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.
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 micromol/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 microgram/mL) and ROCK by Y-27632 (10 micromol/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 micromol/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.
Eto
M
M
Department of Cardiology, University Hospital, Institutes of Physiology, University of Zürich, Switzerland.
Barandiér
C
C
Rathgeb
L
L
Kozai
T
T
Joch
H
H
Yang
Z
Z
Lüscher
T F
TF
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008666:Metalloendopeptidases; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects; D054460:rho-Associated Kinases
D019332:Endothelin-1; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes