Abstract：
Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer\'s disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.
摘要：
在与神经炎症和进行性退行性疾病相关的反应性星形胶质细胞中观察到Ca2/钙调蛋白依赖性磷酸酶钙调磷酸酶（CN）的过度激活，比如老年痴呆症.除了关键转录因子（例如活化t细胞的核因子和核因子-κB）外，星形胶质细胞中很少研究其他CN依赖性途径。半通道蛋白，在星形胶质细胞中发现高水平的连接蛋白43(Cx43),并且在其羧基末端附近含有CN敏感的Ser残基。据报道，Cx43的CN依赖性去磷酸化在用伤害性刺激治疗的原代星形胶质细胞中，但在神经炎症和疾病的背景下，关于CN/Cx43相互作用仍有很多未知。蛋白质印迹用于评估用过度活跃的CN片段处理的大鼠胚胎原代星形胶质细胞中的总Cx43和去磷酸化的Cx43亚型（ΔCN，通过腺病毒），或者用促炎细胞因子鸡尾酒。在类似的处理条件下，使用溴化乙锭(EtBr)吸收试验来评估膜通透性.在存在或不存在CN抑制剂的情况下测试ΔCN和细胞因子的作用，环孢菌素A.一种连接蛋白抑制剂，在EtBr试验中,也使用了甘珀酮来评估连接蛋白参与膜通透性的情况.用ΔCN或细胞因子处理增加了去磷酸化的Cx43水平以及增加的膜通透性(升高的EtBr摄取)。环孢菌素A阻断了ΔCN或细胞因子治疗的作用。加苯唑酮也抑制了治疗诱导的EtBr摄取变化。结果表明，Cx43半通道可能是星形细胞CN破坏与神经退行性疾病相关的神经功能的重要机制。
