RESUMO
Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.
Assuntos
COVID-19 , Interleucina-6 , Feminino , Humanos , Gravidez , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico , Artérias/metabolismo , Citocinas , Espécies Reativas de Oxigênio/metabolismo , Quinases Associadas a rho , Serotonina , Cordão UmbilicalRESUMO
In the present study, we hypothesized that endothelin (ET) receptors (ETA and ETB ) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3-/- and caspase-/- mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ETA receptor antagonist reversed the effect. The ETB receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1ß levels in a concentration-dependent manner (100 nM-10 µM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ETA - and ETB -mediated activation of NLRP3 in mouse CC via Ca2+ -dependent ROS generation.
Assuntos
Endotelina-1 , Disfunção Erétil , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Masculino , Camundongos , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico , Antagonistas dos Receptores de Endotelina , Endotelina-1/metabolismo , Disfunção Erétil/metabolismo , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio , Receptores de EndotelinaRESUMO
We hypothesized that acute stress would induce endothelial dysfunction. Male Wistar rats were restrained for 2 h within wire mesh. Functional and biochemical analyses were conducted 24 h after the 2-h period of restraint. Stressed rats showed decreased exploration on the open arms of an elevated-plus maze (EPM) and increased plasma corticosterone concentration. Acute restraint stress did not alter systolic blood pressure, whereas it increased the in vitro contractile response to phenylephrine and serotonin in endothelium-intact rat aortas. NG-nitro-l-arginine methyl ester (l-NAME; nitric oxide synthase, NOS, inhibitor) did not alter the contraction induced by phenylephrine in aortic rings from stressed rats. Tiron, indomethacin and SQ29548 reversed the increase in the contractile response to phenylephrine induced by restraint stress. Increased systemic and vascular oxidative stress was evident in stressed rats. Restraint stress decreased plasma and vascular nitrate/nitrite (NOx) concentration and increased aortic expression of inducible (i) NOS, but not endothelial (e) NOS. Reduced expression of cyclooxygenase (COX)-1, but not COX-2, was observed in aortas from stressed rats. Restraint stress increased thromboxane (TX)B(2) (stable TXA(2) metabolite) concentration but did not affect prostaglandin (PG)F2α concentration in the aorta. Restraint reduced superoxide dismutase (SOD) activity, whereas concentrations of hydrogen peroxide (H(2)O(2)) and reduced glutathione (GSH) were not affected. The major new finding of our study is that restraint stress increases vascular contraction by an endothelium-dependent mechanism that involves increased oxidative stress and the generation of COX-derived vasoconstrictor prostanoids. Such stress-induced endothelial dysfunction could predispose to the development of cardiovascular diseases.
Assuntos
Aorta/fisiopatologia , Endotélio Vascular/fisiopatologia , Estresse Oxidativo/fisiologia , Estresse Psicológico/fisiopatologia , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprosta/metabolismo , Endotélio Vascular/metabolismo , Ácidos Graxos Insaturados , Glutationa/metabolismo , Hidrazinas/farmacologia , Peróxido de Hidrogênio/metabolismo , Indometacina/farmacologia , Masculino , Proteínas de Membrana/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenilefrina/farmacologia , Prostaglandinas , Ratos , Ratos Wistar , Restrição Física , Serotonina/farmacologia , Estresse Psicológico/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Tromboxano B2/metabolismo , Vasoconstritores/farmacologiaRESUMO
There are many evidences indicating a compensatory mechanism in contralateral carotids following balloon injury. Previously it was observed α1-adrenoceptor-mediated hyper-reactivity and impairment of calcium influx in contralateral carotids 4 days after injury. At a later stage, α1-adrenoceptor-mediated contraction is similar to the control and we hypothesized that downstream signaling was normal. In the present study, we aimed to evaluate α1-adrenoceptor-mediated calcium influx in contralateral carotids 15 days after balloon injury. Concentration-response curves for CaCl2 in presence of the α1-adrenoceptor agonist (phenylephrine), measurement of the intracellular calcium transient and the levels of reactive oxygen species using fluorescent dyes were performed in control and contralateral carotids. Phenylephrine-induced intracellular calcium mobilization in contralateral carotids was not altered, while phenylephrine-induced calcium influx was reduced in the contralateral artery. Nitric oxide synthase inhibitors, L-NAME or L-NNA, restored this response, but nitrite and nitrate levels were decreased in contralateral carotids. Additionally, a rise in oxygen free radicals was observed in contralateral carotids. Furthermore, Tiron, a superoxide anion scavenger, restored α1-adrenoceptor-mediated calcium influx in contralateral carotids to the control level. Similar results were observed with the selective potassium channels blockers 4-aminopyridine and charybdotoxin. In conclusion, data showed that balloon catheter injury resulted in increased superoxide anions levels, activation of potassium channels (Kv and BKCa), inhibition of calcium channels (Cav) and preservation of α1-adrenoceptor-mediated contraction at a later stage after injury.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Cálcio/fisiologia , Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Receptores Adrenérgicos alfa 1/fisiologia , Superóxidos/metabolismo , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Artérias Carótidas/fisiopatologia , Lesões das Artérias Carótidas/fisiopatologia , Sequestradores de Radicais Livres/farmacologia , Masculino , Nitratos/metabolismo , Nitritos/metabolismo , Ratos , Ratos Wistar , Verapamil/farmacologiaRESUMO
A flow system with zone merging and zone trapping in the main reactor was proposed. The sample and reagent inserted aliquots merge together and the resulting zone is directed towards a displaceable reactor inside which its most concentrated portion is trapped. After the pre-set TRAP period, the handled sample is released towards detection. A comparison with an analogous flow system exploiting zone stopping revealed the superior characteristics of sampling rate and system operation; moreover, the sample inserted volume plays little influence on sampling rate. The system was applied to the spectrophotometric determination of cobalt in pastures, and enhanced figures of merit (sampling rate=18 h(-1); peak height r.s.d.=0.7%, detection limit=0.046 µg L(-1) Co; reagent consumption=330 µg of Tiron per measurement; 98%Assuntos
Cobalto/análise
, Poaceae/química
, Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/química
, Catálise
, Peróxido de Hidrogênio/química
, Indicadores e Reagentes/química
, Oxirredução
, Hidróxido de Sódio/química
, Espectrofotometria/métodos
RESUMO
In pathological conditions, such as hypertension, there is impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity. In the present study we tested the hypothesis that acute superoxide scavenging would restore the depressed baroreflex sensitivity (BRS) in spontaneously hypertensive rats (SHR). Male 10-week-old SHR (n = 14) and their controls (Wistar-Kyoto (WKY) rats; n = 14) underwent femoral artery and vein catheterization for conscious blood pressure recording and drug administration. The BRS was obtained by the drug-induced method using phenylephrine (8 µg/kg, i.v.) and sodium nitroprusside (25 µg/kg, i.v.) before and after the administration of tiron (30 mg/kg, i.v.), a superoxide dismutase mimetic, or apocynin (30 µg/kg), an NADPH oxidase inhibitor. Spontaneously hypertensive rats was significantly hypertensive compared with WKY rats (160 ± 7 vs 105 ± 2 mmHg, respectively). However, there was no significant difference in heart rate between the two groups (388 ± 10 vs 370 ± 20 b.p.m.). In addition, SHR exhibited a diminished BRS compared with WKY rats (-1.34 ± 0.11 vs -2.91 ± 0.20 b.p.m./mmHg, respectively). Administration of tiron improved BRS in SHR (from -1.34 ± 0.11 to 2.26 ± 0.21 b.p.m./mmHg), as did apocynin (to -2.14 ± 0.23 b.p.m./mmHg). Serum samples from SHR (n = 20) and WKY rats (n = 20) were collected for thiobarbituric acid-reactive substances assays before and after tiron or apocynin to confirm the reduction in oxidative stress. There was considerably greater oxidative stress in SHR compared with WKY rats (36.2 ± 3.0 vs 13.3 ± 2.6 nmol/L, respectively). Both apocynin and tiron treatment reduced the oxidative stress in SHR (from 36.2 ± 3.0 to 21.5 ± 3.0 nmol/L and from 37.2 ± 3.9 to 21.9 ± 1.6 nmol/L, respectively). The data suggest that acute scavenging of NADPH oxidase-derived superoxide improves baroreflex sensitivity in SHR.
Assuntos
Barorreflexo/fisiologia , Sequestradores de Radicais Livres/metabolismo , Hipertensão/metabolismo , NADPH Oxidases/metabolismo , Superóxidos/metabolismo , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/uso terapêutico , Acetofenonas/farmacologia , Acetofenonas/uso terapêutico , Animais , Barorreflexo/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertensão/enzimologia , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca(2+) signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca(2+)](i) and MagFluo-4AM to measure Ca(2+) transients. Exposure to FCCP (2 microM, 2 min) caused a continuous increase in [Ca(2+)](i) at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca(2+) transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca(2+)](i) = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca(2+) release. FCCP diminished the rate but not the amount of Ca(2+) released, indicating that depletion of Ca(2+) stores did not cause the decrease in Ca(2+) transient amplitude. Ca(2+) transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca(2+)](i) induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca(2+) in the FCCP effect on transient amplitude.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/toxicidade , Mitocôndrias Musculares/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Desacopladores/toxicidade , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Potenciais de Ação , Animais , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Cresóis/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Sequestradores de Radicais Livres/farmacologia , Fura-2/análogos & derivados , Indóis/farmacologia , Cinética , Camundongos , Microscopia de Fluorescência/métodos , Mitocôndrias Musculares/metabolismo , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Vascular response was assessed in rats made diabetic by subtotal pancreatectomy (PPx). Adult male Wistar rats submitted to PPx eight weeks earlier, and exhibiting altered levels of fasting glucose and an abnormal tolerance glucose test, were used. Sham-operated laparotomized rats were employed as controls. Dose-response curves for acetylcholine-induced, endothelium-related relaxation of aortic rings (after previous exposure to phenylephrine) were conducted in a high glucose solution (44 mmol/L). PPx decreased significantly acetylcholine-induced relaxation only in the presence of a high glucose solution (p<0.00001). Tiron, superoxide dismutase (SOD) or melatonin restored altered aortic relaxation. Melatonin, SOD or Tiron were equally effective in restoring the impaired sodium nitroprusside-induced vasorelaxation in endothelium-denuded aortic rings of PPx rats. The results support the evidence about the ability of antioxidants to restore altered vascular reactivity of aortic rings in PPx rats, probably through the scavenging property of superoxide anion accumulation.
Assuntos
Antioxidantes/farmacologia , Aorta Torácica/efeitos dos fármacos , Pancreatectomia , Vasodilatação/efeitos dos fármacos , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Acetilcolina/farmacologia , Animais , Aorta Torácica/fisiologia , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiologia , Glucose/farmacologia , Técnicas In Vitro , Melatonina/farmacologia , Nitroprussiato/farmacologia , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Superóxido Dismutase/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
We examined intra- and extracellular H(2)O(2) and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H(2)O(2) and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p <.05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p <.05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p <.05). Intense electrical stimulation also increased (p <.05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) and NO may constitute an important defense mechanism against the formation of intracellular (*)OH and (*)ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.