Rudolph J G, Lemasters J J, Crews F T
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, USA.
Alcohol Clin Exp Res. 1998 Dec;22(9):2080-5.
Excessive oxidative radical formation has been implicated in a number of neurodegenerative processes, including glutamate-N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. Previous studies have suggested that the formation of reactive oxygen species (ROS) during NMDA exposure is involved in triggering the excitotoxic cascade. Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate NMDA-mediated processes, such as nitric oxide formation and excitotoxicity. It was the objective of this study to investigate the role of NMDA and ROS formation in the development of NMDA supersensitivity after chronic ethanol exposure. The fluorescent dyes dichlorofluorescein diacetate (H2DCF) and propidium iodide, which have been shown to be reliable markers for the detection of cellular oxidation and cell death, respectively, in neuronal culture preparations, were used to examine the relationship between ROS production and NMDA-mediated neuronal death after chronic ethanol administration. Cultures treated chronically with ethanol (100 mM) for 96 hr displayed significantly less H2DCF oxidation when exposed to various concentrations of FeSO4 for 25 min. However, this decrease in intracellular oxidation did not have any apparent inhibitory effects on the amount of cell death observed 12 hr after the 25-min exposure to FeSO4. When NMDA-FeSO4-mediated oxidation was examined in cultures treated chronically with ethanol, dose-dependent increases in H2DCF oxidation were observed, but only in control-treated cultures. This blunting of intracellular H2DCF oxidation did not attenuate the potentiation of NMDA-mediated excitotoxicity observed after chronic ethanol exposure. These results suggest that the observed supersensitivity to NMDA is not due to increases in intracellular ROS formation and that chronic ethanol may induce neuronal factors that reduce ROS formation, but do not protect against normal death.
过量的氧化自由基形成与许多神经退行性过程有关，包括谷氨酸-N-甲基-D-天冬氨酸（NMDA）介导的兴奋性毒性。先前的研究表明，NMDA暴露期间活性氧（ROS）的形成参与触发兴奋性毒性级联反应。已证明原代神经元培养物长期暴露于乙醇会增强NMDA介导的过程，如一氧化氮形成和兴奋性毒性。本研究的目的是调查NMDA和ROS形成在慢性乙醇暴露后NMDA超敏反应发展中的作用。荧光染料二氯荧光素二乙酸酯（H2DCF）和碘化丙啶已被证明分别是神经元培养制剂中检测细胞氧化和细胞死亡的可靠标志物，用于检查慢性乙醇给药后ROS产生与NMDA介导的神经元死亡之间的关系。用乙醇（100 mM）长期处理96小时的培养物在暴露于各种浓度的硫酸亚铁25分钟时，H2DCF氧化显著减少。然而，这种细胞内氧化的减少对25分钟暴露于硫酸亚铁后12小时观察到的细胞死亡量没有任何明显的抑制作用。当在长期用乙醇处理的培养物中检查NMDA-硫酸亚铁介导的氧化时，观察到H2DCF氧化呈剂量依赖性增加，但仅在对照处理的培养物中。细胞内H2DCF氧化的这种减弱并未减弱慢性乙醇暴露后观察到的NMDA介导的兴奋性毒性的增强。这些结果表明，观察到的对NMDA的超敏反应不是由于细胞内ROS形成增加，并且慢性乙醇可能诱导减少ROS形成但不能防止正常死亡的神经元因子。
