Nuclear Ca super(++)-influx, Ca super(++)/Calmodulin-Dependent Protein Kinase IV Activity and CREB Protein Phosphorylation during Post-Hypoxic Reoxygenation in Neuronal Nuclei of Newborn Piglets: The Role of Nitric Oxide

The present study tests the hypothesis that post-hypoxic reoxygenation results in an nitric oxide (NO)-mediated increase in nuclear Ca super(++)-influx, increased calmodulin kinase (CaM kinase) IV activity, and increased Ser super(133) phosphorylation of cyclic AMP response element binding (CREB) protein in neuronal nuclei of the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO sub(2) = 0.07 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox), and Hx + reox injected with 7-nitroindazole sodium salt (7-NINA), a nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Nuclear Ca super(++)-influx was determined using super(45)Ca super(++) and CaM kinase IV activity determined by super(33)P-incorporation into syntide-2. Ser super(133) phosphorylation of CREB protein was determined by Western blot analysis using a specific anti-phosphorylated Ser super(133)-CREB protein antibody. ATP and PCr values in Hx, Hx + reox, and Hx + 7-NINA were significantly different from Nx (P < 0.05 versus Nx). Ca super(++)-influx (pmoles/mg protein/min) was 3.79 plus or minus 0.91 in Nx; 11.81 plus or minus 2.54 in Hx (P < 0.05 versus Nx), 16.55 plus or minus 3.55 in Hx + reox (P < 0.05 versus Nx), and 12.40 plus or minus 2.93 in Hx + 7-NINA (P = NS versus Hx). CaM kinase IV activity (pmoles/mg protein/min) was 1,220 plus or minus 76 in Nx, 2,403 plus or minus 254 in Hx (P < 0.05 versus Nx), 1,971 plus or minus 147 in Hx + reox (P < 0.05 versus Hx), and 1,939 plus or minus 125 Hx + 7-NINA (P < 0.05 versus Hx). Ser super(133) phosphorylated CREB protein expression (OD x mm super(2)) was 87 plus or minus 2 in Nx, 203 plus or minus 24 in Hx (P < 0.05 versus Nx), 186 plus or minus 23 in Hx + reox (P < 0.05 Nx, P = NS versus Hx), and 128 plus or minus 10 in Hx + 7-NINA (P < 0.05 versus Hx and Hx + reox). The results show that post-Hx administration of 7-NINA prevents the increased nuclear Ca super(++)-influx and CREB protein phosphorylation at Ser super(133) during reox. We conclude that post-Hx increase in nuclear Ca super(++)-influx leading to increased phosphorylation of CREB protein is mediated by NO derived from nNOS. However, hypoxia-induced increase in CaM Kinase IV activity decreased during the post-Hx reox. We propose that hypoxia-induced increase in CaM Kinase IV activity leads to increased phosphorylation of CREB protein and transcription of proapoptot