Botulinum toxin inhibits arachidonic acid release associated with acetylcholine release from PC12 cells.
The molecular mechanisms of depolarization-induced calcium-dependent acetylcholine (ACh) release and its inhibition by botulinum neurotoxin type A (BoTx) are not clear. We studied these mechanisms in an in vitro cholinergic neuronal pheochromocytoma PC12 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ng/ml nerve growth factor (NGF) for 4 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 (three to four times) in [3H]ACh 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]AA) from the cell membrane, which was inhibited by BoTx (2 nM). Addition of phospholipase A2 (PLA2) inhibitors (quinacrine, 4-bromophenacyl bromide, manoalide) to the perfusion medium inhibited K(+)-stimulated [3H]ACh and [3H]AA release in a dose-dependent manner. Inclusion of exogenous AA, the PLA2 activator melittin, or PLA2 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.
Department of Biology, Walter Reed Army Institute of Research, Washington, D.C. 20067.
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Ray
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JD
Berman
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Middleton
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Brendle
D000098:Acetophenones / Q000494:pharmacology; D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001905:Botulinum Toxins / Q000494:pharmacology*; D002454:Cell Differentiation; D004305:Dose-Response Relationship, Drug; D004976:Ethacrynic Acid / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D009637:Masoprocol / Q000494:pharmacology; D008555:Melitten / Q000494:pharmacology; D008954:Models, Biological; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism; D054467:Phospholipases A2; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011796:Quinacrine / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology
D000098:Acetophenones; D009414:Nerve Growth Factors; D013729:Terpenes; D008555:Melitten; D016718:Arachidonic Acid; D009637:Masoprocol; C045673:manoalide; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D001905:Botulinum Toxins; D011796:Quinacrine; D004976:Ethacrynic Acid; D000109:Acetylcholine; C006821:4-bromophenacyl bromide; D011188:Potassium; D007213:Indomethacin