A.G. Kanthasamy

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.

Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24‐h period. TMT treatment was accompanied by a low level of PKC down‐regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µM). Phorbol myristate‐induced PKC down‐regulation or inhibition with chelerythrine provided protection against TMT‐induced cytotoxicity. It was concluded that TMT‐induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.

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.

Calcium mediation of cyanide-induced catecholamine release : implications for neurotoxicity

Exposure of rat pheochromocytoma (PC12) cells to KCN (1.0-10 mM) over a 30-min period stimulated secretion of dopamine (DA) and decreased intracellular DA content. Addition of KCN (10 mM) to rat frontal cortex slices preloaded with 1-[7-3H]norepinephrine ([3H]NE) increased secretion of NE over a 10- to 30-min incubation period. In PC12 cells release of DA by KCN was nearly abolished in calcium-free media or by prior addition of diltiazem, a calcium channel antagonist. Release of [3H]NE from rat cortical slices by cyanide was only partly inhibited by diltiazem suggesting that intracellular calcium may be involved in this response. In PC12 cells KCN also produced a dose-related release of the DA precursor dihydroxyphenylalanine, without altering intracellular stores. Levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were enhanced at lower concentrations of KCN. These observations indicate cyanide elicits exocytotic release of neurotransmitters in a calcium-dependent manner and also show that cyanide alters catecholamine metabolism. These actions of cyanide may be important in CNS symptoms of intoxication.

Antagonism of cyanide toxicity by isosorbide dinitrate: possible role of nitric oxide

In a search for improved cyanide antidotes, the efficacy of isosorbide dinitrate (ISDN), was compared with that of the known cyanide antidote, NaNO2. ISDN was as effective as an optimal dose of NaNO2 in protecting mice against cyanide lethality. To study the mechanism involved, the extent of formation of the cyanide scavenger, methemoglobin, in the action of ISDN was determined. ISDN (300 mg/kg, p.o.) increased methemoglobin from 5 to 10% of total hemoglobin, while, in contrast, NaNO2 (100 mg/kg, i.p.) increased methemoglobin levels to 50% of total hemoglobin. Lowering the dose of NaNO2 to 30 mg/kg reduced methemoglobin levels to approximately 10% of total hemoglobin and in turn nearly abolished its antidotal effect. Decreasing methemoglobin to less than control levels using methylene blue failed to abolish cyanide antagonism by ISDN. Thus, methemoglobin formation by ISDN does not account for its antidotal action. Further studies comparing the respiratory depressant effects of cyanide in the presence of ISDN or NaNO2 also indicated that these two antidotes have different mechanisms of action. Efforts to produce tolerance to the antidotal effect of ISDN against cyanide toxicity were unsuccessful. It is suggested that the well-known ability of ISDN to generate nitric oxide may account for the noted cyanide antagonism.

Inhibition of potassium-stimulated dopamine release by the nitric oxide generator isosorbide dinitrate

In PC12 cells, isosorbide dinitrate (ISDN) and S-nitrosol-acetyl-penicillamine (SNAP), both nitric oxide (NO) generators, attenuated K+ (56 mM)-stimulated release of dopamine. The attenuation was not observed with isosorbide, an ISDN analog lacking NO generating capacity. In this model, A23187 (Ca2+ ionophore), Bay K8644 (Ca2+ slow channel agonist) and veratridine (Na+ channel agonist) stimulated dopamine release. Treatment with ISDN enhanced Bay K8644 and veratridine-evoked dopamine release, while ISDN had no significant effect on the A23187 response. Incubation with 8-bromo-cGMP (membrane permeable cGMP analog) had no effect on basal or stimulated dopamine release in these cells, suggesting NOs response was not mediated by cGMP. In additional studies, K+ (56 mM), Bay K8644 and veratridine elevated cytosolic free calcium levels ([Ca2+]i). ISDN reduced K(+)-stimulated increase in [Ca2+]i, but enhanced the increases of [Ca2+]i induced by Bay K8644 or veratridine. These results suggest NO interacts with K(+)-induced membrane depolarization (possibly by inhibiting membrane conductance to K+) to attenuate Ca2+ influx and Ca(2+)-mediated dopamine secretion stimulated by K+.

Plasma membrane hyperpolarization by cyanide in chromaffin cells: role of potassium channels

Exposure of rat pheochromocytoma (PC12) cells to cyanide produces elevation of cytosolic calcium, impaired Na+−H+ exchange, membrane lipid peroxidation and release of neurotransmitters. Since these observations suggested cyanide alters plasma membrane function, the present study examined the effect of NaCN on the membrane potential of undifferentiated PC12 cells in suspension. In PC12 cells loaded with the voltage sensitive fluorescent dye, bis-oxonol, cyanide (2.5–10 mM) elicited an immediate (within seconds), concentration related decrease in fluorescence, indicating hyperpolarization of the plasma membrane. Increasing extracellular K+ concentration to 20 mM blocked the effect of cyanide (5 mM), suggesting cyanide increased K+ efflux. Pretreatment with quinine blocked the cyanide-induced hyperpolarization, whereas glyburide had little effect, showing the hyperpolarization produced by cyanide was due to activation of Ca2+ sensitive K+ channels. Removal of Ca2+ from the media did not influence cyanide-induced hyperpolarization. However, buffering intracellular Ca2+ by loading cells with the Ca2+ chelators, Quin II or BAPTA, abolished the cyanide effect, showing cytosolic Ca2+ is a key factor. These findings suggest that cyanide mobilizes Ca2+ from intracellular stores which leads to hyperpolarization via the activation of Ca2+ sensitive K+ channels.

Use of PC12 Cells as a Neurotoxicological Screen: Characterization of Anticyanide Compounds

A series of six biochemical markers of cyanide toxicity (dopamine release, hydroperoxide generation, cytosolic-free calcium levels, catalase activity, cytochrome oxidase activity, and superoxide dismutase activity) in cultured rat pheochromocytoma (PC12) cells were used to establish a screen for evaluation of potential anticyanide compounds. Thirty-nine substances, including anticonvulsants, adrenergic blockers, antioxidants, and antipsychotics were tested and ranked according to the results. Based on the composite scoring in all six assays, carbamazepine, mannitol, allopurinol, and phenytoin were ranked as the most effective anticyanide compounds. Additionally, known cyanide antidotes (e.g., pyruvate, mercaptopyruvate, alpha-ketoglutarate, naloxone, and flunarizine) obtained relatively high ranking in the PC12 cell screen. Furthermore, a significant correlation was found between protective effects (based on LD50s) of cyanide antidotes in mice and ranking in the in vitro screen. This study illustrates that by assaying a series of biochemical markers in a neuronal-type cell line, a rapid, cost-effective in vitro toxicological screen is possible. Several compounds have been identified which inhibit the biochemical effects of cyanide and may be used to enhance effectiveness of the standard cyanide antidotes.

Role of intracellular Cd2+ in catecholamine release and lethality in PC12 cells

To evaluate the role of intracellular Cd2+ in catecholamine release and lethality in rat pheochromocytoma (PC12) cells the following results were obtained: [1] the presence of Cd2+ intracellularly was demonstrated with the Cd(2+)-sensitive fluorescent dye BTC-5N, [2] Cd2+ entry through Ca(2+)-channels was either blocked with nifedipine or diltiazem or increased with Bay K8644, [3] Cd2+ entry through voltage sensitive Ca2+ channels was related to dopamine release and cell lethality, [4] a calmodulin inhibitor protected against Cd2+ toxicity, and [5] extracellular Ca2+ concentration, altered prior to Cd2+ exposure, was inversely related to dopamine release by Cd2+. The data indicate intracellular effects of Cd2+ rather than cell surface actions are primarily involved in neurotransmitter release and lethality by toxic levels of Cd2+ in adrenomedullary cells. To evaluate the role of intracellular Cd2+ in catecholamine release and lethality in rat pheochromocytoma (PC12) cells the following results were obtained: [1] the presence of Cd2+ intracellularly was demonstrated with the Cd(2+)-sensitive fluorescent dye BTC-5N, [2] Cd2+ entry through Ca(2+)-channels was either blocked with nifedipine or diltiazem or increased with Bay K8644, [3] Cd2+ entry through voltage sensitive Ca2+ channels was related to dopamine release and cell lethality, [4] a calmodulin inhibitor protected against Cd2+ toxicity, and [5] extracellular Ca2+ concentration, altered prior to Cd2+ exposure, was inversely related to dopamine release by Cd2+. The data indicate intracellular effects of Cd2+ rather than cell surface actions are primarily involved in neurotransmitter release and lethality by toxic levels of Cd2+ in adrenomedullary cells.