Expression of Bcl-2 and Bax in Neuronal Nuclei During Severe Acute Hypoxia in Newborn Piglets 359

Abstract

Previous studies have shown that Bcl-2 and Bax proteins are regulators of programmed cell death and survival. Bcl-2 protein promotes cell survival, while Bax protein promotes apoptotic cell death under a variety of pathological and physiological conditions. It also is known that the relative amounts of Bcl-2 and Bax proteins determine cellular susceptibility to apoptotic stimuli. Nuclear memberanes are rich in these proteins. The present study tests the hypothesis that cerebral hypoxia in newborn piglets will result in an up-regulation of Bax protein and altering the Bax to Bcl-2 protein ratio in the nuclear membrane. Anesthetized, ventilated newborn piglets, 3-5 days old, were exposed to either room air (normoxia, n=5) or had the FIO2 gradually lowered to 7% and then adjusted as needed to achieve a PO2 of 18 mmHg (hypoxia, n=5) for 1 hr. Tissue hypoxia was confirmed biochemically by decreased levels of ATP and phosphocreatine. At the end of the experimental period, cerebral cortical neuronal nuclei were immediately isolated using a discontinuous sucrose gradient. Nuclear proteins were analyzed for Bcl-2 and Bax proteins using immunoprecipitation and Western blot analysis. The proteins were separeted by 12% SDS-PAGE and then electrically transferred to nitrocellulose paper where they were probed using specific antibodies directed against Bcl-2 or Bax. The samples were immunostained using horseradish peroxidase conjugates. Western blots were analyzed using an imaging densitometer. Nuclear membrane Bax protein levels increased from 3.4 OD × mm2 during normoxia to 5.2 OD × mm2 during hypoxia. The results show that during acute severe hypoxia. Bax protein generation was up-regulated to 1.5 times control values where the amount of Bcl-2 protein remained the same as control. Since Bcl-2 protein heterodimerizes with Bax to prevent cell death, we speculate that during acute severe hypoxia in newborn piglet cortices, up-regulation of Bax protein and/or a change in the ratio of Bax to Bcl-2 protein concentrations will lead to hypoxia-induced programmed cell death.