Linetska M V, Storchak L G, Himmelreich N H
Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Leontovich Street 9, Kiev 01601, Ukraine.
Neurochem Int. 2003 Jun;42(7):583-90. doi: 10.1016/s0197-0186(02)00158-4.
Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.
磷脂酰肌醇4,5 - 二磷酸参与了多种膜运输过程，包括神经递质的胞吐作用。然而，关于磷脂酰肌醇4 - 激酶抑制剂苯砷酸氧化物（PAO）影响不同类型神经递质胞吐释放能力的研究结果存在矛盾。我们致力于详细分析PAO对大鼠脑突触体暴露于不同浓度α - 银环蛇毒素时产生的钙依赖性和钙非依赖性[3H]GABA释放的作用。我们还比较了PAO对α - 银环蛇毒素和4 - 氨基吡啶（4 - AP）诱发的[3H]GABA释放的作用。实验表明，3 microM PAO作用后，4 - AP去极化诱发的[3H]GABA释放减少了80%，而10 microM PAO则完全抑制了释放。当使用α - 银环蛇毒素作为刺激物时，随着所用毒素浓度从0.5 nM升高到3.0 nM，[3H]GABA释放增加，但同时，毒素诱导的[3H]GABA释放对PAO的反应减弱：10 microM PAO几乎完全抑制了0.5 nMα - 银环蛇毒素的作用，而对3.0 nMα - 银环蛇毒素的反应仅部分降低（降低40%）。为了测试PAO的效力是否取决于毒素诱导的胞质[3H]GABA外流，还利用了胞质[3H]GABA池耗尽的突触体。通过用尼克酸进行胞质[3H]GABA的异源交换来实现耗尽。实验表明，用尼克酸处理负载的突触体导致0.5 nMα - 银环蛇毒素诱发的[3H]GABA释放有所增加，但对3.0 nMα - 银环蛇毒素的反应降低了两倍。无论使用何种α - 银环蛇毒素浓度，PAO基本上都能抑制耗尽的突触体中[3H]GABA的释放。因此，从胞质池中释放的[3H]GABA的量在很大程度上决定了α - 银环蛇毒素作用对PAO的不敏感性。因此，我们的数据表明，亚纳摩尔浓度的α - 银环蛇毒素可用于刺激[3H]GABA的胞吐释放。突触体暴露于纳摩尔浓度的毒素不仅会刺激胞吐作用，还会导致[3H]GABA从胞质池中泄漏。PAO能有效抑制[
