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Novel Insight into the Mechanism of Action of Botulinum Toxin in a Cholinergic Neuronal Cell Culture Model.

ADP008892

Publication Date	1993
Personal Author	Ray, P.; Berman, J. D.; Middleton, W.; Brendle, J.
Page Count	8
Abstract	Botulinum neurotoxin type A (BoTx), which is an identified biological threat agent acts by blocking the Ca 2(+) -dependent release of the neurotransmitter acetylcholine (ACh) from presynaptic nerve terminals at neuromuscular junctions. There is no effective antidote against this deadly poison primarily due to a lack of understanding of the exact sequence of events leading to the neuromuscular blockade. We studied this mechanism in an in vitro cholinergic neuronal pheochromocytoma PC 1 2 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ng/ml nerve growth factor (NGF) for 4 to 5 days to enhance cellular ACh synthesis and release. Stimulation of these cells with high K(+) (80 mM) in the perfusion medium caused a marked increase (3-4X) in 3Hach release in a Ca 2(+) -dependent manner. K(+)-stimulated (3H)ACh release was totally inhibited by pretreatment of cells with BoTx (2 nM) for 2 h. High K(+) also stimulated the release of arachidonic acid ((3H)ACh from the cell membrane, which was inhibited by BoTx (2 nM). Addition of quinacrine, a phospholipase A2 (PLA2) inhibitor, to the perfusion medium inhibited K(+)stimulated (3H)ACh and (3H)AA release in a dose-dependent manner. Inclusion of exogenous AA, the PLA(2), activator mellitin or PLA, itself prevented the effect of BoTx. These results demonstrate that in NGF differentiated PC12 cells, AA release is associated with ACh release, BoTx inhibits both processes, and increased AA can protect against BoTx. Drugs that can modulate PLA(2) activity or the level of intracellular AA or its metabolites may therefore be prospective countermeasures against BoTx poisoning.
Keywords	Clostridium botulinum Cholinergic nerves Acetylcholine Acids Antidotes Drugs Inhibitors Membranes Metabolites Models Nerves Neurotoxins Neurotransmitters Poisoning Sequences In vitro analysis Toxins and antitoxins Botulinum toxin Neurons Cultured cells PC12 cells
Source Agency	Non Paid ADAS
NTIS Subject Category	57Y - Toxicology 57F - Cytology, Genetics, & Molecular Biology 57K - Microbiology
Corporate Authors	Walter Reed Army Inst. of Research, Washington, DC.
Supplemental Notes	This article is from 'Proceedings of the Medical Defense Bioscience Review (1993) Held in Baltimore, Maryland on 10-13 May 1993. Volume 3', AD-A275 669, p1383-1390.
Document Type	Technical Report
NTIS Issue Number	199414

Novel Insight into the Mechanism of Action of Botulinum Toxin in a Cholinergic Neuronal Cell Culture Model.

Novel Insight into the Mechanism of Action of Botulinum Toxin in a Cholinergic Neuronal Cell Culture Model.
ADP008892

Publication Date	1993
Personal Author	Ray, P.; Berman, J. D.; Middleton, W.; Brendle, J.
Page Count	8
Abstract	Botulinum neurotoxin type A (BoTx), which is an identified biological threat agent acts by blocking the Ca 2(+) -dependent release of the neurotransmitter acetylcholine (ACh) from presynaptic nerve terminals at neuromuscular junctions. There is no effective antidote against this deadly poison primarily due to a lack of understanding of the exact sequence of events leading to the neuromuscular blockade. We studied this mechanism in an in vitro cholinergic neuronal pheochromocytoma PC 1 2 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ng/ml nerve growth factor (NGF) for 4 to 5 days to enhance cellular ACh synthesis and release. Stimulation of these cells with high K(+) (80 mM) in the perfusion medium caused a marked increase (3-4X) in 3Hach release in a Ca 2(+) -dependent manner. K(+)-stimulated (3H)ACh release was totally inhibited by pretreatment of cells with BoTx (2 nM) for 2 h. High K(+) also stimulated the release of arachidonic acid ((3H)ACh from the cell membrane, which was inhibited by BoTx (2 nM). Addition of quinacrine, a phospholipase A2 (PLA2) inhibitor, to the perfusion medium inhibited K(+)stimulated (3H)ACh and (3H)AA release in a dose-dependent manner. Inclusion of exogenous AA, the PLA(2), activator mellitin or PLA, itself prevented the effect of BoTx. These results demonstrate that in NGF differentiated PC12 cells, AA release is associated with ACh release, BoTx inhibits both processes, and increased AA can protect against BoTx. Drugs that can modulate PLA(2) activity or the level of intracellular AA or its metabolites may therefore be prospective countermeasures against BoTx poisoning.
Keywords	Clostridium botulinum Cholinergic nerves Acetylcholine Acids Antidotes Drugs Inhibitors Membranes Metabolites Models Nerves Neurotoxins Neurotransmitters Poisoning Sequences In vitro analysis Toxins and antitoxins Botulinum toxin Neurons Cultured cells PC12 cells
Source Agency	Non Paid ADAS
NTIS Subject Category	57Y - Toxicology 57F - Cytology, Genetics, & Molecular Biology 57K - Microbiology
Corporate Authors	Walter Reed Army Inst. of Research, Washington, DC.
Supplemental Notes	This article is from 'Proceedings of the Medical Defense Bioscience Review (1993) Held in Baltimore, Maryland on 10-13 May 1993. Volume 3', AD-A275 669, p1383-1390.
Document Type	Technical Report
NTIS Issue Number	199414