Hrynevich Sviatlana V, Pekun Tatyana G, Waseem Tatyana V, Fedorovich Sergei V
Laboratory of Biophysics and Engineering of Cell, Institute of Biophysics and Cell Engineering, Akademicheskaya St., 27, 220072, Minsk, Belarus.
Neurochem Res. 2015 Jun;40(6):1188-96. doi: 10.1007/s11064-015-1579-0. Epub 2015 Apr 17.
Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.
低血糖可导致神经元细胞死亡，类似于谷氨酸诱导的细胞死亡。在本文中，我们使用荧光染料DiSC3(5)和JC-1，研究了从孵育培养基中去除葡萄糖对大鼠脑突触体中线粒体和质膜电位变化的影响。我们还通过荧光染料吖啶橙监测了突触小泡中的pH梯度及其循环利用。发现葡萄糖剥夺会导致K(+)诱导的Ca(2+)依赖性胞吐作用受到抑制，线粒体和质膜电位向更正的值偏移。这些参数对因去除葡萄糖导致的能量不足的敏感性呈现以下顺序：线粒体膜电位>质膜电位>突触小泡中的pH梯度。与对照组相比，后者几乎不受剥夺的影响。使用pH依赖性染料吖啶橙来研究突触小泡的循环利用。然而，线粒体毒素鱼藤酮（10 µM）和寡霉素（5 µg/mL）的混合物也显示会增强该化合物的荧光。这意味着吖啶橙可能部分分布在线粒体的膜间隙中。从孵育培养基中去除葡萄糖导致吖啶橙对鱼藤酮+寡霉素的反应提高了3.7倍，表明线粒体pH梯度显著增加。我们的结果表明，在低血糖情况下，神经元突触前末梢的生物物理特性不利于过度的、不受控制的神经递质释放。胞吐作用的抑制和线粒体pH梯度的增加（同时保持小泡pH梯度）被认为是补偿机制。
