Characterization of melittin effects in synaptosomes.

Abstract

The effects of melittin at increasing concentrations on: [3H]GABA release from mouse brain synaptosomes; on the radioactivity released from [3H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 microM progressively increases [3H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [3H]GABA induced by melittin below 3 microM is Ca2+ dependent, but not that induced by the higher concentrations. The Ca2+ dependent fraction of the [3H]GABA released by 0.3 microM melittin is selectively inhibited by 10 microM quinacrine and 1 microM nordihydroguaiaretic acid (NDGA) and facilitated by 3 microM indomethacin, whereas the Ca2+ independent fraction of the [3H]GABA released by melittin is not. In the presence of Ca2+, melittin 0.3, 1 and 10 microM progressively increases [3H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 microM treated synaptosomes, but a noticeable disorganization is produced in 10 microM melittin-treated synaptosomes, independently on the presence of external Ca2+. LDH activity only increases over control activity in the supernatant solutions of 10 microM melittin treated synaptosomes, also in a Ca2+ independent manner. Our interpretation of these results is that the Ca2+-dependent, pharmacologic sensitive component of melittin-induced release of [3H]GABA, unmasked when 0.3 microM melittin was used, involves the activation of a Ca2+-dependent type of membrane PLA2. The Ca2+-independent release of [3H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.