Effect of hypoxia on protein tyrosine kinase activity in cortical membranes of newborn piglets - the role of nitric oxide

Previous studies have shown that cerebral hypoxia results in increased tyrosine phosphorylation of cerebral cortical cell membrane proteins as well as nuclear membrane anti-apoptotic protein, Bcl-2. The present study tests the hypothesis that hypoxia results in increased protein tyrosine kinase activity in cortical cell membranes of newborn piglets and that the inhibition of neuronal NOS by administration of 7-nitroindazole sodium salt (7-NINA), a selective inhibitor of nitric oxide synthase (NOS), will prevent the hypoxia-induced increase in protein tyrosine kinase activity. To test this hypothesis, protein tyrosine kinase activity was determined in cerebral cortical membranes of 2- to 4-day-old newborn piglets divided into normoxic (n = 6), hypoxic (n = 5) and 7-NINA-treated hypoxic (n = 5) (7-NINA, 1 mg/kg, i.p., prior to hypoxia) groups. Tissue hypoxia was achieved by exposing the animals to an FiO(2) of 0.07 for 60 min and was documented biochemically by determining tissue ATP and phosphocreatine (PCr) levels. Cortical P-2 membranes were isolated and protein tyrosine kinase activity determined by P-33 incorporation into a specific peptide substrate for 15 min at 37degreesC in a medium containing 100 mM HEPES, pH 7.0, 1 mM EDTA, 125 mM MgCl2, 25 mM MnCl2, 2 mM DTT, 0.2 mM sodium orthovanadate, 2 mM EGTA, 150 muM tyrosine kinase peptide substrate [Lys 19] cdc2(6-20)-NH2, P-33-ATP, and 10 mug of membrane protein. Protein tyrosine kinase activity was determined by the difference between P-33 incorporation in the presence and absence of specific peptide substrate and expressed as pmol/mg protein/h. The ATP values in the normoxic, hypoxic and 7-NINA-treated hypoxic animals were ATP: 4.57 +/- 0.45 mumol/g, 1.29 +/- 0.23 mumol/g (p < 0.05 versus normoxic) and 1.50 +/- 0.14 mumol/g brain (p < 0.05 versus normoxic), respectively. The PCr values in the normoxic, hypoxic and 7-NINA-treated hypoxic animals were: 3.77 +/- 0.36 mumol/g, 0.77 +/- 0.13 mumol/g (p < 0.05 versus normoxic) and 1.02 +/- 0.24 mumol/g brain (p < 0.05 versus normoxic), respectively. Protein tyrosine kinase activity in the normoxic, hypoxic and the 7-NINA-treated groups was 378 +/- 77 pmol/mg protein/h, 854 +/- 169 pmol/mg protein/h (p < 0.05 versus normoxic) and 464 +/- 129 pmol/mg protein/h (p < 0.05 versus hypoxic), respectively. The data show that cerebral tissue hypoxia results in increased protein tyrosine kinase activity in cortical membranes of newborn piglets and pretreatment with 7-NINA prevents the hypoxia-induced increase in protein tyrosine kinase activity. We conclude that the hypoxia-induced increase in protein tyrosine kinase activity is NO-mediated. We propose that the hypoxia-induced increase in protein tyrosine kinase activity leading to increased phosphorylation of Bcl-2 is a critical link to hypoxic neuronal injury pathway. (C) 2004 Elsevier Ireland Ltd. All rights reserved.