González L, Nekrassov V, Castell A, Sitges M
Instituto de Investigaciones Biomédicas, Dpto. de Biología Celular, UNAM and Instituto Mexicano de Psiquiatría, SSA (PUIS).
Neurochem Res. 1997 Feb;22(2):189-99. doi: 10.1023/a:1027319708321.
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.
从小鼠脑突触体释放的[³H]γ-氨基丁酸（GABA）；从[³H]花生四烯酸标记的突触体膜释放的放射性；突触体超微结构以及细胞质标志物乳酸脱氢酶（LDH）的泄漏情况。0.3、1、3、7和10微摩尔的蜂毒素可逐渐增加[³H]GABA的释放，但当暴露于恒定浓度毒素的组织量增加时，蜂毒素的效力会降低。低于3微摩尔的蜂毒素诱导的[³H]GABA释放是钙依赖性的，但较高浓度诱导的释放则不是。0.3微摩尔蜂毒素释放的[³H]GABA的钙依赖性部分被10微摩尔奎纳克林和1微摩尔去甲二氢愈创木酸（NDGA）选择性抑制，并被3微摩尔吲哚美辛促进，而蜂毒素释放的[³H]GABA的非钙依赖性部分则不然。在有钙离子存在的情况下，0.3、1和10微摩尔的蜂毒素相对于对照释放可逐渐增加[³H]花生四烯酸的释放，但随着组织量增加，蜂毒素的效力也会降低。在0.3微摩尔处理的突触体中未观察到突触体超微结构有明显变化，但在10微摩尔蜂毒素处理的突触体中会产生明显的结构紊乱，且与外部钙离子的存在无关。仅在10微摩尔蜂毒素处理的突触体的上清液中，LDH活性相对于对照活性增加，同样也是非钙依赖性的。我们对这些结果的解释是，当使用0.3微摩尔蜂毒素时揭示的蜂毒素诱导释放[³H]GABA的钙依赖性、药理学敏感成分涉及一种钙依赖性膜磷脂酶A2的激活。相比之下，[³H]GABA的非钙依赖性释放很可能是由于蜂毒素/脂质复杂相互作用产生的膜扰动所致。
