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。

S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfide S-oxide (GS (O) SG), glutathione disulfide S-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS (O) SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [35S] cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS (O) SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS (O) SG is one of the most potent glutathiolating agents generated upon oxidative stress.

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.

Previous studies have shown that the level of total conventional protein kinase C (cPKC) membrane translocation (activation) was increased in brain of hypoxic preconditioned mice. In order to find out which isoform of cPKC may participate in the development of cerebral hypoxic preconditioning (HPC), we used Western bolt and immunohistochemistry to observe the effects of repetitive hypoxic exposure (H1-H6, n=6 for each group) on the level of cPKC isoform-specific protein expression and its membrane translocation in cortex and hippocampus of mice. We found that levels of cPKC βII and γ membrane translocation were increased significantly (p<0.05 versus normoxic H0 group, n=6) in response to repetitive hypoxic exposure (H1-H4) at early phase of hypoxic preconditioning, but no significant changes of cPKC α and βI membrane translocation while cPKC α, βI, βII and γ protein expression were found both in hippocampus and cortex. In addition, an extensive subcellular redistribution of cPKC βII and γ was detected by immunohistochemistry staining in cortex after repetitive hypoxic exposures (H3). However, a significant decrease in the expression of cPKC γ protein (p<0.05 versus H0 group) was found only in cortex of delayed hypoxic preconditioned mice (H5-6). These results suggested that the activation of cPKC βII and γ may be involved in the early phase of cerebral hypoxic preconditioning and the changes in cPKC γ protein expression may participate in the development of late phase of cerebral hypoxic preconditioning, as well as selective vulnerability to hypoxia both in cortex and hippocampus.

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.