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

Abstract

Apocynin (aPO, 4′-Hydroxy-3′-methoxyacetophenone) is a cell-permeable, anti-inflammatory phenolic compound that acts as an inhibitor of NADPH-dependent oxidase (NOX). However, the mechanisms through which aPO can interact directly with plasmalemmal ionic channels to perturb the amplitude or gating of ionic currents in excitable cells remain incompletely understood. Herein, we aimed to investigate any modifications of aPO on ionic currents in pituitary GH3 cells or murine HL-1 cardiomyocytes. In whole-cell current recordings, GH3-cell exposure to aPO effectively stimulated the peak and late components of voltage-gated Na+ current (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 Evidence for Sparsentan-Mediated Inhibition of INa and IK(erg): Possibly Unlinked to Its Antagonism of Angiotensin II or Endothelin Type a Receptor

“…In continued presence of Tef, the I Na(R) amplitude at the same level of voltage was further reduced to 331 ± 29 pA ( n = 7, p < 0.05) during cell exposure to Tef (10 μM) plus sparsentan (3 μM). Therefore, the experimental observations presented herein reflected that the instantaneous I Na(R) responding to the abrupt descending ramp pulse was sensitive to activation by Tef, as reported previously [ 28 , 29 , 30 ], and that the further application of sparsentan became capable of attenuating Tef-stimulated I Na(R) in these cells.…”

“…When the whole-cell configuration was established, we voltage-clamped the tested cell at the level of −80 mV, and to ensure complete recovery of I Na , the voltage preceding the depolarizing pulse was set at −100 mV with a duration of 40 ms; thereafter, for the activation of I Na , the 40 ms depolarizing voltage pulse from −100 to −10 mV followed by a return to −50 mV for another 40 ms was imposed on the cell. Upon this protocol, the I Na with a rapid activation, inactivation and deactivation was robustly evoked [ 21 , 28 ]. Of interest, one minute after GH 3 cells were constantly exposed to spansentan (1 or 3 μM), the peak amplitude of I Na was promptly decreased and the inactivation time course of the current concurrently increased ( Figure 1 A,B).…”

“…Earlier reports have demonstrated the existence of I Na(R) in these lactotrophs which were sensitive to stimulation by Tef [ 28 , 29 ]. This type of I Na was noticed to exhibit a nonmonotonic voltage dependency on membrane repolarization [ 28 , 30 ].…”

“…Earlier reports have demonstrated the existence of I Na(R) in these lactotrophs which were sensitive to stimulation by Tef [ 28 , 29 ]. This type of I Na was noticed to exhibit a nonmonotonic voltage dependency on membrane repolarization [ 28 , 30 ]. Hence, we started out examining whether the sparsentan addition can lead to any adjustments on Tef-stimulated I Na(R) present in these cells.…”

“…However, under our experimental protocol, the voltage-activated inward currents were sensitive to stimulation by Tef or β-pompilidotoxin. Tef or β-pompilidotoxin has been broadly reported to be an activator of I Na [ 21 , 28 , 31 , 32 ]. Moreover, in the continued presence of Tef or β-pompilidotoxin, additional exposure of cells to sparsentan was found to reverse the increase in I Na(R) or peak I Na stimulated by these compounds ( Figure 5 and Figure 6 ).…”