RESUMO
KB-R7943, an inhibitor of a reversed Na(+)/Ca(2+) exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Deltapsi(mit)), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca(2+)](i) and on Deltapsi(mit) changes in rat cultured cerebellar granule neurons. 15 micromol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca(2+)](i) overload, KB-R7943 considerably improved the [Ca(2+)](i) recovery and restoration of Deltapsi(mit) after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Deltapsi(mit). KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca(2+) uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca(2+) accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.
Assuntos
Cálcio/metabolismo , Ácido Glutâmico/toxicidade , Fármacos Neuroprotetores/farmacologia , Tioureia/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Ácido Caínico/farmacologia , Lítio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Wistar , Tioureia/farmacologiaRESUMO
In the present work, the forward and/or reversed Na+/Ca2+ exchange in cerebellar granular cells was suppressed by substitution of Na+o by Li+ before, during, and after exposure to glutamate for varied time and also using the inhibitor KB-R7943 of the reversed exchange. After glutamate challenge for 1 min, Na+o/Li+ substitution did not influence the recovery of low [Ca2+]i in a calcium-free medium. A 1-h incubation with 100 microM glutamate induced in the neurons a biphasic and irreversible [Ca2+]i rise (delayed calcium deregulation (DCD)), enhancement of [Na+]i, and decrease in the mitochondrial potential. If Na+o had been substituted by Li+ before the application of glutamate, i.e. the exchange reversal was suppressed during the exposure to glutamate, the number of cells with DCD was nearly fourfold lowered. However, addition of the Na+/K+-ATPase inhibitor ouabain (0.5 mM) not preventing the exchange reversal also decreased DCD in the presence of glutamate. Both exposures decreased the glutamate-caused loss of intracellular ATP. Glucose deprivation partially abolished protective effects of the Na+o/Li+ substitution and ouabain. KB-R7943 (10 microM) increased 7.4-fold the number of cells with the [Ca2+]i decreased to the basal level after the exposure to glutamate. Thus, reversal of the Na+/Ca2+ exchange reinforced the glutamate-caused perturbations of calcium homeostasis in the neurons and slowed the recovery of the decreased [Ca2+]i in the post-glutamate period. However, for development of DCD, in addition to the exchange reversal, other factors are required, in particular a decrease in the intracellular concentration of ATP.
Assuntos
Cálcio/metabolismo , Glutamatos/metabolismo , Neurônios/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Células Cultivadas , Cerebelo/citologia , Citoplasma/metabolismo , Glucose/metabolismo , Compostos de Lítio/farmacologia , Potencial da Membrana Mitocondrial , Neurônios/efeitos dos fármacos , Ouabaína/farmacologia , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
Semax (100 microM) and its Pro-Gly-Pro fragment (20 and 100 microM) delayed the development of calcium dysregulation and reduction of the mitochondrial potential in cultured cerebellar granule cells under conditions of glutamate neurotoxicity. Incubation with these peptides improved neuronal survival by on average 30%. The neuroprotective effect of semax in cerebral ischemia/hypoxia can be due to improvement of mitochondrial resistance to "calcium" stress.
Assuntos
Hormônio Adrenocorticotrópico/análogos & derivados , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ácido Glutâmico/toxicidade , Homeostase/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Prolina/análogos & derivados , Hormônio Adrenocorticotrópico/farmacologia , Animais , Neurônios/metabolismo , Prolina/farmacologia , Ratos , Ratos WistarRESUMO
In primary 7-8-day culture of cerebellar granule cells, glutamate exposure (100 microM, 10-240 min) induced a 60-30% drop in ATP level; during the postglutamate period ATP level completely recovered after 24 h. Inhibition of NO-synthase with L-NAME during glutamate application resulted in less pronounced decrease in ATP level immediately after its application and had no effect on ATP recovery after 24 h. It was found that hyperstimulation of glutamate receptors elevates concentration of NO products (nitrites and nitrates), while NO2(-) ions can increase ATP content.
Assuntos
Trifosfato de Adenosina/metabolismo , Cerebelo/metabolismo , Nitritos/metabolismo , Óxidos de Nitrogênio/metabolismo , Receptores de Glutamato/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Glutamatos/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RatosRESUMO
Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.
Assuntos
Ácido Araquidônico/farmacologia , Cálcio/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Cerebelo/citologia , Dinitrofenóis/farmacologia , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Líquido Intracelular/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligomicinas/farmacologia , Ácido Palmítico/farmacologia , Ratos , Cianeto de Sódio/farmacologiaRESUMO
Rabbit antibodies against GluR1 subunit of AMPA glutamate receptors in a concentration of 1 mug/ml significantly increased intracellular Ca(2+)concentration and decreased mitochondrial potential in hippocampal neurons, i.e. produced changes typical of the influence of glutamate in toxic concentrations. In cerebellar neurons rabbit antibodies potentiated glutamate-induced increase in intracellular Ca(2+)concentration and significantly decreased the mitochondrial potential (compared to the level observed after application of glutamate alone). The exposure of cultured cerebellar neurons to antibodies in a concentration of 0.1 mug/ml for 24 h was followed by a 50% decrease in ATP concentration and development of neuronal necrosis. Our results attest to an important role of autoimmune damage to neurons during hyperstimulation of glutamate receptors.