Cardio-protective effects of angiotensin-(1-5) via mas receptor in rats against ischemic-perfusion injury.

Angiotensin-(1-5) [Ang-(1-5)], which is a metabolite of Ang-(1-7) catalyzed by angiotensin-converting enzyme, is a novel pentapeptide of the renin-angiotensin system. Ang-(1-7), Ang III and Ang IV have a cardio-protective effect via Mas receptor, Ang II type 2 receptor (AT2R) and AT4R, respectively. However, it is not clear whether Ang-(1-5) has cardio-protective effects. The aim of this study is to investigate whether Ang-(1-5) protects the heart against ischemia-reperfusion (I/R) injury. After sacrificing Sprague-Dawley rats, the hearts were perfused with Krebs-Henseleit buffer for a 20 min pre-ischemic period with and without Ang-(1-5) followed by 20 min global ischemia and 50 min reperfusion. Ang-(1-5) (1 µM) improved changes in post-ischemic left ventricular developed pressure (LVDP), ±dP/dt, and post-ischemic left ventricular end-diastolic pressure (LVEDP) induced by reperfusion compared to control hearts. Ang-(1-5) decreased myocardial infarct size and LDH activity, and increased coronary flow and the amount of atrial natriuretic peptide (ANP) in coronary effluent during reperfusion compared to control hearts. Pretreatment with Mas receptor antagonist but not with AT1R or AT2R antagonist attenuated the improvement of changes in I/R-induced ventricular hemodynamics by Ang-(1-5). Ang-(1-5) treatment decreased Bax, caspase-3 and caspase-9 protein levels, and increased Bcl-2 protein level, which were attenuated by A779 pretreatment. Ang-(1-5) treatment increased Mn-superoxide dismutase, catalase, and heme oxygenase-1 protein levels, which was attenuated by A779 pretreatment. These results suggest that the cardio-protective effects of Ang-(1-5) against I/R injury may be partly related to activating anti-oxidant and anti-apoptotic enzymes via Mas receptor.

Similarity and dissimilarity between angiotensin A and angiotensin II in cardiovascular functions in a rat model.

Angiotensin (Ang) A differs from Ang II in a single N-terminal alanine residue. The aim of this study was to investigate whether the effects of Ang A on postischemic cardiac injury and hemodynamics differ from Ang II. After euthanizing Sprague-Dawley rats, hearts were perfused with Krebs-Henseleit buffer for a 20 min preischemic period with or without Ang A or Ang II, followed by 20 min global ischemia and 50 min reperfusion. The blood pressure was measured in anesthetized rats. Ang A (0.1, 1.0, 10 µg/kg) deteriorated the postischemic left ventricular hemodynamics in a dose-dependent manner, which was similar to that by Ang II. Ang A (10 µg/kg) increased the infarct size and the lactate dehydrogenase level, and decreased the coronary flow, which were attenuated by the pretreatment with Ang type 1 receptor (AT1R) antagonist (losartan) but not by AT2R antagonist (PD123319). Ang A increased the expression of apoptotic proteins and decreased the expression of antioxidative proteins. Interestingly, Ang A increased the atrial natriuretic peptide (ANP) level in coronary effluent and in atrial perfusate but Ang II did not increase it. Ang A increased mean arterial blood pressure, which was less potent than Ang II. These results suggest that Ang A has a similar effect on postischemic injury via AT1R and less potent vasopressor effect but opposite effect on ANP secretion as compared to Ang II.

Hypoxia augments NaHS-induced ANP secretion via KATP channel, HIF-1α and PPAR-γ pathway.

It has been reported that sodium hydrosulfide (NaHS) stimulated high stretch induced-atrial natriuretic peptide (ANP) secretion via ATP sensitive potassium (KATP) channel. KATP channel is activated during hypoxic condition as a compensatory mechanism. However, whether NaHS affects ANP secretion during hypoxia remains obscure. The purpose of the present study is to discover the impact of NaHS on ANP secretion during hypoxia and to unravel its signaling pathway. Isolated beating rat atria were perfused with buffer exposed to different O2 tension (to 100% O2, normoxia; to 20% O2, hypoxia). The ANP secretion increased negatively correlated with O2 tension. NaHS (50 µM) did not show any significant effect on low stretch induced-ANP secretion in normoxic condition but augmented low stretch induced-ANP secretion in hypoxic condition. The augmentation of NaHS-induced ANP secretion during hypoxia was blocked by the pretreatment with KATP channel blocker (glibenclamide) and was enhanced by the pretreatment with KATP channel activator (pinacidil). Hypoxia increased the expression of PPAR-γ protein but did not change the expression of HIF-1α protein and eNOS phosphorylation. The NaHS-induced ANP secretion during hypoxia was also blocked by the pretreatment with HIF-1α inhibitor (2-methoxy- estradiol), PPAR-γ inhibitor (GW9662) but not by NOS inhibitor (L-NAME) and endothelin receptor inhibitor (bosentan). The intravenous infusion of NaHS increased plasma ANP level in monocrotaline-treated rats but not in sham rats. These results suggest that hypoxia augmented NaHS-induced ANP secretion partly through KATP channel, HIF-1α, and PPAR-γ pathway.

Hydrogen sulfide donor, NaHS, stimulates ANP secretion via the KATP channel and the NOS/sGC pathway in rat atria.

Hydrogen sulfide (H2S) is normally produced from l-cysteine in mammalian tissues and related to the pathogenesis of cardiovascular diseases. The aim of this study is to investigate the effects of H2S donor on atrial natriuretic peptide (ANP) secretion and define its mechanism using normal and isoproterenol (ISP)-treated rats. Several H2S donors were perfused into isolated beating rat atria, and atrial pressure (AP) and ANP secretion were measured. NaHS augmented high stretch-induced ANP secretion and decreased AP in a dose-dependent manner. The high stretch-induced ANP secretion was stimulated by Na2S but was not changed by GYY4137 and sodium thiosulfate. NaHS and Na2S produced very high amount of H2S rapidly whereas GYY4137 produced very low amount of H2S slowly. NaHS-stimulated ANP secretion was blocked by the pretreatment with inhibitor for KATP channel, nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), phosphoinositol 3 kinase (PI3K) or protein kinase B. H2S synthesis enzyme inhibitor (DL-propargylglycine) did not show any significant changes in atrial parameters. However, the response of ANP secretion to NaHS markedly attenuated and DL-propargylglycine suppressed ANP secretion in ISP-treated rat atria. The expression of eNOS protein was decreased but the expression of cardiomyocyte-specific H2S producing enzyme, cystathione γ-lyase, was not changed in ISP-treated rat atria. The attenuation of NaHS-induced ANP secretion in ISP-treated rat atria may be due to the low expression of eNOS protein. These findings clarify that NaHS stimulates ANP secretion via the KATP channel and the PI3K/Akt/NOS/sGC pathway in rat atria.

Angiotensin-(1-9) ameliorates pulmonary arterial hypertension via angiotensin type II receptor.

Angiotensin-(1-9) [Ang-(1-9)], generated from Ang I by Ang II converting enzyme 2, has been reported to have protective effects on cardiac and vascular remodeling. However, there is no report about the effect of Ang-(1-9) on pulmonary hypertension. The aim of the present study is to investigate whether Ang-(1-9) improves pulmonary vascular remodeling in monocrotaline (MCT)-induced pulmonary hypertensive rats. Sprague-Dawley rats received Ang-(1-9) (576 µg/kg/day) or saline via osmotic mini-pumps for 3 weeks. Three days after implantation of osmotic mini-pumps, 50 mg/kg MCT or vehicle were subcutaneously injected. MCT caused increases in right ventricular weight and systolic pressure, which were reduced by co-administration of Ang-(1-9). Ang-(1-9) also attenuated endothelial damage and medial hypertrophy of pulmonary arterioles as well as pulmonary fibrosis induced by MCT. The protective effects of Ang-(1-9) against pulmonary hypertension were inhibited by Ang type 2 receptor (AT2R) blocker, but not by Mas receptor blocker. Additionally, the levels of LDH and inflammatory cytokines, such as TNF-α, MCP-1, IL-1ß, and IL-6, in plasma were lower in Ang-(1-9) co-treated MCT group than in vehicle-treated MCT group. Changes in expressions of apoptosis-related proteins such as Bax, Bcl-2, Caspase-3 and -9 in the lung tissue of MCT rats were attenuated by the treatment with Ang-(1-9). These results indicate that Ang-(1-9) improves MCT-induced pulmonary hypertension by decreasing apoptosis and inflammatory reaction via AT2R.

Alamandine Protects the Heart Against Reperfusion Injury via the MrgD Receptor.

BACKGROUND: Alamandine differs from angiotensin-(1-7) in a single N-terminal alanine residue. The aim of this study was to investigate whether alamandine protects the heart against reperfusion injury. Methods and Results: After euthanizing Sprague-Dawley rats, hearts were perfused with Krebs-Henseleit buffer for a 20-min pre-ischemic period with or without alamandine, followed by 20 min global ischemia and 50 min reperfusion. Alamandine (0.1 mg/kg) improved the postischemic left ventricular developed pressure and ±dP/dt, decreased the infarct size, and decreased the lactate dehydrogenase levels in the effluent. Alamandine increased the coronary flow and the amount of atrial natriuretic peptide (ANP) in the coronary effluent, and it decreased the expression of apoptotic proteins and increased the expression of antioxidative proteins. Pretreatment with the MrgD receptor antagonist or PD123319, but not the angiotensin type 1 receptor antagonist, attenuated the cardioprotective effects of alamandine. A similar cardioprotective effect with alamandine was also observed with high plasma ANP levels in an in vivo study. Alamandine directly stimulated ANP secretion from isolated atria, which was completely blocked by pretreatment with the MrgD receptor antagonist and was partially blocked by PD123319. CONCLUSIONS: These results suggest that the cardioprotective effects of alamandine against I/R injury are, in part, related to the activation of antioxidant and antiapoptotic enzymes via the MrgD receptor.

Modification of levosimendan-induced suppression of atrial natriuretic peptide secretion in hypertrophied rat atria.

This study aimed to determine the effects of levosimendan, a calcium sensitizer, on atrial contractility and atrial natriuretic peptide (ANP) secretion and its modification in hypertrophied atria. Isolated perfused beating rat atria were used from control and isoproterenol-treated rats. Levosimendan and its metabolite OR-1896 caused a positive inotropic effect and suppressed ANP secretion in rat atria. Similar to levosimendan, the selective phosphodiesterase 3 (PDE3) or PDE4 inhibitor also suppressed ANP secretion. Suppression of ANP secretion by 1 µM levosimendan was abolished by PDE3 inhibitor, but reversed by PDE4 inhibitor. Levosimendan-induced suppression of ANP secretion was potentiated by KATP channel blocker, but blocked by KATP channel opener. Levosimendan alone did not significantly change cyclic adenosine monophosphate (cAMP) efflux in the perfusate; however, levosimendan combined with PDE4 inhibitor markedly increased this efflux. The stimulation of ANP secretion induced by levosimendan combined with PDE4 inhibitor was blocked by the protein kinase A (PKA) inhibitor. In isoproterenol-treated atria, levosimendan augmented the positive inotropic effect and ANP secretion in response to an increased extracellular calcium concentration ([Ca+]o). These results suggests that levosimendan suppresses ANP secretion by both inhibiting PDE3 and opening KATP channels and that levosimendan combined with PDE4 inhibitor stimulates ANP secretion by activating the cAMP-PKA pathway. Modification of the effects of levosimendan on [Ca+]o-induced positive inotropic effects and ANP secretion in isoproterenol-treated rat atria might be related to a disturbance in calcium metabolism.

Comparative effects of angiotensin II and angiotensin-(4-8) on blood pressure and ANP secretion in rats.

Angiotensin II (Ang II) is metabolized from N-terminal by aminopeptidases and from C-terminal by Ang converting enzyme (ACE) to generate several truncated angiotensin peptides (Angs). The truncated Angs have different biological effects but it remains unknown whether Ang-(4-8) is an active peptide. The present study was to investigate the effects of Ang-(4-8) on hemodynamics and atrial natriuretic peptide (ANP) secretion using isolated beating rat atria. Atrial stretch caused increases in atrial contractility by 60% and in ANP secretion by 70%. Ang-(4-8) (0.01, 0.1, and 1 µM) suppressed high stretch-induced ANP secretion in a dose-dependent manner. Ang-(4-8) (0.1 µM)-induced suppression of ANP secretion was attenuated by the pretreatment with an antagonist of Ang type 1 receptor (AT1R) but not by an antagonist of AT2R or AT4R. Ang-(4-8)-induced suppression of ANP secretion was attenuated by the pretreatment with inhibitor of phospholipase (PLC), inositol triphosphate (IP3) receptor, or nonspecific protein kinase C (PKC). The potency of Ang-(4-8) to inhibit ANP secretion was similar to Ang II. However, Ang-(4-8) 10 µM caused an increased mean arterial pressure which was similar to that by 1 nM Ang II. Therefore, we suggest that Ang-(4-8) suppresses high stretch-induced ANP secretion through the AT1R and PLC/IP3/PKC pathway. Ang-(4-8) is a biologically active peptide which functions as an inhibition mechanism of ANP secretion and an increment of blood pressure.

Angiotensin-(1-5), an active mediator of renin-angiotensin system, stimulates ANP secretion via Mas receptor.

Angiotensin-(1-5) [Ang-(1-5)], which is a metabolite of Angiotensin-(1-7) [Ang-(1-7)] catalyzed by angiotensin-converting enzyme (ACE), is a pentapeptide of the renin-angiotensin system (RAS). It has been reported that Ang-(1-7) and Ang-(1-9) stimulate the secretion of atrial natriuretic peptide (ANP) via Mas receptor (Mas R) and Ang II type 2 receptor (AT2R), respectively. However, it still remains unknown whether Ang-(1-5) has a similar function to Ang-(1-7). We investigated the effect of Ang-(1-5) on ANP secretion and to define its signaling pathway using isolated perfused beating rat atria. Ang-(1-5) (0.3, 3, 10µM) stimulated high pacing frequency-induced ANP secretion in a dose-dependent manner. Ang-(1-5)-induced ANP secretion (3µM) was attenuated by the pretreatment with an antagonist of Mas R (A-779) but not by an antagonist of AT1R (losartan) or AT2R (PD123,319). An inhibitor for phosphatidylinositol 3-kinase (PI3K; wortmannin), protein kinase B (Akt; API-2), or nitric oxide synthase (NOS; L-NAME) also attenuated the augmentation of ANP secretion induced by Ang-(1-5). Ang-(1-5)-induced ANP secretion was markedly attenuated in isoproterenol-treated hypertrophied atria. The secretagogue effect of Ang-(1-5) on ANP secretion was similar to those induced by Ang-(1-9) and Ang-(1-7). These results suggest that Ang-(1-5) is an active mediator of renin-angiotensin system to stimulate ANP secretion via Mas R and PI3K-Akt-NOS pathway.

Effects of endothelin family on ANP secretion.

The endothelins (ET) peptide family consists of ET-1, ET-2, ET-3, and sarafotoxin (s6C, a snake venom) and their actions appears to be different among isoforms. The aim of this study was to compare the secretagogue effect of ET-1 on atrial natriuretic peptide (ANP) secretion with ET-3 and evaluate its physiological meaning. Isolated nonbeating atria from male Sprague-Dawley rats were used to evaluate stretch-activated ANP secretion in response to ET-1, ET-2, ET-3, and s6C. Changes in mean blood pressure (MAP) were measured during acute injection of ET-1 and ET-3 with and without natriuretic peptide receptor-A antagonist (A71915) in anesthetized rats. Changes in atrial volume induced by increased atrial pressure from o to 1, 2, 4, or 6cm H2O caused proportional increases in mechanically-stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. ET-1 (10nM) augmented basal and stretch-activated ANP secretion in terms of ECF translocation, which was blocked by the pretreatment with ETA receptor antagonist (BQ123, 1µM) but not by ETB receptor antagonist (BQ788, 1µM). ETA receptor antagonist itself suppressed stretch-activated ANP secretion. As compared to ET-1- induced ANP secretion (3.2-fold by 10nM), the secretagogue effects of ANP secretion by ET-2 was similar (2.8-fold by 10nM) and ET-3 and s6C were less potent (1.7-fold and 1.5-fold by 100nM, respectively). Acute injection of ET-1 or ET-3 increased mean blood pressure (MAP), which was augmented in the presence of natriuretic peptide receptor-A antagonist. Therefore, we suggest that the order of secretagogue effect of ET family on ANP secretion was ET-1≥ET-2>>ET-3>s6C and ET-1-induced ANP secretion negatively regulates the pressor effect of ET-1.

Angiotensin IV protects cardiac reperfusion injury by inhibiting apoptosis and inflammation via AT4R in rats.

Angiotensin IV (Ang IV) is formed by aminopeptidase N from Ang III by removing the first N-terminal amino acid. Previously, we reported that Ang III has some cardioprotective effects against global ischemia in Langendorff heart. However, it is not clear whether Ang IV has cardioprotective effects. The aim of the present study was to evaluate the effect of Ang IV on myocardial ischemia-reperfusion (I/R) injury in rats. Before ischemia, male Sprague-Dawley rats received Ang IV (1mg/kg/day) for 3 days. Anesthetized rats were subjected to 45min of ischemia by ligation of left anterior descending coronary artery followed by reperfusion and then, sacrificed 1 day or 1 week after reperfusion. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, and infarct size were measured. Quantitative analysis of apoptotic and inflammatory proteins in ventricles were performed using Western blotting. Pretreatment with Ang IV attenuated I/R-induced increases in plasma CK and LDH levels, and infarct size, which were blunted by Ang IV receptor (AT4R) antagonist and but not by antagonist for AT1R, AT2R, or Mas receptor. I/R increased Bax, caspase-3 and caspase-9 protein levels, and decreased Bcl-2 protein level in ventricles, which were blunted by Ang IV. I/R-induced increases in TNF-α, MMP-9, and VCAM-1 protein levels in ventricles were also blunted by Ang IV. Ang IV increased the phosphorylation of Akt and mTOR. These effects were attenuated by co-treatment with AT4R antagonist or inhibitors of downstream signaling pathway. Myocardial dysfunction after reperfusion was improved by Ang IV. These results suggest that Ang IV has cardioprotective effect against I/R injury by inhibiting apoptosis via AT4R and PI3K-Akt-mTOR pathway.

Regional heterogeneity of expression of renal NPRs, TonEBP, and AQP-2 mRNAs in rats with acute kidney injury.

To understand the pathophysiology of ischemia/reperfusion (I/R) - induced acute kidney injury (AKI), the present study defined changes in renal function, plasma renotropic hormones and its receptors in the kidney 2, 5, or 7 days after 45 min-renal ischemia in rats. Blood urea nitrogen, plasma creatinine, and osmolarity increased 2 days after I/R injury and tended to return to control level 7 days after I/R injury. Decreased renal function tended to return to control level 5 days after I/R injury. However, plasma concentrations of atrial natriuretic peptide and renin did not change. In control kidney, natriuretic peptide receptor (NPR)-A, -B and -C mRNAs were highly expressed in medulla (ME), inner cortex (IC), and outer cortex (OC), respectively, and tonicity-responsive enhancer binding protein (TonEBP), auqaporin-2 (AQP-2) and eNOS mRNAs were highly expressed in ME. NPR-A and -B mRNA expressions were markedly decreased 2 days after I/R injury. On 5 days after I/R injury, NPR-A mRNA expression increased in OC and recovered to control level in IC but not in ME. NPR-B mRNA expression was increased in OC, and recovered to control level in IC and ME. NPR-C mRNA expression was markedly decreased in OC 2 and 5 days after I/R injury. TonEBP, APQ-2 and eNOS mRNA expressions were markedly decreased 2 days after I/R injury and did not recover in ME 7 days after I/R injury. Therefore, we suggest that there is a regional heterogeneity of regulation of renal NPRs, TonEBP, and APQ-2 mRNA in AKI.

Attenuation of renovascular hypertension by cyclooxygenase-2 inhibitor partly through ANP release.

Angiotensin II (Ang II) is an important inflammatory mediator. Ang II induces cyclooxygenase-2 (COX-2) expression and prostaglandin F2α release followed by cardiac hypertrophy. Inhibition of COX-2 may modulate high blood pressure but controversy still exists. The aim of this study was to determine the role of COX-2 in the regulation of blood pressure and to define the mechanisms in two kidney one-clip hypertensive (2K1C) rats. Chronic treatment with nimesulide or NS-398 (5 mg/kg/day) for 3 weeks lowered high blood pressure and cardiac hypertrophy with decreased expression levels of cardiac hypertrophy markers [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP)], Ang type 1 receptor, urotensin II, and urotensin II receptor in 2K1C rats. Plasma level of ANP was markedly increased and plasma levels of Ang II and aldosterone were decreased by treatment with nimesulide or NS-398. In both in vitro and in vivo experiments, nimesulide or NS-398 augmented ANP release in 2K1C rats. The inhibitory effect of NS-398 on blood pressure was attenuated by the pretreatment with natriuretic peptide receptor-A (NPR-A) antagonist (A71915, 30 µg/kg/day). These results suggest that chronic treatment with nimesulide or NS-398 attenuated hypertension and cardiac hypertrophy partly through ANP release in 2K1C rats.

Angiotensin IV stimulates high atrial stretch-induced ANP secretion via insulin regulated aminopeptidase.

Angiotensin IV (Ang IV) is formed by aminopeptidase N (APN) from angiotensin III (Ang III) by removing the first N-terminal amino acid. Previously, we reported that angiotensin II (Ang II) inhibits atrial natriuretic peptide (ANP) secretion via angiotensin II type 1 receptor (AT1R). In contrast, angiotensin-(1-7) [Ang-(1-7)] and Ang III stimulate ANP secretion via Mas receptor (Mas R) and angiotensin II type 2 receptor (AT2R), respectively. However, it is not known whether there is any relationship between Ang IV and ANP secretion. Therefore, the aim of the present study was to determine the effect of Ang IV on ANP secretion and to find its downstream signaling pathway using in isolated perfused beating atria. Ang IV (0.1, 1 and 10µM) stimulated high atrial stretch-induced ANP secretion and ANP concentration in a dose-dependent manner. The augmented effect of Ang IV (1µM) on high atrial stretch-induced ANP secretion and concentration was attenuated by pretreatment with insulin-regulated aminopeptidase (IRAP) antagonist but not by AT1R or AT2R antagonist. Pretreatment with inhibitors of downstream signaling pathway including phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) blocked Ang IV-induced ANP secretion and concentration. Therefore, these results suggest that Ang IV stimulates ANP secretion and concentration via IRAP and PI3K-Akt-mTOR pathway.

Comparision of secretagogue effects of rosiglitazone and telmisartan on ANP secretion in rats.

Peroxisome proliferator-activated receptor-gamma (PPAR-γ), a nuclear transcription factor, is a key regulator of insulin signaling, and glucose and fat metabolism. In this study, we evaluated the direct effect of PPAR-γ ligand on the secretion of atrial natriuretic peptide (ANP). The isolated perfused beating atria were used and rosiglitazone (0.01, 0.3 and 1 µM) or telmisartan was perfused into atria with and without inhibitors. High frequency stimulation caused a decreased atrial contractility by 40% and an increased ANP secretion by 80%. Rosiglitazone augmented high frequency-induced ANP secretion and concentration in a dose-dependent manner. Rosiglitazone-induced ANP secretion was attenuated by the pretreatment with PPAR-γ antagonist (GW 9662), or inhibitor for phosphoinositol 3-kinase (PI3-kinase, wortmannin), Akt (API-2) or nitric oxide synthase (l-NAME). Telmisartan, a partial agonist of PPAR-γ with angiotensin II type 1 receptor (AT1R) blocker, also stimulated ANP secretion, which was more potent than rosiglitazone or losartan. Infusion of rosiglitazone or telmisartan in anesthetized rats tended to decrease mean arterial pressure and to increase pulse pressure without difference. A plasma ANP level was increased by telmisartan more than by rosiglitazone. In diabetic rats, an increased plasma ANP level was more prominent than sham rats. Therefore, we suggest that rosiglitazone stimulates high frequency-induced ANP secretion through the PPAR-γ receptor-PI3-kinase-Akt-eNOS pathway and telmisartan shows synergistic effect on ANP secretion.

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor.

AIMS: Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion. MAIN METHODS: We examined the effects of Ang-(1-9) on ANP secretion and atrial contractility with and without inhibitors in isolated perfused atria. KEY FINDINGS: Ang-(1-9) stimulated ANP secretion and concentration without change in atrial contractility. Ang-(1-9)-induced-ANP secretion was increased from 5% to 60% by 3 µM Ang-(1-9) during the low-stretch state of the atrium. This stimulatory effect of Ang-(1-9) on ANP secretion was attenuated by pretreatment with an Ang II type 2 receptor (AT2R) antagonist but not by AT1R or Mas receptor antagonist. In addition, pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) blocked Ang-(1-9)-induced ANP secretion. In the high-stretch atrial state, Ang-(1-9)-induced ANP secretion was increased more than in the low-stretch state following addition of 1 µM Ang-(1-9) (from 108% to 170%). In an in vivo experiment, acute infusion of Ang-(1-9) increased plasma ANP level without altering arterial blood pressure. This effect was attenuated by pretreatment with AT2R antagonist but not by Mas receptor antagonist. SIGNIFICANCE: These results suggest that Ang-(1-9) stimulates ANP secretion via the AT2R-PI3K-Akt-NO-cGMP pathway.

Angiotensin AT2 receptor agonist stimulates high stretch induced- ANP secretion via PI3K/NO/sGC/PKG/pathway.

Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the major cardiovascular effects of Ang II. However, the effects mediated via AT2R are still controversial. The aim of the present study is to define the effect of AT2R agonist CGP42112A (CGP) on high stretch-induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP (0.01, 0.1 and 1µM) stimulated high stretch-induced ANP secretion and concentration from isolated perfused rat atria. However, atrial contractility and the translocation of extracellular fluid did not change. The augmented effect of CGP (0.1µM) on high stretch-induced ANP secretion was attenuated by the pretreatment with AT2R antagonist or inhibitor for phosphoinositol 3-kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However, antagonist for AT1R or Mas receptor did not influence CGP-induced ANP secretion. In vivo study, acute infusion of CGP for 10min increased plasma ANP level without blood pressure change. In renal hypertensive rat atria, AT2R mRNA and protein levels were up-regulated and the response of plasma ANP level to CGP infusion in renal hypertensive rats augmented. The pretreatment with AT2R antagonist for 10min followed by CGP infusion attenuated an increased plasma ANP level induced by CGP. However, pretreatment with AT1R or Mas receptor antagonist unaffected CGP-induced increase in plasma ANP level. Therefore, we suggest that AT2R agonist CGP stimulates high stretch-induced ANP secretion through PI3K/NO/sGC/PKG pathway and these effects are augmented in renal hypertensive rats.

Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart.

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor.

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH2O to 7.5 cmH2O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 µM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT2) receptor antagonist but not by AT1 or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81±10.19% but Ang II decreased plasma ANP level by 30.41±7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT2 receptor/PI3K/Akt/nitric oxide/PKG pathway.

Cardioprotective effects of angiotensin III against ischemic injury via the AT2 receptor and KATP channels.

Angiotensin III (Ang III) has similar effects on blood pressure and aldosterone secretion as Ang II, but cardioprotective effects are also proposed. In this study, we investigated whether Ang III protects the heart against ischemia/reperfusion (I/R) injury. After sacrificing Sprague-Dawley rats, the hearts were perfused with Krebs-Henseleit buffer for a 20 min preischemic period with and without Ang III followed by 20-min global ischemia and 50-min reperfusion. Pretreatment with Ang III (1 µmol/L) improved an increased postischemic left ventricular end-diastolic pressure (LVEDP) and a decreased postischemic left ventricular developed pressure (LVDP) induced by reperfusion compared to untreated hearts. Ang III markedly decreased infarct size and lactate dehydrogenase levels in effluent during reperfusion. Ang III increased coronary flow and the concentrations of atrial natriuretic peptide in coronary effluent during reperfusion. Pretreatment with Ang II type 2 receptor (AT2R) antagonist or ATP-sensitive K(+) channel (KATP) blocker for 15 min before ischemia attenuated the improvement of LVEDP, LVDP, and ±dP/dt induced by Ang III. Ang III treatment increased Mn-superoxide dismutase, catalase, and heme oxygenase-1 protein levels, which was attenuated by pretreatment with AT2R antagonist or KATP blocker. Ang III treatment also decreased Bax, caspase-3, and caspase-9 protein levels, and increased Bcl-2 protein level, which were attenuated by pretreatment with AT2R antagonist or KATP blocker. These results suggest that the cardioprotective effects of Ang III against I/R injury may be partly related to activating antioxidant and antiapoptotic enzymes via AT2R and KATP channels.