Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways -: Role of Rho/ROCK and mitogen-activated protein kinase
Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways -: Role of Rho/ROCK and mitogen-activated protein kinase
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DOI：
10.1161/hh1901.097084
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发表时间：
2001-09-28
影响因子：
20.1
通讯作者：
Lüscher, TF
中科院分区：
文献类型：
--
作者：
Eto, M;Barandiér, C;Lüscher, TF
一氧化氮和内皮素的失衡在心血管疾病中起着重要作用。凝血酶对血管内皮细胞功能有深远的影响。本研究探讨了凝血酶调节人内皮细胞内皮型一氧化氮合酶(ENOS)和内皮素转换酶-1表达的分子机制。人脐静脉内皮细胞与凝血酶(0.01~4U/mL)孵育15~24小时后，eNOS表达明显下调，ECE1蛋白表达明显升高，且呈剂量依赖关系。凝血酶还可降低enos基因的表达，提高ECE1基因的表达水平。在mRNA稳定性实验中，凝血酶缩短了enos mRNA的半衰期，但不能缩短ECE1的半衰期。该激动剂(SFLLRN，10~100mU·m o l/L)激活蛋白水解酶激活的受体I对eNOS的表达无影响，但使ECE1作为凝血酶的水平升高。凝血酶激活Rho A和细胞外信号调节激酶(ERK)1和ERK2。用C3胞外酶(20ug/mL)抑制Rho A，用Y-27632(10mU/L)抑制ROCK，可阻断凝血酶对eNOS表达的下调。Y-27632还可阻止凝血酶延长孵育引起的一氧化氮合酶活性的降低。另一方面，PD98059(50 mU/L)可抑制ERK1和ERK2的激活，阻止凝血酶上调ECE1的表达，并阻止ECEs活性的增加和ET-1在培养液中的积累。用凝血酶过夜处理大鼠主动脉会损害内皮依赖性的松弛，但不会损害非内皮依赖性的松弛。因此，凝血酶分别通过Rho/ROCK和ERK途径抑制eNOS和上调ECE1的表达。凝血酶的这些作用可能对心血管疾病的内皮功能障碍很重要，特别是在急性冠脉发作时。
An imbalance of nitric oxide and endothelin plays an important role in cardiovascular disease. Thrombin exerts profound effects on endothelial function. The present study investigated the molecular mechanisms by which thrombin regulates endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE)-1 expression in human endothelial cells. Incubation of human umbilical vein endothelial cells with thrombin (0.01 to 4 U/mL) for 15 to 24 hours markedly downregulated eNOS and increased ECE-1 protein level in a dose-dependent manner. Thrombin also decreased eNOS mRNA and increased ECE-1 mRNA level. In mRNA stability assay, thrombin shortened the half-life of eNOS mRNA but not that of ECE-1 mRNA. Activation of protease-activated receptor I by the agonist (SFLLRN, 10 to 100 mu mol/L) had no effect on eNOS expression but increased ECE-1 level as thrombin. Thrombin activated Rho A and extracellular signal-regulated kinase (ERK)1 and ERK2. Inhibition of Rho A by C3 exoenzyme (20 mug/mL) and ROCK by Y-27632 (10 mu mol/L) prevented the downregulation of eNOS expression by thrombin. Y-27632 also prevented the reduction in NOS activity induced by prolonged incubation with thrombin. On the other hand, inhibition of ERK1 and ERK2 activation by PD98059 (50 mu mol/L) prevented the upregulation of ECE-1 expression by thrombin as well as the increase in ECE activity and ET-1 accumulation in the medium. Treatment of rat aorta with thrombin overnight impaired endothelium-dependent relaxations but not endothelium-independent relaxations. Thus, thrombin suppresses eNOS and upregulates ECE-1 expression via Rho/ROCK and ERK pathway, respectively. These effects of thrombin may be important for endothelial dysfunction in cardiovascular disease, particularly during acute coronary episodes.