Joseph L. Borowitz

NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant‐sensitive fluorescent dye 2,7‐dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ∼70% by MK‐801 (a noncompetitive NMDA‐receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with NG‐nitro‐l‐arginine methyl ester (l‐NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O2−· andH2O2. Concurrent inhibition of O2−· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A2 inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca2+‐free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate‐induced death and oxidant generation. Glutamate‐induced cytotoxicity was blocked by MK‐801 and attenuated by treatment with l‐NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non‐NMDA receptors through a Ca2+‐mediated process. Activation of NO synthase and phospholipaseA2 contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.

Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells.

Abstract: Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA‐induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson‐like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12‐36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100‐500 μM) and KCN (100‐500 μM) induced a concentration‐ and time‐dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end‐labeling) staining, was also concentration‐ and time‐dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA‐induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 μM) and/or KCN (100 μM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60‐70% of the cells from DA‐ and DA plus KCN‐induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA‐ and KCN‐induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA‐induced apoptosis. The nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide‐mediated oxidative stress as an initiator of apoptosis.

Brain lipid peroxidation and antioxidant protectant mechanisms following acute cyanide intoxication.

The status of brain antioxidant enzymes and glutathione levels in mice intoxicated with KCN were correlated with lipid peroxidation in brain membranes. KCN (7 mg/kg, s.c.) rapidly increased conjugated dienes in brain lipids, with peak levels observed 30 min after cyanide treatment. At 60 min post cyanide, conjugated diene levels were only slightly elevated above controls. Temporal changes in activity of most antioxidant enzymes corresponded with the observed time course of cyanide-induced membrane lipid peroxidation. Thirty minutes after KCN, brain catalase (CA), glutathione peroxidase (GPX) and glutathione reductase (GR) activities were significantly reduced (percent inhibition compared to control: CA 44%, GPX 30%, and GR 41%). At 60 min, CA and GPX enzyme activity returned to control levels, whereas GR was elevated 34% above control activity. Superoxide dismutase was not significantly inhibited 30 min after KCN, but declined to 71% of control activity at 60 min. Brain levels of reduced glutathione declined 42% below control 30 min after cyanide and returned to within 9.4% of control at 60 min. At 30 and 60 min after cyanide, oxidize glutathione levels were not significantly changed from control levels. These studies suggest that membrane lipid peroxidation and subsequent membrane dysfunction observed in cyanide intoxication is related in part to a compromised antioxidant defense.

Monitoring intracellular nitric oxide formation by dichlorofluorescin in neuronal cells

A method for rapid fluorometric assay of intracellular nitric oxide (NO) formation was developed for use in cultured neuronal cells. In a cell-free system 2,7-dichlorofluorescin (DCF), a non-fluorescent species, is oxidized by NO to dichlorofluorescein, a fluorescent compound. Addition of NO to a solution containing DCF increased the fluorescent signal within 10 s and continued to increase slowly over a 10-min period. The intensity of the fluorescence was dependent upon the concentration of NO. In DCF-loaded PC12 cells, addition of NO markedly increased fluorescence (limit of detection = 16 microM NO) and pretreatment with reduced hemoglobin (Hb) inhibited the NO-mediated increase of fluorescence in both the cell-free system and PC12 cells. In PC12 cells loaded with DCF, the NO generator sodium nitroprusside (SNP) produced a rapid increase of fluorescence. To rule out the possibility that reactive oxygen species (ROS) mediated the increased of fluorescence, superoxide dismutase (SOD) and catalase were added to the cuvette. The enzymes did not alter the fluorescence generated after addition of NO to PC12 cells. This assay was used to determine the ability of glutamate to stimulate NO production in cerebellar granule cells. When 10 microM glutamate was added to DCF-loaded cerebellar granule cells, a rapid increase in fluorescence was noted. The fluorescence was blocked approximately 50% after addition of either Hb or SOD, or by pretreatment with NG-nitro-L-arginine methyl ester (300 microM), a nitric oxide synthase (NOS) inhibitor. It was concluded that glutamate stimulated intracellular generation of both NO and ROS, and at least 50% of the oxidation of DCF was attributed to intracellular generation of NO. These results demonstrate that oxidation of DCF by NO can be used to measure intracellular generation of NO and by adding either Hb or SOD to the cell system, the extent of oxidation of DCF attributed to NO and ROS can be determined.

NF‐κB‐mediated up‐regulation of Bcl‐XS and Bax contributes to cytochrome c release in cyanide‐induced apoptosis

Cyanide induces apoptosis through cytochrome c activated caspase cascade in primary cultured cortical neurons. The underlying mechanism for cytochrome c release from mitochondria after cyanide treatment is still unclear. In this study, the roles of endogenous Bcl‐2 proteins in cyanide‐induced apoptosis were investigated. After cyanide (100–500 µm) treatment for 24 h, two pro‐apoptotic Bcl‐2 proteins, Bcl‐XS and Bax were up‐regulated as shown by western blot and RT‐PCR analysis. The expression levels of two antiapoptotic Bcl‐2 proteins, Bcl‐2 and Bcl‐XL, remained unchanged after cyanide treatment, whereas the mRNA levels of Bcl‐XS and Bax began to increase within 2 h and their protein levels increased 6 h after treatment. NF‐κB, a redox‐sensitive transcription factor activated after cyanide treatment, is responsible for the up‐regulation of Bcl‐XS and Bax. SN50, which is a synthetic peptide that blocks translocation of NF‐κB from cytosol to nucleus, inhibited the up‐regulation of Bcl‐XS and Bax. Similar results were obtained using a specific κB decoy DNA. NMDA receptor activation and reactive oxygen species (ROS) generation are upstream events of NF‐κB activation, as blockade of these two events by MK801, l‐NAME or PBN inhibited cyanide‐induced up‐regulation of Bcl‐XS and Bax. Up‐regulation of pro‐apoptotic Bcl‐XS and Bax contributed to cyanide‐induced cytochrome c release, because SN50 and a specific Bax antisense oligodeoxynucleotide significantly reduced release of cytochrome c from mitochondria as shown by western blot analysis. It was concluded that NF‐κB‐mediated up‐regulation of Bcl‐XS and Bax is involved in regulating cytochrome c release in cyanide‐induced apoptosis.

Cyanide-induced lipid peroxidation in different organs: subcellular distribution and hydroperoxide generation in neuronal cells

To evaluate hydroperoxide generation as a potential mechanism of cyanide neurotoxicity, mice were treated with KCN (7 mg/kg, subcutaneously (s.c.)) and the level of lipid peroxidation (expressed as conjugated dienes) was measured later in various organs. Brain showed elevated conjugated diene levels after cyanide but the liver, which is not considered a target for cyanide toxicity, showed no increase. The heart also showed no increase, whereas kidney conjugated dienes slowly increased to a peak 1 h after cyanide. In vitro studies show elevation of peroxidized lipids in mouse brain cortical slices following incubation with KCN (0.1 mM). Omission of calcium from the medium or pretreatment of brain slices with diltiazem (a calcium channel blocker) prevented formation of conjugated dienes by KCN. Calcium thus appears to play a critical role in cyanide-induced generation of peroxidized lipids in neuronal cells. Subcellular fractionation of brains from mice treated with cyanide showed that lipid peroxidation increased in the microsomal fraction but not in the mitochondrial fraction. Fluorescent studies using 2,7-dichlorofluorescein (a hydroperoxide sensitive fluorescent dye) show that hydroperoxides are generated rapidly after cyanide treatment of PC12 cells, a neuron-like cell, and hydroperoxide levels remain elevated for many minutes in the presence of cyanide. These results suggest that hydroperoxide generation with subsequent peroxidation of lipids may lead to changes in structure and function of certain membranes and contribute to the neurotoxic damage produced by cyanide.

Actions of GABA, picrotoxin and bicuculline on adrenal medulla

Abstract GABA releases catecholamines from isolated perfused bovine adrenal glands. The secretory response is dependent on calcium and independent of acetylcholine. Tachyphylaxis to the releasing effect is observed. Picrotoxin inhibits the effect of acetylcholine and the effect of low but not high concentrations of GABA. Bicuculline, another reported GABA antagonist, blocks the effect of all concentrations of GABA but not the effect of acetylcholine. Bicuculline is therefore a more specific GABA blocker in adrenal medulla than picrotoxin. These findings suggest the existence of GABA receptors in adrenal medulla which may be analogous in some respects to those reported in autonomic ganglia.

BNIP3 mediates cell death by different pathways following localization to endoplasmic reticulum and mitochondrion

BNIP3 (Bcl‐2/adenovirus E1B 19–kDa interacting protein 3) is a BH3‐only proapoptotic member of the Bcl‐2 family. Because the interaction of Bcl‐2 proteins with intracellular Ca2+ stores has been linked to apoptosis, the role of Ca2+ transfer between endoplasmic reticulum (ER) and mitochondria in BNIP3mediated cell death was determined in a rat dopaminergic neuronal cell line, Mes 23.5. BNIP3 mutants were constructed to target either ER or mitochondria. Localization of BNIP3 to the ER membrane facilitated release of Ca2+ and subsequently increased uptake of Ca2+ into mitochondria. Excessive accumulation of mitochondrial Ca2+ decreased mitochondrial membrane potential (ΔΨm, resulting in execution of a caspaseindependent cell death. Reduction of ER Ca2+ induced by ER‐targeted BNIP3 and the subsequent cell death was blocked by the antiapoptotic protein, Bcl‐2. On the other hand, mitochondria‐targeted BNIP3 initiated apoptosis by aCa2+‐independent mechanism by inducing mitochondrial pore transition and dissipation of ΔΨm. The disruption of and cell death was not blocked by Bcl‐2 overexpression. These findings show that BNIP3 undergoes a dual subcellular localization and initiates different cell death signaling events in the ER and mitochondria. Bcl‐2 counters the BNIP3‐initiated mobilization of ER Ca2+ depletion to reduce the level of apoptosis.—Zhang, L., Li, L., Liu, H., Borowitz, J. L., Isom, G. E. BNIP3 mediates cell death by different pathways following localization to endoplasmic reticulum and mitochondrion. FASEB J. 23, 3405–3414 (2009). www.fasebj.org

Cyanide enhancement of dopamine-induced apoptosis in mesencephalic cells involves mitochondrial dysfunction and oxidative stress

Dopamine (DA)-induced neurotoxicity is potentiated when cellular metabolism is compromised. Since cyanide is a neurotoxin that produces mitochondrial dysfunction and stimulates intracellular generation of reactive oxygen species (ROS), KCN was used to study DA-induced apoptosis in primary cultured mesencephalon cells. Treatment of neurons with DA (300 microM) for 24h produced apoptosis as determined by TUNEL staining, DNA fragmentation and increased caspase activity. Pretreatment with KCN (100 microM) 30min prior to DA increased the number of cells undergoing apoptosis. When added to the cells alone, this concentration of KCN did not induce apoptosis. DA stimulated intracellular generation of ROS, and treatment with KCN enhanced ROS generation. Treatment of cells with glutathione or uric acid (antioxidants/scavengers) attenuated both the increase in ROS generation and the apoptosis, demonstrating that ROS are initiators of the cytotoxicity. Studies on the sequence of events mediating the response showed that DA-induced depolarization of the mitochondrial membrane was dependent on ROS generation and KCN enhanced this action of DA. Following changes in mitochondrial membrane potential, cytochrome c was released from mitochondria, leading to caspase activation and eventually cell death. These results demonstrate that oxidative stress and mitochondrial dysfunction are initiators of DA-induced apoptosis. Subsequent cytochrome c release activates the caspase effector component of apoptosis. Cyanide potentiates the neurotoxicity of DA by enhancing the generation of ROS and impairing mitochondrial function.

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.