Effect of Edaravone on Aβ_1–40 Induced Enhancement of Voltage‐Gated Calcium Channel Current

β‐Amyloid (Aβ) damages nerve cells through a variety of biological effects, including oxidative stress and calcium steady‐state imbalance [1]. Aβ promotes voltage‐gated calcium channels, causing Ca²⁺ influx, resulting in intracellular calcium overload [2]; and free radicals increase voltage‐gated calcium channel (VGCC)‐mediated Ca²⁺ current [3]. These findings suggest that Aβ may achieve this effect via free radicals. Edaravone (EDA) is a hydroxyl radical scavenger, and has certain antioxidant capacity. There has been no report on whether EDA can affect the enhancement effect of VGCC‐currents induced by Aβ. In this study, PC12 cells were coincubated with EDA and Aβ_1–40 to examined potential effects of EDA on the enhancement of VGCC‐currents induced by Aβ.

PC12 cells were exposed to Aβ_1–40 (final concentration of 1μmol/L), in the absence of presence of EDA (1, 10, 100, or 300 μmol/L). 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to examine cell viability. Whole‐cell patch‐clamp recording technique was used to measure calcium current, membrane capacitance. Current density was defined as (current/membrane capacitance). Data were statistically analyzed by one‐way ANOVA.

Aβ_1–40 exposure significantly decreased the OD value at 570 nm (Figure 1). EDA alone did not affect the OD value. EDA attenuated the effects of Aβ_1–40 at 100 and 300 μmol/L, but not at 1 and 10 μmol/L. Aβ_1–40 treatment increased the peak current and current density (from 318.59 pA and 29.52 pA/pF to 580.54 pA and 56.85 pA/pF; P < 0.01 for both). CdCl₂ (150 μmol/L in the extracellular solution) blocked the inward current. Effects of Aβ_1–40 were blocked by EDA (100 μmol/L). EDA treatment alone did not affect current peak or density. Cell inward currents were observed generally larger in the Aβ_1–40 group, and the value was significantly reduced by EDA (Figure 2, panel A). The i–v curves of VGCC were V‐shaped in all groups, and the channel was activated when the membrane potential was approximately –40 mV and the peak inward current appeared at +20 mV (Figure 2, panel B).

Figure 1.

Optical density at 570 nm under different treatment conditions. Cells were exposed to 1 μmol/L Aβ_1–40, in the absence or presence of different concentrations of EDA for 12 h. OD was analyzed by MTT assay. The data showed that EDA significantly increased the OD value at 100 and 300 μmol/L, but not at 1 and 10 μmol/L. *P < 0.01 versus Aβ_1–40 alone.

Figure 2.

Electrophysiological results. Panel A, Characteristics of VGCC. The inward current of cell exposed to 1 μmol/L Aβ_1–40 was highest, followed by coincubation with 1 μmol/L Aβ_1–40 and 100 μmol/L EDA. The inward current was observed decreased, close to the amplitude of control group and the group treated with EDA alone. Using CdCl₂ (150 μmol/L CdCl₂) could block all inward currents. Panel B, VGCC currents and current‐voltage curve. The peak inward current appeared when the membrane potential was +20 mV. The average peak current value of the Aβ_1–40 group was the largest. In comparison with other groups, 100 μmol/L EDA attenuated the effect of Aβ_1–40 (P < 0.01). EDA alone had no effects on the average peak current value.

Aβ directly generates free radicals, but can also combine with metal ions (Fe³⁺, Cu²⁺) to generate hydroxyl radical and other reactive oxygen species (ROS) from hydrogen peroxide (H₂O₂) via Fenton reaction [4]. H₂O₂ can promote the enhancement of L‐VGCC Ca²⁺ current amplitudes [5]. EDA is a radical scavenger, and can reduce the damaging effects of lipid peroxidation. The results from this study showed that EDA could attenuate Aβ_1–40‐induced cytotoxicity in a concentration‐dependent manner. In the whole‐cell recording, Aβ_1–40 markedly enhanced the VGCC currents. Consistent with previous reports [6], the current was blocked with CdCl₂. Using 100 μmol/L EDA incubation alone no obvious change was observed on amplitude of VGCC current that prompted EDA have no effect on normal cell VGCC current. The effect of Aβ_1–40 on VGCC‐current in PC12 cells was abolished by the presence of 100 μmol/L EDA, indicating an important role of Ca²⁺ overload. These results suggest that EDA could attenuate Ca²⁺ overload, and mitigates the changes associated with Aβ generation [7].

Conflict of Interests

The authors have no conflict of interest.