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Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a Bcl-2 homology domain 3 (BH3) domain only protein, has been identified as a mitochondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary rat cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either increased with BNIP3 cDNA (BNIP3+) or knocked down with small interfering RNA (RNAi). In BNIP3+ cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (delta psim), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Ala-Asp-fluoromethyl ketone (zVAD-fmk) suppressed BNIP3+-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knockdown by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3 delta TM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3 delta TM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3+ cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of delta psim and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduce delta psim. This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death.

Cyanide produces degeneration of the nervous system in which different modes of cell death are activated in the vulnerable brain areas. In brain, the mechanism underlying the cell death is not clear. In this study, an immortalized dopaminergic cell line was used to characterize the cell death signaling cascade activated by cyanide. Cyanide-treated cells exhibited a time- and concentration-dependent apoptosis that was caspase independent. Cyanide induced a rapid surge of intracellular reactive oxygen species (ROS) generation, followed by p38 mitogen-activated protein kinase (MAPK) activation and nuclear accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). Activation of p38 MAPK and HIF-1alpha accumulation were attenuated by N-acetyl-L-cysteine (antioxidant), catalase (hydrogen peroxide scavenger), or a selective p38 MAPK inhibitor (SB203580). Cyanide activated the hypoxia response element (HRE) promoter, which was also blocked by the antioxidants and SB203580. HRE activation was followed by increased BNIP3 gene transcription, as reflected by elevated BNIP3 mRNA and protein levels. BNIP3 upregulation was reduced by selective RNAi knockdown of HIF-1alpha. Overexpression of BNIP3 produced mitochondrial dysfunction (reduced membrane potential), caspase-independent apoptosis, and sensitization of the cells to cyanide-induced toxicity. Expression of a dominant-negative mutant or RNAi knockdown of BNIP3 protected the cells from cyanide. It was concluded that cyanide activated the HIF-1alpha-mediated pathway of BNIP3 induction through a redox-sensitive process. Increased BNIP3 expression then served as an initiator of mitochondrial-mediated death.

Dopamine (DA)-induced neurotoxicity is potentiated when cellular metabolism is compromised. Since cyanide is a neurotoxin that produces mitochondrial dysfunction and stimulates intracellular generation of reactive oxygen species (ROS), KCN was used to study DA-induced apoptosis in primary cultured mesencephalon cells. Treatment of neurons with DA (300 microM) for 24h produced apoptosis as determined by TUNEL staining, DNA fragmentation and increased caspase activity. Pretreatment with KCN (100 microM) 30min prior to DA increased the number of cells undergoing apoptosis. When added to the cells alone, this concentration of KCN did not induce apoptosis. DA stimulated intracellular generation of ROS, and treatment with KCN enhanced ROS generation. Treatment of cells with glutathione or uric acid (antioxidants/scavengers) attenuated both the increase in ROS generation and the apoptosis, demonstrating that ROS are initiators of the cytotoxicity. Studies on the sequence of events mediating the response showed that DA-induced depolarization of the mitochondrial membrane was dependent on ROS generation and KCN enhanced this action of DA. Following changes in mitochondrial membrane potential, cytochrome c was released from mitochondria, leading to caspase activation and eventually cell death. These results demonstrate that oxidative stress and mitochondrial dysfunction are initiators of DA-induced apoptosis. Subsequent cytochrome c release activates the caspase effector component of apoptosis. Cyanide potentiates the neurotoxicity of DA by enhancing the generation of ROS and impairing mitochondrial function.

Pyridostigmine is a reversible cholinesterase (ChE) inhibitor that is associated with neurologic dysfunction involving both central and peripheral nervous systems. To determine the neurotoxic potential of pyridostigmine, rats were sacrificed at intervals after drug administration (0.5-1.85 mg/kg, i.p., twice daily for 4 days) and brains examined histologically. ChE inhibition was used as a biomarker of pyridostigmine activity. Using the in situ terminal deoxynucleotidyl transferase nick-end labeling of DNA fragments (TUNEL) method and electron microscopy, apoptotic brain cell death was noted in cerebral cortex over a dose range of 0.5-1.85 mg/kg and at the higher dose (1.85 mg/kg), apoptosis was also noted in striatum and hippocampus. These responses were blocked by pretreatment with atropine. Rat cortical cells in culture also underwent apoptosis when exposed to pyridostigmine (250 microM for 24 hr), indicating that the pyridostigmine can initiate apoptosis, independent of peripheral mechanisms. Pretreatment of cells with atropine (10 microM) inhibited pyridostigmine-induced apoptosis, confirming the response was mediated by muscarinic receptors. Short term treatment of rats with pyridostigmine (1.85 mg/kg twice daily for 4 days) induced a prolonged apoptotic response, which was evident in rat cortex up to 30 days after the last dose. Active apoptosis persisted, despite recovery of serum ChE activity. These in vivo and in vitro observations indicate that pyridostigmine can initiate a prolonged neurodegeneration.

We previously demonstrated that 2-iminothiazolidine-4-carboxylic acid (2-ICA), formed by cyanide reacting with cysteine, caused glutamate antagonist-sensitive seizures when injected i.c.v. (intracerebroventricular) in mice and produced hippocampal CA1 damage following i.c.v. infusion in rats. In this study, the ability of either 2-ICA, glutamate, proline or NMDA (N-methyl-D-aspartate) injected i.c.v. to produce hippocampal lesions sensitive to glutamate antagonists was compared in mice. Hippocampal CA1 damage was observed 5-days following either a seizure (3.2 mumol) or subseizure (1.0 mumol) dose of 2-ICA. Glutamate (3.2 mumol) or proline (10 mumol) also produced hippocampal damage; glutamate damage was primarily to the CA1 subfield, whereas proline damaged neurons throughout the entire hippocampal formation. NMDA (3.2 nmol) caused seizure activity in all animals with a 50% lethality. No hippocampal damage was observed in surviving mice. Neither MK-801 (dizocilpine maleate) nor CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) pretreatment prevented hippocampal lesions produced by 2-ICA. In contrast, MK-801 significantly reduced the frequency of mice displaying glutamate hippocampal lesions, but failed to block seizures produced by glutamate. MK-801 also protected neurons in the CA2-3 zone and the dentate gyrus, but not in the CA1 region of proline-injected mice. Finally, pretreatment with the mixed metabotropic glutamate receptor (mGluR)1/mGluR2 antagonist-agonist (S)-4-carboxy-3-hydroxyphenylglycine (CHPG) prevented hippocampal damage produced by the mGluR1 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), but did not protect against 2-ICA hippocampal lesions. These results show that 2-ICA hippocampal CA1 damage is not mediated through ionotropic or metabotropic glutamate receptors. 2-ICA hippocampal damage may represent a neurotoxicity that is distinct from excitotoxic-mediated cell death.

Excitatory amino acid (EAA)-like and excitotoxic properties of the secondary metabolite of cyanide, 2-iminothiazolidine-4-carboxylic acid, (2-ICA) were evaluated because of its possible role in cyanide-induced neurotoxicity. Intracerebroventricular (i.c.v.) injections of 2-ICA in mice produced wild-running seizures that were qualitatively and quantitatively similar to seizures observed with glutamate. 2-ICA, kainate and proline seizures were prevented by both the NMDA and non-NMDA antagonists, MK-801 and CNQX, respectively. In contrast, NMDA-induced seizures were prevented by MK-801, but not CNQX. When infused i.c.v. in rats over a seven day period, 2-ICA produced extensive and selective loss of CA-1 pyramidal neurons of the hippocampus. In hippocampal slices preloaded with D-[3H]aspartate, 2-ICA superfusion did not evoke release nor significantly augment potassium stimulated release of the radiolabeled transmitter. These findings indicate 2-ICA has excitotoxic properties and its role in cyanide neurotoxicity deserves further study.

Incubation of rat pheochromocytoma (PC12) cells with cyanide (1-10 mM) for 30 min produced a compound which eluted between dopamine (DA) and 3,4-dihydroxyphenylacetic acid in HPLC-EC analysis. Generation of the compound was rapid, concentration-dependent and blocked by pretreatment with clorgyline, a monoamine oxidase A inhibitor. In cell free incubation, the compound formed rapidly when cyanide was added to a mixture of monoamine oxidase and DA, but was not detected when cyanide was included with DA alone. The compound was radiolabelled when 14C-KCN was added to the incubation mixture. Based on these results, it is proposed that the deaminated metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), reacts non enzymatically with cyanide to form the cyanohydrin adduct 2-hydroxy-3-(3,4-dihydroxyphenyl) propionitrile (HPN). HPN was confirmed by spectral analysis and co-elution with synthetic HPN. Incubation of mouse brain slices with cyanide (1 mM) generated 0.98 ng HPN/100 mg wet wt. over a 10 min period and HPN was detected in brains of mice after injection of cyanide (15.6 micrograms) into the lateral brain ventricle. Repeated doses of KCN (6 mg/kg, s.c., five times) produced 0.14 +/- 0.03 ng/100 mg of tissue in striatum. Incubation of PC12 cells for 60 min with 500 microM HPN killed 23% of the cells and increased DA release from the cells by 39.8% over untreated cells. Uptake of HPN into the cells was partially blocked by the catecholamine uptake inhibitor imipramine. These results indicate cyanide reacts with the DA metabolite DOPAL to generate a biologically active cyanohydrin adduct which may contribute to the neurotoxic response to cyanide.

We reported previously that glutamate excitotoxicity may contribute to cyanide-induced neuronal injury. Cyanide stimulates glutamate release which can activate glutamate receptors to initiate excitotoxic processes. This study examines the role of EAA receptor subtypes in mediating cyanide-induced cytotoxicity. Cytotoxicity was assessed in primary rat hippocampal cultures by measuring lactate dehydrogenase (LDH) in the culture media. NaCN (0.1-10 mM) or glutamate (0.01-1 mM) produced concentration-dependent cytotoxicity following 18 hrs of incubation. Glutamate-induced cytotoxicity was partially blocked by the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA antagonist, 2-amino-5-phosphonovalerate (APV). Simultaneous exposure of cultures to both CNQX and APV provided complete protection against glutamate cytotoxicity. NaCN-induced cytotoxicity was not blocked by CNQX, but completely blocked by APV and simultaneous exposure to CNQX and APV did not offer added protection. These results indicate that in hippocampal cultures, both non-NMDA and NMDA receptors mediate glutamate excitotoxicity, whereas NaCN-induced cytotoxicity is mediated primarily by activation of the NMDA receptors.

The ability of atrial natriuretic peptide (ANP) to modulate K+-stimulated release of [3H]norepinephrine ([3H]NE) from rat hypothalamic slices was investigated. ANP-(1-28) significantly decreased K+-stimulated [3H]NE release in a concentration-dependent manner (maximal inhibition = 22% of control with 100 nM, ED50 = 70 pM). Pretreatment with pertussis toxin did not alter the response to ANP. 8Br-cGMP (10 microM), a cGMP analog, significantly decreased [3H]NE release and when combined with 10 nM ANP-(1-28), an additive effect was observed. Additionally, 3-isobutyl 1-methylxanthine (IBMX) (200 microM), a phosphodiesterase inhibitor, combined with ANP-(1-28) 10 nM, significantly decreased [3H]NE release. These results indicate that ANP-(1-28) modulated release of [3H]NE from rat hypothalamic slices and the effect is most likely mediated by elevation of intraneuronal cGMP.

Agents that enhance gamma-aminobutyric acid (GABA) neurotransmission can modulate certain effects of opioids, such as analgesia. In this study, the interaction between morphine and GABAergic agents on the release of [3H]norepinephrine ([3H]NE) from rat frontal cortical slices was examined. GABA (10(-4) M), enhanced potassium-stimulated [3H]NE release and reversed the inhibitory effect of 10(-6) M morphine. GABA and muscimol modulated the inhibitory effect of morphine in a noncompetitive manner. Bicuculline methiodide (10(-4) M) reduced the effect of GABA in the absence of morphine, and appeared to reduce the effect of GABA in the presence of morphine, although the latter effect was not statistically significant from the controls. While the GABAA agonist muscimol mimicked the effect of GABA, the GABAB agonist baclofen did not affect the release of [3H]NE in the absence or the presence of 10(-6) M morphine. These results support the involvement of GABAA receptors in modulating the action of opioids on the noradrenergic system in the cerebral cortex of the rat.

Cyanide is known to initiate release of catecholamines from chromaffin cells and isolated adrenals but the effect of cyanide on plasma catecholamine levels has not been reported. The present study demonstrates that cyanide produces marked elevation of plasma catecholamines in mice. A sublethal dose of KCN (5 mg/kg, sc) significantly increased plasma norepinephrine (NE) and epinephrine (EPI) at 5 min after administration, and the elevated levels returned to normal at 15 min. At a dose of 10 mg/kg, NE and EPI plasma levels remained elevated over a 15 min period. Multiple exposures to sublethal doses of KCN (5 mg/kg, sc, 4 doses at 15 min intervals) produced a steep, sustained rise in plasma NE and EPI. Administration of KCN (15.6 micrograms) directly into the lateral ventricles of the brain caused convulsions and respiratory depression, but did not affect plasma catecholamine levels. Pretreatment with pargyline did not alter the magnitude of the response to KCN. Adrenalectomy prevented the increase in plasma EPI and had no influence on plasma NE levels, indicating cyanide acts directly on the adrenals to stimulate EPI release. It is proposed that KCN directly stimulates the sympathoadrenal axis to increase plasma catecholamine levels.

Gamma-Aminobutyric acid (GABA) and GABAA agonists enhance stimulated release of [3H]norepinephrine [( 3H]NA) in several regions of the rat brain. In this study, the mechanisms by which GABA and GABAergic agonists augment potassium-stimulated release of [3H]NA from rat frontal cortical slices were examined. GABA enhanced potassium-stimulated [3H]NA release, but did not alter release of [3H]NA evoked by the calcium ionophore A23187, 10(-5) M, either in the presence or the absence of extracellular calcium. The effect of GABA on potassium-stimulated [3H]NA release was apparently reduced by the GABAA antagonist bicuculline methiodide, 10(-4) M, and by the selective inhibitor of GABA uptake SKF 89976A, 10(-5) M, but was abolished only when bicuculline methiodide and SKF 89976A were present in combination. The GABAA agonist muscimol enhanced potassium-stimulated release of [3H]NA in a manner similar to GABA. In addition, nipecotic acid, a substrate for GABA uptake, enhanced potassium-stimulated [3H]NA release. Thus, GABA appears to enhance potassium-stimulated [3H]NA release by acting upon both GABA uptake and GABAA receptors. The GABAA receptors involved in this effect may be a subtype of GABAA receptors since they are not modulated by benzodiazepines. These results support the involvement of the GABA uptake carrier and the GABAA receptor in mediating the enhancement by GABA of potassium-stimulated [3H]NA release in the cortex of the rat.

Trimethyltin (TMT) produces selective hippocampal lesions similar to that caused by convulsants which interact with the brain excitatory amino acid transmission. The hippocampal glutamate system was studied because of its possible contribution to neuronal damage produced by TMT. At 24 hr following administration of TMT (3 mg/kg; ip) to mice, hippocampal glutamate levels were decreased, although no changes in levels were observed at 1 hr, 6 hr or 15 hr. In partially depolarized hippocampal slices, TMT (10 microM), produced a release of endogenous glutamate. Continuous monitoring by fluorometry found glutamate release to occur immediately following addition of TMT to partially depolarized slices; this release was dependent on extracellular calcium. These results indicate TMT can activate the endogenous excitotransmitter system, which may contribute to the neuronal damage associated with TMT exposure.

Antibodies that specifically recognize and bind opiate receptors were developed by the anti-idiotypic method. Antimorphine antibodies were generated in rabbits and showed selective binding to various opiate ligands. Guinea pigs were immunized with antimorphine antibodies to produce anti-idiotypic antibodies that cross-reacted with opiate receptors. Guinea pig antiserum inhibited binding of naloxone (0.6 nmol) to mouse brain homogenate. In isolated mouse vas deferens, contraction of electrically stimulated tissue was inhibited by the antiserum. The results indicate that anti-idiotypic antibodies which interact with opiate receptors were developed without isolating receptors.

AC glioma cells, a clonal cell line derived from a rat glioma, responded to 1 mM dibutyryl-cyclic AMP and isobutylmethylxanthine with a change to stellate morphology. A concentration-related morphological change was induced by beta 1- and beta 2-adrenergic agonists with the order of potency being isoproterenol greater than soterenol greater than norepinephrine. Propranolol (nonselective, beta-antagonist), butoxamine (beta 2-antagonist) and metoprolol (beta 1-antagonist) significantly decreased the cell response to isoproternol. Schild analysis of the response, using the competitive antagonist metoprolol, gave pA2 values of 7.5 and 8.5 for the agonists norepinephrine and soterenol, respectively, with slopes of the curves being less than unity. These observations indicate that both beta 1- and beta 2-adrenergic receptors mediate the change in cellular morphology.

The effects of central administration of (D-Ala2)methionine-enkephalinamide (EA) on respiration were studied in the unanesthetized rat. EA induced a dose-dependent depression of respiratory rate and tidal volume which was reversed by the subcutaneous administration of naloxone HCl (10 mg/kg). Increasing atmospheric CO2 concentration stimulated EA-induced respiratory depression and the peptide produced a parallel shift to the right of both the respiratory rate -- PaCO2 and tidal volume -- PaCO2 curves. Following chronic administration of EA tolerance to the respiratory depression was observed and chronic exposure to morphine produced partial tolerance to the respiratory depression induced by EA. It was concluded that EA desensitizes the central chemoreceptors to CO2 and the action of EA on respiration parallels that of morphine.

Previous studies have demonstrated that central administration of dibutyryl cyclic AMP (DBcAMP) produces a dose-dependent rise in blood pressure accompanied by tachycardia. In an attempt to delineate brain sites that mediate these cardiovascular responses, DBcAMP was stereotaxically injected into local brain areas. Injections of 100 micrograms into the right lateral, third, and fourth ventricles produced pressor responses. Perfusion of the brain from the lateral ventricle to the cisterna magna with 15 micrograms/25 microliters per min induced a rise in blood pressure and significant bradycardia. The cyclic nucleotide was injected unilaterally in doses of 10, 25, and 50 micrograms into the posterior hypothalamic nucleus (PHN), anterior hypothalamic area (AHA), locus coeruleus (LC), and nucleus tractus solitarii (NTS). PHN injections induced tachycardia with minimal changes in blood pressure. Bradycardia and a depressor response were observed following injection of the 10 micrograms dose into the LC. Within the AHA, DBcAMP (10 micrograms) did not induce significant changes in cardiovascular function. Additionally, injections into the NTS initiated a biphasic depressor-pressor effect. Within these sites, cardiovascular responses to the 10 micrograms dose of DBcAMP paralleled those observed after adrenergic activation. These results strongly suggest that cyclic AMP functions as a second messenger within select central cardiovascular regulatory sites and plays an important role in cardiovascular regulation.

The effects of the central administration (i.c.v.) of dibutyryl cyclic AMP (DBcAMP) on arterial blood pressure and heart rate were studied in the rat. The cyclic nucleotide produced a dose-dependent rise in blood pressure and an accompanying tachycardia. Maximal pressor effect of a single dose of DBcAMP (350 micrograms) was observed 25 min post injection and the duration of the response was 60 min. Chemical degeneration of central catecholaminergic neurons with 6-hydroxydopamine treatment abolished the pressor response to DBcAMP. Partial chemical degeneration of central serotonin pathways with 5,6-dihydroxytryptamine did not significantly alter the effect. The pressor activity of the cyclic nucleotide was attenuated by central administration of phentolamine. Spinal cord section at the C6-C7 level or peripheral chemical degeneration of catecholaminergic neurons combined with bilateral adrenalectomy abolished the pressor response to DBcAMP. It was concluded that the pressor response to i.c.v. administration of DBcAMP is mediated by central catecholaminergic pathways and is the result of an increase in efferent sympathetic outflow and release of catecholamines from the adrenal medulla.

Anti-idiotypic antibodies which recognize the opiate receptor were generated in guinea pigs following immunization against purified rabbit anti-morphine antibodies. The anti-idiotypic antibodies produced a concentration-dependent inhibition of [3H]naloxone binding to opiate receptors in a membranous mouse brain preparation. Saturation analysis indicated that the antibodies produced a non-competitive inhibition of naloxone binding. The ability of the antibodies to interact with biological systems was investigated in in vitro systems. In both the isolated guinea pig ileal longitudinal muscle and mouse vas deferens, the antibodies produced a concentration-dependent, opiate agonist-like action. The anti-morphine anti-idiotypic antibodies appear to interact specifically with the opiate receptor and may serve as useful tools in characterization of this receptor system.