Abstract 370
Poster Session III, Monday, 5/3 (poster 161)
Previous studies have shown that hypoxia-induced activation of the NMDA receptor ion-channel leads to increased intracellular Ca++ and subsequently to an increase in intranuclear Ca++. Increased Ca++ in the nucleus leads to activation of Ca++-dependent endonucleases resulting in fragmentation of nuclear DNA. The present study tests the hypothesis that Mg++ administration prior to hypoxia will prevent the hypoxia-induced DNA fragmentation by blocking the NMDA receptor mediated influx of Ca++ in the cell, thus preventing the activation of endonucleases. Anesthetized, ventilated newborn piglets were divided into normoxic control (n = 4), untreated hypoxic (n = 4) and Mg++-treated hypoxic (n = 4) groups. Cerebral hypoxia was induced by lowering the FiO2 (0.05-0.07) to achieve a PaO2 of 16-21 mmHg for 60 min, and documented by measurement of tissue ATP and phosphocreatine (PCr) levels. The Mg++-treated group received MgSO4 600 mg/kg over 30 min prior to hypoxia followed by 300 mg/kg infused during 60 min of hypoxia. Cerebral cortical nuclei were isolated using a discontinuous sucrose gradient. Ca++/Mg++-dependent endonuclease activation was determined by incubating cerebral cortical nuclei in the presence of 2 mM CaCl2 and 5 mM MgCl2 at 37°C. DNA was extracted by the phenol/chloroform method. DNA fragments were separated by gel electrophoresis in 1% agarose. DNA fragments with 3′OH ends produced by the endonuclease reaction were measured by the random oligonucleotide-primed synthesis (ROPS) assay. The ROPS assay was performed in a medium containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 7.5 mM DTT, 5 units Klenow polymerase, 1 µCi 3H-dCTP, 33 µM dCTP, and 0.5 mM of dATP, dGTP and dTTP. Following calcium and magnesium activation, incorporation of 3H-dCTP increased by 48 pmoles/mg DNA (from 80 to 128 pmoles/mg DNA)in normoxic nuclei, by 70 pmoles/mg DNA (from 37 to 107 pmoles/mg DNA) in untreated hypoxic nuclei, and by 45 pmoles/mg DNA (from 84 to 129 pmoles/mg DNA) in the Mg++-treated hypoxic nuclei. The data indicate that there is an increase in activation of the Ca++/Mg++-dependent endonuclease of 45% in untreated hypoxic nuclei compared to Mg++-treated hypoxic nuclei. The Ca++/Mg++-dependent endonuclease activity in normoxic and Mg++-treated hypoxic nuclei was similar. As previously demonstrated by gel electrophoresis, there is an increased fragmentation of nuclear DNA in untreated hypoxic nuclei which was not observed in Mg++-treated hypoxic nuclei. We conclude that administration of MgSO4 to newborn piglets prior to hypoxia prevents hypoxia-induced activation of Ca++/Mg++-dependent endonuclease activity and DNA fragmentation. We speculate that MgSO4 administration prior to hypoxia blocks calcium entry at the cell membrane NMDA receptor ion-channel, inhibiting the activation of Ca++/Mg++-dependent nuclear endonuclease, thus preventing genomic DNA fragmentation and hypoxia-induced programmed cell death.
