Decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin-(1-7)

The renin–angiotensin (Ang) system (RAS) plays an important role in the control of glucose metabolism and glycemia. Several studies demonstrated that the effects of angiotensin-(1-7) are mainly opposite to the actions of biological angiotensin II. Recent studies have demonstrated that rats with increased circulating angiotensin-(1-7), acting through the G protein coupled receptor Mas, have enhanced glucose tolerance and insulin sensitivity, presenting improved metabolic parameters. However, there is no data regarding the role of angiotensin-(1-7)–Mas axis in hepatic glycemic metabolism. In the present study, the gluconeogenesis and glycogenolysis was investigated in Sprague–Dawley (SD) and in TGR(A1-7)3292 (TGR) rats which present approximately twofold increase in plasma Ang-(1-7) levels compared to SD. The pyruvate administration in fasted rats showed a decreased synthesis of glucose in TGR compared to the SD rats, pointing to a downregulation of gluconeogenesis. Supporting this data, the mRNA evaluation of gluconeogenic enzymes showed a significant reduction in phosphoenolpyruvate carboxykinase reinforced by a significantly diminished expression of hepatocyte nuclear factor 4α (HNF-4α), responsible for the regulation of gluconeogenic enzymes. In conclusion our data show that the improved glucose metabolism induced by Ang-(1-7) could be due, at least in part, to a downregulation of hepatic gluconeogenesis.     

Expression of an angiotensin-(1-7)-producing fusion protein in rats induced marked changes in regional vascular resistance

We have described a transgenic rat line that expresses an angiotensin-(1-7)-producing fusion protein, the TGR(A1-7)3292. In these rats, testis acts as an angiotensin-(1-7) biological pump, increasing its plasma concentration 2.5-fold. In this study, we performed hemodynamic measurements in TGR(A1-7)3292 and age-matched Hannover Sprague-Dawley (SD) control rats, using fluorescent microspheres. Urethane-anesthetized transgenic rats had similar levels of baseline blood pressure (99 +/- 3 mmHg) as did SD rats (101 +/- 3 mmHg). However, pronounced differences were observed in other hemodynamic measurements. TGR(A1-7)3292 rats presented a significant increase in stroke volume (0.29 +/- 0.01 vs. 0.25 +/- 0.01 ml in SD), increased cardiac index (24.6 +/- 0.91 vs. 21.9 +/- 0.65 ml.min(-1).kg) and decreased total peripheral resistance (3.9 +/- 0.13 vs. 4.5 +/- 0.13 mmHg.ml(-1).min.100 g). The increase in stroke volume in transgenic rats may be partially explained by the small decrease in heart rate (326 +/- 7.0 vs. 359 +/- 6.0 beats/min in SD). Strikingly, TGR(A1-7)3292 rats presented a substantial decrease in the vascular resistance in lung, spleen, kidney, adrenals, brain, testis and brown fat tissue with no significant differences in the left ventricle, mesentery, skin, gastrocnemius muscle and white fat tissue. These results corroborate and extend previous results observed after acute angiotensin-(1-7) infusion, showing that chronic increase in circulating angiotensin-(1-7) produces sustained and important changes in regional and systemic hemodynamics. Moreover, our data suggest a physiological role for angiotensin-(1-7) in the tonic control of regional blood flow.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Captopril intake decreases body weight gain via angiotensin-(1-7)

Angiotensin-(1-7) [Ang-(1-7)] plays a beneficial role in cardiovascular physiology by providing a counterbalance to the function of angiotensin II (Ang II). Although Ang II has been shown to be an adipokine secreted by adipocyte and affect lipid metabolism, the role of Ang-(1-7) in adipose tissue remains to be clarified. The aim of the present study was to investigate whether Ang-(1-7) affects lipid metabolism in adipose tissue. Ang-(1-7) increased glycerol release from primary adipocytes in a dose-dependent manner. A lipolytic effect of Ang-(1-7) was attenuated by pretreatment with A-779, a Mas receptor blocker and with an inhibitor of phosphoinositol 3-kinase (PI3K), or eNOS. However, losartan and PD123319 did not cause any change in Ang-(1-7)-induced lipolysis. Ang-(1-7)-induced lipolysis had an addictive effect with isoproterenol. In normal rats, chronic intake of captopril for 4 wks decreased body weight gain and the amount of adipose tissue and increased plasma Ang-(1-7) level. These effects were attenuated by administration of A-779. The levels of Mas receptor and phosphorylation of hormone-sensitive lipase (p-HSL) were significantly increased by treatment with captopril and these captopril-mediated effects were attenuated by the administration of A-779. There was no difference in diameter of adipocytes among sham, captopril- and captopril+A-779-treated groups. The similar effects of captopril on body weight, expression of Mas receptor, and p-HSL were observed in Ang-(1-7)-treated rats. These results suggest that captopril intake decreased body weight gain partly through Ang-(1-7)/Mas receptor/PI3K pathway.     

The Angiotensin-(1-7)/Mas Receptor Axis Is Expressed in Sinoatrial Node Cells of Rats

The authors’ previous studies have indicated that angiotensin(Ang)-(1-7) protects the heart against reperfusion arrhythmias. The aim of this study was to determine whether a functional angiotensin-converting enzyme2 (ACE2)/Ang-(1-7)/Mas receptor axis is present in the sinoatrial node (SAN) of Wistar rats. SAN cells were identified by Masson’s trichrome staining, HCN4 expression, and lack of connexin43 expression. Immunohistochemistry technique was used to detect the expression of ACE2, Ang-(1-7), and Mas in the SAN. To evaluate the role of this axis in the SAN function, atrial tachyarrhythmias (ATs) were induced in isolated rat atria perfused with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1-7). The specific Mas antagonist, A-779, was used to evaluate the role of Mas in the Ang-(1-7) effects. The findings showed that all components of the ACE2/Ang-(1-7)/Mas branch are present in the SAN of rats. Importantly, it was found that this axis is functional because perfusion of atria with Ang-(1-7) significantly reduced the duration of ATs. Additionally, this anti-arrhythmogenic effect was attenuated by A-779. No significant changes were observed in heart rate, contractile tension, or ±dT/dt. These observations demonstrate that the ACE2/Ang-(1-7)/Mas axis is expressed in SAN cells of rats. They provide the morphological support to the anti-arrhythmogenic effect of Ang-(1-7).     

The ACE2/Ang-(1-7)/Mas Axis Regulates the Development of Pancreatic Endocrine Cells in Mouse Embryos

Angiotensin-converting enzyme 2 (ACE2), its product Angiotensin-(1-7) [Ang-(1-7)], and Ang-(1-7) receptor Mas, have been shown to regulate organogenesis during embryonic development in various species. However, it is not known whether a local ACE2/Ang-(1-7)/Mas axis is present in the fetal pancreas. It is hypothesized that there is a local ACE2/Ang-(1-7)/Mas axis in the embryonic pancreas in mice that is involved in regulating islet cell development. To address this issue, the endogenous expression profile of axis constituents in embryonic mouse pancreata was examined. Involvement of the ACE2 axis in the regulation of pancreatic development was also examined. The present experiments showed in an in vivo animal model that endogenous expression levels of ACE2 and the Mas receptor were upregulated in mouse pancreata in late embryogenesis, peaking on embryonic day E16.5, when it reached 3 folds compared to that seen at E12.5. Consistently, endogenous expression of Ang-(1-7) also peaked at E16.5. Treatment with the ACE2 inhibitor DX600 did not alter islet development. However, prenatal treatment with A779, a Mas receptor antagonist, reduced the β-cell to α-cell ratio in neonatal islets, impaired islet insulin secretory function, and impaired the pups’ glucose tolerance. In ex vivo pancreas explant cultures, A779 again decreased the β-cell to α-cell ratio, apparently through its effects on β-cell proliferation (reduced proliferation shown with Ki67 staining), and also decreased Insulin and Ngn3 mRNA expression. Furthermore, treatment of explant cultures with Ang-(1-7) increased mRNA levels of Insulin and pancreatic progenitor marker Ngn3, as well as Nox4, the ROS generation enzyme; these stimulatory effects were attenuated by co-treatment with A779, suggesting that Ang-(1-7), via Mas receptor signaling, may promote differentiation of pancreatic progenitors into insulin-producing cells via modulation of reactive oxygen species. These data together suggest that a Mas receptor-mediated mechanism may stimulate pancreatic cell development.     

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.     

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats

Background

Angiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.

Methods

To examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.

Results

Angiotensin II (Ang II) and angiotensin type 1 receptor (AT₁R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.

Conclusion

Ang-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT₁R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.     

Beneficial Effects of the Activation of the Angiotensin-(1–7) Mas Receptor in a Murine Model of Adriamycin-Induced Nephropathy

Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT₁ receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT₁ receptor antagonist, Losartan. AT₁ and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas^+/+) and Mas knockout (Mas ^−/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas^+/+, but not in Mas ^−/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT₁ receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.     

Exercise induces renin–angiotensin system unbalance and high collagen expression in the heart of Mas-deficient mice

The renin–angiotensin system (RAS) is involved in the cardiac and vascular remodeling associated with cardiovascular diseases. Angiotensin (Ang) II/AT₁ axis is known to promote cardiac hypertrophy and collagen deposition. In contrast, Ang-(1–7)/Mas axis opposes Ang II effects in the heart producing anti-trophic and anti-fibrotic effects. Exercise training is known to induce cardiac remodeling with physiological hypertrophy without fibrosis. We hypothesize that cardiac remodeling induced by chronic exercise depends on the action of Ang-(1–7)/Mas axis. Thus, we evaluated the effect of exercise training on collagen deposition and RAS components in the heart of FVB/N mice lacking Mas receptor (Mas-KO). Male wild-type and Mas-KO mice were subjected to a moderate-intense swimming exercise training for 6 weeks. The left ventricle (LV) of the animals was sectioned and submitted to qRT-PCR and histological analysis. Circulating and tissue angiotensin peptides were measured by RIA. Sedentary Mas-KO presented a higher circulating Ang II/Ang-(1–7) ratio and an increased ACE2 expression in the LV. Physical training induced in Mas-KO and WT a similar cardiac hypertrophy accompanied by a pronounced increase in collagen I and III mRNA expression. Trained Mas-KO and trained WT presented increased Ang-(1–7) in the blood. However, only in trained-WT there was an increase in Ang-(1–7) in the LV. In summary, we showed that deletion of Mas in FVB/N mice produced an unbalance in RAS equilibrium increasing Ang II/AT₁ arm and inducing deleterious cardiac effects as deposition of extracellular matrix proteins. These data indicate that Ang-(1–7)/Mas axis is an important counter-regulatory mechanism in physical training mediate cardiac adaptations.     

Angiotensin-(1-7) attenuates high glucose-induced proximal tubular epithelial-to-mesenchymal transition via inhibiting ERK1/2 and p38 phosphorylation

AimsThe kidney is an important target for both Angiotensin II and angiotensin-(1–7) [Ang-(1–7)] in the renin–angiotensin system. However, the renal function of Ang-(1–7) remains unclear. This study is aimed at investigating the effect of Ang-(1–7) on high glucose-induced epithelial to mesenchymal transition (EMT) in cultured renal epithelial cells.Main methodsCultured renal epithelial (NRK-52E) cell line was used in the experiment. Fluorescence immunocytochemistry was performed to observe α-smooth muscle actin (α-SMA). Real-time PCR and Western blot were used to determine mRNA and protein levels. Enzyme-linked immunosorbent assay was used to measure the concentration of transforming growth factor-β1 (TGF-β1) in the culture media.Key findingsHigh glucose-induced decreased in both angiotensin-converting enzyme-related carboxypeptidase (ACE2) and Mas mRNA levels. Meanwhile, high glucose induced increases in α-SMA and vimentin, decreases in E-cadherin, elevations in TGF-β1 and fibronectin secretions. Ang-(1–7) partially reversed high glucose-induced changes in α-SMA, vimentin, E-cadherin, TGF-β1 and fibronectin. High glucose stimulated ERK, p38 and JNK phosphorylation and Ang-(1–7) reversed the changes in ERK and p38 but not JNK phosphorylation.SignificanceInhibition and insufficiency in ACE2-Ang-(1–7)–Mas axis under high glucose condition participate EMT. Supplementation of Ang-(1–7) attenuates high glucose-induced EMT. ERK and p38 intracellular signaling pathways, not JNK, mediate the effect of Ang-(1–7) on EMT.     

The antithrombotic effect of angiotensin-(1-7) involves mas-mediated NO release from platelets

The antithrombotic effect of angiotensin(Ang)-(1-7) has been reported, but the mechanism of this effect is not known. We investigated the participation of platelets and receptor Mas-related mechanisms in this action. We used Western blotting to test for the presence of Mas protein in rat platelets and used fluorescent-labeled FAM-Ang-(1-7) to determine the specific binding for Ang-(1-7) and its displacement by the receptor Mas antagonist A-779 in rat platelets and in Mas(-/ -) and Mas(+/+) mice platelets. To test whether Ang-(1-7) induces NO release from platelets, we used the NO indicator DAF-FM. In addition we examined the role of Mas in the Ang-(1-7) antithrombotic effect on induced thrombi in the vena cava of male Mas(-/ -) and Mas(+/+) mice. The functional relevance of Mas in hemostasis was evaluated by determining bleeding time in Mas(+/+) and Mas(-/ -) mice. We observed the presence of Mas protein in platelets, as indicated by Western Blot, and displacement of the binding of fluorescent Ang-(1-7) to rat platelets by A-779. Furthermore, in Mas(+/+) mouse platelets we found specific binding for Ang-(1-7), which was absent in Mas(-/ -) mouse platelets. Ang-(1-7) released NO from rat and Mas(+/+) mouse platelets, and A-779 blocked this effect. The NO release stimulated by Ang-(1-7) was abolished in Mas(-/ -) mouse platelets. Ang-(1-7) inhibited thrombus formation in Mas(+/+) mice. Strikingly, this effect was abolished in Mas(-) (/) (-)mice. Moreover, Mas deficiency resulted in a significant decrease in bleeding time (8.50 +/- 1.47 vs. 4.28 +/- 0.66 min). This study is the first to show the presence of Mas protein and specific binding for Ang-(1-7) in rat and mouse platelets. Our data also suggest that the Ang-(1-7) antithrombotic effect involves Mas-mediated NO release from platelets. More importantly, we showed that the antithrombotic effect of Ang-(1-7) in vivo is Mas dependent and that Mas is functionally important in hemostasis.     

Angiotensin-(1–7) and the G Protein-Coupled Receptor Mas Are Key Players in Renal Inflammation

Angiotensin (Ang) II mediates pathophysiologial changes in the kidney. Ang-(1–7) by interacting with the G protein-coupled receptor Mas may also have important biological activities.In this study, renal deficiency for Mas diminished renal damage in models of renal insufficiency as unilateral ureteral obstruction and ischemia/reperfusion injury while the infusion of Ang-(1–7) to wild-type mice pronounced the pathological outcome by aggravating the inflammatory response. Mas deficiency inhibited NF-κB activation and thus the elevation of inflammation-stimulating cytokines, while Ang-(1–7) infusion had proinflammatory properties in experimental models of renal failure as well as under basal conditions. The Ang-(1–7)-mediated NF-κB activation was Mas dependent but did not involve Ang II receptors. Therefore, the blockade of the NF-κB-activating properties of the receptor Mas could be a new strategy in the therapy of failing kidney.     

Effect and mechanism of the Ang-(1-7) on human mesangial cells injury induced by low density lipoprotein

Hyperlipidemia is an independent risk factor for renal disease, and lipid deposition is associated with glomerulosclerosis. The angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis (ACE2-Ang-(1-7)-Mas axis) has been reported to participate in lipid metabolic regulation but its mechanism remains unclear. We hypothesized Ang-(1-7) would reduce lipid uptake in human mesangial cells (HMCs) by regulating the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr–SREBP2–SCAP) negative feedback system, and improve glomerulosclerosis by regulating the transforming growth factor-β1 (TGF-β1). In this study we found that ACE2 was undetected in HMCs. The administration of LDL caused normal LDLr–SREBPs–SCAP negative feedback effect. Exogenous Ang-(1-7) enhanced this negative feedback effect via down-regulating LDLr, SREBP2, and SCAP expression, and effectively inhibited LDL-induced lipid deposition and cholesterol increases. This enhanced inhibitory effect was reversed by the Mas receptor antagonist A-779. Meanwhile, Ang-(1-7) significantly decreased the high LDL-induced production of TGF-β1, an effect blocked by A-779. Interestingly, HMCs treated with Ang-(1-7) alone activated the TGF-β1 expression. Our results suggested that Ang-(1-7) inhibits LDL accumulation and decreases cholesterol levels via modulating the LDLr–SREBPs–SCAP negative feedback system through the Mas receptor. Moreover, Ang-(1-7) exhibits a dual regulatory effect on TGF-β1 in HMCs.     

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) Via the Mas Receptor Alleviates the Diabetes-Induced Decrease in GFAP and GAP-43 Immunoreactivity with Concomitant Reduction in the COX-2 in Hippocampal Formation: An Immunohistochemical Study

We have previously shown that chronic treatment with angiotensin-(1?C7) [Ang-(1?C7)] can prevent diabetes-induced cardiovascular dysfunction. However, effect of Ang-(1?C7) treatment on diabetes-induced alterations in the CNS is unknown. The aim of this study was to test the hypothesis that treatment with Ang-(1?C7) can produce protection against diabetes-induced CNS changes. We examined the effect of Ang-(1?C7) on the number of cyclooxygenase-2 (COX-2) immunoreactive neurons and the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and assessed the changes in the neuronal growth-associated protein-43 (GAP-43) of the hippocampal formation in streptozotocin-induced diabetes in rats. Animals were sacrificed 30?days after induction of diabetes and/or treatment with Ang-(1?C7). Ang-(1?C7) treatment significantly prevented diabetes-induced decrease in the number of GFAP immunoreactive astrocytes and GAP-43 positive neurons in all hippocampal regions. Co-administration of A779, a selective Ang-(1?C7) receptor antagonist, inhibited Ang-(1?C7)-mediated protective effects indicating that Ang-(1?C7) produces its effects through activation of receptor Mas. Further, Ang-(1?C7) treatment through activation of Mas significantly prevented diabetes-induced increase in the number of the COX-2 immunolabeled neurons in all sub-regions of the hippocampus examined. These results show that Ang-(1?C7) has a protective role against diabetes-induced changes in the CNS.     

Vasopressin V1a, V1b and V2 receptors mRNA in the kidney and heart of the renin transgenic TGR(mRen2)27 and Sprague Dawley rats.

Vasopressin plays significant role in regulation of blood pressure by means of V1 and V2 receptors, however regulation of synthesis of these receptors in hypertension is only poorly recognized. The purpose of the present study was to compare expression of V1a, V1b and V2 vasopressin (R) mRNA in the renal cortex, renal medulla and the heart of hypertensive renin transgenic TGR(mRen2)27 rats (TGR) and of their parent normotensive Sprague Dawley (SD) strain. The study was performed on 12 weeks old TGR and SD rats. Competitive PCR method was used for quantitative analysis of V1a, V1b and V2 receptors mRNA in fragments of renal cortex, renal medulla and apex of the left ventricle of the heart. In both strains expression of V1aR and V2R mRNA was significantly greater in the renal medulla than in the renal cortex. In the renal medulla but not in the cortex expression of V1aR mRNA was significantly greater in TGR than in SD rats. V2R mRNA expression was similar in the renal cortex and renal medulla of both strains. V1aR mRNA was well expressed in the heart of SD and TGR rats, however there was no significant difference between these two strains. V2R mRNA was not present in the heart. V1bR mRNa could not be detected either in the kidney or in the heart. The results provide evidence for specific increase of expression of V1a receptors mRNA in the renal medulla of TGR rats.     

Effects of Exercise Training on Circulating and Skeletal Muscle Renin-Angiotensin System in Chronic Heart Failure Rats

Background

Accumulated evidence shows that the ACE-AngII-AT1 axis of the renin-angiotensin system (RAS) is markedly activated in chronic heart failure (CHF). Recent studies provide information that Angiotensin (Ang)-(1–7), a metabolite of AngII, counteracts the effects of AngII. However, this balance between AngII and Ang-(1–7) is still little understood in CHF. We investigated the effects of exercise training on circulating and skeletal muscle RAS in the ischemic model of CHF.

Methods/Main Results

Male Wistar rats underwent left coronary artery ligation or a Sham operation. They were divided into four groups: 1) Sedentary Sham (Sham-S), 2) exercise-trained Sham (Sham-Ex), sedentary CHF (CHF-S), and exercise-trained CHF (CHF-Ex). Angiotensin concentrations and ACE and ACE2 activity in the circulation and skeletal muscle (soleus and plantaris) were quantified. Skeletal muscle ACE and ACE2 protein expression, and AT1, AT2, and Mas receptor gene expression were also evaluated. CHF reduced ACE2 serum activity. Exercise training restored ACE2 and reduced ACE activity in CHF. Exercise training reduced plasma AngII concentration in both Sham and CHF rats and increased the Ang-(1–7)/AngII ratio in CHF rats. CHF and exercise training did not change skeletal muscle ACE and ACE2 activity and protein expression. CHF increased AngII levels in both soleus and plantaris muscle, and exercise training normalized them. Exercise training increased Ang-(1–7) in the plantaris muscle of CHF rats. The AT1 receptor was only increased in the soleus muscle of CHF rats, and exercise training normalized it. Exercise training increased the expression of the Mas receptor in the soleus muscle of both exercise-trained groups, and normalized it in plantaris muscle.

Conclusions

Exercise training causes a shift in RAS towards the Ang-(1–7)-Mas axis in skeletal muscle, which can be influenced by skeletal muscle metabolic characteristics. The changes in RAS circulation do not necessarily reflect the changes occurring in the RAS of skeletal muscle.     

Hypotensive function of the brain angiotensin-(1-7) in Sprague Dawley and renin transgenic rats.

Angiotensin-(1-7) (Ang-[1-7]) is present in the brain of normotensive Sprague Dawley (SD) rats, and its hypothalamic content is elevated in TGRmRen2(27) rats (TGR) with renin dependent transgenic hypertension. The purpose of the present study was to determine the role of intrabrain Ang-(1-7) in the regulation of cardiovascular functions in SD and TGR rats under resting conditions and during haemodynamic challenge produced by rapid bleeding. Two groups of experiments were performed on conscious SD and TGR rats that were chronically instrumented with a lateral cerebral ventricle (LCV) cannula and an intraarterial catheter. Blood pressure (MAP) and heart rate period (Hp=distance between two systolic peaks) were continuously monitored: 1) under resting conditions during an LCV infusion of either artificial cerebrospinal fluid (aCSF, 5 microl/hr) or Ang-(1-7) in aCSF (100 pmol/5 microl/hr), and 2) before and after haemorrhage performed during LCV infusion of either aCSF or Ang-(1-7) antagonist (A-779, 4 nmol/5 microl/hr). Cerebroventricular infusion of Ang-(1-7) did not affect baseline MAP in the SD rats but it caused a significant decrease in blood pressure in the TGR rats. In the control experiments, haemorrhage significantly reduced MAP in the SD and TGR rats and heart rate in the TGR rats. Cerebroventricular infusion of Ang-(1-7) antagonist eliminated posthaemorrhagic hypotension in both strains and bradycardia in the TGR rats. The results indicate that intrabrain Ang-(1-7) may contribute to posthaemorrhagic hypotension and bradycardia. Moreover, the manner in which it centrally regulates the cardiovascular functions in the SD and TGR rats may be considerably different.     

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.