Blanc E M, Bruce-Keller A J, Mattson M P
Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington 40536-0230, USA.
J Neurochem. 1998 Mar;70(3):958-70. doi: 10.1046/j.1471-4159.1998.70030958.x.
We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18-alpha-glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7-dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4-hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte-astrocyte coupling but no astrocyte-neuron or neuron-neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA-treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L-type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate- and FeSO4-induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.
我们研究了在胚胎大鼠海马细胞培养物中，使星形胶质细胞间隙连接解偶联对神经元氧化损伤易感性的影响。用间隙连接解偶联剂18-α-甘草次酸（GA）处理的混合培养物（神经元生长在星形胶质细胞单层上），在暴露于氧化损伤（硫酸亚铁和4-羟基壬烯醛）后，细胞内过氧化物生成（2,7-二氯荧光素荧光）显著增强、线粒体功能受损[（二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐还原]以及细胞死亡（乳酸脱氢酶释放）。单独使用GA对过氧化物的基础水平、线粒体功能或神经元存活几乎没有影响。细胞间染料转移分析显示，混合培养物中存在广泛的星形胶质细胞-星形胶质细胞偶联，但不存在星形胶质细胞-神经元或神经元-神经元偶联。对纯星形胶质细胞培养物的研究以及对混合培养物中神经元的显微镜分析表明，GA处理的培养物中氧化应激增加和细胞死亡仅发生在神经元中，而不是星形胶质细胞中。抗氧化剂（没食子酸丙酯和谷胱甘肽）可阻止暴露于硫酸亚铁/GA的神经元死亡。在暴露于GA的混合培养物中的神经元中，由硫酸亚铁诱导的神经元细胞内钙水平（[Ca2+]i）升高增强。去除细胞外钙和L型钙通道阻滞剂尼莫地平可预防硫酸亚铁和GA诱导的线粒体功能损害和细胞死亡，而谷氨酸受体拮抗剂无效。最后，GA加剧了器官型海马切片培养物中红藻氨酸和硫酸亚铁诱导的锥体细胞损伤。数据表明，星形胶质细胞间间隙连接通讯通过一种涉及稳定细胞钙稳态和消除氧化应激的机制降低神经元对氧化损伤的易感性。
