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Experimental studies on change of intracellular free calcium concentration following hypoxia and glucose free, and effect of nitric oxide on its changes in rat hippocampal slices.

大鼠海马切片缺氧、无糖后细胞内游离钙浓度变化及一氧化氮对其变化影响的实验研究。

基本信息

批准号：
06671381
负责人：
ANDOH Takashi
金额：
$ 0.96万
依托单位：
GIFU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

It has been commonly assumed that calcium, which normally serves important functions as a membrane stabilizer, metabolic regulator and second messenger, also can mediate anoxic and toxic cell death. In special, changes of the extracellular concentrations in the key cerebral cations under oxygen deprivation have been noticed previously by a number of investigators, in verse, there is a dearth of research on the behavior of intracellular ions because of the difficulties of their precise measurement. In the present study, the change of hypoxia-glucose free induced intracellular free calcium accumulation in rat hippocampal slice was studies by microfluorometry using the CFa^<2+>-sensitive dye fura-2, in which the efficacy of cytoprotection on neuronal damage in relation to hypothermia, agents, and nitric oxide was included.The results obtained were as follows ; 1) Acute and massive increase of calcium accumulations was seen in the CA1 pyramidal neurons 80-170 second after the beginning of … More hypoxia and glucose free, while decrease of calcium accumulations was observed in the CA1 regions by decrease of the external calcium concentration. Increase of intracellular free calcium under a 10 min hypoxia-glucose free was rapidly restored to the original levels of calcium after reperfusion, however, that under a 15 min hypoxia-glucose free was restored slowly and incompletely at 37ﾟ2) The effect of mild hypothermia on the hypoxia-induced calcium accumulation in hippocampal slice was investigated. When the slices were superfused with hypoxic medium at 37ﾟC,35ﾟC,33ﾟC and 31ﾟC,acute increase of intracellular calcium accumulation were temperature-dependently recognized with prolongation of latency. This retardation in calcium accumulation under hypothermia may indicate involvement of the mechanisms by which hypothermia diminishes is chemic injury. In fact, increased intracellular free calcium under the longest 15 min hypoxia-glucose free at 31ﾟC was completely restored to the original levels of calcium by reperfusion.3) To evaluate the efficacies of cytoprotection against is chemic neuronal damage, the effect of thiopental on hypoxia-glucose free-induced calcium accumulation in hippocampal slices was investigated. When slices were superfused with hypoxic-glucose free medium at 37ﾟC that did not contain thiopental, an acute increase in calcium accumulation was detected 75-200s (mean latency of 123s) after the beginning of hypoxia-glucose free. When slices were superfused with hypoxic-glucose free mediums at 37ﾟC that contained 25muM, 50muM and 75muM of thiopental, acute increase of calcium accumulation was seen with prolongation of latency in CA1 pyramidal neurons by 75muM of thiopental.4) Sodium nitroprusside (SNP) that might generate nitric oxide (NO) dose-dependently inhibited N-methyl-D-aspartate (NMDA)-evoked intracellular free calcium accumulation. S-nitroso-N-acetylpenicillamine, an NO-containing compounds that were 100 times more potent than SNP in stimulating acetylpenicillamine, an NO-containing confounds that were 100 times more potent than SNP in stimulating cGMP accumulation dose-independently failed to inhibit NMDA-evoked intracellular free calcium accumulation. Preincubation of hippocampal slices with L-nitro-L-arginine failed to inhibit NMDA-evoked intracellular free calcium accumulation. THerefore, this effect of SNP is independent of its ability to generate NO,thus SNP was no longer considered as a specific tool for mimicking the action of endogeneously produced NO.5) From these results, it is conceivable that large intracellular intracellular calcium accumulation is induced in field CA1 of hippocampal slices under hypoxic condition and its retardation may indicate involvement of the mechanisms of cytoprotective effect against is chemic injury. Although it is difficult to measure the absolute value of intracellular calcium concentration, the method demonstrated here would offer valuable information for the study of the intracellular mechanisms in neural tissue other than hippocampus as well. Less

通常认为，钙通常作为膜稳定剂、代谢调节剂和第二信使发挥重要作用，也可以介导缺氧和毒性细胞死亡。特别是，在缺氧条件下，细胞外的关键脑阳离子的浓度变化已经被许多研究者注意到，反之，由于其精确测量的困难，有一个缺乏对细胞内离子的行为的研究。本研究采用CFa^2+敏感染料Fura-2荧光显微荧光法研究了缺氧无糖诱导的大鼠海马脑片细胞内游离钙的变化，并探讨了低温、药物和一氧化氮对神经元损伤的保护作用。（1）海马CA_1区锥体神经元内钙离子浓度在缺血后80-170秒内急剧增加， ...更多信息缺氧和无葡萄糖，而在CA 1区观察到的钙积累的减少，通过降低外部钙浓度。缺氧无糖10 min时，细胞内游离钙升高迅速恢复到再灌注前水平，而缺氧无糖15 min时，再灌注37 ℃时，细胞内游离钙升高缓慢且不完全恢复。2）观察亚低温对缺氧诱导的海马脑片钙蓄积的影响。在37 ℃、35 ℃、33 ℃和31 ℃的低氧条件下，细胞内钙离子浓度迅速增加，并伴有潜伏期延长。低温下钙积累的延迟可能表明低温减少的机制与化学损伤有关。事实上，在31 ℃最长15 min无糖缺氧条件下增加的细胞内游离钙通过再灌注完全恢复到原始水平。3）为了评价细胞保护对缺血性神经元损伤的有效性，研究了硫喷妥钠对缺氧无糖诱导的海马脑片钙积聚的影响。当用不含硫喷妥钠的无缺氧葡萄糖培养基在37 ℃灌流切片时，在无缺氧葡萄糖开始后75- 200秒（平均潜伏期为123秒）检测到钙积累的急性增加。当脑片在37 ℃下灌流含25 μ M、50 μ M和75 μ M硫喷妥钠的无缺氧葡萄糖培养基时，观察到CA 1锥体神经元内钙积聚的急性增加，并观察到75 μ M硫喷妥钠延长潜伏期。4）可能产生一氧化氮（NO）的硝普钠（SNP）剂量依赖性地抑制N-甲基-D-天冬氨酸（NMDA）诱导的细胞内游离钙积聚。S-亚硝基-N-乙酰青霉胺是一种比SNP刺激乙酰青霉胺强100倍的含NO化合物，一种比SNP刺激cGMP积累强100倍的含NO化合物，但不能剂量依赖性地抑制NMDA诱导的细胞内游离钙积累。用L-硝基-L-精氨酸预孵育海马脑片不能抑制NMDA诱导的细胞内游离钙积累。因此，SNP的这种作用不依赖于其产生NO的能力，因此SNP不再被认为是模拟内源性产生的NO的作用的特异性工具。5）从这些结果来看，可以想象，在缺氧条件下，海马脑片的CA 1区诱导了大量的细胞内钙积累，其延迟可能表明参与了细胞保护作用的机制。化学损伤虽然很难测量细胞内钙离子浓度的绝对值，但本文所展示的方法将为研究海马以外的神经组织的细胞内机制提供有价值的信息。少