Abstract 223
Critical Care Platform, Monday, 5/3
Previous studies have shown that the neuronal high affinity Ca++-ATPase, located on the outer nuclear membrane, is responsible for ATP-dependent calcium entry into the nucleus. We have also shown that, during hypoxia, the activity of the enzyme is increased in cerebral cortical nuclei. Since increased nitric oxide production during hypoxia leads to formation of peroxynitrite radicals and subsequent nitration of amino acid residues, the present study tests the hypothesis that nitration increases the activity of the cerebral nuclear high affinity Ca++-ATPase. Studies were performed in anesthetized ventilated newborn piglets. Following 1 hr of normoxia cerebral cortex was obtained and placed in isolation medium containing 0.32 M sucrose, 1 mM MgCl2 and 10 mM Tris-HCl buffer (pH 6.8). Cerebral cortical nuclei were isolated by discontinuous density gradient centrifugation. The normoxic state of the cortical nuclei was confirmed by measuring brain tissue levels of ATP and phosphocreatine. An aliquot of nuclei from each sample was nitrated with 0.5 mM peroxynitrite in a medium of 100 mM K2HPO4(pH 7.4), 100 µM DTPA, and 25 mM sodium bicarbonate. Nitration of the nuclear membranes was confirmed by Western blot-analysis using antinitrotyrosine antibody. Ca++-ATPase activity was determined in nitrated and non-nitrated samples. The activity of the high affinity Ca++-ATPase was determined in a 1 ml assay medium containing 20 mM HEPES, 100 mM KCl, 250 mM 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 minutes, a period during which the rate of reaction was linear. The reaction was stopped by the addition of 0.5 ml 12.5% trichloroacetic acid. The samples were centrifuged and the supernatant was analyzed for inorganic phosphate. The activity of the enzyme was expressed as nmoles Pi/mg protein/hour. Activity of the high-affinity Ca++-ATPase significantly increased from 764 ± 98 nmoles Pi/mg protein/hr in untreated normoxic nuclear membranes to 1178 ± 118 nmoles Pi/mg protein/hr following nitration (p<0.05). Thus, in vitro, nitration increased enzyme activity by 50%. We speculate that during hypoxia in vivo, the increase in peroxynitrite formation will lead to nitration and increased activity of the nuclear membrane high-affinity Ca++-ATPase, altering intranuclear Ca++ concentrations. Since intranuclear calcium controls nuclear functions including gene transcription, the increased activity of the nuclear membrane high affinity Ca++-ATPase after nitration could alter patterns of gene expression, leading to cell dysfunction and programmed cell death.
