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.
Ray
P
P
Department of Biology, Walter Reed Army Institute of Research, Washington, D.C. 20067.
Berman
J D
JD
Middleton
W
W
Brendle
J
J
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