Abstract 1999
Poster Session II, Sunday, 5/2 (poster 230)
Previous studies have shown that Bax, a proapoptotic gene, and Bcl-2, an anti-apoptotic gene, code for proteins that play a critical role in regulating programmed cell death by forming an heterodimeric complex. In addition phosphorylated Bcl-2 cannot form an heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded cerebral hypoxia results in increased phosphorylation of Bcl-2 with increase in degree of hypoxia, thus altering the heterodimerization of Bcl-2 with Bax. Studies were performed in 10 newborn piglets, age 3-5 days, divided into normoxic (Nx n=3) and hypoxic (Hx n=7) groups. In order to create graded levels of cerebral hypoxia the animals were exposed to a single FiO2 from 0.21 to 0.05 for 1 hour. Tissue hypoxia was determined biochemically by measuring levels of ATP and phosphocreatine (PCr). Cerebral cortical nuclei were isolated using a discontinuous sucrose gradient. The cortical membranes were immunoprecipitated with antiphosphotyrosine, incubated with protein A Sepharose, washed and suspended in 150 µl of Laemmli sample buffer and then heated at 90°C for 3-5 min. The protein A sepharose was centrifuged and the supernatant was analyzed by 12% SDS gel electrophoresis. Proteins were transferred to 0.2 mm nitrocellulose membranes, reacted with Bax and Bcl-2 antibodies and then conjugated with horseradish peroxidase-labeled goat anti-rabbit IgG. After washing, nitrocellulose membranes were incubated with chemiluminescent substrate and exposed to X-ray film. Protein bands were analyzed by imaging densitometry. The density of the protein band was expressed as absorbance OD × mm2. During graded hypoxia, as PCr levels decreased from 4.99, 3.36, 2.80, 1.69, 0.90, 0.81 to 0.61 µmoles/g brain tissue, the precipitated amount of phosphorylated Bcl-2 increased from 1.8, 2.9, 3.4, 3.7, 3.9, 4.1, to 4.7, OD × mm2. The increase in phosphorylated Bcl-2 correlates inversely with tissue ATP and PCr levels (r=0.97, r=0.88, respectively). Phosphorylated Bax expression was not altered during graded hypoxia (2.8 ± 0.98 Hx, 2.6 ± 0.63 Nx). The results show that during hypoxia there is increased phosphorylation of Bcl-2 without a change in Bax phosphorylation. We speculate that, during hypoxia, altered phosphorylation of Bcl-2 will prevent heterodimerization of Bcl-2 with Bax, resulting in caspase-mediated programmed cell death in the hypoxic newborn brain.
