Effect of In Vitro Lipid Peroxidation on High Affinity Ca⁺⁺-ATPase Activity in Neuronal Nuclear Membranes of the Newborn Piglet

Abstract 1703 Poster Session III, Monday, 5/3 (poster 70)

Previous studies have shown that brain tissue hypoxia results in increased activity of the high affinity Ca⁺⁺-ATPase located on the outer nuclear membrane. The present study tests the hypothesis that the mechanism of the hypoxia-induced increase in high affinity Ca⁺⁺-ATPase activity is due to peroxidation of the nuclear membrane lipids. Studies were conducted on cerebral cortical tissue obtained from five anesthetized normoxic newborn piglets. Cerebral cortical tissue was homogenized in a medium containing 0.32 M Sucrose, 1 mM MgCl₂ and 10 mM Tris-HCl buffer, pH 6.8 and centrifuged at 850 × g for 10 min. Cerebral cortical nuclei were subsequently isolated and purified using a discontinuous sucrose gradient. Nuclear membranes from each sample were divided into two aliquots. For peroxidation an aliquot of nuclear membranes was incubated at 37°C for 20 min in the presence of 100 mM ascorbate and 25 µM FeCl₂. Control membranes were incubated in buffer alone. Lipid peroxidation products were analyzed as thiobarbituric acid (TBA) reactive substances. Aliquots from the incubated control and peroxidized membranes were heated at 100°C with 0.67% TBA for 10 min and the absorbance was recorded at 532 nm. The activity of the high affinity Ca⁺⁺-ATPase was determined in a 1 ml assay medium containing 20 mM HEPES, 100 mM KCl, 250 µM MgCl₂, 100 µM EGTA, 95 µM CaCl₂, 1 mM ouabain, 1 mM ATP and 150 µg nuclear membrane protein. The reaction was carried out at 37°C for 30 min, a period during which the reaction was linear. The reaction was stopped by the addition of 0.5 ml 12.5% trichloracetic acid. The sample was centrifuged and the supernatant was analyzed for inorganic phosphate content. Ca⁺⁺-ATPase activity was calculated and expressed as nmoles/mg protein/hr. There was a 3 to 5 fold increase in TBA reactive substances following in vitro lipid peroxidation. High affinity Ca⁺⁺-ATPase activity was 177 ± 43 nmoles/mg protein/hr in control samples and 323 ± 85 nmoles/mg protein/hr in peroxidized samples (p<0.05), a 79% increase in activity. We conclude that lipid peroxidation in vitro results in an increased activity of the high affinity Ca⁺⁺-ATPase in neuronal nuclear membranes. Our previous studies have shown that lipid peroxidation decreases plasma membrane Na⁺, K⁺-ATPase activity; thus lipid peroxidation appears to be a mechanism for hypoxia-induced alteration of enzyme function in both the nuclear and cell (plasma) membranes. The differential effects on the two enzymes could be due to differences in the lipid composition of the membranes or differences in the lipid dependence of Ca⁺⁺-ATPase and Na⁺, K⁺-ATPase activity.

Funded by NIH-HD-20337