Neurogranin (Ng) is a brain-specific, postsynaptically located protein kinase C (PKC) substrate, highly expressed in the cortex, hippocampus, striatum, and amygdala. This protein is a Ca2-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. To investigate the role of Ng in neural function, a strain of Ng knockout mouse (KO) was generated. Previously we reported (Pak, J. H., Huang, F. L., Li, J., Balschun, D., Reymann, K. G., Chiang, C., Westphal, H., and Huang, K. -P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11232- 11237) that these KO mice displayed no obvious neuroanatomical abnormality, but exhibited deficits in learning and memory and activation of Ca2/CaM-dependent protein kinase II. In this report, we analyzed several downstream phosphorylation targets in phorbol 12-myristate 13-acetate-and forskolin-treated hippocampal slices from wild type (WT) and KO mice. Phorbol 12-myristate 13-acetate caused phosphorylation of Ng in WT mice and promoted the translocation of PKC from the cytosolic to the particulate fractions of both the WT and KO mice, albeit to a lesser extent in the latter. Phosphorylation of downstream targets, including mitogenactivated protein kinases, 90-kDa ribosomal S6 kinase, and the cAMP response element binding protein (CREB) was significantly attenuated in KO mice. Stimulation of hippocampal slices with forskolin also caused greater stimulation of protein kinase A (PKA) in the WT as compared with those of the KO mice. Again, phosphorylation of the downstream targets of PKA was attenuated in the KO mice. These results suggest that Ng plays a pivotal role in regulating both PKC-and PKA-mediated signaling pathways, and that the deficits in learning and memory of spatial tasks detected in the KO mice may be the result of defects in the signaling pathways leading to the phosphorylation of CREB.

目的：初步探讨新奇型蛋白激酶C（novel protein kinases C，nPKCs）在脑低氧预适应发生发展过程中的作用。 方法：利用蛋白电泳（SDS2PAGE）和蛋白印迹（Western blot）等生化技术，并结合本室已建立的小鼠低氧预适应模型，观察低氧预适应对小鼠海马和大脑皮层组织内nPKCs（nPKCε、δ、η、μ和θ亚型）膜转位（激活）的影响。结果：随低氧次数（H02H4）或低氧耐受时间的增加，小鼠海马（H0：41.6% ± 1.4% vs H1-H4：46.9% ±4.5%，52. 7 ± 3.9%，58.8% ± 2.7%，61.3% ± 3.7%）和大脑皮层（H0：38. 4% ± 4.5% vs H1-H4：42.4% ± 5.0%，48.7% ± 6.5%，55.3% ± 8.9%，61.2% ± 10.2%）组织内nPKCε膜转位明显增加，且海马和大脑皮层分别在H2 、H3 、H4 和H3 、H4具有统计学的显著意义（P < 0.01）而nPKCδ、η、μ和θ亚型在海马和大脑皮层组织内的膜转位变化均无统计学意义。结论：nPKCε可能在脑低氧预适应的发生发展过程中发挥着重要作用，但需进一步的研究证实。

初步探讨蛋白激酶C（PKC）在脑低氧预适应发生机制中的作用，以我室建立的小鼠低氧预适应模型，结合SDS2聚丙烯酰胺凝胶电泳（SDS2PAGE）、蛋白印迹（Western bolt）等生化技术，观察了低氧预适应对小鼠大脑皮层及海马组织内PKC膜转位的影响。结果表明，1在小鼠大脑皮层和海马组织内的PKC膜转位均随着低氧次数（小鼠低氧耐受时间) 的增加而增高。特别是低氧3次的海马组织和低氧4次的大脑皮层和海马组织内，PKC膜转位的增加有统计学显著意义。结果提示，PKC的激活可能在脑低氧预适应发生机制中具有重要的作用。

本实验拟通过观察重复性低氧对经典型蛋白激本酶C（Cpkc）膜转位水平激活程度的影响。初步探讨cPKC特定亚型在脑低氧预适应发生发展过程中的作用。按我室已建立的小鼠低氧预适应模型方法，制备重复性低氧1～4次的小鼠（H1～H4）。应用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白印迹（Western holt）等生化技术，并结合Gel Doc 凝胶成像系统，半定时量检测小鼠海马和大脑皮层组织内cPKCα和γ的膜转位水平。实验结果表明，随低氧次数（H1～H4）的增加，小鼠海马组织内cPKCγ的膜转位水平明显增高，且在H2、H3和H4组的变化具有统计学显示著意义（P<0.05，n=6）；而cPKCα亚型无论在大脑皮层还在海马组织内的膜转位比均无统计学意义：上述观察结果显示，cPKCγ膜转位可能在脑低氧适应的发生发展过程中发挥着重要作用；但cPKCβ1、β且以及其它新奇型和非典型PCK特定亚型的变化还有待于进一步的研究和探讨。