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Phospholipaise A2 and arachidonic acid‐mediated mechanism of neuroexocytosis: a possible target of botidinum neurotoxin A other then SNAP‐25
Author(s) -
Ray P.,
Ishida H.,
Millard C. B.,
Petrali J. P.,
Ray R.
Publication year - 1999
Publication title -
journal of applied toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.784
H-Index - 87
eISSN - 1099-1263
pISSN - 0260-437X
DOI - 10.1002/(sici)1099-1263(199912)19:1+<s27::aid-jat610>3.0.co;2-a
Subject(s) - mastoparan , exocytosis , acetylcholine , synaptic vesicle , vesicle , neurotoxin , chemistry , microbiology and biotechnology , phospholipase a2 , biochemistry , biophysics , biology , g protein , membrane , endocrinology , signal transduction , enzyme
The vesicular neuroexocytosis process consists of two important steps: fusion of transmitter‐loaded vesicles at release sites on the presynaptic nerve terminal membrane; followed by the release of transmitter molecules into the synaptic cleft. We previously reported that in nerve growth factor (NGF)‐differentiated PC12 cells, arachidonic acid (AA) release is associated with acetylcholine (ACh) release, botulinum neurotoxin A (BoNT/A) inhibits both processes and AA itself or a phospholipase A 2 (PLA 2 ) activator can cause ACh release in BoNT/A‐poisoned cells in which SNAP‐25 has supposedly been hydrolyzed. In the present study, we examined the roles of two endogenous intraterminal components in neuroexocytosis: the membrane fusogenic agent AA; and the vesicle fusion protein SNAP‐25. A PLA 2 activator, mastoparan, was used to induce the release of AA and ACh from NGF‐differentiated PC12 cells. Release depended upon the mastoparan concentration, as well as Ca 2+ influx via the neuronal‐type voltage‐sensitive Ca 2+ channels. Release of ACh followed a rise in intracellular free Ca 2+ concentration; the increased Ca 2+ activated PLA 2 and, thereby, increased the AA level. Scanning and transmission electron microscopy confirmed that mastoparan‐induced ACh and AA release were not due to simple diffusion through damaged plasma membranes. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP‐25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP‐25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K + induced ACh exocytosis. The results support the conclusion that PLA 2 and AA have important roles in neuroexocytosis that are independent of SNAP‐25. Both PLA 2 and AA have been shown to be involved in actin cytoskeletal organization related to vesicle fusion and exocytosis. This mechanism may be an alternative target of BoNT/A other than SNAP‐25.