RESUMO
Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.
Assuntos
Dermatite/fisiopatologia , Receptor Tipo 1 de Angiotensina/fisiologia , Receptor Tipo 2 de Angiotensina/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Células Cultivadas , Dermatite/etiologia , Feminino , Adjuvante de Freund/administração & dosagem , Gânglios Espinais/citologia , Neuritos/fisiologia , Dor/fisiopatologia , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 2 de Angiotensina/análise , Células Receptoras Sensoriais/química , Pele/inervaçãoRESUMO
A insuficiência cardíaca (IC) é uma síndrome de elevada morbimortalidade, correspondendo a um grave problema de saúde pública. Uma das abordagens terapêuticas para IC consiste no uso de antagonistas do receptor de angiotensina II do tipo 1 (AT1R), conhecidos como sartanas. Estudos apontam que uma nova classe de compostos, os agonistas enviesados, é capaz de induzir a sinalização da via da ß-arrestina sem ativação da via da proteína G. Essa seletividade funcional é particularmente interessante, pois a via dependente da proteína G é responsável pelo aumento da pressão arterial, morte celular e fibrose tecidual, levando a hipertrofia cardíaca e progressão da IC. No entanto, a via da ß-arrestina está associada com renovação celular e aumento do inotropismo. Além disso, estudos in vivo sugerem que agonistas enviesados poderiam corresponder a uma terapia superior à dos antagonistas convencionais, que bloqueiam ambas as vias. Apesar do potencial terapêutico, esses compostos possuem estrutura peptídica e, por isso, tem sua administração restrita à via intravenosa. A resolução da estrutura cristalográfica do AT1R permitiu estudos de modelagem molecular mais acurados. Tendo isso em mente, nesse trabalho foram propostos agonistas enviesados de natureza não peptídica para o AT1R por meio de técnicas de modelagem molecular e validação das hipóteses levantadas por ensaios in vitro. Foram realizados estudos de dinâmica molecular com o AT1R (PDB ID: 4YAY) em uma bicamada lipídica e ensaios de ancoramento molecular da angiotensina II (AngII) e do ligante enviesado TRV027. As poses de ancoramento molecular selecionadas foram utilizadas em dinâmicas de complexo, que revelaram diferenças entre os sistemas apo (sem nenhum ligante) e holo (com o ligante no sitio de ligação). Nossos resultados sugerem que o TRV027 induz um padrão exclusivo de ligações de hidrogênio e de estrutura secundária, enquanto que a AngII afeta os resíduos do bolso hidrofóbico do sitio de ligação, principalmente a conformação do Trp2536.48. Com base nas simulações, três farmacóforos foram criados e utilizados de maneira complementar em triagens virtuais na base de dados ZINC15, resultando na seleção de cinco compostos. Um desses compostos apresentou afinidade pelo receptor AT1R e, ainda que estudos complementares de ativação de vias especificas sejam necessários para que o composto possa ser classificado como agonista enviesado, já se constitui em molécula potencialmente promissora. Além disso, esses estudos permitiram a proposição de estruturas inéditas que podem vir a ser hits no processo de desenvolvimento de agonistas enviesados para AT1R. Portanto, como continuidade desse trabalho, essas moléculas serão sintetizadas e investigadas quanto à possível interação com o receptor.
Heart Failure (HF) is a common syndrome with high morbimortality, being considered a serious public health problem. One of the therapeutic approaches for HF consists in the use of the sartan class, which are angiotensin II type 1 receptor (AT1R) antagonists. Recent studies have shown that a new class of compounds, known as biased agonists, is able to induce signaling via ß-arrestin without G-protein activation. This functional selectivity is particularly interesting since G-protein dependent signaling is responsible for cell death and cardiac tissue fibrosis, which leads to cardiac muscle hypertophy and HF progression. On the other hand, ß-arrestin signaling is associated with cellular renewal and increased inotropism. In vivo studies suggests that biased agonists could correspond to a superior therapy over conventional angiotensin II type 1 receptor antagonists, which blocks cell signaling as a whole, however their peptidic structure restricts their use to intravenous administration. Moreover, the AT1R crystal structure determination holds great promise for more accurate molecular modeling studies. With that being said, the aim of this work was to plan and develop new non-peptidic biased agonists for ATR1 employing molecular modeling techniques and in vitro tests for hypothesis validation. Molecular dynamics (MD) simulations of the refined AT1R crystal (PDB ID: 4YAY) embedded in a lipid bilayer and molecular docking studies with angiotensin II (AngII) and TRV027 (biased agonist) were conducted. Selected docking poses from both ligands underwent complex MD simulations revealing differences between apo (ligand free) and holo (ligand in the binding site) systems. Our results suggest that TRV027 induces an exclusive hydrogen bond and secondary structure pattern, while AngII affects the hydrophobic pocket conformation, mainly Trp253. Based on the simulations, three pharmacophore models were created and used in virtual screenings in the ZINC15 database, resulting in the selection of five compounds that were tested in vitro. One of the compounds displayed affinity for AT1R and is a promising molecule. Nonetheless, it needs further pathway activation characterization in order to be a classified as a biased agonist. Furthermore, these results have contributed significantly for the proposition of new structures that could be hits with biased agonist activity for AT1R. Thus, for future works, we point out the necessity for synthesis and characterization of this new compounds
Assuntos
Técnicas In Vitro/métodos , Angiotensina II/agonistas , Insuficiência Cardíaca/patologia , Ligantes , Organização e Administração , Receptores de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/análise , MétodosRESUMO
Besides stimulating vasoconstriction, Angiotensin II is also well known in inducing reactive oxygen species and promoting inflammatory phenotype switch via its type 1 receptor. In clinic, Angiotensin II type 1 (AT1) receptor blocker like candesartan has been widely applied as an antihypertensive medication. We previous have demonstrated that a higher dose of candesartan plays a protective role after kidney injury. However, whether candesartan could exhibit anti-inflammatory effects remains unclear. Here, by stimulating isolated human embryonic kidney epithelial cells with tumor necrosis factor-α (TNF-α), we observed the anti-inflammation capacity of candesartan ex vivo. It was found that pre-treat with candesartan significantly suppressed transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) expression after incubation with TNF-α. Surprisingly, silence of angiotensin II type 1 receptor has little effects on reducing TGF-ß or IL-6 products. Furthermore, candesartan inhibited TNF-α-induced oxidative stress in the primary cultured tubular epithelial cells. Overall, our data indicates that candesartan suppresses TNF-α-induced inflammatory cytokine production by inhibiting oxidative stress, rather than block AT1 receptor activity.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Tetrazóis/farmacologia , Análise de Variância , Compostos de Bifenilo , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-6/análise , Rim/embriologia , Linfotoxina-alfa/análise , Linfotoxina-alfa/efeitos dos fármacos , Espécies Reativas de Oxigênio/análise , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
Angiotensin II (Ang II) acts on Ang II type 1 (AT1) receptors located in the organum vasculosum and subfornical organ (SFO) of the lamina terminalis as a main facilitatory mechanism of sodium appetite. The brain serotonin (5-HT) system with soma located in the dorsal raphe nucleus (DRN) provides a main inhibitory mechanism. In the present study, we first investigated the existence of Ang II AT1 receptors in serotonergic DRN neurones. Then, we examined whether whole body sodium depletion affects the gene expression of the AT1a receptor subtype and the presumed functional significance of AT1 receptors. Using confocal microscopy, we found that tryptophan hydroxylase-2 and serotonin neurones express AT1 receptors in the DRN. Immunofluorescence quantification showed a significant reduction in 5-HT content but no change in AT1 receptor expression or AT1/5-HT colocalisation in the DRN after sodium depletion. Whole body sodium depletion also significantly increased Agtr1a mRNA expression in the SFO and DRN. Oral treatment with the AT1 receptor antagonist losartan reversed the changes in Agtr1a expression in the SFO but not the DRN. Losartan injection into either the DRN or the mesencephalic aqueduct had no influence on sodium depletion-induced 0.3 mol L-1 NaCl intake. The results indicate the expression of Agtr1a mRNA in the DRN and SFO as a marker of sodium depletion. They also suggest that serotonergic DRN neurones are targets for Ang II. However, the function of their AT1 receptors remains elusive.
Assuntos
Núcleo Dorsal da Rafe/metabolismo , Expressão Gênica , Receptor Tipo 1 de Angiotensina/genética , Serotonina/análise , Sódio/deficiência , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Apetite/fisiologia , Núcleo Dorsal da Rafe/química , Imunofluorescência , Expressão Gênica/fisiologia , Losartan/farmacologia , Masculino , Neurônios/química , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/fisiologia , Sódio/sangue , Órgão Subfornical/química , Órgão Subfornical/metabolismo , Triptofano Hidroxilase/análiseRESUMO
O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)
The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)
Assuntos
Humanos , Quimiocinas/metabolismo , Citocinas/metabolismo , Fibroblastos/fisiologia , Interleucina-1beta/fisiologia , Sistema Renina-Angiotensina/fisiologia , Análise de Variância , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Angiotensina II/análise , Angiotensina II/fisiologia , Angiotensina I/análise , Angiotensina I/fisiologia , Western Blotting , Células Cultivadas , Quimiocinas/análise , Citocinas/análise , Gengiva/citologia , Losartan/farmacologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/fisiologia , Peptidil Dipeptidase A/análise , Peptidil Dipeptidase A/fisiologia , Ligamento Periodontal/citologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/fisiologia , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/fisiologiaRESUMO
Cardiovascular disease is often associated with chronic kidney disease and vice versa; myocardial vitamin D receptors (VDRs) are among the probable links between the 2 disorders. The vitamin D receptor activator paricalcitol protects against some renal and cardiovascular complications. However, the structural and electrophysiological effects of myocardial vitamin D receptor modification and its impact on the response to ischemia-reperfusion are currently unknown. This work attempted to determine whether obstructive nephropathy induced myocardial changes (in rats) linked to vitamin D receptor deficiency and to ventricular arrhythmias in Langendorff-perfused hearts. Unilateral ureteral-obstructed and Sham-operated rats were treated with either paricalcitol (30 ng/kg/d intraperitoneal) or vehicle for 15 days. In 5 hearts from each group, we found that obstructed rats showed a reduction in VDRs and an increase in angiotensin II type 1 receptor expression (messenger RNA and protein), suffered fibrosis (determined by Masson trichrome stain) and myofibril reduction with an increase in mitochondrial size, and had dilated crests (determined by electron microscopy). These changes were reversed by paricalcitol. In 8 additional hearts per group, we found that obstructed rats showed a higher incidence of ventricular fibrillation during reperfusion (after 10 minutes of regional ischemia) than did those treated with paricalcitol. The action potential duration was prolonged throughout the experiment in paricalcitol-treated rats. We conclude that the reduction in myocardial vitamin D receptor expression in obstructed rats might be related to myocardial remodeling associated with an increase in arrhythmogenesis and that paricalcitol protects against these changes by restoring myocardial vitamin D receptor levels and prolonging action potentials.
Assuntos
Arritmias Cardíacas/tratamento farmacológico , Ergocalciferóis/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miocárdio/patologia , Receptores de Calcitriol/deficiência , Obstrução Ureteral/complicações , Potenciais de Ação , Animais , Circulação Coronária , Feminino , Miocárdio/metabolismo , Ratos , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/análise , Receptores de Calcitriol/análise , Obstrução Ureteral/metabolismoRESUMO
OBJECTIVE: To validate the importance of the angiotensin II receptor isotypes and the lymphatic vessels in systemic sclerosis and idiopathic pulmonary fibrosis. METHODS: We examined angiotensin II type 1 and 2 receptors and lymphatic vessels in the pulmonary tissues obtained from open lung biopsies of 30 patients with systemic sclerosis and 28 patients with idiopathic pulmonary fibrosis. Their histologic patterns included cellular and fibrotic non-specific interstitial pneumonia for systemic sclerosis and usual interstitial pneumonia for idiopathic pulmonary fibrosis. We used immunohistochemistry and histomorphometry to evaluate the number of cells in the alveolar septae and the vessels stained by these markers. Survival curves were also used. RESULTS: We found a significantly increased percentage of septal and vessel cells immunostained for the angiotensin type 1 and 2 receptors in the systemic sclerosis and idiopathic pulmonary fibrosis patients compared with the controls. A similar percentage of angiotensin 2 receptor positive vessel cells was observed in fibrotic non-specific interstitial pneumonia and usual interstitial pneumonia. A significantly increased percentage of lymphatic vessels was present in the usual interstitial pneumonia group compared with the non-specific interstitial pneumonia and control groups. A Cox regression analysis showed a high risk of death for the patients with usual interstitial pneumonia and a high percentage of vessel cells immunostained for the angiotensin 2 receptor in the lymphatic vessels. CONCLUSION: We concluded that angiotensin II receptor expression in the lung parenchyma can potentially control organ remodeling and fibrosis, which suggests that strategies aimed at preventing high angiotensin 2 receptor expression may be used as potential therapeutic target in patients with pulmonary systemic sclerosis and idiopathic pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática/patologia , Vasos Linfáticos/patologia , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 2 de Angiotensina/análise , Escleroderma Sistêmico/patologia , Idoso , Análise de Variância , Biópsia , Feminino , Fibrose , Humanos , Imuno-Histoquímica , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Testes de Função Respiratória , Fatores de Risco , Estatísticas não ParamétricasRESUMO
OBJECTIVE: To validate the importance of the angiotensin II receptor isotypes and the lymphatic vessels in systemic sclerosis and idiopathic pulmonary fibrosis. METHODS: We examined angiotensin II type 1 and 2 receptors and lymphatic vessels in the pulmonary tissues obtained from open lung biopsies of 30 patients with systemic sclerosis and 28 patients with idiopathic pulmonary fibrosis. Their histologic patterns included cellular and fibrotic non-specific interstitial pneumonia for systemic sclerosis and usual interstitial pneumonia for idiopathic pulmonary fibrosis. We used immunohistochemistry and histomorphometry to evaluate the number of cells in the alveolar septae and the vessels stained by these markers. Survival curves were also used. RESULTS: We found a significantly increased percentage of septal and vessel cells immunostained for the angiotensin type 1 and 2 receptors in the systemic sclerosis and idiopathic pulmonary fibrosis patients compared with the controls. A similar percentage of angiotensin 2 receptor positive vessel cells was observed in fibrotic non-specific interstitial pneumonia and usual interstitial pneumonia. A significantly increased percentage of lymphatic vessels was present in the usual interstitial pneumonia group compared with the non-specific interstitial pneumonia and control groups. A Cox regression analysis showed a high risk of death for the patients with usual interstitial pneumonia and a high percentage of vessel cells immunostained for the angiotensin 2 receptor in the lymphatic vessels. CONCLUSION: We concluded that angiotensin II receptor expression in the lung parenchyma can potentially control organ remodeling and fibrosis, which suggests that strategies aimed at preventing high angiotensin 2 receptor expression may be used as potential therapeutic target in patients with pulmonary systemic sclerosis and idiopathic pulmonary fibrosis. .
Assuntos
Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar Idiopática/patologia , Vasos Linfáticos/patologia , Receptor Tipo 1 de Angiotensina/análise , /análise , Escleroderma Sistêmico/patologia , Análise de Variância , Biópsia , Fibrose , Imuno-Histoquímica , Pulmão/patologia , Modelos de Riscos Proporcionais , Testes de Função Respiratória , Fatores de Risco , Estatísticas não ParamétricasRESUMO
High salt intake is a known cardiovascular risk factor and is associated with cardiac alterations. To better understand this effect, male Wistar rats were fed a normal (NSD: 1.3% NaCl), high 4 (HSD4: 4%), or high 8 (HSD8: 8%) salt diet from weaning until 18 wk of age. The HSD8 group was subdivided into HSD8, HSD8+HZ (15 mg . kg(-1) . d(-1) hydralazine in the drinking water), and HSD8+LOS (20 mg . kg(-1) . d(-1) losartan in the drinking water) groups. The cardiomyocyte diameter was greater in the HSD4 and HSD8 groups than in the HSD8+LOS and NSD groups. Interstitial fibrosis was greater in the HSD4 and HSD8 groups than in the HSD8+HZ and NSD groups. Hydralazine prevented high blood pressure (BP) and fibrosis, but not cardiomyocyte hypertrophy. Losartan prevented high BP and cardiomyocyte hypertrophy, but not fibrosis. Angiotensin II type 1 receptor (AT(1)) protein expression in both ventricles was greater in the HSD8 group than in the NSD group. Losartan, but not hydralazine, prevented this effect. Compared with the NSD group, the binding of an AT(1) conformation-specific antibody that recognizes the activated form of the receptor was lower in both ventricles in all other groups. Losartan further lowered the binding of the anti-AT(1) antibody in both ventricles compared with all other experimental groups. Angiotensin II was greater in both ventricles in all groups compared with the NSD group. Myocardial structural alterations in response to HSD are independent of the effect on BP. Salt-induced cardiomyocyte hypertrophy and interstitial fibrosis possibly are due to different mechanisms. Evidence from the present study suggests that salt-induced AT(1) receptor internalization is probably due to angiotensin II binding.
Assuntos
Pressão Sanguínea/fisiologia , Cardiomegalia/induzido quimicamente , Cardiomegalia/fisiopatologia , Miocárdio/patologia , Cloreto de Sódio na Dieta/administração & dosagem , Aldosterona/sangue , Angiotensina II/análise , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Animais , Anti-Hipertensivos/administração & dosagem , Cardiomegalia/patologia , Colágeno Tipo I/análise , Colágeno Tipo III/análise , Modelos Animais de Doenças , Ingestão de Líquidos , Ingestão de Alimentos , Ecocardiografia , Fibrose , Expressão Gênica , Ventrículos do Coração/química , Ventrículos do Coração/patologia , Hidralazina/administração & dosagem , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Losartan/administração & dosagem , Masculino , Potássio/sangue , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/fisiologia , Receptor Tipo 2 de Angiotensina/análise , Renina/sangue , Sistema Renina-Angiotensina/genética , Sistema Renina-Angiotensina/fisiologia , Sódio/sangue , Sódio/urina , Fator de Crescimento Transformador beta/análise , UrinaRESUMO
Diverse intracoronary agonists cause cardiac effects while acting on coronary endothelial luminal membrane (CELM) receptor. Our data show: a) the presence of AT(1)R in isolated CELM and in all cardiac cell types and b) sustained intracoronary infusions of Ang II-POL, a large sized molecule (approximately 15,000 kDa) confined to the vessel lumen that can only act on CELM's AT(1)R or Ang II (approximately 1 kDa); both exert the same maximum positive inotropic (PIE) and coronary constriction (CPP). The effects of these two agonists are blocked by Losartan and by Sar-POL; a large size antagonist (approximately 15,000 kDa) that acts only on CELM. Ang II effects are transient due to desensitization and cause tachyphylaxis to Ang II and toward Ang II-POL suggesting that both Ang II and Ang II-POL act on the same receptor group. In contrast, Ang II-POL effects are sustained and do not cause tachyphylaxis. The results show that intravascular Ang II and Ang II-POL act differentially by an unknown mechanism on CELM's AT(1)R and suggest that intravascular Ang II and Ang II-POL cause PIE and CCP by activation limited to CELM's AT(1)R through an unknown mechanism that is space-confined to the CELM's AT(1)R.
Assuntos
Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/agonistas , Angiotensina II/farmacologia , Animais , Vasos Coronários/fisiologia , Endotélio Vascular/fisiologia , Losartan/farmacologia , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Saralasina/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
BACKGROUND: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. METHODS: Reverse transcription-polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). RESULTS: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang I (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. CONCLUSION: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro.
Assuntos
Gengiva/metabolismo , Sistema Renina-Angiotensina/fisiologia , Angiotensinogênio/análise , Angiotensinas/análise , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/metabolismo , Epitélio/metabolismo , Fibroblastos/metabolismo , Fluorometria , Gengiva/citologia , Imuno-Histoquímica , Masculino , Oligopeptídeos/metabolismo , Peptidil Dipeptidase A/análise , Periodontite/metabolismo , Periodontite/patologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 2 de Angiotensina/análise , Renina/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Intrarenal concentration of angiotensin II increases after the onset of ureteral obstruction in the obstructed kidney. The effect of pretreatment with losartan, a specific angiotensin II AT1 receptor antagonist, on lipid contents, which were previously modified by unilateral ureteral obstruction (UUO), was studied in renal cortex of rats. Adult Wistar Kyoto rats were subjected to either UUO for 24 hr or control sham operation after being treated with losartan in the drinking water at 10 mg/kg rat/day for 15 days. In the cortex of obstructed kidney the increased free and esterified cholesterol concentrations were associated with the increased cholesterol synthesis measured by incorporation of 14C-acetate (0.001>p), compared with control and contralateral kidneys. The increased amount of phosphatidylcholine was related with the increased incorporation of 14C-choline into phosphatidylcholine (0.01>p). Phosphatidylethanolamine and sphingomyelin decreased slightly but total phospholipid content did not change. The level of AT1 receptor mRNA in obstructed kidney was significantly lower than in control and contralateral kidneys. Losartan pretreatment attenuated (0.01>p) the increase in cholesterol content and synthesis and restored and enhanced the AT1 angiotensin II receptor gene expression. The interference in the renin-angiotensin system before UUO may modify renal cortex cholesterol content.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Córtex Renal/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/análise , Losartan/farmacologia , Obstrução Ureteral/metabolismo , Angiotensina II/metabolismo , Animais , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Córtex Renal/efeitos dos fármacos , Córtex Renal/fisiopatologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/genética , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Esfingomielinas/metabolismoRESUMO
OBJECTIVE: Sexual dimorphism has been observed in arterial hypertension. Blood pressure levels are lower in female than in male spontaneously hypertensive rats (SHR). Angiotensin II (Ang II) plays a major role in the regulation of blood pressure. The aim of this study was to compare Ang II vascular reactivity and AT(1) and AT(2) receptor gene expression in female and male SHR. METHODS: SHR animals were divided into four groups: (I) male, (II) female in physiological estrus, (III) ovariectomized and (IV) ovariectomized treated with estrogen. Arterial blood pressure, AT(1) and AT(2) mRNA expression were determined. Ang II responses in aorta and mesenteric vessels were also evaluated. RESULTS: In female SHR, aorta and mesenteric microvessels were hyporeactive to Ang II in comparison to male SHR. In ovariectomized females, Ang II vasoconstriction was similar to that of males. Estrogen treatment abolished this difference. The mRNA expression for AT(1) was higher in aorta and mesenteric vessels from males than in females. In ovariectomized SHR, mRNA expression for AT(1) was comparable to that of males. Treatment with estrogen reversed the over expression observed. Whereas AT(2) gene expression did not differ, a lower ratio AT(1)/AT(2) was found in female than in male vessels. A higher mRNA expression for AT(1) was observed in kidney from male than in female. Ovariectomy resulted in up-regulation of this subtype receptor. Treatment with estrogen reversed the overexpression. AT(2) gene expression was higher in kidney from female than male SHR. Ovariectomy reduced AT(2) gene expression and estrogen treatment reversed the alteration observed in kidney. CONCLUSION: There is sexual dimorphism in vascular reactivity and in receptor gene expression to Ang II in SHR. We conclude that estrogen modulates AT(1) and AT(2) receptor gene expression and that this might explain at least partially the lower blood pressure observed in female SHR.