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, 1mg/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 (33)P incorporation into a specific
peptide substrate for 15 min at 37 degrees C in a medium containing 100 mM
HEPES, pH 7.0, 1mM
EDTA, 125 mM MgCl(2), 25 mM MnCl(2), 2mM DTT, 0.2 mM
sodium orthovanadate, 2mM
EGTA, 150 microM
tyrosine kinase peptide substrate [Lys 19] cdc2(6-20)-NH(2), (33)P-ATP, and 10 microg of
membrane protein.
Protein tyrosine kinase activity was determined by the difference between (33)P 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 micromol/g, 1.29+/-0.23 micromol/g (p<0.05 versus normoxic) and 1.50+/-0.14 micromol/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 micromol/g, 0.77+/-0.13 micromol/g (p<0.05 versus normoxic) and 1.02+/-0.24 micromol/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 tyrosin
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.