kn-62 and Reperfusion-Injury

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

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