Department of Anesthesiology and Pain Medicine, Cheil General Hospital, Kwandong University, Mukjung-dong, Jun-gu, Seoul, South Korea.
Department of Anesthesiology and Pain Medicine, Seoul National University Hospital, Yongon-dong, Chongno-gu, Seoul, South Korea.
Toxicol Lett. 2014 Feb 10;225(1):147-52. doi: 10.1016/j.toxlet.2013.12.002. Epub 2013 Dec 16.
Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72 h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001-1 μM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03 μM or higher and at an exposure time of 72 h. Vmax on the glutamate response was significantly reduced in the nicotine group (0.03 μM for 72 h), but the Km value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03 μM for 72 h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors+nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors+nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.
尼古丁是烟草的主要成分，会在动物模型中引发癫痫发作，并且吸烟被认为是与癫痫或癫痫发作相关的行为风险因素。在海马体中，尼古丁引起癫痫发作的起源，大多数谷氨酸摄取可能主要由兴奋性氨基酸转运蛋白 3（EAAT3）完成。已经报道了颞叶癫痫与 EAAT3 下调之间的关联。因此，我们假设尼古丁可能通过减弱 EAAT3 活性引发癫痫发作。我们在 Xenopus 卵母细胞表达系统中使用双电极电压钳研究了慢性尼古丁暴露（72 小时）导致 EAAT3 活性降低的情况。还确定了蛋白激酶 C（PKC）和磷脂酰肌醇 3-激酶（PI3K）的作用。尼古丁（0.001-1 μM）导致 EAAT3 活性呈时间和剂量依赖性下降，最大抑制作用出现在尼古丁浓度为 0.03 μM 或更高且暴露时间为 72 小时。在尼古丁组中，谷氨酸反应的 Vmax 显著降低（72 小时的 0.03 μM），但 EAAT3 对谷氨酸的 Km 值没有改变。当用佛波醇 12-肉豆蔻酸 13-乙酸酯（PKC 激活剂）预处理暴露于尼古丁的卵母细胞（72 小时的 0.03 μM）时，尼古丁引起的 EAAT3 活性降低被消除。PKC 抑制剂（Staurosporine、Chelerythrine 和 Calphostin C）显著降低基础 EAAT3 活性，但 PKC 抑制剂、尼古丁和 PKC 抑制剂+尼古丁组之间没有显着差异。PI3K 抑制剂（wortmannin 和 LY294002）、尼古丁和 PI3K 抑制剂+尼古丁之间观察到相似的反应模式。总之，这项研究表明，尼古丁降低 EAAT3 活性，这种抑制似乎依赖于 PKC 和 PI3K。我们的结果可能为尼古丁引起的癫痫发作提供了另一种机制。
