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

已证实，钙离子通过电压依赖性钙离子通道进入突触前神经末梢，在突触前神经末梢去极化诱导的神经递质释放中起重要作用。除了这种去极化诱导的钙内流，一些研究表明，突触前去极化本身可以控制胞吐机制，促进神经递质的释放，即使在没有细胞外钙的情况下也是如此。本研究采用全细胞膜片钳技术，研究了高钾对机械分离的大鼠脊髓背角和海马神经元在无钙条件下自发甘氨酸和GABA释放的增强作用。本文还研究了高K~(++)引起的神经细胞内Ca~(2+)浓度的升高。细胞外K~+浓度升高可逆地增加无细胞外钙时甘氨酸能和GABA能抑制性突触后电流(IPSCs)的频率。阻断电压依赖性钠离子通道(河豚毒素)和钙离子通道(硝苯地平和欧米伽-革兰氏毒素-SIA)对钾诱导的递质释放的增强无影响。在没有细胞外Na+的情况下，也观察到高钾诱导的IPSC频率的增加，尽管在这种情况下恢复到基线水平的时间延长了。高钾溶液对甘氨酸释放的作用可被BAPTA-AM、thapsigargin和磷脂酶C抑制剂U-73122耗尽细胞内储存的Ca~(2+)所阻止。结果提示，细胞外K~(++)浓度升高引起细胞内钙离子释放，这种释放不依赖于细胞外Na~(++)和Ca~(2+)的内流，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.