Effects of angiotensin-(1-7) and other bioactive components of the renin-angiotensin system on vascular resistance and noradrenaline release in rat kidney

OBJECTIVE: Angiotensin (Ang) is broken down enzymatically to several different metabolites which, in addition to Ang II, may have important biological effects in the kidney. This study investigates the role of Ang metabolites on vascular resistance and noradrenaline release in the rat kidney. METHODS AND RESULTS: In rat isolated kidney Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I induced pressor responses and enhanced noradrenaline release to renal nerve stimulation (RNS) in an concentration-dependent manner, with the following rank order of potency (EC(50)): Ang II >or= Ang III > Ang I = des-Asp-Ang I > Ang IV. All effects were blocked by the AT(1)-receptor antagonist EXP 3174 (0.1 micromol/l) but not by the AT(2)-receptor antagonist PD 123319 (1 micromol/l). Angiotensin-converting enzyme (ACE) inhibition by captopril (10 micromol/l) abolished the effect of Ang I and des-Asp-Ang I but had no influence on the effect of the other metabolites. Ang-(1-7) blocked the effects of Ang I and Ang II, being 10 times more potent against Ang I than Ang II. The selective Ang-(1-7) receptor blocker d-Ala7-Ang-(1-7) (10 micromol/l) did not influence the inhibitory effects of Ang-(1-7). Ang-(1-7) (10 micromol/l) by itself had no influence on vascular resistance and RNS-induced noradrenaline release. CONCLUSION: Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I regulate renal vascular resistance and noradrenaline release by activation of AT(1) receptors. In the case of Ang I and des-Asp-Ang I this depends on conversion by ACE. Ang-(1-7) may act as a potent endogenous inhibitor/antagonist of ACE and the AT(1)-receptors, respectively.     

Role of angiotensin-(1-7) in rostral ventrolateral medulla in blood pressure regulation via sympathetic nerve activity in Wistar-Kyoto and spontaneous hypertensive rats

Angiotensin (Ang)-(1-7) Ang-(1-7) is formed from angiotensin II by angiotensin-converting enzyme 2 (ACE2) and modulates the renin-angiotensin system. We evaluated whether the Ang-(1-7)-Mas axis in the rostral ventrolateral medulla (RVLM) contributes to neural mechanisms of blood pressure (BP) regulation. We microinjected Ang-(1-7), Ang-(1-7)-Mas receptor antagonist A-779, and ACE2 inhibitor DX600 into the RVLM of anesthetized Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHRs). Unilateral Ang-(1-7) microinjection induced a significantly greater increase in AP (arterial blood pressure) in SHR than in WKY. Bilateral A-779 microinjection induced a significantly greater decrease in AP and renal sympathetic nerve activity in SHR than in WKY. Bilateral DX600 microinjection induced a significantly greater decrease in AP in SHR than in WKY. Our results suggest that endogenous Ang-(1-7) in the RVLM contributes to maintain AP and renal sympathetic nerve activity both in SHR and WKY and that its activity might be enhanced in SHR.     

Angiotensin II modulates renal sympathetic neurotransmission through nitric oxide in AT2 receptor knockout mice

OBJECTIVE: Angiotensin (Ang) II enhances renal sympathetic neurotransmission and stimulates nitric oxide (NO) release. The present study investigates whether Ang II-mediated modulation of sympathetic neurotransmission is dependent on NO production in the kidney. AT2 -/y receptor-deficient mice are used to identify the Ang II receptor subtype involved. METHODS: Mice kidneys were isolated and perfused with Krebs-Henseleit solution. Drugs were added to the perfusion solution in a cumulative manner. Release of endogenous noradrenaline (NA) was measured by high-performance liquid chromatography (HPLC). AT1 receptor expression was analysed by real-time polymerase chain reaction (PCR). RESULTS: Ang II (0.01-30 nmol/l) dose dependently increased pressor responses in kidneys of AT2 -/y mice and wild-type (AT2 +/y) mice. Maximal pressor responses and EC50 values for Ang II was greater in AT2 -/y than in AT2 +/y mice. L-NAME (N(omega)-nitro-L-arginine methyl ester; 0.3 mmol/l) enhanced Ang II-induced pressor responses in both strains. In AT2 -/y mice, Ang II-induced facilitation of NA release was more pronounced than in AT2 +/y mice. L-NAME reduced Ang II-mediated facilitation of NA release in both strains. This reduction was more potent in AT2 -/y mice. In kidneys of AT2 -/y mice the AT1 receptor expression was significantly upregulated. CONCLUSION: These results suggest that activation of AT1 receptors by Ang II releases NO in mouse kidney to modulate sympathetic neurotransmission. Since AT1 receptors are upregulated in AT2 -/y mice kidneys, NO-dependent effects were greater in these mice. Thus, NO seems to play an important modulatory role for renal sympathetic neurotransmission.     

Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT(1)) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT(1) receptor. Ang-(1-7) competed poorly for [(125)I]Ang II binding to the AT(1) receptor on vascular smooth muscle cells, with an IC(50) of 2.0 micromol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [(125)I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 micromol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [B(max) 437.7+/-261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5+/-301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT(1) receptor. Pretreatment with the AT(1) receptor antagonist L-158,809 blocked the reduction in [(125)I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2+/-6.4%, P<0.01, n=5) only at 1 micromol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT(1) receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT(1) receptor or Ang-(1-7)-mediated downregulation of the vascular AT(1) receptor.     

The renin-angiotensin system and experimental heart failure

Experimental studies suggest that the renin-angiotensin system (RAS) and its primary effector peptide, angiotensin II (Ang II), are involved in the pathophysiology of cardiac hypertrophy and failure. All the components required for Ang II production are present in the heart, and cardiac Ang II formation appears to be regulated independent from the circulating RAS. In animal models and in patients with heart failure, the cardiac RAS is activated and, presumably, local Ang II formation is enhanced. Several cardiac cell types express Ang II type 1 (AT1) and/or type 2 (AT2)-receptors and represent potential targets for Ang II-mediated effects. In neonatal cardiac myocytes, Ang II induces a hypertrophic response via the AT1-receptor. Likewise, activation of the AT1-receptor triggers hypertrophy in terminally differentiated cardiac myocytes and in perfused heart preparations. In the neonatal system, Ang II appears to be a major autocrine/paracrine mediator of cardiac myocyte hypertrophy in response to passive mechanical stretch. By contrast, AT1-receptor activation apparently is not required to trigger load-induced hypertrophy in the adult cardiomyocyte. Recent studies suggest that the AT2-receptor opposes AT1-receptor-mediated growth signals in neonatal and in adult cardiac myocytes. Pharmacological studies have established that a blockade of the RAS at the level of the angiotensin-converting enzyme (ACE) or the AT1-receptor ameliorates the remodeling process of the heart and prolongs long-term survival in animal models of cardiac hypertrophy and failure. The therapeutic effects of ACE inhibitors and AT1-receptor antagonists clearly suggest an important role for the ACE-Ang II-AT1-receptor axis in the development of cardiac hypertrophy and failure. It must be kept in mind, however, that these drugs enhance AT2-receptor and B2-kinin receptor-dependent signaling pathways which may contribute significantly to the beneficial effects observed in vivo. Molecular and physiological analyses of transgenic mice with a cardiac-specific overexpression of the AT1 or AT2-receptor confirm that AT1 and AT2-receptor-dependent signaling cascades potently modulate cardiac myocyte function and growth. However, studies in AT1-receptor knockout mice demonstrate that cardiac hypertrophy in response to hemodynamic overload can occur independent from the AT1-receptor. In this paper, we review recent experimental evidence suggesting a critical role for the RAS in cardiac hypertrophy and failure with special emphasis on the putative role of Ang II and Ang II-receptor signaling in cardiac myocytes.     

Reduced circulating levels of angiotensin-(1--7) in systemic sclerosis: a new pathway in the dysregulation of endothelial-dependent vascular tone control

OBJECTIVE: Systemic sclerosis (SSc) impairs endothelium-dependent vasodilatation. Among angiotensin I (Ang I)-derived compounds, vasoconstrictor angiotensin II (Ang II) and vasodilator angiotensin-(1-7) (Ang-(1-7)), cleaved from ACE and neutral endopeptidase (NEP) 24.11, respectively, play an important role in vascular tone regulation. Ang-(1-7) may act independently or by activating other vasodilating molecules, such as nitric oxide (NO) or prostaglandin I2 (PGI2). Our aim was to assess, in patients with SSc, circulating levels of Ang I, Ang II and Ang-(1-7), with their metabolising enzymes ACE and NEP, and levels of NO and PGI2, and to correlate them to the main characteristics of SSc. METHODS: Levels of Ang I, Ang II, Ang-(1-7), NEP, ACE, NO and PGI2 were measured in 32 patients with SSc, who were also assessed for humoral and clinical characteristics, and 55 controls. RESULTS: Plasma Ang I, Ang II and Ang-(1-7) levels were lower in patients with SSc than in controls (p<0.001in all cases). When Ang II and Ang-(1-7) levels were expressed as a function of the available Ang I, lower Ang-(1-7) levels in patients with SSc than in controls were confirmed (p<0.001), while no difference was found for Ang II levels. In patients with SSc, the Ang II/Ang-(1-7) ratio indicated a prevalence of Ang II over Ang-(1-7), while in controls Ang-(1-7) was prevalent (p<0.001). Levels of ACE, NEP, NO and PGI2 were lower in patients with SSc than in controls (p<0.05 in all cases). CONCLUSION: In patients with SSc, prevalence of the vasoconstricting Ang II over the vasodilator Ang-(1-7) suggests a dysfunction of the angiotensin-derived cascade that may contribute to dysregulation of vascular tone.     

Nitric oxide and the renin-angiotensin system. Is there a physiological interplay between the systems?

Opposed actions for nitric oxide (NO) and angiotensin II (Ang II) in vascular contraction and vascular smooth muscle cell proliferation and apoptosis are well documented. In addition, various experimental approaches have shown that NO negatively modulates the renin-angiotensin system by inhibiting angiotensin-converting enzyme (ACE) activity and down-regulating AT1 receptors. On the other hand, Ang II and Ang-(1-7) positively stimulate NO synthesis and release. In this review, we analyse the data suggesting a mutual regulation between the renin-angiotensin and the nitric oxide-generating systems, and we propose a homeostatic interplay between both factors aimed at regulating cardiovascular function.     

Liver disease and the renin-angiotensin system: recent discoveries and clinical implications

The renin–angiotensin system (RAS) is a key regulator of vascular resistance, sodium and water homeostasis and the response to tissue injury. Historically, angiotensin II (Ang II) was thought to be the primary effector peptide of this system. Ang II is produced predominantly by the effect of angiotensin converting enzyme (ACE) on angiotensin I (Ang I). Ang II acts mainly through the angiotensin II type-1 receptor (AT₁) and, together with ACE, these components represent the 'classical' axis of the RAS. Drug therapies targeting the RAS by inhibiting Ang II formation (ACE inhibitors) or binding to its receptor (angiotensin receptor blockers) are now in widespread clinical use and have been shown to reduce tissue injury and fibrosis in cardiac and renal disease independently of their effects on blood pressure. In 2000, two groups using different methodologies identified a homolog of ACE, called ACE2, which cleaves Ang II to form the biologically active heptapeptide, Ang-(1–7). Conceptually, ACE2, Ang-(1–7), and its putative receptor, the mas receptor represent an 'alternative' axis of the RAS capable of opposing the often deleterious actions of Ang II. Interestingly, ACE inhibitors and angiotensin receptor blockers increase Ang-(1–7) production and it has been proposed that some of the beneficial effects of these drugs are mediated through upregulation of Ang-(1–7) rather than inhibition of Ang II production or receptor binding. The present review focuses on the novel components and pathways of the RAS with particular reference to their potential contribution towards the pathophysiology of liver disease.     

Angiotensin-converting enzyme inhibition, but not AT(1) receptor blockade, in the solitary tract nucleus improves baroreflex sensitivity in anesthetized transgenic hypertensive (mRen2)27 rats

Transgenic hypertensive (mRen2)27 rats overexpress the murine Ren2 gene and have impaired baroreflex sensitivity (BRS) for control of the heart rate. Removal of endogenous angiotensin (Ang)-(1-7) tone using a receptor blocker does not further lower BRS. Therefore, we assessed whether blockade of Ang II with a receptor antagonist or combined reduction in Ang II and restoration of endogenous Ang-(1-7) levels with Ang-converting enzyme (ACE) inhibition will improve BRS in these animals. Bilateral solitary tract nucleus (nTS) microinjections of the AT(1) receptor blocker, candesartan (CAN, 24?pmol in 120?nl, n=9), or a peptidic ACE inhibitor, bradykinin (BK) potentiating nonapeptide (Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro; BPP9α, 9?nmol in 60?nl, n=12), in anesthetized male (mRen2)27 rats (15-25 weeks of age) show that AT(1) receptor blockade had no significant effect on BRS, whereas microinjection of BPP9α improved BRS over 60-120?min. To determine whether Ang-(1-7) or BK contribute to the increase in BRS, separate experiments using the Ang-(1-7) receptor antagonist D-Ala(7)-Ang-(1-7) or the BK antagonist HOE-140 showed that only the Ang-(1-7) receptor blocker completely reversed the BRS improvement. Thus, acute AT(1) blockade is unable to reverse the effects of long-term Ang II overexpression on BRS, whereas ACE inhibition restores BRS over this same time frame. As the BPP9α potentiation of BK actions is a rapid phenomenon, the likely mechanism for the observed delayed increase in BRS is through ACE inhibition and elevation of endogenous Ang-(1-7).     

Increased expression of angiotensin converting enzyme 2 in conjunction with reduction of neointima by angiotensin II type 1 receptor blockade.

Angiotensin converting enzyme 2 (ACE2), a newly recognized homolog of ACE that converts angiotensin II (Ang II) to angiotensin-1-7 (Ang-(1-7)), is found in vascular smooth muscle cells. Expression of ACE2 may be a local determinant of vascular Ang-(1-7) production and, when increased, may augment the increasingly recognized protective effects of this peptide within injured tissues. We previously showed that treatment with the angiotensin II type 1 (AT1) receptor blocker (ARB) olmesartan increased aortic ACE2 and Ang-(1-7) in conjunction with improved vascular remodeling in spontaneously hypertensive rats (SHR). In the present study, we investigated balloon injury-related ACE2 in the vasculature by determining the effect of sustained AT1 blockade on ACE2 protein expression in the carotid arteries of 12-week-old male SHR treated with either vehicle (n=5) or 10 mg/kg olmesartan (n=5) in drinking water for 14 days. Olmesartan treatment caused a 61% reduction in the cross-sectional area of the neointima, from 0.27+/-0.01 mm2 in vehicle-treated rats to 0.11+/-0.01 mm2 in olmesartan-treated rats. In contrast, olmesartan treatment had no effect on the medial area of injured or uninjured carotid arteries compared to that in vehicle-treated rats. Quantitative analysis of ACE2 immunostaining intensity in the carotid artery of SHR was significantly greater (p<0.05) in the neointima of olmesartan-treated SHR compared to that in vehicle-treated animals. In contrast, ACE2 immunostaining intensity was not quantitatively different in uninjured carotid arteries of olmesartan and vehicle-treated animals. These studies suggest that changes in ACE2 within the vascular system of SHR are regulated by a factor other than arterial pressure.     

Renal renin-angiotensin system activity in naturally reared and cross-fostered spontaneously hypertensive rats

BACKGROUND: Young (4 week) spontaneously hypertensive rats (SHR) exhibit greater renal responses to angiotensin II (Ang II) than normotensive Wistar Kyoto (WKY) rats. SHR pups cross-fostering to a WKY dam at birth (SHRX) are less sensitive to Ang II and have lower adult blood pressure. The aim of this study was to compare renal renin-angiotensin system activity in young naturally reared and cross-fostered SHR pups. METHODS: SHR and WKY rats were reared either by their natural mothers or by a foster mother of the opposite strain. At 5, 10, and 15 days of age, renal tissue renin activity and Ang II concentration were measured by radioimmunoassay. Renin-secreting cells were identified by in situ hybridization and AT(1) receptor expression was compared using Western blots. Ang II-mediated cAMP generation was measured in isolated proximal tubules. CONCLUSIONS: Tissue renin activity and numbers of renin-secreting cells did not differ, but Ang II was higher in SHRX. The AT(1) receptor expression was significantly lower in SHRX compared with SHR. Basal and Ang II-stimulated cAMP was lower in SHR tubules compared with WKY and SHRX tubules.Cross-fostering reversed the increased renal sensitivity of the SHR to Ang II. These data suggest that renal AT(1) receptor expression can be manipulated during the postnatal period and that this may affect adult blood pressure.     

Angiotensin-(1-7) does not affect vasodilator or TPA responses to bradykinin in human forearm

Studies in isolated vessels and rat models of hypertension suggest that angiotensin (Ang)-(1-7) potentiates the vasodilator effect of bradykinin, possibly through ACE inhibition. We therefore tested the hypothesis that Ang-(1-7) potentiates the vasodilator or tissue plasminogen activator (TPA) response to bradykinin in the human forearm vasculature. Graded doses of Ang-(1-7) (10, 100, and 300 pmol/min), bradykinin (47, 94, and 189 pmol/min), and Ang I (1, 10, and 30 pmol/min) were administered through the brachial artery to 8 normotensive subjects in random order. Thirty minutes after initiation of a constant infusion of Ang-(1-7) (100 pmol/min), bradykinin and Ang I infusions were repeated. There were no systemic hemodynamic effects of the agonists. Bradykinin significantly increased forearm blood flow (P<0.001, from 3.8+/-0.5 to 13.9+/-3.1 mL/min per 100 mL at 189 pmol/min) and net TPA release (P=0.007, from 1.1+/-1.0 to 23.6+/-6.2 ng/min per 100 mL at 189 pmol/min), whereas Ang I caused vasoconstriction (P=0.003, from 3.3+/-0.4 to 2.5+/-0.3 mL/min per 100 mL at 30-pmol/min dose). There was no effect of Ang-(1-7) on either forearm blood flow (P=0.62, 3.3+/-0.4 to 3.5+/-0.4 mL/min per 100 mL at 300 pmol/min) or TPA release (P=0.52, from 0.7+/-0.8 to 1.0+/-0.7 ng/min/100 mL at 300 pmol/min). Moreover, there was no effect of 100 pmol/min Ang-(1-7) on the vasodilator [P=0.46 for Ang-(1-7) effect] or TPA [P=0.82 for Ang-(1-7) effect] response to bradykinin or the vasoconstrictor response to Ang I [P=0.62 for Ang-(1-7) effect]. These data do not support a role of Ang-(1-7), given at supraphysiological doses, in the regulation of human peripheral vascular resistance or fibrinolysis.     

Prevention of angiotensin II-mediated renal oxidative stress, inflammation, and fibrosis by angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase capable of metabolizing angiotensin (Ang) II into Ang 1 to 7. We hypothesized that ACE2 is a negative regulator of Ang II signaling and its adverse effects on the kidneys. Ang II infusion (1.5 mg/kg?1/d?1) for 4 days resulted in higher renal Ang II levels and increased nicotinamide adenine dinucleotide phosphate oxidase activity in ACE2 knockout (Ace2(-/y)) mice compared to wild-type mice. Expression of proinflammatory cytokines, interleukin-1β and chemokine (C-C motif) ligand 5, were increased in association with greater activation of extracellular-regulated kinase 1/2 and increase of protein kinase C-α levels. These changes were associated with increased expression of fibrosis-associated genes (α-smooth muscle actin, transforming growth factor-β, procollagen type Iα1) and increased protein levels of collagen I with histological evidence of increased tubulointerstitial fibrosis. Ang II-infused wild-type mice were then treated with recombinant human ACE2 (2 mg/kg?1/d?1, intraperitoneal). Daily treatment with recombinant human ACE2 reduced Ang II-induced pressor response and normalized renal Ang II levels and oxidative stress. These changes were associated with a suppression of Ang II-mediated activation of extracellular-regulated kinase 1/2 and protein kinase C pathway and Ang II-mediated renal fibrosis and T-lymphocyte-mediated inflammation. We conclude that loss of ACE2 enhances renal Ang II levels and Ang II-induced renal oxidative stress, resulting in greater renal injury, whereas recombinant human ACE2 prevents Ang II-induced hypertension, renal oxidative stress, and tubulointerstitial fibrosis. ACE2 is an important negative regulator of Ang II-induced renal disease and enhancing ACE2 action may have therapeutic potential for patients with kidney disease.     

Hemodynamic effects of vasorelaxant compounds in mice lacking one, two or all three angiotensin II receptors

The renin-angiotensin system (RAS) has a vital role in regulating the cardiovascular system. The primary effector of the RAS is the octapeptide angiotensin (Ang) II, a potent regulator of blood pressure and water homeostasis. Ang II mediates its functions through the stimulation of two distinct receptors, AT(1) (two subtypes in rodents (AT(1a) and AT(1b))) and AT(2). It was shown that in addition to Ang II, shorter fragments of Ang are also biologically active. Ang-(1-7) came into focus because it opposes many of the detrimental effects of Ang II. However, it is still controversial whether Ang II receptors are involved in Ang-(1-7)-mediated signaling. To characterize the impacts of Ang II receptors on Ang-(1-7)-stimulated vascular relaxation, the effects of acute infusion of the three vasorelaxant compounds, that is, Ang-(1-7), bradykinin (BK) and acetylcholine (ACh), on heart rate (HR) and mean arterial pressure (MAP) were investigated in mice deficient for one, two or all three Ang II receptors. Ang-(1-7) and BK reduced MAP in wild-type, AT(1a)/AT(1b)-deficient and AT(2)-deficient mice. Although the change in absolute MAP values in the hypotensive triple knockouts (KO) could not be further reduced by both peptides, the percent change in MAP was comparable between the triple KO and wild-type mice. Both peptides did not alter the HR in all four genotypes. ACh significantly reduced absolute MAP values in all four genotypes with a similar percentage of reduction. In contrast to Ang-(1-7) and BK, ACh significantly reduced HR without genotypic differences. Our results generate proof that Ang-(1-7)-induced effects on MAP are mediated by a receptor that is independent of AT(1) and AT(2).     

Enalapril attenuates downregulation of Angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat

The early and long-term effects of coronary artery ligation on the plasma and left ventricular angiotensin-converting enzyme (ACE and ACE2) activities, ACE and ACE2 mRNA levels, circulating angiotensin (Ang) levels [Ang I, Ang-(1-7), Ang-(1-9), and Ang II], and cardiac function were evaluated 1 and 8 weeks after experimental myocardial infarction in adult Sprague Dawley rats. Sham-operated rats were used as controls. Coronary artery ligation caused myocardial infarction, hypertrophy, and dysfunction 8 weeks after surgery. At week 1, circulating Ang II and Ang-(1-9) levels as well as left ventricular and plasma ACE and ACE2 activities increased in myocardial-infarcted rats as compared with controls. At 8 weeks post-myocardial infarction, circulating ACE activity, ACE mRNA levels, and Ang II levels remained higher, but plasma and left ventricular ACE2 activities and mRNA levels and circulating levels of Ang-(1-9) were lower than in controls. No changes in plasma Ang-(1-7) levels were observed at any time. Enalapril prevented cardiac hypertrophy and dysfunction as well as the changes in left ventricular ACE, left ventricular and plasmatic ACE2, and circulating levels of Ang II and Ang-(1-9) after 8 weeks postinfarction. Thus, the decrease in ACE2 expression and activity and circulating Ang-(1-9) levels in late ventricular dysfunction post-myocardial infarction were prevented with enalapril. These findings suggest that in this second arm of the renin-angiotensin system, ACE2 may act through Ang-(1-9), rather than Ang-(1-7), as a counterregulator of the first arm, where ACE catalyzes the formation of Ang II.     

Molecular mechanisms of inhibition of vascular growth by angiotensin-(1-7)

Angiotensin (Ang) peptides play a critical role in regulating vascular reactivity and structure. We showed that Ang-(1-7) reduced smooth muscle growth after vascular injury and attenuated the proliferation of vascular smooth muscle cells (VSMCs). This study investigated the molecular mechanisms of the antiproliferative effects of Ang-(1-7) in cultured rat aortic VSMCs. Ang-(1-7) caused a dose-dependent release of prostacyclin from VSMCs, with a maximal release of 277.9+/-25.2% of basal values (P<0.05) by 100 nmol/L Ang-(1-7). The cyclooxygenase inhibitor indomethacin significantly attenuated growth inhibition by Ang-(1-7). In contrast, neither a lipoxygenase inhibitor nor a cytochrome p450 epoxygenase inhibitor prevented the antiproliferative effects of Ang-(1-7). These results suggest that Ang-(1-7) inhibits vascular growth by releasing prostacyclin. Ang-(1-7) caused a dose-dependent release of cAMP, which might result from prostacyclin-mediated activation of adenylate cyclase. The cAMP-dependent protein kinase inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate attenuated the Ang-(1-7)-mediated inhibition of serum-stimulated thymidine incorporation. Finally, Ang-(1-7) inhibited Ang II stimulation of mitogen-activated protein kinase activities (ERK1/2). Incubation of VSMCs with concentrations of Ang-(1-7) up to 1 micromol/L had no effect on ERK1/2 activation. However, preincubation with increasing concentrations of Ang-(1-7) caused a dose-dependent reduction in Ang II-stimulated ERK1/2 activities. Ang-(1-7) (1 micromol/L) reduced 100 nmol/L Ang II-stimulated ERK1 and ERK2 activation by 42.3+/-6.2% and 41.2+/-4.2%, respectively (P<0.01). These results suggest that Ang-(1-7) inhibits vascular growth through the release of prostacyclin, through the prostacyclin-mediated production of cAMP and activation of cAMP-dependent protein kinase, and by attenuation of mitogen-activated protein kinase activation.     

Impaired heart rate baroreflex in older rats: role of endogenous angiotensin-(1-7) at the nucleus tractus solitarii

Age-related baroreflex reductions in function may originate from central neural dysregulation as well as vascular structural/functional changes. We determined the role of 2 angiotensin (Ang) peptides at the nucleus tractus solitarii in age-related baroreflex impairment. Baroreflex sensitivity control of heart rate in response to increases in blood pressure was tested in younger (3 to 5 months) and older (16 to 20 months) anesthetized male Sprague-Dawley rats before and after bilateral solitary tract injections of the Ang II type 1 (AT1) receptor antagonist candesartan (24 pmol) or the Ang-(1-7) antagonist (D-Ala7)-Ang-(1-7) (144 fmol or 24 pmol). Basal reflex sensitivity of older rats was significantly lower than younger rats. In younger rats, the reflex was facilitated by bilateral candesartan injections and attenuated by bilateral (D-Ala7)-Ang-(1-7) injections. In older rats, the reflex was facilitated by AT1 blockade; however, (D-Ala7)-Ang-(1-7) injected into the solitary tract nucleus had no effect. Neprilysin mRNA in the medulla was lower in older rats compared with younger rats, whereas angiotensin-converting enzyme (ACE), ACE2, and mas receptor mRNA levels of older rats did not differ from values of younger rats. Thus, opposing actions of endogenous Ang II and Ang-(1-7) in the solitary tract nucleus contribute to baroreflex function in response to increases in mean arterial pressure of younger rats. The attenuated counterbalancing effect of Ang-(1-7) on baroreflex function is lost in older rats, which may be attributable to diminished production of the peptide from neprilysin.     

Angiotensin II-mediated oxidative stress and inflammation mediate the age-dependent cardiomyopathy in ACE2 null mice

OBJECTIVES: The peptidase action of angiotensin converting enzyme 2 (ACE2) allows it to function as a negative regulator of the renin-angiotensin system. Current pharmacotherapies for human heart failure, such as ACE inhibitors and angiotensin and aldosterone receptor blockers, increase the activity of ACE2 in the heart. In this study, we investigate the mechanism for the age-dependent cardiomyopathy in ACE2 null mice. METHODS AND RESULTS: Ace2(-/y) mutant mice develop a progressive age-dependent dilated cardiomyopathy with increased oxidative stress, neutrophilic infiltration, inflammatory cytokine and collagenase levels, mitogen-activated protein kinase (MAPK) activation and pathological hypertrophy. The angiotensin II receptor-1 (AT1) blocker, irbesartan, prevented the dilated cardiomyopathy in aged Ace2(-/y) mutant mice, confirming a critical role of angiotensin II (Ang II)-mediated stimulation of AT1 receptors. Ang II activation of AT1 receptors triggers G-protein-coupled receptor (GPCR)-activated phosphoinositide 3-kinase gamma (PI3Kgamma) and its downstream pathways. We showed that p110gamma, the catalytic subunit of PI3Kgamma, is a key mediator of NADPH oxidase activation in response to Ang II. The double mutant mice (Ace2(-/y)/p110gamma(-/-)) exhibited marked reductions in oxidative stress, neutrophilic infiltration, and pathological hypertrophy resulting in myocardial protection, suggesting that PI3Kgamma plays a critical role in Ang II-mediated cardiomyopathy. CONCLUSIONS: Our findings demonstrate that the age-dependent cardiomyopathy in ACE2 null mice is related to increased Ang II-mediated oxidative stress and neutrophilic infiltration via AT1 receptors. Our combination of genetic and pharmacological approaches defines a critical role of ACE2 in the suppression of Ang II-mediated heart failure.