β受体激动增加人脐静脉内皮细胞内皮型一氧化氮合酶活性的细胞内机制

Protein kinases involved in the endothelial nitric oxide synthase activation by beta-adrenoceptors stimulation in human umbilical vein endothelial cells

背景:β-肾上腺素能受体激动可增加内皮型一氧化氮合酶活性的细胞信号转导通路,对于揭示β-肾上腺素能系统调控血管张力的生理学机制有重要价值,既往实验显示异丙肾上腺素激活内皮型一氧化氮合酶与内皮型一氧化氮合酶蛋白磷酸化有关。目的:探讨β-肾上腺素能受体激动增加人脐静脉内皮细胞内皮型一氧化氮合酶活性信号转导通路中可能涉及的蛋白激酶。设计:对比观察。单位:郑州大学第一附属医院老年病科。材料:实验于2006-09/2007-06在河南省高等学校临床医学重点学科开放实验室完成。实验材料为郑州大学第一附属医院妇产科住院的健康顺产或剖宫产患者自愿捐献的新生儿无血肿、夹痕的脐带。所有患者对实验均知情同意,实验方案经医院伦理委员会批准。主要试剂:β-肾上腺素能受体激动剂异丙肾上腺素、磷脂酰肌醇3激酶抑制剂(Wortmannin)、蛋白激酶A抑制剂(H-89)、3H-L-arginine(美国SIGMA公司)。方法:设置空白对照组、H-89组、Wortmannin组及H-89+Wortmannin组,每组因素下再分异丙肾上腺素及空白对照两个干预因素组,每小组三复管。观察H-89和Wortmannin分别与人脐静脉内皮细胞孵育10min,再与异丙肾上腺素孵育30min后内皮型一氧化氮合酶活性变化。同位素二步色谱法测定3H-L-citrulline的产量反映内皮型一氧化氮合酶活性。人脐静脉内皮细胞上3H-L-citrulline的产量相对空白对照组以百分比形式表达。主要观察指标:各组内皮型一氧化氮合酶活性变化。结果:①内皮型一氧化氮合酶基础活性为每微克蛋白(3085.62±272.72)mBq,与空白对照组比较,异丙肾上腺素可明显增加内皮型一氧化氮合酶活性(30.72±3.91)%(P<0.001),单纯H-89及Wortmannin不改变内皮型一氧化氮合酶基础活性(0.44±1.00)%,(0.36±1.47)%(P>0.05)。②预先加入H-89及Wortmannin后再加入异丙肾上腺素,可见异丙肾上腺素增加内皮型一氧化氮合酶活性的作用受到明显抑制(4.73±2.19)%,(17.35±3.72)%(P<0.001)。③联合阻断蛋白激酶A和磷脂酰肌醇3激酶对异丙肾上腺素增加内皮型一氧化氮合酶活性的抑制作用(4.92±0.99)%,与单纯阻断蛋白激酶A的作用类似,无累加效应(P>0.05)。结论:β-肾上腺素能受体激动剂异丙肾上腺素能激活内皮型一氧化氮合酶活性的信号通路,在此过程中蛋白激酶A及磷脂酰肌醇3激酶均有参与。

BACKGROUND： Beta-adrenergic activation can enhance signal-transduction pathway of endothelial nitric oxide （NO）. Vascular endothelial NO production in response to β -adrenergic activation is important in the normal control of vessel tone by the sympatheadrenal system. Previous trials have demonstrated that endothelial nitric oxide synthase （eNOS） activated by isoprenaline, β -adrenergic agonist, may be correlated with phosphorylation of eNOS. OBJECTIVE： To investigate the possible protein kinase involved in the signal transduction of the eNOS activation by β -adrenoceptor （β AR） stimulation in human vascular endothelium. DESIGN： Comparative observation. SETTING： Department of Geriatrics, First Affiliated Hospital of Zhengzhou University. MATERIALS： The experiment was performed at the Open Key Clinic Medical Experimental Laboratory of University of Henan Province between September 2006 and June 2007. Fresh umbilical cords were obtained following delivery of healthy babies to healthy normotensive mothers, either by vaginal delivery or by elective Caesarean section. The written informed consent was obtained from all donors. Approval was granted by the Affiliated Hospital of Zhengzhou University. Isoprenaline, H-89, Wortmannin, [^3H]-L-arginine were from Sigma, USA. METHODS： Blank control group, H-89 group （protein kinase inhibitor）, Wortmannin group （phosphatidylinositol 3-kinase inhibitor） and H-89＋Wortmannin group were set up, and isoprenaline subgroup and blank subgroup were subdivided with 3 tubes in each group. Human umbilical vein endothelial cells （HUVECs） were cultured with H-89 and Wortmannin, separately for 10 minutes, then agonist isoprenaline or vehicle was added and the incubation continued for another 30 minutes, eNOS activity was determined by the conversion of [^3H]-L-arginine to [^3H]-L-citrulline. The results were corrected by protein concentration in the corresponding cellular extracts. MAIN OUTCOME MEASURES： Changes in eNOS activity of each group. RESULTS： ①Basal [^3H]-L-citrulline production was （3 085.62±272.72） mBq per 1 lag protein. Isoprenaline increased [^3H]-L-citrulline formation by （30.72±3.91）% compared with the control group （P 〈 0.001）. H-89 or Wortmannin alone did not alter eNOS activity [（0.44± 1.00）%, （0.36± 1.47）%, P 〉 0.05]. ②The increase of eNOS activity by isoprenaline was significantly inhibited by pretreatment of H-89 and Wortmannin [（4.73±2.19）%, （17.35±3.72）%, P 〈 0.001]. ③The inhibition of protein kinase and phosphatidylinositol 3-kinase to isoprenaline-induced increase in eNOS activity [（4.92± 0.99）%] was similar to protein kinase alone, but not in an additive effect （P 〉 0.05）. CONCLUSION： Protein kinase and phosphatidylinositol 3-kinase are involved in the signal transduction of β -adrenoceptors mediated eNOS activation.