RESUMO
Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (ANG II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we used cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were used, alongside confocal microscopy, to detect NO production and NO/Ca2+ cross talk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that ANG II and ANG III generated high levels of NO by activating the angiotensin II type 2 receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to ANG 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.NEW & NOTEWORTHY The renin-angiotensin system plays a crucial role in the production of intracellular nitric oxide within podocytes. This mechanism operates through the activation of the angiotensin II type 2 receptor, leading to dynamic modifications in intracellular calcium levels and the actin filament network. This intricate process is vital for linking the activity of angiotensin receptors to podocyte function.
Assuntos
Angiotensina II , Óxido Nítrico , Podócitos , Receptor Tipo 2 de Angiotensina , Sistema Renina-Angiotensina , Transdução de Sinais , Podócitos/metabolismo , Óxido Nítrico/metabolismo , Sistema Renina-Angiotensina/fisiologia , Animais , Humanos , Angiotensina II/farmacologia , Receptor Tipo 2 de Angiotensina/metabolismo , Células Cultivadas , Angiotensina I/metabolismo , Ratos , Sinalização do Cálcio/efeitos dos fármacos , Masculino , Angiotensina III/metabolismo , Angiotensina III/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologiaRESUMO
The renin-angiotensin system, with the octapeptide angiotensin II as key player, is important in the renal, cardiac and vascular physiology. Prolyl carboxypeptidase (PRCP), prolyl endopeptidase (PREP) and angiotensin converting enzyme 2 (ACE2) are reported to be involved in the conversion of angiotensin II to angiotensin (1-7). Previous investigations showed that the processing of angiotensin II is cell- and species-specific and little is known about its conversion in human endothelial cells. Therefore, we aimed to investigate the C-terminal processing of angiotensin II and III in comparison to the processing of des-Arg9-bradykinin in human endothelial cells. To this end, human umbilical vein and aortic endothelial cells (HUVEC and HAoEC) were incubated with the peptides for different time periods. Mass spectrometry analysis was performed on the supernatants to check for cleavage products. Contribution of PRCP, ACE2 and PREP to the peptide cleavage was evaluated by use of the selective inhibitors compound 8o, DX600 and KYP-2047. The use of these selective inhibitors revealed that the C-terminal cleavage of angiotensin II and III was PRCP-dependent in HUVEC and HAoEC. In contrast, the C-terminal cleavage of des-Arg9-bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. With this study, we contribute to a better understanding of the processing of peptides involved in the alternative renin-angiotensin system. We conclude that PRCP is the main enzyme for the C-terminal processing of angiotensin peptides in human umbilical vein and aortic endothelial cells. For the first time the contribution of PRCP was investigated by use of a selective PRCP-inhibitor.
Assuntos
Angiotensina III/metabolismo , Angiotensina II/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Aorta/metabolismo , Carboxipeptidases/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Angiotensina III/antagonistas & inibidores , Aorta/citologia , Aorta/efeitos dos fármacos , Carboxipeptidases/antagonistas & inibidores , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Peptídeos/farmacologiaRESUMO
Angiotensin III (Ang III) is a heptapeptide derived from Ang II that has been confirmed as the preferred agonist of angiotensin II type 2 receptor (AT2R). Recent studies have revealed AT2R mainly exerts anti-inflammation effects. However, the effects of the Ang III/AT2R pathway on adipocytes remain unknown. Here, the effects of Ang III on glucose uptake were examined. The results showed that AT2R expression was upregulated during adipogenesis in 3T3-L1 preadipocytes, whereas AT1R expression was diminished. Also, Ang III (10 nM) significantly increased glucose uptake by 3T3-L1 adipocytes, which was blocked by PD123319, an AT2R blocker, but not by irbesartan, an AT1R blocker. Ang III also induced the expression of glucose transporter type 1 (GLUT1). These stimulatory effects were inhibited by pretreatment with PD123319, but not with irbesartan. Together, these results indicate that Ang III enhances glucose uptake by upregulating GLUT1 expression via AT2R.
Assuntos
Adipócitos/metabolismo , Angiotensina III/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Células 3T3-L1 , Animais , Desoxiglucose/farmacologia , Transportador de Glucose Tipo 1/genética , Camundongos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Transdução de SinaisRESUMO
In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats without inducing angiotensin II accumulation. We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. We found that the intracerebroventricular (icv) administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.
Assuntos
Enzima de Conversão de Angiotensina 2/efeitos dos fármacos , Anti-Hipertensivos/farmacologia , Dissulfetos/farmacologia , Glutamil Aminopeptidase/antagonistas & inibidores , Hipertensão/fisiopatologia , Ácidos Sulfônicos/farmacologia , Angiotensina III/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Acetato de Desoxicorticosterona/administração & dosagem , Modelos Animais de Doenças , Glutamil Aminopeptidase/metabolismo , Hipertensão/induzido quimicamente , Masculino , Camundongos , Ratos Wistar , Cloreto de Sódio na DietaRESUMO
The brain renin-angiotensin system (RAS) has been recently involved in the homeostatic regulation of energy. Our goal was to analyse the influence of a diet rich in saturated fatty acids (butter) against one enriched in monounsaturated fatty acids (olive oil) on hypothalamic RAS, and their relationship with the metabolism of fatty acids. Increases in body weight and visceral fat, together with an increase in aminopeptidase A expression and reductions in AngII and AngIV were observed in the hypothalamus of animals fed with the butter diet. In this group, a marked reduction in the expression of genes related to lipid metabolism (LPL, CD36, and CPT-1) was observed in liver and muscle. No changes were found in terms of body weight, total visceral fat and the expression of hepatic genes related to fatty acid metabolism in the olive oil diet. The expressions of LPL and CD36 were reduced in the muscles, although the decrease was lower than in the butter diet. At the same time, the fasting levels of leptin were reduced, no changes were observed in the hypothalamic expression of aminopeptidase A and decreases were noted in the levels of AngII, AngIV and AngIII. These results support that the type of dietary fat is able to modify the hypothalamic profile of RAS and the body energy balance, related to changes in lipid metabolism.
Assuntos
Manteiga , Hipotálamo/metabolismo , Metabolismo dos Lipídeos , Azeite de Oliva/metabolismo , Sistema Renina-Angiotensina/fisiologia , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina III/metabolismo , Animais , Peso Corporal , Antígenos CD36/metabolismo , Dieta Hiperlipídica , Metabolismo Energético , Jejum/metabolismo , Expressão Gênica , Glutamil Aminopeptidase/metabolismo , Gordura Intra-Abdominal/crescimento & desenvolvimento , Leptina/metabolismo , Metabolismo dos Lipídeos/genética , Lipase Lipoproteica/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Aumento de PesoRESUMO
The pathophysiology of heart failure (HF) and hypertension are thought to involve brain renin-angiotensin system (RAS) hyperactivity. Angiotensin III, a key effector peptide in the brain RAS, provides tonic stimulatory control over blood pressure (BP) in hypertensive rats. Aminopeptidase A (APA), the enzyme responsible for generating brain angiotensin III, constitutes a potential therapeutic target for hypertension treatment. We focus here on studies of RB150/firibastat, the first prodrug of the specific and selective APA inhibitor EC33 able to cross the blood-brain barrier. We consider its development from therapeutic target discovery to clinical trials of the prodrug. After oral administration, firibastat crosses the gastrointestinal and blood-brain barriers. On arrival in the brain, it is cleaved to generate EC33, which inhibits brain APA activity, lowering BP in various experimental models of hypertension. Firibastat was clinically and biologically well tolerated, even at high doses, in phase I trials conducted in healthy human subjects. It was then shown to decrease BP effectively in patients of various ethnic origins with hypertension in phase II trials. Brain RAS hyperactivity leads to excessive sympathetic activity, which can contribute to HF after myocardial infarction (MI). Chronic treatment with oral firibastat (4 or 8 weeks after MI) has been shown to normalize brain APA activity in mice. This effect is accompanied by a normalization of brain RAS and sympathetic activities, reducing cardiac fibrosis and hypertrophy and preventing cardiac dysfunction. Firibastat may therefore represent a novel therapeutic advance in the clinical management of patients with hypertension and potentially with HF after MI.
Assuntos
Encéfalo/metabolismo , Glutamil Aminopeptidase/antagonistas & inibidores , Insuficiência Cardíaca/prevenção & controle , Hipertensão/tratamento farmacológico , Angiotensina II/metabolismo , Angiotensina III/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Ensaios Clínicos como Assunto , Dissulfetos/farmacologia , Dissulfetos/uso terapêutico , Glutamil Aminopeptidase/metabolismo , Insuficiência Cardíaca/etiologia , Humanos , Hipertensão/metabolismo , Infarto do Miocárdio/complicações , Sistema Renina-Angiotensina , Ácidos Sulfônicos/farmacologia , Ácidos Sulfônicos/uso terapêuticoRESUMO
BACKGROUND: Mitogen-activated protein kinases (MAPKs) are essential molecular transducers of extracellular stimuli into intracellular responses. MAPKs are crucial in mediating actions of the renin-angiotensin-aldosterone system (RAAS), in particular, functions mediated by Angiotensin (Ang) II, the main biological peptide produced by this system. We have shown that another biologically active heptapeptide Ang III also induces MAPKs in the central nervous system. The ability of Ang III to induce MAPKs in the periphery is unknown and was the focus of this study. METHODS: We determined whether Ang III induced p38 MAPK in vascular smooth cells (VSMCs) isolated from Wistar and spontaneously hypertensive rats (SHRs) and compared these actions to those of Ang II. Further, the role of this MAPK in Ang III-mediated VSMC proliferation was also determined. RESULTS: Both Ang peptides similarly induced p38 MAPK phosphorylation in VSMCs of Wistar VSMCs in a concentration- and time-dependent manner. SHR VSMCs were less sensitive to Ang III, which caused less of an effect on p38 MAPK phosphorylation in these cells. The Ang III effect was specific and occurred by activation of the Ang type 1 (AT1) receptor. The p38 MAPK pathway was also involved in Ang III-induced VSMC growth, as measured by DNA synthesis. CONCLUSIONS: These findings suggest that the p38 MAPK signaling pathway is an important cascade in regulating the actions of Ang III in VSMCs. Most importantly, dysregulation of Ang III actions in these cells are apparent and may contribute to pathological conditions associated with dysfunctions in VSMCS.
Assuntos
Angiotensina III/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Angiotensina II/metabolismo , Animais , Proliferação de Células/fisiologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Fosforilação/fisiologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
AIMS: Inhibition of brain angiotensin III by central infusion of aminopeptidase A (APA) inhibitor firibastat (RB150) inhibits sympathetic hyperactivity and heart failure in rats after myocardial infarction (MI). This study evaluated effectiveness of systemic treatment with firibastat compared with AT1R blocker, losartan. METHODS AND RESULTS: MI was induced by ligation of left coronary artery in male Wistar rats. Rats were treated from 1 to 5 weeks after MI in protocol 1 with vehicle, or firibastat at 50 mg/kg/d subcutaneously (s.c.) or 150 mg/kg/d oral, once daily, and in protocol 2, with vehicle, firibastat 150 mg/kg or losartan 50 mg/kg oral twice daily. At 5 weeks, left ventricle function was evaluated by echocardiography and Millar catheter. After MI, rats developed moderate severe heart failure. Both s.c. and oral firibastat inhibited brain APA and attenuated left ventricle dysfunction. Oral firibastat and losartan similarly improved left ventricular end diastolic pressure. However, whereas firibastat improved dP/dtmax, losartan lowered dP/dtmax and left ventricular peak systolic pressure, and increased plasma creatinine by ~50%. On the other hand, losartan more effectively inhibited cardiac fibrosis. CONCLUSION: Inhibition of the brain renin-angiotensin system by oral APA inhibitor is at least as effective as oral AT1R blocker to inhibit cardiac dysfunction after MI but without hypotension or renal dysfunction.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Angiotensina III/metabolismo , Encéfalo/efeitos dos fármacos , Dissulfetos/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Glutamil Aminopeptidase/antagonistas & inibidores , Insuficiência Cardíaca/prevenção & controle , Losartan/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Ácidos Sulfônicos/administração & dosagem , Administração Oral , Animais , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Fibrose , Glutamil Aminopeptidase/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Injeções Subcutâneas , Masculino , Infarto do Miocárdio/complicações , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/fisiopatologia , Ratos Wistar , Transdução de Sinais , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Hyperaldosteronism is associated with hypertension, cardiac hypertrophy, and congestive heart failure. Steroidogenic factors facilitate aldosterone secretion by increasing adrenal blood flow. Angiotensin (Ang) II decreases adrenal vascular tone through release of zona glomerulosa (ZG) cell-derived vasodilatory eicosanoids. However, ZG cell-mediated relaxation of bovine adrenal cortical arteries to Ang II is not altered by angiotensin type 1 or 2 receptor antagonists. Because traditional Ang II receptors do not mediate these vasorelaxations to Ang II, we investigated the role of Ang II metabolites. Ang III was identified by liquid chromatography-mass spectrometry as the primary ZG cell metabolite of Ang II. Ang III stimulated ZG cell-mediated relaxation of adrenal arteries with greater potency than did Ang II. Furthermore, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by aminopeptidase inhibition, and Ang III-stimulated relaxations persisted. Ang IV had little effect compared with Ang II. Moreover, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by an Ang III antagonist but not by an Ang (1-7) antagonist. In contrast, Ang II and Ang III were equipotent in stimulating aldosterone secretion from ZG cells and were unaffected by aminopeptidase inhibition. Additionally, aspartyl and leucyl aminopeptidases, which convert Ang II to Ang III, are the primary peptidase expressed in ZG cells. This was confirmed by enzyme activity. These data indicate that intra-adrenal metabolism of Ang II to Ang III is required for ZG cell-mediated relaxations of adrenal arteries but not aldosterone secretion. These studies have defined an important role of Ang III in the adrenal gland.
Assuntos
Córtex Suprarrenal/irrigação sanguínea , Angiotensina III/metabolismo , Angiotensina II/metabolismo , Arteríolas/metabolismo , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Zona Glomerulosa/metabolismo , Matadouros , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Aminopeptidases/metabolismo , Angiotensina I/antagonistas & inibidores , Angiotensina I/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/química , Angiotensina II/farmacologia , Animais , Arteríolas/citologia , Arteríolas/efeitos dos fármacos , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Vasodilatação/efeitos dos fármacos , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacosRESUMO
Renin angiotensin system (RAS) is an endogenous hormone system involved in the control of blood pressure and fluid volume. Dysregulation of RAS has a pathological role in causing cardiovascular diseases through hypertension. Among several key components of RAS, angiotensin peptides, varying in amino acid length and biological function, have important roles in preventing or promoting hypertension, cardiovascular diseases, stroke, vascular remodeling etc. These peptides are generated by the metabolism of inactive angiotensinogen or its derived peptides by hydrolyzing action of certain enzymes. Angiotensin II, angiotensin (1-12), angiotensin A and angiotensin III bind primarily to angiotensin II type 1 receptor and cause vasoconstriction, accumulation of inflammatory markers to sub-endothelial region of blood vessels and activate smooth muscle cell proliferation. Moreover, when bound to angiotensin II type 2 receptor, angiotensin II works as cardio-protective peptide and halt pathological cell signals. Other peptides like angiotensin (1-9), angiotensin (1-7), alamandine and angiotensin IV also help in protecting from cardiovascular diseases by binding to their respective receptors.
Assuntos
Angiotensina II/metabolismo , Hipertensão/metabolismo , Sistema Renina-Angiotensina/fisiologia , Angiotensina I/metabolismo , Angiotensina II/análogos & derivados , Angiotensina III/metabolismo , Angiotensinogênio/metabolismo , Angiotensinas/metabolismo , Animais , Pressão Sanguínea , Humanos , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Proteção , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais , VasoconstriçãoRESUMO
We have previously demonstrated that the intrathecal (i.t.) administration of angiotensin (Ang) II into mice produces a nociceptive behaviour consisting of scratching, biting and licking accompanied by the phosphorylation of p38 MAPK in the spinal cord, which was mediated through AT1 receptors. Both the p38 MAPK phosphorylation and subsequent nociceptive behaviour were attenuated by the i.t. co-administration of Ang (1-7), an N-terminal fragment of Ang II, that acted via Mas receptors. On the other hand, a C-terminal fragment of Ang II, namely Ang III, was also shown to induce a nociceptive behaviour by acting upon AT1 receptors on spinal astrocytes and neurons, and was found to be more potent than Ang II. However, the inhibitory effect of Ang (1-7) on the Ang III-induced nociceptive behaviour remains unclear. Thus, here we examined whether Ang (1-7) can attenuate the Ang III-induced nociceptive behaviour and activation of spinal p38 MAPK. The i.t. administration of Ang (1-7) (1-100fmol) dose-dependently attenuated the Ang III (1pmol)-induced nociceptive behaviour in mice. Moreover, the inhibitory effect of Ang (1-7) at a dose of 100fmol was prevented by A779 (30fmol), a Mas receptor antagonist. Western blot analysis showed that the phosphorylation of p38 MAPK induced by the i.t. administration of Ang III (1pmol) was also attenuated by Ang (1-7) (100fmol), and this inhibition was prevented by A779 (30fmol). Furthermore, we showed that in the lumbar superficial dorsal horn, Mas receptors are expressed in neurons and microglia but absent from astrocytes. Together, these results suggest that the i.t. administration of Ang (1-7) attenuates the nociceptive behaviour and accompanying p38 MAPK phosphorylation induced by Ang III, and that this effect is likely mediated through Mas receptors on spinal neurons.
Assuntos
Angiotensina III/metabolismo , Angiotensina I/metabolismo , Nociceptividade , Fragmentos de Peptídeos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Angiotensina I/administração & dosagem , Angiotensina III/administração & dosagem , Animais , Masculino , Camundongos , Microglia/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fosforilação , Células do Corno Posterior/metabolismoRESUMO
Hyperactivity of the renin-angiotensin system (RAS) is associated with the pathogenesis of Alzheimer's disease (AD) believed to be mediated by angiotensin-II (Ang-II) activation of the angiotensin type 1 receptor (AT1R). We previously showed that angiotensin-converting enzyme-1 (ACE-1) activity, the rate-limiting enzyme in the production of Ang-II, is increased in human postmortem brain tissue in AD. Angiotensin-III (Ang-III) activates the AT1R and angiotensin type-2 receptor (AT2R), but its potential role in the pathophysiology of AD remains unexplored. We measured Ang-II and Ang-III levels by ELISA, and the levels and activities of aminopeptidase-A (AP-A) and aminopeptidase-N (AP-N) (responsible for the production and metabolism of Ang-III, respectively) in human postmortem brain tissue in the mid-frontal cortex (Brodmann area 9) in a cohort of AD (nâ=â90) and age-matched non-demented controls (nâ=â59), for which we had previous measurements of ACE-1 activity, Aß level, and tau pathology (also in the mid-frontal cortex). We found that both Ang-II and Ang-III levels were significantly higher in AD compared to age-matched controls and that Ang-III, rather than Ang-II, was strongly associated with Aß load and tau load. Levels of AP-A were significantly reduced in AD but AP-A enzyme activity was unchanged whereas AP-N activity was reduced in AD but AP-N protein level was unchanged. Together, these data indicate that the APA/Ang-III/APN/Ang-IV/AT4R pathway is dysregulated and that elevated Ang-III could contribute to the pathogenesis of AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Angiotensina III/metabolismo , Lobo Frontal/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Antígenos CD13/metabolismo , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Glutamil Aminopeptidase/metabolismo , Humanos , MasculinoRESUMO
Synthetic receptors selective for target peptides or proteins have received attention because of their potential applications in the separation of biomolecules and biomedical diagnostics. Herein, a [2]rotaxane-based functional monomer containing monocarboxylated α-cyclodextrin (α-CD) was synthesized, and its crosslinked polymers were evaluated to determine their binding ability to a model peptide, angiotensinâ III (Arg-Val-Tyr-Ile-His-Pro-Phe), containing an arginine (Arg) residue. The binding ability of the resulting polymers toward angiotensinâ III, angiotensinâ IV (Val-Tyr-Ile-His-Pro-Phe), and FMRF-amide (Phe-Met-Arg-Phe) was examined by the batch-binding assay and compared with that of control polymers, in which maleic acid-introduced α-CD was chemically crosslinked. The results suggest that the [2]rotaxane-based functional monomer in the crosslinked polymer contributes to the high affinity toward angiotensin III. The α-CD motion and rotation within the [2]rotaxane-based crosslinked polymer may be applicable for designing molecular recognition materials.
Assuntos
Angiotensina III/metabolismo , Rotaxanos/metabolismo , alfa-Ciclodextrinas/química , Sequência de Aminoácidos , Angiotensina II/análogos & derivados , Angiotensina II/química , Angiotensina II/metabolismo , Angiotensina III/química , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Rotaxanos/síntese química , Rotaxanos/químicaRESUMO
PURPOSE OF REVIEW: This review updates major new findings and concepts introduced during the past year on the role of angiotensin II (Ang II) subtype 2 receptors (AT2Rs) in the control of blood pressure and renal function. RECENT FINDINGS: AT2R activation prevents sodium (Na) retention and lowers blood pressure in the Ang II infusion model of experimental hypertension and prevents salt-sensitive hypertension in the obese Zucker rat model of obesity and the metabolic syndrome. Ang II metabolite, des-aspartyl-Ang II (Ang III) is the predominant AT2R agonist in the kidney and possibly also in the vasculature; a novel synthetic Ang III peptide, ß-Pro-Ang III, is vasodepressor and lowers blood pressure in conscious spontaneously hypertensive rats in the presence of low-level Ang II type 1 receptor (AT1R) blockade. Because nitric oxide is a product of AT2R activation, a potential feed-forward loop, wherein nitric oxide increases AT2R transcription, may reinforce the beneficial actions of AT2R in the long term. AT2R activation also reduces proteinuria and oxidative stress in glomerulosclerotic kidneys of high-salt obese Zucker rats. SUMMARY: Studies during the past year have helped to clarify the physiological and pathophysiological roles of AT2Rs and have enhanced the promise of AT2R agonists in cardiovascular and renal disease.
Assuntos
Pressão Sanguínea/fisiologia , Hipertensão/fisiopatologia , Rim/fisiopatologia , Receptor Tipo 2 de Angiotensina/fisiologia , Angiotensina II/metabolismo , Angiotensina III/análogos & derivados , Angiotensina III/metabolismo , Animais , Humanos , Rim/metabolismo , Óxido Nítrico/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/metabolismo , Sódio/metabolismoRESUMO
AIM: Local levels of angiotensin peptides depend on their rates of production and degradation, which induce proatherogenic or atheroprotective effects. Here, we reveal the kinetics of Angiotensin-I metabolism in paired early and advanced atherosclerotic lesions. METHODS: Lesions were spiked with labeled Ang-I* and supernatants withdrawn after 0, 10, 20, 40 and 80min. The concentration of produced Ang-II*, Ang-III*, Ang-IV* and Ang-(1-7)* peptides were measured using multiple reaction monitoring mass spectrometry coupled to ultra-performance liquid chromatography, normalized to tissue weight and initial [Ang-I*]. RESULTS: Ang-(1-7)* was the major angiotensin peptide produced, showing increased levels in both tissue types, with 2-3 fold lower levels in advanced compared to early lesions. In contrast, Ang-II* was 2-3 fold higher in advanced compared to early lesions, showing a decrease between 0 and 40min then an increase at 80min in both tissue types. The levels of Ang-IV were stable in both tissue types across all time points. Finally, Ang-III was non-detectable in both lesions across all time points. CONCLUSION: Our results suggest that progression of atherosclerosis depends on the increased levels of Ang-II along with the decreased levels of Ang-(1-7), which supports the use of Ang-(1-7) along with Angiotensin type-1 receptor (AT1R) blockers.
Assuntos
Angiotensina I/metabolismo , Doenças das Artérias Carótidas/metabolismo , Fragmentos de Peptídeos/metabolismo , Placa Aterosclerótica/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina III/metabolismo , Doenças das Artérias Carótidas/patologia , Cromatografia Líquida de Alta Pressão/métodos , Progressão da Doença , Humanos , Espectrometria de Massas/métodos , Placa Aterosclerótica/patologia , Fatores de TempoRESUMO
We have previously demonstrated the possibility that angiotensin (Ang) II and its N-terminal metabolite Ang (1-7) act as neurotransmitters and/or neuromodulators in the spinal transmission of nociceptive information. Ang III, which is a C-terminal metabolite of Ang II, can also act on AT1 receptors, but its role in spinal nociceptive transmission remains unclear. Therefore, we examined the role of Ang III on the spinal nociceptive system in comparison with that of Ang II. Intrathecal (i.t.) administration of Ang III into mice produced a nociceptive behavior, which was dose-dependently inhibited by the co-administration of the AT1 receptor antagonist losartan and the p38 MAPK inhibitor SB203580, but not by the AT2 receptor antagonist PD123319, MEK1/2 inhibitor U0126 and JNK inhibitor SP600125. In addition, Ang III increased the phosphorylation of p38 MAPK in the dorsal lumbar spinal cord, which was inhibited by losartan. These effects were similar to those of observed with Ang II. The nociceptive behavior produced by Ang II or III was also attenuated by the administration of the astrocytic inhibitor L-α-aminoadipic acid, but not by the microglial inhibitor minocycline. Double immunohistochemical staining showed that spinal AT1 receptors were expressed on neurons and astrocytes, and that i.t. administration of either Ang II or III phosphorylated p38 MAPK in both spinal astrocytes and neurons. These results indicate that Ang III produces nociceptive behavior similar to Ang II, and suggest that the phosphorylation of p38 MAPK mediated through AT1 receptors on spinal astrocytes and neurons contributes to Ang II- and III-induced nociceptive behavior.
Assuntos
Angiotensina III/metabolismo , Angiotensina II/metabolismo , Dor Nociceptiva/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Medula Espinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Modelos Animais de Doenças , Vértebras Lombares , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Dor Nociceptiva/tratamento farmacológico , Dor Nociceptiva/patologia , Fosforilação , Receptor Tipo 2 de Angiotensina/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
INTRODUCTION: The human esophageal mucosa expresses various components of the renin-angiotensin system (RAS), e.g. the main effector peptide angiotensin II (AngII). The aim of this study was to investigate the esophageal presence of angiotensin III (AngIII) and angiotensin IV (AngIV) forming enzymes and the AngIV receptor (AT4R). The aim was also to study the actions of AngIV and to look for aberrations in patients with gastroesophageal reflux disease (GERD). MATERIALS AND METHODS: Esophageal biopsies were collected from healthy volunteers (n: 19) and individuals with erosive reflux disease (n: 14). Gene transcripts and protein expression of aminopeptidase A, -B and -M, and the AT4R were investigated by reverse transcriptase polymerase chain reaction (rt-PCR), western blot (WB) and immunohistochemistry (IHC). The functional impact of AngIV was examined in an Ussing chamber. RESULTS: Aminopeptidase A, -B and -M and the AT4R were expressed in the esophageal epithelium. The AT4R was less prominent in certain areas in the mucosa of reflux patients. AngIV influenced the esophageal epithelial ion transport. The impact was lower in patients with GERD. CONCLUSION: The AT4R and formation enzymes of AngIII and AngIV are present in the human esophageal epithelium. Moreover, the present results suggest that AngIV exert regulatory impact on the epithelium and that RAS is involved in mucosal aberrations associated with GERD.
Assuntos
Angiotensina II/análogos & derivados , Esôfago/metabolismo , Refluxo Gastroesofágico/metabolismo , Mucosa/metabolismo , Adulto , Idoso , Angiotensina II/metabolismo , Angiotensina III/metabolismo , Western Blotting , Fenômenos Eletrofisiológicos , Esôfago/fisiopatologia , Feminino , Refluxo Gastroesofágico/genética , Refluxo Gastroesofágico/fisiopatologia , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa/fisiopatologia , Reação em Cadeia da Polimerase , Transporte Proteico , Receptores de Angiotensina/metabolismo , Transcrição Gênica , Adulto JovemRESUMO
Hypertension affects one-third of the adult population and is a growing problem due to the increasing incidence of obesity and diabetes. Brain RAS (renin-angiotensin system) hyperactivity has been implicated in the development and maintenance of hypertension in several types of experimental and genetic hypertension animal models. We have identified in the brain RAS that APA (aminopeptidase A) and APN (aminopeptidase N), two membrane-bound zinc metalloproteases, are involved in the metabolism of AngII (angiotensin II) and AngIII (angiotensin III) respectively. The present review summarizes the main findings suggesting that AngIII plays a predominant role in the brain RAS in the control of BP (blood pressure). We first explored the organization of the APA active site by site-directed mutagenesis and molecular modelling. The development and the use in vivo of specific and selective APA and APN inhibitors EC33 and PC18 respectively, has allowed the demonstration that brain AngIII generated by APA is one of the main effector peptides of the brain RAS, exerting a tonic stimulatory control over BP in conscious hypertensive rats. This identified brain APA as a potential therapeutic target for the treatment of hypertension, which has led to the development of potent orally active APA inhibitors, such as RB150. RB150 administered orally in hypertensive DOCA (deoxycorticosteroneacetate)-salt rats or SHRs (spontaneously hypertensive rats) crosses the intestinal, hepatic and blood-brain barriers, enters the brain, generates two active molecules of EC33 which inhibit brain APA activity, block the formation of brain AngIII and normalize BP for several hours. The decrease in BP involves two different mechanisms: a decrease in vasopressin release into the bloodstream, which in turn increases diuresis resulting in a blood volume reduction that participates in the decrease in BP and/or a decrease in sympathetic tone, decreasing vascular resistance. RB150 constitutes the prototype of a new class of centrally acting antihypertensive agents and is currently being evaluated in a Phase Ib clinical trial.
Assuntos
Dissulfetos/uso terapêutico , Glutamil Aminopeptidase/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Ácidos Sulfônicos/uso terapêutico , Angiotensina III/metabolismo , Angiotensina III/fisiologia , Animais , Sítios de Ligação , Pressão Sanguínea , Barreira Hematoencefálica , Encéfalo/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Desenho de Fármacos , Glutamil Aminopeptidase/química , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Inibidores de Proteases/farmacocinética , RatosRESUMO
Aminopeptidase-A (APA) is a less well-studied enzyme of the renin-angiotensin system. We propose that it is involved in cardiac angiotensin (ANG) metabolism and its pathologies. ANG-(1-7) can ameliorate remodeling after myocardial injury. The aims of this study are to (1) develop mass spectrometric (MS) approaches for the assessment of ANG processing by APA within the myocardium; and (2) investigate the role of APA in cardiac ANG-(1-7) metabolism after myocardial infarction (MI) using sensitive MS techniques. MI was induced in C57Bl/6 male mice by ligating the left anterior descending (LAD) artery. Frozen mouse heart sections (in situ assay) or myocardial homogenates (in vitro assay) were incubated with the endogenous APA substrate, ANG II. Results showed concentration- and time-dependent cardiac formation of ANG III from ANG II, which was inhibited by the specific APA inhibitor, 4-amino-4-phosphonobutyric acid. Myocardial APA activity was significantly increased 24 h after LAD ligation (0.82 ± 0.02 vs. 0.32 ± 0.02 ρmol·min(-1)·µg(-1), MI vs. sham, P < 0.01). Both MS enzyme assays identified the presence of a new peptide, ANG-(2-7), m/z 784, which accumulated in the MI (146.45 ± 6.4 vs. 72.96 ± 7.0%, MI vs. sham, P < 0.05). Use of recombinant APA enzyme revealed that APA is responsible for ANG-(2-7) formation from ANG-(1-7). APA exhibited similar substrate affinity for ANG-(1-7) compared with ANG II {Km (ANG II) = 14.67 ± 1.6 vs. Km [ANG-(1-7)] = 6.07 ± 1.12 µmol/l, P < 0.05}. Results demonstrate a novel role of APA in ANG-(1-7) metabolism and suggest that the upregulation of APA, which occurs after MI, may deprive the heart of cardioprotective ANG-(1-7). Thus APA may serve as a potentially novel therapeutic target for management of tissue remodeling after MI.
Assuntos
Angiotensina I/metabolismo , Glutamil Aminopeptidase/metabolismo , Infarto do Miocárdio/enzimologia , Miocárdio/enzimologia , Fragmentos de Peptídeos/metabolismo , Angiotensina II/metabolismo , Angiotensina III/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Glutamil Aminopeptidase/antagonistas & inibidores , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/patologia , Peptidil Dipeptidase A/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Espectrometria de Massas em Tandem , Remodelação VentricularRESUMO
Hyperaldosteronism is linked to the development and progression of several different cardiovascular diseases. Angiotensin (Ang) II increases aldosterone secretion and adrenal blood flow. Ang II peptide fragments are produced by various peptidases, and these Angs have diverse and vital physiologic roles. Due to the uncharacteristic vasorelaxation of adrenal arteries by Ang II, we tested the hypothesis that Ang II metabolism contributes to its relaxant activity in adrenal arteries. Metabolism of Angs by bovine adrenal cortical arteries and isolated bovine adrenal vascular cells was measured by liquid chromatography-mass spectrometry. The primary Ang metabolites of adrenal arteries are Ang III and Ang (1-7), with Ang IV produced to a lesser extent. Bovine microvascular endothelial cells produced a similar metabolic profile to adrenal arteries, whereas bovine adrenal artery smooth muscle cells exhibited less metabolism. In preconstricted adrenal arteries, Ang II caused relaxation in picomolar concentrations and constrictions at 10nM. Ang-converting enzyme 2 inhibition augmented this relaxation response, whereas aminopeptidase inhibition did not. Ang III was equipotent to Ang II in relaxing adrenal arteries. Ang IV did not cause relaxation. Nitric oxide synthase inhibition enhanced Ang II-induced constriction of adrenal arteries. Aminopeptidase inhibition increased the concentration range for Ang II-induced constriction of adrenal arteries. Ang III and Ang IV did not change the basal tone but caused constriction of adrenal arteries with nitric oxide synthase inhibition. These data indicate that Ang II metabolism modulates the vascular effects of Ang II in the adrenal vasculature.