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。

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。

神经颗粒素（Neurogranin，Ng）是一种新发现的由78个氨基酸组成的脑特异性蛋白，主要分布于人类或动物的大脑皮层、海马和嗅球等脑区的神经突触后。作为Calpacitin蛋白家族中的一员，Ng是蛋白激酶C的天然作用底物及钙调蛋白（CaM）的储库。在生理状态下，Ng与CaM结合形成复合体，而在蛋白激酶C或氧化剂的作用下，Ng可被磷酸化、氧化及谷胱甘肽化等化学修饰，降低其与CaM的亲和力，从而参与对CaM及CaM2激活的蛋白酶，如CaM2依赖性NO合酶、CaM2依赖性蛋白激酶II（CaMKII）及CaM2依赖性腺苷酸环化酶的调节。同时，由于CaM2依赖性蛋白酶大多参与长时程增强（LTP）和长时程抑制（LTD）的诱导，并且Ng的基因表达和蛋白质合成与神经元的突触形成、分化同步，因此，Ng可能在学习、记忆、神经系统发育（可塑性）等生理性变化中具有重要作用。此外，一些研究表明，Ng还可能参与甲状腺机能减退、睡眠剥夺、衰老及脑低氧预适应等病理生理学变化所造成的神经系统功能的改变。

Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca21. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter, N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3-5 min and declined to base line in 30min. The response was dose-dependent (EC50 ;100mm) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitor NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. Ng was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione, and H2O2 at concentrations less than 0.5mm. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca21 and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.