Angiotensin-(1–7) stimulates high atrial pacing-induced ANP secretion via Mas/PI3-kinase/Akt axis and Na+/H+ exchanger

Amin Shah, Rukhsana Gul, Kuichang Yuan, Shan Gao, Young-Bin Oh, Uh-Hyun Kim, Suhn Hee Kim

Abstract

Angiotensin-(1–7) [ANG-(1–7)], one of the bioactive peptides produced in the renin-angiotensin system, plays a pivotal role in cardiovascular physiology by providing a counterbalance to the function of ANG II. Recently, it has been considered as a potential candidate for therapeutic use in the treatment of various types of cardiovascular diseases. The aim of the present study is to explain the modulatory role of ANG-(1–7) in atrial natriuretic peptide (ANP) secretion and investigate the functional relationship between two peptides to induce cardiovascular effects using isolated perfused beating rat atria and a cardiac hypertrophied rat model. ANG-(1–7) (0.01, 0.1, and 1 μM) increased ANP secretion and ANP concentration in a dose-dependent manner at high atrial pacing (6.0 Hz) with increased cGMP production. However, at low atrial pacing (1.2 Hz), ANG-(1–7) did not cause changes in atrial parameters. Pretreatment with an antagonist of the Mas receptor or with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), or nitric oxide synthase blocked the augmentation of high atrial pacing-induced ANP secretion by ANG-(1–7). A similar result was observed with the inhibition of the Na+/H+ exchanger-1 and Ca2+/calmodulin-dependent kinase II (CaMKII). ANG-(1–7) did not show basal intracellular Ca2+ signaling in quiescent atrial myocytes. In an in vivo study using an isoproterenol-induced cardiac hypertrophy animal model, an acute infusion of ANG-(1–7) increased the plasma concentration of ANP by twofold without changes in blood pressure and heart rate. A chronic administration of ANG-(1–7) increased the plasma ANP level and attenuated isoproterenol-induced cardiac hypertrophy. The antihypertrophic effect was abrogated by a cotreatment with the natriuretic peptide receptor-A antagonist. These results suggest that 1) ANG-(1–7) increased ANP secretion at high atrial pacing via the Mas/PI3K/Akt pathway and the activation of Na+/H+ exchanger-1 and CaMKII and 2) ANG-(1–7) decreased cardiac hypertrophy which might be mediated by ANP.

  • atrial natriuretic peptide
  • cardiac hypertrophy
  • atrial myocyte
  • Mas
  • signal transduction
  • phosphatidylinositol 3-kinase

angiotensin-(1–7) [ANG-(1–7)] is an active heptapeptide formed from ANG I and/or ANG II. It displays a vasodilatory action via the release of nitric oxide (NO) (38) and prostaglandins (1) or by the amplification of bradykinin action (37). In addition to its potent natriuretic (9) and antifibrotic effects (22), it also inhibits the growth of vascular smooth muscle cells (16) and cardiomyocytes (51). ANG II, another classical peptide of the renin-angiotensin system (RAS), causes vasoconstriction, hypertrophy, fibrosis, and proliferation, which are deleterious to the cardiovascular system. Beneficial cardiovascular functions of ANG-(1–7) are exerted primarily through actions that are antagonistic to ANG II. As recently demonstrated, it acts through the G protein-coupled receptor Mas (43), leading to the downstream activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial NO synthase (eNOS) signaling pathway in endothelial cells (41) and cardiomyocytes (10).

On the other hand, atrial natriuretic peptide (ANP), a cardiac peptide hormone released mainly from the atrium, also plays a crucial role in the cardiovascular system. It activates the guanylyl cyclase-A receptor, which produces a second messenger cGMP from GTP in a variety of tissues, and thereby causing natriuresis (11), hypotension (33), hypovolemia (27), and antiproliferation of vascular smooth muscle cells (21). An extensive study of the mechanisms of ANP secretion has determined that the most important factors in the stimulation of ANP secretion are atrial volume change (28) and endothelin-1 (30). Pacing is also a potent physiological stimulus in the ANP production in cardiac myocytes (46). An increased ANP secretion and gene expression during exercise (15) and cardiac hypertrophy have been reported (34). Therefore, an increased ANP secretion is important for compensatory mechanisms in physiological adjustments and pathological conditions in the cardiovascular system. ANG II has also been reported to increase ANP secretion in minced atrial tissues (54) and isolated rabbit hearts (14) via protein kinase C-dependent prostaglandin (7). However, some investigators found no effect of ANG II on ANP secretion in isolated tissues or primary cell cultures (18). Thus a direct positive or negative effect of ANG II on ANP secretion is still controversial (11). In addition, no available reports exist in regard to the effects of ANG-(1–7) on ANP secretion. Patients treated with the inhibitor for angiotensin-converting enzyme have shown higher plasma levels of ANG-(1–7) and ANP, which contribute to the hypotensive effect of angiotensin-converting enzyme inhibitors (23, 32, 47). We hypothesized that ANG-(1–7) modulates ANP secretion from the heart, and these two peptides have a functional relationship to exhibit the cardiovascular effect in pathophysiological conditions. Therefore, the objectives of this study are 1) to elucidate the role of ANG-(1–7) on ANP secretion and to unravel possible underlying signaling mechanisms and 2) to investigate the functional relationship between two peptides in the heart.

MATERIALS AND METHODS

Animals.

Male Sprague-Dawley (SD) rats, obtained from the Orientbio (Seoungnam, Korea), were housed in a temperature-controlled room with a 12-h:12-h light-dark cycle. The animals were provided with free access to standard laboratory chow (5L79 Purina rat and mouse 18% chow, Charles River, Wilmington, MA) and water. All experimental protocols conformed to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996) and were approved by our institution.

Preparation of perfused beating rat atria.

Isolated perfused beating atria were prepared using a previously described method (20). In brief, the heart was rapidly excised after decapitation; the left atrium was dissected and inserted into a cannula and ligated with silk. The cannulated atrium was kept in an organ chamber perfused with oxygenated HEPES-buffered saline at 36.5°C. Intra-atrial pressure was recorded using a Power lab (ML-820, ADInstruments) via a pressure transducer (Statham P23Db, Oxnard, CA), and pulse pressure was obtained from the difference between systolic and diastolic pressure. The composition of HEPES-buffered saline was as follows: 10 mM HEPES, 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 10 mM glucose, and 0.1% bovine serum albumin (pH 7.4). The pericardial buffer solution, which contained [3H]inulin (Amersham Biosciences) for the measurement of translocation of extracellular fluid (ECF), was also oxygenated via silicone tube coils inside the organ chamber. To stabilize ANP secretion and to maintain a steady-state [3H]inulin level in extracellular space, the atrium was perfused for 90 min. The atrium was paced at 1.2 Hz (duration, 0.3 ms; and voltage, 40 V) from the beginning and high stimulation frequency was applied (6.0 Hz) from 110 to 140 min. Atrial perfusate was collected at 2-min intervals at 4°C from 90 to 140 min.

Radioimmunoassay of cGMP concentration.

cGMP concentrations in perfusates of the first three samples and the last three samples collected from beating atria were measured. For measurement of cGMP in perfusates, 100 μl of perfusate was treated with trichloroacetic acid (300 μl) to a final concentration of 6% for 15 min at room temperature and centrifuged at 4°C. The supernatant (100 μl) was transferred to a polypropylene tube and extracted three times with water-saturated ether. The extract was dried using a Speed-Vac concentrator (Savant, Farmingdale, NY), and the dried samples were resuspended with sodium acetate buffer (100 μl, 50 mM). The production of cGMP was measured with a specific radioimmunoassay (RIA), as described previously (29). Intra- and interassay coefficiency of variation was 4.2% (n = 5) and 7.1% (n = 8), respectively.

Experimental protocols.

Experiments were conducted with sixteen groups. Group 1 included control atria perfused with HEPES buffer (n = 7). Group 2 included ANG-(1–7) (0.01 μM, n = 6; 0.1 μM, n = 8; and 1 μM, n = 7)-perfused atria. Group 3 included atria perfused with Mas receptor antagonist A-779 {d-Ala7-[ANG-(1–7)]; 10 μM} + ANG-(1–7) (0.1 μM, n = 7). In group 4, the atria were perfused with A-779 (10 μM, n = 5) alone. In group 5, the atria were perfused with wortmannin (PI3K inhibitor, 0.1 μM) + ANG-(1–7) (0.1 μM, n = 7), whereas group 6 was comprised of atria treated with wortmannin (0.1 μM, n = 6) alone. Similarly, groups 7 and 8 consisted of atria perfused with Akt/protein kinase B inhibitor [API-2; 21,5-dihydro-5-methyl-1-β-d-ribofuranosyl-1,4,5,6,8-pentaazaacenaphthylen-3-amine, 0.1 μM] + ANG-(1–7) (0.1 μM, n = 7) and API-2 (0.1 μM, n = 5) alone, respectively. Groups 9 and 10 included atria treated with Nω-nitro-l-arginine methyl ester hydrochloride [l-NAME, NO synthase (NOS) inhibitor, 10 μM] + ANG-(1–7) (0.1 μM, n = 5) and l-NAME (10 μM, n = 5) alone, respectively. Groups 11 and 12 consisted of atria perfused with ANG-(1–7) (0.1 μM, n = 5) with pretreatment of amiloride [Na+/H+ exchanger (NHE) inhibitor, 5 μM, n = 5] and amiloride (5 μM, n = 5) alone, respectively. Groups 13 and 14 consisted of atria perfused with ANG-(1–7) (0.1 μM, n = 5) with a pretreatment of cariporide (NHE-1 inhibitor, 1 μM, n = 5) and cariporide (1 μM, n = 5) alone, respectively. Groups 15 and 16 included atria treated with KN-93 [Ca2+/calmodulin-dependent kinase II inhibitor (CaMKII); N-{2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl}-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide phosphate salt (1 μM)] + ANG-(1–7) (0.1 μM, n = 5) and KN-93 (1 μM, n = 5) alone, respectively.

In group 2, ANG-(1–7) was perfused at 75 min after the start of perfusion. When treated alone, all of the inhibitors (Groups 4, 6, 8, 10, 12, 14, and 16) were infused into the atrial lumen at 45 min after the start of perfusion. In groups 3, 5, 7, 9, 11, 13, and 15, atria were pretreated with antagonists at 45 min after the start of perfusion, with simultaneous infusion of ANG-(1–7) at 75 min after the start of perfusion. Perfusate was collected for 50 min at 2-min intervals from 90 to 140 min in all groups.

Acute infusion of ANG-(1–7).

ANG-(1–7) was infused into the control group and the isoproterenol (ISP)-injected group (5 mg·kg−1·day−1 ip for 3 days). Either control (n = 7) or ISP-treated (n = 8) male SD rats, weighing 250–300 g, were anesthetized by an intraperitoneal injection of a mixture of ketamine and xylazine (9:1, 2 ml/kg). Body temperature was maintained at 36–37°C using a heating pad. Following a midline incision in the neck, the jugular vein and carotid artery were carefully dissected, cannulated with a polyethylene tube (PE-50), and secured with ligation. The cannula in the jugular vein was connected to a peristaltic pump (Minipuls 2 Gilson, Villiers le Bel, France) for infusion of vehicle (0.9% NaCl) and ANG-(1–7) (25 μM) in 0.9% NaCl at a constant rate of 60 μl/min, and the cannula in the carotid artery was connected to the pressure transducer (Statham P23Db). Blood pressure and heart rate (HR) were recorded using a Power lab (ML-820, ADInstruments) via the pressure transducer. Animals were stabilized for 5 min after cannulation with 0.9% saline infusion. Blood samples (800 μl) were collected at three time intervals for the measurement of plasma ANP. The first blood sample was collected via the carotid artery following a period of stabilization. The saline was infused for 5 min with subsequent ANG-(1–7) infusion (25 μM) through the jugular vein for 10 min. A second blood sample was then collected. The saline was infused again for 5 min, and a third blood sample was collected. Rats were euthanized at the end of the experiment, and the weight of each cardiac chamber was measured. Blood was centrifuged at 10,000 g at 4°C for 10 min. As described previously, plasma ANP was extracted using a Sep-Pak C18 cartridge (Waters Associates, Milford, MA) (48) and measured using a RIA (6).

Chronic infusion of ANG-(1–7).

For chronic study, SD rats weighing 200–220 g were divided into 5 groups (n = 4 for all groups): 1) control group, 2) ISP-treated group, 3) ISP + ANG-(1–7)-treated group, 4) ISP + ANG-(1–7) + A-71915 [natriuretic peptide receptor-A (NPR-A) antagonist]-treated group, and 5) ISP + A-71915-treated group. Control rats received saline, and ISP-treated rats received ISP intraperitoneally at a dose of 3 mg·kg−1·day−1 for 3 days. In the third group, ANG-(1–7) was infused for 14 days at a dose of 576 μg·kg−1·day−1 via a mini-osmotic pump (Alzet 2002, Cupertino, CA) implanted subcutaneously between the scapula, and ISP (3 mg·kg−1·day−1) was injected in the initial 3 days. In the fourth group, A-71915, a NPR-A antagonist, was infused for 14 days at a dose of 30 μg·kg−1·day−1 via a mini-osmotic pump (Alzet 2002); ANG-(1–7) and ISP were administered by the same method used in the third group. In the fifth group, A-71915 was infused for 14 days at a dose of 30 μg·kg−1·day−1 via a mini-osmotic pump (Alzet 2002); ISP was administered by the same method used in the second group. At the end of the experiment, the rats were euthanized, the weight of heart tissue was measured, and blood was collected for plasma ANP measurement.

RIA of ANP concentrations.

The concentration of ANP in perfusates and plasma was measured using a specific RIA, as described previously (6). The intra- and interassay coefficiency of variation were 6.3% (n = 9) and 7.8% (n = 11), respectively. The amount of secreted ANP was expressed in nanograms per minute per gram of atrial tissue. We previously reported on a two-step sequential mechanism of ANP secretion; first, stored ANP is released from atrial myocytes into the interstitial space by atrial distension, and, second, released ANP is secreted into the atrial lumen, concomitant with ECF translocation by atrial contraction.

Therefore, the molar concentration of ANP release into the interstitium was calculated as follows: [ANP concentration (in μM)] = [ANP (in pg·min−1·g−1)]/[ECF translocation (in μl·min−1·g−1) × 3,060] × 1,000.

Because the ANP secreted was found to be the processed ANP, the denominator 3,060 indicates the molecular mass of ANP(1–28) (in Da) (5).

Measurement of ECF translocation.

The radioactivity of [3H]inulin in atrial perfusate was measured using a liquid scintillation counter (Tris-Carb 23-TR; A Packard Bioscience, Downers Grove, IL). The amount of ECF (μl·min−1·g−1) translocated through the atrial wall was calculated as follows: Total radioactivity in perfusate [in counts per minute (cpm)/min]/radioactivity in the pericardial reservoir (in cpm/μl) × 1,000/atrial wet weight (in mg) (4).

Measurement of intracellular Ca2+ concentration in atrial myocytes.

Atrial myocytes were isolated from male SD rats (200–220 g) using a modified version of the method previously described (19). Briefly, hearts were rapidly excised, cannulated, and subjected to retrograde perfusion on a Langendorff apparatus with Krebs-Henseleit (KH) buffer containing 10 mM HEPES, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM pyruvate, 11 mM glucose, and 1 mM CaCl2 (pH 7.37) for 2 min and then with Ca2+-free KH buffer for 2 min. The perfusion buffer was then changed to Ca2+-free KH buffer containing 0.5 mg/ml of collagenase type II and 0.08 mg/ml protease type XIV for 35 min. Perfusate was gassed with 95% O2-5% CO2 and maintained at 37°C. The atrium was removed, chopped into small pieces, and incubated in a 15-ml Falcon tube at 37°C for 3 min with shaking; undigested tissue was then allowed to settle for ∼1 min. The pellet containing undigested tissue was discarded, and the Ca2+ concentration in the supernatants was gradually increased up to 1 mM. Isolated atrial myocytes were pelleted by centrifugation at 23 g for 1 min at room temperature and resuspended in a stabilizing buffer (pH 7.4) containing 20 mM HEPES, 137 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 15 mM glucose, and 10 mM 2,3-butanedione monoxime and 1 mM Ca2+. The cell preparation was kept in the stabilizing buffer, which contained 1% bovine serum albumin, for about 10 min at 37°C and then washed and resuspended in medium-199 (Invitrogen, Grand Island, NY), supplemented with 2% albumin, 2 mM l-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml gentamycin. Atrial myocytes were seeded on confocal dishes previously coated with laminin for 1 h (10 μg/ml). Following incubation for 20 min at 37°C in an incubator humidified with 5% CO2-95% air, the medium was changed to remove round and unattached cells. The experiments were performed on the same day or 12–16 h after isolation.

Atrial myocytes attached to laminin-coated confocal dishes were loaded with Ca2+ indicator Fluo-3 AM (3 μM) (Molecular Probes, Eugene, OR) and incubated for 20 min at 37°C. After they were washed, basal intracellular Ca2+ concentration ([Ca2+]i) was measured for 30 s, followed by an addition of ANG-(1–7) (0.1, 0.25, 0.5, or 1 μM) or ANG II (0.1 μM). The change of [Ca2+]i in cells was determined at 488 nm excitation/530 nm emission using an air-cooled argon laser system (25). The emitted fluorescence at 530 nm was collected using a photomultiplier. One image was scanned every 3 s using a confocal microscope (Nikon). A calculation of [Ca2+]i was performed using an equation provided by Tsien et al. (53), i.e., [Ca2+]i = Kd(FFmin)/(FmaxF), where Kd is 450 nM for Fluo-3 and F is observed fluorescence levels.

Real-time PCR.

Total RNA was extracted from rat atrial tissues using TRIzol reagent (Invitrogen), and reverse transcription was performed using Superscript II and 18-mers Oligo-dT (Invitrogen). Specific primers were designed using primer express software; primer sequences were as follows: rat Mas receptor (accession NM_012757.21), 5′-GGGCGTCTGGACAAAGAGTCT-3′ (forward), and 5′-CATTTTCTCTCTGGCAGGATGA-3′ (reverse); rat ANP (accession NM_012612.2), 5′-CCGGTACCGAAGATAACAGC-3′ (forward), and 5′-CTCCAGGAGGGTATTCACCA-3′ (reverse); and rat actin (accession NM_031144.2), 5′-ACCAGTTCGCCATGGATGAC-3′ (forward), and 5′-TGCCGGAGCCGTTGTC-3′ (reverse). The real-time PCR reaction was contained in a final volume of 10 μl, 10 ng of reversed transcribed total RNA, 200 nM of forward and reverse primers, and 2× PCR master mix. The PCR reaction was carried out in 384-well plates using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster, CA). All reactions were performed in triplicate.

Statistical analysis.

Results are presented as means ± SE. Statistical significance of differences was assessed using ANOVA followed by the Bonferroni multiple comparison test. Student's t-test was also used. The critical level of significance was set at P < 0.05.

RESULTS

Effects of ANG-(1–7) on atrial hemodynamics and ANP secretion.

To evaluate the effect of ANG-(1–7) on ANP secretion, ANG-(1–7) was perfused into rat atria paced at 1.2 Hz. No changes in ANP secretion were induced by ANG-(1–7) (0.1 μM) (Fig. 1A). In consideration of this finding and the view that ANG-(1–7) plays vital roles primarily in pathophysiological conditions in the heart (50), we tested the effect of ANG-(1–7) on ANP secretion in atria paced at different stimulation frequencies and in a cardiac hypertrophied rat model. When atria were paced from 1.2 to 6.0 Hz, atrial pulse pressure was markedly decreased (Fig. 1B,a) and ECF translocation abruptly increased and then recovered to the control value (Fig. 1B,b). ANP secretion and ANP concentration increased gradually throughout the experiment (Fig. 1B, c and d). ANG-(1–7), at a concentration of 0.01 and 0.1 μM, had no effect on atrial contractility, ECF translocation, ANP secretion, and ANP concentration at 1.2 Hz. However, ANG-(1–7) gradually augmented high atrial pacing-induced ANP secretion and ANP concentration that reached a plateau at 14 min after application of high frequency (Fig. 1B, c and d). None of the concentrations of ANG-(1–7) affected high atrial pacing-induced changes in pulse pressure and ECF translocation at 6.0 Hz.

Fig. 1.

A: effects of angiotensin (1–7) [ANG-(1–7)] (0.1 μM) on pulse pressure (a), extracellular fluid translocation (ECF transloc; b), atrial natriuretic peptide (ANP) secretion (c), and ANP concentration (Conc) (d) as a function of time in isolated perfused atria beating at low frequency (1.2 Hz) throughout the experiment. B: effects of various doses of ANG-(1–7) (0.01 and 0.1 μM) on pulse pressure (a), ECF translocation (b), ANP secretion (c), and ANP concentration (d) in isolated perfused atria beating at 1.2 and 6.0 Hz. ANG-(1–7) was perfused at 75 min after the start of perfusion. After stabilization period of 90 min, perfusate was collected every 2 min as a control (Cont) at low frequency (1.2 Hz) for 20 min (collection period, 0–20 min). Stimulation frequency was then changed to high frequency (6.0 Hz), and perfusate was collected for 30 min (collection period, 20–50 min). Values are expressed as means ± SD. *P < 0.05, significantly different from control group.

Figure 2 shows the effect of ANG-(1–7) on atrial hemodynamics and ANP secretion presented by the relative percent change from the mean of the first five control values at 1.2 Hz and the last five experimental values at 6.0 Hz in the presence of ANG-(1–7). Pulse pressure was decreased at high atrial pacing by 40%, and ANG-(1–7) had no significant effect on it (Fig. 2A). High atrial pacing increased ECF translocation by 15%, which was not affected by ANG-(1–7) (Fig. 2B). ANG-(1–7) augmented high atrial pacing-induced ANP secretion (Fig. 2C) and ANP concentration (Fig. 2D) in a dose-dependent manner. When compared with the control period, ANG-(1–7), 0.1 and 1 μM, increased cGMP level (data not shown) in perfusate by 23.6 ± 5.1 and 58.5 ± 7.8%, respectively.

Fig. 2.

Relative percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by different concentrations of ANG-(1–7) (0.01, 0.1, and 1 μM). Values are expressed as percent changes of the mean of the last 5 experimental values at 6.0 Hz compared with the mean of the first 5 control values at 1.2 Hz in the presence of ANG-(1–7). *P < 0.05, significantly different from the control group; #P < 0.05, significantly different from the corresponding group.

Changes in ANG-(1–7)-induced ANP secretion with inhibitors of the Mas/PI3K/Akt pathway.

To dissect the signaling pathway of ANG-(1–7)-stimulated ANP release, we pretreated atria with receptor Mas antagonist and inhibitors of downstream signaling molecules. A-779 (10 μM), an antagonist of receptor Mas, significantly abrogated ANG-(1–7)-induced ANP secretion (Fig. 3C) and ANP concentration (Fig. 3D). To identify the modulation of ANG-(1–7)-induced ANP secretion by the PI3K-Akt pathway, the respective inhibitors were perfused into the atrial lumen. Pretreatment with wortmannin (PI3K inhibitor; 0.1 μM) or API-2 (Akt inhibitor; 0.1 μM) significantly blocked high atrial pacing-induced ANP secretion (Fig. 3D) and ANP concentration (Fig. 3D) augmented by ANG-(1–7). Pretreatment with l-NAME (NOS inhibitor; 10 μM) decreased ANG-(1–7)-induced ANP secretion and ANP concentration (Fig. 3, C and D). We performed perfusion experiments with antagonists or inhibitors alone to rule out self-inhibitory effects. None of the antagonists or inhibitors affected changes in atrial parameters (Fig. 3).

Fig. 3.

Modification of the effects of ANG-(1–7) on pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by inhibitor for the Mas receptor and downstream signaling molecules. Values are expressed as percent changes of the mean of the last 5 experimental values at 6.0 Hz compared with mean of the first 5 control values at 1.2 Hz in the presence of inhibitors and/or ANG-(1–7). The antagonists [A-779, 10 μM; wortmannin (Wort), 0.1 μM; 1,5-dihydro-5-methyl-1-β-d-ribofuranosyl-1,4,5,6,8-pentaazaacenaphthylen-3-amine (API-2), 0.1 μM; and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), 10 μM] were perfused at 45 min after the start of the experiment. ANG-(1–7) (0.1 μM) was perfused simultaneously at 75 min after the start of experiment. All modulators were perfused alone at 45 min after the start of the experiment to examine their self-effects. A-779, d-Ala7-[ANG-(1–7)]. *P < 0.05, significantly different from the ANG-(1–7)-infused group; #P < 0.05 and ##P < 0.01, significantly different from the corresponding group.

Changes in ANG-(1–7)-induced ANP secretion with Ca2+ modulators.

To determine the role of Ca2+ in the modulation of ANG-(1–7)-induced ANP secretion in high atrial pacing, atria were pretreated with blockers that modify intracellular Ca2+. Diltiazem (L-type Ca2+ channel blocker; 1 μM) did not modify increased ANP secretion (Fig. 4C) and ANP concentration (Fig. 4D) induced by ANG-(1–7). However, when perfused alone, it significantly attenuated high atrial pacing-induced ANP secretion and ANP concentration (Fig. 4, C and D). KN-93 (CaMKII inhibitor; 1 μM) caused significant decreases in ANP secretion and ANP concentration. Moreover, pretreatment with KN-93 also blocked the stimulatory effect of ANG-(1–7) on ANP secretion and ANP concentration (Fig. 4, C and D). Next, as shown in Fig. 4, C and D, pretreatment with amiloride (NHE inhibitor; 0.1 μM) and cariporide (NHE-1 inhibitor; 1 μM) significantly blocked ANG-(1–7)-stimulated ANP secretion and ANP concentration. Atrial parameters were not altered by any of the Ca2+ modulators.

Fig. 4.

Comparison of relative percent changes in pulse pressure (A), ECF translocation (B), ANP secretion (C), and ANP concentration (D) by different calcium modulators with or without ANG-(1–7) (0.1 μM). Values are expressed as percent changes of the mean of the last 5 experimental values at 6.0 Hz compared with mean of the first 5 control values at 1.2 Hz in the presence of inhibitors and/or ANG-(1–7). Calcium modulators [diltiazem (Dilt), 1 μM; N-{2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl}-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide phosphate salt (KN-93), 1 μM; amiloride, 0.1 μM; and cariporide, 1 μM] were pretreated at 45 min after the start of the experiment with simultaneous infusion of ANG-(1–7) (0.1 μM) at 75 min. All modulators were perfused alone at 45 min after the start of the experiment to examine their self-effects. *P < 0.05, significantly different from the ANG-(1–7)-infused group; #P < 0.05 and ##P < 0.01, significantly different from the corresponding group.

[Ca2+]i in atrial myocytes was measured for the determination of whether ANG-(1–7) affects the regulation of basal [Ca2+]i response. Basal [Ca2+]i in quiescent atrial myocytes was 80.9 ± 2.2 nM. No [Ca2+]i signal was produced by the treatment of atrial myocytes with ANG-(1–7) (0.1 and 1 μM) (Fig. 5, A and B). On the contrary, the treatment of atrial myocytes with ANG II (0.1 μM) as a control abruptly increased [Ca2+]i, which was then sustained (Fig. 5C).

Fig. 5.

Effect of ANG-(1–7) on basal intracellular calcium concentration ([Ca2+]i) in rat atrial myocytes. Representative tracings of Ca2+ response to 0.1 and 1 μM ANG-(1–7) (A and B) or 0.1 μM ANG II (C) from individual atrial myocytes (n = 3) are shown. Comparison of mean [Ca2+]i before and after exposure to different concentration of ANG-(1–7) (0.1, 0.25, 0.5, and 1.0 μM) or ANG II (0.1 μM) (D) is shown. Data shown were analyzed at 54 s. Values represent means ± SE of 3 independent experiments. Arrow indicates time of ANG-(1–7) or ANG II treatment. **P < 0.01, significantly different from the control value.

Effect of acute infusion of ANG-(1–7) on plasma ANP concentration in ISP-treated rats.

To identify the role of ANG-(1–7) on hemodynamics and ANP secretion in vivo, ANG-(1–7) was infused intravenously using ISP-treated rats. ISP treatment for 3 days significantly induced cardiac hypertrophy in rats (Fig. 6A). The expression of Mas mRNA in atria was not different between control (n = 6) and ISP-treated (n = 6) rats (Fig. 6B). The expression of ANP mRNA was increased fourfold in the atria of ISP-treated rats (Fig. 6B). The basal concentration of plasma ANP was higher in ISP-treated rats than in control rats (Fig. 6C). ANG-(1–7) significantly increased the plasma ANP level from 105.8 ± 19.7 to 193.7 ± 38.7 pg/ml in ISP-treated rats, whereas ANG-(1–7) caused no significant change in the plasma ANP level in control rats (Fig. 6C). Mean arterial pressure (MAP) and HR did not appear to be affected by ANG-(1–7) in ISP-treated and control rats (Fig. 6, D and E).

Fig. 6.

Changes in cardiac mass (A) and expression of atrial Mas and ANP mRNA (B) in isoproterenol-treated rats (5 mg·kg−1·day−1 ip) for 3 days. Changes in plasma ANP level, mean arterial pressure (MAP), and heart rate (HR) by intravenous infusion of ANG-(1–7) (25 μM) in control rats and isoproterenol-treated rats (C–E) are shown. The horizontal shade represents the period of ANG-(1–7) infusion. Changes in cardiac mass and plasma ANP level by in vivo administration of different drugs for 2 wk period (F and G) are shown. ISP, injection of ISP (3 mg·kg−1·day−1 ip) alone or in combination for the initial 3 days; ANG-(1–7), ANG-(1–7) administration (576 μg·kg−1·day−1) via mini-osmotic pumps; A, A-71915 (natriuretic peptide receptor-A antagonist) infusion (30 μg·kg−1·day−1) via mini-osmotic pumps. Lt Vent, left ventricle; Rt Vent, right ventricle; Lt atrium, left atrium; Rt atrium, right atrium; A, A-71915 (Arg6, β-cyclohexyl-Ala8, d-Tic16, Arg17, Cys18)-atrial natriuretic factor (6–18) amide. *P < 0.05 and **P < 0.01, significantly different from the control group; ##P < 0.01, significantly different from the control value; +P < 0.05, significantly different from the ISP group.

Effect of chronic injection of ANG-(1–7) on ISP-induced cardiac hypertrophy.

For an evaluation of the chronic effect of ANG-(1–7) on ISP-induced cardiac hypertrophy, we injected ISP with simultaneous ANG-(1–7) infusion for 2 wk. The treatment of ISP alone for 3 days significantly increased the mass of all cardiac chambers (Fig. 6F). ANG-(1–7) treatment for 2 wk attenuated the tissue weight (in g/100 g body wt) of whole heart (0.398 ± 0.010 vs. 0.456 ± 0.024), left ventricle (0.263 ± 0.009 vs. 0.286 ± 0.012), left atrium (0.0097 ± 0.0003 vs. 0.0141 ± 0.0010), and right atrium (0.0079 ± 0.0008 vs. 0.0110 ± 0.0004) without a significant decrease in right ventricle (0.058 ± 0.002 vs. 0.064 ± 0.002) (Fig. 6F). In the NPR-A antagonist-infused group, ANG-(1–7) did not prevent ISP-induced cardiac hypertrophy (Fig. 6F). Chronic infusion of ANG-(1–7) significantly increased the plasma ANP level (Fig. 6G).

DISCUSSION

With the discovery of ANG-(1–7) and other related peptides, the classical model of RAS has been recently replaced by the new model of RAS. Among the metabolites in RAS, ANG-(1–7) is considered as an imperative cardiovascular peptide that plays the role of the counterregulatory arm in cardiac pathophysiology. In the present study, we explored the role of ANG-(1–7) in the regulation of ANP secretion using ex vivo high atrial pacing (exercise) and an in vivo cardiac hypertrophy model. Results showed that ANG-(1–7) augmented ANP secretion from isolated perfused atria at high pacing but not at low pacing. ANG-(1–7)-induced ANP secretion was blocked by a pretreatment with an inhibitor of Mas, PI3K, Akt, or NOS. In addition, a blocker of NHE-1 or CaMKII also inhibited ANG-(1–7)-induced ANP secretion. Further examination with a rat model of cardiac hypertrophy induced by ISP revealed that an acute and chronic administration of ANG-(1–7) increases plasma ANP level, and chronic infusion of ANG-(1–7) attenuated ISP-induced cardiac hypertrophy, which was blocked by cotreatment with the NPR-A antagonist. These findings suggest that ANG-(1–7) stimulates ANP secretion through the signaling pathway involving the Mas/PI3K/Akt axis, NHE-1, and CaMKII and that ANG-(1–7) regulates ANP secretion and decreases cardiac hypertrophy under conditions of an increased HR and/or cardiac hypertrophy.

A recent study has demonstrated a selective increase in ventricular ANG-(1–7) levels and Mas mRNA and protein expression in trained spontaneously hypertensive rats, but not in trained Wister-Kyoto rats (13). The involvement of the ANG-(1–7)-Mas axis activation in the beneficial effects of physical training in the heart has been suggested. This finding emphasizes the role of ANG-(1–7) mainly in pathophysiological conditions. The present study demonstrates that ANG-(1–7) elevated high atrial pacing-induced ANP secretion; however, ANG-(1–7) at low atrial pacing had no effects on ANP secretion. Taken together, these observations suggest that ANG-(1–7) may stimulate ANP secretion during increased HR, a condition similar to that of physical exercise. To further evaluate our in vitro findings, we used a cardiac hypertrophied rat model induced by ISP (26). When compared those with control rats, HR and MAP in ISP-treated rats were not significantly different. Although the reasons are not clearly understood, this might be due to the effect of anesthesia (3) or ISP treatment for only 3 days instead of 7 days, as was generally the case in other studies. Strikingly, we observed a significant increase in the plasma ANP level in ISP-treated rats after infusion of ANG-(1–7) for 10 min. Thereafter, the cessation of ANG-(1–7) infusion resulted in a rapid fall of the plasma ANP level almost equal to the baseline value. This might be due to a short half-life of ANG-(1–7) (52). An identification of the mechanism for the acute effect of ANG-(1–7) on ANP release in a hypertrophied rat model will require further examination. ANG-(1–7) had no effect on MAP and HR, which suggests that increased ANP secretion by ANG-(1–7) is probably independent of hemodynamic changes. These observations could be corroborated by the work of Mercure et al. (35) who observe no changes in blood pressure, HR, cardiac geometry, or contractility in transgenic rats that overproduced ANG-(1–7). Our findings strongly support the positive modulation of ANP secretion by ANG-(1–7) in both physiological adjustments and pathological conditions of the heart.

In cardiovascular disease, cardiac hypertrophy, a compensatory phenomenon, ultimately leads to ventricular remodeling and cardiac dysfunction. The therapeutic strategy for a decrease of hypertrophy is the prevention of cardiac remodeling and the improvement of cardiac dysfunction (24a). ANG-(1–7) has numerous cardiac antiremodeling functions, including the inhibition of cardiomyocytes growth (51), the prevention of cardiac fibrosis (22), and the reduction of cardiac hypertrophy (35). ANG-(1–7) also improves cardiac dysfunction (31). In fact, Mas deficiency in mice is indicated by severe cardiac dysfunction and a marked expression of extracellular proteins (44). In the present study, we observed the increase in plasma ANP and the attenuation of ISP-induced cardiac hypertrophy by chronic treatment of ANG-(1–7), reaffirming its antihypertrophic property. Interestingly, the blockage of the NPR-A receptor diminished the antihypertrophic effect of ANG-(1–7), suggesting the existence of a functional relationship between ANP and ANG-(1–7). This finding opens a new avenue for further research into the interaction between new RAS and ANP systems in the heart.

ANG-(1–7) executes its functions via the G protein-coupled receptor Mas (43), which is now widely accepted as a functional receptor for ANG-(1–7). Although the molecular mechanism of Mas signaling has not been completely elucidated, the activation of the PI3K/Akt/eNOS pathway by ANG-(1–7)/Mas axis has been demonstrated with endothelial cells (41), cardiomyocytes, (10) and rat hearts (17). Sampaio et al. (41) have shown eNOS phosphorylation by ANG-(1–7) via Mas, which leads to NO release through PI3K-Akt-dependent pathways in endothelial cells. This molecular signaling triggered by ANG-(1–7) was also confirmed in cardiomyocytes by Dias-Peixoto et al. (10). Furthermore, we demonstrated the attenuation of ANG-(1–7)-induced ANP secretion by blockers of each of the components of the Mas-PI3K-Akt signaling pathway. Based on these findings, the Mas-PI3K-Akt pathway may be delineated as the most common signaling pathway for the exhibition of the physiological effects of ANG-(1–7). Considering the production of NO by ANG-(1–7) (41) and the inhibitory role of NO on ANP secretion through cGMP (11), we further estimated the cGMP level in perfusate. Interestingly, the cGMP level was significantly increased by ANG-(1–7), which is similar to the previous report (12). This increase in cGMP may be due to the activation of the Mas/PI3K/Akt/eNOS signaling axis by ANG-(1–7) or partly by the effect of increased ANP induced by ANG-(1–7). Surprisingly, the pretreatment with l-NAME attenuated the augmentation of high atrial pacing-induced ANP secretion by ANG-(1–7). Using a variety of inhibitors of endothelial-derived relaxation factor, Sanchez-Ferrer et al. (42) showed increased ANP secretion. The discrepancy may be a result of different experimental protocols. Thus this study also reflects controversial and differential roles of NO/cGMP on ANP secretion in different experimental setups. However, the reason for decreased ANG-(1–7)-induced ANP secretion by l-NAME at high stimulation frequency could not be determined. In the present study, ANG-(1–7) caused an increase in both ANP secretion and cGMP level. Therefore, it could be speculated that the increased production of cGMP by ANG-(1–7) may not be important in the modulation of ANP release in high pacing atria for the following reasons: insufficient cGMP production [20% increase by 0.1 μM ANG-(1–7)], the inhibitory effect of cGMP on ANP secretion being masked by a stimulatory effect of ANG-(1–7), the distinct role of pGC-cGMP and sGC-cGMP signaling in the regulation of ANP release (55), or even the absence of the effect of NO on ANP secretion (24).

The involvement of the L-type Ca2+ channel and NHE in regulation of ANP secretion has been demonstrated. Frequency-induced ANP secretion is associated with the activation of NHE, which results in the increase of [Ca2+]i by a reversal of the operation mode of the Na+/Ca2+ exchanger (45). However, pretreatment with L-type Ca2+ channel blocker did not affect ANG-(1–7)-augmented ANP secretion in atria with high pacing. This increase in ANP secretion, independent of L-type Ca2+ channel, could most likely be a result of the CaMKII activation due to the fact that ANG-(1–7) has been shown to increase CaMKII and MAP kinase activity in rabbit aortic smooth muscle cells via the ANG-II type 2 receptor and Mas receptors (36). Moreover, ANP and BNP secretion recruited by pacing has been shown to share the same CaMKII-dependent mechanism (40). Our observation showing the attenuation of ANP secretion by CaMKII inhibitor during high pacing and/or presence of ANG-(1–7) was in agreement with this finding. Therefore, CaMKII is probably the major candidate molecule responsible for ANP release when activated by ANG-(1–7) only in high atrial pacing. When the role of ANG-(1–7) on [Ca2+]i was evaluated, ANG-(1–7) did not alter [Ca2+]i in resting atrial myocytes; these results are similar to those observed by Dias-Peixoto et al. (10) in ventricular myocytes. Nevertheless, it is to be noted that ventricular myocytes from Mas-deficient mice presented reduced peak and slower [Ca2+]i transients in their study. In our context, it would be more plausible to delve into the effect of ANG-(1–7) on [Ca2+]i in beating atrial myocytes at high stimulation frequency, which is a limitation of this study. Akt has been shown to phosphorylate and inhibit cardiac NHE-1 (49). On the other hand, it enhances myocardial [Ca2+]i handling in SD rats with Akt adenoviral gene transfer (8). In the present study, the pretreatment with amiloride, a selective inhibitor of NHE, attenuated ANG-(1–7)-mediated ANP secretion. Furthermore, a similar result was observed with the pretreatment of cariporide, a specific inhibitor of NHE-1. These results indicate that ANG-(1–7) might stimulate NHE-1 during high atrial pacing. Thus ANG-(1–7) may increase [Ca2+]i by the stimulation of NHE-1 or via Akt activation only in high atrial pacing, which in turn activates CaMKII and ultimately causes the release of ANP from atrial myocytes. However, a putative role of ANG-(1–7) in rising [Ca2+]i in high atrial pacing via the ANG-(1–7)/NHE-1 or ANG-(1–7)/PI3K/Akt pathways will require further investigation.

In conclusion, our results have demonstrated that ANG-(1–7)/Mas-induced ANP secretion is mediated by a signaling pathway involving PI3K, Akt, and NHE-1 in beating atria paced with high stimulation frequency and that ANG-(1–7) elevates plasma ANP level and attenuates hypertrophy in cardiac hypertrophic rats. Thus our study provides additional evidence for a beneficial role of ANG-(1–7) in the stimulation of ANP secretion and the attenuation of cardiac hypertrophy, probably mediated by ANP, in both physiological adjustments and pathological conditions of the heart.

GRANTS

This work was supported by Korean Science and Engineering Foundation Grant R13-2008-005-00000-0. A. Shah was supported by the foreigner support program from the Korea Research Foundation (KRF-2008-211-E00001).

DISCLOSURES

No conflicts of interest are declared by the author(s).

ACKNOWLEDGMENTS

Cariporide was kindly provided by Dr. Juergen Puenter (Sanofi-Aventis Deutschland; Frankfurt, Germany).

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View Abstract