The role of Ca^<2+>-dependent protein kinases in central nervous system in health and diseases.

本研究从生物化学和药理学的角度探讨了中枢神经系统Ca^<2+>依赖性蛋白激酶在健康和疾病中的作用。首先，我们利用三种抗蛋白激酶C（PKC）同工酶单克隆抗体建立了亚型特异性酶免疫分析（EIA）。EIA显示PKC在中枢神经系统肿瘤细胞包括胶质母细胞瘤和神经母细胞瘤细胞中的定量分析和亚型特异性表达。结果提示，PKC同工酶在CNS肿瘤中具有细胞类型特异性，检测PKC同工酶有助于了解PKC的不同作用，并有助于肿瘤的诊断。from glia神经胶质or neuronalcells神经细胞.此外，我们还制备了抗PKCS的抗体，并检测了PKCS的组织和细胞特异性表达。第二，我们合成了多种蛋白激酶的特异性抑制剂，包括Ca^<2+>/钙调蛋白激酶II（CaM Idnase II）、cAMP依赖性蛋白激酶（A-kinase）和酪蛋白激酶I。KN-62是一种特异性的CaM激酶11抑制剂，对其它激酶活性无抑制作用。此外，KN-62还能抑制GABA的释放和中枢神经系统的长时程增强，这表明该化合物是一种有用的工具，用于阐明CaM激酶H在许多组织中的功能。H-89（A-激酶抑制剂）也能抑制Folskolin诱导的PC- 12细胞突起生长，抑制即早基因的诱导，并能测定PKC同工酶的细胞型特异性表达，开发多种蛋白激酶的特异性抑制剂。我们应该尝试下一步，以阐明这些蛋白质的功能，使用特定的抑制剂。

To elucidate the functions of Ca^<2+>-dependent protein kinases in central nervous system in health and diseases, biochemical and pharmacological approaches were performed in this study. First, we have developed the subtype specific enzyme immunoassay (EIA) using three anti-protein kinase C (PKC) isozymes monoclonal antibodies. The EIA showed the quantitative analysis and subtype-specific expression of PKC in CNS tumor cells including glioblastoma and neuroblastoma cells. The results suggested that PKC isozymes have a cell-type specificity in CNS tumors, and that detection of PKC isozymes will aid in understanding the diverse effects of PKC and also to diagnose tumors derived. from glia or neuronalcells. In addition, we produced antibodies against the novel PKCS, and examined tissue and cell-type specific expression of the novel PKCS.Second, we have newly synthesized specific inhibitors of various protein ldnases including Ca^<2+>/calmodulin protein kinase II (CaM Idnase 11), cAMp-dependent protein kinase (A-kinase) and casein kidase I. KN-62 is specific CaM kinase 11 inhibitor and dose not inhibit other kinase activities. Furtennore, KN-62 inhibit the releasing of GABA and long term potentiation in central nervous system suggesting that the compound is a useful tool for elucidation of the functions of CaM kinase H in many tissues. H-89 (A-kinase inhibitor) also inhibited neurite growth n PC- 12 cells induced by folskolin, and induction of immediately early gene.We could determined the cell-type specific expression of PKC isozymes and developed specific inhibitors of various protein kinases. We should try on next steps to elucidate the functions of these protein Idnases using specific inhibitors.