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.

钙通常起着膜稳定剂、代谢调节器和第二信使的重要作用，但通常认为它也可以介导缺氧和毒性细胞的死亡。特别是缺氧条件下脑内关键阳离子胞外浓度的变化已被许多研究者注意到，相反，由于其精确测量的困难，对细胞内离子行为的研究还很少。低氧和无糖增加，而CA1区钙积聚减少，外加钙离子浓度降低。用37ﾟC、35ﾟC、33ﾟC和31ﾟC的低氧介质灌流后，细胞内钙积聚的急剧增加随潜伏期的延长呈温度依赖性增加。低温下钙蓄积的这种延迟可能表明低温减少的机制与化学损伤有关。当脑片在37ﾟC下用不含硫喷妥钠的无糖低氧培养液灌流时，在开始无糖缺氧后75-200s(平均潜伏期123s)，钙积聚急剧增加。当脑片在37ﾟC灌流含25muM、50muM和75muM硫喷妥钠的无糖低氧培养液时，75muM的硫喷妥钠可引起CA1区锥体神经元钙积聚的急剧增加和潜伏期的延长。4)硝普钠可剂量依赖性地抑制N-甲基-D-天冬氨酸诱导的细胞内游离钙积聚。含NO化合物S-亚硝基-N-乙酰青霉胺刺激乙酰青霉胺的能力是硝普钠的100倍，而刺激cGMP积累的能力是硝普钠的100倍，但剂量依赖性地不能抑制N-甲基-D-天冬氨酸诱导的细胞内游离钙积聚。用L-硝基-L-精氨酸预先孵育大鼠海马片，不能抑制N-甲基-D-天冬氨酸诱导的细胞内游离钙积聚。因此，SNP的这种作用与其产生NO的能力无关，因此SNP不再被认为是模仿内源性5号产生的作用的特异性工具。从这些结果可以想象，在低氧条件下，海马脑片CA1区诱导了大量的细胞内钙积聚，其抑制可能表明其对化学损伤的细胞保护作用参与了机制。虽然很难测量细胞内钙离子浓度的绝对值，但该方法也将为研究海马区以外的神经组织的细胞内机制提供有价值的信息。较少

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