The present study tests the hypothesis that post-hypoxic reoxygenation results in an
nitric oxide (NO)-mediated increase in nuclear Ca(++)-influx, increased
calmodulin kinase (
CaM kinase) IV activity, and increased Ser(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(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(++)-influx was determined using (45)Ca(++) and
CaM kinase IV activity determined by (33)P-incorporation into
syntide-2. Ser(133) phosphorylation of
CREB protein was determined by Western blot analysis using a specific anti-phosphorylated Ser(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(++)-influx (pmoles/mg
protein/min) was 3.79 +/- 0.91 in Nx; 11.81 +/- 2.54 in Hx (P < 0.05 versus Nx), 16.55 +/- 3.55 in Hx + reox (P < 0.05 versus Nx), and 12.40 +/- 2.93 in Hx + 7-NINA (P = NS versus Hx).
CaM kinase IV activity (pmoles/mg
protein/min) was 1,220 +/- 76 in Nx, 2,403 +/- 254 in Hx (P < 0.05 versus Nx), 1,971 +/- 147 in Hx + reox (P < 0.05 versus Hx), and 1,939 +/- 125 Hx + 7-NINA (P < 0.05 versus Hx). Ser(133) phosphorylated
CREB protein expression (OD x mm(2)) was 87 +/- 2 in Nx, 203 +/- 24 in Hx (P < 0.05 versus Nx), 186 +/- 23 in Hx + reox (P < 0.05 Nx, P = NS versus Hx), and 128 +/- 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(++)-influx and
CREB protein phosphorylation at Ser(133) during reox. We conclude that post-Hx increase in nuclear Ca(++)-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 proapoptotic genes during post-Hx reox resulting in Hx neuronal death.