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Significance: Cardiovascular disease and drug-induced health side effects are frequently associated with-or even caused by-an imbalance between the concentrations of reactive oxygen and nitrogen species (RONS) and antioxidants, respectively, determining the metabolism of these harmful oxidants. Recent Advances: According to the "kindling radical" hypothesis, the initial formation of RONS may further trigger the additional activation of RONS formation under certain pathological conditions. The present review specifically focuses on a dysfunctional, uncoupled endothelial nitric oxide synthase (eNOS) caused by RONS in the setting of transportation noise exposure or chronic treatment with organic nitrates, especially nitroglycerin (GTN). We further describe the various "redox switches" that are proposed to be involved in the uncoupling process of eNOS. Critical Issues: In particular, the oxidative depletion of tetrahydrobiopterin and S-glutathionylation of the eNOS reductase domain are highlighted as major pathways for eNOS uncoupling upon noise exposure or GTN treatment. In addition, oxidative disruption of the eNOS dimer, inhibitory phosphorylation of eNOS at the threonine or tyrosine residues, redox-triggered accumulation of asymmetric dimethylarginine, and l-arginine deficiency are discussed as alternative mechanisms of eNOS uncoupling. Future Directions: The clinical consequences of eNOS dysfunction due to uncoupling on cardiovascular disease are summarized also, providing a template for future clinical studies on endothelial dysfunction caused by pharmacological or environmental risk factors.
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Enfermedades Cardiovasculares , Ruido del Transporte , Enfermedades Vasculares , Humanos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitratos/metabolismo , Enfermedades Cardiovasculares/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enfermedades Vasculares/metabolismo , Oxidación-Reducción , Óxido Nítrico/metabolismo , Endotelio Vascular/metabolismoRESUMEN
Reduced bioavailability of the nitric oxide (NO) signaling molecule has been associated with the onset of cardiovascular disease. One of the better-known and effective therapies for cardiovascular disorders is the use of organic nitrates, such as glyceryl trinitrate (GTN), which increases the concentration of NO. Unfortunately, chronic use of this therapy can induce a phenomenon known as "nitrate tolerance", which is defined as the loss of hemodynamic effects and a reduction in therapeutic effects. As such, a higher dosage of GTN is required in order to achieve the same vasodilatory and antiplatelet effects. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a cardioprotective enzyme that catalyzes the bio-activation of GTN to NO. Nitrate tolerance is accompanied by an increase in oxidative stress, endothelial dysfunction, and sympathetic activation, as well as a loss of the catalytic activity of ALDH2 itself. On the basis of current knowledge, nitrate intake in the diet would guarantee a concentration of NO such as to avoid (or at least reduce) treatment with GTN and the consequent onset of nitrate tolerance in the course of cardiovascular diseases, so as not to make necessary the increase in GTN concentrations and the possible inhibition/alteration of ALDH2, which aggravates the problem of a positive feedback mechanism. Therefore, the purpose of this review is to summarize data relating to the introduction into the diet of some natural products that could assist pharmacological therapy in order to provide the NO necessary to reduce the intake of GTN and the phenomenon of nitrate tolerance and to ensure the correct catalytic activity of ALDH2.
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Enfermedades Cardiovasculares , Óxido Nítrico , Humanos , Nitratos/uso terapéutico , Nitratos/farmacología , Aldehído Deshidrogenasa , Enfermedades Cardiovasculares/tratamiento farmacológico , Nitroglicerina/uso terapéutico , Nitroglicerina/farmacología , Aldehído Deshidrogenasa Mitocondrial , Vasodilatadores/farmacologíaRESUMEN
Glyceryl trinitrate (GTN) is one of the earliest known treatments for angina with a fascinating history that bridges three centuries. However, despite its central role in the nitric oxide (NO) story as a NO-donating compound, establishing the precise mechanism of how GTN exerts its medicinal benefit has proven to be far more difficult. This review brings together the explosive and vasodilatory nature of this three-carbon molecule while providing an update on the likely in vivo pathways through which GTN, and the rest of the organic nitrate family, release NO, nitrite, or a combination of both, while also trying to explain nitrate tolerance. Over the last 20 years the alcohol detoxification enzyme, aldehyde dehydrogenase (ALDH), has undoubtedly emerged as the front runner to explaining GTN's bioactivation. This is best illustrated by reduced GTN efficacy in subjects carrying the single point mutation (Glu504Lys) in ALDH, which is also responsible for alcohol intolerance, as characterized by flushing. While these findings are significant for anyone following the GTN story, they appear particularly relevant for healthcare professionals, and especially so, if administering GTN to patients as an emergency treatment. In short, although the GTN puzzle has not been fully solved, clinical study data continue to cement the importance of ALDH, as uncovered in 2002, as a key GTN activator.
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Consumo de Bebidas Alcohólicas/tratamiento farmacológico , Alcoholismo/tratamiento farmacológico , Aldehído Deshidrogenasa/antagonistas & inhibidores , Nitroglicerina/farmacología , Vasodilatadores/farmacología , Consumo de Bebidas Alcohólicas/metabolismo , Alcoholismo/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Animales , HumanosRESUMEN
Industrial wastewaters usually possess a wide range of nitrate strength. Microalgae-based nitrate-rich wastewater treatment could realize nitrate recovery along with CO2 sequestration for sustainable biomass production, but the low tolerance of the microalgal strains to high-strength nitrate restricted the treatment process. The present study comprehensively evaluated a euryhaline marine microalga Tetraselmis subcordiformis for photosynthetic nitrate removal and biomass production in synthetic wastewater with a broad range of nitrate strength (0.24-7.0 g NO3--N/L). This alga could acclimate to high nitrate strength up to 3.5 g NO3--N/L (HN) without compromising biomass production. Nitrate could be completely removed within four days when low nitrate (0.24 g NO3--N/L, LN) was loaded. The maximum nitrate removal rate of 331 mg N/L/day and specific nitrate removal rate of 360 mg N/day/g cell was obtained under medium nitrate condition (1.8 g NO3--N/L, MN). High-nitrate stress under 7.0 g NO3--N/L (SHN) caused an increased light energy dissipation while decreased the density of photosystem II active reaction center, which partially protect the cells from photodamage and contributed to their acclimation to SHN. The algae also enhanced amino acid/fatty acid proportions essential for maintaining intracellular redox states to cope with the stress caused by LN or SHN. HN and SHN was in favor of protein accumulation and maintenance with enhanced proportion of essential amino acids, which entitled the algal biomass to be of high quality for animal feed applied in livestock graziery and aquaculture. LN facilitated productive starch and lipid accumulation with good quality for biofuels production. The nitrate removal rate and biomass productivity exceeded most of the microalgae reported in literature under similar conditions, which highlighted Tetraselmis subcordiformis as a potent strain for flexible nitrate-rich wastewater remediation coupled with fast CO2 bio-mitigation and high-quality biomass production for sustainable algal biorefinery.
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Chlorophyta , Microalgas , Aclimatación , Animales , Biocombustibles , Biomasa , Nitratos , Aguas ResidualesRESUMEN
OBJECTIVES: One of the most important problems of taking nitroglycerin is the nitrate tolerance phenomenon and endothelial dysfunction. Oxidative stress is a high-emphasized one of tolerance mechanisms. The possible effect of crocin, one of the anti-oxidant ingredients of saffron, on the nitrate tolerance model was investigated. MATERIALS AND METHODS: In the present study, lipid peroxidation and the level of activated and deactivated forms of eNOS were measured. Animals were administered subcutaneously with 25 mg/kg of nitroglycerin, twice a day for 3 days to induce nitrate tolerance model. For evaluation of crocin effects, 20, 40 and 80 mg/kg/day of this compound were injected intraperitoneally in concomitant with nitroglycerin. In the isolated aorta test, after preparation of aorta rings, different concentrations of acetylcholine, sodium nitroprusside and nitroglycerin were added to the organ bath after inducing contraction by phenylephrine and the responsiveness of tissues was recorded. RESULTS: Findings showed that nitroglycerin administration caused a remarkable overproduction of malondialdehyde (MDA) in the cells and crocin treatment significantly decreased the MDA level. In the nitrate tolerance group, the level of activated eNOS decreased and the level of deactivated eNOS increased. Crocin partly alleviated these changes: however, its effects were not remarkable. Nitroglycerin injection for 3 days developed tolerance to nitroglycerin and cross-tolerance to acetylcholine (endothelial dysfunction) and sodium nitroprusside. Crocin failed to influence significantly on the nitrate tolerance. CONCLUSION: Crocin effectiveness is possibly time-dependent; therefore, increasing the duration of treatment with crocin may lead to a significant prevention of nitrate tolerance and endothelial dysfunction.
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BACKGROUND: Aldehyde dehydrogenase 2 (ALDH2) plays a central role in the biotransformation of glyceryl trinitrate (GTN) or nitroglycerin, which is widely used for the treatment of coronary artery disease (CAD). The deficient variant ALDH2 genotype (ALDH2*2) is prevalent among East Asians. This study examined whether there are differences in nitroglycerine-mediated dilation (NMD) and flow-mediated dilation (FMD) response between wildALDH2*1/*1and variantALDH2*2patients with CAD.MethodsâandâResults:The study subjects comprised 55 coronary spastic angina (CSA) patients, confirmed by coronary angiography and intracoronary injection of acetylcholine (42 men and 13 women, mean age 68.0±9.0 years). They underwent NMD and FMD tests in the morning before and after continuous transdermal GTN administration for 48 h. NMD was lower at baseline inALDH2*2than in theALDH2*1/*1group (P=0.0499) and decreased significantly in both groups (P<0.0001 and P<0.0001, respectively) after GTN, with significantly lower levels in theALDH2*2group (P=0.0002). FMD decreased significantly in bothALDH2*1/*1andALDH2*2groups (P<0.0001and P=0.0002, respectively) after continuous GTN administration, with no significant differences between the 2 groups both before and after GTN. CONCLUSIONS: Continuous administration of GTN produced endothelial dysfunction as well as nitrate tolerance in bothALDH2*1/1andALDH2*2patients with CSA.ALDH2*2attenuated GTN response and exacerbated GTN tolerance, but not endothelial dysfunction, as compared toALDH2*1/*1in patients with CSA.
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Aldehído Deshidrogenasa Mitocondrial/genética , Angina de Pecho/tratamiento farmacológico , Angina de Pecho/genética , Pueblo Asiatico/genética , Vasoespasmo Coronario/tratamiento farmacológico , Vasoespasmo Coronario/genética , Resistencia a Medicamentos/genética , Nitroglicerina/administración & dosificación , Polimorfismo Genético , Vasoconstricción/efectos de los fármacos , Vasodilatadores/administración & dosificación , Anciano , Angina de Pecho/etnología , Angina de Pecho/fisiopatología , Vasoespasmo Coronario/etnología , Vasoespasmo Coronario/fisiopatología , Femenino , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Nitroglicerina/efectos adversos , Vasoconstricción/genética , Vasodilatadores/efectos adversosRESUMEN
BACKGROUND: Reactive oxygen and nitrogen species (RONS such as H2O2, nitric oxide) are generated within the organism. Whereas physiological formation rates confer redox regulation of essential cellular functions and provide the basis for adaptive stress responses, their excessive formation contributes to impaired cellular function or even cell death, organ dysfunction and severe disease phenotypes of the entire organism. Therefore, quantification of RONS formation and knowledge of their tissue/cell/compartment-specific distribution is of great biological and clinical importance. METHODS: Here, we used a high-performance/pressure liquid chromatography (HPLC) assay to quantify the superoxide-specific oxidation product of the mitochondria-targeted fluorescence dye triphenylphosphonium-linked hydroethidium (mitoSOX) in biochemical systems and three animal models with established oxidative stress. Type 1 diabetes (single injection of streptozotocin), hypertension (infusion of angiotensin-II for 7 days) and nitrate tolerance (infusion of nitroglycerin for 4 days) was induced in male Wistar rats. RESULTS: The usefulness of mitoSOX/HPLC for quantification of mitochondrial superoxide was confirmed by xanthine oxidase activity as well as isolated stimulated rat heart mitochondria in the presence or absence of superoxide scavengers. Vascular function was assessed by isometric tension methodology and was impaired in the rat models of oxidative stress. Vascular dysfunction correlated with increased mitoSOX oxidation but also classical RONS detection assays as well as typical markers of oxidative stress. CONCLUSION: mitoSOX/HPLC represents a valid method for detection of mitochondrial superoxide formation in tissues of different animal disease models and correlates well with functional parameters and other markers of oxidative stress.
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Glyceryl trinitrate (GTN) has found widespread use for the treatment of angina pectoris, a pathological condition manifested by chest pain resulting from insufficient blood supply to the heart. Metabolic conversion of GTN, a nitric oxide (NO) pro-drug, into NO induces vasodilation and improves blood flow. Patients develop tolerance to GTN after several weeks of continuous use, limiting the potential for long-term therapy. The mechanistic cause of nitrate tolerance is relatively unknown. We developed a cell culture model of nitrate tolerance that utilizes stable isotopes to measure metabolism of 15N3-GTN into 15N-nitrite. We performed global metabolomics to identify the mechanism of GTN-induced nitrate tolerance and to elucidate the protective role of vitamin C (ascorbic acid). Metabolomics analyses revealed that GTN impaired purine metabolism and depleted intracellular ATP and GTP. GTN inactivated xanthine oxidase (XO), an enzyme that is critical for the metabolic bioactivation of GTN into NO. Ascorbic acid prevented inactivation of XO, resulting in increased NO production from GTN. Our studies suggest that ascorbic acid has the ability to prevent nitrate tolerance by protecting XO, but not aldehyde dehydrogenase (another GTN bioactivating enzyme), from GTN-induced inactivation. Our findings provide a mechanistic explanation for the previously observed beneficial effects of ascorbic acid in nitrate therapy.
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Background Postoperative atrial fibrillation (POAF) is a frequent complication of coronary artery bypass graft (CABG) surgery. This arrhythmia occurs more frequently among patients who receive perioperative inotropic therapy (PINOT). Administration of nitrates with antiplatelet agents reduces the conversion rate of cyclic guanosine monophosphate to guanosine monophosphate. This process is associated with increased concentrations of free radicals, catecholamines, and blood plasma volume. We hypothesized that patients undergoing CABG surgery who receive PINOT may be more susceptible to POAF when nitrates are administered with antiplatelet agents. Methods Clinical records were examined from a prospectively maintained cohort of 4,124 patients undergoing primary isolated CABG surgery to identify POAF-associated factors. Results POAF risk was increased among patients receiving PINOT, and the greatest effect was observed when nitrates were administered with antiplatelet therapy. Adjustment for comorbidities did not substantively change the study results. Conclusions Administration of nitrates with certain antiplatelet agents was associated with an increased POAF risk among patients undergoing CABG surgery. Additional studies are needed to determine whether preventive strategies such as administration of antioxidants will reduce this risk.
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Fibrilación Atrial/etiología , Fármacos Cardiovasculares/efectos adversos , Puente de Arteria Coronaria/efectos adversos , Enfermedad de la Arteria Coronaria/cirugía , Nitratos/efectos adversos , Inhibidores de Agregación Plaquetaria/efectos adversos , Adulto , Fibrilación Atrial/inducido químicamente , Fármacos Cardiovasculares/uso terapéutico , Enfermedad de la Arteria Coronaria/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nitratos/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
PURPOSE: We have demonstrated that RuBPY induces hypotensive effect in hypertensive rats, promotes vasodilation at low concentrations, and presents low cytotoxicity. This study aimed to verify whether the NO donor RuBPY synthesized in our laboratory induces in vitro tolerance and cross-tolerance to acetylcholine (ACh) and sodium nitroprusside (SNP) in rat cava vein. METHODS: We compared the maximum relaxing effect (ME) and potency (pD2) of RuBPY and nitroglycerin (GTN) in cava vein rings. Exposure to RuBPY or GTN induced in vitro tolerance. Western Blotting helped to evaluate phosphorylation of endothelial nitric oxide synthase (NOS3/eNOS) at the Ser1177 activation site and at the Thr495 inactivation site and to determine the ratio between active eNOS dimers and inactive eNOS monomers. The NO and ROS ratio was assessed by flow citometry. RESULTS: RuBPY did not induce cross-tolerance with ACh, and this NO donor took longer to induce tolerance than GTN. Only GTN elicited phosphorylation of eNOS at Ser1177 and Thr495. In contrast to results obtained with pre-exposure to GTN, pre-exposure to RuBPY did not reduce the formation of NO. The O2- ratio increased in cells incubated with GTN. CONCLUSIONS: A major contribution of this work has been to evaluate the phenomenon of tolerance induced by GTN and by the new ruthenium complex RuBPY in a venous bed. RuBPY is more advantageous than GTN: RuBPY takes longer to induce tolerance, does not induce endothelial dysfunction or cross-tolerance to ACh, and generates lower amount of ROS.
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Acetilcolina/farmacología , Complejos de Coordinación/farmacología , Tolerancia a Medicamentos/fisiología , Donantes de Óxido Nítrico/farmacología , Rutenio , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/farmacología , Animales , Masculino , Relajación Muscular/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitroglicerina/farmacología , Nitroprusiato/farmacología , Fosforilación/efectos de los fármacos , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Venas/fisiología , Vena Cava Inferior/fisiologíaRESUMEN
Chronic angina pectoris is associated with considerable morbidity and mortality, especially if treated suboptimally. For many patients, aggressive pharmacologic intervention is necessary in order to alleviate anginal symptoms. The optimal treatment of stable ischemic heart disease (SIHD) should be the prevention of angina and ischemia, with the goal of maximizing both quality and quantity of life. In addition to effective risk factor modification with lifestyle changes, intensive pharmacologic secondary prevention is the therapeutic cornerstone in managing patients with SIHD. Current guidelines recommend a multifaceted therapeutic approach with ß-blockers as first-line treatment. Another important pharmacologic intervention for managing SIHD is nitrates. Nitrates can provide both relief of acute angina and can be used prophylactically before exposure to known triggers of myocardial ischemia to prevent angina. Additional therapeutic options include calcium channel blockers and ranolazine, an inhibitor of the late inward sodium current, that can be used alone or in addition to nitrates or ß-blockers when these agents fail to alleviate symptoms. Ranolazine appears to be particularly effective for patients with microvascular angina and endothelial dysfunction. In addition, certain antianginal therapies are approved in Europe and have been shown to improve symptoms, including ivabradine, nicorandil, and trimetazidine; however, these have yet to be approved in the United States. Ultimately, there are several different medications available to the physician for managing the patient with SIHD having chronic angina, when either used alone or in combination. The purpose of this review is to highlight the most important therapeutic approaches to optimizing contemporary treatment in response to individual patient needs.
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Antagonistas Adrenérgicos beta/administración & dosificación , Angina de Pecho/tratamiento farmacológico , Angina de Pecho/epidemiología , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/epidemiología , Nitratos/administración & dosificación , Angina de Pecho/diagnóstico , Animales , Antiarrítmicos/administración & dosificación , Quimioterapia Combinada , Humanos , Isquemia Miocárdica/diagnóstico , Guías de Práctica Clínica como Asunto/normas , Vasodilatadores/administración & dosificaciónRESUMEN
AIMS: To determine the role of reactive oxygen species (ROS) on sodium nitroprusside (SNP)-induced tolerance. Additionally, we evaluated the role of ROS on NF-κB activation and pro-inflammatory cytokines production during SNP-induced tolerance. MAIN METHODS: To induce in vitro tolerance, endothelium-intact or -denuded aortic rings isolated from male Balb-c mice were incubated for 15, 30, 45 or 60min with SNP (10nmol/L). KEY FINDINGS: Tolerance to SNP was observed after incubation of endothelium-denuded, but not endothelium-intact aortas for 60min with this inorganic nitrate. Pre-incubation of denuded rings with tiron (superoxide anion (O2-) scavenger), and the NADPH oxidase inhibitors apocynin and atorvastatin reversed SNP-induced tolerance. l-NAME (non-selective NOS inhibitor) and l-arginine (NOS substrate) also prevented SNP-induced tolerance. Similarly, ibuprofen (non-selective cyclooxygenase (COX) inhibitor), nimesulide (selective COX-2 inhibitor), AH6809 (prostaglandin PGF2α receptor antagonist) or SQ29584 [PGH2/thromboxane TXA2 receptor antagonist] reversed SNP-induced tolerance. Increased ROS generation was detected in tolerant arteries and both tiron and atorvastatin reversed this response. Tiron prevented tolerance-induced increase on O2- and hydrogen peroxide (H2O2) levels. The increase onp65/NF-κB expression and TNF-α production in tolerant arteries was prevented by tiron. The major new finding of our study is that SNP-induced tolerance is mediated by NADPH-oxidase derived ROS and vasoconstrictor prostanoids derived from COX-2, which are capable of reducing the vasorelaxation induced by SNP. Additionally, we found that ROS mediate the activation of NF-κB and the production of TNF-α in tolerant arteries. SIGNIFICANCE: These findings identify putative molecular mechanisms whereby SNP induces tolerance in the vasculature.
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Aorta/metabolismo , Ciclooxigenasa 2/metabolismo , Nitroprusiato/farmacología , Prostaglandina H2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.
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Presión Sanguínea/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Donantes de Óxido Nítrico/farmacología , Triazenos/farmacología , Adolescente , Adulto , Anciano , Animales , GMP Cíclico/metabolismo , Perros , Humanos , Técnicas In Vitro , Túbulos Renales Proximales/citología , Masculino , Persona de Mediana Edad , Donantes de Óxido Nítrico/uso terapéutico , Triazenos/uso terapéutico , Adulto JovenRESUMEN
Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10-1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O (6)-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O (6)-methylguanine-DNA methyltransferase displayed more vascular O (6)-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O (6)-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction.
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Daño del ADN , Tolerancia a Medicamentos/fisiología , Endotelio Vascular/efectos de los fármacos , Nitroglicerina/toxicidad , Vasodilatadores/toxicidad , Animales , Western Blotting , Ensayo Cometa , Modelos Animales de Enfermedad , Immunoblotting , Inmunohistoquímica , Ratones , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors.
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Aterosclerosis/tratamiento farmacológico , Polaridad Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Óxidos de Nitrógeno/administración & dosificación , Animales , Aorta/efectos de los fármacos , Aorta/inmunología , Aorta/fisiopatología , Aterosclerosis/inmunología , Aterosclerosis/fisiopatología , Quimiocina CCL2/inmunología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/inmunología , Interleucina-6/inmunología , Macrófagos/citología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunologíaRESUMEN
The nickel-piperazine/NO donor compound, Ni(PipNONO)Cl, belonging to the family of compounds labelled as "metal-nonoates", due to its promising vasodilating activity, has been considered as a potential drug candidate in anti-hypertensive therapy. Drug efficacy has been evaluated in spontaneously hypertensive rats (SHR) in comparison with normotensive animals (C57BL/6 mice and WKY rats). In normotensive animals the metal-nonoate maintained blood pressure at basal level both following acute administration and after 30 days of treatment. In SHR, Ni(PipNONO)Cl reduced blood pressure in the dose range of 3-10mg/kg. When compared with a commercial NONOate, DETA/NO, used at the same doses, Ni(PipNONO)Cl was more active in reducing blood pressure in SHR than DETA/NO in the first two weeks, while the effect of the two molecules was similar in the third and fourth week. The degradation and control compound Ni(Pip)Cl2 had no effect on blood pressure and heart rate in same animal models. Remarkably, the blood pressure reduction induced by the new NO-donor Ni(PipNONO)Cl does not evoke changes in the heart rate and tolerance. Considering the mechanisms of vascular protection, 30 days of administration of Ni(PipNONO)Cl improved endothelial function in SHR by upregulating endothelial NO synthase (eNOS) through increased eNOS protein levels and downregulated Caveolin-1 (Cav-1), and by increasing superoxide dismutase 1 (SOD1) protein level in aortae. In cultured endothelial cells Ni(PipNONO)Cl restored the cell functions (cytoskeletal protein expression, migration and proliferation) altered by the inflammatory mediator interleukin-1ß (IL-1ß), impairing the endothelial to mesenchimal transition. In conclusion, Ni(PipNONO)Cl maintained unaltered blood pressure in normotensive mice and rats, and it exerted anti-hypertensive effect in SHR through the restoration of vascular endothelial protective functions.
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Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Níquel/uso terapéutico , Donantes de Óxido Nítrico/uso terapéutico , Piperazinas/uso terapéutico , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Presión Sanguínea/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Frecuencia Cardíaca/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Hipertensión/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
The nitric oxide (NO) metabolites nitrite (NO2(-)) and nitrate (NO3(-)) can be quantified as an endpoint of endothelial function. We developed a LC-MS/MS method of measuring nitrite and nitrate isotopologues, which has a lower limit of quantification (LLOQ) of 1 nM. This method allows for isotopic labeling to differentiate newly formed nitrite and nitrate from nanomolar to micromolar background levels of nitrite and nitrate in biological matrices. This method utilizes 2,3-diaminonaphthalene (DAN) derivatization, which reacts with nitrite under acidic conditions to produce 2,3-naphthotriazole (NAT). NAT was chromatographically separated on a Shimadzu LC System with an Agilent Extend-C18 5 µm 2.1 × 150 mm column and detected using a multiple reaction monitoring (MRM) method on an ABSciex 3200 QTRAP mass spectrometer operated in positive mode. Mass spectrometry allows for the quantification of (14)N-NAT (m/z 170.1) and (15)N-NAT (m/z 171.1). Both nitrite and nitrate demonstrated a linear detector response (1 nM - 10 µM, 1 nM - 100 nM, respectively), and were unaffected by common interferences (Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phenol red, and NADPH). This method requires minimal sample preparation, making it ideal for most biological applications. We applied this method to develop a cell culture model to study the development of nitrate tolerance in human endothelial cells (EA.hy926).
Asunto(s)
Cromatografía Liquida/métodos , Células Endoteliales/metabolismo , Marcaje Isotópico/métodos , Nitratos/metabolismo , Nitroglicerina/metabolismo , Espectrometría de Masas en Tándem/métodos , Línea Celular , Células Endoteliales/química , Humanos , Nitratos/química , Nitritos/química , Nitritos/metabolismo , Isótopos de Nitrógeno/análisis , Isótopos de Nitrógeno/metabolismo , Nitroglicerina/químicaRESUMEN
Chronically administered organic nitrates induce nitrate tolerance and endothelial dysfunction, which limit their therapeutic use. eNOS uncoupling, ROS over-production, aldehyde dehydrogenase-2 as well as superoxide dismutase (SOD) oxidative inhibition, and cGMP desensitization are thought to play an important role. Natural polyphenols are effective antioxidants, which might counteract the mechanisms leading to nitrate tolerance. The aim of this work was to verify whether freeze-dried (dealcoholized) red wine (FDRW) was able to revert glyceryl trinitrate (GTN) tolerance and endothelial dysfunction induced in rat aorta rings with either GTN or diethyldithiocarbamate (DETCA), an irreversible inhibitor of Cu/Zn SOD. GTN induced a concentration-dependent relaxation of rings pre-contracted with phenylephrine. GTN spasmolysis was significantly reduced in rings pre-incubated with either GTN or DETCA. FDRW, at 2.8 µg of gallic acid equivalents (GAE)/mL concentration, was able to revert partially, though significantly, GTN-induced tolerance but not tolerance and endothelial dysfunction induced by DETCA. This work provides the first evidence in vitro that red wine components, at concentrations comparable to those achieved in human blood after moderate consumption of red wine, revert tolerance to nitrates with a mechanism possibly mediated by SOD.
Asunto(s)
Tolerancia a Medicamentos , Endotelio Vascular/efectos de los fármacos , Nitroglicerina/farmacología , Polifenoles/farmacología , Vino , Animales , Antioxidantes/farmacología , Aorta/efectos de los fármacos , Endotelio Vascular/fisiopatología , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Superóxido Dismutasa/antagonistas & inhibidoresRESUMEN
Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event in GTN tolerance, an effect counteracted both in vitro and in vivo by novel peroxynitrite decomposition catalyst.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Tolerancia a Medicamentos , Metaloporfirinas/farmacología , Nitroglicerina/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , GMP Cíclico/metabolismo , Endotelio Vascular/efectos de los fármacos , Humanos , Hipotensión/inducido químicamente , Dinitrato de Isosorbide/análogos & derivados , Dinitrato de Isosorbide/farmacología , Masculino , Músculo Liso Vascular/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Nitroglicerina/antagonistas & inhibidores , Agregación Plaquetaria/efectos de los fármacos , Ratas , Trombina/antagonistas & inhibidores , Trombina/farmacología , Tirosina/análogos & derivados , Tirosina/biosíntesis , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND: Continuous long-term treatment with nitrates may cause nitrate tolerance. Nebivolol is a highly selective beta1-adrenergic antagonist with additional nitric oxide (NO)-mediated vasodilatory effects. However, there have been no investigations into whether or not the long-term administration of nebivolol might cause nitrate tolerance. METHODS: We performed a randomized, double-blind, placebo-controlled, cross-over study in 16 healthy men. Subjects received 5 mg nebivolol or placebo once daily for 8 days in random order divided by a drug-free interval of 2 weeks. Forearm blood flow (FBF) was measured by venous occlusion plethysmography 3 h after oral intake of the first and last doses of nebivolol and placebo, respectively. FBF was measured again following the intravenous administration of 4 µg nitroglycerin/kg body weight/min for 5 min. RESULTS: Following 8 days of continuous intake of placebo, nitroglycerin increased FBF by 54% (p < 0.05), whereas nitroglycerin increased FBF by 96% (p < 0.01) following 8 days of continuous intake of nebivolol, and the increase after 8 days of nebivolol was significantly (p < 0.05) more pronounced than after 8 days of placebo. CONCLUSIONS: These findings indicate no evidence of nitrate tolerance caused by long-term administration of nebivolol. On the contrary, long-term intake of nebivolol increases rather than decreases the NO-mediated vasodilating effects.