Bleakman D, Roback J D, Wainer B H, Miller R J, Harrison N L
Department of Pharmacological and Physiological Sciences, University of Chicago, IL 60637.
Brain Res. 1993 Jan 15;600(2):257-67. doi: 10.1016/0006-8993(93)91381-2.
Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).
取自胚胎大鼠的隔区神经元在组织培养中生长。运用微量荧光测定法和电生理技术研究这些神经元中的钙离子稳态。神经元中估计的基础细胞内游离离子钙浓度（[Ca2+]i）较低（50 - 100 nM）。用50 mM K+使神经元去极化导致[Ca2+]i迅速升高至500 - 1000 nM，并在几分钟内恢复到基线[Ca2+]i。去除细胞外Ca2+可完全消除由K+去极化引起的[Ca2+]i升高，而“L型”Ca2+通道阻滞剂尼莫地平（1 microM）可使其降低约80%。兴奋性氨基酸L - 谷氨酸、quisqualate、AMPA和海人藻酸也可使[Ca2+]i升高。对AMPA和海人藻酸的反应被CNQX和DNQX阻断。在没有细胞外Mg2+的情况下，观察到[Ca2+]i有大幅波动，这些波动可被去除细胞外Ca2+、河豚毒素（TTX）或N - 甲基 - D - 天冬氨酸（NMDA）拮抗剂如2 - 氨基 - 5 - 磷酸戊酸（APV）阻断。在零Mg2+和TTX条件下，NMDA引起[Ca2+]i剂量依赖性升高，且被APV阻断。咖啡因（10 mM）在没有细胞外Ca2+的情况下引起[Ca2+]i短暂升高，而毒胡萝卜素可阻止这种升高，这表明存在对咖啡因敏感的ATP依赖性细胞内钙库。毒胡萝卜素（2 microM）对[Ca2+]i或从K+去极化后的恢复影响很小。去除细胞外Na+对基础[Ca2+]i或对高K+的反应影响很小，这表明Na+/Ca2+交换机制在隔区神经元[Ca2+]i的短期控制中不起重要作用。线粒体解偶联剂CCCP使基础[Ca2+]i缓慢升高；然而，在存在CCCP的情况下，[Ca2+]i从高K+刺激中仍能正常恢复，这表明线粒体不参与对适度[Ca2+]i升高的快速缓冲。在同时进行的电生理和微量荧光测定记录中，测量了与动作电位活动相关的[Ca2+]i升高。一串动作电位诱导的[Ca2+]i升高幅度随动作电位串的持续时间以及在5至200 Hz范围内的发放频率增加而增加。动作电位串给神经元带来适度钙负荷后，[Ca2+]i的恢复遵循简单的指数衰减（时间常数 = 3 - 5秒）。
