Hypothalamic cardiovascular effects of angiotensin-(1-7) in spontaneously hypertensive rats

The objective of the present work was to study the cardiovascular actions of the intrahypothalamic injection of Ang-(1-7) and its effects on the pressor response to Ang II in spontaneously hypertensive (SH) rats and Wistar Kyoto (WKY) animals. In anaesthetized SH and WKY rats, a carotid artery was cannulated for mean arterial pressure (MAP) measurement and a stainless-steel needle was inserted into the anterior hypothalamus for drug administration. The cardiovascular effects of the intrahypothalamic administration of Ang-(1-7) were determined in SH and WKY rats. In SH rats, the effect of irbesartan and D-Ala-Ang-(1-7) on Ang-(1-7) cardiovascular effect was also evaluated. Ang II was administered in the hypothalamus of SH and WKY rats and changes in blood pressure and heart rate were measured followed by the administration of Ang II, Ang II+Ang-(1-7) or Ang II+D-Ala-Ang-(1-7). Ang-(1-7) did not the change basal MAP in WKY rats, but induced a pressor response in SH animals. Whilst the co-administration of D-Ala-Ang-(1-7) did not affect the response to Ang-(1-7), the previous administration of irbesartan prevented the effect of the peptide. The intrahypothalamic injection of Ang II induced a significantly greater pressor response in SH animals compared to normotensive rats. The co-administration of Ang-(1-7) with Ang II did not affect the pressor response to Ang II in the WKY group. In SH rats, whilst the co-administration of Ang-(1-7) with Ang II reduced the pressor response to Ang II, the concomitant application of D-Ala-Ang-(1-7) with Ang II increased the pressor response to the octapeptide after 5 and 10 min of intrahypothalamic administration. In conclusion, our result demonstrated that the biologically active peptide Ang-(1-7) did not participate in the hypothalamic blood pressure regulation of WKY animals. In SH rats, Ang-(1-7) exerted pleiotropic effects on blood pressure regulation. High dose of the heptapeptide produced a pressor response because of an unspecific action by activation of AT1 receptors. The concomitant administration of lower doses of Ang-(1-7) with Ang II reduced the pressor response to the octapeptide. Finally, the effect of AT(1-7) antagonist on Ang II pressor response suggested that hypothalamic formed Ang-(1-7) are implicated in the regulation of the cardiovascular effects of Ang II.     

Increased hypothalamic angiotensin-(1-7) levels in rats with aortic coarctation-induced hypertension

Since angiotensin (Ang) (1-7) injected into the brain blocked Ang II pressor actions in rats made hypertensive by aortic coarctation (CH), we examined systemic and tissue angiotensin peptide levels, specifically concentrating on the hypothalamic Ang-(1-7) levels. Plasma, heart and kidney isolated from CH rats showed increased levels of Ang I, Ang II and Ang-(1-7) compared with the normotensive group, with Ang II being the predominant peptide in heart and kidney. In the hypothalamus, equimolar amounts of Ang II and Ang-(1-7) were found in the sham group, whereas only Ang-(1-7) levels increased in CH rats. We conclude that aortic coarctation activates systemic and tissue renin-angiotensin system. The increased central levels of Ang-(1-7) in the CH rats suggest a potential mitigating role of this peptide in central control of the hypertensive process.     

Chronic infusion of angiotensin-(1-7) reduces heart angiotensin II levels in rats

We tested the hypothesis that the actions of Angiotensin (Ang)-(1-7) in the heart could involve changes in tissue levels of Ang II. This possibility was addressed by determining the effect of chronic infusion of Ang-(1-7) on plasma and tissue angiotensins. Ang-(1-7) was infused subcutaneously (osmotic minipumps) in Wistar rats. Angiotensins were determined by radioimmunoassay (RIA) in plasma, heart, and kidney. Tissue and plasma angiotensin-converting enzyme (ACE) activity and plasma renin activity (PRA) were also measured. Cardiac and renal ACE2 mRNA levels and cardiac angiotensinogen mRNA levels were assessed by semi-quantitative polymerase chain reaction (PCR). AT1 receptor number was evaluated by autoradiograph. Chronic infusion of Ang-(1-7) (2 microg/h, 6 days) produced a marked decrease of Ang II levels in the heart. A less pronounced but significant decrease of Ang-(1-7) was also observed. No significant changes were observed for Ang I. Ang II was not altered in the kidney. In this tissue, a significant increase of Ang-(1-7) and Ang I concentration was observed. A significant increase of plasma Ang-(1-7) and Ang II was also observed. Ang-(1-7) infusion did not change ACE activity or PRA. A selective slight significant increase in ACE2 expression in the heart was observed. Heart angiotensinogen mRNA as well as the number of Ang II binding sites did not change. These results suggest that AT1 receptors-independent changes in heart Ang II concentration might contribute for the beneficial effects of Ang-(1-7) in the heart. Moreover, these results reinforce the hypothesis that this angiotensin plays an important site-specific role within the renin-angiotensin system.     

Angiotensin-(1-7) regulates the levels of angiotensin II receptor subtype AT1 mRNA differentially in a strain-specific fashion

Ang-(1-7) is an effector peptide of the renin-angiotensin system with several distinct actions that are likely mediated by a specific receptor. Regulatory effects of angiotensin (Ang) peptides, Ang-(1-7) and Ang II, on Ang receptor subtype 1 (AT1) mRNA expression were investigated in vascular smooth muscle cells (VSMC) from four University of Akron (Akr) rat strains (WKY, SHR and two backcross consomic lines SHR/y and SHR/a), and in SHR and WKY cells from Charles River Laboratories (Crl). In WKY/Akr and SHR/Akr, Ang-(1-7) treatment increased the levels of AT1 mRNA. This effect was inhibited by the specific Ang-(1-7) antagonist, A-779, in WKY/Akr but not SHR/Akr. Ang II had no effect in Akr cells, but it down-regulated AT1 mRNA in WKY/Crl and SHR/Crl VSMC. Ang-(1-7) did not affect AT1 mRNA levels in Crl lines. In conclusion, Ang-(1-7) regulates the AT1 receptor either directly or indirectly in a strain-specific fashion. The Ang-(1-7) antagonist, A-779, blocks the actions of Ang-(1-7) only in VSMC from WKY/Akr rats, suggesting either that the binding sites for Ang-(1-7) have different properties in SHR/Akr and WKY/Akr cell lines, or that some of the effects of Ang-(1-7) are not receptor mediated. Further, we found differences between Akr cells and Crl cells that are consistent with their genetic heterogeneity.     

Different Effects of Angiotensin II and Angiotensin-(1-7) on Vascular Smooth Muscle Cell Proliferation and Migration

Background

Angiotensin (Ang) II and Ang-(1-7) are two of the bioactive peptides of the rennin-angiotensin system. Ang II is involved in the development of cardiovascular disease, such as hypertension and atherosclerosis, while Ang-(1-7) shows cardiovascular protection in contrast to Ang II.

Methodology/Principal Findings

In this study, we investigated effects of Ang II and Ang-(1-7) on vascular smooth muscle cell (SMC) proliferation and migration, which are critical in the formation of atherosclerotic lesions. Treatment with Ang II resulted in an increase of SMC proliferation, whereas Ang-(1-7) alone had no effects. However, preincubation with Ang-(1-7) inhibited Ang II-induced SMC proliferation. Ang II promoted SMC migration, and this effect was abolished by pretreatment with Ang-(1-7). The stimulatory effects of Ang II on SMC proliferation and migration were blocked by the Ang II receptor antagonist lorsartan, while the inhibitory effects of Ang-(1-7) were abolished by the Ang-(1-7) receptor antagonist A-799. Ang II treatment caused activation of ERK1/2 mediated signaling, and this was inhibited by preincubation of SMCs with Ang-(1-7).

Conclusion

These results suggest that Ang-(1-7) inhibits Ang II-induced SMC proliferation and migration, at least in part, through negative modulation of Ang II induced ERK1/2 activity.     

Angiotensin-(1-7) potentiates responses to bradykinin but does not change responses to angiotensin I

Angiotensin-(1-7) (Ang-(1-7)), a bioactive peptide in the renin-angiotensin system, has counterregulatory actions to angiotensin II (Ang II). However, the mechanism by which Ang-(1-7) enhances vasodepressor responses to bradykinin (BK) is not well understood. In the present study, the effects of Ang-(1-7) on responses to BK, BK analogs, angiotensin I (Ang I), and Ang II were investigated in the anesthetized rat. The infusion of Ang-(1-7) (55 pmol/min i.v.) enhanced decreases in systemic arterial pressure in response to i.v. injections of BK and the BK analogs [Hyp3, Tyr(Me)8]-bradykinin (HT-BK) and [Phe8psi (CH2-NH) Arg9]-bradykinin (PA-BK) without altering pressor responses to Ang I or II, or depressor responses to acetylcholine and sodium nitroprusside. The angiotensin-converting enzyme (ACE) inhibitor enalaprilat enhanced responses to BK and the BK analog HT-BK without altering responses to PA-BK and inhibited responses to Ang I. The potentiating effects of Ang-(1-7) and enalaprilat on responses to BK were not attenuated by the Ang-(1-7) receptor antagonist A-779. Ang-(1-7)- and ACE inhibitor-potentiated responses to BK were attenuated by the BK B2 receptor antagonist Hoe 140. The cyclooxygenase inhibitor sodium meclofenamate had no significant effect on responses to BK or Ang-(1-7)-potentiated BK responses. These results suggest that Ang-(1-7) potentiates responses to BK by a selective B2 receptor mechanism that is independent of an effect on Ang-(1-7) receptors, ACE, or cyclooxygenase product formation. These data suggest that ACE inhibitor-potentiated responses to BK are not mediated by an A-779-sensitive mechanism and are consistent with the hypothesis that enalaprilat-induced BK potentiation is due to decreased BK inactivation.     

Opposing actions of angiotensins on angiogenesis

Using the murine sponge model of angiogenesis, associated to functional and morphological parameters we have demonstrated opposing actions of angiotensin II (Ang II) and angiotensin-(1-7;Ang-1-7) in modulating fibrovascular tissue growth. Angiogenesis in the implants was assessed at day 7 postimplantation by extracting the hemoglobin content, by determining the outflow rate of sodium fluorescein applied intraimplant and by histological analysis. Furthermore, the proliferative activity of control and angiotensin-treated implants was established using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4 -sulfonyl)2H-tetrazolium)assay. The hemoglobin content in the control implants was 2.4 +/- 0.14 (microg/mg wet weight) versus 3.6 +/- 0.27(Ang II;100 ng) and 0.86 +/- 0.07 Ang-(1-7); 20 ng. Blood flow in the implants as determined by t1/2 values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation) showed that Ang II stimulated angiogenesis, whereas Ang-(1-7) inhibited it. The proliferative activity of the sponge-induced fibrovascular tissue was enhanced by Ang II and diminished by Ang-(1-7). These results show the pro-versus anti-angiogenic effects of these angiotensin molecules, providing evidence for their opposing effects on vascular tissue growth and wound healing in vivo.     

Angiotensin-(1-7) inhibits the angiotensin II-enhanced norepinephrine release in coarcted hypertensive rats

Since it has been suggested that angiotensin (Ang) (1-7) functions as an antihypertensive peptide, we studied its effect on the Ang II-enhanced norepinephrine (NE) release evoked by K+ in hypothalami isolated from aortic coarcted hypertensive (CH) rats. The endogenous NE stores were labeled by incubation of the tissues with 3H-NE during 30 min, and after 90 min of washing, they were incubated in Krebs solution containing 25 mM KCl in the absence or presence of the peptides. Ang-(1-7) not only diminished the K+-evoked NE release from hypothalami of CH rats, but also blocked the Ang II-enhanced NE release induced by K+. Ang-(1-7) blocking action on the Ang II response was prevented by [D-Ala7]Ang-(1-7), an Ang-(1-7) specific antagonist, by PD 123319, an AT2-receptor antagonist, and by Hoe 140, a B2 receptor antagonist. Ang-(1-7) inhibitory effect on the Ang II facilitatory effect on K+-stimulated NE release disappeared in the presence of Nomega-nitro-L-arginine methylester and was restored by L-arginine. Our present results suggest that Ang-(1-7) may contribute to blood pressure regulation by blocking Ang II actions on NE release at the central level. This inhibitory effect is a nitric oxide-mediated mechanism involving AT2 receptors and/or Ang-(1-7) specific receptors and local bradykinin generation.     

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney

To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 μm) were incubated with 10-1,000 μmol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.     

The Mas receptor mediates modulation of insulin signaling by angiotensin-(1-7)

Angiotensin (Ang)-(1-7) stimulates proteins belonging to the insulin signaling pathway and ameliorates the Ang II negative effects at this level. However, up to date, receptors involved and mechanisms behind these observations remain unknown. Accordingly, in the present study, we explored the in vivo effects of antagonism of the Ang-(1-7) specific Mas receptor on insulin signal transduction in rat insulin-target tissues. We evaluated the acute modulation of insulin-stimulated phosphorylation of Akt, GSK-3β (Glycogen synthase kinase-3β) and AS160 (Akt substrate of 160kDa) by Ang-(1-7) and/or Ang II in the presence and absence of the selective Mas receptor antagonist A-779 in insulin-target tissues of normal rats. Also using A-779, we determined whether the Mas receptor mediates the improvement of insulin sensitivity exerted by chronic Ang-(1-7) treatment in fructose-fed rats (FFR), a model of insulin resistance, dyslipidemia and mild hypertension. The two major findings of the present work are as follows; 1) Ang-(1-7) attenuates acute Ang II-mediated inhibition of insulin signaling components in normal rats via a Mas receptor-dependent mechanism; and 2). The Mas receptor appears to be involved in beneficial effects of Ang-(1-7) on the phosphorylation of crucial insulin signaling mediators (Akt, GSK-3β and AS160), in liver, skeletal muscle and adipose tissue of FFR. These results shed light into the mechanism by which Ang-(1-7) exerts its positive physiological modulation of insulin actions in classical metabolic tissues and reinforces the central role of Akt in these effects.     

PKA-mediated effect of MAS receptor in counteracting angiotensin II-stimulated renal Na-ATPase

We showed previously that angiotensin-(1-7) [Ang-(1-7)] reversed stimulation of proximal tubule Na⁺-ATPase promoted by angiotensin II (Ang II) through a d-ala⁷-Ang-(1-7) (A779)-sensitive receptor. Here we investigated the signaling pathway coupled to this receptor. According to our data, Ang-(1-7) produces a MAS-mediated reversal of Ang II-stimulated Na⁺-ATPase by a Gs/PKA pathway because: (1) the Ang-(1-7) effect is reversed by GDPβS, an inhibitor of trimeric G protein and Gs polyclonal antibody. Cholera toxin, an activator of Gs protein, mimicked it; (2) in the presence of Ang II, Ang-(1-7) increased the PKA activity 10-fold; (3) the peptide inhibitor of PKA blocked the Ang-(1-7) effect on Ang II-stimulated Na⁺-ATPase; (4) Ang-(1-7) reverses the Ang II-stimulated PKC activity; (5) cAMP mimicked the Ang-(1-7) effect on the Ang II-stimulated Na⁺-ATPase. Our results provide new understanding about the signaling mechanisms coupled to MAS receptor-mediated renal Ang-(1-7) effects.     

Angiotensin II and Angiotensin-(1-7) in Paraventricular Nucleus Modulate Cardiac Sympathetic Afferent Reflex in Renovascular Hypertensive Rats

Background

The enhanced cardiac sympathetic afferent reflex (CSAR) is involved in the sympathetic activation that contributes to the pathogenesis and progression of hypertension. Activation of AT₁ receptors by angiotension (Ang) II in the paraventricular nucleus (PVN) augments the enhanced CSAR and sympathetic outflow in hypertension. The present study is designed to determine whether Ang-(1-7) in PVN plays the similar roles as Ang II and the interaction between Ang-(1-7) and Ang II on CSAR in renovascular hypertension.

Methodology/Principal Findings

The two-kidney, one-clip (2K1C) method was used to induce renovascular hypertension. The CSAR was evaluated by the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to epicardial application of capsaicin in sinoaortic-denervated and cervical-vagotomized rats with urethane and α-chloralose anesthesia. Either Ang II or Ang-(1-7) in PVN caused greater increases in RSNA and MAP, and enhancement in CSAR in 2K1C rats than in sham-operated (Sham) rats. Mas receptor antagonist A-779 and AT₁ receptor antagonist losartan induced opposite effects to Ang-(1-7) or Ang II respectively in 2K1C rats, but losartan had no effects in Sham rats. Losartan but not the A-779 abolished the effects of Ang II, while A-779 but not the losartan blocked the effects of Ang-(1-7). PVN pretreatment with Ang-(1-7) dose-dependently augmented the RSNA, MAP, and CSAR responses to the Ang II in 2K1C rats. Ang II level, AT₁ receptor and Mas receptor protein expression in PVN increased in 2K1C rats compared with Sham rats but Ang-(1-7) level did not.

Conclusions

Ang-(1-7) in PVN is as effective as Ang II in enhancing the CSAR and increasing sympathetic outflow and both endogenous Ang-(1-7) and Ang II in PVN contribute to the enhanced CSAR and sympathetic outflow in renovascular hypertension. Ang-(1-7) in PVN potentiates the effects of Ang II in renovascular hypertension.     

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1-7). Parasite infection decreased Ang-(1-7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1-7) decreased the level of infection in an A779 (specific antagonist of Ang-(1-7) receptor, MAS)-sensitive manner. 10(-7) M PD123319, an AT(2) receptor antagonist, partially reversed the effects of Ang-(1-7) and Ang II. However, 10(-6) M losartan, an antagonist of the AT(1) receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10(-8) M Ang II or 10(-8) M Ang-(1-7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10(-7) M A779. 10(-6) M dibutyryl-cAMP increased the level of infection and 10(-7) M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1-7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1-7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus.     

Counteraction between angiotensin II and angiotensin-(1-7) via activating angiotensin type I and Mas receptor on rat renal mesangial cells

In the updated concept of renin-angiotensin system (RAS), it contains the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-angtiogensin type 1 receptor (AT1) axis and the angiotensin-converting enzyme-related carboxypeptidase (ACE2)-Ang-(1-7)-Mas axis. The former axis has been well demonstrated performing the vasoconstrictive, proliferative and pro-inflammatory functions by activation of AT1 receptors, while the later new identified axis is considered counterbalancing the effects of the former. The present study is aimed at observing the interaction between Ang-(1-7) and Ang II on cultured rat renal mesangial cells (MCs). RT-PCR, Western blot and immunofluorescent staining and confocal microscopy results showed that both AT1 and Mas receptor were co-distributed in rat renal MCs. Ang-(1-7) showed similar effects on Ang II in cultured MCs that stimulated phosphorylated extracellular signal-regulated kinase (ERK)1/2 phosphorylation and transforms growth factor-β1 synthesis, and cell proliferation and extracellular matrix synthesis. Co-treatment of the cell with Ang-(1-7) and Ang II, Ang-(1-7) counteracted AngII-induced effects in a concentration dependent manner, but failed to alter the changes induced by endothelin-1. The stimulating effect of Ang II was mediated by AT1 receptor while all the effects of Ang-(1-7) were blocked by Mas receptor antagonist A-779, but not by AT1 receptor antagonist losartan or AT2 receptor antagonist PD123319. These results suggest that Ang-(1-7) and Ang II specifically interact with each other on rat renal MCs via activation of their specific receptors, Mas and AT1 receptor respectively.     

Processing of angiotensin peptides by NG108-15 neuroblastoma x glioma hybrid cell line

The metabolism of angiotensin (Ang) peptides was studied in NG108-15 neuroblastoma x glioma hybrid cells which express Ang II receptors, renin, dipeptidyl carboxypeptidase A (converting enzyme), as well as Ang I and Ang II. In these experiments, 0.2 nM of either 125I-Ang I or 125I-Ang II was incubated with intact cell monolayers and the medium was analyzed for 125I-products by high performance liquid chromatography. The major product generated from the metabolism of labeled Ang I or Ang II was identified as the amino-terminal heptapeptide Ang-(1-7). N-benzyloxycarbonyl-prolyl-prolinal (ZPP), a specific inhibitor of prolyl endopeptidase, inhibited the formation of Ang-(1-7) from Ang I by 35%. Complete inhibition of Ang-(1-7) generation was attained with p-chloromercuriphenyl-sulfonate, which suggests that a sulfhydryl-containing peptidase other than prolyl endopeptidase is also involved in Ang-(1-7) formation. Ang II was observed to be a minor product resulting from Ang I metabolism. Although the converting enzyme inhibitor enalaprilat (MK-422) significantly reduced Ang II formation, it had no effect on the levels of Ang-(1-7). These findings demonstrate a preferential processing of Ang I into Ang-(1-7) which is not dependent on the prior formation of Ang II.     

Angiotensin II-induced mitochondrial Nox4 is a major endogenous source of oxidative stress in kidney tubular cells

Angiotensin II (Ang II)-induced activation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase leads to increased production of reactive oxygen species (ROS), an important intracellular second messenger in renal disease. Recent findings suggest that Ang II induces mitochondrial depolarization and further amplifies mitochondrial generation of ROS. We examined the hypothesis that ROS injury mediated by Ang II-induced mitochondrial Nox4 plays a pivotal role in mitochondrial dysfunction in tubular cells and is related to cell survival. In addition, we assessed whether angiotensin (1-7) peptide (Ang-(1-7)) was able to counteract Ang II-induced ROS-mediated cellular injury. Cultured NRK-52E cells were stimulated with 10(-6) M Ang II for 24 h with or without Ang-(1-7) or apocynin. Ang II simulated mitochondrial Nox4 and resulted in the abrupt production of mitochondrial superoxide (O(2) (-)) and hydrogen peroxide (H(2)O(2)). Ang II also induced depolarization of the mitochondrial membrane potential, and cytosolic secretion of cytochrome C and apoptosis-inducing factor (AIF). Ang-(1-7) attenuated Ang II-induced mitochondrial Nox4 expression and apoptosis, and its effect was comparable to that of the NAD(P)H oxidase inhibitor. These findings suggest that Ang II-induced activation of mitochondrial Nox4 is an important endogenous source of ROS, and is related to cell survival. The ACE2-Ang-(1-7)-Mas receptor axis should be investigated further as a novel target of Ang II-mediated ROS injury.     

Angiotensin-(1-7) reverts the stimulatory effect of angiotensin II on the proximal tubule Na(+)-ATPase activity via a A779-sensitive receptor.

Recently, we demonstrated that the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubules is reversed, in a dose-dependent manner, by Ang-(1-7) [Biochim. Biophys. Acta 1467 (2000) 189]. In the present paper, we characterized the receptor involved in this phenomenon. The preincubation of the Na(+)-ATPase with 10(-8) M Ang II increases the enzyme activity from 7.50+/-0.02 (control) to 12.40+/-1.50 nmol Pi mg(-1) min(-1) (p<0.05). Addition of 10(-9) M Ang-(1-7) completely reverts this effect returning the ATPase activity to the control level. This effect seems to be specific to Ang-(1-7) since Ang III (10(-12)-10(-8) M) does not modify the stimulation of the renal proximal tubule Na(+)-ATPase activity by Ang II. Saralasin abolishes the Ang-(1-7) effect in a dose-dependent manner being the maximal effect obtained at 10(-11) M. The increase in A779 concentration (from 10(-12) to 10(-7) M), a specific Ang-(1-7) antagonist, also abolishes the Ang-(1-7) effect. On the other hand, PD123319 (10(-8)-10(-6) M), an AT(2) antagonist receptor, and losartan (10(-12)-10(-7) M), an AT(1) antagonist receptor, does not modify the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) reverts the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubule through a A779-sensitive receptor.