key: cord-016335-z2movens authors: Ferrario, Carlos M.; Averill, David B.; Brosnihan, K. Bridget; Chappell, Mark C.; Diz, Debra I.; Gallagher, Patricia E.; Neves, Liomar; Tallant, E. Ann title: Regulation of Cardiovascular Control Mechanisms by Angiotensin-(1–7) and Angiotensin-Converting Enzyme 2 date: 2010-06-15 journal: Hypertension and Hormone Mechanisms DOI: 10.1007/978-1-59259-987-5_3 sha: doc_id: 16335 cord_uid: z2movens Among the molecular forms of angiotensin peptides generated by the action of renin on angiotensinogen (Aogen), both angiotensin II (Ang II) and the amino terminal heptapeptide angiotensin-(1–7) [Ang-(1–7)] are critically involved in the long-term control of tissue perfusion, cell-cell communication, development, and growth. Whereas an impressive body of literature continues to uncover pleiotropic effects of Ang II in the regulation of cell function, research on Ang-(1–7) has a shorter history as it was only 16 yr ago that a biological function for this heptapeptide was first demonstrated in the isolated rat neuro-hypophysial explant preparation (1). On the contrary, the synthesis of angiotonin/ hypertensin (now Ang II) was first obtained in 1957 (2), three decades ahead of the discovery of Ang-(1–7) biological properties. carboxy-terminal hydrophobic or basic residues, whereas ACE cleaves two amino acids from its substrate. The ACE2 sequence is similar to those of the testis-specific or germinal form of ACE (tACE) and the Drosophila homolog of ACE (AnCE; sequence identities of 43% and 35%, and similarities of 61% and 55%, respectively) (21) . Conformational differences between the somatic form of ACE and germinal ACE account for the demonstration that ACE2 activity is not blocked by ACE inhibitors (15, 19) . The potential importance of ACE2, not only in the regulation of cardiac function and blood pressure but also in other disease states, has been realized with the discovery that ACE2 also serves as the cellular entry point for the severe acute respiratory syndrome (SARS) virus (21) (22) (23) (24) . Prabakaran et al. (21) built a homology model of the ACE2 structure with a root-mean-square deviation less than 0.5 Å from the aligned crystal structures of tACE and AnCE. According to the authors a prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site (21) . Negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (RBD) of the S-glycoprotein, which they recently identified (25) . Several distinct patches of hydrophobic residues at the ACE2 surface were noted at close proximity to the charged ridges that could contribute to binding. These results may help explain the structure and function of ACE2. We have employed immunocytochemistry to identify the tissues expressing Ang- (1) (2) (3) (4) (5) (6) (7) focusing primarily on the heart, kidney, brain, vascular system, and the utero-placenta complex. Early studies have demonstrated the Ang-(1-7) immunoreactivity in the cell bodies and in the axons of magnocellular neurons of the paraventricular nucleus (PVN) and supra-optic nucleus (SON) of the hypothalamus as well as in the neurons of the nucleus circularis (26) . In fact, Ang-(1-7) immunoreactivity in neurons of the PVN and SON was co-localized with vasopressin-like immunoreactivity whereas Ang-(1-7) was not co-localized in paraventricular neurons immunoreactive for oxytocin (26, 27) . The same pattern of Ang-(1-7) immunoreactivity, seen in Sprague-Dawley rats, was also observed in the brains of (mRen2)27 transgenic rats (28) in which a subpopulation of nitric oxide synthase-containing neurons also contained Ang-(1-7)-like immunoreactivity. Collectively, the co-localization of Ang-(1-7) and vasopressin immunoreactivity in neurons of the magnocellular division of the PVN and the SON are congruent with functional studies demonstrating a role of Ang-(1-7) in the regulation of hydro-mineral balance involving neurons of the hypothalami-neurohypophysial pathway. In addition, the colocalization of Ang-(1-7) immunoreactivity in nitric oxide synthase-containing neurons of the PVN is especially interesting because a number of studies now show that the angiotensin peptides may modulate the disposition of reactive oxygen species in the PVN of animals with heart failure (29) . Our interest in the role of Ang-(1-7) in cardiovascular regulation during heart failure led to the investigation of expression of Ang-(1-7) in the heart (30) . Ang-(1-7) immunoreactivity in Lewis rats was restricted to myocytes of both the right and left ventricles. Fig. 2 shows that Ang-(1-7) staining in myocytes had a granular appearance throughout the cytoplasm. In contrast, there was a distinct absence of staining for the peptide in vascular smooth muscle as well as in interstitial cells of the heart. When Ang-(1-7) was examined in the hearts of rats subjected to ligation of the left main coronary artery, we observed a significant increase in Ang-(1-7) staining in ventricular myocytes that had undergone hypertrophic remodeling. In the region of ischemic damage there was a marked absence of Ang-(1-7) staining in fibroblasts and connective tissue. The increase in Ang-(1-7) staining in rats with congestive heart failure was positively correlated with an increase in left ventricular end-diastolic pressure and negatively correlated with 46 Part I / Ferrario et al. (Fig. 3 ). Fig. 3B shows a granular reaction product for Ang-(1-7) staining in the juxtaglomerular cells of the afferent arteriole (32) . It is interesting that blood vessels (arterioles) of the kidney exhibit Ang-(1-7) staining whereas this was not the observation for blood vessels in the heart or in other organs. Vasodilation produced by Ang-(1-7) was first described in 1993 by Benter et al. (33) in rats that were made areflexic by spinal cord destruction. The vasodilator properties of the peptide were confirmed in isolated canine (34, 35) and porcine (36) coronary artery vessels, rabbit pial arterioles (37) , the rat mesenteric circulation (38) , the spontaneously hypertensive rats (SHR) (39), [mRen2]27 hypertensive transgenic rats (40) , two-kidney one-clip hypertensive dogs (41) , and in the human forearm circulation (42) . As reviewed elsewhere (43, 44) , the vasodilator response is mediated by release of vascular endothelium nitric oxide (NO), prostacyclin, and a receptor-mediated potentiation of bradykinin. The biological action of Ang-(1-7) satisfies the concept that the peptide acts to oppose the vasoconstrictor and hypertensive effects of Ang II by raising the activity of intrinsic vasodilator autocoids. Ang-(1-7) is present in hypothalamus, amygdala, and medulla oblongata at concentrations equivalent to or greater than those of Ang II (45) and evidence of a functional role for Ang-(1-7) exists in brain regions known to respond to other components of the RAS. Ang-(1-7) excites neurons in the PVN and in the nucleus of the solitary tract, dorsal motor nucleus of the vagus, and rostral and caudal ventrolateral medulla. The population of cells excited by Ang-(1-7) and Ang II shows some overlap, but the majority of cells respond to one or the other peptide. In addition to direct cellular actions, Ang-(1-7) releases other neurotransmitters including monoamines, substance P, vasopressin, glutamate, prostaglandins, and NO as reviewed recently (43) . Like Ang II, the Ang-(1-7)mediated release of vasopressin may involve tachykinin peptides (46) (47) (48) . In contrast, intracerebroventricular injections of Ang-(1-7) do not increase blood pressure or promote drinking, which are thought to be mediated by Ang II acting on monoaminergic pathways (49, 50) . An opposing action of Ang-(1-7) on central mechanisms regulating blood pressure is demonstrated by the finding that cerebroventricular injections of antibodies to Ang-(1-7) produce opposite effects to those of Ang II (47) . Evidence from studies using c-fos reveals that Ang-(1-7) activates pathways in the organum vasculosum of the lamina terminalis and median preoptic nucleus, whereas Ang II activates these pathways as well as those involving the PVN and subfornical organ (51, 52) . Pathways in the medulla oblongata appear separate for the two peptides as well, since sino-aortic denervation potentiated the responses to Ang-(1-7) but not those to Ang II in the nucleus of the solitary tract (53) . Important indicators of a role for the endogenous peptide in cardiovascular regulation come from studies using the Ang-(1-7) antagonist D-Ala 7 -Ang-(1-7) (44) . Blockade of endogenous Ang-(1-7) within the solitary tract nucleus augments baroreceptor reflex control of heart rate, providing an independent and opposite effect from those of Ang II (54, 55) . A loss of tonic input by Ang-(1-7) may accompany the age-related decrease in reflex gain (56) . In ASrAogen animals deficient of glial angiotensinogen, a role for endogenous Ang-(1-7) to facilitate baroreflex sensitivity persists, whereas the attenuating effect of Ang II is lost (57) . These data suggest different sources of the two peptides consistent with their divergent functional effects. In the rostral and caudal ventrolateral medulla, Ang-(1-7) exerts excitatory actions mimicking those of Ang II (54, 55) , although clear differences exist in terms of regulation of the responses to the two peptides (58) (59) (60) . In brain, receptor subtypes exhibiting varying degrees of selectivity for either the AT 1 or AT 2 antagonists and D-Ala 7 -Ang-(1-7) appear to mediate the actions of Ang-(1-7) (43, 61, 62) . The pharmacology of the receptor involved may be dependent on the specific transmitter studied, but blockade by D-Ala 7 -Ang-(1-7) is a common feature as reviewed in detail by Ferrario et al. (43) . As an explanation for the fact that either D-Ala 7 -Ang-(1-7), AT 1 or AT 2 receptor antagonists, or some combination of the three antagonists is effective in blocking actions of Ang-(1-7) in brain, there is evidence that distinct subpopulations of classical AT 1 sites in the nucleus tractus solitarius (nTS) and dorsal motor nucleus of the vagus (dmnX) exist showing differential sensitivity/selectivity to Ang-(1-7) and the AT 2 antagonist (43) . Moreover, AT 1 receptor antagonists (either losartan or candesartan) and D-Ala 7 -Ang-(1-7) block the actions of the peptide in the nTS (63) and ventrolateral medulla (64) . It is now recognized that the mas gene codes for a receptor responsible for vascular and renal effects of Ang-(1-7) (65) . Mice deficient in the mas receptor show impairments in the baroreceptor reflex and alterations in responses to Ang II (64, 66) , providing additional evidence of a role for endogenous Ang-(1-7) in these functions. There are few studies about the direct actions of Ang-(1-7) on heart function, although the peptide is highly expressed in rat myocardium (30) and can be detected in larger concentrations in the cardiac interstitium or the coronary sinus blood after acute coronary artery ligation (67) (68) (69) . In the isolated perfused heart of a rat, Ang-(1-7) appears to act as an anti-arrhythmogenic factor during reperfusion injury (70, 71) , although a study from the same group showed that Ang-(1-7) increased reperfusion arrhythmias (72) . Functional data in whole animals suggest that Ang-(1-7) may have cardioprotective functions because an 8 wk infusion of Ang-(1-7) in Sprague-Dawley rats, started 2-wk postmyocardial infarction, attenuated heart failure progression together with restoration of vascular endothelial function (31) . That the heart may be an important site for Ang-(1-7) actions is highlighted by the demonstration that ACE activity in plasma and atrial tissue is inhibited by Ang-(1-7) (73) whereas the peptide enhances tritiated norepinephrine release from isolated atrial tissue at doses comparable to those for Ang II (74) . The studies by us (69, 75) and others on the relation between Ang-(1-7) function and cardiac ACE2 expression are shedding light on an intracardiac role for Ang-(1-7) as counterbalancing the hypertrophic and inotropic actions of Ang-(1-7) following myocardial injury. Ang-(1-7) was formed in the intact human myocardial circulation in patients with postcardiac transplantation and its levels were decreased when Ang II formation was suppressed by enalaprilat (76) . The evolution of myocardial infarction in the rat 28 d after coronary artery ligation was accompanied by large increases in plasma Ang-(1-7) concentrations; further plasma Ang-(1-7) augmentation accompanied by reversal of cardiac remodeling because of continuous blockade of Ang II receptors with either losartan or olmesartan was accompanied by a threefold increase in cardiac ACE2 mRNA (75) . In addition, emerging data suggest that Ang-(1-7) may oppose the atherogenic actions of Ang II through inhibition of smooth muscle proliferation and blockade of inflammatory cytokines. This interpretation is supported by the finding that the chronic effects of losartan in the prevention of fatty streak formation and monocyte activation in monkeys was associated with large increases in plasma Ang-(1-7) concentrations (77-81). Although Ang-(1-7) is processed from either Ang I or Ang II in the circulation and in many tissues, it is important to emphasize that the processing pathways for Ang-(1-7) in the circulation and kidney are distinct. The endopeptidase neprilysin is the primary enzyme forming Ang-(1-7) from Ang I or Ang-(1-9) in the circulation (43, 82) . Although levels of neprilysin are low to undetectable, the enzyme is appropriately localized to the exocellular surface of endothelial and smooth muscle cells to contribute to the formation of Ang-(1-7). In the kidney, Ang-(1-7) is the primary product formed in preparations of isolated proximal tubules and exists in urine at significantly higher levels than Ang II (8) . Neprilysin may contribute to both the formation as well as the degradation of the peptide. Neprilysin cleaves Ang I to Ang-(1-7), but continues to metabolize Ang-(1-7) at the Tyr 5 -Ile 6 bond to form Ang-(1-4) and Ang-(5-7) (8, 9) . Indeed, neprilysin inhibitors increase the urinary levels of Ang-(1-7) in both human and rat (83, 84) . Moreover, the combined ACE/neprilysin inhibitor omapatrilat augmented renal and urinary levels of Ang-(1-7), but produced a blunted Ang-(1-7) response in plasma in comparison to ACE inhibition alone (32, 84) . Omapatrilat was also associated with increased expression of ACE2 and Ang-(1-7) in the proximal tubule (85) . These data suggest that upregulation of ACE2 may contribute to the renal protective effects of ACE or combined vaso-peptidase inhibitors through increased conversion of Ang II to Ang-(1-7), as well as the reduced metabolism of Ang-(1-7). Indeed, ACE2 (-/-) mice exhibit enhanced intrarenal levels of Ang II and pronounced glomerulosclerosis (83) . We found that Ang-(1-7) and ACE2 are present within the proximal tubular regions of the mouse and the rat kidney (86, 87) . These data clearly support the concept of a complete RAS within the proximal tubule including expression of renin, ACE, angiotensinogen, the AT 1 and AT 2 receptors (88, 89) . The presence of Ang II and Ang-(1-7) in kidney supports the concept of important and divergent actions for the two peptides. For Ang II, renal actions include potent vasoconstriction, retention of sodium and water, as well as a stimulus for inflammation, and oxidative stress. In contrast, Ang-(1-7) stimulates diuresis and natriuresis that are associated with modest increases in the glomerular filtration rate (43) . Ang-(1-7) induces vasodilation of afferent arterioles through a NO-dependent pathway (90) . Indeed, immunocytochemical staining for the Ang-(1-7) receptor mas is evident in the afferent arteriole, as well as throughout the proximal tubules of the renal cortex providing biochemical support for the functional actions of the peptide (87) . Ang-(1-7) and its metabolite Ang-(3-7) are potent inhibitors of Na + , K + -ATPase activity in the renal epithelium (91) (92) (93) . Ang-(1-7) also inhibits the transcellular flux of sodium, which was associated with activation of phospholipase A 2 (PLA 2 ) (94). Ang-(1-7) dependent inhibition of sodium transport is potentiated by ACE inhibition suggesting a shift toward the formation and protection of Ang-(1-7) within the proximal tubules. The chronic and pronounced diuresis following omapatrilat treatment was associated with large increases in urinary excretion of Ang-(1-7) and enhanced immunocytochemical staining of the peptide in the kidney (32) . Recent studies have revealed that Ang-(1-7) abolished the Ang II-dependent stimulation of the Na + -ATPase activity in ovine kidney (95, 96) . Furthermore, intrarenal administration of Ang-(1-7) blocked the antinatriuretic actions of Ang II (97) . These studies emphasize that the actions of Ang-(1-7) within the kidney may be particularly relevant in the setting of an activated RAS. Moreover, the discrete localization of Ang- (1-7) , ACE2, and the mas receptor provides evidence for an alternative RAS within the proximal tubule epithelium that may antagonize the actions of Ang II in this renal compartment. Pregnancy is a physiological condition characterized by increased RAS activity (98) that does not manifest in increased blood pressure (99) . Merrill et al. (100) evaluated the effect of pregnancy on Ang-(1-7) in nulliparous preeclamptic patients and in third trimester normotensive pregnant controls (3rd T; matched for parity, race, and gestational age). Plasma Ang-(1-7) was increased by 34% (p < 0.05), whereas plasma Ang II was increased by 50% (p < 0.05). In preeclampsia subjects plasma Ang-(1-7) was reduced (13 ± 2 pg/mL, p < 0.05 vs third T); plasma Ang II was also reduced (32 ± 4 pg/mL, preeclamptic vs 3rd trimester normal pregnant, p < 0.05), but remained elevated as compared to nonpregnant subjects and 50% higher than plasma Ang- (1-7) . Other components of the RAS, with the exception of ACE, were reduced in preeclamptic subjects. Assessment of the relationship between Ang-(1-7) and blood pressure revealed a negative correlation of Ang-(1-7) with systolic (r = -0.4, p < 0.02) and diastolic (r = -0.5, p < 0.02) blood pressures. These data suggested a potential role for reduced production of Ang-(1-7) contributing to the elevated blood pressure. In preeclampsia, the decreased levels of plasma Ang-(1-7) in the presence of persistent elevated plasma Ang II are consistent with the development of hypertension. Additionally, a 24-h urinary excretion of Ang-(1-7) and Ang II was evaluated during the ovulatory menstrual cycle, single normotensive pregnancies, and their subsequent lactation (101) . No significant differences in urinary Ang-(1-7) were observed between the follicular and luteal phase of the normal menstrual cycle. There was a progressive rise of urinary Ang-(1-7) throughout normal human gestation, attaining levels that are 10-fold greater than that of the normal menstrual cycle. Urinary Ang II showed a similar pattern reaching levels that were 25-fold higher than the values at the menstrual cycle. At 35 wk of gestation, Ang-(1-7) was the predominant angiotensin peptide in the urine, reaching levels that were sixfold higher than Ang II. The urinary excretion levels may reflect local kidney production of peptides. Thus, increases in renal Ang-(1-7) levels may play a role in the vasodilatory adaptations of mid and late human pregnancies. To understand the contribution of the RAS during pregnancy, studies were conducted in pregnant rats at late gestation (19th day) and compared to virgin female rats at diestrus phase of the estrous cycle. Twenty-four hour urinary excretion of the angiotensin peptides was significantly increased in pregnant animals by 93% (Ang I), 44% (Ang II), and 60% [Ang-(1-7)] of values found in virgin rats. Kidney Ang I and Ang-(1-7) concentrations were significantly increased by seven-and fivefold, respectively (p < 0.05) in pregnant animals as compared to virgin females. In contrast, there was no significant change in renal Ang II concentrations of pregnant and virgin females. These studies provide evidence that urinary excretion of angiotensin peptides reflect local kidney content of angiotensins during pregnancy. The potential contribution of Ang-(1-7) to vascular control in pregnancy was also documented from increased vasodilator responses to the local application of the peptide in isolated small mesenteric arteries obtained from pregnant rats (102) . In alignment with this interpretation, Ang-(1-7) and ACE2 staining in the kidney of 19 d pregnant Sprague-Dawley rats showed higher intensity when compared with virgin rats (103). Ang-(1-7) is a poor competitor at pharmacologically defined AT 1 or AT 2 receptors (1-7) , did not compete for binding at rat adrenal AT 1 or AT 2 receptors, and did not attenuate pressor or contractile responses to Ang II, demonstrating selectivity for Ang-(1-7). We identified Ang-(1-7) binding sites on bovine aortic endothelial cells (BAEC), canine coronary artery rings, and rat blood vessels that are sensitive to [D-Ala 7 ]-Ang-(1-7) (108) (109) (110) . In addition, a multitude of physiological responses to Ang-(1-7) are selectively blocked by [D-Ala 7 ]-Ang-(1-7) or the sarcosine analogs of Ang II, but not by AT 1 or AT 2 receptor antagonists, including the depressor response to Ang-(1-7) in the pithed rat and the lowering of blood pressure in hypertensive rats (33, 40, 111) . Collectively, these results demonstrate that the hypotensive response to Ang-(1-7) is mediated by a non-AT 1 , non-AT 2 , [D-Ala 7 ]-Ang-(1-7)-sensitive receptor. We refer to this receptor as the AT (1) (2) (3) (4) (5) (6) (7) receptor, as defined by its sensitivity to Ang-(1-7), its antagonism by [Sar 1 -Thr 8 ]-Ang II and [D-Ala 7 ]-Ang-(1-7), and its lack of response to AT 1 or AT 2 receptor antagonists, either functionally or in competition for binding. Identification of an AT (1) (2) (3) (4) (5) (6) (7) receptor is confounded by reports of responses to Ang-(1-7) that are sensitive to AT 1 or AT 2 receptor antagonists or both. Some of the renal and central effects of Ang-(1-7) are mediated by a losartan-sensitive receptor (64, 91, 112) . Arachidonic acid release from rabbit VSMCs and hypothalamic norepinephrine release were blocked by both [D-Ala 7 ]-Ang-(1-7) and the AT 2 antagonist PD123319 (113, 114) . Additional responses to Ang-(1-7) in brain and heart are blocked by both AT 1 and AT 2 receptor antagonists (74, 115) . These results provide evidence for additional subtypes of the AT (1) (2) (3) (4) (5) (6) (7) receptor that are sensitive to losartan and/or PD123319 or suggest an interaction with the AT 1 and/or the AT 2 receptor. Many of the physiological and cellular responses that are mediated by the AT (1) (2) (3) (4) (5) (6) (7) receptor are linked to the production of prostaglandins. Ang-(1-7) stimulates prostaglandin production in endothelial cells, VSMCs, astrocytes, and renal tubular epithelial cells (74, 94, (104) (105) (106) (107) 115) . In addition, physiological responses to Ang-(1-7) are dependent on prostanoid production, based on the studies using the cyclooxygenase inhibitor indomethacin (Fig. 4) . Recently, Santos et al. (65) reported that the orphan G protein-coupled receptor mas is an Ang-(1-7) receptor. Ang-(1-7) bound with high affinity to cells transfected with the mas receptor, which was blocked by [D-Ala 7 ]-Ang-(1-7), and renal or depressor responses to Ang-(1-7) were lost in mas-depleted mice. We recently showed that antisense oligonucleotides or siRNAs to mas prevent the Ang-(1-7)-mediated inhibition of growth in VSMCs, which is also blocked by [D-Ala 7 ]-Ang-(1-7) (116). These results suggest that the mas receptor serves as a selective Ang-(1-7) binding site. Fig. 4 shows the signal transduction pathway by which Ang-(1-7) activates the G protein-coupled mas receptor to increase the production of NO and prostacyclin (PGI 2 ) via increases in cGMP and cAMP, respectively. Ang-(1-7) also reduces the mitogen-activated protein kinases (MAPKs) by either increasing MAPK phosphatases or reducing the MAPK kinase MEK. The increase in cAMP and cGMP and the decrease in MAPK activity cause vasodilation and inhibit cell growth. The mas receptor is predominantly expressed in the testis and the hippocampus and amygdala of the mammalian forebrain with minimal levels in the rodent heart and kidney. This tissue distribution differs from previous reports of Ang-(1-7) binding and functional responses, suggesting the existence of other AT (1) (2) (3) (4) (5) (6) (7) receptors. Ang-(1-7), a product of both Ang I and Ang II metabolisms, functions to antagonize the actions of Ang II by acting primarily through binding to a non-AT 1 /AT 2 receptor and also the mas receptor. Ang-(1-7) stimulates vasodilation through increased production of vasodilator prostaglandins and NO, as well as amplifying the intrinsic actions of bradykinin. In the kidney, Ang-(1-7) promotes natriuresis and diuresis through an effect on transport mechanisms involved in water and electrolyte absorption within the renal tubules and collecting ducts. In addition, Ang-(1-7) modulates the actions of tubular vasopressin via an effect on its V2 receptor. In the heart, Ang-(1-7) counteracts the hypertrophic, pro-fibrotic, and pro-thrombotic effects of Ang II as well as increases myocardial blood flow. Ang-(1-7) contributes to the antihypertensive effects of ACE inhibitors and Ang II antagonists by several mechanisms (84, 117, 118, 119) . Inhibition of ACE increases blood and tissue concentrations of Ang-(1-7) by preventing ACE-mediated Ang-(1-7) degradation and increasing substrate (Ang I) availability. In contrast, blockade of Ang II receptors increases blood and tissue levels of Ang-(1-7) by: (i) increasing Ang I substrate availability through the dis-inhibition of Ang II-mediated renin release, and (ii) augmenting the rate of Ang II conversion into Ang-(1-7) via increased ACE2 expression and activity. Both ACE and ACE2 represent critical steps at which modulation of angiotensin peptide functions are precisely regulated, the specific mechanisms of which need further investigation. Genomic studies are also urgently needed to determine whether polymorphisms in the ACE2 gene or the genes for Ang-(1-7)-forming enzymes exist. Release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-(1-7) heptapeptide Synthesis and pharmacology of the octapeptide angiotonin Angiotensin-(1-7): a new hormone of the angiotensin system Angiotensin-(1-7). A member of circulating angiotensin peptides Evidence that prolyl endopeptidase participates in the processing of brain angiotensin Production of angiotensin-(1-7) by human vascular endothelium Inhibition of angiotensin converting enzyme by the metalloendopeptidase 3.4.24.15 inhibitor c-phenylpropylalanyl-alanyl-phenylalanyl-p-aminobenzoate Pathways for angiotensin-(1-7) metabolism in pulmonary and renal tissues Pathways of angiotensin-(1-7) metabolism in the kidney Metabolism of angiotensin-(1-7) by angiotensin converting enzyme Release of angiotensin-(1-7) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition Converting enzyme determines plasma clearance of angiotensin-(1-7) Vasodepressor actions of angiotensin-(1-7) unmasked during combined treatment with lisinopril and losartan Angiotensin-(1-7) contributes to the antihypertensive effects of blockade of the renin-angiotensin system A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9 ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors. Can The angiotensin-converting enzyme gene family: genomics and pharmacology Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase Heart block, ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins A model of the ACE2 structure and function as a SARS-CoV receptor The secret life of ACE2 as a receptor for the SARS virus Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus ACE2: from vasopeptidase to SARS virus receptor The SARS-CoV S glycoprotein: expression and functional characterization Immunocytochemical localization of angiotensin-(1-7) in the rat forebrain Angiotensin-(1-7) immunoreactivity in the hypothalamus of the (mRen-2d)27 transgenic rat Angiotensin-(1-7) and nitric oxide synthase in the hypothalamoneurohypophysial system Brain angiotensin II: new insights into its role in sympathetic regulation Cardiac angiotensin-(1-7) in ischemic cardiomyopathy Angiotensin-(1-7) attenuates the development of heart failure after myocardial infarction in rats Vasopeptidase inhibition and Ang-(1-7) in the spontaneously hypertensive rat Cardiovascular actions of angiotensin Angiotensin-(1-7) dilates canine coronary arteries through kinins and nitric oxide Effect of the angiotensin-(1-7) peptide on nitric oxide release Release of nitric oxide by angiotensin-(1-7) from porcine coronary endothelium: implications for a novel angiotensin receptor Comparative effects of angiotensin-(1-7) and angiotensin II on piglet pial arterioles Characterization of angiotensin-(1-7) receptor subtype in mesenteric arteries Angiotensin-(1-7) in the spontaneously hypertensive rat Contribution of angiotensin-(1-7) to blood pressure regulation in salt-depleted hypertensive rats Angiotensin-(1-7) and nitric oxide interaction in renovascular hypertension Angiotensin-(1-7) potentiates bradykinin-induced vasodilatation in man Angiotensin-(1-7): its contributions to arterial pressure control mechanisms Angiotensin-(1-7): an update Identification of angiotensin-(1-7) in rat brain. Evidence for differential processing of angiotensin peptides The hypothalamic paraventricular nucleus is a site of action for the central effect of tachykinins on plasma vasopressin Opposing actions of angiotensin-(1-7) and angiotensin II in the brain of transgenic hypertensive rats Vasopressin release induced by intracranial injection of eledoisin is mediated by central angiotensin II Acute depressor actions of angiotensin II in the nucleus of the solitary tract are mediated by substance P Characterization of receptors for angiotensin-induced drinking and blood pressure responses in conscious rats using angiotensin analogs extended at the N-terminal Angiotensin-(1-7) is an antagonist at the type 1 angiotensin II receptor The association of thirst, sodium appetite and vasopressin release with c-fos expression in the forebrain of the rat after intracerebroventricular injection of angiotensin II, angiotensin-(1-7) or carbachol Actions of angiotensin peptides after partial denervation of the solitary tract nucleus Angiotensin peptides and baroreflex control of sympathetic outflow: pathways and mechanisms of the medulla oblongata Angiotensin peptides as neurotransmitters/ neuromodulators in the dorsomedial medulla Reduced baroreflex sensitivity and diminished role for endogenous angiotensin-(1-7) at the nucleus tractus solitarii in older rats may reflect reduced formation of the peptide Glial versus non-glial angiotensin II and angiotensin-(1-7) in the nucleus tractus solitarii independently modulate control of the circulation Alterations of the reninangiotensin system at the RVLM of transgenic rats with low brain angiotensinogen Angiotensin peptides acting at rostral ventrolateral medulla contribute to hypertension of TGR(mREN2)27 rats Potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-(1-7) in normotensive and hypertensive rats Central and peripheral actions of angiotensin-(1-7). Braz Characterization of a new angiotensin antagonist selective for angiotensin-(1-7): evidence that the actions of angiotensin-(1-7) are mediated by specific angiotensin receptors Characterization of the receptors mediating the actions of angiotensin-(1-7) in the nucleus tractus solitarri The cardiovascular effects of angiotensin-(1-7) in the rostral and caudal ventrolateral medulla of the rabbit Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas Interaction between Mas and the angiotensin AT1 receptor in the amygdala Metabolism of angiotensin I in isolated rat hearts. Effect of angiotensin converting enzyme inhibitors The renin-angiotensin system during acute myocardial ischemia in dogs Angiotensin peptides modulate bradykinin levels in the interstitium of the dog heart in vivo Angiotensin-(1-7): cardioprotective effect in myocardial ischemia/reperfusion Angiotensin-(1-7) improves the postischemic function in isolated perfused rat hearts. Braz Effect of angiotensin-(1-7) on reperfusion arrhythmias in isolated rat hearts. Braz Angiotensin-(1-7) is a modulator of the human renin-angiotensin system Effects of angiotensin II and angiotensin-(1-7) on the release of [ 3 H]norepinephrine from rat atria Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors Angiotensin-(1-7) formation in the intact human heart: in vivo dependence on angiotensin II as substrate Renin-angiotensin system as a therapeutic target in managing atherosclerosis Angiotensin-(1-7) reduces smooth muscle growth after vascular injury Inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia Mechanisms linking angiotensin II and atherogenesis Renin-angiotensin system expression in rat bone marrow haematopoietic and stromal cells Effect of reduced angiotensin-converting enzyme gene expression and angiotensin-converting enzyme inhibition on angiotensin and bradykinin peptide levels in mice Angiotensin-converting enzyme 2 is an essential regulator of heart function Effects of omapatrilat on the renin-angiotensin system in salt-sensitive hypertension Evidence for the formation of angiotensin-(1-7) and the inhibitory actions of the peptide on Na + , K + -ATPase activity in rat proximal tubules Omapatrilat treatment is associated with increased ACE-2 and angiotensin-(1-7) in spontaneously hypertensive rats Novel aspects of the renal renin-angiotensin system: angiotensin-(1-7), ace2 and blood pressure regulation Regulation of intrarenal angiotensin II in hypertension Why are angiotensin concentrations so high in the kidney? Vasodilator action of angiotensin-(1-7) on isolated rabbit afferent arterioles Renal actions of angiotensin-(1-7): in vivo and in vitro studies Angiotensin-(1-7) can interact with the rat proximal tubule AT(4) receptor system Autoradiographic analysis and regulation of angiotensin receptor subtypes AT(4) Modulation of phospholipase A 2 activity and sodium transport by angiotensin Angiotensin-(1-7) modulates the ouabain-insensitive Na + -ATPase activity from basolateral membrane of the proximal tubule Angiotensin-(1-7) reverts the stimulatory effect of angiotensin II on the proximal tubule Na(+)-ATPase activity via a A779-sensitive receptor Intrarenal infusion of angiotensin-(1-7) modulates renal functional responses to exogenous angiotensin II in the rat The Renin-Angiotensin System in Pregnancy and Parturition Chesley's Hypertensive Disorders in Pregnancy Angiotensin-(1-7) in normal and preeclamptic pregnancy Urinary vasodilator and vasoconstrictor angiotensins during menstrual cycle, pregnancy, and lactation Pregnancy enhances the angiotensin (Ang)-(1-7) vasodilator response in mesenteric arteries and increases the renal concentration and urinary excretion of Ang Enhanced renal immunocytochemical expression of Ang-(1-7) and ACE2 during pregnancy Characterization of angiotensin receptors mediating prostaglandin synthesis in C6 glioma cells Alterations in prostaglandin production in spontaneously hypertensive rat smooth muscle cells Differential regulation of prostaglandin synthesis by angiotensin peptides in porcine aortic smooth muscle cells: subtypes of angiotensin receptors involved Human astrocytes contain two distinct angiotensin receptor subtypes Counterregulatory actions of angiotensin-(1-7) Evidence that prostaglandins mediate the antihypertensive actions of angiotensin-(1-7) during chronic blockade of the renin-angiotensin system Bovine aortic endothelial cells contain an angiotensin-(1-7) receptor Cardiovascular effects of angiotensin-(1-7) in conscious spontaneously hypertensive rats Angiotensin-(1-7) has a biphasic effect on fluid absorption in the proximal straight tubule Angiotensin-(1-7) reduces norepinephrine release through a nitric oxide mechanism in rat hypothalamus Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones Effects of angiotensin II and angiotensin-(1-7) on the release of [ 3 H] norepinephrine from rat atria Inhibition of MAP kinase activity by angiotensin-(1-7) in vascular smooth muscle cells is mediated by the mas receptor Characterization of angiotensin-(1-7) in the urine of normal and essential hypertensive subjects Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension The study described in this review was supported in part by grants from the National Heart, Lung and Blood Institute of the National Institutes of Health (HL-51952, HL-068258, and HL-56973, HL-67363, HL-42631), and the American Heart Association (AHA 151521). An unrestricted grant from the Unifi Corporation (Greensboro, NC) is also acknowledged.