Gap Junction Protein Cx43,Cx40Phosphorylation Levels And Their Phosphorylation Sites Analysis In The Cerebral Vasospasm

Objective:In cardio-cerebral-vascular system, the gap junction proteins that have the mainexpressions and functions are Cx43and Cx40, Whose phosphorylation act a centralrole in many cardio-cerebral-vascular diseases.In this study, we decide to find out thechanges of their phosphorylation levels and screen out their phosphorylation sites.Itis expected to provide a basic evidence for the important role of Cx43,Cx40phosphorylation in CVS, and discover the potential target for drug screeningresearch.Methods:1. Construction of the CVS model: The basal arteries from homogeneous SDrats were operated, and sheared into several fragments. Then the vessel strips werecultivate in ET-1(400pmol).2. Detection of the CVS model：All SD rats were divided into ET-1stimulatedgroup and the normal group. The tension changes between ET-1stimulated vesseland the normal vessel were compared.3. Analysis of the phosphorylation levels between Cx43and Cx40: The totalphosphorylated protein was gathered with PhosphoPrrotein Purification Kit. Thetotal phosphorylated protein was experimented through WB technique. Then, theconnexin phosphorylation level was analyzed based on the results of WB experimentand was compared with the normal group without ET-1stimulation.4. Detection of the phosphorylated sites of Cx43and Cx40in CVS:Analysis thephosphorylated sites of Cx43and Cx40in CVS by mass spectrometry,and screen outthe phosphorylated sites with mass spectrometric technique.Results:1. The CVS model stimulated by ET-1was successfully constructed. Thetension detection found that the tension changed obviously after it was stimulated byET-1 2. In normal group, Cx43and Cx40are at relatively low phosphorylationlevels,however, as compared to Cx43phosphorylation level, Cx40is at a relativelyhigher level of phosphorylation.3. After CVS, the phosphorylation levels of Cx43and Cx40increased obviously.However, by comparing with the increments of P-Cx43and P-Cx40, it showed thatthe expression of P-GJ protein is mainly Cx40.4. Based on previous research, it showed that the expression of GJ protein isCx40in the normal brain arterial smooth muscle, but it changed into Cx43after CVS.Combining this experimental results found after CVS, the expression of GJ proteinin the brain arterial smooth muscle is Cx43, but the phosphorylated protein is Cx40.5. Nine phosphorylation sites in Cx40and four in Cx43were detected bytesting the phosphorylation sites of Cx43and Cx40in CVS.Conclusion:The inconsistence between the expression of total protein and phosphorylatedprotein may play an important role in the pathological process of CVS. The ninedetected phosphorylation sites in Cx40and four in Cx43may play major roles in GJfunction changes, and finally affect the occurrence and development of CVS.