Tarasenko Alla, Krupko Olga, Himmelreich Nina
Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Leontovich Str. 9, Kyiv 01601, Ukraine.
Biochim Biophys Acta. 2014 Jun;1840(6):1923-32. doi: 10.1016/j.bbagen.2014.01.030. Epub 2014 Jan 28.
Nitric oxide (NO) is an important presynaptic modulator of synaptic transmission. Here, we aimed to correlate the release of the major inhibitory neurotransmitter GABA with intracellular events occurring in rat brain axon terminals during their exposure to NO in the range of nanomolar-low micromolar concentrations.
Using [(3)H]GABA and fluorescent dyes (Fluo 4-AM, acridine orange and rhodamine 6G), the following parameters were evaluated: vesicular and cytosolic GABA pools, intracellular calcium concentration, synaptic vesicle acidification, and mitochondrial membrane potential. Diethylamine NONOate (DEA/NO) and S-nitroso-N-acetylpenicillamine (SNAP) were used as NO donors.
DEA/NO and SNAP (in the presence of dithiothreitol (DTT)) stimulated external Ca(2+)-independent [(3)H]GABA release, which was not attributed to a rise in intracellular calcium concentration. [(3)H]GABA release coincided with increasing GABA level in cytosol and decreasing the vesicular GABA content available for exocytotic release. There was a strong temporal correlation between NO-induced increase in cytosolic [GABA] and dissipation of both synaptic vesicle proton gradient and mitochondrial membrane potential. Dissipation was reversible, and recovery of both parameters correlated in time with re-accumulation of [(3)H]GABA into synaptic vesicles. The molar ratio of DTT to SNAP determined the rate and duration of the recovery processes.
We suggest that NO can stimulate GABA release via GABA transporter reversal resulting from increased GABA levels in cytosol. The latter is reversible and appears to be due to S-nitrosylation of key proteins, which affect the energy status of the pre-synapse.
Our findings provide new insight into molecular mechanism(s) underlying the presynaptic action of nitric oxide on inhibitory neurotransmission.
一氧化氮（NO）是突触传递重要的突触前调质。在此，我们旨在将主要抑制性神经递质γ-氨基丁酸（GABA）的释放与大鼠脑轴突终末在纳摩尔至低微摩尔浓度范围内暴露于NO期间发生的细胞内事件相关联。
使用[³H]GABA和荧光染料（Fluo 4-AM、吖啶橙和罗丹明6G），评估以下参数：囊泡和胞质GABA池、细胞内钙浓度、突触囊泡酸化和线粒体膜电位。二乙胺NONOate（DEA/NO）和S-亚硝基-N-乙酰青霉胺（SNAP）用作NO供体。
DEA/NO和SNAP（在二硫苏糖醇（DTT）存在下）刺激了不依赖细胞外Ca²⁺的[³H]GABA释放，这并非归因于细胞内钙浓度的升高。[³H]GABA释放与胞质中GABA水平升高以及可用于胞吐释放的囊泡GABA含量降低同时发生。NO诱导的胞质[GABA]增加与突触囊泡质子梯度和线粒体膜电位的耗散之间存在强烈的时间相关性。耗散是可逆的，且两个参数的恢复在时间上与[³H]GABA重新积累到突触囊泡中相关。DTT与SNAP的摩尔比决定了恢复过程的速率和持续时间。
我们认为NO可通过胞质中GABA水平升高导致的GABA转运体逆转来刺激GABA释放。后者是可逆的，似乎是由于关键蛋白的S-亚硝基化，这影响了突触前的能量状态。
我们的发现为一氧化氮对抑制性神经传递的突触前作用的分子机制提供了新的见解。
