Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-di-hydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+ -containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 x 10-11 M for synaptosomes, and 1.5 x 10-10 M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+ -free, EGTA-containing medium, provided that high (in the 10-10 M range) concentrations were used; near-maximal responses were observed at 10-5 M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 x 10-5 M Ca2+, and was maximal at 2 x 10-4 M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modifiy the release induced by the toxin in Ca2+ -containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the CA2+ - containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+ -free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, acculmulation of multivesicular bodies, mitochondrial alternations, and decreased number of secretion granules (Ca2+ -containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with α-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites. Because of its profound and exclusively presynaptic effects LPTx promises to be an interesting tool for study of the regulation of exocytotic neurotransmitter release.
|Number of pages||12|
|Journal||Journal of Neurochemistry|
|Publication status||Published - 1985|
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience