Kang Yao1, Biao Xu, Ya-Ping Liu, Albert Ferro. 1. Department of Cardiology, Affiliated Zhong Da Hospital of Southeast University, Nanjing 210009, China.
Abstract
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 micromol/L) for 30 min, in the absence or presence of selective beta1- or beta2 -adrenoceptor antagonists. eNOS was then isolated by immunoprecipitation using a specific eNOS antibody. eNOS activity was determined by the conversion of L-[3H]arginine to L-[3H]citrulline. The levels of eNOS expression and eNOS phosphorylation were measured by Western blotting. RESULTS: Treatment of HUVEC with isoprenaline (1 micromol/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 beta2-adrenoceptor antagonist ICI 118551. CONCLUSION: Isoprenaline increases eNOS activity of HUVEC, this is mediated by beta2-adrenoceptor and associated with an increase of eNOS serine phosphorylation level.
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 micromol/L) for 30 min, in the absence or presence of selective beta1- or beta2 -adrenoceptor antagonists. eNOS was then isolated by immunoprecipitation using a specific eNOS antibody. eNOS activity was determined by the conversion of L-[3H]arginine to L-[3H]citrulline. The levels of eNOS expression and eNOS phosphorylation were measured by Western blotting. RESULTS: Treatment of HUVEC with isoprenaline (1 micromol/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 beta2-adrenoceptor antagonist ICI 118551. CONCLUSION:Isoprenaline increases eNOS activity of HUVEC, this is mediated by beta2-adrenoceptor and associated with an increase of eNOS serine phosphorylation level.