Department of Neurological Surgery, University of California, Davis, USA.
Restor Neurol Neurosci. 2011;29(5):331-46. doi: 10.3233/RNN-2011-603.
Glutamate transporters are responsible for removing glutamate from the extracellular space and have the potential to protect neurons from excitotoxicity. In the present study, the effects of ceftriaxone and (2R, 4R)-APDC (APDC) on the protein expression of GLAST and GLT-1, the rate of glutamate uptake, and neuroprotection were evaluated in a cell culture model of glutamate excitotoxicity.
Mixed neuron/astrocyte cultures were prepared from 1 day old rat pups. Protein levels of GLAST and GLT-1 glutamate transporters were quantified using In-Cell Western techniques after acute or 5-day treatment with either ceftriaxone or APDC. Glutamate uptake was measured using Michaelis-Menten kinetics to evaluate the effects of 5-day treatment with ceftriaxone or APDC. Neuronal cell death in response to a 10-minute 1 mM glutamate challenge was measured following 5-day treatment with either ceftriaxone or APDC.
Five-day treatment with 100 μM ceftriaxone significantly increased both GLAST and GLT-1 protein levels 31.3% and 47.5% above control, respectively, increased the Vmax 29.3%, increased the Km of glutamate uptake 117.9%, and reduced neuronal death 22.0% after a 1 mM glutamate challenge. Five-day treatment with 1 mM APDC significantly increased GLAST protein levels 27.6%, increased the Vmax 92.4%, increased the Km of glutamate transport 118.9%, and decreased neuronal death 36.8% after a 1 mM glutamate challenge.
Chronic treatment with ceftriaxone or APDC provided neuroprotection from glutamate excitotoxicity while increasing GLAST and GLT-1 protein levels and increasing glutamate uptake. These compounds may have therapeutic potential in chronic excitotoxic neurodegenerative diseases.
谷氨酸转运体负责从细胞外空间中去除谷氨酸，并有可能保护神经元免受兴奋毒性的侵害。在本研究中，我们评估了头孢曲松和（2R，4R）-APDC（APDC）对谷氨酸兴奋毒性细胞培养模型中 GLAST 和 GLT-1 蛋白表达、谷氨酸摄取率和神经保护作用的影响。
从 1 天大的大鼠幼崽中制备混合神经元/星形胶质细胞培养物。使用 In-Cell Western 技术在急性或 5 天的头孢曲松或 APDC 处理后定量测定 GLAST 和 GLT-1 谷氨酸转运体的蛋白水平。使用米氏动力学评估 5 天的头孢曲松或 APDC 处理对谷氨酸摄取的影响。在 5 天的头孢曲松或 APDC 处理后，用 1 mM 谷氨酸刺激 10 分钟后，测量神经元细胞死亡。
100 μM 头孢曲松 5 天处理分别使 GLAST 和 GLT-1 蛋白水平增加 31.3%和 47.5%，分别比对照组增加 29.3%，增加谷氨酸摄取的 Vmax 增加 117.9%，并减少 1 mM 谷氨酸刺激后的神经元死亡 22.0%。1 mM APDC 5 天处理使 GLAST 蛋白水平增加 27.6%，增加 Vmax 增加 92.4%，增加谷氨酸转运的 Km 增加 118.9%，减少 1 mM 谷氨酸刺激后的神经元死亡 36.8%。
头孢曲松或 APDC 的慢性治疗提供了对谷氨酸兴奋毒性的神经保护作用，同时增加了 GLAST 和 GLT-1 蛋白水平并增加了谷氨酸摄取。这些化合物在慢性兴奋性神经退行性疾病中可能具有治疗潜力。
