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1.
Brain Res ; 1583: 132-40, 2014 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-25108041

RESUMO

Angiotensin II (Ang II) stimulates water and saline intakes when injected into the brain of rats. This arises from activation of the AT1 Ang II receptor subtype. Acute repeated injections, however, decrease the water intake response to Ang II without affecting saline intake. Previous studies provide evidence that Ang II-induced water intake is mediated via the classical G protein coupling pathway, whereas the saline intake caused by Ang II is mediated by an ERK 1/2 MAP kinase signaling pathway. Accordingly, the different behavioral response to repeated injections of Ang II may reflect a selective effect on G protein coupling. To test this hypothesis, we examined the binding of a radiolabeled agonist ((125)I-sarcosine(1) Ang II) and a radiolabeled antagonist ((125)I-sarcosine(1), isoleucine(8) Ang II) in brain homogenates and tissue sections prepared from rats given repeated injections of Ang II or vehicle. Although no treatment-related differences were found in hypothalamic homogenates, a focus on specific brain structures using receptor autoradiography, found that the desensitization treatment reduced binding of both radioligands in the paraventricular nucleus of the hypothalamus (PVN) and median preoptic nucleus (MnPO), but not in the subfornical organ (SFO). Because G protein coupling is reported to have a selective effect on agonist binding without affecting antagonist binding, these findings do not support a G protein uncoupling treatment effect. This suggests that receptor number is more critical to the water intake response than the saline intake response, or that pathways downstream from the G protein mediate desensitization of the water intake response.


Assuntos
Angiotensina II/farmacologia , Fármacos do Sistema Nervoso Central/farmacologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Área Pré-Óptica/efeitos dos fármacos , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/administração & dosagem , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II/metabolismo , Animais , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Líquidos/fisiologia , Água Potável/administração & dosagem , Radioisótopos do Iodo , Masculino , Núcleo Hipotalâmico Paraventricular/metabolismo , Área Pré-Óptica/fisiopatologia , Ensaio Radioligante , Compostos Radiofarmacêuticos , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/metabolismo , Cloreto de Sódio na Dieta/administração & dosagem , Órgão Subfornical/efeitos dos fármacos , Órgão Subfornical/metabolismo
2.
Atherosclerosis ; 236(1): 108-15, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25016365

RESUMO

BACKGROUND: A number of studies have suggested that angiotensin II (AII) receptor type 1 (ATR1) blocking drugs (ARBs) have anti-inflammatory effects however the mechanisms responsible are poorly investigated. OBJECTIVE: To determine the role of extracellular signal regulated kinase (ERK)1/2 in ARB induced anti-inflammatory effects within human carotid atherosclerosis. METHODS: Atheroma samples obtained from patients undergoing carotid endarterectomy were cultured with and without ATR1 (irbesartan), ERK1/2 (PD98059), AII ([Sar(1), Ile(8)]-AII) and angiotensin converting enzyme (ACE)2 (DX600) blockade. The in vitro effects of ATR1 and ERK1/2 blockade and exogenous AII on serum stimulated healthy, primary vascular cells were also investigated. Outcome was assessed by measuring cytokine, (interleukin (IL)-6, IL-8, C-C motif chemokine (CCL)2, C-X-C motif chemokine (CXCL)5, osteoprotegerin (OPG), osteopontin (OPN), CXCL16), concentrations in supernatants and phosphorylated ERK1/2 in the tissue lysates using ELISA. ERK1/2 expression in the tissue was assessed using Western blotting. RESULTS: Irbesartan reduced concentrations of IL-6, IL-8, CCL2, CXCL5, OPG, OPN and CXCL16 in both atheroma and primary vascular cell culture supernatants. The reduction in cytokine levels in the atheroma supernatant was correlated to a reduction in ERK1/2 expression in the tissue. Inhibition of ERK1/2 downregulated IL-6, IL-8 and CXCL5 in both atheroma and cell culture supernatants. AII and ACE2 blockade had no impact on cytokine or active ERK1/2 levels in the atheroma culture. CONCLUSION: Our findings suggest that ATR1 blockade downregulates atheroma tissue ERK1/2 expression leading to a reduction in cytokine production and that a non-AII agonist ATR1 signalling response may induce expression of these inflammation associated cytokines in the atheroma.


Assuntos
1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Compostos de Bifenilo/farmacologia , Citocinas/metabolismo , Endotélio Vascular/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptídeos/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Tetrazóis/farmacologia , Idoso , Artérias Carótidas/patologia , Células Cultivadas , Quimiocinas/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Inflamação , Irbesartana , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Osteoprotegerina/metabolismo , Placa Aterosclerótica/patologia , Sistema Renina-Angiotensina/fisiologia
4.
Mini Rev Med Chem ; 12(9): 812-6, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22681254

RESUMO

G protein-coupled receptors (GPCRs) can be activated by multiple ligands and exhibit the capacity to couple to numerous intracellular signal transduction pathways. This property allows GPCRs to be modulated by biased agonists that selectively activate specific subsets of GPCR-regulated cellular signaling proteins. The angiotensin II type 1 receptor (AT1R) is a GPCR that endogenously binds to the peptide ligand angiotensin II. More recently it has been demonstrated that a modified peptide, [Sar1I-le4-Ile8]-angiotensin II (SII) acts as a biased agonist towards the AT1R. SII binds to the AT1R without promoting heterotrimeric G protein-coupling, but serves to link the receptor to the beta-arrestin-dependent activation of the mitogen activated protein kinase pathway. The present mini-review summarizes current knowledge regarding the role of biased agonists in stimulating biased AT1R signaling.


Assuntos
1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/agonistas , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Arrestinas/metabolismo , Células HEK293 , Humanos , Ligantes , Losartan/metabolismo , Losartan/farmacologia , Conformação Proteica , Receptor Tipo 1 de Angiotensina/metabolismo , Estresse Mecânico , beta-Arrestinas
5.
J Pharmacol Exp Ther ; 335(3): 754-61, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20861168

RESUMO

We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.


Assuntos
Neurônios/citologia , Neurônios/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 4-Cloromercuriobenzenossulfonato/farmacologia , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Feminino , Glutationa/farmacologia , Dissulfeto de Glutationa/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos , Neurônios/efeitos dos fármacos , Prosencéfalo/citologia , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Temperatura , Ácido p-Cloromercurobenzoico/farmacologia
6.
J Recept Signal Transduct Res ; 30(4): 234-45, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20524779

RESUMO

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing beta cell are not well investigated. In insulin-secreting cell line (INS-1), AT(1) and AT(4) receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of (125)I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at approximately 1 nM. The increase was antagonized by 1 microM losartan or 10 microM divalinal (AT(1) and AT(4) receptor antagonists, respectively). The insulin release was accompanied by a (45)Ca(2+) uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT(4) receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT(1) and AT(4) receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca(2+). The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.


Assuntos
Angiotensina II/análogos & derivados , Glicemia/efeitos dos fármacos , Insulina/sangue , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Animais , Western Blotting , Linhagem Celular , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Losartan/farmacologia , Macrolídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Transfecção
7.
J Comp Physiol B ; 180(7): 1057-65, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20495810

RESUMO

Angiotensin II (Ang II) is an important regulator of cardiovascular function in adult vertebrates. Although its role in regulating the adult system has been extensively investigated, the cardiovascular response to Ang II in embryonic vertebrates is relatively unknown. We investigated the potential of Ang II as a regulator of cardiovascular function in embryonic chickens, which lack central nervous system control of cardiovascular function throughout the majority of incubation. The cardiovascular response to Ang II in embryonic chickens was investigated over the final 50% of their development. Ang II produced a dose-dependent increase in arterial pressure on each day of development studied, and the response increased in intensity as development progressed. The Ang II type-1 receptor nonspecific competitive peptide antagonist [Sar(1) ile(8)] Ang II blocked the cardiovascular response to subsequent injections of Ang II on day 21 only. The embryonic pressure response to Ang II (hypertension only) differed from that of adult chickens, in which initial hypotension is followed by hypertension. The constant level of gene expression for the Ang II receptor, in conjunction with an increasing pressure response to the peptide, suggests that two Ang II receptor subtypes are present during chicken development. Collectively, the data indicate that Ang II plays an important role in the cardiovascular development of chickens; however, its role in maintaining basal function requires further study.


Assuntos
Angiotensina II/fisiologia , Sistema Cardiovascular/embriologia , Desenvolvimento Embrionário/fisiologia , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiologia , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/antagonistas & inibidores , Angiotensina II/sangue , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Sistema Cardiovascular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/embriologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Sistema Renina-Angiotensina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
8.
J Pharmacol Exp Ther ; 330(1): 118-24, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19351865

RESUMO

Angiotensin II (AngII) initiates cellular effects via its G protein-coupled angiotensin 1 (AT(1)) receptor (AT(1)R). Previously, we showed that AngII-induced expression of the prostanoid-producing enzyme cyclooxygenase 2 (COX-2) was dependent upon nuclear trafficking of activated AT(1)R. In the present study, mastoparan (an activator of G proteins), suramin (an inhibitor of G proteins), 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; a specific inhibitor of phospholipase C), and sarcosine(1)-Ile(4)-Ile(8)-AngII (SII-AngII; a G protein-independent AT(1)R agonist) were used to determine the involvement of G proteins and AT(1A)R trafficking in AngII-stimulated COX-2 protein expression in human embryonic kidney-293 cells stably expressing AT(1A)/green fluorescent protein receptors and cultured vascular smooth muscle cells, respectively. Mastoparan alone stimulated release of intracellular calcium and increased COX-2 expression. Preincubation with mastoparan inhibited AngII-induced calcium signaling without altering AngII-induced AT(1A)R trafficking, p42/44 extracellular signal-regulated kinase (ERK) activation, or COX-2 expression. Suramin or U73122 had no significant effect on their own; they did not inhibit AngII-induced AT(1A)R trafficking, p42/44 ERK activation, or COX-2 expression; but they did inhibit AngII-induced calcium responses. SII-AngII stimulated AT(1A)R trafficking and increased COX-2 protein expression without activating intracellular calcium release. These data suggest that G protein activation results in increased COX-2 protein expression, but AngII-induced COX-2 expression seems to occur independently of G protein activation.


Assuntos
Angiotensina II/fisiologia , Aorta/metabolismo , Ciclo-Oxigenase 2/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Animais , Aorta/enzimologia , Aorta/fisiologia , Linhagem Celular , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/fisiologia , Peptídeos/farmacologia , Ratos , Venenos de Vespas/farmacologia
9.
Am J Hypertens ; 22(5): 569-76, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19300422

RESUMO

BACKGROUND: The Angiotensin II (Ang II) type 1 (AT(1)R) and type 2 (AT(2)R) receptors are increased in the heart following myocardial infarction and dilated cardiomyopathy, yet their contribution at a cellular level to compensation and/or failure remains controversial. METHODS: We ectopically expressed AT(1)R and AT(2)R in cultured adult rat cardiomyocytes and cardiac fibroblasts to investigate Ang II-mediated cardiomyocyte hypertrophy and cardiac cell viability. RESULTS: In adult rat cardiomyocytes, Ang II did not induce hypertrophy via the AT(1)R, and no effect of Ang II on cell viability was observed following AT(1)R or AT(2)R expression. In adult rat cardiac fibroblasts, Ang II stimulated cell death by apoptosis via the AT(1)R (but not the AT(2)R), which required the presence of extracellular calcium, and induced a rapid dissipation of mitochondrial membrane potential, which was significant from 8 h. CONCLUSIONS: We conclude that Ang II/AT(1)R triggers apoptosis in adult rat cardiac fibroblasts, which is dependent on Ca2+ influx.


Assuntos
Angiotensina II/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/fisiologia , Receptor Tipo 2 de Angiotensina/fisiologia , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Cálcio/fisiologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução Genética
10.
Life Sci ; 83(11-12): 421-5, 2008 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-18692076

RESUMO

AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.


Assuntos
Angiotensinas/metabolismo , Química Encefálica/fisiologia , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Adulto , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Córtex Cerebral/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Ligantes , Losartan/farmacologia , Masculino , Pessoa de Meia-Idade , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Piridinas/farmacologia , Ensaio Radioligante , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores de Angiotensina/efeitos dos fármacos , Caracteres Sexuais , Ácido p-Cloromercurobenzoico/farmacologia
11.
Horm Metab Res ; 40(11): 760-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18711690

RESUMO

A local paracrine acting angiotensin (ANG) system of preadipocytes and mature adipocytes is involved in metabolic effects and tissue differentiation. The present study reports on the investigation of binding affinities for various angiotensin receptors including their relevance in 3T3-L1 adipocytes and preadipocytes and 3T3-442A preadipocytes. Competitive binding studies using both 125I-ANG II and its more stable analogue 125I-SARILE for investigating AT1/AT2 binding sites in 3T3-L1 preadipocytes reveal a biphasic competition curve with KDs at a low and high nanomolar range. By using the AT2 receptor selective ligand 125I-CGP4112A the presence of high affinity AT2 binding sites in preadipocytes was observed. High nonspecific binding and a low receptor number is characteristic for all these experiments. An AT4 binding site (binding site for ANG IV) exists in 3T3-L1 and F442A preadipocytes and adipocytes with a high nanomolar KD. This low binding affinity was confirmed by a biological assay, the IRAP assay (=insulin regulated aminopeptidase assay). IRAP is associated with the AT4 receptor, which is a binding site at the luminal part of membrane bound IRAP. The curves for competition binding and for inhibition of IRAP activity are superimposable with respect to angiotensin IV. In conclusion, AT1 and AT2 binding sites are present in preadipocytes. AT2 receptor binding affinities are shown in preadipocytes for the first time. The description of a non-AT1/AT2 binding site with low affinity remains speculative albeit of high interest because antidiabetic and obesity related effects of angiotensin peptides and sartanes as antagonists are observed at these high concentrations. Local concentrations of ANG II and their degradation products may be extremely high. The low amounts of AT1 and AT2 binding sites emphasize the relevance of other binding sites in adipose tissue development and metabolic effects. The AT4 binding site seems to be one of the predominant receptors in adipose cells. Other degraded, but still bioactive peptides like ANG III, IV and ANG(1-7), activating receptors not influenced by ANG II, could be of importance.


Assuntos
Adipócitos/química , Receptores de Angiotensina/análise , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Angiotensina II/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Diferenciação Celular , Linhagem Celular , Cistinil Aminopeptidase/metabolismo , Radioisótopos do Iodo , Camundongos , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/análise , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores de Angiotensina/metabolismo
12.
Basic Clin Pharmacol Toxicol ; 100(5): 289-95, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17448113

RESUMO

The angiotensin II (AngII) type 1 receptor (AT(1)R) has been shown to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) through G proteins or G protein-independently through beta-arrestin2 in cellular expression systems. As activation mechanisms may greatly influence the biological effects of ERK1/2 activity, differential activation of the AT(1)R in its native cellular context could have important biological and pharmacological implications. To examine if AT(1)R activates ERK1/2 by G protein-independent mechanisms in the heart, we used the [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) analogue in native preparations of cardiac myocytes and beating hearts. We found that [SII] AngII does not activate G(q)-coupling, yet stimulates the beta-arrestin2-dependent ERK1/2. The G(q)-activated pool of ERK1/2 rapidly translocates to the nucleus, while the beta-arrestin2-scaffolded pool remains in the cytosol. Similar biased agonism was achieved in Langendorff-perfused hearts, where both agonists elicit ERK1/2 phosphorylation, but [SII] AngII induces neither inotropic nor chronotropic effects.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Receptor Tipo 1 de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Arrestinas/metabolismo , Núcleo Celular/enzimologia , Células Cultivadas , Circulação Coronária/efeitos dos fármacos , Citosol/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Perfusão , Ratos , Ratos Sprague-Dawley , Ratos Wistar , beta-Arrestinas
13.
Basic Clin Pharmacol Toxicol ; 100(5): 296-301, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17448114

RESUMO

The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains obscure. Application of the modified analogue [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) allowed us to dissect the two pathways of ERK1/2 activation in native cardiac myocytes. Although cytosol-retained, the beta-arrestin2-bound pool of ERK1/2 represents an active signalling component that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus, the beta-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling by the AT(1)R. The opportunity to separate desirable cardiac myocyte division from detrimental hypertrophy holds promise that novel pharmacological approaches will allow targeting of pathway-specific actions.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Miócitos Cardíacos/enzimologia , Receptor Tipo 1 de Angiotensina/fisiologia , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Proliferação de Células , Células Cultivadas , Sistema de Sinalização das MAP Quinases , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Regul Pept ; 137(3): 140-6, 2006 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-16928404

RESUMO

The effects of losartan on angiotensin receptors in hypertrophic rat hearts were studied. The study was prompted by inconsistent findings of either an increase or decrease in the mRNA of the AT1 receptor in the hearts of cardiac hypertrophic rats treated with losartan, and a paucity of information on the effects of losartan on functional angiotensin receptors in the heart. Losartan, administered i.p. to aortic coarcted rats, dose-dependently attenuated the cardiac hypertrophy. Significant effect was observed with a dose of 2.72 micromol/kg/day for four days. Hypertrophy was accompanied by an increase in [125I]-Sar1-Ile8-angiotensin II binding sites (due mainly to an increase in AT2 binding) and AT2 receptor protein in cardiac ventricles of aortic coarcted rats. Treatment with effective anti-hypertrophic doses of losartan dose-dependently downregulated the [125I]-Sar1-Ile8-angiotensin II binding sites, constitutive AT1 receptor protein, and the over expressed AT2 receptor protein. It was suggested that the anti-cardiac hypertrophic action of losartan resulted from its ability to suppress the expression of both the basal and enhanced cardiac angiotensin receptors. This raises the question as to whether such drastic action could form the therapeutic basis for the use of losartan in cardiac pathologies.


Assuntos
Cardiomegalia/tratamento farmacológico , Cardiomegalia/metabolismo , Losartan/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Sequência de Bases , Cardiomegalia/genética , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Masculino , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/genética
15.
Hypertension ; 47(1): 122-7, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16330679

RESUMO

Angiotensin II plays an important role in vascular remodeling through effects that involve, in part, interactions of vascular smooth muscle cells with extracellular matrix via integrins, which belong to a family of transmembrane receptors. We hypothesized that angiotensin (Ang) II regulates expression of vascular integrins and their ligands in experimental hypertension. Rats were infused subcutaneously with Ang II and received angiotensin type-1 (AT1) receptor blocker losartan, the AT1/angiotensin type-2 (AT2) [Sar1-Ile8]-Ang II, or the vasodilator hydralazine for 7 days. Osteopontin and integrin subunit expression were evaluated immunohistochemically. Ang II enhanced vascular alpha8, beta1, beta3 integrins and osteopontin expression, which were significantly reduced by losartan, [Sar1-Ile8]-Ang II, and hydralazine. Although Ang II increased vascular alpha5 subunit expression, this was additionally increased by losartan. Losartan was the only treatment that induced alpha1 subunit expression. These results demonstrate that AT1 and AT2 receptors have countervailing effects on vascular integrin subunit expression that may influence their effects on vascular remodeling and extracellular matrix composition.


Assuntos
Angiotensina II/administração & dosagem , Vasos Sanguíneos/metabolismo , Hipertensão/metabolismo , Integrinas/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Vasoconstritores/administração & dosagem , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Hidralazina/farmacologia , Hipertensão/induzido quimicamente , Bombas de Infusão , Losartan/farmacologia , Masculino , Osteopontina , Ratos , Ratos Sprague-Dawley , Sialoglicoproteínas/metabolismo , Vasodilatadores/farmacologia
16.
Hypertension ; 46(3): 598-606, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16043661

RESUMO

We investigated the role of angiotensin II type 1 (AT1) and AT2 receptors, matrix metalloproteinases (MMPs), and extracellular matrix (ECM) components involved in vascular remodeling of resistance arteries induced by angiotensin II (Ang II). Sprague-Dawley rats received Ang II (120 ng/kg per minute SC) +/- the AT1 antagonist losartan (10 mg/kg per day PO), the AT1/AT2 antagonist Sar1-Ile8-Ang II (Sar-Ile; 10 microg/kg per minute SC), or hydralazine (25 mg/kg per day PO) for 7 days. Structure and mechanical properties of small mesenteric arteries were evaluated on a pressurized myograph. Ang II increased growth index (+21%), which was partially decreased by losartan (-11%) and abrogated by Sar-Ile. Hydralazine markedly increased growth index (+32%) despite systolic blood pressure (BP) lowering, suggesting a BP-independent effect of Ang II on vascular growth. Elastic modulus was increased by Sar-Ile compared with Ang II and control. Vascular type I collagen was reduced (P<0.05), whereas fibronectin increased significantly with Sar-Ile. Vascular tissue inhibitor of metalloproteinase-2 binding to MMP-2 was abrogated by Sar-Ile, but MMP-2 activity was significantly increased compared with losartan, Ang II, and controls. Thus, AT1 blockade exerted antigrowth effects and reduced stiffness of small resistance arteries by decreasing nonelastic fibrillar components (collagen and fibronectin). Concomitant AT1/AT2 blockade prevented growth, reduced collagen type I and elastin deposition but increased vascular stiffness, fibronectin, and MMP-2 activity. These results demonstrate opposing roles of AT1 receptors that increase fibronectin and vascular stiffness and AT2 receptors that decrease MMP-2 and increase elastin. Changes in vascular wall mechanics, ECM deposition, and MMP activity are thus modulated differentially by Ang II receptors.


Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Artéria Mesentérica Superior/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Receptor Tipo 2 de Angiotensina/fisiologia , Resistência Vascular , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II , Animais , Western Blotting , Colágeno Tipo I/metabolismo , Elasticidade , Elastina/metabolismo , Fibronectinas/metabolismo , Hidralazina/farmacologia , Losartan/farmacologia , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/crescimento & desenvolvimento , Artéria Mesentérica Superior/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo
17.
Eur J Pharmacol ; 513(1-2): 35-45, 2005 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-15878707

RESUMO

Chinese Hamster Ovary Cells (CHO-K1) were transiently and stably transfected to express the human angiotensin AT(1) receptor. Cell surface receptor expression was maximal 2 days after transient transfection. Their pharmacological and signalling properties differed from stably expressed receptors. Receptor reserve was significant in the transient cells but not in stable cells, explaining the higher potency of angiotensin II and the lower degree of insurmountable inhibition by candesartan in the transient cells. [Sar(1)Ile(8)]angiotensin II (sarile) is a potent angiotensin AT(1) receptor antagonist for the stable cells but is a partial agonist, producing 19% of the maximal response by angiotensin II, in transient cells. Internalization of [(3)H]angiotensin II and [(125)I]sarile (i.e., acid-resistant binding) was more pronounced in stable cells. CHO-K1 cells were also transiently transfected with the enhanced green fluorescence-AT(1) receptor gene. Confocal microscopy revealed rapid internalization induced by angiotensin II and sarile but not by candesartan. The above disparities may result from differences in receptor maturation and/or cellular surrounding.


Assuntos
Receptor Tipo 1 de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Ligantes , Microscopia Confocal , Ensaio Radioligante , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tetrazóis/metabolismo , Tetrazóis/farmacologia , Transfecção , Trítio
18.
Hypertension ; 45(1): 115-9, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15569855

RESUMO

Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT1a-, AT1b-, and AT2-receptors. Because of a high degree of similarity among these G-protein-coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT1a receptor (AT1aR) subtype. A dsRNA, AT1 47, was found to be highly selective and efficient in reducing the levels of AT1aR subtype. Transfection of AT1aR-expressing CHO cells with dsRNA AT1 47 resulted in an 80% decrease in the AT1aR expression. In contrast, dsRNA AT1 47 showed no significant effects on both AT1bR and AT2R subtypes. Thus, AT1 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.


Assuntos
Inativação Gênica , Interferência de RNA , RNA de Cadeia Dupla/farmacologia , RNA Interferente Pequeno/farmacologia , Receptor Tipo 1 de Angiotensina/genética , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Transporte de Íons/efeitos dos fármacos , Ligação Proteica , RNA Mensageiro/antagonistas & inibidores , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
19.
Circ Res ; 94(11): 1451-7, 2004 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-15117826

RESUMO

Angiotensin IV (Ang IV) is a metabolite of the potent vasoconstrictor angiotensin II (Ang II). Because specific binding sites for this peptide have been reported in numerous tissues including the brain, it has been suggested that a specific Ang IV receptor (AT4) might exist. Bolus injection of Ang IV in brain ventricles has been implicated in learning, memory, and localized vasodilatation. However, the functions of Ang IV in a physiological context are still unknown. In this study, we generated a transgenic (TG) mouse model that chronically releases Ang IV peptide specifically in the brain. TG mice were found to be hypertensive by the tail-cuff method as compared with control littermates. Treatment with the angiotensin-converting enzyme inhibitor captopril had no effect on blood pressure, but surprisingly treatment with the Ang II AT1 receptor antagonist candesartan normalized the blood pressure despite the fact that the levels of Ang IV in the brains of TG mice were only 4-fold elevated over the normal endogenous level of Ang peptides. Calcium mobilization assays performed on cultured CHO cells chronically transfected with the AT1 receptor confirm that low-dose Ang IV can mobilize calcium via the AT1 receptor only in the presence of Ang II, consistent with an allosteric mechanism. These results suggest that chronic elevation of Ang IV in the brain can induce hypertension that can be treated with angiotensin II AT1 receptor antagonists.


Assuntos
Angiotensina II/análogos & derivados , Angiotensina II/fisiologia , Encéfalo/metabolismo , Hipertensão/genética , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Regulação Alostérica , Angiotensina II/biossíntese , Angiotensina II/genética , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Benzimidazóis/farmacologia , Células CHO/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Cricetinae , Cricetulus , Expressão Gênica , Genes Sintéticos , Proteína Glial Fibrilar Ácida/genética , Globinas/genética , Humanos , Hipertensão/tratamento farmacológico , Imidazóis/farmacologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Piridinas/farmacologia , Coelhos , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Renina/genética , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Tetrazóis/farmacologia , Transgenes
20.
Curr Top Med Chem ; 4(4): 385-401, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14965308

RESUMO

Peptidomimitism is applied to the medicinal chemistry in order to synthesize drugs that devoid of the disadvantages of peptides. AT1 antagonists constitute a new generation of drugs for the treatment of hypertension designed and synthesized to mimic the C-terminal segment of Angiotensin II and to block its binding action on AT1 receptor. An effort was made to understand the molecular basis of hypertension by studying the conformational analysis of Ang II and its derivatives as well as the AT1 antagonists belonging to SARTANs class of molecules. Such studies offer the possibility to reveal the stereoelectronic factors responsible for bioactivity of AT1 antagonists and to design and synthesize new analogs. An example will be given which proves that drugs with better pharmacological and financial profiles may arise based on this rational design.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Angiotensina II/análogos & derivados , Anti-Hipertensivos/química , Desenho de Fármacos , 1-Sarcosina-8-Isoleucina Angiotensina II/análogos & derivados , 1-Sarcosina-8-Isoleucina Angiotensina II/química , Angiotensina II/química , Angiotensina II/farmacologia , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacologia , Humanos , Hipertensão/tratamento farmacológico , Losartan/análogos & derivados , Losartan/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Mimetismo Molecular , Peptídeos/química , Peptídeos/farmacologia , Relação Estrutura-Atividade
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