The Dep. of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine, 9 Leontovicha St., Kiev 01601, Ukraine.
Neurochem Int. 2009 Dec;55(8):724-31. doi: 10.1016/j.neuint.2009.07.003. Epub 2009 Jul 22.
Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO ( approximately 10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[(14)C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca(2+) or Mg(2+)/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.
在暴露于超重力（10G，1 小时）的大鼠脑突触体中，研究了谷氨酸释放和突触小泡异型/同型融合。与对照相比，超重力条件下，刺激的囊泡胞吐作用（通过 pH 敏感染料吖啶橙（AO）的 KCl 诱发荧光尖峰来确定）降低了两倍。一系列测量结果表明，突触小泡积累 AO 的能力降低（AO 荧光的稳态基线水平约高 10％）。用预先加载的 l-[(14)C]谷氨酸进行的实验表明，突触小体积累的总谷氨酸量相同，环境谷氨酸浓度相同，但在超重力下从通透的去氧胆酸钠处理的突触小体中测量的细胞质谷氨酸水平增大。因此，可以认为+G 诱导的刺激的囊泡胞吐作用变化是细胞内谷氨酸池重新分布的结果，即突触小泡中谷氨酸含量降低和细胞质谷氨酸水平富集。为了研究超重力对胞吐作用的最后一步即膜融合的影响，使用了由大鼠脑突触体分离的突触小泡、质膜小泡和胞质蛋白组成的无细胞系统。发现超重力降低了突触小泡和质膜小泡的融合能力，而突触小体胞质蛋白变得更加活跃，以促进膜融合。在超重力条件下，由 Ca(2+)或 Mg(2+)/ATP 引发的同种型和异型融合反应的总速率保持不变。因此，超重力可能会引起突触病，其特征是神经递质不完全填充突触小泡和胞吐释放的变化。
