Effect of divalent metals on the neuronal proteasomal system, prion protein ubiquitination and aggregation.

The role of normal cellular prion protein (PrP) remains to be fully elucidated; however, the protein is crucial for the infection and progression of prion diseases. Recent evidence indicates that PrP is a metalloprotein since the octapeptide repeat sequences in the protein have high affinity for various divalent cations and the binding sites appear to play a role in the pathogenesis of prion diseases. In our present study, we tested several divalent metals including manganese and cadmium and determined their effects on protein degradation and protein aggregation in mouse neuronal cells expressing PrP. Cadmium was more neurotoxic than manganese following 24h exposure. Manganese did not show any significant effect on the inhibition of proteasomal activity or formation of high molecular weight ubiquitinated PrPs. Interestingly, treatment with cadmium profoundly inhibited proteasomal activity, which resulted in greatly increased formation of high molecular weight ubiquitinated PrPs. Immunohistochemical analysis also revealed a dramatic increase in formation of oligomers after cadmium treatment. Cadmium also increased the formation of ubiquitinated PrP, but it did not lead to the formation of proteinase-K resistant PrP. Collectively, our results show that a divalent metal, cadmium affects proteasomal function and PrP aggregation, which promote neurotoxicity.

Methamphetamine-induced neurotoxicity linked to ubiquitin-proteasome system dysfunction and autophagy-related changes that can be modulated by protein kinase C delta in dopaminergic neuronal cells.

A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and ubiquitin-proteasome system (UPS) dysfunction are not well understood. The present study investigates the contributions of protein kinase C delta (PKCδ)-mediated signaling events in MA-induced autophagy, UPS dysfunction, and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased PKCδ activation, caspase-3 activation, accumulation of ubiquitin-positive aggregates and microtubule-associated light chain-3 (LC3-II) levels. Interestingly, siRNA-mediated knockdown of PKCδ or overexpression of cleavage-resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA-mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA-treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria-mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3-mediated apoptotic cell death in the nigrostriatal dopaminergic system.

Evidence of oxidative injury of the spinal cord in 2 horses with equine degenerative myeloencephalopathy.

The cervical spinal cords of 2 horses with equine degenerative myeloencephalopathy (EDM) were evaluated for evidence of oxidative damage to the central nervous system (CNS) using immunohistochemical staining for 3-nitrotyrosine (3-NT) and 4-hydroxynonenol (4-HNE). Neurons of the CNS from horses with EDM had positive immunohistochemical staining, whereas control samples did not, thus supporting the theory that oxidative damage is a potential underlying factor in horses with EDM. In addition, serum vitamin E concentration was low in both EDM-affected horses, and vitamin E concentration was also deficient in the cerebrospinal fluid in 1 EDM horse, further supporting the association between low vitamin E concentrations and oxidative damage to the CNS. Continued research is necessary to further define the pathophysiologic mechanisms of EDM.

Paraquat induces epigenetic changes by promoting histone acetylation in cell culture models of dopaminergic degeneration.

Environmental neurotoxic exposure to agrochemicals has been implicated in the etiopathogenesis of Parkinson's disease (PD). The widely used herbicide paraquat is among the few environmental chemicals potentially linked with PD. Since epigenetic changes are beginning to emerge as key mechanisms in neurodegenerative diseases, herein we examined the effects of paraquat on histone acetylation, a major epigenetic change in chromatin that can regulate gene expression, chromatin remodeling, cell survival and cell death. Exposure of N27 dopaminergic cells to paraquat induced histone H3 acetylation in a time-dependent manner. However, paraquat did not alter acetylation of another core histone H4. Paraquat-induced histone acetylation was associated with decreased total histone deacetylase (HDAC) activity and HDAC4 and 7 protein expression levels. To determine if histone acetylation plays a role in paraquat-induced apoptosis, the novel HAT inhibitor anacardic acid was used. Anacardic acid treatment significantly attenuated paraquat-induced caspase-3 enzyme activity, suppressed proteolytic activation and kinase activity of protein kinase C delta (PKCδ) and also blocked paraquat-induced cytotoxicity. Together, these results demonstrate that the neurotoxic agent paraquat induced acetylation of core histones in cell culture models of PD and that the inhibition of HAT activity by anacardic acid protects against apoptotic cell death, indicating that histone acetylation may represent key epigenetic changes in dopaminergic neuronal cells during neurotoxic insults.

Environmental neurotoxic pesticide increases histone acetylation to promote apoptosis in dopaminergic neuronal cells: relevance to epigenetic mechanisms of neurodegeneration.

Pesticide exposure has been implicated in the etiopathogenesis of Parkinson's disease (PD); in particular, the organochlorine insecticide dieldrin is believed to be associated with PD. Emerging evidence indicates that histone modifications play a critical role in cell death. In this study, we examined the effects of dieldrin treatment on histone acetylation and its role in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. In mesencephalic dopaminergic neuronal cells, dieldrin induced a time-dependent increase in the acetylation of core histones H3 and H4. Histone acetylation occurred within 10 min of dieldrin exposure indicating that acetylation is an early event in dieldrin neurotoxicity. The hyperacetylation was attributed to dieldrin-induced proteasomal dysfunction, resulting in accumulation of a key histone acetyltransferase (HAT), cAMP response element-binding protein. The novel HAT inhibitor anacardic acid significantly attenuated dieldrin-induced histone acetylation, Protein kinase C delta proteolytic activation and DNA fragmentation in dopaminergic cells protected against dopaminergic neuronal degeneration in primary mesencephalic neuronal cultures. Furthermore, 30-day exposure of dieldrin in mouse models induced histone hyperacetylation in the striatum and substantia nigra. For the first time, our results collectively demonstrate that exposure to the neurotoxic pesticide dieldrin induces acetylation of core histones because of proteasomal dysfunction and that hyperacetylation plays a key role in dopaminergic neuronal degeneration after exposure of dieldrin.

Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death.

Autophagy is a phylogenetically conserved process that plays a critical role in the degradation of oxidatively damaged proteins and organelle turnover. The role of oxidative stress and apoptosis in methamphetamine (METH)-induced neurotoxicity is well known; however, the potential contribution of autophagy to METH-induced oxidative damage in dopaminergic neuronal systems remains unclear. The goals of the present article were twofold: (a) to develop an in vitro dopaminergic cell culture model to study cellular and molecular mechanisms underlying METH-induced autophagy and apoptosis, and (b) to determine whether lysosomal protease cathepsin-D activation, resulting from the loss of lysosomal membrane integrity, contributes to METH-induced apoptosis. To accomplish these goals, we characterized morphological and biochemical changes in an immortalized mesencephalic dopaminergic neuronal cell line (N27 cells) following treatment with METH. Exposure of METH (2 mM) to N27 cells resulted in the appearance of cytoplasmic vacuolar structures reminiscent of autophagic vacuoles within 3 h. In order to ascertain the identity of the vacuolar structures that are formed following METH exposure, immunohistochemical staining for markers of autophagy were performed. LAMP 2, a classical marker of autophagolysosomes, revealed an extensive punctuate pattern of distribution on the vacuolar membrane surface, with exclusive localization in the cytoplasm. Additionally, using DNA fragmentation analysis we showed a dose-dependent increase in fragmented DNA in METH treated N27 cells. Since METH-induced autophagy preceded DNA fragmentation, we tested whether dysfunction of the autophagolysosomal system contributes to nuclear damage. Immunofluorescence studies with cathepsin-d demonstrated a granular pattern of staining in untreated cells, whereas an increased cathepsin- D immunoreactivity with a globular pattern of staining was observed in METH-treated cells. Nevertheless, blockade of cathepsin-D activation by pepstatin-A, cathepsin-D inhibitor, failed to alter METH-induced DNA fragmentation. Collectively, these results demonstrate that N27 dopaminergic neuronal cell model may serve as an excellent in vitro model to study the mechanisms of METH-induced autophagy and apoptosis. Furthermore, it is less likely that cathepsin-D may serve as a trigger for the induction of apoptosis subsequent to exposure of N27 dopaminergic neuronal cells to METH.

Blockade of PKCdelta proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death.

The use of methylcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 microM MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced proteolytic cleavage of protein kinase Cdelta (PKCdelta), resulting in persistently increased kinase activity. The proteolytic activation of PKCdelta was suppressed by treatment with 100 microM Z-VAD-FMK and 100 microM Z-DEVD-FMK, suggesting that caspase-3 mediates the proteolytic activation of PKCdelta. Pretreatment with 100 microM Z-DEVD-FMK and 5 microM rottlerin (a PKCdelta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKCdelta(K376R) mutant or the caspase cleavage resistant PKCdelta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via proteolytic activation of PKCdelta in MMT-induced dopaminergic degeneration and suggest that PKCdelta may serve as an attractive therapeutic target in Parkinson-related neurological diseases.

Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration.

We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly activates caspase-3 in PC12 cells. Finally, overexpression of the kinase-inactive PKCdelta(K376R) mutant in rat mesencephalic dopaminergic neuronal cells attenuates dieldrin-induced caspase-3 activity and DNA fragmentation, further confirming the pro-apoptotic function of PKCdelta in dopaminergic cells. Together, we conclude that caspase-3-dependent proteolytic activation of PKCdelta is a critical event in dieldrin-induced apoptotic cell death in dopaminergic cells.

Proteolytic activation of proapoptotic kinase PKCdelta is regulated by overexpression of Bcl-2: implications for oxidative stress and environmental factors in Parkinson's disease.

We previously demonstrated that the organochlorine pesticide dieldrin, a potential chemical risk factor for development of Parkinson's disease (PD), impairs mitochondrial function and promotes apoptosis in dopaminergic PC12 cells. We further demonstrated that caspase-3-dependent proteolytic activation of a member of the novel PKC family, protein kinase Cdelta (PKCdelta), contributes to apoptotic cell death in dopaminergic cells. In the present study, we report that the proapoptotic function of PKCdelta can be regulated by overexpression of the mitochondrial anti-apoptotic protein Bcl2 in dieldrin-treated dopaminergic cells. Exposure to dieldrin (30 or 100 micro M) for 3 h produced a dose-dependent increase in caspase-3 activation and DNA fragmentation in vector-transfected PC12 cells. Overexpression of human Bcl-2 in PC12 cells completely suppressed dieldrin-induced caspase-3 activation and DNA fragmentation. Furthermore, dieldrin-induced proteolytic activation of PKCdelta was also remarkably reduced in Bcl-2-overexpressed cells. Together, these results suggest that the proapoptotic function of PKCdelta can be regulated by mitochondrial redox modulators during neurodegenerative processes.

Dieldrin-induced oxidative stress and neurochemical changes contribute to apoptopic cell death in dopaminergic cells.

We examined the acute toxicity of dieldrin, a possible environmental risk factor of Parkinson's disease, in a dopaminergic cell model, PC12 cells, to determine early cellular events underlying the pesticide-induced degenerative processes. EC(50) for 1 h dieldrin exposure was 143 microM for PC12 cells, whereas EC(50) for non-dopaminergic cells was 292-351 microM, indicating that dieldrin is more toxic to dopaminergic cells. Dieldrin also induced rapid, dose-dependent releases of dopamine and its metabolite, DOPAC, resulting in depletion of intracellular dopamine. Additionally, dieldrin exposure caused depolarization of mitochondrial membrane potential in a dose-dependent manner. Flow cytometric analysis showed generation of reactive oxygen species (ROS) within 5 min of dieldrin treatment, and significant increases in lipid peroxidation were also detected following 1 h exposure. ROS generation was remarkably inhibited in the presence of SOD. Dieldrin-induced apoptosis was significantly attenuated by both SOD and MnTBAP (SOD mimetic), suggesting that dieldrin-induced superoxide radicals serve as important signals in initiation of apoptosis. Furthermore, pretreatment with deprenyl (MAO-inhibitor) or alpha-methyl-L-p-tyrosine (TH-inhibitor) also suppressed dieldrin-induced ROS generation and DNA fragmentation. Taken together, these results suggest that rapid release of dopamine and generation of ROS are early cellular events that may account for dieldrin-induced apoptotic cell death in dopaminergic cells.

Animal models of myoclonus: an overview.

Attempts to characterize the mechanism(s) associated with myoclonus have led to the development of several naturally occurring and pharmacologically based animal models of myoclonus. Congenital disorders in animals that result in myoclonic seizures have been found in subpopulations of baboons that exhibit photoresponsive myoclonus and in herds of Hereford cattle that possess a fatal, autosomal-inherited imbalance in spinal glycine neurotransmission. Pharmacologically based models of myoclonus use a variety of approaches to product myoclonic seizures in test animals.

Animal model of posthypoxic myoclonus: II. Neurochemical, pathologic, and pharmacologic characterization.

The sudden, brief, shock-like, involuntary movements caused by active muscular contractions or inhibitions characterize myoclonus. It is manifested in a wide variety of pathologic conditions affecting the brain, spinal cord, or peripheral nerves, and is thought to be related to neuronal hyperexcitability. The pathology, physiology, and pharmacology of myoclonus are not well understood as a result of the rarity of the disorder in people and the lack of a suitable animal model. Posthypoxic myoclonus is a major myoclonus syndrome which occurs as a result of severe cerebral ischemia/hypoxia. There has been tremendous interest in the development of a suitable animal model that reflects the etiology and clinical pathology of posthypoxic myoclonus. Recently, we have developed a new animal model of posthypoxic myoclonus in which rats were subjected to a mechanically induced cardiac arrest procedure. Herein, we describe the neurochemical, pharmacologic, and pathologic characteristics of this animal model of posthypoxic myoclonus.

Effect of riluzole on the neurological and neuropathological changes in an animal model of cardiac arrest-induced movement disorder.

Posthypoxic myoclonus and seizures precipitate as secondary neurological consequences in ischemic/hypoxic insults of the central nervous system. Neuronal hyperexcitation may be due to excessive activation of glutamatergic neurotransmission, an effect that has been shown to follow ischemic/hypoxic events. Therefore, riluzole, an anticonvulsant that inhibits the release of glutamate by stabilizing the inactivated state of activated voltage-sensitive sodium channels, was tested for its antimyoclonic and neuroprotective properties in the cardiac arrest-induced animal model of posthypoxic myoclonus. Riluzole (4-12 mg/kg i.p.) dose-dependently attenuated the audiogenic seizures and action myoclonus seen in this animal model. Histological examination using Nissl staining and the novel Fluoro-Jade histochemistry in cardiac-arrested animals showed an extensive neuronal degeneration in the hippocampus and cerebellum. Riluzole treatment almost completely prevented the neuronal degeneration in these brain areas. The neuroprotective effect was more pronounced in hippocampal pyramidal neurons and cerebellar Purkinje cells. These effects were seen at therapeutically relevant doses of riluzole, and the animals tolerated the treatment well. These findings indicate that the pathogenesis of posthypoxic myoclonus and seizure may involve excessive activation of glutamate neurotransmission, and that riluzole may serve as an effective pharmacological agent with neuroprotective potential for the treatment of neurological conditions associated with cardiac arrest in humans.

Neuroprotective effects of the strychnine-insensitive glycine site NMDA antagonist (R)-HA-966 in an experimental model of Parkinson's disease.

The neuroprotective effects of (R)-HA-966 and (S)-HA-966 (3-amino-1-hydroxy-2-pyrrolidinone) were examined in an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced animal model of Parkinson's disease. Systemic pretreatment of C57 black mice with the strychnine-insensitive glycine site antagonist, (R)-HA-966 (3-30 mg/kg, i.p.), dose-dependently attenuated MPTP-induced depletion of striatal dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC). Pretreatment with (R)-HA-966 also significantly protected the degeneration of tyrosine hydroxylase-positive neurons in the substantia nigra of mice treated with MPTP and alleviated the acute behavioral changes caused by the neurotoxin. In contrast, the other racemic form, (S)-HA-966, neither prevented the neurochemical depletions nor the neuronal injury caused by MPTP. These results indicate that excitatory mechanisms of neurodegeneration are involved in the pathophysiology of Parkinson's disease, and that strychnine-insensitive glycine site NMDA antagonists may serve as dopaminoprotective agents which intervene in the progressive neurodegeneration in Parkinson's disease.

Subchronic administration of N-[2-(3,4-dichlorophenyl) ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) alters sigma 1 receptor binding.

BD1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine) is known to bind with high affinity and selectivity to sigma sites in vitro. In prior in vivo studies, it has been shown to attenuate the dystonic postures and orofacial dyskinesias that are produced by sigma receptor ligands, including the neuroleptic haloperidol. Since abnormal movements, such as dystonic postures and orofacial dyskinesias, are side effects that are associated with many sigma-active neuroleptics, compounds such as BD1047 may have therapeutic potential for preventing and treating these unwanted movements. A possible limitation to the therapeutic potential of BD1047, however, is that at least in cell culture and albeit weak, it can be cytotoxic. Therefore, the present study analyzed the possible neurotoxic effects of in vivo subchronic intracerebroventricular infusion of BD1047 (10 nmol/h) or artificial cerebrospinal fluid (CSF) into rat brains using osmotic minipumps for 7 or 14 days. Following a 24 h wash-out period, the animals were killed, the brains removed, and P2 membranes prepared. Membranes from rats treated for 7 or 14 days with BD1047 showed a marked decrease in [3H](+)-pentazocine binding as compared to membranes from CSF-treated animals, suggesting a loss of sigma 1 receptor binding. Histological examination of brain sections processed for Nissl stains and glial fibrillary acidic protein (GFAP) immunohistochemistry excluded the possibility of a cytotoxically induced down-regulation, suggesting possible receptor internalization or desensitization mediated via sigma 1 sites. Under the conditions used in our study, BD1047 does not appear to be neurotoxic, and the data, when taken together with other studies, suggest that BD1047 acts as a partial agonist at sigma sites.

Reactive oxygen species generated by cyanide mediate toxicity in rat pheochromocytoma cells.

Peroxide formation has been implicated in impairment of motor function by cyanide which occurs in both animals and man. The present study employs the neuronal model, rat pheochromocytoma (PC12) cells to evaluate peroxidation as a toxic mechanism of cyanide. Confocal imaging shows that peroxides form within a few seconds in cell cytoplasm after cyanide exposure and continue to accumulate over a period of several minutes. Peroxide generation by cyanide is decreased to about 50% by phospholipase A2 inhibitors indicating involvement of arachidonic acid in the oxidative process. Also antioxidant defense enzymes (CuZn superoxide dismutase and especially catalase) in PC12 cells are inhibited by cyanide. It appears that peroxide accumulation after cyanide treatment involves both inhibition of breakdown and increased production. Furthermore, both peroxide accumulation and cell death induced by cyanide in PC12 cells are blocked by an antioxidant (ascorbate). These data support the hypothesis that the cytotoxic action of cyanide is related in part to an oxidative process.

Novel NMDA/glycine site antagonists attenuate cocaine-induced behavioral toxicity.

N-Methyl-D-aspartate (NMDA)/glycine site antagonists were tested for their ability to prevent cocaine-induced convulsions and lethality in Swiss Webster mice. Pre-treatment of mice with the novel NMDA/glycine site antagonists ACEA-1021 (5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione) or ACEA-1328 (5-nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione) attenuated cocaine-induced convulsions; these effects were pharmacologically antagonized with D-cycloserine. The structurally-related NMDA/glycine site antagonist DCQX (6,7-dichloroquinoxaline-2,3-dione) and the structurally-unrelated NMDA/glycine site partial agonist HA-966 (3-amino-1-hydroxy-2-pyrrolidinone) also attenuated cocaine-induced convulsions, with the R(+)-isomer of HA-966 being more effective than the S(-)-isomer. In contrast, the selective alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor antagonist, NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) , failed to provide statistically significant protection although it shares the 2,3-quinoxalinedione structure of DCQX and the ACEA compounds. Pre-treatment with ACEA-1021, ACEA-1328, DCQX, or R(+)-HA-966 also attenuated cocaine-induced lethality in mice. Significantly, post-treatment with ACEA-1021, immediately prior to or after the onset of seizures, prevented death in up to 86% of mice receiving a lethal dose of cocaine; post-treatment with vehicle resulted in death of all mice. The results suggest the utility of targeting excitatory mechanisms for the treatment of cocaine overdose and offer a novel base structure from which effective pharmacotherapies can be developed.

Cyanide-induced neurotoxicity involves nitric oxide and reactive oxygen species generation after N-methyl-D-aspartate receptor activation.

To study oxidative mechanisms in cyanide toxicity, cyanide-induced generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescence dye 2,7-dichlorofluorescin. KCN produced a concentration-dependent (25-200 microM) generation of intracellular oxidant species that was blocked by N-methyl-D-aspartate receptor antagonists (MK-801 or AP5) or by removal of extracellular Ca++ from the incubation medium. To determine the relative contribution of NO and reactive oxygen species (ROS) to the increase of cellular fluorescence after KCN, a selective inhibitor of nitric oxide synthase, a NO scavenger and enzymes that metabolize ROS were added to the incubation medium. Interference with the nitric oxide system (reduced hemoglobin as a NO scavenger or [N(G)-nitro-L-arginine methyl ester [L-NAME] reduced fluorescence by 50%). Addition of enzymes that metabolize peroxide (catalase or superoxide dismutase [SOD]) also reduced fluorescence by nearly 50%. Combination of SOD with hemoglobin or L-NAME provided additional attenuation of the fluorescence and it was concluded that both NO and ROS are generated concurrently after KCN. Furthermore a correlation was observed between NO and ROS formation and levels of malonaldehyde (MDA), a marker of lipid peroxidation. Pretreatment with MK-801 blocked KCN-induced MDA formation, whereas L-NAME partially diminished MDA production. Treatment with a combination of SOD/catalase and L-NAME blocked the KCN-induced lipid peroxidation. In cytotoxicity studies cyanide-induced cell death was blocked by MK-801, whereas partial attenuation was produced by L-NAME; SOD/catalase treatments did not protect the cells. However, significant protection from cyanide-induced cytotoxicity was observed when L-NAME was combined with SOD/catalase. It is concluded that cyanide activates N-methyl-D-aspartate receptors to simultaneously generate both NO and ROS, which may lead to formation of the cytotoxic peroxynitrite anion.

Antimyoclonic effect of gabapentin in a posthypoxic animal model of myoclonus.

The antimyoclonic property of the novel antiepileptic drug, gabapentin (1-(aminomethyl) cyclohexane acetic acid), was tested in cardiac arrest-and p,p'-DDT(1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane)-induced animal models of myoclonus. Gabapentin dose-dependently attenuated myoclonus in posthypoxic rats for more than 3 h. The drug was also found to be effective in controlling the early stages of seizures following the anoxic insult. In contrast, the drug was ineffective in controlling either myoclonus or seizures in p,p'-DDT-treated animals. These results suggest that gabapentin can be used used as an effective therapeutic agent in an acute hypoxia/ischemia-induced neurological disorder. The data further indicate that distinct neurological mechanisms may be operating in the expression of myoclonus among posthypoxic and p,p'-DDT-induced animal models.

Antagonism of cyanide toxicity by isosorbide dinitrate: possible role of nitric oxide.

In a search for improved cyanide antidotes, the efficacy of isosorbide dinitrate (ISDN), was compared with that of the known cyanide antidote, NaNO2. ISDN was as effective as an optimal dose of NaNO2 in protecting mice against cyanide lethality. To study the mechanism involved, the extent of formation of the cyanide scavenger, methemoglobin, in the action of ISDN was determined. ISDN (300 mg/kg, p.o.) increased methemoglobin from 5 to 10% of total hemoglobin, while, in contrast, NaNO2 (100 mg/kg, i.p.) increased methemoglobin levels to 50% of total hemoglobin. Lowering the dose of NaNO2 to 30 mg/kg reduced methemoglobin levels to approximately 10% of total hemoglobin and in turn nearly abolished its antidotal effect. Decreasing methemoglobin to less than control levels using methylene blue failed to abolish cyanide antagonism by ISDN. Thus, methemoglobin formation by ISDN does not account for its antidotal action. Further studies comparing the respiratory depressant effects of cyanide in the presence of ISDN or NaNO2 also indicated that these two antidotes have different mechanisms of action. Efforts to produce tolerance to the antidotal effect of ISDN against cyanide toxicity were unsuccessful. It is suggested that the well-known ability of ISDN to generate nitric oxide may account for the noted cyanide antagonism.