Cosi C, Suzuki H, Milani D, Facci L, Menegazzi M, Vantini G, Kanai Y, Skaper S D
Fidia Research Laboratories, Abano Terme, Italy.
J Neurosci Res. 1994 Sep 1;39(1):38-46. doi: 10.1002/jnr.490390106.
Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.
谷氨酸神经毒性与胞质游离Ca2+的增加相关。在一些细胞系统中，Ca2+依赖性核酸内切酶的激活或自由基的形成可损伤DNA并激活与染色质结合的酶聚（ADP - 核糖）聚合酶（pADPRP）。我们研究了pADPRP是否可能参与体外谷氨酸神经毒性。培养12天的小脑颗粒细胞用毒性剂量的谷氨酸（100 microM）处理时，显示出聚ADP - 核糖免疫反应性迅速且短暂增加。细胞免疫染色不均匀，在洗去谷氨酸后恢复到对照水平。在相同的细胞制剂中，谷氨酸引起酶蛋白免疫反应性显著增加，且在随后的时间持续存在。无毒剂量的谷氨酸不影响免疫染色。在另一组实验中，谷氨酸处理30分钟后pADPRP mRNA增加。为了研究pADPRP在谷氨酸介导的神经毒性中的作用，在该模型中测试了结构不同的pADPRP抑制剂（3 - 氨基苯甲酰胺、苯甲酰胺、3 - 氨基邻苯二甲酰肼）及其无活性类似物（苯甲酸和邻苯二甲酰亚胺）。在谷氨酸处理前60分钟及处理期间的30分钟向培养物中添加抑制剂，可使24小时后评估的神经元死亡减少60 - 100%。谷氨酸诱导的Ca2+内流不受影响。无活性类似物未能提供神经保护作用。这些数据表明，不仅pADPRP可由谷氨酸引发的早期可能由Ca(2+)介导的机制激活，而且它可能也积极促成随后的神经元死亡。
