The Mechanism Of Angiotensin At₁ Receptor Pathway Activates PP2A Leading To Down-regulation Of P-eNOS Ser1177 In Human Umbilical Vein Endothelial Cells

Objective: To investigate the molecular mechanism of angiotensin II(AngII)/ angiotensin II type 1 receptor(AT1R)pathway activated protein phosphatase 2A(PP2A)leading to down-regulation of eNOS Ser1177 phosphorylation level in human umbilical vein endothelial cells(HUVECs).Methods: Firstly,we determined the concentration and time-dependent effect of angiotensin II on down regulation of eNOS Ser1177 phosphorylation.HUVECs cells were were randomly divided into normal control(Control)group and AngII treatment group.AngII concentration were 1×10-9mol/L,1×10-8mol/L,1×10-7mol/L,1×10-6mol/L and 1×10-5mol/L.And the treatment time were12 h,24h and 36 h.Then the molecular mechanism of down-regulation of eNOS Ser1177 was further investigated according to the concentration and time effect of AngII downregulation of eNOS Ser1177.HUVECs were randomly divided into 4 groups:normal control group(Control),AngII group(AngII final concentration were 1×10-7mol/L,1×10-6mol/L and treatment time was 12h),CAN group[The AT1 R blocker candesartan(CAN)at the concentration of 1×10-6mol/L] and CAN+AngII Group(CAN final concentration of pretreatment was 1×10-6mol/L and pretreatment time was 3h).The protein expression of eNOS,eNOS Ser1177,PP2A-Cα,I2PP2 A,and PP2Ac-Tyr307 were measured by Western blot.The content of NO in cell culture medium of each group was detected by chemical colorimetry.Results:(1)The cells were respectively treated with1×10-9mol/L,1×10-8mol/L,1×10-7mol/L,1×10-6mol/L,1×10-5mol/L AngII for 12 h,24h,36 h.Compared with control group,the protein expression of eNOS Ser1177 down-regulated(p<0.05).There was no significant difference in the level of eNOS Ser1177 phosphorylation between AngII groups.But the protein expression of eNOS showed no significant difference.(2)After CAN pretreatment,compared with the same concentration of AngII group,the protein expression of eNOS Ser1177 up-regulated(p<0.05).(3)Compared with controlgroup,the protein expression of PP2Ac-Tyr307 down-regulated(p<0.05).After CAN pretreatment,compared with the same concentration of AngII group,the protein expression of PP2Ac-Tyr307 up-regulated(p<0.05).But the protein expression of PP2A-Cα showed no significant difference.(4)Compared with control group,the protein expression of I2PP2Adecreased(p<0.05).After CAN pretreatment,compared with the same concentration of AngII group,the protein expression of I2PP2 A increased(p<0.05).(5)Compared with control group,the content of NO decreased(p<0.05).After CAN pretreatment,compared with the same concentration of AngII group,the content of NO down-regulated(p<0.05).Conclusion: AngII down-regulates eNOS Ser1177,decreases the production of NO in human umbilical vein endothelial cells via AT1 R pathway.This effect may be associated with decreasing the protein expression of PP2Ac-Tyr307 and I2PP2 A,which results in the enhancement of PP2 A activity.Pretreatment with AT1 R blocker CAN increases the protein expression of I2PP2 A and PP2Ac-Tyr307,reduces the activity of PP2 A,thus restoring ultimately eNOS activity.