OBJECTIVETo study the effects of calcium and calmodulin dependent kinase against hypoxic neuronal injury and its possible mechanisms.

METHODSEmbryonic cortical neurons of 17-day pregnant embryo Sprague-Dawley rats were cultured in vitro and the cultured neurons were randomly allocated into different groups that exposed to hypoxia or hypoxia +calcium channel antagonist. Nimodipine and MK-801 were used to block the L-voltage sensitive calcium channel and NMDA receptor respectively before hypoxia. The methyl thiazolyl tetrazolium (MTT) method was used to determine the cell viability. Fluo-4AM, an intracellular calcium indictor, was used to detect the changes of intracellular calcium after hypoxia. The expressions of CaMKII and CaMKIV were detected by Western blot.

RESULTSThe cell viability of the nimodipine or MK-801-treated groups was significantly higher than that of the untreated hypoxia group. The intracellular calcium level of the nimodipine-treated group decreased rapidly after hypoxia. Compared to nimodipine treatment, MK-801 treatment could inhibit hypoxia-induced calcium influx for a longer time. Nimodipine treatment decreased the CaMKII expression while MK-801 treatment decreased the CaMKIV expression.

CONCLUSIONSNimodipine and MK-801 protect neurons from hypoxic injury possibly by the inhibition of CaMKII and CaMKIV expressions respectively.

Animals ; Calcium ; analysis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinase Type 4 ; Calcium-Calmodulin-Dependent Protein Kinases ; antagonists & inhibitors ; physiology ; Cell Hypoxia ; Dizocilpine Maleate ; pharmacology ; Female ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Nimodipine ; pharmacology ; Rats ; Rats, Sprague-Dawley

The aim of this study is to investigate the neurotoxic effect and mechanism of 1-methyl-4-phenylpyridinium (MPP+) on PC12 cells. MTT assay was used to investigate cell viability, Western blotting assay was performed to observe the protein level and phosphorylation, and dual-luciferase assay was used to study the transactivation. The experiment showed that MPP+ could decrease cell viability significantly in a dose-dependent manner and could decrease BDNF protein level, depress the phosphorylation of ERK, and attenuate the phosphorylation and transactivation of CREB, which is one of transcription factors of BDNF, but did not affect the activity of CaMK II in PC12 cells. So MPP+ might decrease BDNF protein level through MAPK/ERK signal pathway.
1-Methyl-4-phenylpyridinium ; administration & dosage ; pharmacology ; Animals ; Brain-Derived Neurotrophic Factor ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cell Survival ; drug effects ; Cyclic AMP Response Element-Binding Protein ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; drug effects ; PC12 Cells ; Phosphorylation ; Rats ; Signal Transduction

Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular Ca2+ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular Ca2+ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a Ca2+ activated protein, and Ca2+/ calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of Ca2+/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular Ca2+ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and Ca2+/calmodulin-dependent protein kinase II in HepG2 cells.
Apoptosis* ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calmodulin* ; Carcinoma, Hepatocellular* ; Estrogen Receptor Modulators ; Hep G2 Cells ; Hepatoblastoma ; Humans* ; Protein Kinases ; Reactive Oxygen Species* ; Receptors, Estrogen ; Tamoxifen*

Brain-derived neurotrophic factor (BDNF)is the richest neurophin in brain tissue and may act as an activity-dependent neuronal survival factor. In vitro, BDNF plays an important role in preventing cortical neurons from hypoxia-induced neurotoxicity. It could induce a variety of cellular responses such as cell growth, survival, differentiation, and anti-apoptosis mainly via activating mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways. And among these multiple signaling pathways there is growing evidence of complicated cross talk.
Animals ; Brain-Derived Neurotrophic Factor ; pharmacology ; physiology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; drug effects ; physiology ; Cells, Cultured ; Humans ; Mitogen-Activated Protein Kinases ; drug effects ; physiology ; Neurons ; cytology ; drug effects ; enzymology ; Signal Transduction

To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca2+/CaMK II expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death, concentrations of cytosolic free calcium, and the expression of CaMK II by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 10(-7), 10(-6) and 10(-5) mol/L of CORT was added to culture medium, The evident effect of 10(-6) and 10(-5) mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca2+]i increased in the neurons treated with 10(-6) and 10(-5) mol/L of CORT, but no change was observed in the neuron treated with 10(-7) mol/L of CORT. The death was either by way of apoptosis or necrosis in the cells treated with 10(-6) and 10(-5) mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca2+]i and the expression of CaMK II. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca2+]i is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMK II expression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; biosynthesis ; genetics ; Cells, Cultured ; Corticosterone ; pharmacology ; Hippocampus ; cytology ; enzymology ; Neurons ; cytology ; Rats ; Rats, Wistar

OBJECTIVETo reconstitute an Alzheimer's disease model by administering bradykinin (BK) or cyclosporine A (CSA) to the rat hippocampus.

METHODSBK or CSA was administered to the rat hippocampus using a stereotaxic apparatus. The behavior of the rats was observed with an electronic attack jump platform. The phosphorylation of Tau protein was examined through immunohistochemical assay.

RESULTSBehavior studies showed that an obvious disturbance in learning and memory was seen in BK injected rats.No obvious dysfunction was observed in CSA injected rats. The results obtained by immunohistochemical assay indicated that the staining of M4, 12E8, paired helical filament-1 (PHF-1) and calcium/calmodulin-dependent protein kinase II (CaMKII) was stronger, and that of Tau-1 was weaker in BK injected rats compared with the control group. We also found that the binding of M4 and PHF-1 but not 12E8 to Tau was significantly increased in CSA injected rats. As for BK injection, binding of Tau-1 to Tau was decreased after CSA injection.

CONCLUSIONTo our knowledge, this is the first data showing in vivo that the activation of CaMKII induces both Alzheimer-like Tau phosphorylation and behavioral disturbances.

Alzheimer Disease ; etiology ; Animals ; Bradykinin ; toxicity ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; metabolism ; Cyclosporine ; toxicity ; Disease Models, Animal ; Hippocampus ; drug effects ; metabolism ; Immunohistochemistry ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; tau Proteins ; analysis ; metabolism

Kaempferol exerts cardioprotective actions through incompletely understood mechanisms. This study investigated the molecular mechanisms underlying the cardioprotective effects of kaempferol in sinus node dysfunction (SND) heart. Here, we demonstrate that angiotensin II (Ang II) infusion causes SND through oxidized calmodulin kinase II (CaMKII). In contrast to this, kaempferol protects sinus node against Ang II-induced SND. Ang II evoked apoptosis with caspase-3 activation in sinus nodal cells. However, kaempferol lowered the CaMKII oxidization and the sinus nodal cell death. To block the CaMKII oxidization, gene of p47phox, a cytosolic subunit of NADPH oxidase, was deleted using Cas9 KO plasmid. In the absence of p47phox, sinus nodal cells were highly resistance to Ang II-induced apoptosis, suggesting that oxidized-CaMKII contributed to sinus nodal cell death. In Langendorff heart from Ang II infused mice, kaempferol preserved normal impulse formation at right atrium. These data suggested that kaempferol protects sinus node via inhibition of CaMKII oxidization and may be useful for preventing SND in high risk patients.
Angiotensin II ; Animals ; Apoptosis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2* ; Calcium-Calmodulin-Dependent Protein Kinases ; Caspase 3 ; Cell Death ; Cytosol ; Heart ; Heart Atria ; Humans ; Mice ; NADPH Oxidase ; Plasmids ; Sick Sinus Syndrome* ; Sinoatrial Node*

According to the widely accepted concept that contraction of arterial smooth muscle is dependent on the Ca2+/calmodulin and protein kinase C(PKC) systems, it has been recently proposed that activation of these two systems in vascular smooth muscle cells has a bearing on the pathogenesis of cerebral vasospasm. However, most of the reported PKC inhibitors display a poor selectivity. In a rabbit "vasospasm" model, we tested the effect of a new PKC inhibitor(Bisindolylmaleimides; BIS) which has been known for a relatively high degree of selectivity, and also investigated the possible role of a inhibitor of Ca2+/calmodulin-dependent protein kinase(CAM kinase II inhibitor; KN-62). Finally, the vasodilating action of BIS was compared to KN-62. The basilar artery was visualized using transclival exposure, and its diameter monitored using videomicroscopy. Rabbit basilar artery was constricted in vivo by topical application of phorbol dibutyrate. Application of phorbol dibutyrate elicited an acute constriction, reducing arterial diameter to 46.5% of baseline diameter. Treatment with BIS (5micrometer) attenuated phorbol dibutyrate-induced vasoconstriction to 96.2% of baseline. Subsequently, application of oxyhemoglobin(oxy-Hb; 10 -4M) reduced vascular diameter to 68.5% of baseline. This vasospastic response was reversed by 91.4% or 81.3% using BIS(5micrometer) or KN-62(5micrometer), respectively. Combined applications of BIS and KN-62 to investigate the enhancing effect produced basilar arterial constriction of 94.9% of baseline. These findings demonstrate that BIS is a potent PKC inhibitor, and PKC system, rather than Ca2+/calmodulin system, appears to play a major role in the occurrence of cerebral vasospasm. Additionally, we suggest that combination of PKC with CAM kinase II inhibitor may be more useful therapeutic agents for treating cerebral vasospasm.
Basilar Artery* ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Constriction* ; Microscopy, Video ; Muscle, Smooth ; Muscle, Smooth, Vascular ; Phosphotransferases ; Protein Kinase C* ; Protein Kinases* ; Vasoconstriction ; Vasospasm, Intracranial

The most important molecular mechanisms of intraneuronal signal transduction are those mediated by calcium and reversible protein phosphorylation. Although many studies pursued the activation of the protein kinases in the nervous system, there are only few reports focused on the protein phosphatases. In this article, the authors report the effects of cyclosporin A (CSA), an inhibitor of calcineurin, on the calcium signaling-related molecules such as ERKs, calmodulin-dependent kinase II (CaMKII) and CREB in the rat hippocampus. The authors also report the effects of cyclosporin A on the electroconvulsive shock (ECS)-induced seizure and the activation of ERKs. Calcineurin is a protein phosphatase that is abundant in the brain and regulated by calcium and calmodulin. It is proposed that calcineurin plays central roles in the synaptic plasticity and neuronal apoptosis. CSA (50 mg/kg) increased the phosphorylation of ERK, CaMKII and CREB. The treatment of of CSA increased the duration of tonic phase of seizure induced by ECS and augmented the phosphorylation of ERKs after ECS. These results suggested the protective role of calcineurin against the excessive electrical and molecular activities in the brain.
Animals ; Apoptosis ; Brain* ; Calcineurin ; Calcium ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calmodulin ; Cyclosporine* ; Electroshock ; Hippocampus ; Nervous System ; Neurons ; Phosphoprotein Phosphatases ; Phosphorylation ; Phosphotransferases ; Plastics ; Protein Kinases ; Rats* ; Seizures ; Signal Transduction*

Reducing [Mg2+]o to 0.1 mM can evoke repetitive [Ca2+]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg2+]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca2+ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg2+]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca2+ channel antagonist nimodipine, which blocked 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca2+]i spikes. The intracellular Ca2+ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca2+]i spikes. While Go6976, a specific inhibitor of PKCalpha had no effect on the tolerance, both the PKCepsilon translocation inhibitor and the PKCzeta pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca2+]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg2+]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca2+]i spike-induced activation of PKCepsilon and PKCxi, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.
Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Cell Death ; Cycloheximide ; N-Methylaspartate ; Neurons* ; Nimodipine ; Phosphatidylinositol 3-Kinases ; Phosphotransferases ; Rats* ; Receptors, AMPA ; Seizures