Camk Ⅱ Can Be Activated by Ginsenoside Rb1 But Not Involved In Rb1 Enhanced Synapsin Phosphorylation and Glutamate Releasea

摘要

Aim:previously we had proved that Rb1 could enhance PC12 cell to release glutamate by facilitating synapsin phosphorylation.This work aims to investigate the effects of Rb1 on Ca (2+)-calmodulin -dependent protein kinase (CaMKⅡ) and whether CaMKⅡ involves in Rb1 promoted synapsin phosphorylation and glutamate release.Methods:firstly,a fluorescent imaging system for monitoring CaMKⅡ activity in living cells loaded with a fluorescence-labeled peptide as a substrate and an immunoprecipitation (IP) were used to investigate Rb1 effects on intracellular CaMKⅡ activity.Then cultured PC12 cell were treated with or without CaM blocker W7 or PKA inhibitor H89 before Rb1 and Rg1 stimulation.Glutamate release and synapsin phosphorylation of PC12 cell promoted by Rb1 and Rg1 were determined by HPLC with fluorescence detector and immunoblotting respectively.Results:1) Fluorescence intensity increased immediately after exposure to 10μM Rb1 for 20 minutes,which demonstrated that CaMKⅡ was transiently activated;2)Compared with control,a significant increase in phospho-Threonine286 CaMKⅡ was found after exposure to 10μM Rb1;3) CaM blocker W7 could not inhibit Rb1 enhanced glutamate release and synapsin phosphorylation while Rb1 enhanced glutamate release could be abolished by PKA inhibitor H89.Conclusion:Although Rb1 can significantly active CaMKⅡ by enhancing its phosphorylation,the current study indicates that Rb1 exert its glutamate release facilitation maybe mainly by cAMP dependent protein kinase mediated synapsin phosphorylation not by Ca (2+)-calmodulin-dependent protein kinase pathway.