The Regulation And Mechanism Of Akt Phosphorylation On Endothelial Nitric Oxide Synthase In Physiological Conditions

PART ? THE EFFECT OF GSK2334470 AND PP242 ON ENDOTHELIAL NITRIC OXIDE SYNTHASE ACTIVATIONObjective: Endothelial nitric oxide synthase(eNOS)plays an important role in maintaining the function of the cardiovascular system.The activation and abnormal expression of eNOS is related to many cardiovascular diseases.Phosphorylation of eNOS Ser1177 is an important indicator of functional activation of eNOS.The activation of eNOS is regulated by different phosphorylation sites of protein kinase B(PKB/Akt)which is the main upstream regulatory protein of eNOS.Akt activation requires phosphorylation at both Thr308 and Ser473 which can be inhibited by GSK2334470 and PP242,respectively.In this part,the main purpose is to detect the effect of GSK2334470 and PP242 on eNOS activation.Methods: eNOS-HEK293 cells(HEK293 cells transfected with human eNOS plasmid),primary human umbilical vein endothelial cells(HUVECs)and bovine aortic endothelial cells(BAECs)were cultured and treated with different concentrations and period of GSK2334470 and PP242 respectively.GSK2334470 is a specific inhibitor of phosphoinositide dependent kinase-1(PDK1)and P242 is the inhibitor of mammalian target of rapamycin(mTOR).Western Blot was used to detect the expression of phosphorylation of Akt and eNOS.Results:(1)eNOS-HEK293 cells successfully expressed eNOS protein after transfecting with human eNOS plasmid.HUVECs and BAECs were arranged like cobblestones and paving stones under the light microscope.CD31 and factor ? fluorescence staining were positive in HUVECs and BAECs.(2)Compared with the control group,phosphorylation of Akt Thr308 dose-dependently decreased with GSK2334470 treatment(P<0.05),but phosphorylation of Akt Ser473 was not affected(P>0.05);p-Akt Thr308 and p-eNOS Ser1177/1179 time-dependently decreased with GSK2334470(P<0.05),but p-Akt Ser473 was not significantly changed(P>0.05).(3)The phosphorylation of Akt Ser473 dose-dependently decreased with PP242 treatment(P<0.05),Akt Thr308 was not affected(P>0.05);p-Akt Ser473 time-dependently decreased with PP242(P<0.05),p-Akt Thr308 and p-eNOS Ser1177/1179 were not significantly changed(P>0.05).The results of three types of cells are consistent.Conclusion: The phosphorylation site of Akt Thr308 compared with Ser473 may be a more important regulator of p-eNOS Ser1177/1179.PART ? THE EFFECT OF PDK1 AND SIN1 KNOCKDOWN ON ENDOTHELIAL NITRIC OXIDE SYNTHASE ACTIVATIONObjective: Protein kinase PDK1 is the upstream kinase of phosphatidylinositol-3-kinase(PI3K)/Akt/eNOS signal transduction pathway activates Akt by phosphorylating Akt Thr308.Mammalian target of rapamycin complex 2(mTORC2)activates Akt by regulating the phosphorylation of Akt Ser473,SIN1 is one of the subunits of it.This part mainly observes the impact of PDK1 and SIN1 knockdown on Akt and eNOS activation.Methods: Small interferingRNA(siRNA)targeting PDK1,SIN1 and negative control(NC)were constructed to transfect into eNOS-HEK293 cells,respectively.Cells were divided into 4 groups in this part: control group,siRNA-PDK1 group,siRNA-SIN1 group and siRNA-NC group.Western blot was used to detect the expression of PDK1,SIN1 and the phosphorylation of Akt and eNOS.Results:(1)siRNA targeting PDK1 and SIN1 significantly inhibited the protein levels of PDK1 and SIN1 in eNOS-HEK293 cells(P<0.001),but siRNA-NC had no effect on the protein expression(P>0.05).(2)Compared with siRNA-NC group,knockdown of PDK1 significantly reduced the phosphorylation of Akt Thr308(P<0.001),but didn't affect Akt Ser473(P>0.05);knockdown of SIN1 obviously inhibited the phosphorylation of Ser473(P<0.001),but had no effect on Thr308(P>0.05).(3)Compared with the siRNA-NC group,knocking down PDK1 decreased the expression of eNOS Ser1177 phosphorylation(P<0.001),while knockdown of SIN1 didn't change it(P>0.05).Neither PDK1 nor SIN1 knockdown had significant effect on p-eNOS Thr495(P>0.05).Conclusion: Akt Thr308 has a regulatory effect on p-eNOS Ser1177 at the genetic level.PART ? THE EFFECT OF AKT PHOSPHORYLATION ON ENDOTHELIAL NITRIC OXIDE SYNTHASE AND NITRIC OXIDE HOMEOSTASIS IN MOUSE VASCULARObjective: Endothelial-derived relaxing factor nitric oxide(NO)plays a crucial role in maintaining vascular tone and controlling blood pressure in the cardiovascular system.Phosphorylation of eNOS Ser1177 is essential for the production of NO in vascular endothelial cells.Akt is a vital participant that catalyzes NO production through the PI3K/Akt/eNOS signaling pathway.This part mainly explores the effects of Akt phosphorylation sites on eNOS activation and NO concentration of plasma in mouse artery.Methods: Male C57BL/6J mice were divided into 3 groups(4 in each group)randomly: control group(treated with same amount of dimethyl sulfoxide),GSK2334470 group(40 mg/kg)and PP242 group(5 mg/kg).Then intraperitoneal injection of DMSO,GSK2334470 and PP242 were administrated in mice.Plasma,thoracic aortas and mesenteric arteries of mice were collected after treatment for 6 hours.Western blot was used to detect the phosphorylation of Akt and eNOS in mouse artery,and enzyme-linked immunosorbent assay was used to detect the plasma NO concentration in mice.Results:(1)Compared with the control group after treatment with GSK2334470,the phosphorylation level of Akt Thr308 was down-regulated significantly(P<0.01)while Ser473 was not affected(P>0.05)in the mouse artery,and the phosphorylation of eNOS Ser1177 was also inhibited(P<0.01).(2)After injected with PP242,the phosphorylation of Akt Ser473 was decreased obviously(P<0.001),Thr308 and Ser1177 were not significantly affected(P>0.05).(3)The NO concentration of plasma in mice was decreased after treatment with GSK2334470(P<0.05)but not PP242(P>0.05).Conclusion: Inhibition of p-Akt Thr308 significantly reduced the expression of p-eNOS Ser1177 and NO concentration of plasma in mouse artery.Phosphorylation of Akt Thr308 is closely related to the eNOS/NO homeostasis in mouse vascular.PART ? THE EFFECT OF LOW TEMPERATURE ON AKT AND ENDOTHELIAL NITRIC OXIDE SYNTHASE PHOSPHORYLATIONObjective: Human body maintains homeostasis through energy metabolism at low temperature environment.Akt plays an important role in signal transduction of cellular energy metabolism.This part mainly studies the effect of different temperature on Akt phosphorylation and eNOS Ser1177/1179 phosphorylation.Methods: eNOS-HEK293 cells,HUVECs and BAECs were exposed to different temperatures(37?,22? and 4?)for 1 hour respectively.Western Blot was used to detect the expression of Akt and eNOS phosphorylation levels.Results:(1)The protein expression of p-Akt Ser473 was diminished with the low temperature in eNOS-HEK293 cells(P<0.01),p-Akt Thr308 and p-eNOS Ser1177 didn't change significantly(P>0.05).(2)The phosphorylation of Ser473 was inhibited with the decrease of temperature in primary endothelial cells(P<0.05),but the phosphorylation of Thr308 and Ser1177/1179 were increased(P<0.05).Conclusion: The phosphorylation of Akt Thr308 is closely correlated with the expression of p-eNOS Ser1177/1179 in different temperatures.p-Akt Thr308 has an important regulatory effect on eNOS activation in vascular endothelial cells.