Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition

Abstract

Previous studies have shown that hypoxia results in increased phosphorylation of CREB protein that mediates gene expression including that of the pro-apoptotic gene bax. We also have shown that hypoxia-induced expression of Bax protein is prevented by blocking nitric oxide synthase (NOS). The present study tests the hypothesis that inhibition of NOS by N-nitro- L-arginine (NNLA) will prevent the hypoxia-induced increased phosphorylation of CREB protein in neuronal nuclei of newborn piglets. To test this hypothesis, phosphorylation of CREB protein was assessed by immunoblotting neuronal nuclear proteins from five normoxic (Nx), 10 hypoxic (Hx) and five Hx-NNLA-treated 3–5-day-old piglets. NNLA (40 mg/kg) or saline was infused over 60 min prior to induction of hypoxia. Hypoxia was achieved by reducing the FiO 2 (0.15 to 0.05) for 60 min and documented biochemically by ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were separated on 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, reacted with anti-phosphorylated CREB protein antibody and conjugated with horseradish peroxidase antibody. Protein bands were detected using the enhanced chemiluminescence method and quantitated by imaging densitometry. Protein density was expressed as absorbance (OD)×mm 2. ATP levels (μmol/g brain) were 4.3±0.6 in the Nx group, 1.3±0.5 in the Hx group ( P<0.001) and 1.1±0.2 in the Hx-NNLA group ( P<0.001 vs. Nx and Hx). Similarly, PCr levels (μmol/g brain) were 3.8±0.6 in the Nx group, 0.7±0.2 in the Hx group ( P<0.001) and 0.6±0.1 in the Hx-NNLA group ( P<0.001 vs. Nx and Hx). Density of phosphorylated CREB protein (OD×mm 2) was 134.2±52.4 in the Nx group compared to 746.0±76.8 in the Hx group ( P<0.05) and 491.1±40.9 in the Hx-NNLA group ( P<0.05 Hx). The data show that NOS inhibition attenuates the hypoxia-induced increase in CREB protein phosphorylation in the cerebral cortex of newborn piglets.