Calcium ion flux through the N-methyl-D-aspartate (NMDA) receptor ion-channel has been proposed as a mechanism of hypoxia-induced neuronal injury in the newborn and fetal brain. The present study tests the hypothesis that increased free intrasynaptosomal calcium ([Ca2+]i) during hypoxia is mediated by an influx of calcium via the NMDA receptor and is associated with the release of calcium from intracellular stores. Studies were conducted in 12 term guinea pig fetuses, 6 normoxic and 6 whose mothers were exposed to an FiO2 of 7% for 1 hr. Tissue hypoxia was confirmed by decreased ATP and phosphocreatine levels. Cortical synaptosomes were prepared and[Ca2+]i was measured fluorometrically with Fura-2 after NMDA receptor activation by 100μM NMDA and 100μM glutamate in the presence of 100μM glucine. The relative contribution of the NMDA receptor and intrasynaptosomal stores to increased [Ca2+]i was assessed by determining the effect of MK-801, a non-competitive NMDA receptor antagonist, and thapsigardin, an intracellular Ca2+-ATPase inhibitor. For the normoxic group, the increase in [Ca2+]i after NMDA addition was 79 ± 39 nM for untreated synaptosomes (U), 32 ± 38 nM in the presence of MK-801(M), p<0.05, and 21 ± 11 nM in the presence of thapsigardin (T), p<0.01. The increase in [Ca2+]i after the addition of glutamate was 55 ± 32 nM (U), 15 ± 16 nM (M), p<0.05, and 17 ± 18 nM (T), p<0.01. Similarly, in the hypoxic group, the increase in [Ca2+]i after NMDA addition was 110± 38 nM (U), 26 ± 13 nM (M), p<0.05 and 13 ±24 nM (T), p<0.01. The increase in [Ca2+]i after glutamate addition was 52 ± 22 nM (U), 15 ± 16 nM (M), p<0.05 and 17 ± 18 nM(T), p<0.01. The results show that both MK-801 and thapsigardin inhibit the increase in [Ca2+]i during normoxia and hypoxia, demonstrating that the increase in [Ca2+]i during cerebral hypoxia is both NMDA receptor-mediated and Ca2+-ATPase-dependent. We speculate that hypoxia-induced modification of the NMDA receptor leading to a calcium influx may initiate the inositol phosphate pathway and the release of Ca2+ from intrasynaptosomal stores. (Funded NIH # HD-20337)
