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.     

Angiotensin-(1-7) suppresses oxidative stress and improves glucose uptake via Mas receptor in adipocytes

Although reactive oxygen species (ROS) contribute to glucose intolerance induced by the renin-angiotensin system (RAS) is well documented, the role of the newly discovered pathway of RAS, angiotensin (Ang)-(1-7)/Mas axis, in this process remains unknown. Here, we examined the effect of Ang-(1-7) on oxidative stress and glucose uptake in adipocytes. We used primary cultured epididymal adipocytes from C57 mice to study Ang-(1-7) effects on glucose uptake. We also treated fully differentiated 3T3-L1 adipocytes with exogenous Ang-(1-7) or overexpression of angiotensin-converting enzyme 2 (ACE2) to induce endogenous generation of Ang-(1-7) to clarify its effects on ROS production. Intracellular ROS was measured by flow cytometry, dihydroethidium (DHE), and nitroblue tetrazolium assay. Levels of NADPH oxidase and adiponectin mRNA were measured by real-time PCR. Ang-(1-7) improved glucose uptake both in basal and insulin-stimulated states. ROS production was slightly but significantly decreased in adipocytes treated with Ang-(1-7). Additionally, Mas receptor antagonist D-Ala7-Ang-(1-7) (A779) reversed the effect of Ang-(1-7) on glucose uptake and oxidative stress. Furthermore, treatment of adipocytes with Ang-(1-7) decreased NADPH oxidase mRNA levels. We also found that oxidative stress induced by glucose oxidase-suppressed expression of adiponectin, an insulin-sensitive protein. However, the suppression of oxidative stress by Ang-(1-7) restored adiponectin expression, while A779 agonists these changes induced by Ang-(1-7). In conclusion, Ang-(1-7) can protect against oxidative stress and improve glucose metabolism in adipocytes. These results show that Ang-(1-7) is a novel target for the improvement of glucose metabolism by preventing oxidative stress.     

Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6

EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [(32)P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [(32)P]SPP was saturable, demonstrating high affinity (K(D) = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a G(i/o) protein, resulting in the activation of growth-related signaling pathways. (Blood. 2000;95:2624-2629)     

Nonpeptide AVE 0991 is an angiotensin-(1-7) receptor Mas agonist in the mouse kidney

It has been described recently that the nonpeptide AVE 0991 (AVE) mimics the effects of angiotensin-(1-7) [Ang-(1-7)] in bovine endothelial cells. In this study, we tested the possibility that AVE is an agonist of the Ang-(1-7) receptor Mas, in vitro and in vivo. In water-loaded C57BL/6 mice, AVE (0.58 nmol/g body weight) produced a significant reduction in urinary volume (0.06+/-0.03 mL/60 min [n=9] versus 0.27+/-0.05 [n=9]; P<0.01), associated with an increase in urinary osmolality. The Ang-(1-7) antagonist A-779 completely blocked the antidiuretic effect of AVE. As observed previously for Ang-(1-7), the antidiuretic effect of AVE after water load was blunted in Mas-knockout mice (0.37+/-0.10 mL/60 min [n=9] versus 0.27+/-0.03 mL/60 min [n=11] AVE-treated mice). In vitro receptor autoradiography in C57BL/6 mice showed that the specific binding of 125I-Ang-(1-7) to mouse kidney slices was displaced by AVE, whereas no effects were observed in the binding of 125I-angiotensin II or 125I-angiotensin IV. Furthermore, AVE displaced the binding of 125I-Ang-(1-7) in Mas-transfected monkey kidney cells (COS) cells (IC50=4.75x10(-8) mol/L) and of rhodamine-Ang-(1-7) in Mas-transfected Chinese hamster ovary (CHO) cells. It also produced NO release in Mas-transfected CHO cells blocked by A-779 but not by angiotensin II type-1 (AT1) and AT2 antagonists. Contrasting with these data, the antidiuretic effect of AVE was totally blocked by AT2 antagonists and partially blocked (approximately 60%) by AT1 antagonists. The binding data, the results obtained in Mas-knockout mice and in Mas-transfected cells, show that AVE is a Mas receptor agonist. Our data also suggest the involvement of AT2/AT1-related mechanisms, including functional antagonism, oligomerization or cross-talk, in the renal responses to AVE.     

Opportunities for Targeting the Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Receptor Pathway in Hypertension

It is well known that the renin-angiotensin system (RAS) plays a pivotal role in the pathophysiology of cardiovascular diseases. This is well illustrated by the great success of ACE inhibitors and angiotensin (Ang) II AT₁ blockers in the treatment of hypertension and its complications. In the past decade, the classical concept of RAS orchestrated by a series of enzymatic reactions culminating in the linear generation and action of Ang II has expanded and become more complex. From the discoveries of new components such as the angiotensin converting enzyme 2 and the receptor Mas emerged a novel concept of dual opposite branches of the RAS: one vasoconstrictor and pro-hypertensive composed of ACE/Ang II/AT1; and other vasodilator and anti-hypertensive composed of ACE2/Ang-(1-7)/Mas. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1-7)/Mas arm in the cardiovascular system and highlight the initiatives to develop potential therapeutic strategies based on this axis for treating hypertension.     

血管紧张素-(1-7)与心血管疾病

血管紧张素系统（RAS）作为一种循环激素参与血压调节与盐类代谢,并维持内环境稳态.血管紧张素-（1-7）[Ang-（1-7）]是RAS系统中的一个独立代谢物,且是具有生物活性的七肽.Ang-（1-7）分别由血管紧张素Ⅰ及血管紧张素Ⅱ转化而来.近年来的研究发现其作为血管紧张素Ⅱ的拮抗因子,具有舒张血管、利钠利尿、抑制血管平滑肌增生等多种效应,对心血管基础研究及临床实践有重要意义[1].本文就近年血管紧张素-（1-7）与心血管疾病的研究现状作一简要综述.     

血管紧张素(1-7)研究新进展

肾素-血管紧张素系统(RAS)一直是高血压病因研究中的焦点问题. 迄今为止,已从整体动物平,细胞水平直至生物分子水平充分论证了这一系统在高血压发病过程中所起的重要作用. 近年来,随着实践的积累,人们对RAS有了更加完善,全面的了解.血管紧张素(1-7)(Ang( 1 -7))便是血管紧张素家族中被重新认识和评价的一个成员.本文现将近年来有关Ang(1-7 )的一些研究情况简述如下.     

Angiotensin-(1-7) as an antihypertensive, antifibrotic target

Over the past two decades, enormous progress has been made in understanding the possible physiological significance of alternate renin-angiotensin system processing pathways and angiotensin fragments, such as angiotensin (Ang)-(1-7). Evidence from in vivo and ex vivo studies in humans and various animal models suggests a possible role for this heptapeptide in blood pressure regulation, although the mechanisms involved are most likely indirect, involving some combination of bradykinin and nitric oxide signaling. In contrast, a growing body of in vivo and in vitro evidence supports direct cardioprotective (antihypertrophic, antifibrotic) actions of Ang-(1-7). Here, we review key studies investigating the blood pressure and tissue-protective roles of Ang-(1-7), and summarize potential genomic and pharmacologic therapeutic strategies previously advanced by our group and others.     

血管紧张素-(1-7)的心血管调节作用

血管紧张素-(1-7)是肾素血管紧张素系统家族中的新成员,近来研究提示其有抗高血压、抑制血管平滑肌增殖和心肌细胞肥大、影响心脏神经电生理、抗血栓形成、调节水、电解质代谢平衡等作用,是血管紧张素Ⅱ(AngⅡ)的内源性拮抗剂。     

Angiotensin-(1-7) is a modulator of the human renin-angiotensin system.

The renin-angiotensin system is important for cardiovascular homeostasis. Currently, therapies for different cardiovascular diseases are based on inhibition of angiotensin-converting enzyme (ACE) or angiotensin II receptor blockade. Inhibition of ACE blocks metabolism of angiotensin-(1-7) to angiotensin-(1-5) and can lead to elevation of angiotensin-(1-7) levels in plasma and tissue. In animal models, angiotensin-(1-7) itself causes or enhances vasodilation and inhibits vascular contractions to angiotensin II. The function of angiotensin-(1-5) is unknown. We investigated whether angiotensin-(1-7) and angiotensin-(1-5) inhibit ACE or antagonize angiotensin-induced vasoconstrictions in humans. ACE activity in plasma and atrial tissue was inhibited by angiotensin-(1-7) up to 100%, with an IC(50) of 3.0 and 4.0 micromol/L, respectively. In human internal mammary arteries, contractions induced by angiotensin I and II and the non-ACE-specific substrate [Pro(11),D-Ala(12)]-angiotensin I were antagonized by angiotensin-(1-7) (10(-5) mol/L) in a noncompetitive way, with a 60% inhibition of the maximal response to angiotensin II. Contractions to ACE-specific substrate [Pro(10)]-angiotensin I were also inhibited, an effect only partly accounted for by antagonism of angiotensin II. Angiotensin-(1-5) inhibited plasma ACE activity with a potency equal to that of angiotensin I but had no effect on arterial contractions. In conclusion, angiotensin-(1-7) blocks angiotensin II-induced vasoconstriction and inhibits ACE in human cardiovascular tissues. Angiotensin-(1-5) only inhibits ACE. These results show that angiotensin-(1-7) may be an important modulator of the human renin-angiotensin system.     

血管紧张素-(1-7)研究进展

血管紧张素-(1-7)是肾素-血管紧张素系统中具有重要生物学作用的终末活性产物,可通过与其特异性受体结合而发挥改善心功能、降低血压、抑制细胞增殖、抗血栓、调节水电解质平衡等作用。现就血管紧张素-(1-7)的生成、生物学作用及其作用的受体作一综述。     

ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor

The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca(2+) concentration ([Ca(2+)](i)) remains unknown. We measured [Ca(2+)](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary β-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca(2+)](i) in the form of a peak followed by a plateau dependent on extracellular Ca(2+). NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca(2+)](i). The ADPr-induced [Ca(2+)](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP(3) receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPr-induced [Ca(2+)](i) increase. ADPr increased [Ca(2+)](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors.     

Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT(1)) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT(1) receptor. Ang-(1-7) competed poorly for [(125)I]Ang II binding to the AT(1) receptor on vascular smooth muscle cells, with an IC(50) of 2.0 micromol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [(125)I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 micromol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [B(max) 437.7+/-261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5+/-301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT(1) receptor. Pretreatment with the AT(1) receptor antagonist L-158,809 blocked the reduction in [(125)I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2+/-6.4%, P<0.01, n=5) only at 1 micromol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT(1) receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT(1) receptor or Ang-(1-7)-mediated downregulation of the vascular AT(1) receptor.     

Angiotensin-(1-7) in normal and preeclamptic pregnancy

Angiotensin-(1–7) (Ang-[1–7]) is a bioactive component of the renin-angiotensin system, which has depressor, vasodilatory, and antihypertensive actions. In normal pregnancy, we questioned whether the known rise in plasma angiotensin II (Ang II) is counterbalanced by an increase in plasma Ang-(1–7) and whether Ang-(1–7) levels are decreased in preeclampsia and may thus be a factor involved in the development of hypertension. Nulliparous preeclamptic subjects, third-trimester normotensive pregnant subjects, and a nonpregnant group were enrolled (n=15/group). Preeclamptic subjects had no previous history of hypertension or renal, connective-tissue, or metabolic disease, but at the time of delivery had significant hypertension (159±3/98±3 mmHg) and ≥3+ proteinuria. Plasma Ang-(1–7) was increased by 51% in normal pregnancy (p<0.05). Plasma Ang I, Ang II, and renin activity were also significantly elevated in normal pregnancy. In preeclamptic subjects, Ang-(1–7) was significantly decreased (p<0.01) compared with normal pregnant subjects. All other components of the renin-angiotensin-aldosterone system, except serum angiotensin-converting enzyme, were reduced in preeclamptic subjects compared with normal pregnant subjects; only plasma Ang II remained elevated in preeclamptic compared with nonpregnant subjects. These studies demonstrate, for the first time, increased plasma Ang-(1–7) in normal pregnant subjects compared with nonpregnant subjects and decreased Ang-(1–7) in preeclamptic subjects compared with normal pregnant subjects. In preeclampsia the decreased plasma Ang-(1–7) in the presence of elevated Ang II is consistent with the development of hypertension.     

血管紧张素-(1-7)在心血管系统中的作用

血管紧张素-(1-7)是肾素-血管紧张素系统中具有重要生物学作用的终末活性产物,可通过直接作用于心血管中枢调节心血管活动以及与心脏和血管上特异性受体结合而发挥改善心功能、降低血压、抑制心脏和血管重构作用.现就血管紧张素-(1-7)对心血管系统中的作用综述如下.     

Angiotensin-(1-7) inhibits in vitro endothelial cell tube formation in human umbilical vein endothelial cells through the AT(1-7) receptor

Angiotensin-(1–7) is increased in the circulation during human pregnancy, but its functional role is unknown. Recent studies suggested that it opposes angiotensin II mediated vascular growth. Because angiogenesis is critical to normal embryonic development during human pregnancy, this study assessed the in vitro effects of angiotensin-(1–7) on human umbilical vein endothelial cell tube formation. The blocking effects of the angiotensin-(1–7) receptor antagonist, D-[Alanine⁷]-Ang-(1–7), and angiotensin II receptor AT₁ and AT₂ antagonists, losartan and PD123319, on tube formation were measured by counting tube branch points. Human umbilical vein endothelial cells were cultured in EGM-2 medium and treated with angiotensin-(1–7) (0.17 nM–17 μM) for 18 h. Angiotensin-(1–7) inhibited tube formation by 24% (P < 0.01) at all doses tested. Treatment with 1.7 μM angiotensin-(1–7) plus 17 μM D-[Alanine⁷]-Ang-(1–7) resulted in the reversal of angiotensin-(1–7) mediated inhibition of tube formation (P < 0.05). Losartan (17 μM) also reversed the angiotensin-(1–7) mediated inhibition of tube formation (P < 0.05). Tube formation was unaffected by PD123319. These results suggest that angiotensin-(1–7) has an anti-angiogenic effect on human umbilical vein endothelial cells through a unique AT_1–7 receptor that is sensitive to losartan, indicating that angiotensin-(1–7) may play an important role in the regulation of vascular growth in the placenta during pregnancy.     

Angiotensin-(1-7): a novel peptide in the ovary

The present study was undertaken to investigate the presence of angiotensin-(1-7) [Ang-(1-7)] in the ovary and a possible role for it. Cycling female rats were killed in each phase of the estrous cycle, and ovarian Ang II and Ang-(1-7) were separated by HPLC and measured by RIA. The mean levels of Ang-(1-7) in proestrus and estrus were significantly higher than those in metestrus and diestrus (P < 0.05). Ang-(1-7) was also significantly higher in equine chorionic gonadotropin (eCG)-treated immature rats. Ang-(1-7) induced a significant increase in estradiol and progesterone production (P < 0.05) in the ovary of immature rats (24-25 d old) pretreated with eCG and perfused in a closed circuit system. This effect was blocked by A-779, a specific Ang-(1-7) antagonist (P < 0.05). The present data demonstrate the presence and physiological role of a novel renin-Ang system peptide in the ovary. The higher level of Ang-(1-7) in proestrus and estrus as well as in eCG-treated rats suggests the involvement of this renin-Ang system peptide in pre- and postovulatory events.     

D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor

Functional activity of N-methyl-D-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although D-serine is a more potent agonist. Localizations of D-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of d-serine with D-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch-clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied D-serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus, D-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.     

Angiotensin-(1-7): new perspectives in atherosclerosis treatment

Angiotensin (Ang)-(1-7) is recognized as a new bioactive peptide in renin-angiotensin system (RAS). Ang-(1-7) is a counter-regulatory mediator of Ang-II which appears to be protective against cardiovascular disease. Recent studies have found that Ang-(1-7) played an important role in reducing smooth muscle cell proliferation and migration, improving endothelial function and regulating lipid metabolism, leading to inhibition of atherosclerotic lesions and increase of plaque stability. Although clinical application of Ang-(1-7) is restricted due to its pharmacokinetic properties, identification of stabilized compounds, including more stable analogues and specific delivery compounds, has enabled clinical application of Ang-(1-7). In this review, we discussed recent findings concerning the biological role of Ang-(1-7) and related mechanism during atherosclerosis development. In addition, we highlighted the perspective to develop therapeutic strategies using Ang-(1-7) to treat atherosclerosis.     

Vascular responses to Angiotensin-(1-7) during the estrous cycle

Mesenteric arteries (230–290 μm) were isolated from virgin female rats at diestrous and proestrous phases of the estrous cycle and from ovariectomized (OVX) rats with or without estrogen (E₂) replacement (17β-estradiol, 7.5 + 5 mg pellets, 21 d release). Arteries were mounted in a pressurized myograph system. Angiotensin-(1–7) [Ang-(1–7)] concentration-dependent responses (10⁻¹⁰–10⁻⁵ M) were determined in arteries preconstricted with endothelin-1 (10⁻⁷ M). Mesenteric arteries were pretreated with the specific Ang-(1–7) antagonist, d-[Ala⁷]-Ang-(1–7) (10⁻⁷ M) to assess the Ang-(1–7) receptor-mediated dilator effect. Ang-(1–7) did not dilate mesenteric arteries from virgin rats at diestrus and placebo-treated OVX female rats as compared to the time control; however, Ang-(1–7) elicited a modest dilation at proestrus as compared to diestrus, which reached statistical significance at 10⁻⁸ M concentrations. Ang-(1–7) caused a concentration-dependent vasodilation in mesenteric arteries of females with E₂ replacement, with an EC₅₀ of 21 nM. d-[Ala⁷]-Ang-(1–7) blocked the vasodilator effect of Ang-(1–7). Our results demonstrate that during proestrus Ang-(1–7) elicits modest vasodilation as compared to diestrus, but lacks vasodilatory properties in vessels from diestrous and ovariectomized rats. Estrogen replacement restores a significant dilator response to Ang-(1–7) in OVX rats that is mediated by a d-[Ala⁷]-Ang-(1–7) sensitive site.