Study on the mechanisms underlying the depolarization induced intracellular calcium release in CNS neurons

已经确定，通过电压依赖性Ca^<2 +>通道的Ca^<2 +>进入在突触前神经末梢去极化诱导的神经递质释放中起着至关重要的作用。除了这种去极化诱导的Ca^2+内流之外，一些研究表明，突触前去极化本身可以控制胞吐机制，甚至在细胞外Ca^2+缺乏的情况下也能促进神经递质的释放。本研究采用全细胞膜片钳技术，分别研究了高钾对大鼠脊髓背角和海马神经元在无Ca^2+条件下自发性甘氨酸和GABA释放的增强作用。我们还研究了高K^+诱导的分离神经元细胞内Ca^2+浓度升高。在细胞外Ca 2+缺乏的情况下，细胞外K^+浓度升高可可逆地增加自发性甘氨酸能和GABA能抑制性突触后电流（IPSC）的频率。阻断电压依赖性Na+通道（河豚毒素）和Ca^<2+>通道（硝苯地平和omega-grammotoxin-SIA）对钾诱导的递质释放增强没有影响。在缺乏细胞外Na^+的情况下，也观察到了高钾诱导的IPSC频率增加，尽管在这些条件下恢复到基线水平的释放时间延长。BAPTA-AM、毒胡萝卜素和磷脂酶C抑制剂U-73122耗尽细胞内Ca^<2+>储备，阻止了高钾溶液对甘氨酸释放的作用。结果表明，细胞外K^+浓度升高引起的内钙释放与细胞外Na^+和Ca^<2+>-内流无关，可能揭示了钾诱导的突触前神经末梢去极化调节细胞内Ca^<2+浓度和胞吐作用的新机制。

It is well established that entry of Ca^<2+> through voltage-dependent Ca^<2+> channels plays a crucial role in the depolarization-induced release of neurotransmitters from presynaptic nerve terminals. In addition to this depolarization-induced Ca^<2+>-influx, several studies have suggested that presynaptic depolarization per se can control the exocytotic machinery and promote neurotransmitter release even in the absence of extracellular Ca^<2+>. In the present study, the high potassium-induced potentiation of spontaneous glycine and GABA release in extracellular Ca^<2+>-free conditions was studied in mechanically dissociated rat spinal dorsal horn and hippocampal neurons using whole-cell patch-clamp technique, respectively. The high K^+-induced elevation of intracellular Ca^<2+> concentration in the isolated neurons was also studied. Elevating extracellular K^+ concentration reversibly increased the frequency of spontaneous glycinergic and GABAergic inhibitory postsynaptic currents (IPSCs) in the absence of extracellular Ca2+. Blocking voltage-dependent Na+ channels (tetrodotoxin) and Ca^<2+> channels (nifedipine and omega-grammotoxin-SIA) had no effect on this potassium-induced potentiation of transmitter release. The high potassium-induced increase in IPSC frequency was also observed in the absence of extracellular Na^+, although the recovery back to baseline levels of release was prolonged under these conditions. The action of high potassium solution on glycine release was prevented by BAPTA-AM, by depletion of intracellular Ca^<2+> stores by thapsigargin and by the phospholipase C inhibitor U-73122. The results suggest that the elevated extracellular K^+ concentration causes Ca2+ release from internal stores which is independent of extracellular Na^+-and Ca^<2+>-influx, and may reveal a novel mechanism by which the potassium-induced depolarization of presynaptic nerve terminals can regulate intracellular Ca^<2+> concentration and exocytosis.