Previous studies have shown that
hyperoxia results in cerebral cortical neuronal apoptosis. Studies have also shown that phosphorylation of
anti-apoptotic proteins Bcl-2 and Bcl-xl results in loss of their anti-apoptotic potential leading to alteration in mitochondrial membrane permeability and the release of apoptogenic
proteins in the neuronal cell of the newborn piglets. The present study tests the hypothesis that cerebral
hyperoxia will result in increased
serine phosphorylation of apoptotic
proteins Bcl-2, Bcl-xl, Bax, and Bad in the mitochondrial membranes of the cerebral cortex of newborn piglets. Twelve newborn piglets were divided into normoxic (Nx, n = 6) exposed to an FiO(2) of 0.21 for 1 h and hyperoxic (Hyx, n = 6) exposed to FiO(2) of 1.0 for 1 h. In the Hyx group, PaO(2) was maintained above 400 mmHg while the Nx group was kept at 80-100 mmHg. Cerebral cortical tissue was harvested and mitochondrial fractions were isolated. Mitochondrial membrane
proteins were separated using 12% SDS-PAGE, and probed with anti-
serine phosphorylated Bcl-2, Bcl-xl, Bax, and Bad
antibodies.
Protein bands were detected, analyzed by imaging densitometry and density expressed as absorbance (OD x mm(2)). Phosphorylated Bcl-2 (p-Bcl-2)
protein density (OD x mm(2)) was 81.81 +/- 9.24 in Nx and 158.34 +/- 10.66 in Hyx (P < 0.05). Phosphorylated Bcl-xl (p-
Bcl-xl) protein density was 52.98 +/- 3.59 in Nx and 99.62 +/- 18.22 in Hyx (P < 0.05). Phosphorylated Bax (p-
Bax) protein was 161.13 +/- 6.27 in Nx and 174.21 +/- 15.95 in Hyx (P = NS). Phosphorylated Bad (p-
Bad) protein was 166.24 +/- 9.47 in Nx 155.38 +/- 12.32 in Hyx (P = NS). The data show that there is a significant increase in
serine phosphorylation of Bcl-2 and Bcl-xl
proteins while phosphorylation of Bad and Bax
proteins were not altered during
hyperoxia in the mitochondrial fraction of the cerebral cortex of newborn piglets. We conclude that
hyperoxia results in differential post-translational modification of
anti-apoptotic proteins Bcl-2 and Bcl-xl as compared to
pro-apoptotic proteins Bax and Bad in mitochondria. We speculate that phosphorylation of Bcl-2 and Bcl-xl will result in loss of their anti-apoptotic potential by preventing their dimerization with Bax leading to activation of the
caspase cascade of neuronal death.