Accession Number : ADP008892

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

Corporate Author : WALTER REED ARMY INST OF RESEARCH WASHINGTON DC

Personal Author(s) : Ray, Prabhati ; Berman, Johnathan D. ; Middleton, Wilbert ; Brendle, James

Report Date : 13 MAY 1993

Pagination or Media 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.

Descriptors :   *METABOLITES, *NEUROTOXINS, *CLOSTRIDIUM BOTULINUM, *CHOLINERGIC NERVES, ACETYLCHOLINE, ACIDS, ADDITION, ANTIDOTES, BLOCKING, CELLS, COUNTERMEASURES, DRUGS, INCLUSIONS, INHIBITORS, MEMBRANES, MODELS, NERVES, NEUROTRANSMITTERS, PERFUSION, POISONING, POISONS, RELEASE, SEQUENCES, SYNTHESIS, TERMINALS, THREATS, CULTURES(BIOLOGY), IN VITRO ANALYSIS, TOXINS AND ANTITOXINS.

Subject Categories : Microbiology
      Medicine and Medical Research
      Biochemistry

Distribution Statement : APPROVED FOR PUBLIC RELEASE