Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

Abstract

Leptinotarsin, a toxin found in the hemolymph of the beetle Leptinotarsa haidemani, can stimulate release of acetylcholine from synaptic termini. Leptinotarsin causes an increase in the frequency of miniature end plate potentials (mepps) of the rat phrenic nerve-diaphragm preparation. The increase in the frequency of mepps induced by leptinotarsin is biphasic: about 10% of the total mepps are released in an initial burst that lasts about 90 sec, after which the remaining mepps are released over a period of 10-20 min. Tetrodotoxin has no effect upon the release induced by leptinotarsin, but low-Ca2+ conditions abolish the first phase. The two phases of release may represent two presynaptic pools of acetylcholine, both of which can be released in quantized form. In a second study, rat brain synaptosomes were incubated with [3H]choline and were immobilized on Millipore filters. Leptinotarsin induced release of [3H]acetylcholine from this preparation, confirming the release seen by using neurophysiological methods. The ability of leptinotarsin to induce release from either intact nerve terminals or synaptosomes was abolished when the toxin was heated. The releasing activity of leptinotarsin from synaptosomes was also partially dependent upon the presence of Ca2+ in the perfusing solution. Release from synaptosomes followed first-order kinetics, and was not inhibited by commercial antibodies to black widow spider antigens. The data suggest that leptinotarsin acts as a presynaptic neurotoxin and may be of value as a mechanistic probe in understanding the storage and release of neurotransmitters.The mechanisms mediating the release of neurotransmitter have not been resolved. Very little is known about the sequence of events after the entrance of Ca2+ into the presynaptic terminal and the eventual release of neurotransmitter. One approach to this problem has been the identification of toxins that interact with the presynaptic terminal to modify the process of release. By examining the mechanism of action of the toxin, as well as the mechanism of the step(s) upon which the toxin acts, a more detailed description of the process of release may be obtained.Extracts of black widow spider venom glands (BWGE) will stimulate a massive increase in the frequency of miniature end plate potentials (mepps) from the frog neuromuscular junction (1). When Ca2+ is removed from the perfusing medium, BWGE were still able to stimulate release, although at reduced rates, in a variety of preparations (1-4). These data suggest that the action of BWGE was not simply to depolarize the membrane, thereby allowing Ca2+ to enter and elicit release of acetylcholine (AcCho), but was a more specific interaction with the release mechanism. Purification (5,6) indicates that the protein responsible for the releasing activity in BWGE could not be recovered in large amounts, and suggests that the lack of availability of pure protein may limit the usefulness of the toxin as a mechanistic probe. The present study was directed towards the search for a...

Beetle and plant arrow poisons of the Ju|’hoan and Hai||om San peoples of Namibia (Insecta, Coleoptera, Chrysomelidae; Plantae, Anacardiaceae, Apocynaceae, Burseraceae)

“…Because of the high toxin concentration in the “pupa” compared to that in the adult, De la Harpe et al (1983) suggested that diamphotoxin must have some functional role in this life stage, but it was unclear how autolysis was prevented. Diamphotoxin protein may be similar to toxic insect proteins found in tiger moths (Rothschild et al 1970; Hsiao et al 1980) and to leptinotarsin from the beetle, Leptinotarsa Chevrolat (Hsiao and Fraenkel 1969; Snyder 1971; Parker 1971, 1972; Satin et al 1978; Hsiao 1978; McClure et al 1980; Madeddu et al 1985a, b; Miljanich et al 1988). Leptinotarsa (subfamily Chrysomelinae) is phylogenetically distantly related to the galerucine Blepharida -group.…”

Calcium channels: Molecular pharmacology, structure and regulation

“…Beta-leptinotarsin-D and -H, polypeptide neurotoxins isolated from the hemolymph of Colorado potato beatles, also have been proposed to act at Ca channels because they stimulated Ca-dependent release of nenrotransmitters from mammalian nerve terminals, pheochromocytoma cells and neuromuscular junctions (Yoshino, Baxter & McClure, 1980;Crosland, Hsiao & McClure, 1984;Madedder et al, 1985;McClure et al, 1985). A comparison of the characteristics of transmitter release evoked by depolarization or leptinotarsins led to the proposal that the toxins are direct activators of presynaptic voltage-sensitive Ca channels (McClure et al, 1985). Other toxins that are potential Ca channel activators include a polypeptide toxin that has recently been isolated from Goniopora coral , and atrotoxin, a polypeptide partially purified from snake venom .…”

Leptinotoxin‐h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release

“…, 1980; Yoshino et al, 1980; Crosland et al, 1984). Since at the neuromuscular junction the toxin-induced release of acetylcholine w a s quantized, it was assumed t o be due t o stimulated discharge of synaptic vesicles (exocytosis) (McClure et al, 1980). Here we report on the secretory responses induced by LPTx in brain cortex synaptosomes of the guinea pig and in PC12 cells, investigated by both biochemical measurements and electron microscopy.…”

“…The ultimate effect of LPTx on synapses (up t o now investigated only in preparations of the rat diaphragm neuromuscular junction a n d brain cortex synaptosomes) consists of a massive stimulation of neurotransmitter release (McClure e t a]. , 1980; Yoshino et al, 1980; Crosland et al, 1984). Since at the neuromuscular junction the toxin-induced release of acetylcholine w a s quantized, it was assumed t o be due t o stimulated discharge of synaptic vesicles (exocytosis) (McClure et al, 1980).…”