Abstract 1062 Poster Session II, Sunday, 5/2 (poster 64)

Previous studies have shown that nuclear calcium signals control a variety of nuclear functions including gene transcription, DNA synthesis, DNA repair and nuclear envelope breakdown. We have previously demonstrated that the activity of the cerebral nuclear high-affinity Ca++-ATPase increases under hypoxic conditions. The present study tests the hypothesis that the activity of the neuronal nuclear high affinity Ca++-ATPase increases as a function of decreasing energy metabolism in the cerebral cortex. Studies were performed in 7 newborn piglets, age 3-5 days, divided into normoxic and hypoxic (n=5) groups The normoxic group was exposed to room air. The hypoxic group was exposed to a single FiO2 in the range from 0.21 to 0.05 for one hr. Tissue hypoxia was confirmed biochemically by determining brain tissue ATP and phosphocreatine levels. Neuronal nuclei were isolated by using a discontinuous sucrose gradient from the cortex of the normoxic and hypoxic piglets. High-affinity Ca++-ATPase activity was determined in a medium containing 20 mM HEPES buffer (pH 7.0), 100 mM KCl, 95 µM CaCl2, 250 µM MgCl2, 100 µM EGTA, 1 mM ouabain, 1 mM ATP and 250 µg nuclear membrane protein. The reaction was carried out at 37°C for 30 minutes, a period during which the reaction was linear. The reaction was stopped by the addition of 0.5 ml of 12.5% of trichloroacetic acid. The sample was centrifuged and the supernatant analyzed for the amount of inorganic phosphate generated. High affinity Ca++-ATPase activity was determined and expressed as nmoles Pi/mg protein/hr. During graded hypoxia, as ATP decreased from 5.43 to 0.61 µmoles/g brain and PCr decreased from 4.99 to 0.50 µmoles/g brain, total high affinity Ca++-ATPase activity ranged from 569 to 675 nmoles/mg protein/hr, compared to activity in normoxia of 445 nmoles/mg protein/hr. The level of Ca++-ATPase activity correlated inversely with ATP and PCr levels (r = 0.87, r = 0.85 respectively), with activity increasing as tissue high energy phosphates decreased during hypoxia. The results demonstrate that the decrease in cerebral energy metabolism during hypoxia is linearly correlated with an increase in activity of high affinity Ca++-ATPase in cerebral cortical nuclei from immature brain. We speculate that increased nuclear membrane high affinity Ca++-ATPase activity, leading to increased nuclear Ca++, will result in altered gene expression and that could initiate programmed cell death.

(Funded by NIH HD- 20337)