Introduction

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Cyanide is a rapid-acting toxicant in which the CNS is one of the major target organs. Neurological dysfunction induced by cyanide includes respiratory distress, seizures and convulsions (Way, 1984; Borowitz et al., 1992), and in some cases a Parkinson-like condition may develop as a post-toxicity sequela (Uitti et al., 1985; Carella et al., 1988; Valenzuela et al., 1992; Rosenow et al., 1995). In animals, cyanide can produce a Parkinson-like response in which striatal neurodegeneration occurs (Kanthasamy et al., 1994).

Recent studies have shown that an interaction between cyanide and the glutamate neurotransmitter system plays an important part in cyanide neurotoxicity. Cyanide induces the release of glutamate from neuronal stores and alters the brain levels of glutamate (Persson et al., 1985; Patel et al., 1991). The increased extracellular levels of glutamate may result in overstimulation of glutamate receptors, leading to excitotoxic responses (Rothman, 1984). NMDA receptor-mediated Ca⁺⁺ influx appears to be a key event in the excitotoxic process initiated by cyanide. In neuronal cells, specific NMDA receptor antagonists such as APV and MK-801 block cyanide-induced [Ca⁺⁺]_i elevation (Michaels and Rothman, 1990; Cai and McCaslin, 1992; Patel et al., 1992) and prevent neuronal cytotoxicity (Goldberg et al., 1987; Pauwels et al., 1989; Patel et al., 1993). Neither non-NMDA receptor antagonists nor VSCC blockers alter the cytotoxic response to cyanide.

The purpose of the present study was to characterize further the interaction between cyanide and NMDA receptor-mediated responses using cerebellar granule cells. It was proposed that cyanide exerts a direct effect on the NMDA receptor channel complex to modulate the response to NMDA. It was shown that cyanide enhanced the response to NMDA by increasing the probability of NMDA receptor channel opening, resulting in an increased amplitude and duration of the NMDA response.

	Materials and Methods

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Cell culture. Primary cultures of rat cerebellar granule cells were prepared as described previously by Gunasekar et al., (1996). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 20 mM glucose, 25 mM KCl and 5000 units/l penicillin/streptomycin at pH 7.4 on poly-L-lysine (MW 30,000-70,000)-coated sterile cover glass in 6-well 35-mm culture dishes. Non-neuronal cell proliferation was prevented by adding cytosine arabinonucleoside (10 µM) 18 hr after plating. Cultures generated by this method contain more than 95% granule cells. Mature cells (9-12 days in vitro) were used in all experiments.

[Ca⁺⁺]_i measurement. Cytosolic free Ca⁺⁺ levels were determined in granule cells as previously described (Patel et al., 1994). Briefly, cells grown on glass coverslips were loaded with fura-2 by incubation with 5 µM fura-2 AM for 45 min at room temperature in Locke's solution containing (in mM) NaCl, 154; KCl, 5.6; MgCl₂, 1.0; CaCl₂, 2.3; NaHCO₃, 3.6; HEPES, 5.0 and D-glucose, 5.6; pH, 7.4. Dye loading was terminated by replacing the loading solution with fresh Locke's buffer. For microfluorescence measurement of [Ca⁺⁺]_i, the coverslips were mounted in a thermostatically controlled cell chamber maintained at 37°C (Medical System Inc., Greenvale, NY) and placed on an inverted stage Nikon Diaphot-TMD microscope connected to a SLM 8000 C spectrofluorometer (SLM-AMINCO, Inc., Urbana, IL). [Ca⁺⁺]_i measurements were made on a small group (1-3 cells) of fura-2-loaded neurons. After basal [Ca⁺⁺]_i levels were obtained, test compounds were added at the various time points specified via an inlet port connected to syringes for media replacement (the drug solution equilibrated in the chamber within 10 sec of addition), and fluorescence was monitored at 510 nm while excitation wavelength was alternated between 340 nm and 380 nm every 15 sec. Except for studying the effect of extracellular Mg⁺⁺, we routinely omitted MgCl₂ from the solution during the drug treatments. The fluorescence was converted on a real-time basis to Ca⁺⁺ concentrations by use of the SLM 8100 software Intracellular Probe measurement system (SLM-AMINCO, Inc., Urbana, IL) according to the fluorescence ratio method of Grynkiewicz et al., (1985).

Patch-clamp recordings. Patch-clamp experiments were conducted in primary cerebellar granule cells 10 to 12 days old grown on 25-mm glass coverslips. NMDA receptor-mediated currents were recorded in both whole-cell and single-channel configurations. The same external and internal solutions were used for whole-cell and single-channel recordings. The external solution contained (in mM) NaCl, 150; KCl, 2.8; CaCl₂, 1 and Na-HEPES, 10, pH 7.2, and the pipette solution contained CsCl, 150; NaCl, 4; CaCl₂, 0.5; K-EGTA, 5 and K-HEPES, 10, pH 7.2. All test compounds were dissolved in the bath solution and diluted to final concentrations as indicated. Drugs were applied to whole cells or excised membrane patches by close distance (< 20 µM) pressure ejection from blunt-tipped (10-20 µm), fire-polished micropipettes. Upon application of pressure, the concentration of drug bathing the cell rises to greater than 90% of the concentration in the micropipette in less than 1 sec (Choi et al., 1977; Dunlap and Fischbach, 1981). For experiments, cultures on glass coverslips were transferred into a chamber made from a 35-mm plastic culture dish containing 1 ml of external solution, which was then placed on the stage of an inverted phase-contrast microscope at room temperature. High-resistance seals were obtained with borosilicate glass patch pipettes (4-7 M). Recordings were performed with an Axo-patch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 3 kHz with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA). Data acquisition and analysis were performed as previously described (Twitchell and Rane, 1994) with Pulse (Instrutech Corp., Great Neck, NY) and TAC (HEKA Elektronik, Göttingen, Germany) software.

Chemicals. The following drugs and chemicals were used in this study: Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin/streptomycin (Gibco, Grand Island, NY), tissue culture dishes (Costar, Cambridge, MA), glass coverslips (25 mm) (Fisher Scientific, Fair Lawn, NJ), fura-2/AM (Molecular Probes, Eugene, OR), diltiazem hydrochloride (Marion Laboratories, Inc., Kansas City, MO) and MK-801 (Research Biochemicals Inc., Natick, MA). All other chemicals were from Sigma Chemical Co. (St. Louis, MO).

Statistics. Statistical differences between treatments were determined by Student's t test or by one-way analysis of variance (ANOVA) with a Newman-Keuls procedure for multiple comparisons. Differences were considered significant at P < .05.

	Results

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Enhancement of NMDA-stimulated [Ca⁺⁺]_i elevation by cyanide. NMDA treatment in cultured neurons has been shown to induce [Ca⁺⁺]_i elevation due to activation of the NMDA receptor-coupled ion channels (MacDermott et al., 1986). In cerebellar granule cells, NMDA treatment (50 µM, in the presence of 10 µM glycine) in Mg⁺⁺-free medium induced a rapid biphasic increase of [Ca⁺⁺]_i followed by a maintained plateau (fig. 1A). Addition of 50 µM NaCN at the NMDA plateau level resulted in a further increase of [Ca⁺⁺]_i. The enhancement of NMDA-induced [Ca⁺⁺]_i elevation by NaCN was not dependent on the order in which NaCN was added.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of cyanide on NMDA-induced [Ca++]i elevation. A) Representative fluorometric tracing illustrating the effect of NaCN (50 µM) on NMDA (50 µM)-induced [Ca++]i elevation. B) Summary of the effect of varying concentrations of NaCN on NMDA-induced [Ca++]i elevation. Cells were stimulated with NMDA (50 µM in the presence of 10 µM glycine), and NaCN (20, 50 or 100 µM) was added after the NMDA response reached a plateau. [Ca++]i values were determined at 300 sec after the addition of NaCN. Bars represent means ± S.E.M. of 8 to 11 experiments, and asterisks denote significant difference from NMDA response (without NaCN) (*P < .05, ***P < .001).

Effect of cyanide on K⁺-depolarization-induced [Ca⁺⁺]_i elevation. The effect of cyanide on K⁺-depolarization-induced [Ca⁺⁺]_i elevation was also determined. Depolarization-induced Ca⁺⁺ influx is mediated primarily by activation of VSCCs. In the present study, K⁺ depolarization induced a rapid increase in [Ca⁺⁺]_i that peaked at about 10-fold of basal [Ca⁺⁺]_i. The peak then declined and stabilized at about 4-fold of the basal [Ca⁺⁺]_i, which could be further decreased by the VSCC blocker diltiazem (fig. 2A). When NaCN (50 µM) was added to the K⁺ response plateau, there was no significant change in [Ca⁺⁺]_i (fig. 2, B and C).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of cyanide on KCl-depolarization-induced [Ca++]i elevation. A) Representative fluorometric tracing illustrating KCl-depolarization-induced [Ca++]i elevation. Cells were stimulated with 56 mM KCl, and 10 µM diltiazem was added (at arrows) when the KCl response reached a plateau. B) Representative fluorometric tracing illustrating the effect of NaCN (50 µM) on KCl-depolarization-induced [Ca++]i elevation. C) Summary of the effect of NaCN on KCl-depolarization-induced [Ca++]i elevation. Cells were stimulated with 56 mM KCl, and NaCN (50 µM) was added when the NMDA response reached a plateau. [Ca++]i values were determined at peak and plateau levels after KCl and 300 sec after the addition of NaCN. Bars represent means ± S.E.M. of six experiments. There was no significant difference between the KCl plateau and the KCl plateau + NaCN groups.

Effects of APV, MK-801 and extracellular Mg⁺⁺ on cyanide-induced enhancement of NMDA responses. The effects of extracellular Mg⁺⁺ and the NMDA receptor antagonists APV and MK-801 on NaCN-induced enhancement of NMDA response were examined. As demonstrated in figure 3A, the addition of 100 µM APV, a competitive NMDA receptor antagonist, induced a rapid decline in [Ca⁺⁺]_i from the NMDA plateau response. Subsequent application of 50 µM NaCN resulted in no further [Ca⁺⁺]_i elevation. MK-801 (1 µM), a noncompetitive NMDA receptor antagonist, showed a similar inhibitory effect on the NaCN action. APV and MK-801 resulted in 59% and 51% attenuation of NMDA-evoked [Ca⁺⁺]_i, respectively, and cyanide did not affect [Ca⁺⁺]_i in the presence of these antagonists (fig. 3B).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of NMDA receptor antagonists on the cyanide action. A) Representative fluorometric tracing illustrating the effect of NaCN (50 µM) on NMDA-induced [Ca++]i changes in the presence of APV (100 µM). B) Summary of the effect of NaCN on NMDA-induced [Ca++]i changes in the presence or absence of MK-801, APV or MgCl2. Cells were stimulated with NMDA (50 µM in the presence of 10 µM glycine), and APV (100 µM), MK-801 (1 µM) or MgCl2 (1.2 mM) was added 300 sec after NMDA stimulation. NaCN (50 µM) was added subsequently when [Ca++]i reached a plateau. Control represents the NMDA and the NMDA + NaCN treatment groups. [Ca++]i values were determined at 300 sec after each addition. Bars represent means ± S.E.M. of six or more experiments. Values with different letters indicate a significant difference from one another (P < .05).

Effect of receptor modulators on cyanide-induced enhancement of NMDA responses. In order to gain insight into the mechanism of cyanide enhancement of the NMDA response, the cells were pretreated with compounds known to modulate the NMDA-induced Ca⁺⁺ influx. PMA did not alter the response to cyanide, whereas DTT and pregnenolone enhanced the response (fig. 4). Arachidonic acid, which is known to potentiate NMDA-induced calcium influx, enhanced the response to NaCN in the presence of NMDA.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of NMDA receptor modulators on the cyanide action. Summary of the effect of NaCN on NMDA-induced [Ca++]i changes in the presence of DDT (2 mM), PMA (200 mM), pregnenolone sulfate (100 µM) or arachidonate (AA, 10 µM). Cells were treated with the modulators, and 2 min later, 50 µM NMDA (in the presence of 10 µM glycine) was added. NaCN (50 µM) was added when [Ca++]i reached a plateau. Bars represent means ± S.E.M. of responses at 300 sec after addition of NaCN of 4 to 5 experiments, and the asterisk indicates a significant difference from the NaCN group (P < .05).

Effects of diltiazem and nifedepine on cyanide-induced enhancement of NMDA responses. NMDA receptor activation promotes the influx of cations such as Na⁺ and Ca⁺⁺, which can lead to membrane depolarization. This, in turn, can activate membrane VSCCs, resulting in additional Ca⁺⁺ influx. To examine the possibility that secondary opening of VSCCs was involved in the NaCN-induced [Ca⁺⁺]_i elevation, we used two VSCC blockers, diltiazem (10 µM) and nifedepine (1 µM). These compounds had no effect on the NMDA response plateau. Additionally, the NaCN response was not affected by treatment with diltiazem or nifedepine (fig. 5, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of voltage-sensitive Ca++ channel blockers on the cyanide action. A) Representative fluorometric tracing illustrating the effect of NaCN on NMDA-induced [Ca++]i changes in the presence of diltiazem. B) Summary of the effect of NaCN on NMDA-induced [Ca++]i changes in the presence or absence of nifedepine or diltiazem. Cells were stimulated with NMDA (50 µM in the presence of 10 µM glycine), and diltiazem (10 µM) or nifedepine (1 µM) was added 300 sec after NMDA stimulations. NaCN (50 µM) was added subsequently when [Ca++]i reached a plateau. Control represents the NMDA and the NMDA + NaCN treatment groups. Bars represent means ± S.E.M. of 4 to 5 experiments. Values with different letters indicate a significant difference from one another (P < .05).

Effects of tetrodotoxin on cyanide-induced enhancement of NMDA response. A possible mechanism for the NaCN enhancement of the NMDA response may involve presynaptic glutamate release, which can then activate NMDA and non-NMDA receptors to promote Ca⁺⁺ influx. NaCN has been shown previously to induce glutamate release in brain slices (Patel et al., 1991), and NaCN-induced neuronal cytotoxicity can be prevented by tetrodotoxin, which blocks Na⁺ channel-mediated glutamate release (Rothman, 1984). In the present study, tetrodotoxin treatment did not affect the NaCN-induced enhancement of the NMDA response (fig. 6).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Summary of the effect of cyanide on NMDA-induced [Ca++]i changes in the presence or absence of tetrodotoxin. Cells were stimulated with NMDA (50 µM in the presence of 10 µM glycine), and tetrodotoxin (10 µM) was added 300 sec after NMDA. NaCN (50 µM) was added after the [Ca++]i reached a plateau. Bars represent means ± S.E.M. of six or more experiments. Values with different letters indicate a significant difference from one another (P < .05).

Whole-cell recordings. The microfluorescence studies indicated that NaCN specifically enhanced the NMDA-stimulated elevation of [Ca⁺⁺]_i. To study the underlying mechanism in more detail, we conducted patch-clamp experiments. In patch-clamp studies, the effect of NaCN on NMDA receptor-mediated response was measured directly as changes in receptor-mediated currents.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of cyanide on NMDA-stimulated whole-cell currents. A) Representative records illustrating the effect of NaCN on NMDA-stimulated whole-cell currents. Successive whole-cell NMDA currents induced by 50 µM NMDA (in the presence of 10 µM glycine) were recorded in the presence (lower trace) or absence (upper trace) of 100 µM NaCN, in the same cell, at 60 mV. B) Summary of the effect of NaCN on the amplitude (peak level) and duration of NMDA whole-cell currents. The duration of the response was defined as the time for 80% recovery from peak-level currents. Bars represent means ± S.E.M. of 10 experiments, and asterisks denote significant difference from NMDA treatment (*P < .05, **P < .01).

Single-channel measurement. The effect of NaCN on the NMDA receptor response was examined at the single-channel level in excised outside-out membrane patches, a condition in which the involvement of cytoplasmic factors is minimized. Each patch was exposed to both NMDA and NMDA/CN, and changes in channel activity were determined by analysis of 3 to 12 sec of continuous recording for each condition. In the patch shown in figure 8, the presence of NaCN (100 µM) increased the number of NMDA-stimulated channel openings observed for a given recording period. As a result, increased from 2.9% to 15.2%. For the patches studied, the mean increase of N · P induced by NaCN was 208% (fig. 9A). Amplitude and open-time histograms obtained from the patch in figure 8 are shown in figure 9, C and D, respectively. In the absence of NaCN, the mean amplitude of NMDA-stimulated single-channel current is 2.3 ± 0.1 at 80 mV. In the presence of NaCN, the mean amplitude is 2.2 ± 0.1 pA. The estimated single-channel conductance was calculated as 43 pS, which is within the range observed for the main conductance state of the NMDA channel (Nowak et al., 1984). The mean channel open time of the NMDA receptor was not significantly changed by NaCN (fig. 9C); rather, the number of single-channel events for a given time increased in the presence of CN. Similar observations were obtained in two other patches.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   .Effect of cyanide on NMDA-stimulated single-channel currents in cerebellar granule cells. Representative records illustrating the effect of cyanide on NMDA-stimulated single-channel currents recorded in an excised outside-out patch held at 80 mV. Left, sequential traces of continuous NMDA treatment (50 µM plus 10 µM glycine). Right, sequential traces of continuous NMDA + NaCN (50 µM NMDA, 10 µM glycine and 100 µM NaCN).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Effect of cyanide on NMDA-stimulated single-channel currents. A) Effect of NaCN (100 µM) on NMDA receptor channel activity defined as N  ·  P. Bars represent means ± S.E.M. of three experiments, and the asterisk denotes significant difference from NMDA treatment (*P < .05). Amplitude (panel B) and open-time histograms (panel C) of NMDA-stimulated single-channel currents in the presence or absence of NaCN. NMDA single-channel recordings from figure 8 were analyzed. Analysis of the patch was selected for presentation to emphasize the large increase in event number with NMDA + NaCN (in this case, the largest observed) in the absence of significant changes in event amplitude or mean channel open time.

	Discussion

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In cerebellar granule cells, the NMDA-stimulated elevation of [Ca⁺⁺]_i was enhanced 15% to 60% by cyanide at concentrations of 20 to 100 µM, which in the absence of NMDA had no effect on basal [Ca⁺⁺]_i. As demonstrated by patch-clamp studies, cyanide enhanced the NMDA receptor-mediated currents. In the whole-cell configuration, cyanide increased the amplitude and duration of NMDA-stimulated inward currents, and in outside-out patches, it increased the NMDA receptor single-channel opening frequency without affecting the unitary conductance or mean channel open time. Because the effect of cyanide persists in cell-free patches, activation of intracellular second messengers does not appear to be a prerequisite for cyanide's action. It is concluded that cyanide exerts a direct effect on the NMDA receptor to modulate positively the response to NMDA.

This study confirms and extends the observations of Cai and McCaslin (1992), who concluded that cyanide interacts with the excitatory amino acid receptor. It was observed that cyanide enhanced the influx of Ca⁺⁺ after activation of the NMDA receptor and that VSCC blockers did not alter the cyanide response. Our previous study showed, in cultured hippocampal neurons, that cyanide in high concentrations (1 mM) enhanced NMDA mediated Ca⁺⁺ influx and inward current by interacting with the Mg⁺⁺ block of the receptor (Patel et al., 1994). That effect appeared to be energy-independent and could be explained by a direct interaction of cyanide with an allosteric regulatory site on the receptor.

Activation of NMDA receptor channels and concomitant Ca⁺⁺ influx have been shown to be key events in cyanide-induced excitotoxicity. In a number of neuronal models, cyanide-induced [Ca⁺⁺]_i elevation and cytotoxicity were prevented by selective NMDA receptor antagonists (Michaels and Rothman, 1990; Dubinsky and Rothman, 1991; Cai and McCaslin, 1992; Patel et al., 1992, 1993). It has also been shown that cyanide enhanced MK-801 binding, reflecting an increased NMDA receptor activity and channel opening (Akira et al., 1994; Patel et al., 1994). The present study provides direct evidence that cyanide at biologically relevant concentrations (micromolar range) can potentiate the NMDA receptor-mediated response (Ballantyne, 1983; MacMillan, 1989).

To determine whether the response to cyanide resulted from a direct interaction with the NMDA receptor channel, we determined the effect of cyanide on other processes that regulate Ca⁺⁺ influx and cytosolic levels. Even though NMDA- and K⁺-depolarization-induced [Ca⁺⁺]_i elevation reached similar levels, cyanide affected only the NMDA response. This indicates that [Ca⁺⁺]_i elevation per se is not a prerequisite for the enhancement and that it is not due to an altered ability of the cell to buffer changes in cytosolic [Ca⁺⁺]_i levels.

K⁺-depolarization-induced elevation of [Ca⁺⁺]_i results from opening of VSCCs, followed by their inactivation. The long-lasting plateau results from a small proportion of L-type VSCCs remaining activated over a prolonged period (Murphy et al., 1987). In the present study, L-channels mediated the response to KCl, because diltiazem rapidly lowered the K⁺ plateau response. The failure of cyanide to affect the K⁺-induced plateau indicates that cyanide did not influence L-type VSCC-mediated Ca⁺⁺ influx.

Activation of the NMDA receptor promotes the influx of cations, including Na⁺ and Ca⁺⁺, which can lead to membrane depolarization. In turn, depolarization can activate membrane VSCCs, leading to additional Ca⁺⁺ influx (Mayer and Miller, 1990). Selective pharmacological antagonists were used to differentiate between Ca⁺⁺ influx through NMDA-gated channels and through VSCCs. The cyanide-induced enhancement of [Ca⁺⁺]_i was blocked by the NMDA receptor antagonists APV and MK-801 and by Mg⁺⁺. The VSCC blockers nifedipine and diltiazem did not interfere with the cyanide action. It was concluded that the cyanide-induced enhancement of Ca⁺⁺ influx is mediated primarily by NMDA receptor-coupled channels.

Enhancement of whole-cell currents can arise from changes in NMDA receptor single-channel properties, including single-channel conductance, mean channel open time and the frequency of channel openings. In the present study, we found that cyanide increased NMDA receptor channel opening frequency without affecting mean channel open time or single-channel conductance. Like cyanide, a number of other NMDA receptor allosteric modulators also affect the NMDA receptor channel opening probability. The allosteric modulators that increase the opening probability include glycine (Johnson and Ascher, 1987), the polyamine spermine (Rock and MacDonald, 1992), the sulfhydryl reducing agent DTT (Tang and Aizenman, 1993), protein kinase C (Chen and Huang, 1992), arachidonate (Miller et al., 1992) and the neurosteroid pregnenolone sulfate (Bowlby, 1993). Other modulators have been reported to decrease the receptor channel opening probability, including hydrogen ions (Tang et al., 1990), Zn⁺⁺ (Legendre and Westbrook, 1990), the opioid peptide dynorphin (Chen et al., 1995) and nitric oxide (Fagni et al., 1995). It is interesting to note that in this study, arachidonate and pregnenolone increased the response to cyanide. It is possible that cyanide directly interacts with one or more of the receptor modulatory sites to enhance the NMDA response.

Cyanide neurotoxicity is associated with activation of NMDA receptors and sequent influx of [Ca⁺⁺] (Dubinsky and Rothman, 1991; Patel et al., 1992, 1993). In neuronal models, cytotoxicity is mediated exclusively by the NMDA receptor, because selective antagonists prevent cell death (Patel et al., 1991). This is caused in part by release of endogenous glutamate by cyanide, leading to receptor activation (Patel et al., 1991). Activation of the receptor by glutamate would be enhanced by a direct interaction of cyanide with the receptor, leading to cytotoxic elevation of [Ca⁺⁺]. Recently, we have shown in cerebellar granule cells that cyanide-induced activation of the NMDA receptor produces simultaneous generation of nitric oxide and reactive oxygen species, leading to cellular oxidative stress and cytotoxicity (Gunasekar et al., 1996). It is apparent that activation of the receptor is an initiating event in the cytotoxic response to cyanide that is independent of the effect of cyanide on the cell's energy reserves.

The potentiation of NMDA-induced Ca⁺⁺ influx by cyanide has important toxicological implications. Many physiological functions of NMDA receptors are mediated by Ca⁺⁺, including activation of the Ca⁺⁺-dependent signal transduction cascade involved in nerve cell development and synaptic plasticity (Mayer and Miller, 1990; Gozlan, et al., 1995). In pathological conditions, such as in excitotoxic neuronal injury, excessive NMDA receptor activation and subsequent intracellular Ca⁺⁺ overload lead to cellular injury (Choi, 1987; Garthwaite and Garthwaite, 1987; Choi et al., 1988). Enhancement of the NMDA response would probably accelerate or potentiate the excitotoxic response and hence may play an important role in cyanide-induced neurotoxicity.

In summary, the interaction of cyanide with NMDA receptor-mediated responses was characterized. Cyanide enhanced NMDA receptor-mediated inward currents as well as [Ca⁺⁺]_i elevation. Because the potentiation of NMDA response was seen in excised membrane patches, the cyanide modulation is the result of a direct action on the NMDA receptor channel complex and does not require an intact cell or activation of intracellular second messengers. It is possible that modulation of NMDA receptor-mediated responses is involved in the acute manifestations of cyanide intoxication due to CNS dysfunction (respiratory distress, seizures and convulsions) and the post-intoxication sequelae associated with selective neurodegeneration.