Phospholipaise A2 and arachidonic acid-mediated mechanism of neuroexocytosis: a possible target of botidinum neurotoxin A other then SNAP-25.
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.
Ray
P
P
Biology Department, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. ray@wrsmtp-ccmail.army.mil
Ishida
H
H
Millard
C B
CB
Petrali
J P
JP
Ray
R
R
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000502:physiology*; D016716:PC12 Cells; D010455:Peptides; D010741:Phospholipases A / Q000502:physiology*; D051381:Rats; D050825:Synaptosomal-Associated Protein 25; D014862:Wasp Venoms / Q000494:pharmacology
D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D014862:Wasp Venoms; D016718:Arachidonic Acid; C037500:mastoparan; D010741:Phospholipases A; D019274:Botulinum Toxins, Type A; D000109:Acetylcholine; D002118:Calcium