The Effect Of Cx43Ser368Phosphorylation Levels In ET-1Induced Cerebral Vasospasm

Objective:Cx43phosphorylation at Ser368play an important role in Cx43gap junctionfunction, which results in change in protein metabolism and channel behavior thatcontributes to intercellular communication. Examination of Cx43phosphoserine368(pSer368) using a phosphorylation site specific antibody, in spastic cerebral vesselwhich produced by artificial subarachnoid hemorrhage(SAH), show the cerebralarteries have a dramatic increase in Cx43phosphorylation levels at Ser368. Thisstudy is aiming to alter the Cx43phosphorylation level at Ser368, and to observe thechange of cerebralvascular spasm(CVS) pathology process produced by ET-1stimulate. we wish to ascertain the key pathophysiology mechanism of cerebralvas-cular spasm, and may thus open new therapeutic horizonsMethods:1、Construction lentiviral vector(LV): Using the pCMV-Cx43cDNA plasmid astemplate to clone full length Cx43cDNA, and mutate the Cx43cDNA at1102-1104site via PCR. Using Gateway technology to clone Cx43cDNA into PLV.Des3d.p/neoto constructe the eukatyotic expression vector PLV.Ex3d.p/neo-EF1A<Cx43> IRES/EGFP. Screen and using RT-PCR and DNA sequencing technology to confirm thefinally plasmid. The recombinant plasmid was used to contransfect293T cell withauxiliary plasmid. The virus titer was determined by crystal violet dye.2、Construction the animal model: Rats were anesthetized with chloralic hydras.Injection LV into cistema magna via double injection method.14and28days afteroperation, animals were euthanized by an overdose of chloralic hydras. Observe theEnhence green fluorescent protein(EGFP) express in basilar artery(BA) by rapidfrozen section, and determine the vascular morphometric by hematoxylin-eosinstaining.3、The influence of LV infection in CVS: Animal models were euthanized at thebest efficiency of infection. Its BAs were maded to arterial rings and arterial strips.Using different concentration of ET-1to stimulate arterial rings, Application biological information conversion device to recorder vascular ring tension changes.The arterial strips were stimulated the same as arterial rings, and the Cx43proteinexpression and its phosphorylation levels were examined by Wersten blotting.Result:1、DNA sequencing demonstrated that the recombinant LV content mutationalCx43on Ser368was constructed successfully. Under the fluorescence microscopy,abundant of green fluorescent can be observe in293T cells. The recombinant LV waspackaged in293T cells with a viral titer of9.7×107TU/ml.2、Animal models test: BA intimal and medial area can observed EGFP expressin animal models compared with normal BA. HE staining shows that the vascularwall have no inflammatory cell infiltration, did not form scar tissue. The shape ofendothelium and smooth muscle cell were normal compare with normal BA. Itindicate that the animal model was established successfully.3、In vitro, ET-1caused constant contractions in all experimental BA rings, andthe contractions were dose-dependent. With1×10-8mol/L ET-1, the BA rings thatexpress Cx43Ser368A gene have stronger contractions than nomal BA(P<0.05).Accompanied by ET-1concentration increases(1×10-7mol/L、1×10-6mol/L), thecontractions of BA rings that express Cx43Ser368A gene were significantlyenhanced compared with normal BA rings(P<0.05).4、In normal BA, the vessel wall cells exist Cx43protein expression and havelower phosphorylation level at Ser368. After treatment with ET-1, the total Cx43protein expression and its phosphorylation level at Ser368were significantlyenhanced(P<0.05). In BA vessel wall cells,after infected by LV which carry Cx43Ser368A gene, phosphorylation levels of Cx43at Ser368can be inhibited obviouslycompared with normal BA(P<0.05),and after stimulated by ET-1, the phosphorylationlevel was remain at a low level compared with normal BA rings（P<0.05）.Conclusion:Inhibition BA Cx43phosphorylation level at Ser368can enhanced thecontraction of BA in ET-1induced CVS.