RESUMO
(1) The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes. (2) Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier. (3) The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively. (4) In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.
Assuntos
Astrócitos/fisiologia , Barreira Hematoencefálica/fisiologia , Células Endoteliais/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Técnicas In VitroRESUMO
Nitronyl nitroxides (NN) effectively decompose free radicals (. As brain endothelium, forming the blood-brain barrier (BBB), is both the main source and the target of reactive species during cerebral oxidative stress, we studied the effect of NN on brain endothelial cells injured by the mediator of oxidative stress H(2)O(2) (. H(2)O(2) caused hydroxyl radical generation, lipid peroxidation, membrane dysfunction, membrane leak and cell death, concentration dependently. Due to 0.5 mM H(2)O(2), oxy-radical-induced membrane phospholipid peroxidation (malondialdehyde) increased to 0.61+/-0.04 nmol/mg protein vs control (0.32+/-0.03, p<0.05), cells lost cytosolic proteins into the medium and viability decreased to 28+/-2% of control (p<0.05). Permeability through the endothelial monolayer (measure for the tightness of the BBB) rose to 250+/-40% after 0.15 mM H(2)O(2) (p<0.001). Addition of 10 microM of the NN 5,5-dimethyl-2,4-diphenyl-4-methoxy-2-imidazoline-3-oxide-1-oxyl (NN-2), 1 mM phenylbutyl nitrone (PBN), or 10 microM of the lazaroid U83836E improved cell viability during incubation with 0.5 mM H(2)O(2) to 57+/-1%, 49+/-2%, and 42+/-3% (p<0.05, vs drug-free H(2)O(2) group). The permeability enhancement by 0.15 mM H(2)O(2) was reduced to 171+/-21%, 170+/-25%, and 118+/-32% (p<0.05 vs drug-free H(2)O(2) group). Generally, the assumption is supported that during cerebral oxidative stress the protection should also be directed to the cells of the BBB, which can be provided by antioxidative approaches. NN represent a new group of antioxdatively acting cytoprotectiva improving the survival and function of the endothelium against oxidative stress.
Assuntos
Barreira Hematoencefálica/fisiologia , Cromanos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/análise , Estresse Oxidativo/efeitos dos fármacos , Piperazinas/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Endotélio Vascular/fisiologia , Fluoresceína , Sequestradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , L-Lactato Desidrogenase/efeitos dos fármacos , Malondialdeído/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/análise , Óxidos de Nitrogênio/farmacologia , RatosRESUMO
To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.
Assuntos
Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Linhagem Celular , Endotélio Vascular/citologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Biomarcadores , Encéfalo/citologia , Encéfalo/metabolismo , Capilares/citologia , Divisão Celular , Citocinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Epoprostenol/metabolismo , Radicais Livres/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Ratos , Ratos Wistar , Tromboxano A2/metabolismoRESUMO
The cytosolic 4Fe-4S protein aconitase can be converted under the influence of reactive oxygen species into an iron-regulatory protein (IRP1). Therefore, the IRP1 level is considered as an indirect marker of oxidative stress. An experimental approach is presented here to detect the concentration of this marker protein by surface plasmon resonance. The optical method exploits the natural binding affinity of IRP1 to an iron-responsive element (IRE) which was in vitro transcribed with a linker sequence and subsequently immobilized on a BIACORE sensor chip. The detection was found to be reproducible and sensitive in the range 20-200 nM IRP. Conditions of the binding process, such as pH and thiol concentration, were characterized. Feasibility of the method to detect and quantify IRP1 in physiological media was demonstrated.
Assuntos
Proteínas Ferro-Enxofre/análise , Estresse Oxidativo , Proteínas de Ligação a RNA/análise , RNA/química , Aconitato Hidratase/química , Animais , Biomarcadores/análise , Linhagem Celular , Citosol/química , Proteína 1 Reguladora do Ferro , Proteínas Reguladoras de Ferro , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ressonância de Plasmônio de SuperfícieRESUMO
There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.
Assuntos
Barreira Hematoencefálica/fisiologia , Moléculas de Adesão Celular/metabolismo , GMP Cíclico/metabolismo , Endotélio Vascular/enzimologia , Óxido Nítrico/metabolismo , Fosfoproteínas/metabolismo , Animais , Astrócitos/química , Astrócitos/citologia , Astrócitos/enzimologia , Proteínas Sanguíneas/metabolismo , Capilares/química , Capilares/citologia , Capilares/enzimologia , Moléculas de Adesão Celular/análise , Comunicação Celular/fisiologia , Células Cultivadas , GMP Cíclico/análise , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Endotélio Vascular/química , Endotélio Vascular/citologia , Regulação Enzimológica da Expressão Gênica , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Proteínas dos Microfilamentos , Nitratos/análise , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/análise , Fosfoproteínas/análise , Fosforilação , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 +/- 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 +/- 0.17 nmol/mg of protein after reoxygenation vs control (0.41 +/- 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 +/- 0.01 to 0.11 +/- 0.02 nmol/mg of protein (P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E [(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)] were effective scavengers of .OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard (EC50: 12, 5 and 15 microM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 microM U83836E, and 5 microM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 +/- 7, 51 +/- 10 and 48 +/- 6%, respectively (P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.
Assuntos
Amifostina/farmacologia , Cromanos/farmacologia , Citoproteção , Endotélio Vascular/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Piperazinas/farmacologia , Animais , Antioxidantes/farmacologia , Aorta/citologia , Aorta/efeitos dos fármacos , Bovinos , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Oxigênio/farmacologiaRESUMO
Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trapping technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (aN = 1.39 mT, aHbeta = 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7+/-0.5 U/ml, p < 0.001) or 5 microM vitamin E (11.7+/-0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7+/-4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 +/-20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5+/-15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10+/-0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6+/-11.8%; VF 19.42+/-3.64 min/30 min). The Ca antagonist verapamil (0.1 microM), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4+/-6.8% (p < 0.05), 39.6+/-12.7% (p < 0.05) and 52.4+/-2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3+/-6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited.
Assuntos
Traumatismo por Reperfusão Miocárdica/metabolismo , Óxidos de Nitrogênio , Marcadores de Spin , Animais , Antioxidantes/farmacologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Técnicas In Vitro , Cinética , Peroxidação de Lipídeos , Masculino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Verapamil/farmacologia , Vitamina E/farmacologiaRESUMO
We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
Assuntos
Aconitato Hidratase/metabolismo , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Nitratos/farmacologia , Aconitato Hidratase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Hidrólise , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma , Células Tumorais CultivadasRESUMO
A cell culture model of blood-brain barrier (BBB, coculture of rat brain endothelial cells with rat astrocytes) was used to investigate the effect of nitric oxide (.NO) on the damage of the BBB induced by hypoxia/reoxygenation (H/R). Permeability coefficient of fluorescein across the endothelium was used as a marker of BBB tightness. The permeability coefficient increased 5.2 times after H/R indicating strong disruption of the BBB. The presence of the .NO donor S-nitroso-N-acetylpenicillamine (SNAP, 30 microM), authentic .NO (6 microM) or superoxide dismutase (50 units/ml) during H/R attenuated H/R-induced increase in permeability. 30 microM SNAP or 6 microM .NO did not influence the function of BBB during normoxia, however, severe disruption was observed using 150 microM of SNAP and more than 24 microM of .NO. After H/R of endothelial cells, the content of malondialdehyde (MDA) increased 2.3 times indicating radical-induced peroxidation of membrane lipids. 30 microM SNAP or 6 microM authentic .NO completely prevented MDA formation. The results show that .NO may effectively scavenge reactive oxygen species formed during H/R of brain capillary endothelial cells, affording protection of BBB at the molecular and functional level.
Assuntos
Barreira Hematoencefálica/fisiologia , Hipóxia/metabolismo , Óxido Nítrico/fisiologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/citologia , Permeabilidade da Membrana Celular , Células Cultivadas , Endotélio/citologia , Fluoresceína/farmacocinética , Guanilato Ciclase/genética , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão , Transdução de Sinais , Superóxido Dismutase/farmacologiaRESUMO
In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.
Assuntos
Óxido Nítrico/química , Compostos de Sulfidrila/química , Vasodilatadores/síntese química , Animais , Óxidos N-Cíclicos/síntese química , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/farmacologia , Artérias Mesentéricas , Ratos , Vasodilatação , Vasodilatadores/química , Vasodilatadores/farmacologiaAssuntos
Astrócitos/efeitos dos fármacos , Citocinas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Animais , Astrócitos/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Hipóxia Celular , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Interleucina-1/farmacologia , Óxido Nítrico Sintase/biossíntese , Penicilamina/análogos & derivados , Penicilamina/farmacologia , S-Nitroso-N-Acetilpenicilamina , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Spin trapping compounds are used frequently to detect free radicals released by cells. Their cytotoxicity has to be considered in order to prevent perturbations of normal cell growth and viability. Eleven spin traps (eight nitrones and three nitroso traps) have been tested for their effects on bovine aortic endothelial cells (toxicity range, 50% survival rate). The lowest cytotoxicity was found for 5,5-dimethylpyrroline-1-oxide and 2,2,4-trimethyl-2H-imidazole-1-oxide whereas nitrosobenzene and 2-methyl-2-nitrosopropane exerted the strongest cytotoxic effects. In addition, three nitronyl nitroxides were tested. Their cytotoxicity was found to be dependent on substitution, and the toxic concentration of a lipophilic derivative was found to be more than two orders lower as compared to a hydrophilic derivative. The results of this study indicate that most spin traps can be used in cell cultures at customary (i.e. millimolar) concentrations; caution is recommended when nitroso spin traps are applied to cells.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos/toxicidade , Endotélio Vascular/efeitos dos fármacos , Óxidos de Nitrogênio/toxicidade , Compostos Nitrosos/toxicidade , Marcadores de Spin , Animais , Aorta , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.
Assuntos
Endotélio Vascular/metabolismo , Molsidomina/análogos & derivados , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/farmacologia , Superóxidos/metabolismo , Animais , Calcimicina/farmacologia , Bovinos , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Mimetismo Molecular , Molsidomina/metabolismo , Marcadores de Spin , Detecção de Spin , Superóxido Dismutase/metabolismoRESUMO
A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.
Assuntos
Imidazóis/síntese química , Marcadores de Spin/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Marcação por Isótopo , Modelos QuímicosRESUMO
Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated .NO chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without detectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN concentration. Model experiments using hypoxanthine/xanthine oxidase in aqueous solution and KO2 in dimethylsulfoxide as sources of O2.- revealed that there is a rapid reduction of nitronyl nitroxides by superoxide. The second order rate constant for the reaction of the water soluble NN with O2.- was determined to be 8.8 x 10(5) M-1s-1, which is more than two orders of magnitude higher than the value reported previously for reaction with .NO (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the presence of glutathione, ascorbic acid or rabbit liver microsomes. Incorporation of both nitronyl and imino nitroxides into liposomes strongly decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposomal encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.
Assuntos
Óxidos N-Cíclicos/química , Imidazóis/química , Óxido Nítrico/análise , Superóxidos/química , Animais , Calcimicina/farmacologia , Bovinos , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Cinética , Lipossomos/química , Medições Luminescentes , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , NAD/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Coelhos , Superóxido Dismutase/metabolismoRESUMO
1. Hypoxic alterations in isolated rat hepatocytes were demonstrated by a 90% ATP loss during 60 min of ischaemia and temporary increases of nucleotide degradation products. 2. The oxidative stress during reoxygenation was demonstrated in these cells by a decrease in reduced glutathione (GSH) concentration (30%) and a threefold increase in lipid peroxidation products such as 4-hydroxynonenal and thiobarbituric acid-reactive substances (TBA-RSs). The tremendous GSH loss could not be balanced by the slight oxidized glutathione (GSSG) increase during reoxygenation. 3. For the first time the involvement of free radicals was directly demonstrated using electron spin resonance (ESR) spectroscopy in reoxygenated liver cells. Using the spin trap 5,5-dimethylpyrroline-1-oxide (DMPO), a carbon-centred radical and the adduct of the hydroxyl radical could be detected during early reoxygenation. 4. Morphological alteration of cells was observed, beginning during hypoxia and increasing during post-hypoxic reoxygenation. Electron microscopic findings of hypoxic and post-hypoxic cell damage included pyknosis of nuclei, spherical transformation of mitochondria and increased number of vesicles.
Assuntos
Hipóxia/metabolismo , Peroxidação de Lipídeos/fisiologia , Fígado/metabolismo , Nucleotídeos de Purina/metabolismo , Aldeídos/metabolismo , Animais , Sobrevivência Celular/fisiologia , Reagentes de Ligações Cruzadas/metabolismo , Metabolismo Energético/fisiologia , Radicais Livres/metabolismo , Glutationa/metabolismo , Fígado/citologia , Fígado/ultraestrutura , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Detecção de Spin , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
4-Hydroxynonenal (HNE) has been proposed as an important marker of radical-induced lipid peroxidation (LPO) during postischemic reperfusion injury of the myocardium. Therefore, the liberation of HNE into the effluent of isolated perfused rat hearts was investigated. For the first time, the formation of the aldehyde is demonstrated in myocardium. During control perfusion, 1.28 +/- 0.33 pmol HNE.min-1.mg protein-1 were formed by the hearts of 18-mo-old Wistar-Kyoto (WKY) rats and 2.74 +/- 1.12 pmol.min-1.mg protein-1 by those of 18-mo-old spontaneously hypertensive (SHR) rats, respectively. In the WKY group, HNE release increased to 3.35 +/- 1.13 pmol.min-1.mg protein-1 2 min after the onset of reperfusion following 30 min of total and global ischemia compared with the preischemic control period (P < 0.05). In the SHR group, HNE liberation was higher during reperfusion (8.66 +/- 1.33 pmol.min-1.mg protein-1, maximum at 2 min reperfusion) compared with both the respective preischemic control and the respective reperfusion interval of the WKY group (P < 0.05 each). The SHR rats showed signs of congestive cardiac failure of a decompensated hypertrophy in comparison to the normotensive WKY rats. Moreover, the SHR rat hearts exhibited a lower release of adenine nucleotide degradation products (adenine, inosine, hypoxanthine plus uric acid: 48.1 +/- 10.2 nmol.30 min-1.mg protein-1; P < 0.05) and a diminished functional recovery (left ventricular developed pressure, 32 +/- 16 mmHg; P < 0.05) during 30 min of reperfusion compared with the WKY group (77.9 +/- 14.4 nmol.30 min-1.mg protein-1; 90 +/- 21 mmHg).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aldeídos/metabolismo , Peróxidos Lipídicos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Animais , Biomarcadores , Cardiomegalia/fisiopatologia , Coração/fisiopatologia , Frequência Cardíaca , Técnicas In Vitro , Masculino , Isquemia Miocárdica/fisiopatologia , Reperfusão Miocárdica , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Nucleotídeos/metabolismo , Tamanho do Órgão , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating system in vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275 +/- 84% of pre-ischaemic baseline values vs 1260 +/- 413%, p < 0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9 +/- 6 mmHg vs 42 +/- 8 mmHg in the absence of WR-2721, p < 0.02). An accelerated recovery of creatine phosphate levels (78 +/- 5% of pre-ischaemia values vs 41 +/- 5% within 60 min reperfusion: p < 0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)