Effects of homocysteine on activity and mRNA expression of endothelial nitric oxide synthase in cultured endothelial cell

OBJECTIVE To observe the effects of homocysteine(Hcy) at different concentrations on activity and mRNA expression of endothelial nitric oxide synthase(eNOS) in cultured human umbical vein endothelial cell(HUVEC).METHODS HUVEC were cultured with Hcy at different concentrations(0,10,30,100,300 μmol·L-1) for 24,48 and 72 hours respectively.The content of asymmetric dimethylarginine(ADMA) was measured by RP-HPLC.RT-PCR was performed to detect the expression of eNOS mRNA.The activity of dimethylarginine dimethylaminohydrolase(DDAH),eNOS and the production of NO were analyzed at the same time.RESULTS After HUVEC were exposed to different concentrations of Hcy for 24,48 and 72 hours,ADMA concentration in the cell culture medium increased,mean while the activity of DDAH decreased in a dose-time-dependent fashion.The eNOS activity was inhibited,and the content of NO in the cell culture medium was significantly reduced compared with the control group(P 0.05),but only when HUVEC was treated to 100 μmol·L-1 Hcy,did the expression of eNOS mRNA decrease.The expression of eNOS mRNA decreased.CONCLUSION The mechanism of homocysteine-induced endothelial dysfunction may exert on inhibiting DDAH expression,causing ADMA to accumulate,and lead to the decrease in eNOS activity and NO output.Its additional mechanism is the inhibition of the expression of eNOS mRNA.