Gary E. Isom

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.

Cyanide-induced neurotoxicity: Role of neuronal calcium

The effect of cyanide on whole-brain calcium levels was determined in mice administered KCN and correlated with the neurotoxic signs manifested during acute cyanide poisoning. KCN (10mg/kg, sc) significantly increased whole-brain total calcium levels from 48.1 +/- 1.8 to 66.5 +/- 3.9 micrograms/g dry wt within 15 min after administration. The levels remained elevated for 3 hr and returned to control readings after 12 hr. Dose-response studies revealed KCN, at doses of 10-15 mg/kg, produced significant elevations of whole-brain calcium 30 min after administration. No measurable effect was obtained from lower doses which suggested a threshold effect. Pretreatment 15 min before KCN with diltiazem, a calcium channel blocker, prevented the cyanide-induced rise in whole-brain total calcium. Cyanide-induced tremors, which are centrally mediated symptoms of intoxication, were quantified and correlated with the observed changes in whole-brain calcium. Tremors were detected at 10 and 12 mg/kg KCN and peak intensity was observed at 15 min postcyanide. Pretreatment with diltiazem markedly attenuated the cyanide-induced tremors. It appears that a correlation exists between cyanide-induced change in whole-brain calcium and tremors. This study suggests that intraneuronal calcium may play an important role in mediating cyanide neurotoxicity and calcium channel blocking agents may be useful in limiting the severity of the centrally mediated symptoms of acute cyanide intoxication.

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.

Cyanide-Induced Apoptosis and Oxidative Stress in Differentiated PC12 Cells

Abstract: Terminally differentiated PC12 cells are a useful neuron‐like model for studying programmed cell death in response to nerve growth factor (NGF) deprivation. This in vitro model was used to investigate the mechanism by which cyanide‐induced histotoxic hypoxia produces neuronal degeneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN for 24 h did not produce cell death. In contrast, treatment of differentiated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blue dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg2+‐dependent endonuclease inhibitor aurintricarboxylic acid and the transcriptional inhibitor actinomycin D partially attenuated hypoxic toxicity, suggesting roles for endonuclease activation and transcription in this model of neuronal death. Extracted DNA from cyanide‐treated neurons demonstrated cleavage into oligonucleosomal fragments on gel electrophoresis. Transmission electron microscopic analysis showed morphological changes consistent with apoptotic cell death, including membrane blebbing and convolution, as well as chromatin condensation and margination to the nuclear membrane. Addition of either ascorbate or catalase to the cultures partially attenuated the loss of cell viability induced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The ability of cyanide to elevate intracellular oxidant species was determined by microfluorescence in differentiated PC12 cells loaded with the oxidant‐sensitive dye 2′,7′‐dichlorofluorescin. Exposure of cells to 0.1 mM KCN produced a rapid generation of oxidants that was blocked ∼50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undifferentiated, PC12 cells, and that antioxidants significantly reduce the incidence of cyanide‐induced apoptosis.

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.

Effects of oxygen on the antagonism of cyanide intoxication: cytochrome oxidase, in vitro.

Since oxygen was reported to be an effective cyanide antagonist in vivo, particularly in the presence of the classic antidotal combination of sodium nitrite and sodium thiosulfate, in vitro studies were initiated in an attempt to investigate the mechanism of oxygen-mediated cyanide antagonism. The effect of oxygen on cyanide-inhibited cytochrome oxidase with and without cyanide antagonist(s) was investigated in a purified membraneous enzyme system prepared from rat liver mitochondria. Cyanide produced a concentration dependent inhibition of cytochrome oxidase, and 100% oxygen did not alter the inhibition produced by KCN either in the presence or absence of sodium thiosulfate. However, the addition of sodium thiosulfate and rhodanese to the assay reactivated the cyanide-inhibited cytochrome oxidase. Kinetic analysis indicated rhodanese competes with cytochrome oxidase for cyanide, and oxygen had no effect on this coupled reaction. In conclusion, the in vivo antidotal properties of oxygen cannot be attributed to oxygen-mediated reactivation of cyanide-inhibited cytochrome oxidase or an oxygen-mediated acceleration of rhodanese detoxification.

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.

Alteration of cytosolic calcium levels in pc12 cells by potassium cyanide

The effect of KCN on cytosolic Ca2+ levels was measured in PC12 cells using Quin II/AM, a fluorometric calcium indicator. The resting cytosolic Ca2+ concentration, 115.0 +/- 4.9 nm, increased gradually and steadily over a 30-min time period following addition of 10(-4), 10(-3), or 10(-2)M KCN to the cells. After 15 min, 10(-3) and 10(-2)M KCN produced a three- and sixfold increase in the cytosolic Ca2+ concentration, respectively. In K+-depolarized cells, KCN induced a more rapid rise of intracellular calcium than in cells treated with KCN or KCI alone. KCN and/or K+-induced accumulation of cytosolic Ca2+ was blocked when the cells were pretreated with 10(-5)M diltiazem (a calcium channel blocker). These results demonstrate in a cell model that cyanide induces an accumulation of cytosolic Ca2+ and this additional cytosolic Ca2+ load appears to originate primarily from the extracellular compartment. This study supports previous reports implicating calcium as an intracellular mediator of cyanide toxicity.

Peroxidation of brain lipids following cyanide intoxication in mice

Lipid peroxidation of brain lipids as determined by the conjugated diene method was observed in mice following administration of sublethal doses of potassium cyanide (KCN). Conjugated diene production was dose- and time-dependent; 10 mg/kg KCN produced detectable levels of conjugated dienes at 30 min post cyanide, whereas, 15 mg/kg produced marked levels of conjugated dienes over a 10-60-min period after KCN. Pretreatment of mice with either diltiazem (600 micrograms/kg, i.v.) or allopurinol (25 mg/kg, i.v.) blocked the generation of conjugated dienes. These results suggest lipid peroxidation of neuronal membranes play a role in cyanide intoxication and this action is related to altered regulation of neuronal calcium homeostasis and activation of xanthine oxidase.