Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide.

Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), p<0.05 vs. Hx. The relative level p-ERK was 44.91+/-4.20 (Nx), 135.12+/-17.02 (Hx), 58.37+/-9.5 (Hx+L-NAME), p<0.05 vs. Hx. The relative level of p-JNK was 34.86+/-6.77 (Nx), 97.36+/-19.24 (Hx), 46.65+/-12.81 (Hx+L-NAME), p<0.05 vs. Hx. The data show that administration of l-NAME prior to hypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK in the guinea pig fetus at term.