Circulation Research. 2001;89:583-590
Published online before print September 13, 2001, doi: 10.1161/hh1901.097084
Published online before print September 13, 2001, doi: 10.1161/hh1901.097084
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© 2001 American Heart Association, Inc.
Molecular Medicine |
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
From the Department of Cardiology, University Hospital (M.E., T.K., T.F.L.) and Department of Cardiovascular Research, Institutes of Physiology, University of Zürich (M.E., L.R., T.K., H.J., T.F.L.), Zürich, and Department of Vascular Biology, Institutes of Physiology, University of Fribourg (C.B., Z.Y.), Fribourg, Switzerland.
Correspondence to Dr Thomas F. Lüscher, MD, Department of Cardiology, University Hospital, Ramistrasse 100,CH-8091 Zürich, Switzerland. E-mail cardiotfl{at}gmx.ch
Abstract — 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 1 by the agonist (SFLLRN, 10 to 100 µmol/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 µg/mL) and ROCK by Y-27632 (10 µmol/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 µmol/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.
Key Words: cell signaling • mitogen-activated protein kinase • endothelial dysfunction • protease-activated receptor
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