in the right upper side respectively denote the 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, ±0.19 or ±0.094 mV/ms)). Purple asterisks in the middle part of (A) shows Tef-induced augmentation in the amplitude of INa(P) elicited by the upsloping and downsloping ends of the triangular ramp. (B) Representative instantaneous I–V relation of INa(P) in response to isosceles-triangular ramp pulse with a duration of 0.8 s (black color) or 3.2 s (blue color). The current traces in the upper part are controls, while those in the middle or lower part were respectively acquired from the presence of Tef (10 μM) alone or Tef (10 μM) plus ESAX (10 μM). The dashed orange arrows in the middle part and the right side of (B) show the direction of INa(P) trajectories in which time passes during the elicitation by the upright isosceles-triangular ramp pulse with a duration of 0.8 s (black color) or 3.2 s (blue color). The graph in the right side shows an expanded record of the dashed box in the left middle part. The asterisks in the middle part of (B) indicate the figure-of-eight (or infinity-shaped: ∞) 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 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 level of −30 mV in situations where the forward (upsloping) limb of triangular pulse was applied to evoke INa(P) (i.e., high-threshold INa(P)), while that in the right side (i.e., low-threshold INa(P)) was at −80 mV during the backward (downsloping) end of the pulse. * Significantly different from control (p < 0.05) and + significantly different from Tef (10 μM) alone group (p < 0.05).
Figure 6
Figure 6
Inhibitory effect of ESAX on Tef-mediated increase in persistent INa (INa(P)) activated by isosceles-triangular ramp pulse in GH3 cells. In these whole-cell current recordings, the potential applied to the examined cell was held at −50 mV and the isosceles-triangular ramp voltage with varying durations of 0.4 to 3.2 s (i.e., ramp speed of ± 0.094 to 0.75 to mV/ms) to activate INa(P) in response to the forward (from −100 to +50 mV) and backward (from +50 to −100 mV) ramp voltage-clamp command was delivered to it. (A) Representative current traces obtained in the control period (upper), and during cell exposure to Tef (10 μM) (middle), or to Tef (10 μM) plus ESAX (10 μM) (lower). The black, red, or blue color indicated in the right upper side respectively denote the 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, ±0.19 or ±0.094 mV/ms)). Purple asterisks in the middle part of (A) shows Tef-induced augmentation in the amplitude of INa(P) elicited by the upsloping and downsloping ends of the triangular ramp. (B) Representative instantaneous I–V relation of INa(P) in response to isosceles-triangular ramp pulse with a duration of 0.8 s (black color) or 3.2 s (blue color). The current traces in the upper part are controls, while those in the middle or lower part were respectively acquired from the presence of Tef (10 μM) alone or Tef (10 μM) plus ESAX (10 μM). The dashed orange arrows in the middle part and the right side of (B) show the direction of INa(P) trajectories in which time passes during the elicitation by the upright isosceles-triangular ramp pulse with a duration of 0.8 s (black color) or 3.2 s (blue color). The graph in the right side shows an expanded record of the dashed box in the left middle part. The asterisks in the middle part of (B) indicate the figure-of-eight (or infinity-shaped: ∞) 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 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 level of −30 mV in situations where the forward (upsloping) limb of triangular pulse was applied to evoke INa(P) (i.e., high-threshold INa(P)), while that in the right side (i.e., low-threshold INa(P)) was at −80 mV during the backward (downsloping) end of the pulse. * Significantly different from control (p < 0.05) and + significantly different from Tef (10 μM) alone group (p < 0.05).
Figure 7
Figure 7
Effect if ESAX on INa is present in MMQ pituitary lactotrophs. In this series of experiments, we kept cells bathed in Ca2+-free Tyrode’s solution and the electrodes were backfilled with Cs+-containing solution. As whole-cell configuration was firmly established, we voltage-clamped the cell at −80 mV and the depolarizing voltage step to −10 mV followed by return to −50 mV was delivered to it. (A) Representative INa traces in response to depolarizing command pulses (indicated in the upper part). (a): control; (b): 1 μM ESAX; (c): 3 μM ESAX. Inset in the right side indicates an expanded record from dashed box. (B) Summary of the data showing effect of ESAX (1 or 3 μM) on peak amplitude of INa in MMQ cells (mean ± SEM; n = 8 for each bar). Current amplitude was measured at the start of 40 ms depolarizing pulse from −80 to −10 mV. * Significantly different from control (p < 0.05).
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