Previous studies have shown that maximal spermine-dependent activation of the NMDA receptor-ion channel complex is increased during hypoxia. The aim of the present study is to investigate the mechanism by which spermine activates the NMDA receptor. 3H-MK-801 binding was used to assess receptor affinity (Kd) and density (Bmax). Twelve piglets (<7 days old) were exposed for 60 min to either FiO2 0.21 (normoxia, n=7) or an FiO2 0.05-0.07 (PaO2≤ 20 mmHg, hypoxia, n=7). Brain tissue hypoxia was documented biochemically by a decrease in ATP and phosphocreatine. P2 membrane fractions were prepared from cerebral cortices and 3H-MK-801 binding was performed at 32°C in 200 μl of medium containing either buffer or 10 μM spermine plus 100 μM glutamate, 100 μM glycine, 10 mM HEPES/1 mM EDTA buffer (pH 7.0), increasing concentrations of 3H-MK-801(2.5 to 50 nM), and 75 μg protein. Saturation curves and Scatchard plots were constructed to determine the Bmax and Kd. Bmax was not significantly different among the 4 groups (1.5±0.3 pmol/mg protein in normoxia to 1.5±0.3 pmol/mg protein in normoxia+spermine, and from 1.3±0.5 pmol/mg protein in hypoxia to 1.3±0.5 pmol/mg protein in hypoxia+spermine). Kd (mean±SD) decreased from 5.4±0.7 nM in normoxia to 3.6±1.9 nM in normoxia+spermine, and from 6.4±0.7 nM in hypoxia to 3.1±1.2 nM in hypoxia+spermine (p<0.001). The results demonstrate that spermine increases the affinity of the NMDA receptor ion channel for MK-801 in both normoxia and hypoxia without changing the number of binding sites. We speculate that the spermine dependent activation of the NMDA receptor ion-channel occurs by alteration of the ion-channel's affinity through the glutamate recognition site and/or acts as a polycationic molecule to influence the micro environment of the channel. (Funded by MOD 6-FY94-0135, NIH #HD-20337)
