Balázs R, Jørgensen O S, Hack N
Netherlands Institute for Brain Research, Amsterdam.
Neuroscience. 1988 Nov;27(2):437-51. doi: 10.1016/0306-4522(88)90279-5.
Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K+-containing media without N-methyl-D-aspartate), and the potency of N-methyl-D-aspartate (half-maximal effective concentration, EC50, about 20 microM) was similar to its known affinity in binding to cerebral membranes. The effect of N-methyl-D-aspartate was blocked by the specific receptor antagonist 2-amino-5-phosphonovalerate, which also reduced the limited survival of cells in cultures grown in 'low' K+ in the absence of N-methyl-D-aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前关于细胞外高浓度钾离子（[K⁺]e）对培养的小脑颗粒细胞促存活影响的研究表明，体外去极化模拟了颗粒细胞在体内最早接收的传入输入的作用。反过来，这可能是通过刺激兴奋性氨基酸受体介导的，特别是对N - 甲基 - D - 天冬氨酸具有偏好性的亚型，其门控离子通道对Ca²⁺也具有通透性。在此我们报告，N - 甲基 - D - 天冬氨酸确实对源自7 - 8日龄大鼠解离小脑并在含有“低”[K⁺]e（5 - 15 mM）的培养基中培养的细胞在培养中的存活有显著影响。除了对培养物进行肉眼观察外，还使用与活细胞数量相关的估计值（DNA测定和四氮唑盐还原率测定）对N - 甲基 - D - 天冬氨酸的作用进行了定量评估。此外，还测量了在神经细胞（神经细胞黏附分子、D3蛋白和突触素）或胶质细胞（谷氨酰胺合成酶）中相对富集的蛋白质。研究结果表明，N - 甲基 - D - 天冬氨酸对细胞的挽救主要涉及神经细胞，并且N - 甲基 - D - 天冬氨酸的存活需求与高钾一样，在体外2至4天之间形成。该作用取决于N - 甲基 - D - 天冬氨酸的浓度和细胞的去极化程度：随着[K⁺]e从5 mM升高到15 mM，N - 甲基 - D - 天冬氨酸的效力和效能均增加，在此范围内钾离子自身对颗粒细胞存活几乎没有影响。这些特征与通过N - 甲基 - D - 天冬氨酸受体连接通道的离子电导的电压依赖性一致。在存在15 mM K⁺的情况下获得了N - 甲基 - D - 天冬氨酸最显著的作用，此时细胞存活率接近在“对照”培养物（在不含N - 甲基 - D - 天冬氨酸的含25 mM K⁺培养基中生长）中获得的存活率，并且N - 甲基 - D - 天冬氨酸的效力（半数最大有效浓度，EC50，约20 μM）与其已知的与脑膜结合的亲和力相似。N - 甲基 - D - 天冬氨酸的作用被特异性受体拮抗剂2 - 氨基 - 5 - 膦酰戊酸阻断，该拮抗剂也降低了在无N - 甲基 - D - 天冬氨酸的“低”钾培养物中细胞的有限存活率。（摘要截断于400字）
