Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes

Abstract

The presence of P-type calcium channels in synaptosomes prepared from electric organ of Torpedo marmorata was investigated by using the venom of Agelenopsis aperta, a toxin purified from it, FTX, and its synthetic analog. We analysed the action of these agents on acetylcholine release which was continuously followed using a chemiluminescent assay. Agelenopsis aperta venom, FTX and synthetic FTX inhibit acetylcholine release from synaptosomes induced by a presynaptic membrane depolarization with 60 mM KCl. A stronger inhibition of acetylcholine release was observed with the venom than with FTX (70 and 50%, respectively). Another way of triggering acetylcholine release from Torpedo synaptosomes is to insert in the presynaptic membrane a calcium ionophore A23187 which allows the bypass of the natural calcium channels. The venom of Agelenopsis aperta inhibits A23187-evoked acetylcholine release. Purified and synthetic FTX does not possess this property, suggesting that this inhibition of acetylcholine release was due to other toxins of the venom. Another type of pharmacological sensitivity of Torpedo calcium channels was also demonstrated using ω-conotoxin GVIA. At a concentration of 20 μM, this toxin was able to inhibit about 35% of KCl-evoked acetylcholine release. When FTX + ω-conotoxin GVIA were applied together, the inhibitory effect on KCl-evoked acetylcholine release was not significantly increased in comparison with the one observed with FTX alone. In conclusion, we examined the effect of different agents on acetylcholine release from Torpedo marmorata electric organ synaptosomes; acetylcholine release was elicited with KCl depolarization and followed continuously with a chemiluminescent assay. It allowed us to distinguish different calcium fluxes in Torpedo synaptosomes. The existence of P-type voltage-dependent calcium channels was supposed upon observing the inhibitory effect on KCl-evoked acetylcholine release of the venom of Agelenopsis aperta, of FTX (a purified toxin), and of a synthetic FTX. Moreover acetylcholine release from Torpedo synaptosomes was moderately inhibited by ω-conotoxin GVIA, a blocker of N-type calcium channels.