Phosphorylation of Bcl-2 and Bax Proteins During Acute Hypoxia in Cerebral Cortical Nuclei of the Guinea Pig Fetus • 1889

The Proto-oncogene bcl-2 is a critical regulator of programmed cell death in a number of physiological and pathological conditions. Bcl-2 protein, the cell death repressor, makes a heterodimer with Bax, the cell death effector, and the ratio of these two nuclear membrane proteins determines cell survival. Recent studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypotheses that cerebral tissue hypoxia increases the expression of Bax and induces the phosphorylation of Bcl-2 thus altering the heterodimerization of Bcl-2 with Bax. Guinea pig fetuses were obtained from anesthetized, normoxic (21% oxygen, n=6) and hypoxic (7% oxygen for 60 min, n=6) mothers. Fetal brains were removed immediately after hypoxic exposure. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphocreatine. Cerebral cortical nuclei were isolated and purified using a discontinuous sucrose gradient. Using the cortical nuclear membranes, Bcl-2 and Bax proteins were immunoprecipitated, separated on 10% SDS-PAGE, transblotted to nitrocellulose papers, probed with antiphosphoserine antibody, developed using horseradish peroxidase conjugates. The protein bands for phosphorylated and non-phosphorylated Bcl-2 and Bax proteins were analyzed by imaging densitometry. The density of the proteins was expressed as absorbance (OD × mm²). In hypoxic nuclei there was a 1.9-fold increase in Bax expression as compared to normoxic nuclei (0.309 ± 0.67, hypoxic vs 0.163 ± 0.033, mormoxic). Bcl-2 expression was not altered during hypoxia. However, the phosphorylated form of Bcl-2 was 1.7-fold higher in the hypoxic nuclei than is normoxic nuclei (0.165 ± 0.030, hypoxic vs 0.096 ± 0.021 mormoxic). Bax phosphorylation was not affected by hypoxia. The data suggest that, during hypoxia, phosphorylation of Bcl-2 will prevent its heterodimerization with Bax leading to hypoxia-induced programmed cell death. We speculate that altered expression of Bax and phosphorylation of Bcl-2 are potential mechanisms of increased programmed cell death in the hypoxic fetal brain.