The effect of MgSO4 Administration on Phosphorylation of the Tyrosine Residues of the NR2A and NR2B Subunits of the NMDA Receptor During Hypoxia in the Cerebral Cortex of Newborn Piglets † 327

Abstract

Cerebral N-methyl-D-aspartate (NMDA) receptors are heteromeric complexes of NR1 and NR2 (NR2A-NR2B) subunits, the expression of which regulates the receptor ion-channel function. Previous studies have shown that phosphorylation regulates NMDA receptor ion-channel function. It has also been shown that Mg2+ blocks activation of cerebral NMDA receptors during hypoxia. The present study tests the hypothesis that MgSO4 administration inhibits the hypoxia-induced dephosphorylation of the tyrosine residues of the NR2A and NR2B subunits of the cerebral NMDA receptor. The study was performed in 3 groups of anesthetized, ventilated newborn piglets: normoxic controls (n=2), untreated hypoxic (n=2) and Mg2+ -treated hypoxic (n=2) piglets. Cerebral hypoxia was induced by lowering the FiO2 (0.05-0.07) to achieve a PaO2 of 16-21 mmHg for 60 min, and was documented by decreased tissue ATP and phosphocreatine (Pcr) levels. Prior to hypoxia the Mg2+-treated group received MgSO4 (600 mg/kg) over 30 min followed by 300 mg/kg infused during the 60 min of hypoxia. Cortical P2 membranes were prepared. NR2A and NR2B NMDA receptor subunits were immunoprecipitated with antiphosphotyrosine antibody and separated by 8% SDS-PAGE. The subunits were quantified with an imaging densitometer and expressed as optical density (OD × mm2). During normoxia, the phosphorylated tyrosine residues of theNR2A and NR2B subunits had an OD× mm2 of 3.7±0.3 and 6.9±0.4 respectively, and during hypoxia 0.8±0.05 and 2.0±0.2, respectively. In cerebral cortices of MgSO4-treated piglets the OD × mm2 was 1.0±0.3 and 3.3±0.5 for the NR2A and NR2B subunits, respectively. The data demonstrate that during hypoxia there was a 78% and 71% decrease in phosphorylation of the tyrosine residues in the NR2A and NR2B subunits, respectively, compared to normoxia. During hypoxia, MgSO4-treatment resulted in an increase of 25% and 65% in phosphorylation of the tyrosine residues in the NR2A and NR2B subunits, respectively, compared to the untreated hypoxic group. The data show that Mg2+ inhibits the dephosphorylation of the tyrosine residues of the NR2A and NR2B subunits of the cerebral NMDA receptor during hypoxia. We speculate that by inhibiting the dephosphorylation of the tyrosine residues of the NR2A and NR2B subunits of the cerebral NMDA receptor during hypoxia, Mg2+ modifies the function of the ion-channel complex.