The Effects Of Caveolae On The Changes Of Intracellular Calcium Concentrations In Vascular Smooth Muscle Cells

BACKGROUND AND PURPOSES:Subarachnoid hemorrhage (SAH) is a group of clinical symptoms due to bloodcomponents,which are caused by trauma or intracranial aneurysm rupture, flow into thesubarachnoid space mixed with cerebrospinal fluid, and cerebral vasospasm (CVS) is themost serious complication after SAH with a high clinical incidence. CVS can cause severebrain ischemia or delayed ischemic brain damage and even lead to infarction, and it hasbeen considered one of the main reasons of death or disability. After subarachnoidhemorrhage a variety of harmful humoral factors have changed the number and the activityof calcium channel of VSMCs, and then rise intracellular calcium concentration continuesly,through activating the myosin light chain kinase (MLCK) pathway induced smooth musclecontraction.There is a50-100nm flask-shaped special micro invagination domain on thecytoplasmic membrane surface. It is relatively stable and less fluided micro area on the cellmembrane, which provide exchange of the inner and outer material and signals of the cells.Caveolin-1is the surface coat protein of caveolae,which has a caveolin scaffolding domain(CSD) containing a variety of important biological role of membrane receptors, includingthe platelet-derived growth factor receptor, Insulin receptor, angiotensin receptor, et al.Butthere is little reports about that caveolin-1/caveolae regulate the activity of calcium channelsin smooth muscle cells, and cause the changes of intracellular calcium concentration.Therefore,in this study,we decrease or increase VSMCs caveolin-1protein expression,andthen detect the changes of VSMCs [Ca2+]i.The research provided the possible effects ofcaveolin-1/caveolae in CVS.METHODS:1. Separate SD rats carotid artery, then primary culture VSMCs with tissue piecemethod and identification of VSMCs by morphology and immunofluorescence; 2. treat cultured VSMCs with1mmol/L,2.5mmol/L and5mmol/L concentration ofMβCD,and detect caveolin-1expression with western blot method;3. treat cultured VSMCs with20ug/L,50ug/L,100ug/L concentration of TNF-α,and detect caveolin-1expression with western blot method;4. VSMCs were divided into control (A group), MβCD treated(B group),TNF-αtreated (C group), both TNF-αand MβCD treated (D group).The four group cells werethen stimulated by endothelin(ET)-1(100nmol/L) in physiological saline solution(PSS).Study was performed by the Ca2+-sensitive dye Fluo-2/AM and The TILL Photonicsspectrofluorometer was used to measure the fluorescence ratio(R,F340/F380) which standedfor the intracellular calcium changes．RESULTS:1. successfully separate SD rats carotid artery, and primary culture VSMCs in vitrothrough identification by morphology and immunofluorescence;2. caveolin-1expression of VSMCs was decreased when treated with the increaseconcentrations of MβCD,and caveolin-1expression of5mmol/L MβCD treatment groupwas decreased significantly compared with the control group (P <0.01);3. caveolin-1expression of VSMCs was increased when treated with the increaseconcentrations of TNF-α,and caveolin-1expression of100ug/L TNF-α treatment group wasincreased significantly compared with the control group (P <0.01);4. without ET-1sitimulation,there is no difference on the fluorescence ratio of VSMCs inthe four groups(A:0.5916±0.0820,B:0.5753±0.0790, C:0.6551±0.0837,D:0.6105±0.0804，P>0.05); with ET-1sitimulation the fluorescence ratio(R) of A group cells could beincreased by (115±17)％after100nmol/L ET-1stimimulation; the R of B group cells couldbe increased by (67±15)％after the same stimimulation(P<0.05vs A group); the R of Bgroup cells could be increased by (279±21)％after the same stimimulation(P<0.01vs Agroup); the R of B group cells could be increased by (88±16)％after the samestimimulation(P<0.01vs C group).CONCLUSIONS:1.5mmol/L MβCD treatment could disrupt caveolae structure effectively anddecrease caveolin-1expression significantly;2.100ug/L TNF-α treatment could increase caveolin-1expression significantly; 3. caveolin-1expression’ upregulation can significantly increase the elevated degree ofVSMCs [Ca2+]i, and after MβCD effectively disrupt caveolae structure, can significantlydecrease the elevated degree of VSMCs [Ca2+]i, caveolin-1/caveolae may be importanttarget for inhibiting the occurrence of cerebral vasospasm.