Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.Endothelium-derived nitric oxide (NO), synthesized from L-arginine by endothelial nitric oxide synthase (eNOS), plays a critical role in the regulation of vascular tone and the maintenance of vascular integrity by modulating various processes. It promotes vasodilatation, inhibits platelet activation and prevents vascular smooth muscle cell proliferation and/or migration (4). Abnormalities in eNOS gene expression or activation, which result in decreased NO production, are thought to contribute to the pathogenesis of various cardiovascular disorders such as atherosclerosis and hypertension (49).eNOS-mediated NO generation is a highly regulated cellular event. The mechanisms controlling eNOS activity involve multiple regulatory steps including gene expression, co-and posttranslational modification, intracellular localization, cofactors and phosphorylation (20). Although eNOS was originally described as a constitutive enzyme with enzyme activation achieved via calmodulin (CaM) binding in response to increased Ca 2ϩ , recent studies indicate that a variety of stimuli can modulate its expression at the transcriptional (15) and/or posttranscriptional level (6). Moreover, it is evidenced that phosphorylation of eNOS also modulates eNOS activity independent of Ca 2ϩ /CaM. Studies showed that Ser-1177 of human eNOS (Ser-1179 of bovine sequence) is phosphorylated directly by protein kinase B (PKB)/Akt (10, 18, 37), which results in an increase in electron flux through the reductase domain and increase in NO production (36). Shear stress and insulin have been shown to activate eNOS through the activation of PKB (14,41). eNOS phosphorylation at Ser-1177 by PKB therefore represents another important regulatory mechanism of eNOS activation in addition to Ca 2ϩ /CaM-depe...

Background-Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate L-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis. Methods and Results-In human endothelial cells isolated from umbilical veins, thrombin concentration-and timedependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (PϽ0.001) at 1 U/mL for 24 hours. The effect of thrombin was prevented by C3 exoenzyme or the HMG-CoA reductase inhibitor fluvastatin, which inhibit RhoA, or by the ROCK inhibitors Y-27632 and HA-1077. Adenoviral expression of constitutively active RhoA or ROCK mutants enhanced arginase activity (Ϸ3-fold, PϽ0.001), and the effect of active RhoA mutant was inhibited by the ROCK inhibitors. Neither thrombin nor the active RhoA/ROCK mutants affected arginase II protein level, the only isozyme detectable in the cells. Moreover, a significantly higher arginase II activity (1.5-fold, not the protein level) and RhoA protein level (4-fold) were observed in atherosclerotic aortas of apoE Ϫ/Ϫ compared with wild-type mice. Interestingly, L-arginine (1 mmol/L), despite a significantly higher eNOS expression in aortas of apoE Ϫ/Ϫ mice, evoked a more pronounced contraction, which was reverted to a greater vasodilation by the arginase inhibitor L-norvaline (20 mmol/L) compared with the wild-type animals (nϭ5, PϽ0.001). Conclusions-Thrombin

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 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 g/mL) and ROCK by Y-27632 (10 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 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. (Circ Res. 2001;89:583-590.)Key Words: cell signaling Ⅲ mitogen-activated protein kinase Ⅲ endothelial dysfunction Ⅲ protease-activated receptor A therosclerosis is the leading cause of death and accounts for half of the morbidity and mortality in Western countries. 1 An imbalance of endothelium-derived relaxing and contracting factors is a hallmark of cardiovascular disease. 2 Indeed, in human atherosclerosis, the production of nitric oxide (NO) is decreased because of reduced endothelial NO synthase (eNOS) expression. 3 On the other hand, endothelin-1 (ET-1) production and endothelinconverting enzyme-1 (ECE-1) expression are increased in atherosclerosis and restenosis. 4,5 However, the risk factors or mechanisms that lead to this imbalance of endothelial function in human atherosclerosis have not been completely elucidated.Thrombin, the multifunctional enzyme generated in the context of vascular injury from the circulating zymogen prothrombin, is focused in atherosclerosis and its complications. 6 Thrombin plays an important role in platelet activation, modulation of vasomotion, and vascular smooth muscle proliferation or migration 7-12 and in turn contributes to vasospasm and vascular remodeling. 13,14 Acutely, thrombin stimulates eNOS ac...

Adipocytes and perivascular adipose tissue are emerging as regulators of vascular function. The effects of adipocytes and perivascular adipose tissue on human smooth muscle cell (SMC) proliferation were investigated. Conditioned medium was prepared from cultured premature and differentiated 3T3-L1 adipocytes and from periaortic adipose tissue from young (3 mo) and old (24 mo) Wistar-Kyoto (WKY) rats, lean and obese Zucker rats (3 mo), and WKY rats fed normal chow or a high-fat diet for 3 mo. Conditioned medium from differentiated (but not premature) adipocytes stimulated SMC proliferation, which was abolished by charcoal and proteinase K treatment but was resistant to heat, trypsin, or phospholipase B (to hydrolyze lysophosphatidic acid). Further experiments demonstrated that the growth factor(s) are hydrosoluble and present in the fraction of molecular mass >100 kDa. Moreover, conditioned medium from periaortic adipose tissue stimulated SMC proliferation, which was significantly enhanced in aged rats and in rats fed a high-fat diet but not in obese Zucker rats deficient in functional leptin receptors. In conclusion, mature adipocytes release hydrosoluble protein growth factor(s) with a molecular mass >100 kDa for SMCs. Perivascular adipose tissue stimulates SMC proliferation, which is enhanced in aged WKY and in high-fat, diet-induced obesity but not in leptin receptor-deficient obese Zucker rats. These adipocyte-derived growth factor(s) and the effect of perivascular adipose tissue may be involved in vascular disease associated with aging and obesity.