Mechanism of Post-Translational Modification by Tyrosine Phosphorylation of Apoptotic Proteins During Hypoxia in the Cerebral Cortex of Newborn Piglets

Abstract

The present study aims to investigate the mechanism of phosphorylation of apoptotic proteins and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of apoptotic proteins Bcl-2 and Bcl-xl is Ca(2+)-influx-dependent. Piglets were divided in normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-pretreated with clonidine (Clo + Hx, n = 4) groups. Hypoxic animals were exposed to an FiO(2) of 0.06 for 1 h. Clonidine (12.5 microg/kg, IV) was administered to piglets 30 min prior to hypoxia. Hypoxia was confirmed by ATP and phosphocreatinine (PCr) levels. Cytosol was isolated and separated by 12% SDS-PAGE and probed with tyrosine phosphorylated (p) -Bax, Bad, Bcl-2 and Bcl-xl antibodies and bands were detected. The ATP levels (micromol/g brain) in the Nx, Hx, Clo + Hx were 4.3 +/- 1.0 (P < 0.05 vs. Hx, Clo-Hx), 0.9 +/- 0.8 and 1.5 +/- 0.3, respectively. The PCr levels in the Nx, Hx, Clo + Hx were 2.7 +/- 0.7 (P < 0.05 vs. Hx, Clo-Hx), 0.9 +/- 0.2 and 0.9 +/- 0.9, respectively. Ca(2+)-influx (pmoles/mg protein) was 4.96 +/- 0.94 in Nx, 11.11 +/- 2.38 in Hx, and 6.23 +/- 2.07 in Clo + Hx (P < 0.05 Nx vs. Hx and Hx vs. Clo + Hx). p-Bcl-2 density was 21.1 +/- 1.1 Nx, 58.9 +/- 9.6 Hx and 29.5 +/- 6.4 Clo + Hx (P < 0.05 vs. Hx). p-Bcl-xl density was 29.6 +/- 1.5 Nx, 50.6 +/- 7.4 Hx and 32.1 +/- 0.1 Clo + Hx (P < 0.05 vs. Hx). p-Bax density was 38.6 +/- 16.2 Nx, 46.1 +/- 5.5 Hx and 41.6 +/- 1.9 Clo + Hx groups (P = NS). p-Bad was 66.7 +/- 12.8 Nx, 71.2 +/- 6.8 Hx and 78.7 +/- 22.5 Clo + Hx groups (P = NS). Results showed that clonidine administration prior to hypoxia prevents the hypoxia-induced increased nuclear Ca(2+)-influx and increased phosphorylation of Bcl-2 and Bcl-xl while phosphorylation of Bad and Bax was not altered. We conclude that post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl during hypoxia is nuclear Ca(2+)-influx-dependent. We propose that blockade of nuclear Ca(2+)-influx that prevents phosphorylation of antiapoptotic proteins may become a neuroprotective strategy.