Angiotensin ? Decreases ENOS Phosphorylation Through AT₁R Nox/ROS/PP2A Pathway

Objective:To investigate the molecular mechanism of angiotensin ?(Ang ?)/ angiotensin ? type 1 receptor(AT1R)pathway mediated NADPH oxidase(Nox)/ reactive oxygen species(ROS)signaling pathway activating protein phosphatase 2A(PP2A)which down-regulating endothelial nitric oxide synthase(e NOS).Methods: According to the previous experimental results,the concentration and time of Ang ? were selected(1×10-7M,12h).HUVECs were pretreated with candesartan(the specific AT1 R blocker,CAN)(1×10-6M,3h),cells were randomly divided into Control group,Ang ? group,CAN +Ang ? group and CAN group.The levels of protein expression were measured by Western blot.The activity of PP2 A was detected by PP2 A activity kit and the content of ROS was detected by DCFH-DA fluorescent probe.Cells were respectively pretreated with N-acetylcysteine(ROS scavenger,NAC,1×10-3M)and Apocynin(Nox inhibitor,APO,2×10-5M)for 1 h,HUVECs were randomly divided into Control group,Ang ? group,NAC +Ang ? group,NAC group,APO +Ang ? group and APO group,at the same time,the p22 phox si RNA was transferred into HUVECs.Finally,cells were pretreated with PP2(Src inhibitor)(1×10-5M,2h),cells were randomly divided into Control group,Ang ? group,PP2 group and PP2 +Ang ? group.The levels of protein expression were measured by Western blot.Results:(1)After treated with 1×10-7M Ang ? for 12 h,the phosphorylation level of PP2 Ac Tyr307 was down-regulated,the activity of PP2 A increased,and the production of ROS increased(P<0.05),the above results could be reversed by CAN treatment(P<0.05).(2)Pretreatment with NAC and APO could be reversed Ang ? down-regulated the phosphorylatione of e NOS Ser1177 and PP2 Ac Tyr307(P<0.05).(3)Compared with control group,the protein expression of p22 phox was increased after treatment with Ang ?(P<0.05).Compared with the Si-control group,the phosphorylation levels of PP2 Ac Tyr307 and e NOS Ser1177 were increased,the protein expression of p22 phox was reduced in p22 phox si RNA group(P<0.05).After treatment with Ang ?,the effects of Ang?on protein expression of p22 phox and the phosphorylation levels of PP2 Ac Tyr307 and e NOS Ser1177 were significantly reduced by knockdown p22phox(P<0.05).(4)Compared with control group,the phosphorylation level of Src Tyr418 was down-regulated,APO pretreatment could be reversed the result(P<0.05).(5)Compared with control group,Ang ? and PP2 lowered the levels of Src Tyr418,accordingly resulted in the phosphorylation levels of PP2 Ac Tyr307 and e NOS Ser1177 were decreased(P<0.05),after treatment with Ang ?,reduction of Src Tyr418,PP2 Ac Tyr307 and e NOS Ser1177 phosphorylation became more significant than Ang ? group and PP2 group(P<0.05).Conclusion: This study demonstrates that Ang ? depresses Src Tyr418 and its downstream PP2 A Tyr307 phosphorylation via AT1R-mediated Nox/ROS signaling pathway to activate PP2 A,which dephosphorylates e NOS Ser1177 and leads to endothelial dysfunction.