Mechanism of Ca2+-influx and Ca2+/calmodulin-dependent protein kinase IV activity during in utero hypoxia in cerebral cortical neuronal nuclei of the guinea pig fetus at term

Previously we showed that following hypoxia there is an increase in nuclear Ca2+-influx and Ca2+/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 Ca2+-influx and increased CaMK IV activity, by blocking high-affinity Ca2+-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 FiO2 of 0.07 for 60 min. Clonidine, an imidazoline inhibitor of high-affinity Ca2+-ATPase, was administered 12.5 μg/kg IP 30 min prior to hypoxia. Hypoxia was determined biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and ATP-dependent 45Ca2+-influx was determined. CaMK IV activity was determined by 33P-incorporation into syntide 2 for 2 min at 37 °C in a medium containing 50 mM HEPES (pH 7.5), 2 mM DTT, 40 μM syntide 2, 0.2 mM 33P-ATP, 10 mM magnesium acetate, 5 μM PKI 5-24, 2 μM PKC 19–36 inhibitor peptides, 1 μM microcystine LR, 200 μM sodium orthovanadate and either 1 mM EGTA (for CaMK IV-independent activity) or 0.8 mM CaCl2 and 1 mM calmodulin (for total activity). ATP (μmoles/g brain) values were significantly different in the Nx (4.62 ± 0.2), Hx (1.65 ± 0.2, p < 0.05 vs. Nx), and Hx + Clo (1.92 ± 0.6, p < 0.05 vs. Nx). PCr (μmoles/g brain) values in the Nx (3.9 ± 0.1), Hx (1.10 ± 0.3, p < 0.05 vs. Nx), and Hx + Clo (1.14 ± 0.3, p < 0.05 vs. Nx). There was a significant difference between nuclear Ca2+-influx (pmoles/mg protein/min) in Nx (3.98 ± 0.4), Hx (10.38 ± 0.7, p < 0.05 vs. Nx), and Hx + Clo (7.35 ± 0.9, p < 0.05 vs. Nx, p < 0.05 vs. Hx), and CaM KIV (pmoles/mg protein/min) in Nx (1314.00 ± 195.4), Hx (2315.14 ± 148.5, p < 0.05 vs. Nx), and Hx + Clo (1686.75 ± 154.3, p < 0.05 vs. Nx, p < 0.05 vs. Hx). We conclude that the mechanism of hypoxia-induced increased nuclear Ca2+-influx is mediated by high-affinity Ca2+-ATPase and that CaMK IV activity is nuclear Ca2+-influx-dependent. We speculate that hypoxia-induced alteration of high-affinity Ca2+-ATPase is a key step that triggers nuclear Ca2+-influx, leading to CREB protein-mediated increased expression of apoptotic proteins and hypoxic neuronal death.