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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: β-adrenergic receptor activation can increase the signal transduction pathway of endothelial nitric oxide synthase, which is of great value in revealing the physiological mechanism of β-adrenergic system regulating vascular tone. Previous experiments showed that isoproline Adrenergic activation of endothelial nitric oxide synthase is associated with endothelial nitric oxide synthase protein phosphorylation. AIM: To investigate the effects of β-adrenergic receptors on the protein kinase involved in the signal transduction pathway of endothelial nitric oxide synthase in human umbilical vein endothelial cells. Design: comparative observation. Unit: First Affiliated Hospital of Zhengzhou University Geriatrics. MATERIALS: Experiments were performed at the Open Laboratory of Clinical Medicine Key Disciplines of Henan Province from September 2006 to June 2007. Experimental materials for the first Affiliated Hospital of Zhengzhou University, obstetrics and gynecology obstetric delivery of cesarean section or voluntary donation of neonatal no hematoma, the umbilical cord. All patients were informed consent of the experiment, the experimental protocol approved by the hospital ethics committee. The main reagents: β-adrenergic receptor agonist isoproterenol, phosphatidylinositol 3 kinase inhibitor (Wortmannin), protein kinase A inhibitor (H-89), 3H-L-arginine (SIGMA USA) . Methods: The blank control group, H-89 group, Wortmannin group and H-89 + Wortmannin group were set up. Each group was divided into two groups: re-isoproterenol group and blank control group. The changes of endothelial nitric oxide synthase activity after H-89 and Wortmannin were respectively incubated with human umbilical vein endothelial cells for 10 min and then with isoproterenol for 30 min were observed. Isotope two-step chromatography determination of 3H-L-citrulline production reflects endothelial nitric oxide synthase activity. 3H-L-citrulline production on human umbilical vein endothelial cells relative blank control group expressed as a percentage. MAIN OUTCOME MEASURES: Changes of endothelial nitric oxide synthase activity in each group. Results: ① The basal activity of endothelial nitric oxide synthase was 3085.62 ± 272.72 mBq. Isoproterenol significantly increased the activity of endothelial nitric oxide synthase (30.72 ± 3.91)% compared with the blank control group (P <0.001). H-89 and Wortmannin alone did not change the basic activity of endothelial nitric oxide synthase (0.44 ± 1.00)%, (0.36 ± 1.47)% (P> 0.05). ② Pre-adding H-89 and Wortmannin followed by isoproterenol, we found that isoprenaline increased the activity of endothelial nitric oxide synthase (4.73 ± 2.19)%, (17.35 ± 3.72)% (P <0.001). ③ The combined inhibition of protein kinase A and phosphatidylinositol 3 kinase inhibited isoproterenol-induced endothelial nitric oxide synthase (4.92 ± 0.99)%, which was similar to that of protein kinase A alone. Cumulative effect (P> 0.05). CONCLUSION: Isoprenaline, a beta-adrenergic receptor agonist, activates the signaling pathway of endothelial nitric oxide synthase activity, both of which involve protein kinase A and phosphatidylinositol 3 kinase.