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.

钙作为膜稳定剂、代谢调节剂和第二信使具有重要的功能，但也可以介导缺氧和毒性细胞死亡。特别是，缺氧条件下脑内关键阳离子胞外浓度的变化已经被许多研究者注意到，而胞内离子的行为由于难以精确测量而缺乏研究。本研究采用CFa^<2+>-敏感染料fura-2，采用显微荧光法研究了缺氧-无葡萄糖诱导的大鼠海马切片细胞内游离钙积累的变化，其中包括细胞保护对与低温、药物和一氧化氮相关的神经元损伤的作用。所得结果如下：(1) CA1锥体神经元的钙积累在开始后80 ~ 170秒内急剧增加，缺氧和无糖增多，而CA1区域的钙积累则随着外钙浓度的降低而减少。低氧无糖处理10 min后，细胞内游离钙迅速恢复到再灌注前的水平，而低氧无糖处理15 min后，细胞内游离钙在37 2时恢复缓慢且不完全。在37、35、33、31个低氧条件下，细胞内钙积累的急剧增加与潜伏期的延长呈温度依赖性。低温下钙积累的迟滞可能表明低温减少化学损伤的机制的参与。事实上，在31°C的缺氧-无葡萄糖条件下，最长15 min的细胞内游离钙通过再灌注完全恢复到原来的水平。3)为了评价硫喷妥钠对化学神经元损伤的细胞保护作用，我们研究了硫喷妥钠对缺氧-无糖诱导的海马片钙积累的影响。当切片用不含硫贲妥的无氧-无糖培养基（37 C）处理时，在无氧-无糖开始后75-200s（平均潜伏期为123s）检测到钙积累的急性增加。用含25muM、50muM和75muM硫喷妥钠的37 oc低氧无糖培养基浸泡后，CA1锥体神经元的钙积累急剧增加，潜伏期延长。4)硝普钠（SNP）可能产生一氧化氮（NO）的剂量依赖性抑制n -甲基-d -天冬氨酸（NMDA）诱导的细胞内游离钙积累。s -亚硝基-n -乙酰青霉胺，一种含no的化合物，在刺激乙酰青霉胺方面比SNP强100倍，一种含no的混杂物，在刺激cGMP积累方面比SNP强100倍，剂量独立地未能抑制nmda引起的细胞内游离钙积累。用l -硝基- l -精氨酸预孵育海马片未能抑制nmda引起的细胞内游离钙积累。因此，SNP的这种作用是独立于其产生NO的能力的，因此SNP不再被认为是模仿内源性NO作用的特异性工具。5)从这些结果可以想象，缺氧条件下海马切片CA1区诱导了大量细胞内钙的积累，其延迟可能表明参与了细胞保护作用对抗化学损伤的机制。虽然很难测量细胞内钙浓度的绝对值，但本文所展示的方法也将为研究海马以外神经组织的细胞内机制提供有价值的信息。少

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