kn-62 and Brain-Ischemia

Although studies have shown that cerebral ischemic preconditioning (IPC) can ameliorate ischemia/reperfusion (I/R) induced brain damage, but its precise mechanisms remain unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of IPC against ischemic brain damage induced by cerebral I/R and to explore whether the Calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway contributed to the protection provided by IPC. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with 3 min of IPC alone or KN62 (selective antagonist of CaMKII) treatment before IPC, after reperfusion for 3 days, 6 min ischemia was induced. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of CaMKII, nNOS, c-Jun and the expression of FasL. Immunoprecipitation was used to examine the binding between PSD95 and nNOS. The results showed that IPC could significantly protect neurons against cerebral I/R injury, furthermore, the combination of PSD95 and nNOS was increased, coinstantaneously the phosphorylation of CaMKII and nNOS (ser847) were up-regulated, however the activation of c-Jun and FasL were reduced. Conversely, KN62 treatment before IPC reversed all these effects of IPC. Taken together, the results suggest that IPC could diminish ischemic brain injury through CaMKII-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Apoptosis; Brain Ischemia; CA1 Region, Hippocampal; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disks Large Homolog 4 Protein; Fas Ligand Protein; Intracellular Signaling Peptides and Proteins; Ischemic Preconditioning; JNK Mitogen-Activated Protein Kinases; Male; Membrane Proteins; Neurons; Nitric Oxide Synthase Type I; Phosphorylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

Recent studies have shown that GluR6 is involved in the modulation of neuronal cell death. It has been shown that PKA can phosphorylate recombinant GluR6 homomeric receptors and that this phosphorylation of GluR6 was suggested to underlie an enhancement of whole-cell current responses. Here, we try to find out whether brain ischemia and reperfusion could induce any change in the serine phosphorylation of GluR6. Our results showed that the serine phosphorylation of GluR6 increased in hippocampus during brain ischemia and early reperfusion period. Then, we used several drugs to investigate the mechanism of modulating the serine phosphorylation of GluR6. KT5720, a specific cell-permeable inhibitor of protein kinase A (PKA), had no effect on the increase in serine phosphorylation of GluR6 induced by brain ischemia or reperfusion. On the other hand, KN-62, a selective inhibitor of rat brain Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), diminished the increase in serine phosphorylation of GluR6. Moreover, our results showed that either MK801 (a NMDA receptor antagonist) or Nifedipine (a L-type Ca2+ channel (L-VGCC) blocker) decreased the increase in serine phosphorylation. In conclusion, our results suggest that CaMKII, activated through NMDA receptors and L-VGCCs, mediated the serine phosphorylation of GluR6 during brain ischemia and early reperfusion period.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analysis of Variance; Animals; Brain Ischemia; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; GluK2 Kainate Receptor; Hippocampus; Male; Nifedipine; Phosphorylation; Phosphoserine; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Reperfusion

To investigate the interactions among postsynaptic density 95 (PSD-95), Ca2+-calmodulin dependent protein kinase IIalpha (CaMKIIalpha), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats.. Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKIIalpha to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKII) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins.. The alternations of the binding level of PSD-95 and CaMKIIalpha to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10 min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKIIalpha to NR2B.. PSD-95 competes with CaMKII to bind to NR2B during ischemia and reperfusion in rat hippocampus.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Binding, Competitive; Brain Ischemia; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Disks Large Homolog 4 Protein; Hippocampus; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nerve Tissue Proteins; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury

To investigate whether brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase (nNOS) by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the interaction between CaMKII? and nNOS in rat hippocampus.. Brain ischemia was induced by bilateral carotid artery occlusion procedure. Phosphorylation and the interaction of proteins were studied by immunoprecipitation and immunoblotting. We investigated during brain ischemia serine phosphorylation and amount of nNOS in crude membranes fraction (P) and cytosolic fraction (S), interaction between CaMKIIalpha and nNOS, and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62, a selective inhibitor of CaMKII) on phosphorylation and the interaction of proteins in P.. Serine phosphorylation of nNOS in P increased persistently during brain ischemia, and 15 min ischemia-induced serine phosphorylation of nNOS was attenuated significantly by KN-62. But there was no serine phosphorylation of nNOS in S. The distributions of nNOS were not affected by ischemia and KN-62. However, the binding levels of both CaMKIIalpha with nNOS and Thr(286) autophosphorylated CaMKIIalpha with nNOS increased after ischemia, and were diminished by KN-62.. CaMKII interacted with nNOS and regulated serine phosphorylation of nNOS during brain ischemia.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain Ischemia; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Hippocampus; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phosphorylation; Rats; Rats, Sprague-Dawley; Serine

The interaction between translocated calcium/calmdulin-dependent protein kinase IIalpha (CaMK IIalpha) and SynGAP during brain ischemia was investigated by Western blotting and immunoprecipitation. Brain ischemia was induced by the four-vessel occlusion method on Sprague-Dawley rats. After 3 min global ischemia, both the binding of CaMK IIalpha to SynGAP and the serine phosphorylation of SynGAP all dramatically increased. Administrating KN-62 through cerebral ventricle (20 min before ischemia) not only remarkably decreased the binding of CaMK IIalpha to SynGAP but also attenuate the elevated serine phosphorylation of SynGAP following 20 min ischemia in hippocampus. These results suggest that CaMK IIalpha is responsible for the serine phosphorylation of SynGAP and a consequent phosphorylation and inhibition of SynGAP may result in activation of mitogen-activated protein kinase pathway which could serve a protective function in brain ischemia.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain Ischemia; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; GTPase-Activating Proteins; Hippocampus; Male; MAP Kinase Signaling System; Phosphorylation; ras Proteins; Rats; Rats, Sprague-Dawley; Reaction Time; Serine; Synapses; Up-Regulation