Various Nitric Oxide Donors Protect Chick Embryonic Neurons from Cyanide-Induced Apoptosis

Mads Skak Jensen*,1, Niels Chresten Berg Nyborg{dagger} and Erling Sonnich Thomsen*

* Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark; and {dagger} Safety Pharmacology, Drug Safety, Health Care, Research and Development, Preclinical Development, Novo Nordisk A/S, Denmark

Received May 25, 2000; accepted August 1, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The discovery of numerous biochemical effects of cyanide not directly related to the inhibition of the respiratory chain, including the involvement of apoptosis, has challenged the basis of traditional antidote treatment, which primarily depends on nitrite-induced conversion of hemoglobin into methemoglobin, releasing the blockade of cytochrome c oxidase by high-affinity binding of cyanide as cyanmethemoglobin. The fact that amyl nitrite has antidotal effects not related to methemoglobin formation has unfolded new mechanism of actions of nitrites including release of nitric oxide (NO). In this study, we characterized the effect of various NO donor compounds on cyanide-induced cell death in cultured chick embryonic neurons. Apoptosis was induced by treating the neuronal cultures with 1 mM NaCN for 1 h, followed by a cyanide-free incubation period of 23 h. Using this treatment protocol, we showed that cyanide-induced apoptosis was blocked in the presence of the different NO donors sodium nitroprusside, S-nitrosoglutathione, S-nitroso-N-acetylpenicillamin, nitroglycerin, 3-morpholinosydnonimine, and diethylamine nitric oxide, indicating independence of the redox-related species of NO released. The effect was confirmed to be mediated by NO, since exhausted NO donors did not afford protection, and the mechanism likely involved chemical modification of thiol groups, since the effect was completely reversed by dithiothreitol. Furthermore, NMDA antagonists protected against cyanide-induced cell death, whereas inhibitors of nitric oxide synthase increased cyanide-induced apoptotic damage, indicating a protective effect of endogenously generated NO, at least in cell cultures.

Key Words: apoptosis; cyanide; nitric oxide; neurons; neuroprotection.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The toxic effects of cyanide have traditionally been attributed to inhibition of cytochrome c oxidase, the terminal enzyme of the respiratory chain, which compromises oxidative phosphorylation leading to cytotoxic hypoxia. In the last decade, numerous other effects of cyanide have been identified, including enhancement of NMDA receptor responses (Arden et al., 1998Go; Patel et al., 1993Go; Sun et al., 1997Go, 1999Go), elevation of the extracellular glutamate concentration (Patel et al., 1991Go; Zeevalk and Nicklas, 1992Go), activation of voltage-sensitive calcium channels (Johnson et al., 1987Go), and mobilization of calcium from intracellular stores (Yang et al., 1996Go). Furthermore, the involvement of apoptosis in cyanide-induced neuronal death has recently been identified, characterized by apoptotic nuclear changes and dependence of protein synthesis (Jensen et al., 1999Go; Mills et al., 1996Go). The new facets of cyanide toxicity expand the number of potential antidote candidates and help to explain some of the peculiarities of standard antidote treatment. Traditionally, antidote treatment consists of administration of nitrites, i.e., sodium nitrite and amyl nitrite, and sodium thiosulphate, the substrate of rhodanese, which converts cyanide to thiocyanate. The general action of nitrites is believed to be a conversion of hemoglobin into methemoglobin, which subsequently binds cyanide as cyanmethemoglobin with high affinity, thereby releasing cyanide from cytochrome c oxidase. Studies have shown that amyl nitrite has antidotal effects before plasma methemoglobin reaches clinically effective levels, implying that amyl nitrite has additional effects not related to methemoglobin formation (Vick and Froehlich, 1985Go).

Amyl nitrite releases nitric oxide (NO), which is an important mediator and messenger in the cardiovascular system as well as in the central nervous system. NO activates guanylate cyclase, leading to an increase in the concentration of cyclic GMP and resulting in vascular relaxation (reviewed by Hobbs, 1997). It acts as a neurotransmitter, as well as enhancing the release of glutamate and norepinephrine in the central nervous system (reviewed by Dawson and Dawson, 1996; Oh, 1995). NO has been attributed a myriad of roles ranging from cytotoxic, i.e., by inhibiting the respiratory chain (Brown, 1995Go) and contributing to neuronal injury following NMDA receptor overstimulation (Dawson and Dawson, 1996Go) to cytoprotective properties as described below.

NO inhibits NMDA receptor responses (Hoyt et al., 1992Go; Lei et al., 1992Go), supposedly by affecting redox-sensitive sites on one or more NMDA receptor subunits, but the mechanism is still under debate (Aizenman et al., 1998Go; Aizenman and Potthoff, 1999Go; Choi et al., 2000Go). Additionally, NO reduces apoptotic damage either by inhibiting caspases by S-nitrosylation (Haendeler et al., 1997Go; Kim et al., 1997Go; Tenneti et al., 1997Go) or by upregulating the level of the antiapoptotic protooncogene Bcl-2 (Genaro et al., 1995Go; Suschek et al., 1999Go). Thus, the antidotal effect of amyl nitrite could be mediated by one or both of the cytoprotective effects of NO described above, which was supported by studies in which donors of NO provided protection from cyanide toxicity independently of methemoglobin formation (Baskin et al., 1996Go; Sun et al., 1995Go). Conceivably, NO donors in general antagonize the toxic effects of cyanide, but different NO donors cannot be expected to provide identical effects, since the biological effects depend on the redox-related species of NO released; nitric oxide (NO), nitrosonium ion (NO+), or nitroxyl anion (NO) (Lipton et al., 1998Go; Stamler et al., 1992Go).

The aim of this study was to characterize the effects of NO donors with different modes of NO release on cyanide-induced cell death in primary cultures of chick embryonic neurons. In addition, we examined the effects of reducing agents, nitric oxide-synthase inhibitors, and glutamate receptor antagonists on cyanide-induced toxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Gibco BRL, Life Technologies, Denmark. Poly-L-lysine (Mw 70,000–150,000), bisbenzimide (Hoechst 33258), sodium nitroprusside, penicillin G, L-glutamate and trypan blue were from Sigma Chemicals, Denmark. NaCN, dithiothreitol (DTT), and streptomycin sulphate were purchased from Merck, KEBO Lab, Denmark. Diethylamine nitric oxide (DEANO), NG-nitro-L-arginine methyl ester (L-NAME), S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamin (SNAP), and 3-morpholinosydnonimine (SIN-1) were from Molecular Probes Europe, The Netherlands. Kynurenic acid was from Alexis Corporation, Switzerland and nitroglycerin (NTG) was from H-S Apoteket, Rigshospitalet, Denmark.

Cell culture.
Chick neurons were prepared as described by Pettmann et al. (1979). Briefly, telencephalons from 7-day-old chicken embryos were removed and the cerebral hemispheres were disaggregated by sieving through 48-µm nylon mesh. The cell suspension was seeded in poly-L-lysine-coated culture flasks (25 cm2) at a density of 2 x 105 cells/cm2. Cells were maintained in DMEM supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO2. Medium was changed after 3 days in vitro. Cells cultured by this method were reported to contain 98% neurons (Pettmann et al., 1979Go). Cells were used for experiments on day 5, in vitro.

Drug exposure.
Apoptosis was induced by treating the cells with 1 mM NaCN for 1 h, followed by 23 h of recovery to allow sufficient time for execution of the apoptotic program, similar to the method of Jensen et al. (1999). This protocol resulted in approximately 50% cell death. Similarly, cells were treated with 1 mM L-glutamate for 1 h followed by a recovery period of 23 h. When the effect of NO donors were tested, cells were incubated with 1 mM NaCN for 1 h in the presence of freshly prepared NO donor, followed by a 23-h recovery. In the experiments in which the NOS inhibitor L-NAME or the NMDA antagonist kynurenic acid was used, cells were treated with 1 mM NaCN or 1 mM L-glutamate, together with L-NAME or kynurenic acid, for 1 h, followed by a 23-h incubation with L-NAME or kynurenic acid alone.

When the effect of DTT was examined, cells were treated with NO donor or medium for 1 h, after which the medium was removed. Immediately afterward, the cells were incubated with DTT or medium for 30 min and, after medium removal, incubated with 1 mM NaCN/L-glutamate or medium for 1 h, followed by 23-h recovery.

To correlate the in vitro concentration of NaCN used in this study with in vivo data, it may be mentioned that post-mortem brain concentrations following fatal cyanide poisoning in humans have been reported to be as high as 140 µM CN (Hall et al., 1987Go).

NO donors.
Sodium nitroprusside (SNP) belongs to the NO donor class of iron nitrosyls. The mechanism of NO release remains incompletely understood (Feelisch, 1998Go) but SNP is thought to primarily release NO and NO+ equivalents (Feelisch and Stamler, 1996Go).

Diethylamine nitric oxide (DEANO) belongs to the NO donor class of NONOates. It generates NO spontaneously with a half-life of 2.1-min (37°C, pH 7.4).

S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamin (SNAP) belong to the NO donor class of S-nitrosothiols, which can act as donors of NO, NO, or NO+ depending on redox conditions (Feelisch and Stamler, 1996Go).

3-Morpholinosydnonimine (SIN-1) belongs to the NO donor class of sydnonimines. They liberate NO spontaneously, and during the breakdown, superoxide anions (O2•–) are formed, some of which inevitably will react with NO to form peroxynitrite (ONOO) (Feelisch and Stamler, 1996Go).

Nitroglycerin (NTG) belongs to the NO donor class of organic nitrates. Enzymatic or non-enzymatic bioactivation is required for NO release to occur (Feelisch, 1998Go). NTG is known to react readily with thiol groups forming derivative thionitrites (RS-NO) or thionitrates (RS-NO2)(Lipton et al., 1998Go).

Amyl nitrite was not included due to its very slight solubility in water.

Assessment of cell death and apoptosis.
Neuronal cell death was assessed by phase-contrast microscopy (Nikon TMS) using the trypan blue exclusion assay. The medium was removed and the cells were incubated for 5 min in a 0.4% trypan blue solution. The trypan blue solution was removed and the cells were washed with phosphate-buffered saline (PBS; 130 mM NaCl, 10 mM Na2HPO4, and 10 mM NaH2PO4, pH 7.4) at 37°C and immediately assessed for cell viability by counting viable (nonstained) and non-viable (blue) cells. Neuronal viability was expressed as the percent ratio of viable to the total number of neurons counted. The experiments were reproduced independently in 2 cultures with 2 flasks per treatment. Eight microscopic fields and approximately 300 cells were counted per flask in trypan blue staining.

The percentage of apoptotic neurons was assessed by Hoechst-33258 staining. The medium was removed and the cells were incubated in 10 µg/mL Hoechst-33258 in methanol for 15 min, after which the cells were washed in methanol and PBS was added. Nuclear morphology was observed under a fluorescence microscope (Leica DM IRB). Viable cells showed a normal nucleus and chromatin, whereas apoptotic cells showed condensed chromatin with a very intense fluorescence or nuclear fragmentation. Neuronal apoptosis was expressed as the percent ratio of apoptotic neurons to total number of neurons counted. The experiments were reproduced independently in 2 cultures, with 2 flasks per treatment. Nine microscopic fields and approximately 500 cells were counted per flask in Hoechst-33258 staining.

Statistics.
Data are presented as means ± standard deviation (SD). Statistical comparisons were made by one-way ANOVA or Kruskal-Wallis one-way ANOVA on ranks followed by Student-Newman-Keuls multiple comparison procedure.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To evaluate the neuroprotective effect of NO donors on cyanide-induced cell death, chick neurons were exposed to 1 mM NaCN in combination with various NO donor compounds for 1 h, followed by a 23-h recovery. Cell death was subsequently characterized by the trypan blue-exclusion assay and nuclear staining with Hoechst 33258 (Fig. 1A–1FGoGo). Membrane damage and apoptotic nuclear changes developed similarly irrespective of treatment.



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FIG. 1. Effect of NO donors on cyanide-induced neurotoxicity. Chick embryonic neurons were exposed for 1 h to 1 mM NaCN alone or in the presence of increasing concentrations of NO donor, followed by a 23-h recovery. (A) Sodium nitroprusside (SNP); (B) S-nitrosoglutathione (GSNO); (C) S-nitroso-N-acetylpenicillamin (SNAP); (D) nitroglycerin (NTG); (E) 3-morpholinosydnonimine (SIN-1); and (F) diethylamine nitric oxide (DEANO). Cell death was evaluated by trypan blue-exclusion assay (open bars) and nuclear staining with Hoechst 33258 (solid bars), and was expressed as the percent ratio of damaged neurons to the total number of neurons. Data are means ± SD (n = 4 if not otherwise indicated). ***p < 0.001, **p < 0.01, *p < 0.05, significantly different from NaCN-induced injury alone, using ANOVA followed by Student-Newman-Keuls multiple comparisons procedure, except for the trypan blue-exclusion assay in F, where *p < 0.05 is significantly different from NaCN-induced injury alone, using Kruskal-Wallis ANOVA on ranks, followed by Student-Newman-Keuls multiple-comparisons procedure.

 
The percentage of damaged neurons in cultures treated with 1 mM NaCN in combination with sodium nitroprusside (SNP, 500 nM–50 µM) was lower than in cultures treated with NaCN alone (Fig. 1AGo). The protective effect at 10 µM SNP was significant both when using membrane damage (p < 0.001) and apoptotic nuclear changes (p < 0.01) as endpoints. 5 and 50 µM SNP also provided significant protection (p < 0.001 and p < 0.01, respectively) when using the trypan blue-exclusion assay. If the concentration of SNP was raised to 250 µM, a significantly higher toxicity was observed (p < 0.01) compared to NaCN alone, due to the toxic effects of NO.

Protective effects of some concentrations were also observed using S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamin (SNAP), nitroglycerin (NTG), 3-morpholinosydnonimine (SIN-1), or diethylamine nitric oxide (DEANO) as NO donors. Treatment with NO donors alone did not induce neuronal death (data not shown). GSNO-reduced cyanide-induced toxicity in all concentrations tested (1–100 µM, p < 0.001), but the protective effect decreased with increasing concentrations of NO donor (Fig. 1BGo). A similar pattern was observed for DEANO (10–250 µM, p < 0.05; Fig. 1FGo). When SNAP was used, the maximal effect was observed using 10 µM (p < 0.01), but also 1 and 100 µM SNAP produced significant protection (p < 0.001 and p < 0.05, respectively) when apoptotic nuclear change was used as endpoint (Fig. 1CGo).

The protective effect of NTG increased with increasing concentrations (1–50 µM, p < 0.05; Fig. 1DGo), but 50 µM was the highest concentration we were able to test because NTG was dissolved in ethanol. One µM SIN-1 afforded protection (p < 0.001) and in Hoechst 33258 staining also 10 µM (p < 0.01), whereas 100 µM did not change the toxicity of 1 mM NaCN significantly.

In order to ensure that the effects were mediated by NO, experiments with exhausted NO donors were performed. The NO donor-containing solution was allowed to be in direct contact with air and exposed to light overnight which would allow NO to exhaust. Additionally, potassium ferrocyanide and potassium ferricyanide were tested because of the similarity with expired SNP. Neither expired NTG, GSNO, SIN-1, SNP, nor potassium ferrocyanide or potassium ferricyanide reproduced the protective effects of the NO-containing compounds (Fig. 2Go).



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FIG. 2. Effect of exhausted NO donors on cyanide-induced neurotoxicity. Chick embryonic neurons were exposed for 1 h to 1 mM NaCN, alone or in the presence of exhausted NO donors in the maximal protective concentration, followed by a 23-h recovery. (A) Nitroglycerin (NTG), S-nitrosoglutathione (GSNO), and 3-morpholinosydnonimine (SIN-1); (B) sodium nitroprusside (SNP), potassium ferrocyanide, and potassium ferricyanide. Cell death was evaluated by trypan blue-exclusion-assay (open bars) and nuclear staining with Hoechst 33258 (solid bars), and was expressed as the percent ratio of damaged neurons to the total number of neurons. Data are means ± SD (n = 4). No difference was observed from NaCN-induced injury alone using ANOVA followed by Student-Newman-Keuls multiple-comparisons procedure.

 
To investigate whether endogenously generated NO played a role in the antagonism of cyanide toxicity, the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was tested. When the effect was evaluated by Hoechst 33258 staining, a significant increase in the toxicity was observed when cells were treated with NaCN in the presence of L-NAME (p < 0.001), indicating a protective role of endogenously generated NO (Fig. 3Go). When membrane damage was used as the endpoint, no protection was observed. If the protective effects of NO donors were mediated by inhibition of the NMDA receptor, a similar effect would be expected when cells were treated with L-glutamate. This was not observed in the present study.



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FIG. 3. Effect of NOS inhibitor on cyanide- and glutamate-induced neurotoxicity. Chick embryonic neurons were exposed for 1 h to 1 mM NaCN or 1 mM L-glutamate, alone or in the presence of the NOS inhibitor, L-NAME, followed by a 23-h incubation with L-NAME. Cell death was evaluated by trypan blue-exclusion assay (open bars) and nuclear staining with Hoechst 33258 (solid bars), and was expressed as the percent ratio of damaged to the total number of neurons. Data are means ± SD (n = 4). ***p < 0.001 significantly different from NaCN-induced injury alone using ANOVA followed by the Student-Newman-Keuls multiple-comparisons procedure.

 
To test if inhibition of the NMDA receptor was able to antagonize cyanide toxicity, neurons were treated with NaCN or L-glutamate in combination with the NMDA receptor antagonist kynurenic acid. As shown in Figure 4Go, kynurenic acid produced a concentration-dependent protective effect, with respect to both L-glutamate and NaCN. The maximal protective effect was obtained with 500 µM kynurenic acid dosage.



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FIG. 4. Effect of NMDA receptor antagonist on cyanide- and glutamate-induced neurotoxicity. Chick embryonic neurons were exposed for 1 h to 1 mM NaCN or 1 mM L-glutamate, alone or in the presence of the NMDA receptor antagonist, kynurenic acid, followed by a 23-h incubation with kynurenic acid. Cell death was evaluated by trypan blue-exclusion assay (open bars) and nuclear staining with Hoechst 33258 (solid bars) and was expressed as the percent ratio of damaged to the total number of neurons. Data are means ± SD (n = 4). ***p < 0.001, *p< 0.05 significantly different from NaCN- or glutamate-induced injury alone, using ANOVA followed by Student-Newman-Keuls multiple-comparisons procedure, except with neurons treated with glutamate and stained with Hoechst 33258, where *p < 0.05 significantly different from glutamate-induced injury alone using Kruskal-Wallis ANOVA on ranks followed by Student-Newman-Keuls multiple comparisons procedure.

 
Next, we explored the effect of the sulfhydryl reducing agent, dithiothreitol (DTT), on NO-mediated neuroprotection. Treatment with NO donor for 1 h and medium for 30 min, followed by incubation with 1 mM NaCN, resulted in a similar protective effect, as described earlier (Fig. 5A pGo, < 0.001). However, when the cells were incubated with DTT instead of medium during the second treatment, the protective effect was eliminated, which strongly indicates the involvement of thiol groups in the NO-mediated neuroprotection. Similar results were obtained if NaCN was substituted by L-glutamate (Fig. 5BGo). Treatment with NO donors alone or NO donors followed by DTT did not induce neuronal death. Furthermore, incubation with DTT followed by L-glutamate increased the percentage of apoptotic neurons, indicating an increased activity of glutamate receptors (Fig. 5B pGo, < 0.001).



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FIG. 5. Effect of DTT on NO-mediated neuroprotection. Chick embryonic neurons were exposed to 3 succeeding treatments, all followed by medium removal. (A) 1. Medium or NO donor for 1 h; 2. medium or DTT for 30 min; 3. medium or 1 mM NaCN for 1 h, followed by 23 h recovery. (B) 1. Medium or NO donor for 1 h; 2. medium or DTT for 30 min; 3. medium or 1 mM glutamate for 1 h followed by 23-h recovery. Cell death was evaluated by trypan blue-exclusion assay (open bars) and nuclear staining with Hoechst 33258 (solid bars) and expressed as the percent ratio of damaged to the total number of neurons. Data are means ± SD (n = 4). ***p < 0.001 significantly different from NaCN- or glutamate-induced injury alone using ANOVA followed by Student-Newman-Keuls multiple-comparisons procedure.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the first series of experiments, we determined the concentration-dependent effect of various NO donors on cyanide-induced cell death. The type of cell death was characterized by the trypan blue-exclusion assay to detect membrane damage, and by nuclear staining with Hoechst 33258 to estimate the percentage of apoptotic neurons.

Evaluation of neuronal cell death revealed a significant protection from cyanide-induced toxicity by all NO donors tested. Lack of protection by exhausted NO donors verified that the effect was mediated by NO and not by a metabolic breakdown product. The maximal effective concentration of NO donor varied from donor to donor, probably due to different mechanisms of NO release. Furthermore, the concentration of NO seemed to play a pivotal role since high concentrations exacerbated cyanide-induced neuronal death, whereas too low concentrations were unlikely to produce any effect. The augmented toxicity observed in cultures treated with high concentrations of NO could probably be attributed to toxic reaction products of NO, e.g., formation of the highly toxic peroxynitrite (reviewed by Kröncke et al., 1997; Wink and Mitchell, 1998). Previous studies have found a protective effect if NO donors released >2 µM (Vidwans et al., 1999Go), but the exact concentration of NO in the present study would be difficult to estimate because of the differences in the mechanisms of NO release. The fact that all donors provided protection indicated that protection was independent of the redox state of NO. This is in line with results describing protective effects of donors of NO+ and NO as well as ONOO on tumor necrosis factor {alpha}-induced apoptosis (Haendeler et al., 1997Go), and with a recent study describing inhibitory effects of various NO donors on NMDA-stimulated Ca2+ accumulation in cortical neurons independently of the NO/NO+ characteristics of the NO donor compound (Vidwans et al., 1999Go).

The ability of the NOS inhibitor, L-NAME, to significantly increase the percentage of apoptotic neurons suggests that endogenous NO plays a protective role, at least in cell cultures. The results are not conclusive, since the effect was not apparent if membrane damage was used as endpoint and if L-glutamate was substituted for NaCN. If the neuroprotective effect of NO was mediated through NMDA receptors, a similar effect of L-NAME on glutamate toxicity would be expected.

Involvement of the NMDA receptor in cyanide toxicity is unambiguous. The present study shows a significant protective effect of kynurenic acid on cyanide toxicity, indicating that at least part of the toxic effect of cyanide is mediated by the NMDA receptor. This is consistent with previous findings that inhibition of NMDA receptors protect cells from cyanide-induced damage (Goldberg et al., 1987Go; Patel et al., 1992Go; Sturm et al., 1993Go).

The sulfhydryl reducing agent DTT was able to completely block the protection conferred by NO donors, suggesting involvement of thiol groups in the mechanism of protection. Furthermore, the mechanism of action seems to involve chemical modification of the target, since the effect persists, even in the absence of NO. This is consistent with either of the proposed mechanisms of NO-induced neuroprotection: inhibition of the NMDA receptor by S-nitrosylation of critical thiol groups (Hoyt et al., 1992Go; Lei et al., 1992Go), or inhibition of caspases by S-nitrosylation in vitro as well as in vivo (Kim et al., 1997Go; Rossig et al., 1999Go). Based on the present results, we are not able to distinguish the two effects, since both can be reversed by DTT (Haendeler et al., 1997Go; Lei et al., 1992Go) and both cyanide and L-glutamate have been shown to induce apoptotic cell death (Bonfoco et al., 1995Go; Jensen et al., 1999Go; Mills et al., 1996Go).

The protective effect of NO could also be explained in terms of preconditioning. NO have been shown to confer neuroprotection by mediating rapid anoxic preconditioning (Centeno et al., 1999Go) and delayed preconditioning by activation of the p21ras/erk cascade after oxygen-glucose deprivation, independently of activation of soluble guanylate cyclase and Ca2+ influx (Gonzalez-Zulueta et al., 2000Go). NO activates p21ras by inducing a conformational change due to S-nitrosylation of cysteine 118, which is a critical site of redox regulation of p21ras (Lander et al., 1995Go,1996,1997), and this S-nitrosylation has been shown to be reversed by DTT-treatment (Ji et al., 1999Go), which would explain the inhibitory action of DTT on NO-induced neuroprotection in this study.

Extrapolation of the in vitro data to the in vivo situation is complicated by a number of factors. In cyanide poisoning nitrites, e.g. amyl nitrite and sodium nitrite, are used primarily to generate methemoglobin. The mechanism has been questioned and the pronounced hemodynamic effects have been suggested as contributors to the antidotal activity (Way et al., 1988Go). Neither of these mechanisms can account for the observed protective effects of NO donors in vitro in this study, and thus, the antidotal effect of nitrites observed in vivo might very well be the sum of methemoglobin, and hemodynamic and specific NO-related mechanisms. This is supported by previous findings in which ISDN was found to protect against cyanide toxicity in vivo even when the mice were co-treated with methylene blue, stimulating methemoglobin reductase. Furthermore, no difference in the protective effect was observed after repeated dosing with ISDN, suggesting that tolerance to the antidotal effect does not occur (Sun et al., 1995Go). These results indicate that methemoglobin formation and vasodilation are probably not involved in the antidotal effect of ISDN.

Another point to be remembered when extrapolating from the present in vitro investigation to the in vivo situation is that neonatal chick neurons possibly would be more sensitive to apoptosis than adult neurons.

In conclusion, the present results provide evidence for a protective effect of various NO donors on cyanide-induced neuronal death, which seems to be independent of the redox-related species of NO released and independent of methemoglobin formation. The effect is mediated by NO and likely involves thiol groups, since the effect is completely reversed by dithiothreitol.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Fax: +45 35 30 60 10. E-mail: msj{at}dfh.dk. Back


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 DISCUSSION
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