Interaction of Angiotensin-(1-7) with kinins in the kidney circulation: Role of B1 receptors.

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1-7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1-7) (1.0-25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 µM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 µM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1-7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1-7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1-7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1-7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Intrathecal injections of angiotensin IV and oxytocin conjugates induce antihyperalgesia and antiallodynia in both sexes of rats.

Our previous studies have established that intrathecal oxytocin (OT) and angiotensin IV (Ang IV) injections induce antihyperalgesia and antiallodynia in rodents. Ang IV, a renin-angiotensin system hexapeptide, acts as an endogenous inhibitor that inhibits the oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP). The pain inhibitory effects by Ang IV were found to be through its inhibition on IRAP to potentiate the effect of OT. However, these effects were found to be with a significant sex difference, which could be partially due to the higher expression of IRAP at the spinal cords of female. Therefore, we synthesized Ang IV and OT conjugates connected with a peptide bond and tested for their effects on hyperalgesia and allodynia. Carrageenan-induced hyperalgesia and partial sciatic nerve ligation (PSNL) were performed using rat models. Conjugates Ang IV-OT (Ang IV at the N-terminal) and OT-Ang IV (OT at the N-terminal) were synthesized and intrathecally injected into male and female rats. Our results showed that Ang IV-OT exhibited prominent antihyperalgesia in male rats, particularly during hyperalgesia recovery, whereas OT-Ang IV was more effective during development stage. Ang IV-OT showed clear antihyperalgesia in female rats, but OT-Ang IV had no significant effect. Notably, both conjugates alleviated neuropathic allodynia in male rats; however, OT-Ang IV had no effect in female rats, whereas Ang IV-OT induced significant antiallodynia. In conclusion, Ang IV-OT has greater therapeutic potential for treating hyperalgesia and allodynia than OT-Ang IV. Its effects were not affected by sex, unlike those of OT and OT-Ang IV, extending its possible clinical applications.

Continuous Infusion of Angiotensin IV Protects against Acute Myocardial Infarction via the Inhibition of Inflammation and Autophagy.

Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. Angiotensin (Ang) IV possesses many biological properties that are not yet completely understood. Therefore, we investigated the function and mechanism of Ang IV in AMI in in vivo and in vitro conditions. AMI was performed by ligation of the left anterior descending coronary artery (LAD) in male C57 mice. Ang IV was continuously infused by a minipump 3 d before AMI for 33 d. The neonatal rat ventricular myocytes (NRVCs) were stimulated with Ang IV and cultured under hypoxic conditions. In vivo, Ang IV infusion significantly reduced the mortality after AMI. By the 7th day after AMI, compared with the AMI group, Ang IV reduced the inflammatory cytokine expression. Moreover, terminal deoxyribonucleotidyl transferase- (TDT-) mediated dUTP nick-end labeling (TUNEL) assay showed that Ang IV infusion reduced AMI-induced cardiomyocyte apoptosis. Compared with AMI, Ang IV reduced autophagosomes in cardiomyocytes and improved mitochondrial swelling and disarrangement, as assessed by transmission electron microscopy. By 30th day after AMI, Ang IV significantly reduced the ratio of heart weight to body weight. Echocardiography showed that Ang IV improved impaired cardiac function. Hematoxylin and eosin (H&E) and Masson staining showed that Ang IV infusion reduced the infarction size and myocardial fibrosis. In vitro, dihydroethidium (DHE) staining and comet assay showed that, compared with the hypoxia group, Ang IV reduced oxidative stress and DNA damage. Enzyme-linked immunosorbent assay (ELISA) showed that Ang IV reduced hypoxia-induced secretion of the tumor necrosis factor- (TNF-) ɑ and interleukin- (IL-) 1ß. In addition, compared with the hypoxia group, Ang IV reduced the transformation of light chain 3- (LC3-) I to LC3-II but increased p62 expression and decreased cardiomyocyte apoptosis. Overall, the present study showed that Ang IV reduced the inflammatory response, autophagy, and fibrosis after AMI, leading to reduced infarction size and improved cardiac function. Therefore, administration of Ang IV may be a feasible strategy for the treatment of AMI.

Angiotensin IV attenuates diabetic cardiomyopathy via suppressing FoxO1-induced excessive autophagy, apoptosis and fibrosis.

Rationale: The rennin-angiotensin-aldosterone system (RAAS) plays a critical role in the pathogenesis of diabetic cardiomyopathy, but the role of a member of RAAS, angiotensin IV (Ang IV), in this disease and its underlying mechanism are unclear. This study was aimed to clarify the effects of Ang IV and its downstream mediator forkhead box protein O1 (FoxO1) on diabetic cardiomyopathy. Methods:In vivo, diabetic mice were treated with low-, medium- and high-dose Ang IV, AT4R antagonist divalinal, FoxO1 inhibitor AS1842856 (AS), or their combinations. In vitro, H9C2 cardiomyocytes and cardiac fibroblasts were treated with different concentrations of glucose, low-, medium- and high-dose Ang IV, divalinal, FoxO1-overexpression plasmid (FoxO1-OE), AS, or their combinations. Results: Ang IV treatment dose-dependently attenuated left ventricular dysfunction, fibrosis, and myocyte apoptosis in diabetic mice. Besides, enhanced autophagy and FoxO1 protein expression by diabetes were dose-dependently suppressed by Ang IV treatment. However, these cardioprotective effects of Ang IV were completely abolished by divalinal administration. Bioinformatics analysis revealed that the differentially expressed genes were enriched in autophagy, apoptosis, and FoxO signaling pathways among control, diabetes, and diabetes+high-dose Ang IV groups. Similar to Ang IV, AS treatment ameliorated diabetic cardiomyopathy in mice. In vitro, high glucose stimulation increased collagen expression, apoptosis, overactive autophagy flux and FoxO1 nuclear translocation in cardiomyocytes, and upregulated collagen and FoxO1 expression in cardiac fibroblasts, which were substantially attenuated by Ang IV treatment. However, these protective effects of Ang IV were completely blocked by the use of divalinal or FoxO1-OE, and these detrimental effects were reversed by the additional administration of AS. Conclusions: Ang IV treatment dose-dependently attenuated left ventricular dysfunction and remodeling in a mouse model of diabetic cardiomyopathy, and the mechanisms involved stimulation of AT4R and suppression of FoxO1-mediated fibrosis, apoptosis, and overactive autophagy.

Hypothalamic Renin-Angiotensin System and Lipid Metabolism: Effects of Virgin Olive Oil versus Butter in the Diet.

The brain renin-angiotensin system (RAS) has been recently involved in the homeostatic regulation of energy. Our goal was to analyse the influence of a diet rich in saturated fatty acids (butter) against one enriched in monounsaturated fatty acids (olive oil) on hypothalamic RAS, and their relationship with the metabolism of fatty acids. Increases in body weight and visceral fat, together with an increase in aminopeptidase A expression and reductions in AngII and AngIV were observed in the hypothalamus of animals fed with the butter diet. In this group, a marked reduction in the expression of genes related to lipid metabolism (LPL, CD36, and CPT-1) was observed in liver and muscle. No changes were found in terms of body weight, total visceral fat and the expression of hepatic genes related to fatty acid metabolism in the olive oil diet. The expressions of LPL and CD36 were reduced in the muscles, although the decrease was lower than in the butter diet. At the same time, the fasting levels of leptin were reduced, no changes were observed in the hypothalamic expression of aminopeptidase A and decreases were noted in the levels of AngII, AngIV and AngIII. These results support that the type of dietary fat is able to modify the hypothalamic profile of RAS and the body energy balance, related to changes in lipid metabolism.

Tubular Mas receptor mediates lipid-induced kidney injury.

Obesity-related kidney diseases are becoming serious health problems worldwide, yet the mechanism by which obesity causes kidney injury is not fully understood. The purpose of current study was to investigate the role of Mas receptor in lipid-induced kidney injury. In mice fed with high-fat diet (HFD), the protein abundance of markers of autophagy, endoplasmic reticulum stress (ER stress) and apoptosis was dramatically increased in the kidney cortex, which was markedly prevented by Mas deletion (Mas-/-) or Mas receptor antagonist A779. Palmitic acid (PA) induced persistently increased autophagy, ER stress, and apoptosis as well as mitochondrial injuries in primary cultured proximal tubular cells from wild type, but not from Mas-/- mice. In human proximal tubular HK2 cells, PA-induced autophagy and ER stress was aggravated by Mas agonists Ang (1-7) or AVE0991, but attenuated by A779 or Mas knockdown. Stimulation of Mas resulted in elevated intracellular calcium levels [Ca2+]i in HK2 cells treated with PA, whereas inhibition or knockdown of Mas decreased [Ca2+]i. Mitochondrial outer membrane located voltage-dependent anion channel (VDAC1) was markedly upregulated in HK2 cells treated with PA, which was associated with impaired mitochondrial morphology and depolarization. These were enhanced by AVE0991 and suppressed by A779 or Mas knockdown. Mas knockdown in HK2 cells prevented impaired interactions among VDAC1, autophagy adaptor P62, and ubiquitin, induced by PA, leading to a potential ubiquitination of VDAC1. In conclusion, Mas receptor-mediated lipid-induced impaired autophagy and ER stress in the kidney, likely contributing to tubular injuries in obesity-related kidney diseases.

Metabolism of angiotensin peptides by angiotensin converting enzyme 2 (ACE2) and analysis of the effect of excess zinc on ACE2 enzymatic activity.

After decades of notoriety for its adverse cardiovascular, proinflammatory and profibrotic actions, the renin-angiotensin system (RAS) began to be cast in a more favorable light with the discovery of angiotensin-converting enzyme-2 (ACE2) in 2000. This monocarboxypeptidase, best known for its ability to metabolize angiotensin (Ang) II to Ang 1-7, counteracts the adverse effects of Ang II mediated by the AT1 Ang II receptor. Ang peptides are classically considered to be metabolized by aminopeptidases, by which the nomenclature Ang III (des-Asp1Ang II, 2-8 heptapeptide) and Ang IV (des-Asp1des-Arg2Ang II, 3-8 hexapeptide) are derived. This report compares the ability of recombinant human ACE2 (rhACE2) to metabolize Ang III, Ang IV and Ang V, (4-8 pentapeptide) relative to Ang II to form corresponding des-omega-Phe metabolites. rhACE2 has highest affinity (lowest Km) for Ang III, followed by Ang II ∼ Ang V, followed by Ang IV. However, rhACE2 has the highest Kcat for metabolising Ang IV followed by Ang V, Ang III and Ang II. The enzymatic efficiency (Kcat/Km) is highest for Ang V and Ang III followed by Ang IV and is lowest for Ang II. As a gluzincin metallopeptidase, ACE2 requires a zinc molecule at its active site for catalysis. This report also documents inhibition of ACE2 activity by concentrations of zinc exceeding 10 µM. These observations extend the functional significance of ACE2 to include the metabolic inactivation of Ang III, Ang IV and Ang V, reemphasizing the importance of monitoring zinc intake to maintain metabolic homeostasis.

Mas Receptor Activation Contributes to the Improvement of Nitric Oxide Bioavailability and Vascular Remodeling During Chronic AT1R (Angiotensin Type-1 Receptor) Blockade in Experimental Hypertension.

Angiotensin (1-7) production increases during AT1R (angiotensin type-1 receptor) blockade. The contribution of Ang (1-7) (angiotensin [1-7]) and its receptor (MasR) to the favorable effect of angiotensin receptor blockers on remodeling and function of resistance arteries remains unclear. We sought to determine whether MasR contributes to the improvement of vascular structure and function during chronic AT1R blockade. Spontaneously hypertensive rats were treated with Ang (1-7) or olmesartan ± MasR antagonist A-779, or vehicle, for 14 days. Blood pressure was measured by tail cuff methodology. Mesenteric arteries were dissected and mounted on a pressurized micromyograph to evaluate media-to-lumen ratio (M/L) and endothelial function. Expression of MasR and eNOS (endothelial nitric oxide synthase) was evaluated by immunoblotting, plasma nitrate by colorimetric assay, and reactive oxygen species production by dihydroethidium staining. Independently of blood pressure, olmesartan significantly reduced M/L and improved NO bioavailability, A-779 prevented these effects. Likewise, Ang (1-7) significantly reduced M/L and NO bioavailability. MasR expression was significantly increased by Ang (1-7) as well as by olmesartan, and it was blunted in the presence of A-779. Both Ang (1-7) and olmesartan increased eNOS expression and plasma nitrite which were reduced by A-779. Superoxide generation was attenuated by olmesartan and Ang (1-7) and was blunted in the presence of A-779. These MasR-mediated actions were independent of AT2R activation since olmesartan and Ang (1-7) increased MasR expression and reduced M/L in Ang II (angiotensin II)-infused AT2R knockout mice, independently of blood pressure control. A-779 prevented these effects. Hence, MasR activation may contribute to the favorable effects of AT1R antagonism on NO bioavailability and microvascular remodeling, independently of AT2R activation and blood pressure control.

Effect of spinal angiotensin-converting enzyme 2 activation on the formalin-induced nociceptive response in mice.

We have previously demonstrated that the phosphorylation of p38 MAPK, through spinal AT1 receptor activation, is involved in formalin-induced nociception and follows accompanied by the increase in spinal angiotensin (Ang) II levels. We have also found that Ang (1-7), an N-terminal fragment of Ang II generated by ACE2, prevents the Ang II-induced nociceptive behavior via spinal MAS1 and the inhibition of p38 MAPK phosphorylation. Here, we examined whether the ACE2 activator diminazene aceturate (DIZE) can prevent the formalin-induced nociception in mice. The i.t. administration of DIZE attenuated the second, but not the first phase of formalin-induced nociceptive response. An increase in the activity of spinal ACE2 was measured following DIZE administration. The inhibitory effect of DIZE on nociception was abolished by the i.t. co-administration of the MAS1 antagonist A779. The i.t. administration of Ang (1-7) showed a similar effect on the second phase of the response which was also attenuated by A779. Furthermore, DIZE and Ang (1-7) each inhibited the formalin-induced phosphorylation of p38 MAPK on the dorsal lumbar spinal cord. This inhibition was again prevented by A779. ACE2 was expressed in neurons and microglia but absent from astrocytes in the superficial dorsal horn. Our data show that the i.t.-administered DIZE attenuates the second phase of the formalin-induced nociception which is accompanied by the inhibition of p38 MAPK phosphorylation. They also suggest the involvement of MAS1 activation on spinal neurons and microglia in response to the increase in Ang (1-7) following ACE2 activation.

Role of Angiotensin-(1-7) via MAS receptor in human sperm motility and acrosome reaction.

Rennin-angiotensin system (RAS) has been involved in sperm function, even so, little is known about the implication of one of the RAS axis formed by Ang-(1-7) (angiotensin-(1-7)) and MAS receptor. Hence, in the present work, we focused on elucidating the function of the MAS receptor in human spermatozoa. We analyzed the expression and localization of MAS receptor in human spermatozoa and we observed if its activation is able to modulate the sperm motility of normal motility and/or asthenozoospermic patients, as well as, the acrosome reaction of the spermatozoa. MAS receptor is present in human mature spermatozoa, not only at the mRNA level but also at protein level. MAS is localized at the acrosome region, as well as, in the tail of spermatozoa. The sperm incubation with MAS agonist Ang-(1-7) activates at dose-dependent manner the PI3K/AKT pathway (P < 0.01 vs control) and improves the motility of asthenozoospermic patients (P < 0.01 vs control), which is blocked by the specific antagonist (A779) (P < 0.01), but it do not modulate the acrosome reaction. These findings suggest that the ACE2/Ang-(1-7)/Mas axis may be a useful biochemical tool for the treatment of male infertility related to sperm mobility.

Fast relaxation and desensitization of angiotensin II contraction in the pulmonary artery via AT1R and Akt-mediated phosphorylation of muscular eNOS.

Angiotensin II (AngII) triggers a transient contraction of pulmonary arteries (PAs) followed by protracted desensitization. Based on the unconventional eNOS expression in PA smooth muscle cells (PASMCs), we hypothesized that activation of smooth muscle eNOS by AngII might be responsible for fast relaxation and tachyphylaxis. Using dual-wire myograph, mechanically endothelium-denuded rat PA [E(-)PA] showed AngII concentration-dependent transient contractions (ΔTAngII, 95% decay within 1 min), which were abolished by losartan (AT1R antagonist). Neither PD123319 (AT2R antagonist) nor A779 (MasR antagonist) affected ΔTAngII. When the vessels were pretreated with L-NAME (NOS inhibitor), ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), ΔTAngII of E(-)PA became larger and sustained, whereas nNOS or iNOS inhibitors had no such effect. Immunoblotting of human PASMCs (hPASMCs) also showed eNOS expression, and AngII treatment induced activating phosphorylations of Ser1177 in eNOS and of Ser473 in Akt (Ser/Thr protein kinase B), an upstream signal of eNOS phosphorylation. In addition, L-NAME co-treatment promoted AngII-induced Ser19 phosphorylation of myosin light chain. In hPASMCs, AngII abolished plasma membrane expression of AT1R, and recovery by washout took more than 1 h. Consistent with the data from hPASMCs, the second application of AngII to E(-)PA did not induce contraction, and significant recovery of ΔTAngII required prolonged washout (> 2 h) in the myography study. L-NAME treatment before the second application facilitated recovery of ΔTAngII. Muscular eNOS plays an auto-inhibitory role in ΔTAngII of PAs. The molecular changes investigated in hPASMCs revealed eNOS phosphorylation and internalization of AT1R by AngII. We propose that the rat PA smooth muscle eNOS-induced lusitropy and slow recovery of AT1R from tachyphylaxis might counterbalance the excessive contractile response to AngII, contributing to the distinctive low-pressure pulmonary circulation.

Ang-(1-7) treatment attenuates lipopolysaccharide-induced early pulmonary fibrosis.

Early pulmonary fibrosis is the leading cause of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, whether the renin-angiotensin system (RAS) can serve as a therapeutic target is unknown. In this study, an animal model of early pulmonary fibrosis was established via the LPS three-hit regimen. Afterwards, the animals were treated with intraperitoneal injections of Ang-(1-7), AVE0991, or A779 once per day for 20 days. The plasma and BALF AngII levels of the animals were increased, while there were no significant changes in Ang-(1-7) levels in lung tissue after LPS treatment. Furthermore, the AT1R protein levels were significantly increased and the Mas levels were significantly decreased on days 14 and 21. Administration of Ang-(1-7) downregulated LPS-induced AT1R mRNA expression, which was upregulated by A779. The expression of Mas mRNA responded in the opposite direction relative to AT1R. Moreover, LPS caused decreased levels of Mas and E-cadherin and increased AT1R, Vimentin, and Src phosphorylation levels. Ang-(1-7) or AVE0991 blocked these effects but was counteracted by A779 treatment. Our findings suggested that AngII and AT1R levels exhibit opposite dynamic trends during LPS-induced early pulmonary fibrosis, as do Ang-(1-7) and Mas. Ang-(1-7) exerts protective effects against early pulmonary fibrosis, mainly by regulating the balance between AngII and AT1R and between Ang-(1-7) and Mas and by inhibiting Src kinase activation.

Decreased behavioural and neurochemical effects of angiotensin IV following prenatal alcohol exposure in the mouse.

Angiotensin IV (ang IV) is known to improve learning and memory in animal models but the mechanism is unclear. We have previously demonstrated sex differences in the pro-cognitive effects of ang IV, and that prenatal alcohol exposure (PAE) abolishes these effects. This study aimed to explore a possible mechanism underlying the sex differences and the effects of PAE in male mice. Mouse breeding harems received 5% ethanol in drinking water throughout pregnancy and lactation in a two-bottle schedule. The effects of ang IV were assessed in offspring at 4 months of age using the open field test, novel object recognition test and elevated plus maze. Aminopeptidase activity of brain insulin-regulated aminopeptidase (IRAP), a putative target of ang IV, was determined. As seen in a previous similar study, ang IV administered immediately after the second training trial significantly improved novel object recognition 24 h later in male mice but not female. PAE abolished this pro-cognitive effect in males. PAE also increased anxiety-like behaviour in male but not female offspring. Ang IV decreased the aminopeptidase activity of brain IRAP in control male, but not female, mice; PAE abolished this inhibitory effect. Ang IV improved memory consolidation in male but not female mice and PAE abolished this effect in the males. While the effects of PAE may be related to increased anxiety; ang IV decreased the aminopeptidase activity in male but not female mice and PAE abolished this inhibitory effect. The results therefore suggest that improvements in learning and memory induced by peripheral administration of ang IV correlate with a reduction of the enzyme activity of IRAP. This is the first demonstration that ang IV administered peripherally can induce long-term (24 h) changes in IRAP function which are probably not simple competitive inhibition and the first demonstration that PAE alters IRAP activity.

Egg White-Derived Antihypertensive Peptide IRW (Ile-Arg-Trp) Reduces Blood Pressure in Spontaneously Hypertensive Rats via the ACE2/Ang (1-7)/Mas Receptor Axis.

SCOPE: It is found in the previous study that egg-white-derived antihypertensive peptide Ile-Arg-Trp (IRW) upregulated angiotensin converting enzyme 2 (ACE2) in spontaneously hypertensive rats (SHRs). The objective of this study is to evaluate the contribution of ACE2 activation by IRW to blood-pressure-lowering activity in vivo. METHODS AND RESULTS: Adult male SHRs (13-15 week old) are assigned into four groups: 1) untreated with saline infusion; 2) IRW administration (15 mg per kg body weight) with saline infusion; 3) Mas receptor (MasR) antagonist A779 (48 µg per kg body weight per h) infusion; 4) A779 infusion and IRW. Animals are implanted with telemetry transmitter first, and then an osmotic pump filled with saline or A779 is implanted. A779/saline is infused for 7 days, continued with an additional 7 days of treatments. Results indicate that blocking MasR abolished the blood-pressure-lowering effect of IRW. Akt/eNOS signaling in aorta is upregulated by IRW treatment but deactivated by A779 infusion. Circulating levels of interleukin 6 and monocyte chemoattractant protein 1, along with cyclooxygenase 2 in aorta are reduced by IRW but restored by A779 infusion. CONCLUSION: IRW reduces blood pressure of SHR via the ACE2/Ang (1-7)/MasR axis. Mechanisms pertaining to IRW as an ACE2 activator in vivo include enhanced endothelium-dependent vasorelaxation and reduced vascular inflammation.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV.

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Anti-hypersensitive effect of angiotensin (1-7) on streptozotocin-induced diabetic neuropathic pain in mice.

BACKGROUND: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain. METHODS: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit. RESULTS: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779. CONCLUSIONS: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation. SIGNIFICANCE: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients.

Anti-inflammatory effects of Ang-(1-7) via TLR4-mediated inhibition of the JNK/FoxO1 pathway in lipopolysaccharide-stimulated RAW264.7 cells.

Targeting inflammation is considered a challenging pharmacological strategy to prevent or delay the development of inflammatory diseases, such as severe asthma, Crohn's disease, and rheumatoid arthritis. The angiotensin-(1-7) -Mas axis ((Ang-(1-7)-Mas axis) was confirmed to antagonize the effects of the Angiotensin II-AT1 receptor axis and the latter is reported to regulate cardiovascular and renal function, as well as contribute to the inflammatory process. In this paper, we aim to explore the crucial effect of Ang-(1-7) in inflammation and disclose the mechanisms in lipopolysaccharide (LPS)-induced murine macrophages RAW264.7. We found that Ang-(1-7) inhibited the production and secretion of tumor necrosis factor-α and interleukin-6 in a concentration-dependent manner in LPS-induced macrophages. The overexpression of TLR4, phospho-JNK, and FoxO1 induced by LPS were also inhibited by incubation with Ang-(1-7). These inhibitory effects were reversed by A-779. Moreover, we also used a selective JNK inhibitor Sp600125 to further corroborate the involvement of TLR4, JNK, and FoxO1 in the anti-inflammatory action of Ang-(1-7). Our research reveals a new mechanism that Ang-(1-7) may drive anti-inflammatory effects via the Mas receptor through inhibition of the TLR4-mediated JNK/FoxO1 signaling pathway in LPS-induced macrophages. Our findings open new perspectives of Ang-(1-7)-Mas axis in local inflammation.

Angiotensin-(1-7) contributes to insulin-sensitizing effects of angiotensin-converting enzyme inhibition in obese mice.

Angiotensin-converting enzyme (ACE) inhibitors reduce body weight, lower blood pressure (BP), and improve insulin sensitivity in animal models of cardiometabolic syndrome. These effects are generally attributed to reduced angiotensin (ANG) II formation; however, these therapies also increase levels of ANG-(1-7), a beneficial hormone opposing ANG II actions. We hypothesized that this ANG-(1-7) generation contributes to the insulin-sensitizing effects of ACE inhibition in obese mice. Adult male C57BL/6J mice were placed on a 60% high-fat diet for 11 wk. During the last 3 wk of diet, mice received normal water or water containing the ACE inhibitor captopril (50 mg/l) as well as the ANG-(1-7) mas receptor antagonist A779 (400 or 800 ng·kg-1·min-1) or saline vehicle via subcutaneous osmotic minipumps. At the end of treatment, arterial BP was measured, and hyperinsulinemic-euglycemic clamps were performed in conscious obese mice receiving vehicle, captopril, captopril plus A779, or A779 ( n = 6-13/group). Captopril reduced body weight (28 ± 2 vs. 41 ± 2 g saline; P = 0.001), lowered systolic BP (109 ± 6 vs. 144 ± 7 mmHg saline; P = 0.041), and improved whole-body insulin sensitivity (steady-state glucose infusion rate: 31 ± 4 vs. 16 ± 2 mg·kg-1·min-1 saline; P = 0.001) in obese mice. A779 attenuated captopril-mediated improvements in insulin sensitivity (23 ± 2 mg·kg-1·min-1; P = 0.042), with no effect on body weight (32 ± 2 g; P = 0.441) or BP (111 ± 7 mmHg; P = 0.788). There was no effect of A779 alone on cardiometabolic outcomes. These data suggest that insulin-sensitizing effects of ACE inhibition are in part due to activation of ANG-(1-7)/ mas receptor pathways and provide new insight into mechanisms underlying the positive metabolic effects of these therapies.

Involvement of angiotensin-(1-7) in the neuroprotection of captopril against focal cerebral ischemia.

Accumulating evidence suggests that brain angiotensin-converting enzyme (ACE)/angiotensin II/angiotensin II type I receptor axis is activated and thus contributes to the neuronal injury during ischemic stroke. Conversely, inhibition of this axis using centrally active ACE inhibitor captopril was proven neuroprotective in rodents with focal cerebral ischemia. Interestingly, captopril was able to increase angiotensin-(1-7) [Ang-(1-7)] levels in the peripheral organs. As the main component of the alternative renin-angiotensin system axis in the brain, Ang-(1-7) was revealed to protect against focal cerebral ischemia via a MAS1 receptor-dependent manner. Based on this evidence, we hypothesized that Ang-(1-7) might contribute to the neuroprotection of captopril during ischemic stroke. In this study, we evaluated this hypothesis using a rat model of focal cerebral ischemia. We revealed that brain ACE2 activity and Ang-(1-7) levels were significantly elevated following captopril treatment in rats with focal cerebral ischemia. More importantly, we showed that the neuroprotection provided by captopril was partially reversed by A-779, an antagonist for Ang-(1-7) receptor MAS1, indicating that Ang-(1-7) was involved in the neuroprotection of captopril. These findings have uncovered new mechanisms by which captopril protects against focal cerebral ischemia and further suggest that captopril may have practical clinical use for stroke prevention and treatment in addition to its antihypertensive effect.

Lipoxin A4 attenuates LPS-induced acute lung injury via activation of the ACE2-Ang-(1-7)-Mas axis.

Previous studies have reported that lipoxin A4 (LXA4) and the angiotensin I-converting enzyme 2 (ACE2), angiotensin-(1-7) [Ang-(1-7)], and its receptor Mas [ACE2-Ang-(1-7)-Mas] axis play important protective roles in acute lung injury (ALI). However, there is still no direct evidence of LXA4-mediated protection via the ACE2-Ang-(1-7)-Mas axis during ALI. This work was performed using an LPS-induced ALI mouse model and the data indicated the following. First, the animal model was established successfully and LXA4 ameliorated LPS-induced ALI. Second, LXA4 could increase the concentration and activity of ACE2 and the levels of Ang-(1-7) and Mas markedly. Third, LXA4 decreased the levels of TNF-α, IL-1ß, and reactive oxygen species while increasing IL-10 levels. Fourth, LXA4 inhibited the activation of the NF-κB signal pathway and repressed the degradation of inhibitor of NF-κB, the phosphorylation of NF-κB, and the translocation of NF-κB. Finally, and more importantly, BOC-2 (LXA4 receptor inhibitor), MLN-4760 (ACE2 inhibitor), and A779 (Mas receptor antagonist) were found to reverse all of the effects of LXA4. Our data provide evidence that LXA4 protects the lung from ALI through regulation of the ACE2-Ang-(1-7)-Mas axis.