CALCIUM-MEDIATED DAMAGE FOLLOWING HYPOXIA IN CEREBRAL-CORTEX EX-VIVO STUDIED BY NMR-SPECTROSCOPY - EVIDENCE FOR DIRECT INVOLVEMENT OF VOLTAGE-GATED CA2+-CHANNELS

Calcium plays, a prominent role in the neuronal degeneration which accompanies stroke and there has been much conjecture about the possible source of this Ca2+. The transmembrane Ca2+ transporting processes are considered likely candidates for the ischemia-induced rise in intracellular Ca2+. In the present paper we have monitored metabolism in the cerebral cortex in vitro before, during and after aglycaemic hypoxia using P-31 and H-1 NMR spectroscopy. We used the recovery of cellular metabolites phosphocreatine, ATP, lactate, glutamate and N-acetyl aspartate determined by NMR as an indicator of cell damage caused by hypoxia. Phosphocreatine concentration recovered to only -58% of its control level following 15 min of aglycaemic hypoxia in the presence of 1.2 mM Ca2+. The ratios of phosphocreatine/ATP, lactate/N-acetyl aspartate and glutamate/N-acetyl aspartate did not differ at 1 h of recovery from the prehypoxia levels showing that the hypoxia resistant cells were metabolically viable. In the absence of external Ca2+, phosphocreatine recovery improved to approximately 80%. Ten mM Mg2+ or 25 muM diltiazem in the presence of 1.2 mM Ca2+ improved recovery of phosphocreatine to approximately 85%. Two other antagonists of L-type voltage-gated Ca2+-channels, verapamil and nifedipine, did not protect the cerebral cortex from hypoxic damage. N-methyl-D-aspartate (100 muM) applied during hypoxia with 1.2 mM Ca2+ did not augment the loss of phosphocreatine indicating that the cellular damage was not potentiated by the drug, even when 30 mM K+ was present. The presence of N-methyl-D-aspartate did not weaken the protective effect of diltiazem. Blockade of N-methyl-D-aspartate or non-N-methyl-D-aspartate channels did not alleviate cellular damage caused by hypoxic insult. The present results suggest that the immediate, Ca2+-mediated neuronal damage in the cerebral cortex may be mediated by Ca2+ influx through L-type voltage-gated Ca2+-channels.