Adenoviral Delivery of Angiotensin-(1-7) or Angiotensin-(1-9) Inhibits Cardiomyocyte Hypertrophy via the Mas or Angiotensin Type 2 Receptor

The counter-regulatory axis of the renin angiotensin system peptide angiotensin-(1-7) [Ang-(1-7)] has been identified as a potential therapeutic target in cardiac remodelling, acting via the mas receptor. Furthermore, we recently reported that an alternative peptide, Ang-(1-9) also counteracts cardiac remodelling via the angiotensin type 2 receptor (AT₂R). Here, we have engineered adenoviral vectors expressing fusion proteins which release Ang-(1-7) [RAdAng-(1-7)] or Ang-(1-9) [RAdAng-(1-9)] and compared their effects on cardiomyocyte hypertrophy in rat H9c2 cardiomyocytes or primary adult rabbit cardiomyocytes, stimulated with angiotensin II, isoproterenol or arg-vasopressin. RAdAng-(1-7) and RAdAng-(1-9) efficiently transduced cardiomyocytes, expressed fusion proteins and secreted peptides, as demonstrated by western immunoblotting and conditioned media assays. Furthermore, secreted Ang-(1-7) and Ang-(1-9) inhibited cardiomyocyte hypertrophy (Control = 168.7±8.4 µm; AngII = 232.1±10.7 µm; AngII+RAdAng-(1-7) = 186±9.1 µm, RAdAng-(1-9) = 180.5±9 µm; P<0.05) and these effects were selectively reversed by inhibitors of their cognate receptors, the mas antagonist A779 for RAdAng-(1-7) and the AT₂R antagonist PD123,319 for RAdAng-(1-9). Thus gene transfer of Ang-(1-7) and Ang-(1-9) produces receptor-specific effects equivalent to those observed with addition of exogenous peptides. These data highlight that Ang-(1-7) and Ang-(1-9) can be expressed via gene transfer and inhibit cardiomyocyte hypertrophy via their respective receptors. This supports applications for this approach for sustained peptide delivery to study molecular effects and potential gene therapeutic actions.     

The Angiotensin-(1-7)/Mas Receptor Axis Is Expressed in Sinoatrial Node Cells of Rats

The authors’ previous studies have indicated that angiotensin(Ang)-(1-7) protects the heart against reperfusion arrhythmias. The aim of this study was to determine whether a functional angiotensin-converting enzyme2 (ACE2)/Ang-(1-7)/Mas receptor axis is present in the sinoatrial node (SAN) of Wistar rats. SAN cells were identified by Masson’s trichrome staining, HCN4 expression, and lack of connexin43 expression. Immunohistochemistry technique was used to detect the expression of ACE2, Ang-(1-7), and Mas in the SAN. To evaluate the role of this axis in the SAN function, atrial tachyarrhythmias (ATs) were induced in isolated rat atria perfused with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1-7). The specific Mas antagonist, A-779, was used to evaluate the role of Mas in the Ang-(1-7) effects. The findings showed that all components of the ACE2/Ang-(1-7)/Mas branch are present in the SAN of rats. Importantly, it was found that this axis is functional because perfusion of atria with Ang-(1-7) significantly reduced the duration of ATs. Additionally, this anti-arrhythmogenic effect was attenuated by A-779. No significant changes were observed in heart rate, contractile tension, or ±dT/dt. These observations demonstrate that the ACE2/Ang-(1-7)/Mas axis is expressed in SAN cells of rats. They provide the morphological support to the anti-arrhythmogenic effect of Ang-(1-7).     

G蛋白偶联受体转激活酪氨酸激酶受体机制

G蛋白偶联受体(G-protien coupled receptors,GPCRs)和酪氨酸激酶受体(receptor tyrosine kinases,RTKs)是体内两类重要的受体家族,介导着绝大多数信号事件。GPCRs能够"绑架"RTKs进行信号转导,即GPCRs能够在没有外加RTKs配体的情况下激活RTKs,这种现象称为转激活。作为转激活的核心过程,GPCR调控RTK磷酸化主要采取RTK配体依赖模式和非RTK配体依赖模式。不同的G蛋白亚型、酪氨酸磷酸激酶、酪氨酸磷酸酶(protein-tyrosine phosphatases,PTPs)以及活性氧自由基(reactiveoxygen species,ROS)均在此过程中具有重要作用。GPCR和RTK还能形成信号复合体(signaling complex)从而实现蛋白质之间的动态相互作用。对转激活的研究为GPCR靶点药物开发提供了新思路。     

Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases

Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed α-helical bundle through their N-terminal double GG4-like motif T⁶⁴⁸G⁶⁴⁹X₂G⁶⁵²A⁶⁵³ and glycine zipper motif T⁶⁵²X₃S⁶⁵⁶X₃G⁶⁶⁰, respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Huntingtin-Interacting Protein 1 Phosphorylation by Receptor Tyrosine Kinases

Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine “HIP1 phosphorylation motif” (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1''s only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1''s role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival.     

Leucine-rich Repeat and Immunoglobulin Domain-containing Protein-1 (Lrig1) Negative Regulatory Action toward ErbB Receptor Tyrosine Kinases Is Opposed by Leucine-rich Repeat and Immunoglobulin Domain-containing Protein 3 (Lrig3)

Lrig1 is the founding member of the Lrig family of transmembrane leucine-rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of the mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3''s positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.     

Rational Design and Cyclization of MIG6 Peptide to Restore its Binding Affinity for ErbB Family Receptor Tyrosine Kinases

The ErbB receptor tyrosine kinase family consists of four members: EGFR/Her1, Her2, Her3 and Her4; they have been involved in a variety of malignant tumors. The mitogen-inducible gene 6 (MIG6) is a natural tumor-suppressor protein that can inactivate ErbB signaling by directly binding to the catalytic domain of ErbB kinases. Here, a peptide segment s2p was stripped from the MIG6 interaction interface with ErbB, which exhibited a very low or no affinity to the four ErbB kinases. Structural dynamics simulations revealed that the linear peptide is highly flexible in unbound state and would incur a considerable entropy penalty upon binding to kinases. In this respect, the s2p peptide was cyclized by rational design of a disulfide bond across its two termini, resulting in a cyclic peptide s2p-c. Integration of computational analysis and experimental assay found that the cyclization can largely constrain s2p conformation, thus minimizing the entropy penalty and restoring the binding affinity of s2p-c to kinases.     

Angiotensin-(1-7): an update

The renin-angiotensin system is a major physiological regulator of arterial pressure and hydro-electrolyte balance. Evidence has now been accumulated that in addition to angiotensin (Ang) II other Ang peptides [Ang III, Ang IV and Ang-(1-7)], formed in the limited proteolysis processing of angiotensinogen, are importantly involved in mediating several actions of the RAS. In this article we will review our knowledge of the biological actions of Ang-(1-7) with focus on the puzzling aspects of the mediation of its effects and the interaction Ang-(1-7)-kinins. In addition, we will attempt to summarize the evidence that Ang-(1-7) takes an important part of the mechanisms aimed to counteract the vasoconstrictor and proliferative effects of Ang II.     

Angiotensin-(1-7) receptor Mas is an essential modulator of extracellular matrix protein expression in the heart

In this study we investigated the effects of genetic deletion of the Angiotensin-(1-7) receptor Mas or the Angiotensin II receptor AT(2) on the expression of specific extracellular matrix (ECM) proteins in atria, right ventricles and atrioventricular (AV) valves of neonatal and adult mice. Quantification of collagen types I, III and VI and fibronectin was performed using immunofluorescence-labeling and confocal microscopy. Picrosirius red staining was used for the histological assessment of the overall collagen distribution pattern. ECM proteins, metalloproteinases (MMP), ERK1/2 and p38 levels were quantified by western blot analysis. Gelatin zymography was used to evaluate the activity of MMP-2 and MMP-9. We observed that the relative levels of collagen types I and III and fibronectin are significantly higher in both the right ventricle and AV valves of neonatal Mas(-/-) mouse hearts (e.g., collagen type I: 85.28±6.66 vs 43.50±4.41 arbitrary units in the right ventricles of Mas(+/+) mice). Conversely, the level of collagen type VI was lower in the right ventricle and AV valves of Mas(-/-) mice. Adult Mas(-/-) mouse hearts presented similar patterns as observed in neonates. No significant differences in ECM protein level were detected in atria. Likewise, no changes in ECM levels were observed in AT(2) knockout mouse hearts. Although deletion of Mas induced a significant reduction in the level of the active form of MMP-2 in neonate hearts and a reduction of both MMP-2 and MMP-9 in adult Mas(-/-) mice, no significant differences were observed in MMP enzymatic activities when compared to controls. The levels of the active, phosphorylated forms of ERK1/2 and p38 were higher in hearts of both neonatal and adult Mas(-/-) mice. These observations suggest that Mas is involved in the selective expression of specific ECM proteins within both the ventricular myocardium and AV valves. The changes in the ECM profile may alter the connective tissue framework and contribute to the decreased cardiac performance observed in Mas(-/-) mice.     

Angiotensin-(1-7) increases neuronal potassium current via a nitric oxide-dependent mechanism

Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.     

Angiotensin-(1-7) prevents diabetes-induced cardiovascular dysfunction

The aim of this study was to test the hypothesis that treatment with angiotensin-(1-7) [ANG-(1-7)] or ANG-(1-7) nonpeptide analog AVE-0991 can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(1-7) (576 microg.kg(-1).day(-1) ip) or AVE-0991 (576 microg.kg(-1).day(-1) ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(1-7) or AVE-0991. There was a significant increase in urine protein (231 +/- 2 mg/24 h) in diabetic animals compared with controls (88 +/- 6 mg/24 h). Treatment of diabetic animals with ANG-(1-7) or AVE-0991 resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (183 +/- 16 and 149 +/- 15 mg/24 h, respectively). Treatment with ANG-(1-7) or AVE-0991 also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-1, angiotensin II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or AVE-0991-treated animals. These results suggest that activation of ANG-(1-7)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.     

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, 10 μM) stimulated high pacing frequency-induced ANP secretion in a dose-dependent manner. Ang-(1-5)-induced ANP secretion (3 μM) 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.     

Stabilization of Angiotensin-(1-7) in Cardioprotective Solutions

Angiotensin-(1-7) (Ang 1-7) has been previously studied in combination with an antioxidant containing preparation as a cardioprotective reperfusion solution. In this study a stability improvement of aqueous Ang 1-7 solutions was observed. However, no data was provided on the responsibilities and causes of the noticed stability enhancement. Therefore, the influence of pH and pharmaceutical additives as well as the effect of the single specific agents present in the antioxidant preparation such as α-ketoglutaric acid (α-KG), 5-hydroxymethylfurfural (5-HMF), N-acetyl-seleno-l-methionine (NASeLM) and N-acetyl-l-methionine (NALM) on the stability was evaluated. Analyses were performed by an HPLC method with fluorescence detection. Crucial instability was found in a pH range of 5.0–7.5 without addition of the antioxidative mixture. Zetasizing confirmed the presence of microparticles and MS studies showed no degradation products within 25 days. 5-HMF was identified as main component for stability enhancement of Ang 1-7 solution. By adding this substance the stability of the cardioprotective peptide solution can be prolonged and appears as a promising approach for transplant purposes.

     

血管紧张素(1-7)的受体MAS与PSD-95的相互作

经过对蛋白质MAS的氨基酸序列进行分析、比对发现,其羧基末端含有一段高度保守的PDZ结合模序.据此推测,MAS通过此模序可能会与某些PDZ蛋白质发生相互作用.将MAS羧基末端27个氨基酸所对应的DNA序列,克隆到原核表达载体pGEX-4T-1中,构建重组质粒pGEX-MAS-CT.将重组质粒转入大肠杆菌BL21内,经IPTG诱导,并用glutathione-Sepharose 4B纯化,得到纯化的融合蛋白GST-MAS-CT.以GST-MAS-CT为饵蛋白,利用GST pull down方法,在兔脑组织中筛选与MAS特异结合的蛋白质.结果表明,在兔脑组织中有多种蛋白质可以与MAS-CT特异结合,经Western印迹检验其中之一为突触后致密物质-95 (postsynaptic density protein 95, PSD -95).PSD-95与MAS的相互作用的研究结果,为研究完整的MAS与PSD-95的相互作用以及这种相互作用对MAS受体的功能的影响奠定了基础.     

Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.     

Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases

The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery.At the cellular level, receptor tyrosine kinases (RTKs) need to be properly localized to function as signal-receiving and signal-transmitting devices (Lemmon and Schlessinger 2010). To receive signals (i.e., to bind extracellular ligands), RTKs have to be exposed at the surface of the plasma membrane. To transmit signals after ligand binding by RTKs, appropriate signaling components have to be available within intracellular compartments: in the cytoplasm, in association with membrane-bound organelles and in the cell nucleus. Importantly, the intracellular distribution of RTKs and their associated partners is not static but undergoes dynamic changes in different phases of signaling, as reflected for example by endocytic internalization of activated RTKs (Scita and Di Fiore 2010). Therefore, to function properly, the whole RTK signaling machinery within the cell has to be organized and tightly controlled both in space and in time. This organization and control are ensured by intracellular trafficking machineries, mainly by membrane transport systems such as endocytosis and secretion but also by other distribution systems (e.g., responsible for nucleocytoplasmic shuttling of proteins).Recent years have brought increasing evidence that intracellular membrane trafficking, in particular endocytic internalization, degradation, and recycling, can profoundly affect the signaling properties of RTKs (Mukherjee et al. 2006; Abella and Park 2009; Lemmon and Schlessinger 2010; Scita and Di Fiore 2010; Grecco et al. 2011; Sigismund et al. 2012). The changes in the amounts of RTKs at the cell surface can alter the cellular responses when ligands are abundant (Grecco et al. 2011). In turn, the presence of a given RTK at the plasma membrane is determined by the rates of three trafficking processes: delivery of newly synthesized molecules by the secretory pathway, their internalization (occurring for both ligand-bound and ligand-free molecules), and endocytic recycling. Although the molecular details concerning the regulation of RTK delivery to the plasma membrane are not well known, numerous studies document various mechanisms by which internalization and recycling of RTKs can be modulated, thus affecting the signaling outputs (Le Roy and Wrana 2005). In addition to the regulation of RTKs at the cell surface, trafficking processes control the intracellular fate of endocytosed RTKs. Following internalization, RTKs can be either targeted for lysosomal degradation, or recycled back to the plasma membrane (Mukherjee et al. 2006; Abella and Park 2009; Scita and Di Fiore 2010). The first route results in the termination of signaling, whereas the second allows for sustained signaling if the ligand is available. Usually degradation and recycling of a given RTK can occur simultaneously but the balance between them is crucial to determine the net signaling output. Again, the molecular mechanisms that can shift the fate of internalized RTKs between degradation and recycling, thus changing RTK signaling, have begun to emerge in recent years (Polo and Di Fiore 2006; von Zastrow and Sorkin 2007; Sorkin and von Zastrow 2009; Sigismund et al. 2012). Finally, in contrast to an early view that only RTKs present at the plasma membrane are signaling competent, it is now accepted that in many cases activated RTKs can emit signals also after internalization into intracellular compartments (Miaczynska et al. 2004b; Miaczynska and Bar-Sagi 2010; Platta and Stenmark 2011). In some cell types (e.g., in neurons), such “signaling endosomes” are crucial for signal propagation within the cell and for the final cellular response. Moreover, endosomes can serve as platforms for amplification and compartmentalization of signals emitted by RTKs (Sadowski et al. 2009; Platta and Stenmark 2011).In this article, I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. As the membrane trafficking system of a cell is highly interconnected and can be considered a global dynamic continuum, it is important to note that often one primary alteration at a given stage of RTK trafficking may affect other transport steps or compartments, thus causing generalized changes in the intracellular routing and signaling of RTKs.     

The Mas receptor mediates modulation of insulin signaling by angiotensin-(1-7)

Angiotensin (Ang)-(1-7) stimulates proteins belonging to the insulin signaling pathway and ameliorates the Ang II negative effects at this level. However, up to date, receptors involved and mechanisms behind these observations remain unknown. Accordingly, in the present study, we explored the in vivo effects of antagonism of the Ang-(1-7) specific Mas receptor on insulin signal transduction in rat insulin-target tissues. We evaluated the acute modulation of insulin-stimulated phosphorylation of Akt, GSK-3β (Glycogen synthase kinase-3β) and AS160 (Akt substrate of 160kDa) by Ang-(1-7) and/or Ang II in the presence and absence of the selective Mas receptor antagonist A-779 in insulin-target tissues of normal rats. Also using A-779, we determined whether the Mas receptor mediates the improvement of insulin sensitivity exerted by chronic Ang-(1-7) treatment in fructose-fed rats (FFR), a model of insulin resistance, dyslipidemia and mild hypertension. The two major findings of the present work are as follows; 1) Ang-(1-7) attenuates acute Ang II-mediated inhibition of insulin signaling components in normal rats via a Mas receptor-dependent mechanism; and 2). The Mas receptor appears to be involved in beneficial effects of Ang-(1-7) on the phosphorylation of crucial insulin signaling mediators (Akt, GSK-3β and AS160), in liver, skeletal muscle and adipose tissue of FFR. These results shed light into the mechanism by which Ang-(1-7) exerts its positive physiological modulation of insulin actions in classical metabolic tissues and reinforces the central role of Akt in these effects.     

Angiotensin-(1-7) stimulates oxidative stress in rat kidney

The effect of two different doses of angiotensin-(1-7) and angiotensin II on the oxidative stress generation was analyzed in rat kidney. Animals were injected intraperitoneally with a single dose of angiotensin-(1-7) or angiotensin II (20 or 50 nmol/kg body weight) and killed 3 h after injection. Production of thiobarbituric acid reactive substances (TBARS), measured as indicator of oxidative stress induction, was significantly increased in rat kidney after Ang-(1-7) administration up to 30% and 50% over controls, at 20 and 50 nmol/kg, respectively. Reduced glutathione (GSH), the most important soluble antioxidant defense in mammalian cells, showed a significant decrease of 13% and 20% at 20 and 50 nmol/kg of angiotensin-(1-7), respectively. When the antioxidant enzyme activities were determined, it was found that catalase activity was not altered by the assayed angiotensin-(1-7) doses while superoxide dismutase and glutathione peroxidase activities were significantly reduced by injection of 20 nmol/kg (34% and 13%, with respect to controls) and 50 nmol/kg of angiotensin-(1-7) (54% and 22%, respectively). In contrast, angiotensin II injections did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two doses used in this work. These results suggest that angiotensin-(1-7) is undoubtedly related to oxidative stress induction.