RESUMO
Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.
Assuntos
Neurônios/metabolismo , Óxido Nítrico Sintase Tipo III/fisiologia , Fator de Transcrição RelA/metabolismo , Animais , Células Cultivadas , Córtex Cerebelar , Embrião de Mamíferos , Hipocampo , Neurônios/citologia , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-DawleyRESUMO
Endothelial dysfunction is characterised by the low bioavailability of nitric oxide with a relevant negative impact on the nitric oxide/cGMP pathway. The loss of nitric oxide/cGMP signaling may be caused by an increased arginase activity. Plant-derived substances, especially polyphenols, are compounds that have the potential to inhibit arginase activity and they may represent an attractive therapeutic option to combat clinical outcomes related to endothelial dysfunction. An extensive review was carried out using all available data published in English in the Pubmed database, and without restriction regarding the year of publication. Despite the increased number of new substances that have been tested as arginase inhibitors, it is rare to find a compound that satisfies all the toxicological criteria to be used in the development of a new drug. On the other hand, recent data have shown that substances from plants have great potential to be applied as arginase inhibitors, most of which are polyphenols. Of the relevant mechanisms in this process, the inhibition of arginase by natural products seems to act against endothelial dysfunction by reestablishing the vascular function and elevating nitric oxide levels (by increasing the amounts of substrate (L-arginine, and endothelial nitric oxide synthase activation and stabilisation) as well as decreasing the generation of reactive species (formed by uncoupledendothelial nitric oxide synthase). This review summarises several topics regarding arginase inhibition by natural substances as well as indicating this pathway as an emergent strategy to elevate nitric oxide levels in disorders involving endothelial dysfunction. In addition, some aspects regarding structural activity and future perspectives are discussed.
Assuntos
Arginase/antagonistas & inibidores , Produtos Biológicos/farmacologia , Óxido Nítrico/metabolismo , Plantas/química , Polifenóis/farmacologia , Doenças Vasculares/tratamento farmacológico , Arginase/metabolismo , Produtos Biológicos/química , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Estrutura Molecular , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/fisiologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Polifenóis/química , Relação Estrutura-Atividade , Doenças Vasculares/fisiopatologiaRESUMO
Cardiovascular benefits from estradiol activation of nitric oxide endothelial production may depend on vascular wall and on estrogen receptor alpha (ESR1) and nitric oxide synthase (NOS3) polymorphisms. We have evaluated the microcirculation in vivo through nailfold videocapillaroscopy, before and after acute nasal estradiol administration at baseline and after increased sheer stress (postocclusive reactive hyperemia response) in 100 postmenopausal women, being 70 controls (healthy) and 30 simultaneously hypertensive and diabetic (HD), correlating their responses to PvuII and XbaI ESR1 polymorphisms and to VNTR, T-786C and G894T NOS3 variants. In HD women, C variant allele of ESR1 Pvull was associated to higher vasodilatation after estradiol (1.72 vs 1.64 mm/s, pâ=â0.01 compared to TT homozygotes) while G894T and T-786C NOS3 polymorphisms were connected to lower increment after shear stress (15% among wild type and 10% among variant alleles, pâ=â0.02 and 0.04). The G variant allele of ESR1 XbaI polymorphism was associated to higher HOMA-IR (3.54 vs. 1.64, pâ=â0.01) in HD and higher glucose levels in healthy women (91.8 vs. 87.1 mg/dl, pâ=â0.01), in which increased waist and HOMA-IR were also related to the G allele in NOS3 G894T (waist 93.5 vs 88.2 cm, pâ=â0.02; HOMA-IR 2.89 vs 1.48, pâ=â0.05). ESR1 Pvull, NOS3 G894T and T-786C polymorphism analysis may be considered in HD postmenopausal women for endothelial response prediction following estrogen therapy but were not discriminatory for endothelial response in healthy women. ESR1 XbaI and G894T NOS3 polymorphisms may be useful in accessing insulin resistance and type 2 diabetes risks in all women, even before menopause and occurrence of metabolic disease.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/fisiologia , Receptor alfa de Estrogênio/fisiologia , Resistência à Insulina , Óxido Nítrico Sintase Tipo III/fisiologia , Pós-Menopausa , Doenças Cardiovasculares/genética , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/genética , Fatores de RiscoRESUMO
We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration-response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1-100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.
Assuntos
Insulina/fisiologia , Óxido Nítrico Sintase Tipo III/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Bexiga Urinária/fisiologia , Adolescente , Adulto , Animais , Dieta Hiperlipídica , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mucosa/metabolismo , Relaxamento Muscular/efeitos dos fármacos , Obesidade/fisiopatologia , Resposta a Proteínas não Dobradas , Bexiga Urinária Hiperativa/fisiopatologia , Adulto JovemRESUMO
Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT(1) , eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes.
Assuntos
Gorduras na Dieta/toxicidade , Proteínas do Olho/fisiologia , Obesidade/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Degeneração Retiniana/etiologia , Animais , Apoptose , Astrócitos/patologia , Glicemia/análise , Retinopatia Diabética , Modelos Animais de Doenças , Ácidos Graxos/sangue , Proteínas Substratos do Receptor de Insulina/fisiologia , Resistência à Insulina , Peroxidação de Lipídeos , Lipídeos/sangue , Fígado/patologia , Masculino , Óxido Nítrico Sintase Tipo I/fisiologia , Óxido Nítrico Sintase Tipo III/fisiologia , Obesidade/sangue , Obesidade/complicações , Fosfatidilinositol 3-Quinases/fisiologia , Ratos , Ratos Wistar , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/sangue , Degeneração Retiniana/fisiopatologia , Transdução de SinaisRESUMO
Placental vascular tone is critically influenced by nitric oxide (NO) derived from endothelial NO synthase (eNOS) activity. Placental vessels from pregnancies complicated with intrauterine growth restriction present altered NOS-dependent vasodilation. Arginase-2 competes with eNOS for l-arginine and counteracts the NOS-dependent relaxation in umbilical vessels from normal pregnancies. However there is no data regarding the contribution of arginase activity on the impaired endothelial function in IUGR placenta. We studied whether arginase-2 participates in IUGR-related placental vascular dysfunction counteracting eNOS-dependent relaxation, and the regulation of arginase-2 and eNOS expression in endothelial cells from IUGR umbilical arteries (HUAEC) and veins (HUVEC). In IUGR-derived umbilical arteries (UA) and veins (UV), and chorionic arteries (CA), NOS-dependent vasoactive response in the presence and absence of BEC (arginase inhibitor) was studied. Protein levels of eNOS (total and Ser(1177)-P-eNOS), arginase-2 and arginase activity were determined in IUGR HUAEC and HUVEC. In IUGR vessels eNOS-dependent relaxation was reduced, being improved by BEC. This effect was higher in arteries than veins, and in chorionic compared with umbilical vessels. In cultured IUGR endothelial cells, arginase-2 protein expression and activity were increased in HUVEC, without changes in HUAEC. In IUGR-derived endothelium there was a generalized reduction in the in vitro eNOS activation (Ser(1177)-P-eNOS/eNOS), and therefore a decreased eNOS/arginase activity ratio. Here we provide ex vivo and in vitro evidence for a vascular role of arginase throughout placental vasculature, negatively controlling NOS activity. This effect seems to be crucial in the pathophysiology of endothelial dysfunction present in IUGR feto-placental vessels.
Assuntos
Arginase/metabolismo , Vasos Sanguíneos/fisiopatologia , Células Endoteliais/metabolismo , Retardo do Crescimento Fetal/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Placenta/irrigação sanguínea , Artérias Umbilicais/fisiopatologia , Adulto , Arginase/fisiologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Recém-Nascido , Masculino , Óxido Nítrico Sintase Tipo III/fisiologia , Placenta/metabolismo , Placenta/patologia , Circulação Placentária/fisiologia , Gravidez , Artérias Umbilicais/metabolismo , Artérias Umbilicais/patologiaRESUMO
INTRODUCTION: In recent years, new tools for the study of molecular biology and genetics have resulted in significant contributions to the scientific community. The potential use of genetic variations as biomarkers in the management of current and future conditions is generating considerable excitement in health care for disorders such as erectile dysfunction (ED). AIM: This review briefly describes the molecular and genetic mechanisms involved in ED and provides an overall view of the literature relevant to possible relationships between genetic factors and ED. METHODS: This is a narrative review of studies on the potential influence of polymorphisms on the risk of developing ED. MAIN OUTCOME MEASURE: We reviewed genetic association studies involving polymorphisms and the ED phenotype. RESULTS: There is growing evidence for the influence of genetic polymorphisms on the risk of ED and on the interindividual variability in sildenafil treatment. CONCLUSIONS: Although this field is still in its infancy, genetic association studies aimed at defining a molecular basis for ED have provided some important evidence that a patient's genotype may be used in the future to assess risk, as well as to plan treatment and prevention programs in the clinic.
Assuntos
Disfunção Erétil/genética , Polimorfismo Genético/fisiologia , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/fisiopatologia , Marcadores Genéticos/efeitos dos fármacos , Marcadores Genéticos/genética , Marcadores Genéticos/fisiologia , Humanos , Masculino , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/fisiologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/fisiologia , Inibidores da Fosfodiesterase 5/uso terapêutico , Piperazinas/uso terapêutico , Polimorfismo Genético/efeitos dos fármacos , Polimorfismo Genético/genética , Purinas/uso terapêutico , Citrato de Sildenafila , Sulfonas/uso terapêuticoRESUMO
Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.
Assuntos
Citosol/enzimologia , Óxido Nítrico Sintase Tipo III/fisiologia , Calibragem , Membrana Celular/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Humanos , Inflamação , Microscopia de Fluorescência/métodos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Permeabilidade , Fosforilação , Fator de Ativação de Plaquetas/metabolismo , Transporte Proteico , Frações SubcelularesRESUMO
BACKGROUND AND PURPOSE: Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H(2)O(2) is an important endothelium-derived relaxant factor in the mouse aorta. The role of H(2)O(2) and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H(2)O(2) contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE(-/-)). EXPERIMENTAL APPROACH: Changes in isometric tension were recorded on a myograph; simultaneously, NO and H(2)O(2) were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms. KEY RESULTS: Aortas from ApoE(-/-) mice showed impaired vasodilatation paralleled by decreased NO and H(2)O(2) production. Inhibition of nNOS with L-Arg(NO2) -L-Dbu, knockdown of nNOS and catalase, which decomposes H(2)O(2) into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H(2)O(2) in wild-type animals, but had no effect in ApoE(-/-) mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE(-/-) mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE(-/-) mice. CONCLUSIONS AND IMPLICATIONS: Our data show that endothelial nNOS-derived H(2)O(2) production is impaired and contributes to endothelial dysfunction in ApoE(-/-) aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.
Assuntos
Aterosclerose/fisiopatologia , Endotélio Vascular/fisiopatologia , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico Sintase Tipo I/fisiologia , Acetilcolina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiopatologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Catalase/farmacologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo I/deficiência , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/fisiologia , Vasodilatação/efeitos dos fármacosRESUMO
Nitric oxide (NO), primarily identified as an endothelium-derived relaxing factor, is a free radical that signals different biological processes. The identification of NO synthase (NOS) isoforms and the subsequent characterization of the mechanisms of cell activation of the enzymes permitted the partial understanding of both the physiological interactions and of the mechanisms of the diseases in which NO is involved. Mainly expressed in the vascular endothelium, the endothelial NOS isoform (eNOS) plays an important role in the regulation of vascular reactivity and in the development and progression of atherosclerosis. The purpose of this review is to contextualize the reader about the eNOS structure and its mechanisms of cell activation. In view of the advances in molecular biology, we will also address the known mechanisms of gene expression regulation and the role of variants on the genetic code of eNOS associated with cardiovascular phenotypes. Although the importance of NO as an atheroprotective molecule is recognized, our focus will be the review of the literature on NO and its participation in the modulation of the muscle vasodilatation phenotype.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/fisiopatologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/fisiologia , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Músculos/irrigação sanguínea , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo III/genética , Fenótipo , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
O óxido nítrico (NO), primariamente identificado como um fator relaxante derivado do endotélio, é um radical livre atuante na sinalização de diferentes processos biológicos. A identificação das isoformas das sintases do NO (NOS) e a subsequente caracterização dos mecanismos de ativação celulares das enzimas possibilitaram tanto a compreensão de parte das interações fisiológicas como a compreensão de parte dos mecanismos de doença, na qual o NO está envolvido. A isoforma endotelial da NOS (eNOS), expressa principalmente no endotélio vascular, desempenha importante papel na regulação da reatividade vascular e no desenvolvimento e na progressão da aterosclerose. Esta revisão tem o propósito de contextualizar o leitor sobre a estrutura da eNOS e seus mecanismos de ativação celular. Tendo em vista os avanços da biologia molecular, trataremos ainda dos conhecidos mecanismos de regulação da expressão gênica e do papel de variantes no código genético da eNOS associados a fenótipos cardiovasculares. Embora se reconheça a importância do NO como molécula ateroprotetora, nossa atenção estará voltada à revisão de literatura envolvendo NO e sua participação na modulação do fenótipo de vasodilatação muscular.
Nitric oxide (NO), primarily identified as an endothelium-derived relaxing factor, is a free radical that signals different biological processes. The identification of NO synthase (NOS) isoforms and the subsequent characterization of the mechanisms of cell activation of the enzymes permitted the partial understanding of both the physiological interactions and of the mechanisms of the diseases in which NO is involved. Mainly expressed in the vascular endothelium, the endothelial NOS isoform (eNOS) plays an important role in the regulation of vascular reactivity and in the development and progression of atherosclerosis. The purpose of this review is to contextualize the reader about the eNOS structure and its mechanisms of cell activation. In view of the advances in molecular biology, we will also address the known mechanisms of gene expression regulation and the role of variants on the genetic code of eNOS associated with cardiovascular phenotypes. Although the importance of NO as an atheroprotective molecule is recognized, our focus will be the review of the literature on NO and its participation in the modulation of the muscle vasodilatation phenotype.
El óxido nítrico (NO), primariamente identificado como un factor relajante derivado del endotelio, es un radical libre actuante en la señalización de diferentes procesos biológicos. La identificación de las isoformas de las sintasas del NO (NOS) y la subsecuente caracterización de los mecanismos de activación celulares de las enzimas posibilitaron tanto la comprensión de parte de las interacciones fisiológicas como la comprensión de parte de los mecanismos de enfermedad, en la cual el NO está envuelto. La isoforma endotelial de la NOS (eNOS), expresada principalmente en el endotelio vascular, desempeña importante papel en la regulación de la reactividad vascular y en el desarrollo y en la progresión de la aterosclerosis. Esta revisión tiene el propósito de contextualizar al lector sobre la estructura de la eNOS y sus mecanismos de activación celular. Teniendo en vista los avances de la biología molecular, trataremos aun de los conocidos mecanismos de regulación de la expresión génica y del papel de variantes en el código genético de la eNOS asociados a fenotipos cardiovasculares. Aunque se reconozca la importancia del NO como molécula ateroprotectora, nuestra atención estará volcada a la revisión de literatura envolviendo NO y su participación en la modulación del fenotipo de vasodilatación muscular.
Assuntos
Humanos , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/fisiopatologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/fisiologia , Ativação Enzimática , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Músculos/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico/genética , Fenótipo , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
In large vessels, endothelium-dependent vasodilation is mainly attributed to endothelial nitric oxide synthase (eNOS)-derived NO production. However, we have recently shown that neuronal nitric oxide synthase (nNOS)-derived H(2)O(2) is also an endothelium-dependent relaxing factor in the mouse aorta. The relative contribution of nNOS/eNOS, H(2)O(2)/NO remains to be characterized. This work was undertaken to determine the relative contribution of NO versus H(2)O(2), and eNOS versus nNOS to endothelium-dependent vasodilation in the mouse aorta. We used carbon microsensors placed next to the lumen of the vessels to simultaneously measure NO, H(2)O(2) and vascular tone. Acetylcholine produced a concentration-dependent increase in NO and H(2)O(2) production with a good coefficient of linearity with acetylcholine-induced relaxation (R(2)=0.93 and 0.96 for NO and H(2)O(2), respectively). L-NAME, a non-selective inhibitor of nitric oxide synthase, abolished NO and H(2)O(2) production, and impaired vasodilation. Selective pharmacological inhibition of nNOS with L-Arg(NO2)-L-Dbu-NH(2) 2TFA and specific knock-down of nNOS abrogated H(2)O(2) and decreased by half acetylcholine-induced vasodilation. Catalase, which specifically decomposes H(2)O(2), did not interfere with NO, but impaired H(2)O(2) and decreased vasodilation to the same level as those obtained with nNOS inhibition or knocking down. Specific knocking down of eNOS had no effect on H(2)O(2) production but greatly reduced NO and decreased vasodilation to levels similar to those found with nNOS inhibition. In eNOS knocked-down mice, pharmacological nNOS inhibition dramatically reduced H(2)O(2) production and further reduced the residual acetylcholine-induced vasodilation. It is concluded that nNOS/eNOS and H(2)O(2)/NO both contribute in a significant way to relaxation in the mouse aorta.
Assuntos
Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico Sintase Tipo I/fisiologia , Vasodilatação/efeitos dos fármacos , Animais , Catalase/metabolismo , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Nitroarginina/farmacologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Concentração Osmolar , Vasodilatadores/farmacologiaRESUMO
OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECS WERE DIVIDED INTO FOUR GROUPS: control, treatment with 180 microM hydrogen peroxide (H(2)O(2)), treatment with 150 microg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H(2)O(2) for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.
Assuntos
Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Piper/química , Extratos Vegetais/farmacologia , Células Cultivadas , Células Endoteliais/enzimologia , Ensaio de Imunoadsorção Enzimática , Humanos , Estresse Oxidativo/fisiologia , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Fatores de Tempo , Veias Umbilicais/enzimologiaRESUMO
OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were divided into four groups: control, treatment with 180 ìM hydrogen peroxide (H2O2), treatment with 150 ìg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.
Assuntos
Humanos , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Piper/química , Extratos Vegetais/farmacologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Células Endoteliais/enzimologia , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro , Estatísticas não Paramétricas , Fatores de Tempo , Veias Umbilicais/enzimologiaRESUMO
Gene therapy has become an important tool for understanding several cardiovascular diseases. In the present study we investigated the effects of endothelial nitric oxide synthase (eNOS) overexpression on renovascular hypertension. Experiments were carried out in C57BL/6 mice randomly assigned to either a two-kidney one-clip (2K1C) hypertension group or a sham-operated group. At the same time surgery was carried out, both 2K1C and sham mice received an intravenous injection of recombinant adenovirus expressing the functional gene eNOS or the reporter gene beta-galactosidase (beta-gal). Fourteen days later, arterial pressure, baroreflex sensitivity, and cardiac sympathetic and parasympathetic tone were evaluated in conscious mice. Measurement of mean arterial pressure showed arterial hypertension in 2K1C-betagal mice compared with sham-betagal mice (121 +/- 3 vs. 96 +/- 2 mm Hg, p < 0.01), which was prevented by eNOS overexpression (2K1C-eNOS 100 +/- 4 vs. sham-eNOS 99 +/- 3 mm Hg). Linear regression analysis of the reflex tachycardia response to sodium nitroprusside-induced hypotension showed that baroreflex sensitivity was significantly attenuated in 2K1C-betagal mice (5.8 +/- 0.5 vs. sham-betagal 8.0 +/- 0.8 beats.min-1 x mm Hg-1, p < 0.05), but this decrease was not prevented by eNOS overexpression (2K1C-eNOS 7.2 +/- 0.5 vs. sham-eNOS 8.8 +/- 0.7 beats x min-1 x mm Hg-1, p < 0.05). The cardiac sympathetic tone was augmented and the vagal tone was reduced in 2K1C-betagal (152 +/- 17 and 45 +/- 12 beats.min-1, respectively) compared with sham-betagal mice (112 +/- 6 and 89 +/- 7 beats.min-1, respectively), and similar results were observed in 2K1C-eNOS mice compared with sham-eNOS. The data indicate that eNOS overexpression was able to prevent the development of 2K1C renovascular hypertension in mice, without affecting other characteristic cardiovascular dysfunctions.
Assuntos
Hipertensão Renovascular/enzimologia , Hipertensão Renovascular/prevenção & controle , Óxido Nítrico Sintase Tipo III/fisiologia , Adenoviridae/genética , Animais , Sistema Nervoso Autônomo/fisiologia , Barorreflexo/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Coração/fisiologia , Frequência Cardíaca/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Nitroprussiato/farmacologia , Circulação Renal/fisiologia , Vasodilatadores/farmacologiaRESUMO
The endothelium is responsible for the maintenance of vascular homeostasis. In physiological conditions it acts keeping vascular tonus, laminar blood flow, plasmatic membrane fluidity, the balance between coagulation and fibrinolysis and the inhibition of cellular proliferation, migration and the inflammatory response. Endothelial dysfunction is defined as an alteration of vascular relaxation induced by reduction of endothelium-derived relaxing factors (ERRFs), mainly nitric oxide. These abnormal vasomotor responses occur in the presence of various risk factors for atherosclerosis. The metabolic syndrome is considered a state of chronic inflammation accompanied of endothelial dysfunction causing an increased incidence of ischemic cardiovascular events and high mortality. This revision will encompass the physiological process of vascular function regulation, methods for in vivo assessment of endothelial dysfunction and therapies capable to improve vascular function and consequently minimize the cardiovascular risk due to metabolic syndrome.