Effects of β-adrenoceptor stimulation on endothelial nitric-oxide synthase phosphorylation of human umbilical vein endothelial cells

AIM: To investigate the effects of isoprenaline on the phosphorylation level of endothelial nitric oxide synthase (eNOS) of human umbilical vein endothelial cells (HUVEC) and to elucidate the regulation mechanisms of beta-adrenoceptor (beta-AR) on eNOS activity. METHODS: HUVEC were exposed to isoprenaline (1 mumol/L) for 30 min, in the absence or presence of selective beta(1) or beta(2) -adrenoceptor antagonists. eNOS was then isolated by immunoprecipitation using a specific eNOS antibody. eNOS activity was determined by the conversion of L-[(3)H] arginine to L- [(3)H]citrulline. The levels of eNOS expression and eNOS phosphorylation were measured by Western blotting. RESULTS: Treatment of HUVEC with isoprenaline (1 mumol/L) for 30 min caused no measureable change in eNOS expression. However, eNOS activity and serine phosphorylation level were significantly increased (P<0.01). This effect could be abolished by a selective beta(2)-adrenoceptor antagonist ICI 118551. CONCLUSION: Isoprenaline increases eNOS activity of HUVEC, this is mediated by beta(2)-adrenoceptor and associated with an increase of eNOS serine phosphorylation level.