Effect of Cerebral Ischemia on Calcium/Calmodulin-Dependent Protein Kinase II Activity and Phosphorylation

Abstract

Summary:The effects of cerebral ischemia on calcium/ calmodulin-dependent kinase II (CaM kinase II) were in vestigated using the rat four-vessel occlusion model. In agreement with previous results using rat or gerbil models of cerebral ischemia or a rabbit model of spinal cord isch emia, this report demonstrates that transient forebrain ischemia leads to a reduction in CaM kinase II activity within 5 min of occlusion onset. Loss of activity from the cytosol fractions of homogenates from the neocortex, striatum, and hippocampus correlated with a decrease in the amount of CaM kinase Ct and � isoforms detected by immunoblotting. In contrast, there was an apparent in crease in the amount of CaM kinase Ct and � in the par ticulate fractions. The decrease in the amount of CaM kinase isoforms from the cytosol but not the particulate fractions was confirmed by autophosphorylation of CaM kinase II after denaturation and renaturation in situ of the blotted proteins. These results indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the cyCalcium/calmodulin-dependent kinase II (CaM kinase II) is a multifunctional protein kinase critical to multiple signal transduction pathways responsive to changes in intracellular Ca2 + concentrations. The a, 13, and 13' (a splice variant) isoforms of the enzyme are highly enriched in neuronal tissue and compose 0.1-2% of the total protein in various brain 450to sol and particulate fractions. CaM kinase II is a multi functional protein kinase, and the loss of activity may play a critical role in initiating the changes leading to ischemia-induced cell death.To identify a structural basis for the decrease in en zyme activity, tryptic peptide maps of CaM kinase II phosphorylated in vitro were compared. Phosphopeptide maps of CaM kinase Ct from particulate fractions of con trol and ischemic samples revealed not only reduced in corporation of phosphate into the protein but also the absence of a limited number of peptides in the ischemic samples. This suggested that certain sites are inaccessi ble, possibly due to a conformational change, a covalent modification of CaM kinase II, or steric hindrance by an associated molecule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia induced modulation of CaM kinase II.

Calcium-independent CaMKII activity is involved in ginsenoside Rb1-mediated neuronal recovery after hypoxic damage

“…Imbalance in the regulation of CaMKII activity is related to the extent of neuronal loss by ischemic injury (Waxham et al, 1996;Shackelford et al, 1995). Since Rb1 protected neuronal cells from ischemic damage, it is possible that Rb1 in conjunction with CaMKII activity might be involved in the process of regulating synaptic excitability after neuronal damage.…”

“…As one of the key molecular components in relation to ischemic injury, unique regulatory properties of Ca 2+ / calmodulin-dependent protein kinase II (CaMKII) activity have been called special attention (Colbran, 1992;Churn et al, 1992). Protein levels and activity of CaMKII were observed to be re-distributed between cytosolic and membrane fractions in hippocampal tissues, and also showed overall decreases following global ischemic injury (Aronowski et al, 1992;Kolb et al, 1995;Shackelford et al, 1995). Studies also showed that time-dependent loss of synaptic responses was more intense in CA1 pyramidal cells than granule cells in hypoxia-induced hippocampal slices, and these changes were positively correlated with the levels of CaMKII activity (Westgate et al, 1994).…”

The Caenorhabditis elegansSex-determining Protein FEM-2 and Its Human Homologue, hFEM-2, Are Ca2+/Calmodulin-dependent Protein Kinase Phosphatases That Promote Apoptosis

“…Dephosphorylation appears to be an important regulatory step with respect to the kinase activity of CaM kinases (22)(23)(24)(25). Indeed, autophosphorylation of CaM kinase II at Thr 286 has been shown to be up-regulated in ischemic tolerance (44), and loss of CaM kinase activity has been suggested to play a role in initiating the changes leading to ischemia-induced cell death (45). KN-93, a specific inhibitor of CaM kinase II, induces apoptosis in NIH 3T3 cells (46).…”

Cellular and Molecular Pathways of Ischemic Neuronal Death

“…The activity of CaMK II is decreased in a way that is sensitive to NMDA antagonists that rapidly follow transient focal or global cerebral ischemia (Hanson et al, 1994;Shackelford et al, 1995). While homozygous knock-out mice that lack the alpha subunit of CaMK II enhances sensitivity to hypoxic-ischemic insults in vivo (Waxham et al, 1996), a selective cellpermeable inhibitor of CaMK II (KN62) attenuates neuronal death, following exposure to NMDA, or the deprivation of oxygen-glucose in vitro (Hajimohammadreza et al, 1995).…”