Biomedical subjects

G E Isom

Publications and source records attributed to G E Isom.

At least 55 records · Page 3Linked to original sources

PubMed1994

Cyanide induces protein kinase C translocation: blockade by NMDA antagonists.

Activation and translocation of protein kinase C (PKC) during KCN-induced histotoxic hypoxia was studied in rat brain slices prepared from cerebellum, hippocampus, and cortex. Treatment with 1-10 mM KCN produced a significant increase in PKC translocation and enzyme activity in the particulate fraction of cerebellar and hippocampal slices. In cortical slices, PKC activity was not affected by cyanide treatment. The membrane-associated PKC activity reached a maximum 30 minutes after incubation with KCN and remained elevated up to 60 minutes in both the hippocampus and cerebellum. Pretreatment with MK-801 and APV, specific NMDA receptor antagonists, blocked the cyanide-stimulated translocation in the hippocampus and cerebellum, whereas CNQX, an AMPA/kainate receptor antagonist, did not alter the response. These results demonstrate that cyanide stimulates PKC activation and translocation from the cytosol to membranes in select brain areas and NMDA receptor activation mediates this process.

2-Amino-5-phosphonovalerate↗

PubMed1994

Activation of a calcium- and pH-dependent phospholipase A2 by cyanide in PC12 cells.

Previous studies suggested that alterations in phospholipid composition of plasma membranes may contribute to neuronal injury associated with cyanide-induced histotoxic hypoxia. This prompted a study of the effects of KCN on phospholipase A2 (PLA2), an enzyme which catalyzes breakdown of membrane phospholipids. PLA2 activity was measured by quantitating the release of [3H]arachidonic acid ([3H]AA) from rat pheochromocytoma (PC12) cells. KCN produced a time (1-15 min)- and concentration (0.5-10 mM)-dependent release of [3H]AA from the cells. When cells were incubated in Ca(2+)-free buffer, KCN (5 mM) was still able to release [3H]AA. In cells loaded with BAPTA, an intracellular Ca2+ chelator, cyanide-induced release of [3H]AA was blocked, indicating that mobilization of intracellular Ca2+ can activate the enzyme. The PLA2 inhibitors dibucaine (50 microM) and mepacrine (50 microM) inhibited KCN-mediated [3H]AA release. Incubation of PC12 cells in an extracellular pH of 6.50 reduced the KCN effect, whereas incubation at pH 7.90 enhanced [3H]AA release. These data indicate that in PC12 cells KCN activates a Ca(2+)- and pH-dependent PLA2 which may contribute to cyanide-induced cell damage.

Animals↗

PubMed1994

Dopaminergic neurotoxicity of cyanide: neurochemical, histological, and behavioral characterization.

Previous reports have shown that dopamine (DA) is depleted in the brains of animals treated with cyanide. To develop a model for studying the mechanisms of cyanide-induced changes in dopaminergic systems, mice were treated with cyanide (KCN, 6 mg/kg, sc) twice a day for 7 days and 16 hr after the last dose neurochemical, histological, or behavioral parameters were evaluated. DA levels in KCN-treated animals decreased in the striatum (41%), hippocampus (30%), and cerebral cortex (13%) as compared to saline-treated controls. In striatal and hippocampal tissues, but not in cerebral cortex, malondialdehyde levels increased 43 and 57%, respectively, as compared to controls, indicating that peroxidation of lipids occurred in these brain areas. Over 30% of the treated mice exhibited decreased locomotor activity and akinesia, which were suppressed by l-DOPA (100 mg/kg, ip). Tyrosine hydroxylase (TH) immunohistochemical examination of brains from cyanide-treated animals showed a reduced number of TH-positive cells in substantia nigra, indicating a loss of dopaminergic neurons. In contrast, acute cyanide (KCN, 6 mg/kg, sc) did not produce significant neurochemical or behavioral changes. Under these treatment conditions, cyanide produces a central dopaminergic toxicity which is characterized by decreased DA levels in select brain areas, impaired locomotor activity, and neuronal damage.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1994

Accumulation of labeled cyanide in neuronal tissue.

Since cyanide is a reactive chemical substance and has the potential of forming a variety of adducts in biological systems, the rate of accumulation of labeled cyanide was studied in neural tissue, a major target for the toxic action of cyanide. Accumulation of 14CN in mouse brain slices and in rat pheochromocytoma (PC12) cells was about five times more rapid in the first few minutes than at later times. In both tissues, incubation at low temperature (4 degrees C) decreased the secondary phase without affecting the initial phase. In PC12 cells, determination of the subcellular distribution of cyanide revealed that the cytoplasmic fraction was the least sensitive to temperature and therefore may represent the initial rapid phase of cyanide accumulation. Accumulation of cyanide in mitochondrial and microsomal fractions was temperature sensitive. Cyanide accumulation is proportional to the concentration in the medium (0.1-1 mM) in both mouse brain and PC12 cells suggesting that the cyanide sinks in neural tissue have a large capacity and are nonspecific. Cyanide interaction with neural tissue is not uniform since cyanide accumulated more in hypothalamus than in other brain areas. Despite this, mitochondrial enzyme activity was no greater in hypothalamus than in other brain areas. No increase in thiocyanate, the main metabolite of cyanide, could be detected in brain tissue incubated 30 min with 1 mM cyanide, showing that accumulated 14C is not in the form of thiocyanate. Cyanide appears to equilibrate rapidly across the plasma membrane and then slowly accumulates in mitochondria and membrane elements of the neuronal cell. Competition for cyanide among subcellular elements may be a factor in the toxic action of cyanide.

Analysis of Variance↗

PubMed1994

MK-801 prevents cyanide-induced changes of Fos levels in rat brain.

The effect of acute cyanide intoxication on levels of transcriptional regulatory proteins Fos and c-Jun in rat cortex, hippocampus, cerebellum and brain stem was studied. Western blot analysis showed a differential effect of cyanide on Fos levels in the selected brain areas. The most prominent changes were seen 60 min. following ip. injection of KCN in all brain areas except the brain stem, which showed the maximal change 120 min. following cyanide. Fos levels were doubled in cortex and cerebellum and decreased to below 70% of the control levels in hippocampus. Levels of c-Jun were not altered 60 min. following cyanide treatment. Pretreatment with the NMDA receptor antagonist, MK-801, prevented the cyanide-induced changes of Fos. The differential effect of cyanide on Fos levels in different brain areas and the blockade of these changes by MK-801 suggest involvement of multiple neuronal pathways, including the excitatory amino acid (EAA) neurotransmitter system. It is concluded that cyanide alters levels of the transcriptional regulatory protein Fos through activation of the EAA neurotransmitter system and, thus, may affect gene expression in neuronal or glia cells.

Animals↗

PubMed1994

Enhancement of NMDA-mediated responses by cyanide.

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1-5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Interaction of cyanide with a dopamine metabolite: formation of a cyanohydrin adduct and its implications for cyanide-induced neurotoxicity.

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.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1993

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.

Animals↗

PubMed1993

Lysosomal delivery of ANP receptors following internalization in PC12 cell.

Internalization and intracellular processing of ANP-B and C receptors play an important role in regulating cell responsiveness to atrial natriuretic peptides (ANP). Receptor internalization was indirectly monitored with 125I labelled ligand. When 125I-ANP(99-126) was internalized by the cells at 37 degrees C, 55% of the internalized radioactivity was localized in the lysosomal fraction. When receptors were affinity-labelled with 125I-ANP(99-126) and allowed to internalize for varying time periods, two radiolabelled proteins in the m.wt range of 56 and 52 KDa were detected in the cytosolic extract. These proteins appear to be the hydrolytic products of the ANP-C receptor expressed on the plasma membrane. In addition to lysosomal delivery, shedding of the ANP-C receptor from the cell surface was detected following incubation of cells with 125I-ANP(99-126). The dual processes may function to clear exogenous ANP from the extracellular compartments.

Animals↗

PubMed1993

N-methyl-D-aspartate receptors mediate cyanide-induced cytotoxicity in hippocampal cultures.

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.

2-Amino-5-phosphonovalerate↗

PubMed1992

Interaction of cadmium with atrial natriuretic peptide receptors: implications for toxicity.

Atrial natriuretic peptide (ANP) is a diuretic and vascular smooth muscle relaxant which plays a pivotal role in cardiovascular regulation. Since cadmium (Cd) produces cardiovascular toxicity and alters ANP levels in atria and hypothalamus, its effect on ANP receptors were studied in rats and in PC12 cells exposed to Cd. Male rats were injected with CdCl2 (0.01, 0.1, 0.5 or 1.0 mg/kg, i.p.) twice a day for 7 days and then maintained for a period of 30 days. On experimental day 37 ANP receptor binding in the adrenal cortex, aorta and kidney cortex was studied by saturation isotherm analysis. In Cd-treated animals a non-dose related decrease in receptor affinity and density was observed in the kidney and aorta with the aortic ANP receptors being the most sensitive. Cellular regulation of the receptor was studied in PC12 cells, a cell line that expresses functional ANP receptors. Incubation of PC12 cells with Cd reduced both the affinity of the receptor for ANP and decreased the number of binding sites on the cell plasma membrane. The ratio of ligand-receptor complex internalized in the cell to ligand bound to the plasma membrane was significantly decreased following Cd pretreatment (500 microM). A significant decrease in the internalization rate of [125I]ANP was observed in cells incubated concurrently with Cd and ligand. In photoaffinity labelling studies with [125I]ANP, binding of ANP to B and C receptors subtypes was decreased following treatment of either intact cells or plasma membranes with Cd. It was concluded that Cd produces significant alterations in the ANP receptor, both in in vitro and in vivo models and it is proposed these effects play a role in the cardiovascular toxicity of this heavy metal.

Animals↗

PubMed1992

Potentiation of cyanide neurotoxicity by blockade of ATP-sensitive potassium channels.

Exposure of primary hippocampal cultures to NaCN (2 mM) or glyburide (5 microM) alone for 3 h did not produce a rise in extracellular lactic dehydrogenase (LDH) activity. Coincubation with NaCN and glyburide produced a significant efflux of LDH from the neurons. Diazoxide or D-2-amino-5-phosphovalerate (APV) partially reversed the release of LDH by the combination of NaCN and glyburide. These observations indicate ATP-sensitive potassium channels (KATP) are activated by nonlethal concentrations of cyanide and their blockade with glyburide unmasks cyanide's toxicity. The cytotoxicity of cyanide appears to result from a combination of processes resulting in altered ion handling and excitotoxicity.

Adenosine Triphosphate↗

PubMed1992

Modulation of hypothalamic norepinephrine release by atrial natriuretic peptide: involvement of cyclic GMP.

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.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Blockade of N-methyl-D-aspartate receptors prevents cyanide-induced neuronal injury in primary hippocampal cultures.

Cyanide-induced alterations of cytosolic calcium levels and cytotoxicity were examined in primary cultures of rat hippocampus. Cytosolic free Ca2+ ([Ca2+]i) levels were measured in hippocampal neurons using the fluorescent probe, fura 2. A concentration-dependent rise in [Ca2+]i occurred rapidly following exposure of cells to 0.5-10 mM NaCN. In normal medium (1.3 mM Ca2+), 2 mM NaCN produced an increase in [Ca2+]i (172 +/- 27% of control), 45 sec following exposure. Ca2+ elevation produced by NaCN was blocked by removal of Ca2+ from the external medium or by pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovalerate (APV). The cytotoxicity of cyanide, assessed by measuring the efflux of lactate dehydrogenase, was blocked by APV. These results indicate that in hippocampal neurons, cytosolic Ca2+ accumulation induced by cyanide originates from the extracellular compartment and the NMDA receptor ionophore is a significant route for Ca2+ entry. It is proposed that excitotoxic mechanisms may contribute to altered neuronal homeostasis and injury associated with cyanide.

2-Amino-5-phosphonovalerate↗

PubMed1991

Gamma-aminobutyric acidA (GABAA) receptor modulation of morphine inhibition of norepinephrine release.

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.

Animals↗

PubMed1991

Cyanide induces Ca(2+)-dependent and -independent release of glutamate from mouse brain slices.

The effect of cyanide-induced histotoxic hypoxia on endogenous glutamate release from mouse cortical, cerebellar and hippocampal slices was studied. Incubation of slices with cyanide over a 30 min period resulted in extracellular accumulation of glutamate which was decreased in the absence of Ca2+ in the incubation media. When glutamate release was continuously monitored by fluorometry, cyanide initiated a rapid release of glutamate. This initial release was found to be independent of extracellular calcium. Depolarizing concentrations of potassium chloride produced a predominantly Ca(2+)-dependent release. It is concluded that cyanide exposure induced a rapid release of endogenous glutamate mediated by both Ca(2+)-dependent and Ca(2+)-independent mechanisms.

Animals↗

PubMed1991

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.

3,4-Dihydroxyphenylacetic Acid↗