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

Abstract

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 60 min. Clonidine, an imidazoline inhibitor of high-affinity Ca(2+)-ATPase, was administered 12.5 microg/kg IP 30 min 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 2 min 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, 5 microM PKI 5-24, 2 microM PKC 19-36 inhibitor peptides, 1 microM microcystine LR, 200 microM 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, p<0.05 vs. Nx), and Hx+Clo (1.92+/-0.6, p<0.05 vs. Nx). PCr (mumoles/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 Ca(2+)-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 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.

Effect of Concurrent Src Kinase Inhibition with Short-Duration Hypothermia on Ca²⁺/Calmodulin Kinase IV Activity and Neuropathology after Hypoxia-Ischemia in the Newborn Swine Brain

“…Inhibition of Src kinase prevented the hypoxia-induced increase in tyrosine phosphorylation of calmodulin at Tyr99 and CaM kinase IV showing that it is the Src kinase that mediates the increased Tyr phosphorylation of calmodulin and CaM kinase IV in neuronal nuclei of the cerebral cortex of newborn piglets. [12]…”

“…Ca ++/ calmodulin-dependent kinase IV (CaM Kinase IV), located in the nucleus, phosphorylates the cyclic-AMP response element binding (CREB) protein, a regulator of the transcription of genes of cell survival or cell death pathways. [11,12]…”

Epidermal Growth Factor Receptor Inhibition Reverses Cellular and Transcriptomic Alterations Induced by Hypoxia in the Neonatal Piglet Brain

“…It has been shown that EGFR overexpression induces apoptosis through molecular alterations of the glutamate ionotropic receptor N-methyl-D-aspartate (NMDA)-type subunit 2B (GluN2B) ( Tang et al., 2015 ). Modification of the NMDA receptor by oxygen free radicals promotes calcium influx into the cytosol, deactivation of protein tyrosine phosphatases, and activation of EGFR kinase ( Maulik et al., 2008 ; Vibert et al., 2008 ). This series of events causes downstream activation of nuclear CaMKIV, CREB transcription, and the formation of the apoptosome and caspase activation, ultimately leading to DNA fragmentation and cell death ( Mishra et al., 2009 , 2010 ).…”

Effect of Src Kinase inhibition on Cytochrome c, Smac/DIABLO and Apoptosis Inducing Factor (AIF) Following Cerebral Hypoxia-Ischemia in Newborn Piglets

“…Prolonged cerebral HI leads to a state of primary energy failure, characterized by the depletion of cellular reserves of high energy compounds such as adenosine triphosphate (ATP) and phosphocreatine (PCr), as well as by mitochondrial dysfunction. Electrochemical disruption of the neuronal membrane results in lipid peroxidation, prolonged “open” state of the N-methyl-D-aspartate (NMDA) receptor and a subsequent influx of calcium into the cytosol with the formation of free radicals 1 – 4 . In addition, calcium influx triggers the formation of focal adhesions through the deactivation of cytosolic phosphatases and activation of protein complexes that extend from the membrane to the nucleus and mitochondria 5 , 6 .…”