Mechanism of Ca(2+)-influx and Ca(2+)/calmodulin-dependent protein kinase IV activity during in utero hypoxia in cerebral cortical neuronal nuclei of the guinea pig fetus at term.

摘要

Previously we showed that following hypoxia there is an increase in nuclear Ca(2+)-influx and Ca(2+)/calmodulin-dependent protein kinase IV activity (CaMK IV) in the cerebral cortex of term guinea pig fetus. The present study tests the hypothesis that clonidine administration will prevent hypoxia-induced increased neuronal nuclear Ca(2+)-influx and increased CaMK IV activity, by blocking high-affinity Ca(2+)-ATPase. Studies were conducted in 18 pregnant guinea pigs at term, normoxia (Nx, n=6), hypoxia (Hx, n=6) and clonidine with Hx (Hx+Clo, n=6). The pregnant guinea pig was exposed to a decreased FiO(2) of 0.07 for 60min. Clonidine, an imidazoline inhibitor of high-affinity Ca(2+)-ATPase, was administered 12.5mug/kg IP 30min prior to hypoxia. Hypoxia was determined biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and ATP-dependent (45)Ca(2+)-influx was determined. CaMK IV activity was determined by (33)P-incorporation into syntide 2 for 2min at 37 degrees C in a medium containing 50mM HEPES (pH 7.5), 2mM DTT, 40muM syntide 2, 0.2mM (33)P-ATP, 10mM magnesium acetate, 5muM PKI 5-24, 2muM PKC 19-36 inhibitor peptides, 1muM microcystine LR, 200muM sodium orthovanadate and either 1mM EGTA (for CaMK IV-independent activity) or 0.8mM CaCl(2) and 1mM calmodulin (for total activity). ATP (mumoles/gbrain) values were significantly different in the Nx (4.62+/-0.2), Hx (1.65+/-0.2, p0.05 vs. Nx), and Hx+Clo (1.92+/-0.6, p0.05 vs. Nx). PCr (mumoles/g brain) values in the Nx (3.9+/-0.1), Hx (1.10+/-0.3, p0.05 vs. Nx), and Hx+Clo (1.14+/-0.3, p0.05 vs. Nx). There was a significant difference between nuclear Ca(2+)-influx (pmoles/mg protein/min) in Nx (3.98+/-0.4), Hx (10.38+/-0.7, p0.05 vs. Nx), and Hx+Clo (7.35+/-0.9, p0.05 vs. Nx, p0.05 vs. Hx), and CaM KIV (pmoles/mg protein/min) in Nx (1314.00+/-195.4), Hx (2315.14+/-148.5, p0.05 vs. Nx), and Hx+Clo (1686.75+/-154.3, p0.05 vs. Nx, p0.05 vs. Hx). We conclude that the mechanism of hypoxia-induced increased nuclear Ca(2+)-influx is mediated by high-affinity Ca(2+)-ATPase and that CaMK IV activity is nuclear Ca(2+)-influx-dependent. We speculate that hypoxia-induced alteration of high-affinity Ca(2+)-ATPase is a key step that triggers nuclear Ca(2+)-influx, leading to CREB protein-mediated increased expression of apoptotic proteins and hypoxic neuronal death.