Department of Pathological Physiology, Faculty of Medical Sciences, University of Kragujevac, 34000 Kragujevac, Serbia
Institute for Pathological Physiology, School of Medicine, University of Belgrade, 11000 Belgrade, Serbia.
Biol Open. 2016 Apr 15;5(4):381-8. doi: 10.1242/bio.014936.
Oxidative stress and the generation of reactive oxygen species (ROS) play an important role in cellular damage. Electrophysiological analyses have shown that membrane transport proteins are susceptible to ROS. In the present study, oxidative stress was induced in Retzius nerve cells of the leechHaemopis sanguisugaby bath application of 1 mM of hydrogen peroxide (H2O2) and 0.02 mM of copper (Cu) for 20 min. The H2O2/Cu(II) produced considerable changes in the electrical properties of the Retzius nerve cells. Intracellular recording of the resting membrane potential revealed that the neuronal membrane was depolarized in the presence of H2O2/Cu(II). We found that the amplitude of action potentials decreased, while the duration augmented in a progressive way along the drug exposure time. The combined application of H2O2and Cu(II) caused an initial excitation followed by depression of the spontaneous electrical activity. Voltage-clamp recordings revealed a second effect of the oxidant, a powerful inhibition of the outward potassium channels responsible for the repolarization of action potentials. The neurotoxic effect of H2O2/Cu(II) on the spontaneous spike electrogenesis and outward K(+)current of Retzius nerve cells was reduced in the presence of hydroxyl radical scavengers, dimethylthiourea and dimethyl sulfoxide, but not mannitol. This study provides evidence for the oxidative modification of outward potassium channels in Retzius nerve cells. The oxidative mechanism of the H2O2/Cu(II) system action on the electrical properties of Retzius neurons proposed in this study might have a wider significance, referring not only to leeches but also to mammalian neurons.
氧化应激和活性氧（ROS）的产生在细胞损伤中起着重要作用。电生理分析表明，膜转运蛋白易受 ROS 影响。在本研究中，通过 1mM 过氧化氢（H2O2）和 0.02mM 铜（Cu）浴处理 20 分钟，诱导水蛭 Haemopis sanguisuga 的 Retzius 神经细胞发生氧化应激。H2O2/Cu(II) 产生了 Retzius 神经细胞电特性的显著变化。静息膜电位的细胞内记录显示，神经元膜在 H2O2/Cu(II)存在下去极化。我们发现动作电位的幅度减小，而随着药物暴露时间的延长，持续时间逐渐增加。H2O2 和 Cu(II) 的联合应用导致初始兴奋，随后自发电活动抑制。电压钳记录显示了氧化剂的第二种效应，即强烈抑制负责动作电位复极化的外向钾通道。在羟基自由基清除剂二甲基硫脲和二甲基亚砜存在下，H2O2/Cu(II) 对 Retzius 神经细胞自发尖峰电发生和外向 K(+)电流的神经毒性作用降低，但甘露醇没有降低。这项研究为 Retzius 神经细胞外向钾通道的氧化修饰提供了证据。本研究提出的 H2O2/Cu(II) 系统对 Retzius 神经元电特性作用的氧化机制可能具有更广泛的意义，不仅涉及水蛭，还涉及哺乳动物神经元。
