Acute cyanide toxicity is attributed to inhibition of cytochrome c oxidase (CcOX), the oxygen-reducing component of mitochondrial electron transport; however, the mitochondrial action of cyanide is complex and not completely understood. State-3 oxygen consumption and CcOX activity were studied in rat N27 mesencephalic cells to examine the functional interaction of cyanide and nitric oxide (NO). KCN produced a concentration-dependent inhibition of cellular respiration. Cyanide's median inhibitory concentration (IC50) of oxygen consumption (13.2 +/- 1.8microM) was higher than the CcOX IC50 (7.2 +/- 0.1microM). Based on respiratory threshold analysis, 60% inhibition of CcOX was necessary before oxygen consumption was decreased. Addition of high levels of exogenous NO (100microM S-nitroso-N-acetyl-DL-penicillamine) attenuated cyanide inhibition of both respiration and CcOX. On the other hand, when endogenous NO generation was blocked by an NOS inhibitor (N(omega)-monomethyl-L-arginine ester), the cyanide IC50 for both respiration and CcOX increased to 59.6 +/- 0.9microM and 102 +/- 10microM, respectively, thus showing constitutive, low-level NO production enhanced cyanide inhibition. Laser scanning cytometry showed that cyanide elevated mitochondrial NO, which then was available to interact with CcOX to enhance the inhibition. It is concluded that the rapid, potent action of cyanide is due in part to mitochondrial generation of NO, which enhances inhibition of CcOX. At low mitochondrial oxygen tensions, the cyanide-NO interaction would be increased. Also, the antidotal action of sodium nitrite is partly explained by generation of high mitochondrial levels of NO, which antagonizes the CcOX inhibition.

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 (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 M) and KCN (100 -500 M) 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 M) and/or KCN (100 M) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60 -70% of the cells from DAand 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 N G -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 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 lM) treatment for 24 h, two pro-apoptotic Bcl-2 proteins, Bcl-X S 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-X L , remained unchanged after cyanide treatment, whereas the mRNA levels of Bcl-X S and Bax began to increase within 2 h and their protein levels increased 6 h after treatment. NF-jB, a redox-sensitive transcription factor activated after cyanide treatment, is responsible for the up-regulation of Bcl-X S and Bax. SN50, which is a synthetic peptide that blocks translocation of NF-jB from cytosol to nucleus, inhibited the up-regulation of Bcl-X S and Bax. Similar results were obtained using a specific jB decoy DNA. NMDA receptor activation and reactive oxygen species (ROS) generation are upstream events of NF-jB activation, as blockade of these two events by MK801, L-NAME or PBN inhibited cyanide-induced up-regulation of Bcl-X S and Bax. Up-regulation of pro-apoptotic Bcl-X S 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-jB-mediated up-regulation of Bcl-X S and Bax is involved in regulating cytochrome c release in cyanideinduced apoptosis.