Angiotensin - (1-7) On Rat Aortic Endothelial Cell Function And Its Mechanism

Partâ… The Effects of Angiotensin-(1-7)on function of Aortic Endothelial Cells of Wistar Rats.Objective:To investigate the effects of angiotensin-(1-7)on vasoactive substances release in aortic endothelial cells of Wistar rats.Methods:The rat Aortic endothelial cells(RAEC)were were obtained from thoracic aortas and cultured by tissue explant method.The morphology of RAEC was studied by phase contramicroscope.Its molecular markers were observed byâ…§factor immunocytochemistry. Passage 3～5 cells were used in experiment,and They were randomly divided into 3 groups: groupâ… :treated without intervention as the control;groupâ…¡:Ang-(1-7)group;groupâ…¢: Ang-(1-7)+A-779(Mas receptor blocker)group.The experiment was stopped at the time of 30 minites,12 hours and 24 hours respectively.The levels of NO in supernatant of culture cells were detected by enzymatic assay with nitrate reductase;the levels of ET-1 in supernatant of culture cells were detected by the method of double mono-cloned antibody ELISA.;the expression of eNOS in cytoplasm was detected by the method of immuno- cytochemistry.Results:(1)The indentification of endothelial cells:most cells displayed green fluorescence under fluorescent microscopy,which accords with the characteristics of EC.(2)Comparison of the levels of NO in different groups and time:When treated for 30 min, groupâ… (39.20±2.79);groupâ…¡:(43.06±3.22,P＜0.05),which was increased;groupâ…¢(12.53±2.72,P＜0.05),which was decreased significantly.When treated for 12h,groupâ… (35.55±2.94);groupâ…¡:(49.74±5.28,P＜0.05),which was also increased;groupâ…¢:(31.35±3.52, P＞0.05),which did not change significantly.When treated for 24h,groupâ… (36.78±1.92);groupâ…¡:(76.35±0.91,P＜0.05),which was significantly increased;groupâ…¢:(37.62±1.26,P＞0.05), which did not change significantly too.Among different time points,the level of NO gradually increased from 30min-24h in groupâ…¡.The same phenomenon could not be found in groupâ… or groupâ…¢.(3)Comparison of the expression of eNOS in different groups and time:When treated for 30 min,there were not significant differences among the three groups;When treated for 12h,groupâ… (0.476±0.021);groupâ…¡(0.601±0.01,P＜0.05),which increased slightly;groupâ…¢(0.464±0.011,P＞0.05),which did not change significantly.When treated for 24h,groupâ… (0.467±0.015);groupâ…¡(0.816±0.021,P＜0.05),which increased significantly;groupâ…¢(0.472±0.013, P＞0.05),which did not change significantly too.Among different time points,the expression of eNOS gradually increased from 30min-24h in groupâ…¡,which is same as the levels of NO. (4)Comparison of the levels of ET-1 in different groups and time:When treated for 30 min, groupâ… (6.88±0.13);groupâ…¡(8.50±0.61,P＜0.05),which increased surprisely;groupâ…¢(6.73±0.19,P＞0.05),Which did not chang significantly.When treated for 12h,there were not significant differences among the three groups;When treated for 24h,groupâ… (7.02±0.21); groupâ…¡(6.034±0.24,P＜0.05),which decreased slightly;groupâ…¢(7.11±0.12,P＞0.05),which did not chang too.Among different time points,the levels of ET-1 gradually decreased from 30min-24h in groupâ…¡.Conclusion:Ang-(1-7)can promote the expression of eNOS and the release of NO in RAEC,which is time-depended.Surprisely,Ang-(1-7)can promote the release of ET-1 at 30 min and reduce at 24h.Moreover,A-779 can block the action of Ang-(1-7),and reverse the effects of the releasing of NO at 30 min.Partâ…¡The Mechanism of Angiotensin-(1-7)promoting expression of eNOSin Aortic Endothelial Cells of Wistar Rats.Objective:To investigate the change of the expression of eNOS which in order to imply the possible signal pathway by interrupting the PI3-K/Akt and Ca²⁺/CaM respectively.Methods:The rat Aortic endothelial cells(RAEC)were cultured and divided into 6 groups when cultured in the passage 3.Groupâ… :treated without intervention as the control;groupâ…¡: Ang-(1-7);groupâ…¢:Ang-(1-7)+ LY294002(PIP)3 blocker);groupâ…£:LY294002;groupâ…¤: Ang-(1-7)+ U-73122(PLC blocker);groupâ…¥:U-73122.After 24 hours,we tested the levels of eNOS mRNA by the method of RT-PCR,which were adjusted by GAPDH.Then we compared the values of OD among the groups.Results:There were ladders in the locus of 452bp in all the groups.The value of OD was 0.310±0.010 in control.The expression of eNOS were increased significantly in groupâ…¡(0.52±0.017,P＜0.05)and groupâ…¤(0.5094±0.021,P＜0.05),but insignificantly in groupâ…¢(0.347±0.013,P＞0.05),groupâ…£(0.291±0.014,P＞0.05)or groupâ…¥(0.304±0.019,P＞0.05).Conclusion:Ang-(1-7)can promote the expression of eNOS in RAEC,which can be inhibited by LY294002 completely,but not by U-73122.It suggests that the one signal pathway of Ang-(1-7)is PI-3K/Akt,but not the Ca2+/CaM.