Antihypertensive effects of angiotensin-(1-7)

Accumulating evidence suggests that angiotensin-(1-7) (Ang-(1-7)) is an important component of the renin-angiotensin system and that the actions of the peptide may either contribute to or oppose those of Ang II. Ang-(1-7) can be converted directly from Ang I bypassing prerequisite formation of Ang II. Formation of Ang-(1-7) is under the control of at least three endopeptidases depending on the tissue compartment and include neprilysin, thimet oligopeptidase and prolyl oligopeptidase. Both neprilysin and thimet oligopeptidase are also involved in the metabolism of bradykinin and the atrial natriuretic peptide. Moreover, recent studies suggest that in addition to Ang I and bradykinin, Ang-(1-7) is an endogenous substrate for angiotensin converting enzyme. These enzymatic pathways may contribute to a complex relationship between the hypertensive actions of Ang II and various vasodepressor peptides from either the renin-angiotensin system or other peptide systems. Ang-(1-7) is devoid of the vasoconstrictor, central pressor, or thirst-stimulating actions associated with Ang II. In fact, new findings reveal depressor, vasodilator, and antihypertensive actions that may be more apparent in hypertensive animals or humans. Thus, Ang-(1-7) may oppose the actions of Ang II directly or as a result of increasing prostaglandins or nitric oxide. In this review, we examine the mechanisms by which Ang-(1-7) may contribute to cardiovascular regulation.

Effect of selective angiotensin antagonists on the antidiuresis produced by angiotensin-(1-7) in water-loaded rats

In the present study we evaluated the nature of angiotensin receptors involved in the antidiuretic effect of angiotensin-(1-7) (Ang-(1-7)) in water-loaded rats. Water diuresis was induced in male Wistar rats weighing 280 to 320 g by water load (5 ml/100 g body weight by gavage). Immediately after water load the rats were treated subcutaneously with (doses are per 100 g body weight): 1) vehicle (0.05 ml 0.9% NaCl); 2) graded doses of 20, 40 or 80 pmol Ang-(1-7); 3) 200 nmol Losartan; 4) 200 nmol Losartan combined with 40 pmol Ang-(1-7); 5) 1.1 or 4.4 nmol A-779; 6) 1.1 nmol A-779 combined with graded doses of 20, 40 or 80 pmol Ang-(1-7); 7) 4.4 nmol A-779 combined with graded doses of 20, 40 or 80 pmol Ang-(1-7); 8) 95 nmol CGP 42112A, or 9) 95 nmol CGP 42112A combined with 40 pmol Ang-(1-7). The antidiuretic effect of Ang-(1-7) was associated with an increase in urinary Na+ concentration, an increase in urinary osmolality and a reduction in creatinine clearance (CCr: 0.65 ± 0.04 ml/min vs 1.45 ± 0.18 ml/min in vehicle-treated rats, P<0.05). A-779 and Losartan completely blocked the effect of Ang-(1-7) on water diuresis (2.93 ± 0.34 ml/60 min and 3.39 ± 0.58 ml/60 min, respectively). CGP 42112A, at the dose used, did not modify the antidiuretic effect of Ang-(1-7). The blockade produced by Losartan was associated with an increase in CCr and with an increase in sodium and water excretion as compared with Ang-(1-7)-treated rats. When Ang-(1-7) was combined with A-779 there was an increase in CCr and natriuresis and a reduction in urine osmolality compared with rats treated with Ang-(1-7) alone. The observation that both A-779, which does not bind to AT1 receptors, and Losartan blocked the effect of Ang-(1-7) suggests that the kidney effects of Ang-(1-7) are mediated by a non-AT1 angiotensin receptor that is recognized by Losartan.

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33% of the initial values, P<0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 µmol/100 g body weight as a bolus, iv, plus a 30-min infusion of 0.018 µmol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P<0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 ± 0.28 mM, angiotensin II, N = 9 vs 6.4 ± 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.

Role of angiotensin II and vasopressin receptors within the supraoptic nucleus in water and sodium intake induced by the injection of angiotensin II into the medial septal area

In this study we investigated the effects of the injection into the supraoptic nucleus (SON) of non-peptide AT1- and AT2-angiotensin II (ANG II) receptor antagonists, DuP753 and PD123319, as well as of the arginine-vasopressin (AVP) receptor antagonist d(CH2)5-Tyr(Me)-AVP, on water and 3% NaCl intake induced by the injection of ANG II into the medial septal area (MSA). The effects on water or 3% NaCl intake were assessed in 30-h water-deprived or in 20-h water-deprived furosemide-treated adult male rats, respectively. The drugs were injected in 0.5 µl over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. Antagonists were injected at doses of 20, 80 and 180 nmol. Water and sodium intake was measured over a 2-h period. Previous administration of the AT1 receptor antagonist DuP753 into the SON decreased water (65%, N = 10, P<0.01) and sodium intake (81%, N = 8, P<0.01) induced by the injection of ANG II (10 nmol) into the MSA. Neither of these responses was significantly changed by injection of the AT2-receptor antagonist PD123319 into the SON. On the other hand, while there was a decrease in water intake (45%, N = 9, P<0.01), ANG II-induced sodium intake was significantly increased (70%, N = 8, P<0.01) following injection of the V1-type vasopressin antagonist d(CH2)5-Tyr(Me)-AVP into the SON. These results suggest that both AT1 and V1 receptors within the SON may be involved in water and sodium intake induced by the activation of ANG II receptors within the MSA. Furthermore, they do not support the involvement of MSA AT2 receptors in the mediation of these responses.

Differential effects of isoproterenol on the activity of angiotensin-converting enzyme in the rat heart and aorta

The excessive stimulation of beta-adrenergic receptors in the heart induces myocardial hypertrophy. There are several experimental data suggesting that this hypertrophy may also depend, at least partially, on the increase of local production of angiotensin II secondary to the activation of the cardiac renin-angiotensin system. In this study we investigated the effects of isoproterenol on the activity of angiotensin-converting enzyme (ACE) in the heart and also in the aorta and plasma. Male Wistar rats weighing 250 to 305 g were treated with a dose of (±)-isoproterenol (0.3 mg kg-1 day-1, N = 8) sufficient to produce cardiac hypertrophy without deleterious effects on the pumping capacity of the heart. Control rats (N = 7) were treated with vehicle (corn oil). The animals were killed one week later. ACE activity was determined in vitro in the four cardiac chambers, aorta and plasma by a fluorimetric assay. A significant hypertrophy was observed in both ventricular chambers. ACE activity in the atria remained constant after isoproterenol treatment. There was a significant increase (P<0.05) of ACE activity in the right ventricle (6.9 ± 0.9 to 8.2 ± 0.6 nmol His-Leu g-1 min-1) and in the left ventricle (6.4 ± 1.1 to 8.9 ± 0.8 nmol His-Leu g-1 min-1). In the aorta, however, ACE activity decreased (P<0.01) after isoproterenol (41 ± 3 to 27 ± 2 nmol His-Leu g-1 min-1) while it remained unchanged in the plasma. These data suggest that ACE expression in the heart can be increased by stimulation of beta-adrenoceptors. However, this effect is not observed on other local renin-angiotensin systems, such as the aorta. Our data also suggest that the increased sympathetic discharge and the elevated plasma concentration of catecholamines may contribute to the upregulation of ACE expression in the heart after myocardial infarction and heart failure.

Bi-directional actions of estrogen on the renin-angiotensin system

Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogen's inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the renin-angiotensin vasoconstrictor/vasodilator system is to promote the antihypertensive effect.

Angiotensin II-mediated vascular smooth muscle cell growth signaling

The mechanism by which Ang II stimulates the growth of vascular smooth muscle cells was investigated by measuring the phosphorylation of mitogen-activated protein kinases ERK 1 and ERK 2. Ca2+ ionophore was found to have effects practically analogous to Ang II. We found that the signaling pathway involves the activation of epidermal growth factor receptor (EGFR) kinase, activation of the adaptor proteins Shc and Grb2, and the small G-protein Ras. Although the mechanism of AT1- (or Ca2+)-induced activation of EGFR is not yet clear, we have found that calcium-dependent protein kinase CAKß/PYK2 and c-Src are involved in this process. These studies indicate a transactivation mechanism that utilizes EGFR as a bridge between a Gq-coupled receptor and activation of phosphotyrosine generation.

Oxidative stress may explain how hypertension is maintained by normal levels of angiotensin II

It is well known that essential hypertension evolves in most patients with "near normal" levels of plasma renin activity. However, these levels appear to be responsible for the high levels of arterial pressure because they are normalized by the administration of angiotensin II converting inhibitors or angiotensin receptor antagonist. In experimental animals, hypertension can be induced by the continuous intravenous infusion of small doses of angiotensin II that are not sufficient to evoke an immediate pressor response. However, this condition resembles the characteristics of essential hypertension because the high levels of blood pressure exist with normal plasma levels of angiotensin II. It is suggested that small amounts of angiotensin whose plasma levels are inappropriate for the existing size of extracellular volume stimulate oxidative stress which binds nitric oxide forming peroxynitrite. The latter compound oxidizes arachidonic acid producing isoprostaglandin F2a (an isoprostane) which is characterized by a strong antinatriuretic vasoconstrictor renal effect. In this chain of reactions the vasoconstrictor effects derived from oxygen quenching of nitric oxide and increased isoprostane synthesis could explain how hypertension is maintained with normal plasma levels of renin.

Stimulatory effect of dexamethasone on angiotensin-converting enzyme in neonatal rat cardiac myocytes

Angiotensin-converting enzyme (ACE) plays a central role in cardiac remodeling associated with pathological conditions such as myocardial infarction. The existence of different cell types in the heart expressing components of the renin-angiotensin system makes it difficult to evaluate their relative role under physiological and pathological conditions. Since myocytes are the predominant cellular constituent of the heart by mass, in the present study we studied the effects of glucocorticoids on ACE activity using well-defined cultures of neonatal rat cardiac myocytes. Under steady-state conditions, ACE activity was present at very low levels, but after dexamethasone treatment ACE activity increased significantly (100 nmol/l after 24 h) in a time-dependent fashion. These results demonstrate the influence of dexamethasone on ACE activity in rat cardiac myocytes. This is consistent with the idea that ACE activation occurs under stress conditions, such as myocardial infarction, in which glucocorticoid levels may increase approximately 50-fold.

Angiotensin-(1-7) potentiates the coronary vasodilatatory effect of bradykinin in the isolated rat heart

It has been shown that angiotensin-(1-7) (Ang-(1-7)) infusion potentiates the bradykinin (BK)-induced hypotensive response in conscious rats. The present study was conducted to identify Ang-(1-7)-BK interactions in the isolated rat heart perfused according to the Langendorff technique. Hearts were excised and perfused through the aortic stump under a constant flow with Krebs-Ringer solution and the changes in perfusion pressure and heart contractile force were recorded. Bolus injections of BK (2.5, 5, 10 and 20 ng) produced a dose-dependent hypotensive effect. Ang-(1-7) added to the perfusion solution (2 ng/ml) did not change the perfusion pressure or the contractile force but doubled the hypotensive effect of the lower doses of BK. The BK-potentiating Ang-(1-7) activity was blocked by pretreatment with indomethacin (5 mg/kg, ip) or L-NAME (30 mg/kg, ip). The Ang-(1-7) antagonist A-779 (50 ng/ml in Krebs-Ringer) completely blocked the effect of Ang-(1-7) on BK-induced vasodilation. These data suggest that the potentiation of the BK-induced vasodilation by Ang-(1-7) can be attributed to the release of nitric oxide and vasodilator prostaglandins through an Ang-(1-7) receptor-mediated mechanism.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Angiotensin-(1-7) increases osmotic water permeability in isolated toad skin

Angiotensin-(1-7) (Ang-(1-7)) increased osmotic water permeability in the isolated toad skin, a tissue with functional properties similar to those of the distal mammalian nephron. Concentrations of 0.1 to 10 µM were effective, with a peak at 20 min. This effect was similar in magnitude to that of frog skin angiotensin II (Ang II) and oxytocin but lower than that of human Ang II and arginine-vasotocin. The AT2 angiotensin receptor antagonist PD 123319 (1.0 µM) fully inhibited the response to 0.1 µM Ang-(1-7) but had no effect on the response to Ang II at the same concentration. The specific receptor antagonist of Ang-(1-7), A-779, was ineffective in blocking the response to Ang-(1-7) and to frog skin Ang II. The AT1 receptor subtype antagonist losartan, which blocked the response to frog skin Ang II, was ineffective in blocking the response to Ang-(1-7). The present results support the view of an antidiuretic action of Ang-(1-7) in the mammalian nephron.

Angiotensin-converting enzyme inhibition by lisinopril enhances liver regeneration in rats

Bradykinin has been reported to act as a growth factor for fibroblasts, mesangial cells and keratinocytes. Recently, we reported that bradykinin augments liver regeneration after partial hepatectomy in rats. Angiotensin-converting enzyme (ACE) is also a powerful bradykinin-degrading enzyme. We have investigated the effect of ACE inhibition by lisinopril on liver regeneration after partial hepatectomy. Adult male Wistar rats underwent 70% partial hepatectomy (PH). The animals received lisinopril at a dose of 1 mg kg body weight-1 day-1, or saline solution, intraperitoneally, for 5 days before hepatectomy, and daily after surgery. Four to six animals from the lisinopril and saline groups were sacrificed at 12, 24, 36, 48, 72, and 120 h after PH. Liver regeneration was evaluated by immunohistochemical staining for proliferating cell nuclear antigen using the PC-10 monoclonal antibody. The value for the lisinopril-treated group was three-fold above the corresponding control at 12 h after PH (P<0.001), remaining elevated at approximately two-fold above control values at 24, 36, 48 (P<0.001), and at 72 h (P<0.01) after PH, but values did not reach statistical difference at 120 h after PH. Plasma ACE activity measured by radioenzymatic assay was significantly higher in the saline group than in the lisinopril-treated group (P<0.001), with 81% ACE inhibition. The present study shows that plasma ACE inhibition enhances liver regeneration after PH in rats. Since it was reported that bradykinin also augments liver regeneration after PH, this may explain the liver growth stimulating effect of ACE inhibitors.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Effect of race, genetic population structure, and genetic models in two-locus association studies: clustering of functional renin-angiotensin system gene variants in hypertension association studies

Previous genetic association studies have overlooked the potential for biased results when analyzing different population structures in ethnically diverse populations. The purpose of the present study was to quantify this bias in two-locus association studies conducted on an admixtured urban population. We studied the genetic structure distribution of angiotensin-converting enzyme insertion/deletion (ACE I/D) and angiotensinogen methionine/threonine (M/T) polymorphisms in 382 subjects from three subgroups in a highly admixtured urban population. Group I included 150 white subjects; group II, 142 mulatto subjects, and group III, 90 black subjects. We conducted sample size simulation studies using these data in different genetic models of gene action and interaction and used genetic distance calculation algorithms to help determine the population structure for the studied loci. Our results showed a statistically different population structure distribution of both ACE I/D (P = 0.02, OR = 1.56, 95% CI = 1.05-2.33 for the D allele, white versus black subgroup) and angiotensinogen M/T polymorphism (P = 0.007, OR = 1.71, 95% CI = 1.14-2.58 for the T allele, white versus black subgroup). Different sample sizes are predicted to be determinant of the power to detect a given genotypic association with a particular phenotype when conducting two-locus association studies in admixtured populations. In addition, the postulated genetic model is also a major determinant of the power to detect any association in a given sample size. The present simulation study helped to demonstrate the complex interrelation among ethnicity, power of the association, and the postulated genetic model of action of a particular allele in the context of clustering studies. This information is essential for the correct planning and interpretation of future association studies conducted on this population.