Calcium-activated NO production plays a role in neuronal death induced by β-bungarotoxin in primary cultures of cerebellar granular neurons

Wen-Pei Tseng, Shoei Yn Lin-Shiau

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Abstract

The aim of this study was to elucidate the mechanism underlying the neurotoxic effect of β-bungarotoxin (β-BuTX) on cultured cerebellar granular neurons (CGN). β-BuTX had a potent time- and concentration-dependent neurotoxic effect on mature CGN. β-BuTX appeared to destroy initially the neurites and then caused neuronal death by both apoptotic and necrotic processes. Inspection using Nomarski optics showed that these neurons displayed morphological features of necrotic cells, including cell swelling, loss of membrane integrity and eventual dissolution of the cell. Staining with the fluorescent dye Hoechst 33258 showed that β-BuTX-treated neuron bodies stained more densely with smaller apoptotic bodies. Using microspectrofluorimetry and fura-2 to measure cytosolic [Ca2+] ([Ca2+]i), β-BuTX markedly increased [Ca2+]i. BAPTA-AM, EGTA, MK 801 and diltiazem not only attenuated the β-BuTX-mediated rise in [Ca2+]i but also attenuated β-BuTX-mediated neurotoxicity. In addition, these Ca2+ inhibitors prevented the β-BuTX-induced generation of reactive nitrogen species. The NO synthase inhibitor NG-methyl-L-arginine) also exhibited neuroprotection. This is the first report showing that β-BuTX-induced CGN death is mediated, at least in part, by excessive generation of NO triggered by [Ca2+]i overloading. Activation of NMDA receptors and L-type calcium channels is apparently involved in the increase in [Ca2+]i induced by this neurotoxin. This potent neurotoxin will be a useful tool for studying neurotoxic processes and using this model system will allow us to find neuroprotective agents.

Original languageEnglish
Pages (from-to)451-461
Number of pages11
JournalNaunyn-Schmiedeberg's Archives of Pharmacology
Volume367
Issue number5
DOIs
Publication statusPublished - 2003 May 1

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Bungarotoxins
Calcium
Neurons
Neurotoxins
Bisbenzimidazole
Reactive Nitrogen Species
L-Type Calcium Channels
Dizocilpine Maleate
Diltiazem
Fura-2
Egtazic Acid
Neuroprotective Agents
Neurites
N-Methyl-D-Aspartate Receptors
Fluorescent Dyes
Nitric Oxide Synthase
Arginine
Staining and Labeling
Membranes

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

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abstract = "The aim of this study was to elucidate the mechanism underlying the neurotoxic effect of β-bungarotoxin (β-BuTX) on cultured cerebellar granular neurons (CGN). β-BuTX had a potent time- and concentration-dependent neurotoxic effect on mature CGN. β-BuTX appeared to destroy initially the neurites and then caused neuronal death by both apoptotic and necrotic processes. Inspection using Nomarski optics showed that these neurons displayed morphological features of necrotic cells, including cell swelling, loss of membrane integrity and eventual dissolution of the cell. Staining with the fluorescent dye Hoechst 33258 showed that β-BuTX-treated neuron bodies stained more densely with smaller apoptotic bodies. Using microspectrofluorimetry and fura-2 to measure cytosolic [Ca2+] ([Ca2+]i), β-BuTX markedly increased [Ca2+]i. BAPTA-AM, EGTA, MK 801 and diltiazem not only attenuated the β-BuTX-mediated rise in [Ca2+]i but also attenuated β-BuTX-mediated neurotoxicity. In addition, these Ca2+ inhibitors prevented the β-BuTX-induced generation of reactive nitrogen species. The NO synthase inhibitor NG-methyl-L-arginine) also exhibited neuroprotection. This is the first report showing that β-BuTX-induced CGN death is mediated, at least in part, by excessive generation of NO triggered by [Ca2+]i overloading. Activation of NMDA receptors and L-type calcium channels is apparently involved in the increase in [Ca2+]i induced by this neurotoxin. This potent neurotoxin will be a useful tool for studying neurotoxic processes and using this model system will allow us to find neuroprotective agents.",
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AB - The aim of this study was to elucidate the mechanism underlying the neurotoxic effect of β-bungarotoxin (β-BuTX) on cultured cerebellar granular neurons (CGN). β-BuTX had a potent time- and concentration-dependent neurotoxic effect on mature CGN. β-BuTX appeared to destroy initially the neurites and then caused neuronal death by both apoptotic and necrotic processes. Inspection using Nomarski optics showed that these neurons displayed morphological features of necrotic cells, including cell swelling, loss of membrane integrity and eventual dissolution of the cell. Staining with the fluorescent dye Hoechst 33258 showed that β-BuTX-treated neuron bodies stained more densely with smaller apoptotic bodies. Using microspectrofluorimetry and fura-2 to measure cytosolic [Ca2+] ([Ca2+]i), β-BuTX markedly increased [Ca2+]i. BAPTA-AM, EGTA, MK 801 and diltiazem not only attenuated the β-BuTX-mediated rise in [Ca2+]i but also attenuated β-BuTX-mediated neurotoxicity. In addition, these Ca2+ inhibitors prevented the β-BuTX-induced generation of reactive nitrogen species. The NO synthase inhibitor NG-methyl-L-arginine) also exhibited neuroprotection. This is the first report showing that β-BuTX-induced CGN death is mediated, at least in part, by excessive generation of NO triggered by [Ca2+]i overloading. Activation of NMDA receptors and L-type calcium channels is apparently involved in the increase in [Ca2+]i induced by this neurotoxin. This potent neurotoxin will be a useful tool for studying neurotoxic processes and using this model system will allow us to find neuroprotective agents.

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