dizocilpine-maleate and peroxynitric-acid

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.

Topics: Age Factors; Animals; Animals, Newborn; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Membrane Glycoproteins; Mercury Poisoning, Nervous System; Methylmercury Compounds; Nerve Degeneration; Neurons; Nitrates; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Tyrosine

The present study tests the hypothesis that nitration is a potential mechanism of N-methyl-D-aspartate (NMDA) receptor modification, by assessing the effect of peroxynitrite in vitro on the glutamate and ion-channel sites of the NMDA receptor in the fetal guinea pig. Nitration of NMDA receptor subunits was confirmed by Western blot. Following peroxynitrite exposure, (3)H-MK-801 bindings show an increase in the B(max) and a decrease in the K(d), while (3)H-glutamate bindings show a decrease in the K(d) with no change in the B(max). We conclude that peroxynitrite regulates the NMDA receptor function by increasing the affinity of the ion-channel and glutamate sites, and by exposing additional ion-channel sites. We propose that nitration of the NMDA receptor is a potential mechanism for the regulation of the receptor during hypoxia.

Topics: Animals; Cerebral Cortex; Dizocilpine Maleate; Fetus; Glutamic Acid; Guinea Pigs; Hypoxia; Ion Channels; Kinetics; Nitrates; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

The peroxynitrite contributions to hypoxic damage in brain slices that arise from N-methyl-D-aspartate (NMDA) receptor activation were studied by following the temporal-spatial course of nitrotyrosine (NT) formation during six conditions: hypoxia (pO(2)<5 mmHg) with or without 10 microM MK-801 treatment; with exposure to 10, 100 and 1000 microM NMDA; and no treatment (control). In each experiment, twenty 350-micrometer thick cerebrocortical slices, obtained from the parietal lobes of ten 7-day-old Sprague-Dawley rats, were metabolically recovered and allowed to respire in a well-oxygenated perfusion system. Thirty minutes exposures to hypoxia or NMDA were followed by 2 h of oxygenated reperfusion. MK-801 administration began 15 min prior to hypoxia and was discontinued during reperfusion. Anti-NT serum immunohistochemistry stains in 20-micrometer frozen sections of slices taken during oxygenated reperfusion, after hypoxia or NMDA exposure, were positive in both neurons and endothelial cells. NT-positive neurons were detected sooner after hypoxia than after NMDA exposure, suggesting that mechanisms of superoxide generation were different in both groups. After hypoxia and even more so after NMDA exposure, more intense NT-positive staining was observed in endothelial cells than in neurons. Cell damage after hypoxia was attenuated by MK-801. MK-801 decreased post-hypoxia counts of NT-stained endothelial cells by 78.5% (p<0. 001) and NT-stained neurons by 54.1% (p<0.05). Our findings suggest that NMDA receptor activation in hypoxic brain slices is associated with increased post-hypoxic peroxynitrite production that contributes to acute neuronal death and endothelial cell injury. Peroxynitrite injury to endothelial cells, caused either by increased peroxynitrite from within or from increased vulnerability to peroxynitrite from without, might play an important role in hypoxic-ischemic brain injury and NMDA-induced brain injury.

Topics: Animals; Brain; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hypoxia-Ischemia, Brain; N-Methylaspartate; Neurons; Nitrates; Nitric Oxide; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Superoxides; Tyrosine

Functional roles of peroxynitrite in N-methyl-D-aspartate (NMDA)- and sodium nitroprusside (SNP)-evoked releases of acetylcholine (ACh) from cerebral cortical neurons in primary culture have been investigated. NMDA increased the release of ACh in a dose-dependent manner, which was significantly suppressed by (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801), a non-competitive antagonist specific for the NMDA receptor complex, and NO synthase inhibitors. SNP also showed a concentration-dependent increase in ACh release. Hemoglobin significantly abolished the stimulatory effects of both NMDA and SNP on ACh release. In addition, superoxide anion scavengers such as superoxide dismutase and ceruloplasmin significantly reduced the increased ACh release evoked by NMDA and SNP. Synthesized peroxynitrite dose-dependently elevated the release of ACh. These results indicate that the increased release of ACh by NMDA and SNP is mediated through peroxynitrite formed in the reaction of superoxide anion with nitric oxide produced by NMDA receptor activation and liberated from SNP rather than nitric oxide itself.

Topics: Acetylcholine; Animals; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Free Radical Scavengers; Mice; Mice, Inbred Strains; N-Methylaspartate; Neurons; Nitrates; Nitric Oxide Synthase; Nitroprusside; Receptors, N-Methyl-D-Aspartate; Superoxides