Angiotensin-(1-7) Through Receptor Mas Mediates Endothelial Nitric Oxide Synthase Activation via Akt-Dependent Pathways
Angiotensin-(1-7) [Ang-(1-7)] causes endothelial-dependent vasodilation mediated, in part, by NO release. However, the molecular mechanisms involved in endothelial NO synthase (eNOS) activation by Ang-(1-7) remain unknown. Using Chinese hamster ovary cells stably transfected with Mas cDNA (Chinese hamster ovary-Mas), we evaluated the underlying mechanisms related to receptor Mas–mediated posttranslational eNOS activation and NO release. We further examined the Ang-(1-7) profile of eNOS activation in human aortic endothelial cells, which constitutively express the Mas receptor. Chinese hamster ovary-Mas cells and human aortic endothelial cell were stimulated with Ang-(1-7; 10−7 mol/L; 1 to 30 minutes) in the absence or presence of A-779 (10−6 mol/L). Additional experiments were performed in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin (10−6 mol/L). Changes in eNOS (at Ser1177/Thr495 residues) and Akt phosphorylation were evaluated by Western blotting. NO release was measured using both the fluorochrome 2,3-diaminonaphthalene and an NO analyzer. Ang-(1-7) significantly stimulated eNOS activation (reciprocal phosphorylation/dephosphorylation at Ser1177/Thr495) and induced a sustained Akt phosphorylation (P<0.05). Concomitantly, a significant increase in NO release was observed (2-fold increase in relation to control). These effects were blocked by A-779. Wortmannin suppressed eNOS activation in both Chinese hamster ovary-Mas and human aortic endothelial cells. Our findings demonstrate that Ang-(1-7), through Mas, stimulates eNOS activation and NO production via Akt-dependent pathways. These novel data highlight the importance of the Ang-(1-7)/Mas axis as a putative regulator of endothelial function.
The renin–angiotensin system is a crucial regulator of cardiovascular homeostasis. Most physiological effects of angiotensin (Ang) II are mediated via Ang II type 1 (AT1) receptors (AT1R), with Ang II type 2 (AT2) receptors (AT2R) counteracting AT1R actions.1 Growing evidence indicates that the Ang peptide Ang-(1-7) plays an important role in the renin–angiotensin system.2 This heptapeptide is formed by Ang-converting enzyme–dependent and Ang-converting enzyme–independent pathways. Much attention has been given recently to its formation through hydrolysis of Ang II by the ectoenzyme Ang-converting enzyme 2, which is present in many organs.3 Ang-(1-7) opposes many Ang II–stimulated actions. Ang-(1-7), acting through the G protein-coupled receptor (GPCR) Mas, releases NO and prostaglandins causing vasodilation, inhibition of cell growth, and opposition of AT1R-mediated Ang II vasoconstrictor and proliferative effects.2 Overactivity of the renin–angiotensin system, as observed in cardiovascular diseases, and the lack of balance among its peptides may reduce NO bioavailability leading to endothelial dysfunction.4,5 NO plays a critical role in endothelial function, maintaining vasodilator tone, inhibiting platelet aggregation and adhesion, and modulating vascular smooth muscle cell contraction and proliferation.6 Many risk factors implicated in cardiovascular diseases seem to be associated with impairment in the NO system with a reduction in endothelial generation of NO by endothelial NO synthase (eNOS).5,6
eNOS activity is regulated by various kinases, which induce coordinated phosphorylation of specific sites.7 Phosphorylation of eNOS at Ser1177 by the phosphatidylinositol 3′-kinase (PI3K)/protein kinase B/Akt pathway results in a 2-fold increase in eNOS catalytic activity and NO production. eNOS phosphorylation at Ser1177 is a well-characterized pathway activated by vascular endothelial growth factor,8 insulin-like growth factor-1,9 fluid shear stress,10 and bradykinin.11 Phosphorylation also involves a decrease in the dependence of the enzyme for Ca-2+–calmodulin.12 In contrast, eNOS phosphorylation at threonine 495 reduces enzyme activity and NO generation.13
Previous studies demonstrated that endothelial cells are capable of generating Ang-(1-7) from its precursors Ang I and Ang II because they apparently express the required converting enzymes.14 Endothelial cells possess Ang-(1-7) receptors,15 and Ang-(1-7) stimulates NO production in these cells.16 Recently, our group reported that short-term stimulation of the receptor Mas by Ang-(1-7) improves endothelial function through facilitation of NO release.17 The lack of vasodilatory response in isolated aorta of Mas knockout mice to Ang-(1-7) stimulation strongly suggests the Mas receptor as a likely mediator of NO production.18 Unexpectedly, the Ang-(1-7)–stimulated NO generation was not accompanied by alterations in intracellular calcium concentration suggesting that a calcium-independent pathway could be involved in this action.16
Although many Ang-(1-7) actions involve NO release, little is known about the molecular mechanisms that mediate this response. Here we sought to determine, in human endothelial cells, whether Ang-(1-7) regulates eNOS expression/activity and whether this involves the GPCR Mas. Furthermore, we questioned whether Ang-(1-7) differentially regulates ser1179 and thr495 phosphorylation sites of eNOS and assessed whether the phosphatidylinositol 3-kinase/Akt pathway plays a role in this process.
Two cell models were studied: Chinese hamster ovary (CHO) cells (American Type Culture Collection), which were stably transfected with receptor Mas cDNA or bradykinin type 2 (B2) receptors driven by a cytomegalovirus promoter and selected by neomycin, and human aortic endothelial cells ([HAEC] Cascade Biologics).
Production of Mas Antibody
Polyclonal antisera directed against Mas was produced in Mas knockout (Mas−/−) male C57BL/6 mice, using as antigen, a 12-amino acid peptide (LAEEKAMNTSSR) corresponding with the N-terminal domain of the mouse Mas protein. A second anti-human Mas antibody was obtained from Novus Biologicals, Inc. Both antibodies gave a band corresponding to 33 kDa in HAECs.
Cell stimulation was carried out at 37°C in serum-free medium. Cells were stimulated with increasing concentrations of Ang-(1-7) (10−9 to 10−5 mol/L) in the absence or presence of the Ang-(1-7) inhibitor, A-779 (10−6 mol/L; 10 minutes of preincubation). In some experiments, cells were pre-exposed to the Akt inhibitor, wortmannin (10−6 mol/L; 30 minutes). Immunoblotting was performed as described previously.19 Specific antibodies used included AT1R and AT2R, at 1:500 dilution, and MAS, Akt, phospho-Akt, eNOS, phospho-eNOS Ser1177 and phospho-eNOS Thr495, at 1:1000 dilution.
Confluent CHO and CHO-Mas cells cultured in 6-well plates were stimulated with Ang-(1-7) (10−9 to 10−5 mol/L) in the presence or absence of a receptor Mas antagonist, A-779 (10−6 mol/L; 10 minutes). The nitrite in the supernatant was measured by the 2,3-diaminoftalen fluorometric assay as described previously.20 Some sets of experiments were performed in the presence of NG-nitro-l-arginine methyl ester (10−7 mol/L), an eNOS inhibitor. NO release in CHO-Mas cells was also measured by an NO analyzer as described previously.21 For fluorescence images, confluent cells between the second and fourth passages were plated in 6-well plates. CHO-transfected and CHO-nontransfected cells were preincubated in Hanks’ balanced salt solution containing 4,5-diaminofluorescein-diacetate (Molecular Probes; 10−6 mol/L; 20 minutes). Washed cells were incubated with Ang-(1-7) (10−6 mol/L; 30 minutes) at 37°C in a humidified incubator under an atmosphere with 5% CO2. Fluorescence images were obtained using a Zeiss 510 metalaser scanning confocal microscope equipped with an oil-immersion objective lens (×63).
To determine the potential role of B2 receptors in Ang-(1-7)–mediated NO effects, we also examined actions of Ang-(1-7) in B2 receptor-expressing CHO cells using 2,3-diaminoftalen fluorescence. An expanded Methods section is available online at http://hyper.ahajournals.org.
Experiments were repeated 3 to 5 times in duplicate. Results are presented as mean±SEM and compared by Student’s paired t test. Multiple comparisons were compensated by Turkey–Kramer’s correction. A value of P<0.05 was considered significant.
Receptor Characterization of Cell Models
To characterize the cell models, we evaluated the presence of AT1R, AT2R, receptor Mas, and eNOS in CHO-transfected cells (CHO-Mas), nontransfected CHO cells, and HAECs. As shown in Figure 1, CHO-Mas express receptor Mas and eNOS but do not constitutively express AT1R or AT2R. AT1R, AT2R, and Mas are not present in nontransfected cells. HAECs express AT1R, AT2R, receptor Mas, and eNOS. Testis, heart (left ventricle), and A10 cells were used as positive controls for Mas, AT1R, and AT2R, respectively.
Ang-(1-7) Stimulates NO Release via Mas in CHO-Mas Cells
Figure 2 shows nitrite measurements using the 2,3-diaminoftalen fluorescence (Figure 2A), NO analyzer (Figure 2B), and 4,5-diaminofluorescein fluorescence methods (Figure 2C). Ang-(1-7) stimulated NO-release in CHO-transfected cells in a dose-dependent manner (10−8 to 10−6 mol/L; 30 minutes). The Mas antagonist A-779 blocked NO release. The eNOS inhibitor NG-nitro-l-arginine methyl ester also inhibited Ang-(1-7)–stimulated NO generation, indicating that Ang-(1-7) stimulates NO formation through receptor Mas, which is coupled to eNOS. Figure 2C shows CHO-transfected (left) and CHO-nontransfected (right) cells treated with Ang-(1-7) at 10−6 mol/L for 30 minutes. To test whether B2 receptors could mediate NO production by Ang-(1-7), we used CHO cells stably transfected with B2 receptors. As demonstrated in supplemental Figure I (available online), whereas bradykinin dose-dependently increased NO production, Ang-(1-7) had no effect.
Ang-(1-7) Coordinately Regulates Ser1177/Thr495 Phosphorylation of eNOS
The role of Ang-(1-7) in eNOS activation was evaluated by measuring phosphorylation of 2 regulatory sites of the enzyme: Ser1177 (stimulatory site) and Thr495 (inhibitory site). As shown in Figure 3A, Ang-(1-7) (10−7 mol/L) significantly increased Ser1177 phosphorylation (P<0.05) in CHO-Mas cells in a time-dependent manner. Responses were rapid with significant effects occurring within 5 minutes of stimulation. This was blocked by receptor Mas antagonist A-799. As also shown in Figure 3A, A-779 had a slight agonistic effect on the Ser1177 phosphorylation.
In contrast to that observed for the Ser1177 site, Ang-(1-7) stimulation induced a rapid dephosphorylation of the inhibitory site (Thr495), which is normally phosphorylated in baseline conditions (Figure 3B and 3C). Although we have observed a trend for an increase above the control of p-eNOS Thr495 after the initial dephosphorylation, the differences did not reach statistical significance. In HAECs, Ang-(1-7) dose-dependently increased eNOS phosphorylation at Ser1177, with maximal effects obtained at 10−6 mol/L (Figure 4A). This effect was associated with reduced eNOS phosphorylation at the inhibitory site (Thr495; Figure 4B). Similar to CHO-Mas cells, A-779 abrogated Ang-(1-7) effects in HAECs (Figure 4C).
Receptor Mas Mediates Ang-(1-7)–Stimulated NO Production via Akt-Dependent Pathways
To assess whether Ang-(1-7)/Mas influences eNOS activation via Akt, CHO-Mas and HAEC cells were preincubated with wortmannin (10−5 mol/L; 20 minutes), and effects on eNOS phosphorylation (Ser1177) were determined. As shown in Figure 5A and 5B, wortmannin blocked Ang-(1-7)–stimulated Ser1177 phosphorylation, suggesting Akt participation in Ang-(1-7)–stimulated eNOS activation.
Ang-(1-7) Stimulates Phosphorylation of Akt
To verify that Ang-(1-7) induces activation of Akt and that this effect is mediated via Mas, we assessed effects of Ang-(1-7) on Akt phosphorylation in CHO-Mas cells. As demonstrated in Figure 6, within 1 minute of stimulation, Ang-(1-7) significantly increased phosphorylation of Akt (P<0.05). These effects were blocked by A-779.
Major findings from the present study demonstrate the following: (1) human endothelial cells possess GPCR Mas through which Ang-(1-7) mediates activation of eNOS; (2) Ang-(1-7) differentially regulates phosphorylation of Ser1177 and Thr495; and (3) the PI3K/Akt pathway plays an important role in Ang-(1-7)–mediated eNOS activation and NO release. These data suggest that Ang-(1-7) regulates endothelial cell function through functionally active Mas, which stimulates eNOS activity and NO production through Akt-dependent pathways.
Dual phosphorylation of Ser1177 and Thr495 determines the active state of eNOS in endothelial cells.13 eNOS is basally phosphorylated on Thr495 and only weakly phosphorylated on Ser1177 in resting conditions. Rapid changes in the phosphorylation of eNOS at these residues precede its activation in response to stimulation of endothelial cells with agonists such as bradykinin, histamine, estrogen, and ionomycin.11,22,23 However, effects of Ang-(1-7) on eNOS phosphorylation and the role of Mas are unknown. Our data show for the first time that Ang-(1-7) regulates Ser1177/Thr495 phosphorylation and increases NO synthase activity and NO production. Phosphorylation of eNOS at Ser1177 by agonists acting through G protein–coupled receptors is transient.11,12 In contrast, our results indicate that the pronounced phosphorylation of Ser1177 in Ang-(1-7)–stimulated cells was sustained for ≥30 minutes, indicating possible long-lasting eNOS activation.
The eNOS phosphorylation and NO release induced by Ang-(1-7) in Mas-transfected cells were abrogated by the Mas receptor antagonist A-779. A-779 had a slight agonistic effect on the Ser1177 phosphorylation. Partial agonism is a common feature of peptidic antagonists and has also been observed for A-779 in some preparations.24 Studies in Mas-transfected cells ruled out involvement of the other Ang receptors, because these cells do not express AT1R or AT2R constitutively. Evidence for the involvement of receptor Mas in the NO-releasing activity has also been suggested in in vivo studies using Mas knockout mice. In aorta of Mas-deficient mice, endothelium-dependent vasodilator effects of Ang-(1-7) are abolished.18 In support of this, our study demonstrated that Ang-(1-7) did not induce NO release or eNOS phosphorylation in nontransfected cells, which do not possess the GPCR Mas.
Novel findings from our study demonstrate that human endothelial cells constitutively express Mas, which provides a physiological basis for the effects of Ang-(1-7) in human vessels. In support of the results obtained in Mas-transfected cells, we found that pretreatment of HAECs with the Mas receptor antagonist A-779 also inhibited the eNOS phosphorylation. Thus, as demonstrated previously in Mas knockout mice, this receptor seems to be essential for vascular biological actions of Ang-(1-7).18,25,26 Although we cannot exclude an interaction of Mas with Ang II type 1 or type 2 receptors,27,28 we have obtained enough evidence in this study to suggest a primary role for Mas, at least, in Ang-(1-7)–induced NO release.
In keeping with our results, Santos et al14 demonstrated that human endothelial cells can generate Ang-(1-7) from radiolabeled Ang I or Ang II. Heitsch et al showed that Ang-(1-7) increases NO production in endothelial cells.16 Moreover, Faria-Silva et al17 recently demonstrated in vivo that NO is involved in Ang-(1-7)–mediated improvement of endothelial function. Thus, human endothelium is capable of generating Ang-(1-7) and responds functionally to this peptide by releasing NO through Mas.
Several studies have shown that Ang-(1-7) does not increase intracellular calcium concentration in many cell types, including endothelial cells.16 Thus, we hypothesized that its effect on NO production could be mediated by the calcium-independent pathway PI3K/Akt. Consistent with our hypothesis, we found that Akt is rapidly activated by Ang-(1-7)/Mas and that it is an upstream signaling modulator for Ang-(1-7)–stimulated eNOS activation. Wortmannin, a PI3K inhibitor, blocked the Ang-(1-7)–induced Akt and eNOS phosphorylation both in CHO-Mas cells and HAECs. The Akt/protein kinase B pathway is important for phosphorylation of eNOS Ser1177 as observed in response to fluid shear stress, vascular endothelial growth factor, and estrogen.9,10,23 Although bradykinin-induced phosphorylation of eNOS is partially dependent on the PI3K/Akt pathway, it has been suggested that eNOS regulation by GPCRs, such as B2-bradykinin receptor, is more dependent on calmodulin KII and a calcium-dependent pathway.11 In contrast, our results clearly suggest that Ang-(1-7) regulates eNOS through a PI3K/Akt-sensitive pathway. In contrast to what was reported by Heitsch et al,16 the response to Ang-(1-7), in both CHO and HAECs was completely blocked by A779, which does not interfere with B2 receptor-mediated responses.29 Furthermore, Ang-(1-7) failed to increase NO production in B2-expressing cells. Therefore, unlike what was reported in bovine endothelial cells, we have no evidence in CHO cells or HAECs that Ang-(1-7) effects are mediated through B2 receptors. Previous studies demonstrated that Ang-(1-7)–stimulated NO release occurs at baseline concentrations of intracellular calcium.16 Hence, it can be postulated that the Ang-(1-7) stimulation of eNOS at Ser1177 by Akt, in addition to its concomitant effect on thr495 dephosphorylation, which facilitates calmodulin binding, may be a major mechanism for endothelial NO production.
In summary, findings from the present study suggest that the GPCR-Mas mediates Ang-(1-7)–stimulated NO production in human endothelial cells via PI3K/Akt-dependent pathways, which induce changes in eNOS phosphorylation. Our results highlight the importance of the Ang-(1-7)/Mas axis as a potential regulator of endothelial function.
Growing evidence indicates that Ang-(1-7) causes endothelial-dependent vasodilation mediated, in part, by NO release. In addition, Ang-(1-7) seems to counteract many of the effects elicited by Ang II. These events may be important in maintaining endothelial function and vascular integrity. Molecular mechanisms involved in endothelial NO generation by Ang-(1-7) are elusive. In the present study, we demonstrate that in human endothelial cells, which possess GPCR Mas, Ang-(1-7) plays an important role in eNOS activation mediated, in part, through PI3K/Akt-dependent pathways. Our findings highlight the potential importance of the Ang-(1-7)–Mas–Akt–eNOS cascade in the regulation of endothelial function. Dysregulation of this pathway in pathological conditions, such as in hypertension, may contribute to endothelial dysfunction and vascular remodeling.
Dr Jean-Phillipe Gratton is thanked for his help with the NO measurements. Dr João Bosco Pesquero is thanked for the transfected cells.
Sources of Funding
This study was supported by grant 44018 from the Canadian Institutes for Health Research. W.O.S. was supported by a fellowship from the CNPq.
- Received June 26, 2006.
- Revision received July 17, 2006.
- Accepted October 27, 2006.
Berry C, Touyz R, Dominiczak AF, Webb RC, Johns DG. Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide. Am J Physiol Heart Circ Physiol. 2001; 281: H2337–H2365.
Fulton D, Gratton JP, Sessa WC. Post-translational control of endothelial nitric oxide synthase: why isn’t calcium/calmodulin enough? J Pharmacol Exp Ther. 2001; 299: 818–824.
Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC. Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem. 2001; 276: 16587–16591.
Tallant EA, Lu X, Weiss RB, Chappell MC, Ferrario CM. Bovine aortic endothelial cells contain an angiotensin-(1-7) receptor. Hypertension. 1997; 29: 388–393.
Faria-Silva R, Duarte FV, Santos RA. Short-term angiotensin(1-7) receptor MAS stimulation improves endothelial function in normotensive rats. Hypertension. 2005; 46: 948–952.
Santos RA, Simões e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I, Heringer-Walther S, Pinheiro SV, Lopes MT, Bader M, Mendes EP, Lemos VS, Campagnole-Santos MJ, Schultheiss HP, Speth R, Walther T. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc Natl Acad Sci U S A. 2003; 100: 8258–8263.
Diep QN, Intengan HD, Schiffrin EL. Endothelin-1 attenuates omega-3 fatty acid–induced apoptosis by inhibition of caspase 3. Hypertension. 2000; 35: 287–291.
Nakatsubo N, Kojima H, Sakurai K, Kikuchi K, Nagoshi H, Hirata Y, Akaike T, Maeda H, Urano Y, Higuchi T, Nagano T. Improved nitric oxide detection using 2,3-diaminonaphthalene and its application to the evaluation of novel nitric oxide synthase inhibitors. Biol Pharm Bull. 1998; 21: 1247–1250.
Sessa WC, Garcia-Cardena G, Liu J, Keh A, Pollock JS, Bradley J, Thiru S, Braverman IM, Desai KM. The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide. J Biol Chem. 1995; 270: 17641–17644.
Santos RA, Castro CH, Gava E, Pinheiro SV, Almeida AP, de Paula RD, Cruz JS, Ramos AS, Rosa KT, Irigoyen MC, Bader M, Alenina N, Kitten GT, Ferreira AJ. Impairment of in vitro and in vivo heart function in angiotensin-(1-7) receptor Mas knockout mice. Hypertension. 2006; 47: 996–1002.
Kostenis E, Milligan G, Christopoulos A, Sanchez-Ferrer CF, Heringer-Walther S, Sexton PM, Gembardt F, Kellett E, Martini L, Vanderheyden P, Schultheiss HP, Walther T. G-protein-coupled receptor Mas is a physiological antagonist of the angiotensin II type 1 receptor. Circulation. 2005; 111: 1806–1813.
Castro CH, Santos RA, Ferreira AJ, Bader M, Alenina N, Almeida AP. Evidence for a functional interaction of the angiotensin-(1-7) receptor Mas with AT1 and AT2 receptors in the mouse heart. Hypertension. 2005; 46: 937–942.
Santos RA, Campagnole-Santos MJ, Baracho NC, Fontes MA, Silva LC, Neves LA, Oliveira DR, Caligiorne SM, Rodrigues AR, Gropen Junior C. Characterization of a new angiotensin antagonist selective for angiotensin-(1-7): evidence that the actions of angiotensin-(1-7) are mediated by specific angiotensin receptors. Brain Res Bull. 1994; 35: 293–298.