Abstract-In the C57BL/6J mice model, we investigated whether obesity affects the function or expression of components of the tissue renin-angiotensin system and whether endothelin (ET)-1 contributes to these changes. ACE activity (nmol ⅐ L His-Leu ⅐ mg protein Ϫ1 ) was measured in lung, kidney, and liver in control (receiving standard chow) and obese animals treated for 30 weeks with a high-fat, low cholesterol diet alone or in combination with LU135252, an orally active ET A receptor antagonist. ACE mRNA expression was measured in the kidney, and the effects of LU135252 on purified human ACE were determined. Aortic and renal tissue ET-1 protein content was measured, and the vascular contractility to angiotensin II was assessed. Obesity was associated with a tissue-specific increase in ACE activity in the kidney (55Ϯ4 versus 33Ϯ3 nmol/L) but not in the lung (34Ϯ2 versus 32Ϯ2 nmol/L). Long-term LU135252 treatment completely prevented this activation (13.3Ϯ0.3 versus 55Ϯ4 nmol/L, PϽ0.05) independent of ACE mRNA expression, body weight, or renal ET-1 protein but did not affect pulmonary or hepatic ACE activity. Obesity potentiated contractions in response to angiotensin II in the aorta (from 6Ϯ2% to 33Ϯ5% KCl) but not in the carotid artery (4Ϯ1% to 3.6Ϯ1% KCl), an effect that was completely prevented with LU135252 treatment (6Ϯ0.4% versus 33Ϯ5% KCl). No effect of LU135252 on purified ACE was observed. Thus, obesity is associated with the activation of renal ACE in vivo independent of its mRNA expression and enhanced vascular contractility to angiotensin II. These effects are regulated by ET in an organ-specific manner, providing novel mechanisms by which ET antagonists may exert organ protection.
Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.
Abstract-To shed light on mechanisms of angiotensin-converting enzyme (ACE) upregulation, we used a rabbit endothelial cell model to characterize intracellular pathways of -adrenergic stimulation. In these cells, ACE activity is increased by isoproterenol (ISO). The stably transfected 1273-bp ACE promoter is stimulated by ISO in the presence of isobutyl methylxanthine. This effect is abolished by propranolol. Promoter stimulation is mimicked by cholera toxin, forskolin, and 8BrcAMP, but not by 8BrcGMP. Promoter stimulation by ISO and isobutyl methylxanthine is blocked by protein kinase A inhibitors, indicating that -adrenergic stimulation of the ACE gene depends on phosphorylation of protein kinase A targets. Activation by cAMP, resistance to phorbol ester, and lack of synergism between cAMP and phorbol ester suggest that promoter regulation is due to cAMP responsive element rather than to activating protein-2 sequences. Okadaic acid potentiation of 8BrcAMP induction indicated that promoter activation by cAMP is regulated by phosphatases controlling activation of typical cAMP responsive element regulated genes. In summary, -adrenergic activation of rat ACE promoter is specific; uses G s proteins, adenylyl cyclase, protein kinase A; and probably includes cAMP responsive element-like sequences. (Hypertension. 1999;34:31-38.)Key Words: angiotensin-converting enzyme Ⅲ endothelium Ⅲ receptors, adrenergic, beta Ⅲ cyclic AMP Ⅲ luciferase A ngiotensin-converting enzyme (ACE) is the most important enzyme controlling the activation of angiotensin peptides and the inactivation of kinins. 1 Tissue ACE distribution is wide, 1 which suggests that control of ACE gene expression depends on the particular cell type and environment or, alternatively, that ACE gene expression is controlled by a restricted array of non-cell-specific mechanisms. In cultured cells, expression of the endothelial ACE variant is modulated by hormones, 2-4 ionophores, 5 second messengers, 5 and growth factors. 6 Cardiac hypertrophy is one of the most likely pathophysiological conditions in which tissuespecific regulation of endothelial ACE might play an important role. 7-9 Strong evidence implicates the renin-angiotensin system, and ACE in particular, in the development of hypertrophy. 10 It is known that angiotensin II acts either as a hypertrophic or growth factor agent 9 ; that ACE and ACE mRNA (as well as angiotensinogen and its mRNA) are increased in early cardiac hypertrophy 7,8 ; and that ACE inhibitors prevent the development, ameliorate the course, and reduce the mortality of ventricular hypertrophy. 10 Recently, a histochemical study suggested that endothelial cells are the main cell type expressing ACE in the heart. 11 However, only a few studies have been done to understand the molecular basis of renin-angiotensin system activation and, in particular, of ACE upregulation in endothelial cells. 2,4,12,13 Surprisingly, the molecular mechanisms underlying cAMP induction of ACE activity have not yet been explored. The cAMP regulated pathway is ...
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