Zingerone Modulates Neuronal Voltage-Gated Na+ and L-Type Ca2+ Currents

Abstract

Zingerone (ZO), a nontoxic methoxyphenol, has been demonstrated to exert various important biological effects. However, its action on varying types of ionic currents and how they concert in neuronal cells remain incompletely understood. With the aid of patch clamp technology, we investigated the effects of ZO on the amplitude, gating, and hysteresis of plasmalemmal ionic currents from both pituitary tumor (GH₃) cells and hippocampal (mHippoE-14) neurons. The exposure of the GH₃ cells to ZO differentially diminished the peak and late components of the I_Na. Using a double ramp pulse, the amplitude of the I_Na(P) was measured, and the appearance of a hysteresis loop was observed. Moreover, ZO reversed the tefluthrin-mediated augmentation of the hysteretic strength of the I_Na(P) and led to a reduction in the I_Ca,L. As a double ramp pulse was applied, two types of voltage-dependent hysteresis loops were identified in the I_Ca,L, and the replacement with BaCl₂-attenuated hysteresis of the I_Ca,L enhanced the I_Ca,L amplitude along with the current amplitude (i.e., the I_Ba). The hysteretic magnitude of the I_Ca,L activated by the double pulse was attenuated by ZO. The peak and late I_Na in the hippocampal mHippoE-14 neurons was also differentially inhibited by ZO. In addition to acting on the production of reactive oxygen species, ZO produced effects on multiple ionic currents demonstrated herein that, considered together, may significantly impact the functional activities of neuronal cells.

Keywords: L-type Ca2+ current; hysteresis; persistent Na+ current; vanillylacetone); voltage-gated Na+ current; zingerone (gingerone.

Figure 1

Effect of zingerone (ZO) on voltage-gated Na⁺ current (I_Na) recorded from pituitary tumor (GH₃) cells. This set of measurements was undertaken in cells bathed in Ca²⁺-free Tyrode’s solution in which 10 mM TEA was present, where the recording pipette was backfilled with a Cs⁺-containing solution. (A) Representative current traces obtained in (a) the control situation (i.e., ZO was not present) and during cell exposure to 3 μM ZO (b) or 10 μM ZO (c). Inset is the applied voltage pulse protocol. (B) Expanded records from the dashed box in (A). (C) Concentration-dependent inhibition of ZO on the peak (○) and late (■) amplitude of I_Na measured from GH₃ cells (mean ± SEM; n = 7 for each point). Peak or late amplitude with or without ZO addition, respectively, taken at the beginning or end of a rapid depolarizing pulse ranging from −80 to −10 mV. Solid smooth lines are fits to the modified Hill equation (as elaborated in the Section 4).

Figure 2

Inhibitory effect of ZO on Tef-mediated augmentation in persistent I_Na (I_Na(P)) activated by a double ramp pulse in GH₃ cells. In this set of whole-cell current recordings, we held the potential applied to the examined cell at −50 mV, and a triangular ramp voltage with a duration of 1.5 s (i.e., a ramp speed of ±0.2 mV/msec) was applied to elicit I_Na(P). That is, the whole-cell currents were evoked in response to the forward (ascending from −100 to +50 mV) and backward (descending from +50 to −100 mV) ramp voltage-clamp command. (A) Representative current traces obtained in the control period (upper) and during exposure to Tef (10 μM) (middle) or to Tef (10 μM) plus ZO (10 μM). The uppermost inset is the applied pulse protocol, while the broken arrows in each panel are the direction of the current trajectory over time. The figure-eight pattern in the voltage-dependent hysteresis of I_Na(P) elicited by double ramp voltage with a duration of 1.5 s (or ramp speed of ±0.2 mV/msec) should be noted. Panels (B,C), respectively, show the effects of Tef (10 μM) and Tef (10 μM) plus ZO (3 or 10 μM) on the I_Na(P) amplitude activated by the upsloping (ascending) and downsloping (descending) limb of a 1.5 s triangular ramp pulse (mean ± SEM; n = 7 for each bar). The current amplitude in (B) or (C) was taken at either the −20 mV (i.e., high-threshold I_Na(P)) or at −60 mV (i.e., low-threshold I_Na(P)), respectively. * Significantly different from controls (p < 0.05) and † significantly different from the 10 μM Tef alone group (p < 0.05). Panel (A): (One-way ANOVA, F(4,30) = 3.693, p = 0.01) and panel (B): (One-way ANOVA, F(4,30) = 3.822, p = 0.01).

Figure 3

Inhibitory effect of ZO on the L-type Ca²⁺ current (I_Ca,L) identified in GH₃ cells. In these experiments, the cells were kept immersed in a normal Tyrode’s solution containing 1.8 mM CaCl₂. The recording electrode was filled with Cs⁺-containing solution. (A) Representative current traces obtained under (a) the control situation (i.e., ZO was not present), and in the presence of 3 μM ZO (b) or 10 μM ZO (c). The inset shows the applied voltage-clamp protocol. (B) Mean current vs. voltage (I–V) relationships of peak I_Ca,L in the absence (■) and presence (□) of 10 μM ZO (mean ± SEM; n = 7 for each point). The current amplitude was measured at the start of each membrane depolarization to voltages ranging between −40 and +40 mV from a holding potential of −50 mV. (C) Concentration-dependent effect of ZO on the amplitude (□) of I_Ca,L evoked by membrane depolarization to +10 mV from a holding potential of −50 mV (mean ± SEM; n = 8 for each point). The current amplitude was measured at the start of the depolarizing pulse during exposure to various concentrations of ZO. The continuous smooth line indicates the goodness-of-fit to the modified Hill equation, as stated in the Section 4.

Figure 4

Characterization of voltage-dependent hysteresis (i.e., an instantaneous current–voltage relationship) of I_Ca,L identified in GH₃ cells. Cells were bathed in a normal Tyrode’s solution containing 10 mM TEA and 1 μM TTX, and the electrode was filled with a solution containing Cs⁺. (A) Current traces were evoked by a 1.5 s double ramp voltage (as indicated in the inset). The ascending limb is shown in blue, and the descending one is shown in orange. The arrow denotes the direction of the current trajectory over time, while the asterisk notes the appearance of I_Na(P) inhibited by replacement with BaCl₂. The grey area labeled as a and b, respectively, illustrates the hysteretic loop of I_Ca,L (i.e., high- and low-threshold loops). (B,C) show the hysteretic loop of the current trace obtained in the control period (i.e., I_Ca,L) and the BaCl₂ substitution (i.e., Ba²⁺ inward current [I_Ba]), respectively. The representative current trace in (B) is the control (i.e., ZO was not present), while that in (C) was obtained when 2 mM BaCl₂ was substituted for CaCl₂. It should be noted that the hysteretic strength (i.e., both loops) of I_Ca,L elicited by the double ramp pulse (indicated in the inset of (B)) was diminished as the replacement of CaCl₂ with BaCl₂ was made.

Figure 5

Inhibitory effect of ZO on I_Ca,L activated by a double ramp pulse in GH₃ cells. (A) Representative current traces obtained in the control period (a) and in the presence of 3 μM ZO (b) or 10 μM ZO (c). The inset in the upper part illustrates the voltage protocol. (B,C), respectively, illustrate the effects of ZO (3 and 10 μM) or BaCl₂ replacement on the hysteretic area (high- and low-threshold loop) of I_Ca,L (mean ± SEM; n = 7 for each bar). * Significantly different from the controls (p < 0.05) and ** significantly different from the ZO (3 μM)-alone groups (p < 0.05). Panel (A): (One-way ANOVA, F(4,30) = 4.156, p = 0.01) and panel (B): (One-way ANOVA, F(4,30) = 3.692, p = 0.01).

Figure 6

Inability of ZO to perturb the hyperpolarization-activated cation current (I_h) in GH₃ cells. The experiments were conducted in cells bathed in a Ca²⁺-free Tyrode’s solution, with the internal solution backfilled with a K⁺-containing solution. (A) Representative current trace obtained in the control period (a) and cell exposure to 10 μM ZO (b) or 10 μM ZO plus 3 μM cilobradine (Cil) (c). The upper part is the applied voltage-clamp protocol. (B) Summary bar graph showing the effects of ZO or ZO plus cilobradine (Cil) on I_h amplitude (mean ± SEM; n = 7 for each bar). The current amplitude was obtained at the endpoint of a 2 s hyperpolarizing pulse ranging from −40 to −110 mV. * Significantly different from the control or the 10 μM ZO-alone group (p < 0.05). (One-way ANOVA, F(2,18) = 5.085, p = 0.02).

Figure 7

Mild inhibition of a Delayed-Rectified K⁺ Current (I_K(DR)) caused by ZO in GH₃ cells. In these experiments, we kept cells bathed in Ca²⁺-free Tyrode’s solution, and the recording pipette was filled with K⁺-containing solution. As the whole-cell configuration was commenced, we voltage-clamped the examined cell at −50 mV and applied various voltage pulses ranging between −60 and +50 mV at 10 mV. (A) Representative current traces obtained in the control period (upper) and after application of 10 μM OZ (lower). The uppermost part indicates the applied voltage-clamp protocol. (B) The mean I–V relationship of I_K(DR) taken without (■) or with (○) the addition of 10 μM ZO (mean ± SEM; n = 8 for each point). The current amplitude was taken at the endpoint of each voltage command. * Significantly different from the controls taken at the same potential (p < 0.05).

Figure 8

Inhibitory effect of ZO on I_Na on mHippoE-14 hippocampal neurons. (A) Representative current traces obtained in the absence (a) or presence of 1 μM ZO (b). The inset shows the pulse protocol used in this study. Panel (B,C) are summary bar graphs demonstrating the effects of ZO and ZO plus Tef on the peak amplitude or τ_inact(S) of depolarization-activated I_Na, respectively (mean ± SEM; n = 7 for each bar). * Significantly different from the control (p < 0.05), ** significantly different from the 1 μM ZO-alone group (p < 0.05), and + significantly different from the 3 μM ZO-alone group (p < 0.05). Panel (A): (One-way ANOVA, F(3,24) = 3.351, p = 0.03) and panel (B): (One-way ANOVA, F(3,24) = 4.012, p = 0.02).