RESUMO
AIM: We determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. MAIN METHODS: O-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration-response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 µmol/L) in the presence of L-NAME (NOS inhibitor, 100 µmol/L). KEY FINDINGS: The content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SIGNIFICANCE: Increased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.
Assuntos
Acetilglucosamina/química , Aorta Torácica/fisiopatologia , Hipertensão/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Processamento de Proteína Pós-Traducional , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Aorta Torácica/enzimologia , Feminino , Glicosilação , Hipertensão/enzimologia , Artérias Mesentéricas/enzimologia , N-Acetilglucosaminiltransferases , Gravidez , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , beta-N-Acetil-Hexosaminidases/químicaRESUMO
Essential hypertension is considered to be a result of the interaction between genetic and environmental factors, including perinatal factors. Different advantageous perinatal factors proved to have beneficial long-lasting effects against an abnormal genetic background. Taurine is a ubiquitous sulphur-containing amino acid present in foods such as seafood. The antihypertensive effects of taurine have been reported in experimental studies and in human hypertension. We aimed to investigate the effects of perinatal treatment with taurine in spontaneously hypertensive rats (SHR), a known model of genetic hypertension. Female SHR were administered with taurine (3 g/L) during gestation and lactation (SHR-TAU). Untreated SHR and Wistar-Kyoto rats (WKY) were used as controls. Long-lasting effects in offspring were investigated. Addition of taurine to the mother's drinking water reduced blood pressure in adult offspring. No differences were observed in cardiac hypertrophy. Findings on morphometric evaluations suggest that perinatal treatment with taurine would be partially effective in improving structural alterations of the aorta. Modifications in gene expression of Bcl-2 family members and upregulation of endothelial nitric oxide synthase in the aorta of 22-week-old male offspring were found. No differences were observed on relative telomere length in different cardiovascular tissues between SHR and SHR-TAU. Altogether results suggest that taurine programming, albeit sex specific, is associated with gene expression changes which ultimately may lead to improvement of aortic remodelling and enhanced endothelial function because of augmented nitric oxide (NO) production.
Assuntos
Anti-Hipertensivos/farmacologia , Aorta Torácica/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Hipertensão Essencial/tratamento farmacológico , Óxido Nítrico Sintase Tipo III/metabolismo , Taurina/farmacologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/fisiopatologia , Modelos Animais de Doenças , Regulação para Baixo , Hipertensão Essencial/enzimologia , Hipertensão Essencial/genética , Hipertensão Essencial/fisiopatologia , Feminino , Idade Gestacional , Lactação , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Fatores Sexuais , Transdução de SinaisRESUMO
Susceptibility to develop hypertension may be established during early stages of life that include the intrauterine period, infancy and childhood. We recently showed that blood pressure increased when rats reached adulthood when sucrose was ingested for a short-term critical window from postnatal day 12 to 28 in the rat, which corresponds to days around weaning. Here, we studied several factors that might participate in the increased susceptibility to hypertension when adulthood is reached by analyzing the changes produced at the end of the sucrose ingestion during this critical period. Body weight of the rats at the end of the sucrose period was decreased even if there was an increased ingestion in Kcal. We found an increase in blood pressure accompanied by a decrease in endothelial nitric oxide synthase (eNOS) expression in the aorta. When insulin was administered to rats receiving sucrose, glucose in plasma diminished later than in controls and this slight insulin resistance may reduce nitric oxide synthase action. Oleic acid that modulates eNOS expression was increased, lipoperoxidation was elevated and total non-enzymatic anti-oxidant capacity was decreased. There was also a decrease in SOD2 expression. We also studied the expression of Sirt1, which regulates eNOS expression and Sirt3, which regulates SOD2 expression as possible epigenetic targets of enzyme expression involved in the long- term programming of hypertension. Sirt3 was decreased but we did not find an alteration in Sirt1 expression. We conclude that these changes may underpin the epigenetic programming of increased susceptibility to develop hypertension in the adults when there was exposure to high sucrose levels near weaning in rats.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Hipertensão/fisiopatologia , Sirtuína 1/metabolismo , Sirtuínas/metabolismo , Sacarose/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Ácidos Graxos/sangue , Ácidos Graxos/metabolismo , Hipertensão/metabolismo , Resistência à Insulina , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Sacarose/administração & dosagemRESUMO
Hypertension is characterized by maladaptive vascular remodeling and enhanced oxidative stress in the vascular wall. Peroxynitrite may directly activate latent matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMC) by its S-glutathiolation. MMP-2 may then proteolyze calponin-1 in aortas from hypertensive animals, which stimulates VSMC proliferation and medial hypertrophy. Calponin-1 is an intracellular protein which helps to maintain VSMC in their differentiated (contractile) phenotype. The present study therefore investigated whether aortic MMP-2 activity is increased by oxidative stress in early hypertension and then contributes to hypertrophic arterial remodeling by reducing the levels of calponin-1. Male Wistar rats were submitted to the two kidney, one clip (2â¯K-1C) model of hypertension or sham surgery and were treated daily with tempol (18â¯mg/kg/day) or its vehicle (water) by gavage from the third to seventh day post-surgery. Systolic blood pressure (SBP) was daily assessed by tail-cuff plethysmography. After one week, aortas were removed to perform morphological analysis with hematoxylin and eosin staining and to analyze reactive oxygennitrogen species levels by dihydroethidium and immunohistochemistry for nitrotyrosine. MMP-2 activity was analyzed by in situ and gelatin zymography and its S-glutathiolation was analyzed by Western blot for MMP-2 of anti-glutathione immunoprecipitates. Calponin-1 levels were identified in aortas by immunofluorescence. SBP increased by approximately 50â¯mmHg at the first week in 2â¯K-1C rats which was unaffected by tempol. However, tempol ameliorated the hypertension-induced increase in arterial media-to-lumen ratio and hypertrophic remodeling. Tempol also decreased hypertension-induced aortic oxidative stress and the enhanced MMP-2 activity. S-glutathiolation may be a potential mechanism by which oxidative stress activates MMP-2 in aortas of 2â¯K-1C rats. Furthermore, calponin-1 was decreased in aortas from 2â¯K-1C rats and tempol prevented this. In conclusion, oxidative stress may contribute to the increase in aortic MMP-2 activity, possibly by S-glutathiolation, and this may result in calponin-1 loss and maladaptive vascular remodeling in early hypertension.
Assuntos
Aorta Torácica/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Hipertensão Renovascular/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Proteínas dos Microfilamentos/metabolismo , Estresse Oxidativo , Remodelação Vascular , Animais , Aorta Torácica/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Ativação Enzimática , Glutationa/metabolismo , Humanos , Hipertensão Renovascular/patologia , Hipertrofia , Masculino , Ratos Wistar , Transdução de Sinais , Fatores de Tempo , CalponinasRESUMO
Liver cirrhosis is associated with a wide range of cardiovascular abnormalities including hyperdynamic circulation and cirrhotic cardiomyopathy. The pathogenic mechanisms of these cardiovascular changes are multifactorial and include vascular dysregulations. AIM: The present study tested the hypothesis that the systemic vascular hyporesponsiveness in thioacetamide (TAA)-induced liver injury model is dependent on nitric oxide (NO) and cyclooxygenase (COX) derivatives. MAIN METHODS: Wistar rats were treated with TAA for eight weeks to induce liver injury. KEY FINDINGS: The maximal contractile response in concentration-effect curves to phenylephrine was decreased in aorta from TAA-treated rats, but no differences were found in aorta without endothelium, suggesting an endothelium-dependent mechanism in decreased contractile response. There was no difference in the contractile response with and without L-NAME (N(ω)-nitro-l-arginine methyl ester) in rats with liver injury, showing that the TAA treatment impairs NO synthesis. Pre-incubation of the aorta with indomethacin, a COX-inhibitor, normalized the reduced contractile response to phenylephrine in arteries from TAA group. Also, COX-2 and iNOS (inducible nitric oxide syntase) protein expression was increased in aorta from TAA group compared to control group. Animals submitted to TAA treatment had a reduction in systolic blood pressure. Our findings demonstrated that liver injury induced by TAA caused a decrease in aortic contractile response by a COX-dependent mechanism but not by NO release. Also, it was demonstrated an inflammatory process in the aorta of TAA-treated rats by increased expression of COX-2 and iNOS. SIGNIFICANCE: Therefore, there is an essential contribution of COX-2 activation in extra-hepatic vascular dysfunction and inflammation present in cirrhosis induced by TAA.
Assuntos
Aorta Torácica/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Ciclo-Oxigenase 2/metabolismo , Endotélio Vascular/patologia , Tioacetamida/toxicidade , Doenças Vasculares/etiologia , Animais , Aorta Torácica/enzimologia , Pressão Sanguínea , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Endotélio Vascular/enzimologia , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Doenças Vasculares/enzimologiaRESUMO
Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas calponin-1 expression was increased in 2K-1C mesenteric arteries, calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.
Assuntos
Aorta Torácica/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Hipertensão Renovascular/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Artérias Mesentéricas/enzimologia , Proteínas dos Microfilamentos/metabolismo , Remodelação Vascular , Resistência Vascular , Vasoconstrição , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Modelos Animais de Doenças , Feminino , Hipertensão Renovascular/patologia , Hipertensão Renovascular/fisiopatologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/patologia , Artérias Mesentéricas/fisiopatologia , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Ratos Wistar , Transdução de Sinais , Remodelação Vascular/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , CalponinasRESUMO
AIMS: We determined whether decreased reactive oxygen species (ROS) production in the aorta of pregnant spontaneously hypertensive rats (SHR) resulted in increased nitric oxide (NO) bioavailability and hyporeactivity to phenylephrine (PE). MAIN METHODS: Systemic and aortic oxidative stress were measured in pregnant and non-pregnant Wistar rats and SHR. Furthermore, the hypotensive effects of apocynin (30 mg/kg) and Tempol (30 mg/kg) were analyzed. Intact aortic rings of pregnant and non-pregnant rats were stimulated with PE in the absence of or after incubation (30 min) with apocynin (100 µmol/L). The effect of apocynin on the concentrations of NO and ROS were measured in aortic endothelial cells (AEC) using DAF-2DA (10 mmol/L) and DHE (2.5 mmol/L), respectively. Western blotting was performed to analyze eNOS, NOX1, NOX2, NOX4 and SOD expression. ROS production was analyzed by the lucigenin chemiluminescence method. KEY FINDINGS: Aortic oxidative stress and ROS concentration in AEC were reduced in pregnant Wistar rats and SHR, when compared to non-pregnant rats. ROS production and NOX1, NOX2 and NOX4 expression in the aortas were decreased in pregnant SHR, but not in pregnant Wistar rats. Increased eNOS expression in aortas and NO concentration in AEC were observed in pregnant Wistar rats and SHR. Apocynin reduced PE-induced vasoconstriction in the aortas of non-pregnant Wistar rats and SHR, and pregnant Wistar rats, but not in the aortas of pregnant SHR. SIGNIFICANCE: Taken together, these results suggest that ROS production was decreased in the aortas of pregnant SHR and could contribute to higher NO bioavailability and hyporeactivity to PE in the aortas of pregnant SHR.
Assuntos
Aorta Torácica/enzimologia , Cardiotônicos/farmacologia , Glicoproteínas de Membrana/biossíntese , NADH NADPH Oxirredutases/biossíntese , NADPH Oxidases/biossíntese , Fenilefrina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Aorta Torácica/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Técnicas In Vitro , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Vasoconstrição/efeitos dos fármacosRESUMO
The effects of angiotensin II (Ang II) on vascular smooth muscle cells (VSMC) are modulated by reactive oxygen species (ROS) and also involve integrin engagement. However, the potential link between alpha1beta1 integrin signaling with NOX system and their combined contribution to Ang II effects on VSMC have not been investigated. We aimed to elucidate the moslecular mechanisms underlying the activation of these two pathways in Ang II effects on VSMC. Ang II-induced VSMC migration (2-fold increase) and proliferation (2.5-fold increase) is modulated by alpha1beta1 integrin, being inhibited by obtustatin, a specific alpha1beta1 integrin blocker. Ang II also stimulates ROS production in VSMC (140%) that is NOX1 dependent, being completely inhibited in NOX1 silenced cells. The ROS production develops in two peaks, and the second peak is maintained by NOX2 activation. Apocynin and obtustatin inhibit the NOX2-associated second peak, but not the first peak of ROS production, which is related to NOX1 activation. Corroborating the involvement of alpha1beta1 integrin, the pretreatment of VSMC with obtustatin impaired Ang II-induced FAK phosphorylation, AKT activation, p21 degradation and the increase of ILK expression. Silencing of ILK blocked cell migration, AKT phosphorylation and the second peak of ROS, but partially inhibits (70%) VSMC proliferation induced by Ang II. The data demonstrate a novel role for NOX2 in Ang II effects on VSMC, and suggest alpha1beta1 integrin and ILK as target molecules to the development of more effective therapeutic interventions in cardiovascular diseases.
Assuntos
Angiotensina II/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Integrina alfa1beta1/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Acetofenonas/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Integrina alfa1beta1/antagonistas & inibidores , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Venenos de Víboras/farmacologiaRESUMO
This work investigated the blood pressure (BP)-lowering effect of the flavanol (-)-epicatechin in a model of metabolic syndrome. Rats were fed a regular chow diet without (Control) or with 10% (w/v) fructose in the drinking water (high fructose, HF) for 8 weeks. A subgroup of the HF-fed rats was supplemented with (-)-epicatechin 20 mg/kg body weight (HF-EC). Dietary (-)-epicatechin reverted the increase in BP caused by the fructose treatment. In aorta, superoxide anion production and the expression of the NADPH oxidase (NOX) subunits p47(phox) and p22(phox) were enhanced in the HF-fed rats. The increase was prevented by (-)-epicatechin. Similar profile was observed for NOX4 expression. The activity of aorta nitric oxide synthase (NOS) was increased in the HF group and was even higher in the HF-EC rats. These effects were paralleled by increased endothelial NOS phosphorylation at the activation site Ser1177. Among the more relevant mitogen-activated protein kinase pathways in vascular tissue, c-Jun-N-terminal kinase was shown to be activated in the aorta of the HF-fed rats, and (-)-epicatechin supplementation mitigated this activation. Thus, the results suggest that dietary (-)-epicatechin supplementation prevented hypertension in HF-fed rats, decreasing superoxide anion production and elevating NOS activity, favoring an increase in NO bioavailability.
Assuntos
Anti-Hipertensivos/uso terapêutico , Catequina/uso terapêutico , Suplementos Nutricionais , Endotélio Vascular/enzimologia , Hipertensão/prevenção & controle , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/agonistas , Animais , Antioxidantes/uso terapêutico , Aorta Torácica/enzimologia , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Carboidratos da Dieta/efeitos adversos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Frutose/efeitos adversos , Hipertensão/etiologia , Hipertensão/metabolismo , Hipertensão/patologia , Sistema de Sinalização das MAP Quinases , Masculino , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Fosforilação , Processamento de Proteína Pós-Traducional , Distribuição Aleatória , Ratos Sprague-Dawley , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismoRESUMO
The role of oxidative stress in different aortopathies is evaluated. Thirty-two tissue samples from 18 men and 14 women were divided into: 4 control (C) subjects, 11 patients with systemic arterial hypertension (SAH), 4 with variants of Marfan's syndrome (MV), 9 with Marfan's syndrome (M), 2 with Turner's syndrome, and 2 with Takayasu's arteritis (TA). Aorta fragments were homogenized. Lipoperoxidation (LPO), copper-zinc and manganese superoxide dismutase (Mn and Cu-Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), endothelial nitric oxide synthase (eNOS), nitrates and nitrites (NO3(-)/NO2(-)), and type IV collagen, and laminin were evaluated. There was an increase in Mn- and Cu-Zn-SOD activity in SAH, MV, M, and Turner's syndrome. There was also an increase in CAT activity in M and Turner' syndrome. GPx and GST activity decreased and LPO increased in all groups. eNOS was decreased in SAH, MV, and M and NO3 (-)/NO2 (-) were increased in SAH and TA. Type IV collagen was decreased in Turner's syndrome and TA. Laminin γ-1 was decreased in MV and increased in M. In conclusion, similarities and differences in oxidative stress in the different aortopathies studied including pathologies with aneurysms were found with alterations in SOD, CAT, GPx, GST, and eNOS activity that modify subendothelial basement membrane proteins.
Assuntos
Aorta Torácica/enzimologia , Doenças da Aorta/enzimologia , Oxirredutases/análise , Adolescente , Adulto , Idoso , Aorta Torácica/diagnóstico por imagem , Doenças da Aorta/diagnóstico por imagem , Doenças da Aorta/patologia , Catalase/análise , Colágeno Tipo IV/metabolismo , Feminino , Glutationa Peroxidase/análise , Glutationa Transferase/análise , Humanos , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo III/análise , Superóxido Dismutase/análise , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
BACKGROUND: Acute exercise increases reactive oxygen species (ROS) levels, including hydrogen peroxide (H2O2). H2O2 promotes endothelial nitric oxide synthase (eNOS) activation and phosphorylation in endothelial cells. With this in mind, the present study was designed to evaluate ex vivo eNOS phosphorylation in rat aortas incubated with H2O2 and to test this hypothesis in vivo in the aortas of rats submitted to acute exercise. METHODS: For ex vivo studies, six groups of aortic tissue were formed: control, H2O2, N-acetylcysteine (NAC), LY294002, compound C, and LY294002 plus compound C. While incubation with H2O2 increased Akt, AMPK and eNOS phosphorylation, pre-incubation with NAC strongly reduced the phosphorylation of these enzymes. For in vivo studies, male Wistar rats were divided into four groups: control, cont+NAC, exercise, and exer+NAC. After a 3h swimming session, animals were decapitated and aortas were excised for biochemical and immunoblotting analysis. RESULTS: Acute exercise increased superoxide levels and dichlorofluorescein (DCF) concentrations, and this increase was related to phosphorylation of Akt, AMPK and eNOS. On the other hand, use of NAC reduced superoxide levels and DCF concentration. Reduced superoxide levels and DCF in the exer+NAC group were associated with decreased Akt, AMPK and eNOS phosphorylation. These results appear to be connected with vascular function because VASP phosphorylation increased in acute exercise and decreased in exer+NAC. CONCLUSION: Our results indicate that ROS induced by acute exercise play the important role of activating eNOS, a process apparently mediated by Akt and AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aorta Torácica/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Condicionamento Físico Animal/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Masculino , Fosforilação/fisiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Fatores de TempoRESUMO
This study investigated the endothelium-dependent vasorelaxant effects of the essential oil of Ocimum gratissimum (EOOG) in aortas and mesenteric vascular beds isolated from rats. EOOG (3-300 µg/mL) relaxed the tonic contractions induced by phenylephrine (0.1 µmol/L) in isolated aortas in a concentration-dependent manner in both endothelium-containing and endothelium-denuded preparations. This effect was partially reversed by L-NAME (100 µmol/L) but not by indomethacin (10 µmol/L) or TEA (5 mmol/L). In mesenteric vascular beds, bolus injections of EOOG (30, 50, 100, and 300 ng) decreased the perfusion pressure induced by noradrenaline (6 µmol/L) in endothelium-intact preparations but not in those treated with deoxycholate. L-NAME (300 µmol/L) but not TEA (1 mmol/L) or indomethacin (3 µmol/L) significantly reduced the vasodilatory response to EOOG at all of the doses tested. Our data showed that EOOG exerts a dose-dependent vasodilatory response in the resistance blood vessels of rat mesenteric vascular beds and in the capacitance blood vessel, the rat aorta. This action is completely dependent on endothelial nitric oxide (NO) release in the mesenteric vascular beds but only partially dependent on NO in the aorta. These novel effects of EOOG highlight interesting differences between resistance and capacitance blood vessels.
Assuntos
Aorta Torácica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Veias Mesentéricas/efeitos dos fármacos , Ocimum/química , Óleos Voláteis/farmacologia , Vasodilatadores/farmacologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio Vascular/fisiologia , Técnicas In Vitro , Masculino , Artérias Mesentéricas/enzimologia , Artérias Mesentéricas/metabolismo , Veias Mesentéricas/enzimologia , Veias Mesentéricas/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Norepinefrina/antagonistas & inibidores , Norepinefrina/metabolismo , Óleos Voláteis/química , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , Capacitância Vascular/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Vasoconstritores/antagonistas & inibidores , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/antagonistas & inibidoresRESUMO
Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.
Assuntos
Hipertensão Renovascular/patologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Angiotensina II/metabolismo , Animais , Aorta Torácica/enzimologia , Aorta Torácica/fisiopatologia , Pressão Sanguínea , Colágeno/metabolismo , Elastina/metabolismo , Ativação Enzimática , Imunofluorescência , Hipertensão Renovascular/metabolismo , Imuno-Histoquímica , Masculino , Inibidores de Metaloproteinases de Matriz , NADPH Oxidases/metabolismo , Estresse Oxidativo , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Artéria Renal/metabolismo , Artéria Renal/fisiopatologia , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
OBJECTIVES: We tested the effects of liver reperfusion in the immunohistochemical expression of nitric oxide synthase on the thoracic aorta and the heart. MATERIALS AND METHODS: We randomized 24 male Wistar rats into 3 groups: (1) control; (2) R2 group, with 60 minutes of partial (70%) liver ischemia and 2 hours of global liver reperfusion; (3) and R6 group, with 60 minutes of partial liver ischemia and 6 hours of global liver reperfusion. RESULTS: In the heart, there was little, diffuse immunohistochemical endothelial staining; immunohistochemical inducible nitric oxide synthase staining was expressed in the adventitia layer of intramyocardial vessels in both cases, with a time-dependent but not statistically significant increase. In the thoracic aorta, a time-dependent decrease in endothelial nitric oxide synthase expression in the muscular layer after reperfusion, which was statistically significant in R6 versus the control. Positive immunostaining for inducible nitric oxide synthase was seen in the muscular and endothelial layers, and this varied from moderate in the control group, to light in the endothelium in groups R2 and R6. CONCLUSIONS: We observed changes that may be implicated in heart injury and impairment of aortal tone after liver ischemia and reperfusion injury.
Assuntos
Aorta Torácica/enzimologia , Fígado/irrigação sanguínea , Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Traumatismo por Reperfusão/enzimologia , Alanina Transaminase/metabolismo , Animais , Aorta Torácica/patologia , Aspartato Aminotransferases/metabolismo , Fígado/enzimologia , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Modelos Animais , Miocárdio/patologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Fatores de TempoRESUMO
LASSBio-985 is a sulfonamide compound designed as a simplified structure of a nonselective phosphodiesterase type 4 (PDE-4) inhibitor that promotes vasodilatory activity in vitro. PDE are enzymes responsible for the hydrolysis of cyclic adenosine 3',5'- monophosphate and cyclic guanosine 3',5'-monophosphate. Five different isozymes of PDE are found in vascular smooth muscle (PDE1-PDE5). Aortic rings, with or without endothelium, from male normotensive and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. Blood pressure was measured in Wistar Kyoto (WKY) rats and SHR during intravenous infusion of LASSBio-985 (10 mg/kg/min) during 15 min. LASSBio-985 induced a concentration-dependent vasodilation in aortic rings from normotensive and SHR, which was almost completely inhibited in endothelium-denuded vessels. Vasodilatory activity was also reduced in endothelium-intact aortic rings that had been pretreated with N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase inhibitor and 1H-[1,2,4]oxadiazolod[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. LASSBio-985-induced vasodilation was also inhibited by sildenafil (100 µm) and SQ 22536, a PDE5 inhibitor and adenylate cyclase inhibitor, respectively. To evaluate the involvement of some endothelial receptors, atropine, diphenhydramine, HOE 140, naloxone, propranolol, indomethacin, and wortmannin were tested, but none inhibited the effects of LASSBio-985. The residual effect observed on endothelium-denuded aortic rings was abolished by nicardipine, a voltage-sensitive-Ca(2+)-channel blocker. Intravenous infusion of LASSBio-985 (10 mg/kg/min) significantly reduced systolic and diastolic pressures in both WKY and SHR. LASSBio-985 is a compound with vasodilatory activity, which could be consequent to PDE1 inhibition and voltage-sensitive-Ca(2+)-channel blockade.
Assuntos
Anti-Hipertensivos/uso terapêutico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Inibidores de Fosfodiesterase/uso terapêutico , Sulfonamidas/uso terapêutico , Vasodilatadores/uso terapêutico , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Aorta Torácica/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Estrutura Molecular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiopatologia , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacocinética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Sulfonamidas/administração & dosagem , Sulfonamidas/química , Sulfonamidas/farmacologia , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Vasodilatadores/química , Vasodilatadores/farmacologiaRESUMO
Cardiovascular disease is less frequent in premenopausal women than in age-matched men or postmenopausal women. Moreover, the marked age-related decline in serum dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized rats. At 8 weeks of age, female Wistar rats were ovariectomized (OVX) or sham (SHAM) operated and 8 weeks after surgery both groups were treated with vehicle or DHEA (10mg kg⻹ week⻹) for 3 weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine (PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP). Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein expression were analysed. PHE-induced contraction was increased in aortic rings from OVX compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did not change. The incubation of aortic rings with SOD or apocynin restored the enhanced PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment restores the reduced Cu/Zn-SOD protein expression and eNOS phosphorylation and the increased NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an important action of DHEA, improving endothelial function in OVX rats by acting as an antioxidant and enhancing the NO bioavailability.
Assuntos
Desidroepiandrosterona/farmacologia , Endotélio Vascular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Acetofenonas/farmacologia , Acetilcolina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Fármacos Cardiovasculares/farmacologia , Endotélio Vascular/enzimologia , Feminino , NADPH Oxidases/biossíntese , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/biossíntese , Nitroprussiato/farmacologia , Ovariectomia , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/biossínteseRESUMO
AIM OF STUDY: We have assessed the influence of water restriction stress on the nitric oxide (NO) synthase in heart and aorta tissues in young 2-month-old and middle-aged 12-month-old rats. METHODS: Animals were divided into control and 24- and 72-h water-deprived groups. We evaluated systolic blood pressure (SBP), biochemical parameters, nitrate and nitrite urinary excretion (UNOx), NADPH-diaphorase activity, and protein levels of NOS in the right atria, left ventricle, and thoracic aorta tissues. RESULTS: Water restriction during 72 h increased SBP (16%) in 2-month-old rats but decreased it after 24 and 72 h (9 and 15%, respectively) in 12-month-old rats. Atria, aorta endothelium, and smooth muscle NOS activity increased (32, 63, and 88%, respectively) only after 72 h of water restriction in 2-month-old rats. It also increased not only after 72 h but also after 24 h in atria (27 and 18%, respectively) and in ventricle (39 and 67%, respectively) in 12-month-old rats. Meanwhile, in this group's aorta smooth muscle, the enzyme activity decreased (16 and 7%, respectively). A major difference seen between ages was the changes in UNOx excretion, which decreased in the younger in 24 and 72 h (47 and 81%, respectively) and increased in the middle-aged rats (193 and 389%, respectively). Water restriction did not change cardiovascular endothelial and neuronal NOS protein levels in any group. CONCLUSION: NO pathways could contribute to the development of age-related cardiovascular adaptation to volume depletion induced by water restriction.
Assuntos
Envelhecimento/patologia , Ventrículos do Coração/enzimologia , Hipovolemia/patologia , Óxido Nítrico Sintase/metabolismo , Água/fisiologia , Adaptação Fisiológica , Animais , Aorta Torácica/enzimologia , Pressão Sanguínea , Endotélio Vascular/enzimologia , Hipovolemia/enzimologia , Masculino , Modelos Animais , NADPH Desidrogenase/metabolismo , Nitratos/urina , Nitritos/urina , Ratos , Ratos Sprague-Dawley , Estresse FisiológicoRESUMO
AIMS: Na(+), K(+)-ATPase activity contributes to the regulation of vascular contractility and it has been suggested that vascular Na(+), K(+)-ATPase activity may be altered during the progression of diabetes; however the mechanisms involved in the altered Na(+), K(+)-ATPase activity changes remain unclear. Thus, the aim of the present study was to evaluate ouabain-sensitive Na(+), K(+)-ATPase activity and the mechanism(s) responsible for any alterations on this activity in aortas from 1- and 4-week streptozotocin-pretreated (50 mg kg(-1), i.v.) rats. MAIN METHODS: Aortic rings were used to evaluate the relaxation induced by KCl (1-10mM) in the presence and absence of ouabain (0.1 mmol/L) as an index of ouabain-sensitive Na(+), K(+)-ATPase activity. Protein expression of COX-2 and p-PKC-betaII in aortas were also investigated. KEY FINDINGS: Ouabain-sensitive Na(+), K(+)-ATPase activity was unaltered following 1-week of streptozotocin administration, but was increased in the 4-week diabetic aorta (27%). Endothelium removal or nitric oxide synthase inhibition with l-NAME decreased ouabain-sensitive Na(+), K(+)-ATPase activity only in control aortas. In denuded aortic rings, indomethacin, NS-398, ridogrel or Gö-6976 normalized ouabain-sensitive Na(+), K(+)-ATPase activity in 4-week diabetic rats. In addition, COX-2 (51%) and p-PKC-betaII (59%) protein expression were increased in 4-week diabetic aortas compared to controls. SIGNIFICANCE: In conclusion, diabetes led to a time-dependent increase in ouabain-sensitive Na(+), K(+)-ATPase activity. The main mechanism involved in this activation is the release of TxA(2)/PGH(2) by COX-2 in smooth muscle cells, linked to activation of the PKC pathway.
Assuntos
Aorta Torácica/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Ouabaína/farmacologia , Prostaglandinas/metabolismo , Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Western Blotting , Ciclo-Oxigenase 2/biossíntese , Diabetes Mellitus Experimental/enzimologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Masculino , Prostaglandina H2/metabolismo , Ratos , Ratos Wistar , Tromboxano A2/metabolismo , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Antecedentes: Recientemente hemos propuesto en un modelo experimental de infarto al miocardio una significativa interregulación entre los niveles de la enzima convertidora de angiotensina I (ECA) y su homóloga (ECA-2), junto con que angiotensina (Ang)-(1-9) más que Ang-(1-7) actuaría como uncontrarregulador de Ang II. Sin embargo tal relación no se ha investigado en el remodelado aórtico hipertensivo. Objetivo: Determinar la expresión de ECA y ECA-2, los niveles de Angs I, II, (1-7) y (1-9) y los parámetros de remodelado de la pared aórtica de ratas hipertensas. Métodos: Ratas normotensas Lewis (n=18) fueron randomizadas a hipertensión (HTA) por sobrecarga de presión (modelo Goldblatt, GB, 2 riñones-1 pinzado, n=9). Ratas pseudo-operadas se usaron como controles (S, n=9). A las 6 semanas post cirugía, se determinó la masa cardíaca relativa (MCR) y la presión arterial sistólica (PAS). En la aorta torácica se determinó el grosor de la túnica media (GTM), área de la TM (ATM), niveles de mRNA de ECA y ECA-2, factor de crecimiento transformante tipo beta (TGF-beta), inhibidor del activador de plasminógeno (PAI-1) y de la proteína quimioatractante de monocitos (MCP-1) por RT-PCR. La actividad y niveles proteicos de ECA y ECA-2 por fluorimetría y Western blot y los niveles de Angs I, II, (1-7) y (1-9) por HPLC y radioinmunoensayo. Resultados: La MCR y la PAS aumentaron significativamente (p<0,05) en el grupo GB respecto a su control S. Las ratas hipertensas mostraron un aumento significativo (p<0.05) del GTM (18 por ciento), ATM (31 por ciento), niveles de mRNA de ECA (164 por ciento), TGF-beta (105 por ciento), PAI-1(51 por ciento), MCP-1 (53 por ciento) junto con mayor actividad (89 por ciento), niveles proteicos de ECA (130 por ciento) y Ang II (48 por ciento). Esos efectos se asociaron a una significativa disminución del mRNA, los niveles proteicos y actividad...
Background: In experimental models of myocardial infarction we have recently proposed a significantinter-regulation between levels of Angiotensin I converting enzyme (ACE) and its homologous, ACE-2; in addition, we have proposed that Angiotensin 1-9 (Ang-(1-9)) rather than Ang-(1-7) counter regulates Ang II. These relations have not been investigated in hypertensive aortic wall remodeling. Aim: To measure de expression of ACE and ACE-2, the aortic wall levels of Ang I, Ang II, Ang-(1-7) and Ang-(1-9), along with parameters of aortic wall remodeling in hypertensive rats. Methods: 18 Lewis rats were randomized to Goldblatt (2 kidneys, 1 clamped) induced hypertension (n=9) or sham operation (controls, n=9). Six weeks after surgery, relative cardiac mass (RCM), systolic blood pressure (SBP), medial layer aortic wall thickness (MLT) and ML area (MLA) were measured. The aortic wall levels of ACE and ACE-2, tissue growth factor beta (TGF- beta), plasminogen activator inhibitor (PAI-1) and monocyte chemoattractant protein (MCP-1) were determined by RT-PCR. Activity and protein levels of ACE and ACE-2 were measured by fluorometry and Western Blot and ANG I, Ang II, Ang-(1-7) and Ang-(1-9) levels were determined using HPLC and radioimmunoassay. Results: RCM and SBP increased significantly in hypertensive as opossed to sham operated rats...
Assuntos
Animais , Ratos , Angiotensina I/fisiologia , Angiotensina II/fisiologia , Aorta Torácica/enzimologia , Hipertensão , Peptidil Dipeptidase A/fisiologia , Sistema Renina-AngiotensinaRESUMO
Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and re-couples NOS function.