Nitric oxide-mediated activation of extracellular signalregulated kinase (ERk) and c-Jun n-Terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of newborn piglets

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro-L-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic (N-nitro-L-arginine [NNLA]-Hx) 3-5 day old piglets. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance OD x mm(2). ATP levels were 4.57 +/- 0.45 mumoles/g brain in the Nx group, 1.29 +/- 0.23 mumoles/g brain in the Hx group (P<0.05 vs Nx) and 1.50 +/- 0.14 mumoles/g brain in the NNLA-Hx group (P<0.05 vs Nx). PCr levels were 3.77 +/- 0.36 mumoles/g brain in the Nx group, 0.77 +/- 0.13 mumoles/g brain in the hypoxic group (P<0.05) and 1.02 +/- 0.24 in the NNLA-Hx group (P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5 +/- 53.7 OD x mm(2) in the Nx group as compared with 419.6 +/- 63.9 OD x mm(2) in the hypoxic group (P<0.001 vs Nx) and 270.0 +/- 28.7 in the NNLA-Hx group (P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8 +/- 42.8 OD x mm(2) in the normoxic group as compared with 364.6 +/- 60.1 OD x mm(2) in the Hx group (P<0.002) and 254.8 +/- 24.8 in the NNLA-Hx group (P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration NNLA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated (C) 2003 IBRO. Published by Elsevier Ltd. All rights reserved.