RESUMO
MK-801, a non-competitive antagonist of N-methyl-D-aspartate-type glutamate receptors, was tested for its ability to antagonize excitotoxic actions of N-methyl-D-aspartate or quisqualic acid injected into the brains of seven-day-old rats. Stereotaxic injection of N-methyl-D-aspartate (25 nmol/0.5 microliters) or quisqualic acid (100 nmol/1.0 microliter) into the corpus striatum under ether anesthesia consistently produced severe unilateral neuronal necrosis in the basal ganglia, dorsal hippocampus and overlying neocortex. The distribution of the damage corresponded to the topography of glutamate receptors in the vulnerable regions demonstrated by previous autoradiographic studies. N-Methyl-D-aspartate produced severe, confluent neuronal destruction while quisqualic acid typically caused more selective neuronal necrosis. Intraperitoneal administration of MK-801 (0.1-1.0 mg/kg) 30 min before N-methyl-D-aspartate injection had a prominent dose-dependent neuroprotective effects as assessed morphometrically by comparison of bilateral striatal, hippocampal and cerebral hemisphere cross-sectional areas five days later. A 1 mg/kg dose of MK-801 given as pre-treatment completely protected the infant brain. The same dose of MK-801 was also completely protective when administered 30 or 40 min after N-methyl-D-aspartate and afforded partial protection when given 2 h later. MK-801 pre-treatment also prevented the electrically confirmed behavioral seizures induced by N-methyl-D-aspartate. The drug significantly reduced striatal but not hippocampal or neocortical injury when given as two doses (1 mg/kg) 30 min prior to and immediately following quisqualic acid injection. The data indicate that systemic administration of MK-801 can prevent N-methyl-D-aspartate-induced neuronal injury in perinatal rat brain even when administered after the initial insult. MK-801 also partially antagonized quisqualic acid-mediated neurotoxicity, suggesting that quisqualic acid-induced toxicity is, in part, mediated through N-methyl-D-aspartate receptor activation. The sensitivity of the developing brain to the toxicity of N-methyl-D-aspartate provides a sensitive and reproducible in vivo model for exploring these issues and for screening prospective neuroprotective drugs that act at the N-methyl-D-aspartate receptor-channel complex.
Assuntos
Maleato de Dizocilpina/farmacologia , N-Metilaspartato/antagonistas & inibidores , Neurotoxinas/farmacologia , Ácido Quisquálico/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Eletroencefalografia , Feminino , Injeções , Injeções Intraperitoneais , Masculino , N-Metilaspartato/intoxicação , Ácido Quisquálico/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Kavapyrone extracts of the pepper plant Piper methysticum Forst. have been reported to be pharmacologically active in the brain by modulating the function of several ionotropic receptor systems and voltage-sensitive ion channels. While kavapyrones have previously demonstrated neuroprotective effects against several forms of neurotoxicity, the possibility remains that perturbed function of neuronal ion transport may prove to be neurotoxic in some instances. The present studies were designed to examine the effects of the kavapyrone, (+/-) kavain, on viability of organotypic hippocampal explants exposed to the excitotoxin N-methyl-D-aspartate (NMDA). Exposure to (+/-) kavain (1-600 microM) for 24 h did not alter neuronal viability in the CA1, CA3, or dentate gyrus regions of hippocampal explants. However, higher concentrations of (+/-) kavain (> or =300 microM) produced marked neurotoxicity in the lacunosum moleculare layer of the hippocampus. One hour of exposure to NMDA (20 microM) produced significant neuronal death in both the CA3 and CA1 pyramidal cell regions, effects prevented by co-exposure to MK-801 (30 microM). Co-exposure of explants to (+/-) kavain (1-100 microM) with NMDA did not alter the severity of NMDA-induced neurotoxicity. However, exposure of NMDA-treated explants to (+/-) kavain (> or =10 microM) for 24 h after insult produced significant increases in neurotoxicity in the CA1 and dentate gyrus regions of explants. In conclusion, while the kavapyrone (+/-) kavain is neurotoxic only at high concentrations when exposed alone to the developing hippocampus, it appears to adversely affect neuronal recovery following excitotoxic insults.
Assuntos
Animais Recém-Nascidos/fisiologia , Agonistas de Aminoácidos Excitatórios/intoxicação , Hipocampo/efeitos dos fármacos , N-Metilaspartato/intoxicação , Neurotoxinas/farmacologia , Pironas/farmacologia , Animais , Sinergismo Farmacológico , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7-10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5-10 microM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25-0.5 mM). GABA (10 microM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 microM) and vapreotide (RC 160; 10 microM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 microM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 microM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5-10 microM). Finally, both specific (Rp-8-bromoguanosine 3',5'-monophosphate, 10 microM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 microM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca2+ level.
Assuntos
GMP Cíclico/fisiologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Somatostatina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/intoxicação , N-Metilaspartato/intoxicação , Ratos/embriologiaRESUMO
Statin is a 57-kDa protein exclusively expressed in nuclei of nonproliferating mammalian cells. Recent studies have suggested that statin may play a role in the maintenance of growth arrest. Several lines of evidence also support the notion that a variety of genes and gene products are modulated during cell proliferation and cell death. The present study examined the possibility that statin expression could be modulated during neuronal injury using N-methyl-Daspartate (NMDA)-induced toxicity to rat embryonic hippocampal cultures as a model. Immunocytochemical studies using a monoclonal antibody to statin revealed a prominent nuclear localization of statin in cultured hippocampal cells. Western blot analysis showed that this antibody recognizes a 57-kDa protein band, indicative of the presence of statin in this preparation. Brief exposure of hippocampal neurons to NMDA (500 microM) produced severe neuronal degeneration over the subsequent hours. NMDA-treated neurons markedly overexpressed statin. Both NMDA-induced neuronal toxicity and statin overexpression were prevented by the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohept-5,10-imine hydrogen maleate (MK-801). Interestingly, time course studies indicate that the increased expression of statin observed following NMDA exposure clearly preceded the appearance of the first signs of neuronal death as determined by vital staining. In addition, exposure of hippocampal neurons to the Ca2+ ionophore, A23187, produced a marked increase in statin immunodetection, indicating that statin expression is likely regulated in a Ca(2+)-dependent manner. Thus, these results show that statin, which is expressed at low levels in embryonic rat cultured hippocampal neurons, is rapidly overexpressed following a toxic insult produced by the activation of the NMDA receptor. The observation that statin overexpression occurs prior to neuronal death raises the possibility that the up-regulation of statin could be used as an early index of neuronal injury.
Assuntos
Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , N-Metilaspartato/intoxicação , Proteínas/metabolismo , Animais , Calcimicina/farmacologia , Proteínas de Ciclo Celular , Morte Celular , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Hipocampo/patologia , N-Metilaspartato/antagonistas & inibidores , Neurônios/metabolismo , Neurônios/fisiologia , Proteínas Nucleares , Proteínas/antagonistas & inibidores , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Human immunodeficiency virus (HIV-1) infection often results in central nervous system (CNS) dysfunction, yet the mechanism(s) of action for HIV-1 in the CNS are not fully understood. In the present study gp120, the HIV-1 envelope glycoprotein, was shown to selectively inhibit N-methyl-D-aspartate (NMDA) receptor function. In addition to inhibiting radioligand binding to rat NMDA receptors, gp120 inhibited NMDA-induced currents in Xenopus oocytes, attenuated NMDA-stimulated calcium flux and cytotoxicity in cultured cerebellar granule cells, and provided partial protection against NMDA-induced lethality in vivo. These findings suggest that NMDA receptor complex is a possible site of action of HIV-1 within the CNS.
Assuntos
Proteína gp120 do Envelope de HIV/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Maleato de Dizocilpina/antagonistas & inibidores , Maleato de Dizocilpina/metabolismo , Eletrofisiologia , N-Metilaspartato/antagonistas & inibidores , N-Metilaspartato/fisiologia , N-Metilaspartato/intoxicação , Oócitos/metabolismo , Fenciclidina/análogos & derivados , Fenciclidina/antagonistas & inibidores , Fenciclidina/metabolismo , Ácidos Pipecólicos/antagonistas & inibidores , Ácidos Pipecólicos/metabolismo , Ensaio Radioligante , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Proteínas Recombinantes , XenopusRESUMO
Activation of group I metabotropic glutamate receptors (mGlu1 or -5 receptors) is known to either enhance or attenuate excitotoxic neuronal death depending on the experimental conditions. We have examined the possibility that these receptors may switch between two different functional modes in regulating excitotoxicity. In mixed cultures of cortical cells, the selective mGlu1/5 agonist, 3,5-dihydroxyphenylglycine (DHPG), amplified neurodegeneration induced by a toxic pulse of NMDA. This effect was observed when DHPG was either combined with NMDA or transiently applied to the cultures prior to the NMDA pulse. However, two consecutive applications of DHPG consistently produced neuroprotection. Similar effects were observed with DHPG or quisqualate (a potent agonist of mGlu1/5 receptors) in pure cultures of cortical neurons virtually devoid of astrocytes. In cultures of hippocampal pyramidal neurons, however, only protective effects of DHPG were seen suggesting that, in these particular cultures, group I mGlu receptors were endogenously switched into a "neuroprotective mode". The characteristics of the activity-dependent switch from facilitation to inhibition were examined in mixed cultures of cortical cells. The switch in the response to DHPG was observed when the two applications of the drug were separated by an interval ranging from 1-45 min, but was lost when the interval was extended to 90 min. In addition, this phenomenon required the initial activation of mGlu5 receptors (as indicated by the use of subtype-selective antagonists) and was mediated by the activation of protein kinase C. We conclude that group I mGlu receptors are subjected to an activity-dependent switch in regulating excitotoxic neuronal death and, therefore, the recent "history" of these receptors is critical for the response to agonists or antagonists.
Assuntos
Inibição Neural/fisiologia , Neurotoxinas/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Ativação Enzimática/fisiologia , Agonistas de Aminoácidos Excitatórios/intoxicação , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Camundongos , N-Metilaspartato/intoxicação , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Fármacos Neuroprotetores/farmacologia , Proteína Quinase C/metabolismo , Ácido Quisquálico/farmacologia , Receptor de Glutamato Metabotrópico 5 , Resorcinóis/farmacologia , Fatores de TempoRESUMO
This study was designed to examine the effects of d-amphetamine (D-AMPH) and D1- and D2-selective dopaminergic drugs on the concentration of the broad-spectrum excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the striatum of developing and adult rats. At all ages, KYNA levels were significantly reduced 1 h after the systemic administration of D-AMPH (5 mg/kg). SKF 38393 (5 mg/kg) and quinpirole (2 mg/kg) also caused a rapid decrease in striatal KYNA, but only in postnatal day (PND) 7 and 14 rats. All these effects were readily prevented by specific dopamine receptor antagonists. The possible functional significance of the reduction in KYNA levels was tested in PND 14 animals. When pretreated with D-AMPH (5 mg/kg), these rats showed markedly increased vulnerability to an intrastriatal injection of the excitotoxin NMDA. These data suggest that KYNA plays a role as a mediator of dopamine-glutamate interactions in the rat striatum.
Assuntos
Envelhecimento/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/farmacologia , Agonistas de Aminoácidos Excitatórios/intoxicação , Ácido Cinurênico/metabolismo , N-Metilaspartato/intoxicação , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/fisiologia , Corpo Estriado/crescimento & desenvolvimento , Dextroanfetamina/farmacologia , Dopaminérgicos/farmacologia , Antagonistas de Dopamina/farmacologia , Haloperidol/farmacologia , Injeções , Ligantes , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismoRESUMO
Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Doadores de Óxido Nítrico/farmacologia , Animais , Antioxidantes/farmacologia , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Hidrazinas/farmacologia , Membranas Intracelulares/metabolismo , Camundongos , N-Metilaspartato/intoxicação , Neurotoxinas/farmacologia , Óxido Nítrico/farmacologia , Óxidos de Nitrogênio , Ratos , Ratos EndogâmicosRESUMO
Ebselen is a seleno-organic compound currently in clinical trials for the treatment of ischemic stroke and subarachnoid hemorrhage. Its putative mode of action as a neuroprotectant is via cyclical reduction and oxidation reactions, in a manner akin to glutathione peroxidase. For this reason, we have investigated the effects of ebselen on the redox-sensitive NMDA receptor. We have found that ebselen readily reversed dithiothreitol (DTT) potentiation of NMDA-mediated currents in cultured neurons and in Chinese hamster ovary (CHO) cells expressing wild-type NMDA NR1/NR2B receptors. In contrast, ebselen was unable to modulate NMDA-induced currents in neurons previously exposed to the thiol oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or in CHO cells expressing a mutant receptor lacking the NR1 redox modulatory site, suggesting that ebselen oxidizes the NMDA receptor via this site. In addition, ebselen was substantially less effective in modifying NMDA responses in neurons exposed to alkylating agent N-ethylmaleimide (NEM) following DTT treatment. Ebselen also reversed DTT block of carbachol-mediated currents in Cos-7 cells expressing the alpha(2)beta delta epsilon subunits of the acetylcholine receptor, an additional redox-sensitive ion channel. Ebselen was observed to significantly increase cell viability following a 30-min NMDA exposure in cultured neurons. In contrast, other more typical antioxidant compounds did not afford neuroprotection in a similar paradigm. We conclude that ebselen may be neuroprotective in part due to its actions as a modulator of the NMDA receptor redox modulatory site.