Specificity of action of beta-bungarotoxin on acetylcholine release from synaptosomes

Presynaptically acting phospholipase A2 (PLA2) neurotoxins such as beta-bungarotoxin (beta-BuTX) specifically modify the release of acetylcholine (ACh) in the periphery, whereas in the central nervous system (CNS) the release of other neurotransmitters such as norepinephrine (NE) and serotonin (5-HT) are also modified. In addition, ACh release in the periphery is modified in a triphasic manner (decrease, then increase, then block), while in the CNS only the increase has been demonstrated. To determine the specificity of the central effects of beta-BuTX we compared the effects of beta-BuTX and N. n. atra PLA2 on the release from rat cerebrocortical synaptosomes of ACh, NE, and 5-HT. We also measured the leakage of lactate dehydrogenase (LDH) in order to determine whether membrane permeablization was responsible for neurotransmitter leakage. Both the PLA2 neurotoxin (5.0 nM) and the non-neurotoxic enzyme (0.5 nM) stimulated the loss of NE and 5-HT, but only at concentrations which induced leakage of LDH. Conversely, beta-BuTX stimulated the release of ACh at a concentration (0.5 nM) which caused no leakage of LDH, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release. beta-Bungarotoxin thus exerts a specific effect on cholinergic nerve terminals, while the leakage of NE and 5-HT induced by beta-BuTX and N. n. atra PLA2 correlates with membrane disruption due to their PLA2 activities. Within 20 min, 0.5 nM beta-BuTX increased the resting release of ACh and decreased the stimulated release induced by depolarization with 4-aminopyridine, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release and required 45 min to exert an inhibitory effect. beta-BuTX (5.0 nM) also exerted an inhibitory effect on ACh release stimulated by veratridine, but not by high KCl. It is concluded that in low concentrations that do not disrupt membrane permeability, beta-BuTX acts specifically on cholinergic terminals in rat synaptosomes, where it exerts both stimulatory and inhibitory effects.