CHARACTERIZATION OF MELITTIN EFFECTS IN SYNAPTOSOMES

The effects of melittin at increasing concentrations on: [H-3]GABA rel ease from mouse brain synaptosomes; on the radioactivity released from [H-3]arachidonic acid labelled synaptosomal membranes; on synaptosome s ultrastructure and on the leakage of the cytoplasmic marker, lactate -dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 m u M progressively increases [H-3]GABA release, but the efficacy of mel ittin is decreased when the amount of tissue exposed to a constant con centration of the toxin increases. The release of [H-3]GABA induced by melittin below 3 mu M is Ca2+ dependent, but not that induced by the higher concentrations. The Ca2+ dependent fraction of the [H-3]GABA re leased by 0.3 mu M melittin is selectively inhibited by 10 mu M quinac rine and 1 mu M nordihydroguaiaretic acid (NDGA) and facilitated by 3 mu M indomethacin, whereas the Ca2+ independent fraction of the [H-3]G ABA released by melittin is not. Ln the presence of Ca2+, melittin 0.3 , 1 and 10 mu M progressively increases [H-3]arachidonic acid release over control release, but the effectiveness of melittin is also decrea sed as the amount of tissue increases. No apparent changes in synaptos omes ultrastructure are observed in 0.3 mu M treated synaptosomes, but a noticeable disorganization is produced in 10 mu M melittin-treated synaptosomes, independently on the presence of external Ca2+. LDH acti vity only increases over control activity in the supernatant solutions of 10 mu M 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 [H- 3]GABA, unmasked when 0.3 mu M melittin was used, involves the activat ion of a Ca2+-dependent type of membrane PLA(2). The Ca2+-independent release of [3H]GABA is in contrast, highly probable to be due to the m embrane perturbation produced by complex melittin/lipid interactions.