Yamakawa T, Fukunaga K, Higashida H, Miyamoto E
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
Brain Res. 1992 Dec 4;597(2):220-6. doi: 10.1016/0006-8993(92)91477-v.
To elucidate the mechanisms of the intracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca2+/calmodulin-dependent manner. The results suggest that the 50-kDa protein is the subunit of CaM kinase II in NG108-15 cells. The Ca2+/calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 microM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 microM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca2+ with 1 mM EGTA or by the pretreatment with 1 microM nifedipine. Stimulation of 32P-labeled NG108-15 cells with 1 microM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. These results suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.
为阐明缓激肽在NG108 - 15神经母细胞瘤x胶质瘤杂交细胞中引发的细胞内信号转导机制，我们检测了缓激肽刺激对Ca2+/钙调蛋白依赖性蛋白激酶II（CaM激酶II）的激活作用。当用针对脑CaM激酶II的亲和纯化抗体对NG108 - 15细胞提取物进行免疫沉淀时，免疫沉淀物中的一种50 kDa蛋白主要以Ca2+/钙调蛋白依赖性方式发生自身磷酸化。结果表明，该50 kDa蛋白是NG108 - 15细胞中CaM激酶II的亚基。在用1 μM缓激肽刺激细胞后，该酶的Ca2+/钙调蛋白非依赖性活性（自主活性）在10秒内增加了两倍。酶自主活性的增加有两个阶段：短暂的早期峰值阶段和持久的晚期平台阶段。前者在用10 mM咖啡因或20 μM BAPTA - AM预处理细胞后被消除，后者在用1 mM EGTA去除细胞外Ca2+或用1 μM硝苯地平预处理后被消除。用1 μM缓激肽刺激32P标记的NG108 - 15细胞增加了CaM激酶II的自身磷酸化，而这种增加在用咖啡因或BAPTA - AM预处理后被消除。这些结果表明，在NG108 - 15细胞中，CaM激酶II是通过缓激肽诱导的肌醇磷脂信号通路被激活的。
