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.

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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