RESUMO
We introduce a method to monitor the integrity of micellar nanocarriers using a novel fluorescent dye, IR-780-PDMS and Förster resonance energy transfer (FRET) as a readout. In addition, these dye-loaded nanocarriers can be used as a phototoxic agent in vitro. Mainly, a nanocarrier was designed, based on a previously described amphiphilic ABA-copolymer (Pip-PMOXA-PDMS-PMOXA-Pip) scaffold that incorporates the fluorescent FRET dye partners IR-780-PDMS (donor) and IR-780 (acceptor). The confirmation of FRET (that only occurs when donor and acceptor are in the required close proximity of less than â¼10 nm) in the nanocarriers is used to prove that the acceptor dye (IR-780) is still contained in its hydrophobic core. We measured such FRET signals of the nanocarriers also upon cellular uptake into HeLa cells using fluorescence-lifetime imaging microscopy (FLIM). Confocal laser scanning microscopy after incubation with nanocarriers demonstrated the intracellular uptake of the particles and their localization in an intracellular granular pattern. To demonstrate the intactness of the nanocarriers by detection of FRET we measured the fluorescence lifetime (FLIM) of the donor dye. FLIM showed that both types of lifetimes, that of the quenched donor, and that of the unquenched donor were present, in a granular pattern and homogeneously in the cytosol, respectively, indicating the presence of intracellular intact and disintegrated micellar nanocarriers. These data show that the herewith-described FRET method allows monitoring the intactness of nanocarriers while en route to the target, and also that the cargo is delivered and released within a potential target cell. In addition, near-infrared (NIR) irradiation of IR-780-loaded micellar nanocarriers leads to photocytotoxicity, which we demonstrated in in vitro experiments. Our findings open potential avenues in photodynamic therapy (PDT) of cancer.
Assuntos
Portadores de Fármacos , Corantes Fluorescentes/química , Indóis/química , Nanopartículas/uso terapêutico , Dimetilpolisiloxanos/química , Dimetilpolisiloxanos/uso terapêutico , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/uso terapêutico , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Indóis/uso terapêutico , Proteínas Luminescentes/química , Nanopartículas/química , Neoplasias/terapia , Nylons/química , Fotoquimioterapia/tendênciasRESUMO
The permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel of mitochondria are known to play key roles in mitochondrially mediated apoptosis. We investigated how modulation of the permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel, either as single elements or in combination, affects the proapoptotic intracellular calcium ([Ca(2+)](i)) transients and the mitochondrial membrane potential (psi(m)). For this purpose a model was established exploring the [Ca(2+)](i) transients in N2A cells using continuous application of ATP that causes a biphasic [Ca(2+)](i) response. This response was sensitive to endoplasmatic reticulum (ER) Ca(2+) depletion and a smooth ER Ca(2+)-ATPase (SERCA) antagonist. PTP inhibition by cyclosporine A (CsA) or its non-immunosuppressive derivative NIM811 caused an amplification of the secondary [Ca(2+)](i) peak and induced a hyperpolarization of psi(m). Both the putative mtK-ATP channel inhibitor 5-hydroxydecanoate (5-HD) and the opener diazoxide ameliorated the ATP-induced secondary [Ca(2+)](i) peak. The effect of diazoxide was accompanied by a depolarization of psi(m) whereas 5-HD had no effect on psi(m). When diazoxide and CsA or NIM811 were applied together the secondary [Ca(2+)](i) rise did not return to baseline and a not significant hyperpolarization of psi(m) was observed. So, simultaneous inhibition of PTP and activation of the mtK-ATP channel prevents the increased slope of the secondary [Ca(2+)](i) peak induced by CsA (or NIM811) and also the depolarization after diazoxide application. Hence, we propose that modulation of one of these channels leads to functional changes of the other channel by means of Delta[Ca(2+)](i) and Deltapsi(m).
Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Canais de Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Modelos Biológicos , Neuroblastoma , Rodaminas/metabolismo , Rodaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
We investigated to what extent the antioxidative hydroxystilbene oxyresveratrol (trans-2,3',4,5'-tetrahydroxystilbene, OXY), that we showed earlier to be strongly neuroprotective in a stroke model, may cross the blood-brain barrier (BBB) in healthy rats and in subjects submitted to focal infarction. Tissue extraction and in vivo microdialysis in the striatum show that systematically applied OXY is able to penetrate the BBB in control animals, but to a low extent. Microdialysis samples from animals that were subjected to a middle cerebral artery occlusion (MCAO) displayed strongly increased OXY levels (more than six-fold) in the infarct region as compared to sham-operated rats. Our data show that OXY may exert direct protective effects in the brain by crossing the BBB and may prove an excellent complementary drug for the treatment of neurodegenerative disorders that causally involve oxidative/nitrosative stress, especially in stroke.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Infarto Cerebral/prevenção & controle , Fármacos Neuroprotetores/farmacocinética , Estilbenos/farmacocinética , Animais , Área Sob a Curva , Antígeno CD11b/metabolismo , Infarto Cerebral/etiologia , Cromatografia Líquida de Alta Pressão/métodos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Fluoresceína/farmacocinética , Fluoresceínas , Indóis , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Masculino , Microdiálise/métodos , Fármacos Neuroprotetores/uso terapêutico , Compostos Orgânicos/farmacocinética , Permeabilidade/efeitos dos fármacos , Ratos , Reperfusão/métodos , Estilbenos/uso terapêutico , Fatores de Tempo , Distribuição TecidualRESUMO
We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.
Assuntos
Arginina/metabolismo , Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Espécies Reativas de Nitrogênio/biossíntese , Animais , Células Cultivadas , Citometria de Fluxo , Humanos , Camundongos , Microscopia ConfocalRESUMO
ATP-sensitive potassium channels of the inner mitochondrial membrane (mtKATP) are blocked by ATP. They are suggested to be involved in protective mechanisms such as ischemic preconditioning (IPC). Here we identify this channel type for the first time in a human cell line (Jurkat cells). Vesicles of the inner mitochondrial membrane (mitoplasts) were prepared by hypoosmotic shock. Single-channel currents were measured by means of the patch-clamp technique. We identified an outward-rectifying channel with a slope conductance of 15 and 82 pS at negative and positive potentials, respectively. The block by 5-hydroxydecanoic acid and inhibition by ATP characterize this channel as the mtKATP channel. ATP also increased the frequency of events within the burst. This effect was modulated by the Ca2+-bath concentration. We also show that the human mtKATP channel is a direct target for nitric oxide that blocked the channel activity. Although the molecular structure of this channel type is still unknown, its characterization as an outward-rectifying channel and modulation by calcium ions and nitric oxide may help to elucidate its functional significance, which possibly implicates a role in cell survival after IPC.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Canais de Potássio/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/farmacologia , Ácidos Decanoicos/farmacologia , Diazóxido/farmacologia , Humanos , Hidroxiácidos/farmacologia , Células Jurkat , Cinética , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacosRESUMO
Nitric oxide (NO) is produced in mammals by different isoforms of NO synthase (NOS), including the constitutive mitochondrial enzyme (mtNOS). Here we demonstrate that the concentration of NO resulting from a mitochondrial NOS activity increases under hypoxic conditions in isolated rat liver mitochondria. We show that mitochondrially derived NO mediates the impairment of active (state 3) respiration as measured in the presence of the substrates glutamate and malate after reoxygenation. Simultaneously, NO induces oxidative stress in mitochondria, characterized by an increase in the amount of protein carbonyls and a decrease in glutathione (GSH). Both the accumulation of oxidative stress markers during and the impaired respiration after reoxygenation were prevented by blocking NO production with the NOS inhibitor L-NAME. These observations suggest that mitochondria are exposed to high amounts of NO generated by a mitochondrial NOS upon hypoxia/reoxygenation. Such increased NO levels, in turn, inhibit mitochondrial respiration and may cause oxidative stress that leads to irreversible impairment of mitochondria.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Óxido Nítrico/biossíntese , Estresse Oxidativo , Animais , Hipóxia Celular , Respiração Celular , Inibidores Enzimáticos/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Consumo de Oxigênio , RatosRESUMO
Melatonin, the secretory product of the pineal gland, is known to be neuroprotective in cerebral ischemia, which is so far mostly attributed to its antioxidant properties. Here we show that melatonin directly inhibits the mitochondrial permeability transition pore (mtPTP). mtPTP contributes to the pathology of ischemia by releasing calcium and cytochrome c (cyt c) from mitochondria. Consistently, NMDA-induced calcium rises were diminished by melatonin in cultured mouse striatal neurons, similar to the pattern seen with cyclosporine A (CsA). When the mouse striatal neurons were subjected to oxygen-glucose deprivation (OGD), melatonin strongly prevented the OGD-induced loss of the mitochondrial membrane potential. To assess the direct effect of melatonin on the mtPTP activity at the single channel level, recordings from the inner mitochondrial membrane were obtained by a patch-clamp approach using rat liver mitoplasts. Melatonin strongly inhibited mtPTP currents in a dose-dependent manner with an IC50 of 0.8 microM. If melatonin is an inhibitor of the mtPTP, it should prevent mitochondrial cyt c release as seen in stroke models. Rats underwent middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. Melatonin (10 mg/kg ip) or vehicle was given at the time of occlusion and at the time of reperfusion. Indeed, infarct area in the brain sections of melatonin-treated animals displayed a considerably decreased cyt c release along with less activation of caspase-3 and apoptotic DNA fragmentation. Melatonin treatment diminished the loss of neurons and decreased the infarct volume as compared with untreated MCAO rats. Our findings suggest that the direct inhibition of the mtPTP by melatonin may essentially contribute to its anti-apoptotic effects in transient brain ischemia.
Assuntos
Apoptose/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Canais Iônicos/antagonistas & inibidores , Ataque Isquêmico Transitório/tratamento farmacológico , Melatonina/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Sinalização do Cálcio/efeitos dos fármacos , Hipóxia Celular , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Concentração Inibidora 50 , Ataque Isquêmico Transitório/metabolismo , Masculino , Melatonina/fisiologia , Melatonina/uso terapêutico , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Modelos Animais , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismoRESUMO
Overproduction of nitric oxide by NMDA receptor stimulation is implicated in calcium deregulation and neurodegeneration of striatal neurons. We investigated the involvement of nitric oxide (NO) in inducing intracellular calcium release and in modifying calcium transients evoked by NMDA. NO application (4-10 microM) reversibly and repeatedly increased the intracellular calcium concentration [Ca2+]i in Fura-2- or fluo-3-loaded cultured mouse striatal neurons. NO-induced [Ca2+]i responses persisted in the absence of extracellular calcium, indicating that Ca2+ was released from intracellular stores. The source of calcium was distinct from [Ca2+]i-activated (ruthenium red and ryanodine sensitive) or IP3-activated (thapsigargin-sensitive) Ca2+ stores and was not dependent on cGMP production because a cell permeant analog, 8-bromo-cGMP, did not increase basal [Ca2+]i. Glucose removal potentiated the NO-induced release of [Ca2+]i. In contrast, pretreatment with either the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone or cyclosporin A, a blocker of the mitochondrial permeability transition pore, prevented the [Ca2+]i increase after NO. The rise in [Ca2+]i during NO exposure was preceded by a decrease in mitochondrial membrane potential that was partly reversible during washout. Repeated applications of NMDA induced irreversible [Ca2+]i responses in a subpopulation of striatal cells that were greatly reduced by the NOS inhibitor N omega-nitro-l-arginine. Calcium transients were prolonged by conjoint application of NMDA and NO. We conclude that NMDA-evoked [Ca2+]i transients are modulated by endogenous NO production, which leads to release of calcium from the mitochondrial pool. An NO-activated mitochondrial permeability transition pore may lead to cell death after overstimulation of NMDA receptors.
Assuntos
Cálcio/metabolismo , Corpo Estriado/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Óxido Nítrico/farmacologia , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/metabolismo , GMP Cíclico/metabolismo , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Glucose/metabolismo , Camundongos , Microscopia Confocal , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Nitritos/metabolismo , Tapsigargina/farmacologiaRESUMO
Nitric oxide (NO) is a molecule that plays a prominent role in neurotoxic as well as neuroprotective pathways. Here, we investigated the effects of NO on potentially excitotoxic glutamate-induced intracellular calcium ([Ca2+]i) dynamics. Our hypothesis was that pre- and coexposure to NO in conjunction with glutamate receptor stimulation modulates [Ca2+]i responses differentially. [Ca2+]i transients, assessed by the fluorescent cytosolic Ca2+ indicator dye fluo-4, were elicited in mouse striatal neurons by consecutive NMDA applications (200 microM for 100 s each). Subgroups of neuronal cultures were additionally exposed to a NO donor (S-nitroso-N-acetyl-d,l-penicillamine, SNAP, 50-500 microM), either by pre- (for 6 h prior to NMDA) or cotreatment (for 30 min during NMDA). Pretreatment with NO led to dramatically decreased NMDA-evoked [Ca2+]i rises in comparison to controls (NMDA alone). Annexin V/propidium iodide staining showed consistently that NO pretreatment is protective against NMDA-induced cell death. In contrast, NO/NMDA cotreatment caused a potentiation of [Ca2+]i rises, whereby the duration of [Ca2+]i transients following NMDA application was prolonged and remained at an increased plateau level. Simultaneous application of the mitochondrial permeability transition pore (mtPTP) blocker cyclosporin A (2 microM) during the NO/NMDA cotreatment prevented the deregulation of [Ca2+]i. The observed [Ca2+]i deregulation was accompanied by a decrease in the mitochondrial membrane potential as indicated by tetramethylrhodamine methylester (TMRM) fluorescence. These findings suggest that NO can act in a protective way due to preconditioning or can have a possibly detrimental impact in case of acute release. They provide a possible explanation for the ambivalence of NO in neurodegenerative processes where glutamate receptor stimulation and mitochondrial [Ca2+]i sequestration are causally involved.
Assuntos
Cálcio/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Sequestradores de Radicais Livres/farmacologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Óxido Nítrico/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Ciclosporina/farmacologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Camundongos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/metabolismoRESUMO
We investigated the antioxidant and radical scavenging activity of polyphenolic isochromans. To assess the relation between structure and scavenging properties the natural occurring 1-(3'-methoxy-4'-hydroxy)phenyl-6,7-dihydroxy-isochroman (ISO-3, three OH groups) was compared with three newly synthesized derivatives that differ in their degree of hydroxylation by substitution with methoxy-groups (ISO-4: four OH groups; ISO-2: two OH groups and ISO-0: fully methoxylated). We found that ISO-4 is a 2-fold better scavenger for the artificial radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, 100 microM) with an EC50=10.3 microM compared to the natural ISO-3 (EC50=22.4 microM) and to ISO-2 (EC50=25.1 microM), while ISO-0 did not react with DPPH. The scavenging capacity for superoxide enzymatically generated in a hypoxanthin-xanthinoxidase reaction was the highest for ISO-4 (EC50=34.3 microM) compared to those of ISO-3 (EC50=84.0 microM) and ISO-2 (EC50=91.8 microM), while ISO-0 was inactive. In analogy, ISO-4 scavenged peroxynitrite (ONOO-, EC25=23.0 microM) more effective than ISO-3, ISO-2 and ISO-0. When C6 rat glioma cells loaded with the reactive oxygen/nitrogen (ROS/RNS)-sensitive fluorochrome 2,7-dichlorodihydrofluorescein, were exposed to hydrogen peroxide, the lowest stress level as indicated by the fluorescence signal was detected when the cells were pretreated with ISO-4 or ISO-2 but to a much lesser extent with ISO-3, while ISO-0 did not show any effect. All tested hydroxyisochromans superceded the scavenging effect of trolox.The excellent radical and ROS/RNS scavenging features of the hydroxy-1-aryl isochromans and their simple synthesis let these compounds appear to be interesting candidates for pharmaceutical interventions that protect against the deleterious action of ROS/RNS.
Assuntos
Antioxidantes/química , Antioxidantes/síntese química , Cromanos/química , Cromanos/síntese química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/síntese química , Animais , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Cromanos/metabolismo , Cromatografia Líquida de Alta Pressão , Sequestradores de Radicais Livres/metabolismo , Radicais Livres/química , Radicais Livres/metabolismo , Espectrometria de Massas , Microscopia Confocal , Estresse Oxidativo/efeitos dos fármacos , RatosRESUMO
Different lines of studies suggest that both the corticotropin-releasing hormone-related peptide Urocortin I (Ucn) and the neuromodulator nitric oxide (NO) are involved in the regulation of the complex mechanisms controlling feeding and anxiety-related behaviors. The aim of the present study was to investigate the possible interaction between Ucn and NO in the hypothalamic paraventricular nucleus (PVN), an area known to be involved in the modulation of these particular behaviors. Therefore, we mapped local mRNA and peptide/protein presence of both Ucn and the NO producing neuronal NO synthase (nNOS). This investigation was extended to include the hypothalamic supraoptic nucleus (SON) and the Edinger-Westphal nucleus area (EW), the latter being one of the major cellular Ucn-expressing sites. Furthermore, we compared the two predominantly used laboratory rat strains, Wistar and Sprague-Dawley. Ucn mRNA and immunoreactivity were detected in the SON and in the EW. A significant difference between Wistar and Sprague-Dawley rats was found in mRNA levels in the EW. nNOS was detected in all brain areas analyzed, showing a significantly lower immunoreactivity in the PVN and EW of Sprague-Dawley versus Wistar rats. Contrary to some previous reports, no Ucn mRNA and only a very low immunoreactivity were detectable in the PVN of either rat strain. Interestingly, double-labeling immunofluorescence revealed that in the SON approximately 75% of all cells immunoreactive for Ucn were colocalized with nNOS, whereas in the EW only approximately 2% of the Ucn neurons were found to contain nNOS. These findings suggest an interaction between Ucn and NO signaling within the SON, rather than the PVN, that may modulate the regulation of feeding, reproduction, and anxiety-related behaviors.
Assuntos
Hormônio Liberador da Corticotropina/biossíntese , Hipotálamo/metabolismo , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico Sintase/metabolismo , Tegmento Mesencefálico/metabolismo , Animais , Transtornos de Ansiedade/metabolismo , Transtornos de Ansiedade/fisiopatologia , Comportamento Alimentar/fisiologia , Hipotálamo/citologia , Imuno-Histoquímica , Masculino , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo I , Nervo Oculomotor/citologia , Nervo Oculomotor/metabolismo , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Especificidade da Espécie , Núcleo Supraóptico/citologia , Núcleo Supraóptico/metabolismo , Tegmento Mesencefálico/citologia , UrocortinasRESUMO
Oxidative stress is one of the major pathological factors in the cascade that leads to cell death in cerebral ischemia. Here, we investigated the neuroprotective effect of a naturally occurring antioxidant, oxyresveratrol, to reduce brain injury after cerebral stroke. We used the transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia to induce a defined brain infarction. Oxyresveratrol was given twice intraperitoneally: immediately after occlusion and at the time of reperfusion. Oxyresveratrol (10 or 20 mg/kg) significantly reduced the brain infarct volume by approximately 54% and 63%, respectively, when compared to vehicle-treated MCAO rats. Also, the neurological deficits as assessed by different scoring methods improved in oxyresveratrol-treated MCAO rats. Histological analysis of apoptotic markers in the ischemic brain area revealed that oxyresveratrol treatment diminished cytochrome c release and decreased caspase-3 activation in MCAO rats. Also, staining for apoptotic DNA showed that the number of apoptotic nuclei in ischemic brain was reduced after oxyresveratrol treatment as compared to the vehicle-treated MCAO rats. This dose-dependent neuroprotective effect of oxyresveratrol in an in vivo stroke model demonstrates that this drug may prove to be beneficial for a therapeutic strategy to limit brain injury in acute brain ischemia.
Assuntos
Isquemia Encefálica/prevenção & controle , Epoprostenol/análogos & derivados , Ataque Isquêmico Transitório/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Estilbenos/uso terapêutico , Análise de Variância , Animais , Morte Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/patologia , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica/métodos , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Ataque Isquêmico Transitório/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Exame Neurológico/métodos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The effect of direct intrahypothalamic nitric oxide (NO) administration on the release of selected amino acids in the hypothalamic supraoptic nucleus (SON) with and without concomitant forced swimming was investigated. Adult male Wistar rats were chronically fitted with U-shaped microdialysis probes in the SON and forced to swim for 10-min in 20-degree C warm water. Thirty-min microdialysis samples were collected before, during and after the forced swimming period while NO was administered into the SON via microdialysis. The results show that NO administration in combination with forced swimming affects the release of aspartate, glutamate, taurine, and gamma aminobutyric acid (GABA) in different patterns. Whereas the release of the excitatory amino acids aspartate and glutamate was triggered only during NO administration and forced swimming, the inhibitory amino acids GABA and taurine were found in the extracellular fluid in increased concentrations also after the treatment. These data indicate that NO administration differently affects the release of excitatory and inhibitory amino acids within the SON. Thus, SON neurons which contain high concentrations of neuronal NO synthase, might contribute to the regulation of their own secretory activity by releasing NO that controls the orchestrated release of excitatory and inhibitory amino acids from axon terminals of afferences and interneurons as well as release from glial cells.
Assuntos
Sistema Hipotálamo-Hipofisário/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Óxido Nítrico/metabolismo , Estresse Fisiológico/metabolismo , Núcleo Supraóptico/metabolismo , Transmissão Sináptica/fisiologia , Animais , Ácido Aspártico/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Masculino , Microdiálise , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Óxido Nítrico/farmacologia , Ratos , Ratos Wistar , Estresse Fisiológico/fisiopatologia , Núcleo Supraóptico/efeitos dos fármacos , Natação/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Taurina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Leber's hereditary optic neuropathy (LHON) is a retinal neurodegenerative disorder caused by mitochondrial DNA point mutations. Complex I of the respiratory chain affected by the mutation results in a decrease in ATP and an increase of reactive oxygen species production. Evaluating the efficacy of minocycline in LHON, the drug increased the survival of cybrid cells in contrast to the parental cells after thapsigargin-induced calcium overload. Similar protection was observed by treatment with cyclosporine A, a blocker of the mitochondrial permeability transition pore (mPTP). Ratiometric Ca(2+) imaging reveals that acetylcholine/thapsigargin triggered elevation of the cytosolic calcium concentration is alleviated by minocycline and cyclosporine A. The mitochondrial membrane potential of LHON cybrids was significantly conserved and the active-caspase-3/procaspase-3 ratio was decreased in both treatments. Our observations show that minocycline inhibits permeability transition induced by thapsigargin in addition to its antioxidant effects. In relation with its high safety profile, these results would suggest minocycline as a promising neuroprotective agent in LHON.
Assuntos
Células Híbridas/efeitos dos fármacos , Minociclina/farmacologia , Mutação , Fármacos Neuroprotetores/farmacologia , Atrofia Óptica Hereditária de Leber/genética , Acetilcolina/farmacologia , Análise de Variância , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Colinérgicos/farmacologia , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Tapsigargina/farmacologia , Fatores de TempoRESUMO
An organotypic cell culture (OCC) model of the rat hypothalamic paraventricular nucleus (PVN) was established to monitor intracellular calcium levels ([Ca(2+)](i)) of magnocellular neurons in response to glutamate and nitric oxide (NO). The histoarchitectural organization of these cultures was characterized either by immunohistochemical labeling of vasopressin, neuronal nitric oxide synthase (nNOS) and the neuronal marker NeuN or by the enzyme histochemical NADPH-diaphorase staining. A distinct NeuN positive cell population in 14-days old OCC's was confirmed as being the PVN by its vasopressin- and nNOS-immunostained neurons as well as by its NADPH-diaphorase labeling. Life cell imaging was performed using the [Ca(2+)](i) sensor Fluo-4 to measure [Ca(2+)](i) transients in response to bath applications of glutamate, high potassium (60 mM), and ATP. The glutamate-induced [Ca(2+)](i) response was mimicked by AMPA but not NMDA in the PVN. NMDA, however, elicited a [Ca(2+)](i) transient in a different area of the OCC that corresponds to the suprachiasmatic nucleus indicating the potential effectiveness of the stimulus. The AMPA-receptor blocker NBQX abolished the glutamate-induced response in the PVN. An inhibition of endogenous NO production by the NOS inhibitor L-NAME decreased the amplitude of AMPA- and glutamate-induced [Ca(2+)](i) rises. Taken together, these data suggest that AMPA mediates the glutamate-induced [Ca(2+)](i) rises within the PVN, where endogenous NO is able to modulate such glutamate signaling in OCC.
Assuntos
Cálcio/metabolismo , Modelos Biológicos , Óxido Nítrico/farmacologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Receptores de AMPA/metabolismo , Trifosfato de Adenosina/farmacologia , Compostos de Anilina/química , Animais , Técnicas de Cultura de Células , Ácido Glutâmico/farmacologia , Histocitoquímica , N-Metilaspartato/metabolismo , NADPH Desidrogenase/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Núcleo Hipotalâmico Paraventricular/metabolismo , Potássio/farmacologia , Ratos , Coloração e Rotulagem , Xantenos/químicaRESUMO
Hydroxystilbenes are naturally occurring polyphenols with protective effects against reactive oxygen and nitrogen species (ROS/RNS). Here, we investigated oxyresveratrol (OXY), which is contained in high amounts in mulberry wood, in comparison to the antioxidant resveratrol (RES). We found that OXY is a more effective scavenger for 2,2-diphenyl-1-picryl-hydrazyl (DPPH, 100 microM) used as a general free radical model, compared to RES or trans-4-hydroxystilbene (IC(50)=28.9, 38.5, and 39.6 microM, respectively). When primary glial cell cultures were loaded with the ROS/RNS-sensitive fluorochrome 2,7-dichlorodihydrofluorescein, the lowest rise in the fluorescence signal after H(2)O(2) exposure was seen when the cells were pretreated with OXY. Using 4,5-diaminofluorescein (DAF-2) to monitor free nitric oxide levels (7.7 microM NO) in a spectrofluorimetric cell-free assay, we found again that OXY (at 5 microM) is a more effective scavenger. Accordingly, cultures of the murine microglial cell line N9 and primary mixed glial cultures were used to test the drug effects of NO production upon expression of the inducible isoform of nitric oxide synthase (iNOS). We found that both compounds considerably diminished NO (nitrite) levels, RES more effectively than OXY (IC(50)=22.36 and 45.31 microM). RES but not OXY down-regulated the expression of iNOS protein, but both did not alter iNOS activity. Furthermore, OXY displayed a generally lower cytotoxicity than RES. The radical and ROS scavenging properties, as well as the lower cytotoxicity towards microglia and the known good water solubility suggest OXY as a potential protectant against ROS/RNS.
Assuntos
Antioxidantes/farmacologia , Sequestradores de Radicais Livres/farmacologia , Microglia/efeitos dos fármacos , Extratos Vegetais/farmacologia , Estilbenos/farmacologia , Animais , Compostos de Bifenilo/metabolismo , Western Blotting , Células Cultivadas , Fluoresceína/química , Fluoresceínas/química , Hidrazinas/metabolismo , Camundongos , Microglia/citologia , Microglia/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo/efeitos dos fármacos , Picratos , Ratos , Resveratrol , Espectrometria de FluorescênciaRESUMO
4,5-diaminofluorescein diacetate (DAF-2DA) is widely used as a fluorescent probe to detect endogenously produced nitric oxide (NO). Recent reports that refer to the high sensitivity of DAF-2 toward NO prompted us to test its efficiency and specificity in a mixed murine primary glial culture model, in which the NO-synthesizing enzyme inducible nitric oxide synthase (iNOS) is expressed by stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Cultures were loaded with DAF-2DA and the fluorescence was measured using confocal microscopy. NO production in the cultures was determined using the ozone/chemiluminescence technique. Due to the extremely high photosensitivity of DAF-2, low laser intensities were used to avoid artifacts. No difference in DAF-2 fluorescence was observed in NO-producing cultures compared to control cultures, whereas the NO/peroxynitrite-sensitive dye 2,7-dihydrodichlorofluorescein (DCF) showed a significant fluorescence increase specifically in microglia cells. A detectable gain in fluorescence was seen when NO-containing buffer was added to the DAF-2DA-loaded cells with a minimum NO concentration at 7.7 microM. An additional gain of DAF-2 fluorescence was obtained when the cells were depleted of glutathione (GSH) with L-buthionine S,R-sulfoximine (BSO). Hence, we monitored the change in DAF-2 fluorescence intensity in the presence of NO and O(-*)(2) in a cell-free solution. The fluorescence due to NO was indeed larger when O(-*)(2) was added, implying a higher sensitivity of DAF-2 for peroxynitrite. Nevertheless, our results also indicate that measurement of DCF fluorescence is a better tool for monitoring intracellular changes in the levels of NO and/or peroxynitrite than DAF-2.
Assuntos
Microscopia de Fluorescência/métodos , Neuroglia/metabolismo , Estresse Oxidativo/fisiologia , Ácido Peroxinitroso/metabolismo , Animais , Antineoplásicos/farmacologia , Sistema Livre de Células , Células Cultivadas , Fluoresceína , Fluoresceínas , Glutationa/metabolismo , Indicadores e Reagentes , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Microscopia Confocal/métodos , Neuroglia/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo/efeitos dos fármacos , Ácido Peroxinitroso/análise , Sensibilidade e Especificidade , Superóxidos/metabolismoRESUMO
Glutathione (GSH), the major cellular protectant against reactive oxygen and nitrogen species, is compartmentalized in a cytosolic (c) and a mitochondrial (mt) pool. We investigated how c-GSH and mt-GSH are differentially affected by endogenously produced nitric oxide (NO). Microglial cell line (N9) cultures were immunostimulated with lipopolysaccharide/interferon-gamma to elicit the inducible isoform of NO synthase (iNOS). Despite a significant reduction in total GSH, the mt-GSH remained nearly unaffected by iNOS-mediated NO production. To investigate possible consequences of GSH depletion on the mitochondrial membrane potential, we used buthionine sulfoximine (BSO) to reduce separately the c-GSH, whereas ethacrynic acid (EA) was applied to deplete both mt-GSH and c-GSH. The mitochondrial membrane potential was more vulnerable to NO exposure in EA-pretreated cultures than in BSO-pretreated cultures, indicated by a potentiated release of tetramethylrhodamine from mitochondria into the cytosol. To relate the EA-mediated decrease in mitochondrial membrane potential to the oxidant buildup after GSH depletion, we loaded the cells with the oxidant-sensitive fluorochrome 2',7'-dihydrodichlorofluorescein (DCF) diacetate. EA treatment caused an increase in DCF fluorescence over time that was potentiated when the iNOS expression was stimulated. Inhibition of NO production abolished this effect. We conclude that endogenous NO production in microglial cells does not compromise the mt-GSH pool which, in turn, might explain the ability of these cells to combat high-output NO production.
Assuntos
Citosol/metabolismo , Glutationa/metabolismo , Microglia/citologia , Microglia/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Butionina Sulfoximina/farmacologia , Técnicas de Cultura de Células , Citosol/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Ácido Etacrínico/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Recently we reported that a single social defeat experience triggers the release of oxytocin (OXT) from somata and dendrites, but not axon terminals, of neurons of the hypothalamic-neurohypophysial system. To further investigate the regulatory mechanisms underlying this dissociated release, we exposed male Wistar rats to a 30-min social defeat and monitored release of the inhibitory amino acids gamma amino butyric acid (GABA) and taurine within the hypothalamic supraoptic nucleus (SON) using microdialysis. Social defeat caused a significant increase in the release of both GABA and taurine within the SON (up to 480%; P < 0.01 vs. prestress release). To reveal the physiological significance of centrally released GABA, the specific GABAA-receptor antagonist bicuculline (0.02 mm) was administered into the SON via retrodialysis. This approach caused a significant increase in the release of OXT both within the SON and into the blood under basal conditions and during stress (up to 300 and 200%, respectively; P < 0.05 vs. basal values), without affecting plasma vasopressin. Electrophysiological studies confirmed the selective action of bicuculline on the firing activity of OXT neurons in the SON. Taken together, our data demonstrate that GABA is released within the SON during emotional stress to act as a selective inhibitor of both central and peripheral OXT secretion.
Assuntos
Neurônios/metabolismo , Ocitocina/metabolismo , Núcleo Supraóptico/citologia , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Comportamento Animal , Bicuculina/farmacologia , Colecistocinina , Cromatografia Líquida de Alta Pressão/métodos , Vias de Administração de Medicamentos , Eletrofisiologia/métodos , Fluorometria/métodos , Antagonistas GABAérgicos/farmacologia , Ácido Glutâmico/metabolismo , Masculino , Microdiálise/métodos , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Estresse Psicológico/metabolismo , Núcleo Supraóptico/efeitos dos fármacos , Taurina/metabolismo , Vasopressinas/metabolismoRESUMO
Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF-fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark-type NO-sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5-ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS-dependent mechanism.