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

Genistein is a kind of isoflavone compounds， also called phytoestrogens， with clinical effects on cardiovascular disease， cancer and postmenopausal-related gynecological diseases， and also has the potentiality in the prevention and treatment of Alzheimer's disease(AD). In this study， the protective effect of genistein on Aβ₂₅₋₃₅-induced PC12 cell injury and effect on CaM-CaMKIV signaling pathway were observed to investigate its mechanism for AD. PC12 cells were cultured and then the safe concentration of genistein and the modeling concentration and optimal time point of administration of Aβ₂₅₋₃₅ were screened by MTT assay. After being pretreated with different concentrations of genistein(25， 50， 100 μmol·L⁻¹) on PC12 cells， the AD model of PC12 cells was induced by Aβ₂₅₋₃₅. Then the survival rate of cells was detected by MTT assay; morphological change of cells was observed under the inverted microscope， and apoptosis of cells was assessed by AO/EB fluorescence staining; the neuroprotective effects of genistein on AD cell model were observed and the optimal concentration of genistein was determined. Expressions of mRNA and protein levels of CaM， CaMKK， CaMKIV and tau were detected by qRT-PCR and Western blot assay， respectively. The results showed that as compared with the blank group， the cell survival rate was decreased; the cell damage and apoptosis were increased; and the expressions of mRNA and protein levels of CaM， CaMKK， CaMKIV and tau were increased in AD model group. Genistein could significantly improve the cell survival rate， reduce the cell damage and apoptosis of AD cell model， and significantly down-regulate the expressions of mRNA and protein levels of CaM， CaMKK， CaMKIV and tau of AD cell model. These results indicated that genistein has obviously neuroprotective effect on the AD cell model induced by Aβ₂₅₋₃₅， and the mechanism may be related to the down-regulation of CaM-CaMKIV signaling pathway and Tau protein expression.
Amyloid beta-Peptides ; Animals ; Apoptosis ; Calcium-Calmodulin-Dependent Protein Kinase Type 4 ; metabolism ; Calmodulin ; metabolism ; Cell Survival ; Genistein ; pharmacology ; PC12 Cells ; Peptide Fragments ; Protective Agents ; pharmacology ; Rats ; Signal Transduction ; drug effects

OBJECTIVE: To evaluate the association between single nucleotide polymorphisms of calcium/calmodulin-dependent kinase 4 (CAMK4) and the therapeutic effect of amlodipine in essential hypertensive patients in Chinese Han nationality.
 METHODS: A total of 108 mild-to-moderate essential hypertension patients in Chinese Han nationality were treated with amlodipine for 8 weeks at a dosage of 5 mg/d. Polymerase chain reaction-restriction fragment length polymorphism was performed to detect the genotypes (rs10491334). Blood pressure was measured and analyzed.
 RESULTS: The result of rs10491334 polymorphism of CAMK4 was consistent with Hardy-Weinberg equilibrium distribution and the frequencies of C allele and T allele were 88.89% and 11.11%, respectively. The systolic blood pressure and diastolic blood pressure before amlodipine treatment were not statistically different among different genotype carriers (P>0.05). The blood pressure was significantly reduced in all patients after amlodipine treatment (P<0.05). Systolic blood pressure was significantly decreased in patients with rs10491334 CC genotype and CT genotype compared with those patients with rs10491334 TT genotype. Total effective rates of CT and TT carriers were higher than those of the CC genotype carriers (P<0.01).
 CONCLUSION The CAMK4 gene polymorphism might be associated with the efficacy of calcium channel blocker in treating mild-to-moderate essential hypertension patients.
Alleles ; Amlodipine ; Asian Continental Ancestry Group ; Blood Pressure ; Calcium-Calmodulin-Dependent Protein Kinase Type 4 ; Calmodulin ; Essential Hypertension ; Ethnic Groups ; Gene Frequency ; Genotype ; Humans ; Hypertension ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca concentration with a simultaneous increase in the phosphorylation of Ca/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.
Alzheimer Disease ; metabolism ; pathology ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 4 ; metabolism ; Cell Nucleus ; metabolism ; Enzyme Activation ; physiology ; HEK293 Cells ; Humans ; Neurons ; metabolism ; pathology ; Phosphorylation ; Signal Transduction ; physiology ; tau Proteins ; metabolism

OBJECTIVETo observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo.

METHODSGene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: naïve group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively.

RESULTSThe siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated.

CONCLUSIONPre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cells, Cultured ; Disks Large Homolog 4 Protein ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; genetics ; Neuralgia ; therapy ; Neurons ; physiology ; Phosphorylation ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

Sustained activation of β adrenergic receptor (βAR) leads to pathologic cardiac hypertrophy. However, the related mechanisms still remain unclear. In this study, we observe how N-acetylcysteine (NAC) affects the oxidative stress and calcium/calmodulin-dependent protein kinase II (CaMKII) expression in heart of isoproterenol (ISO)-stimulated rats, and investigate whether oxidative stress and CaMKII contribute to the development of sustained βAR-stimulated cardiac hypertrophy. Healthy male Wistar rats were randomly separated into 4 groups: control (CTRL), ISO-treated (ISO), control with NAC supplement (CTRL+NAC) and ISO-treated with NAC supplement (ISO+NAC) groups (6 rats in each group). Systolic blood pressure (SBP) was measured in awake rats with the tail-cuff method every week for two weeks. Heart weight/body weight ratio (HW/BW) and HE staining were used for the detection of myocardial hypertrophy. Myocardial mitochondrial reactive oxygen species (ROS) levels were measured by DCF fluorometry. The expressions of activated-CaMKII (p-CaMKII/CaMKII) and NADPH oxidase 4 (NOX(4)) were determined by Western blot analysis. The results showed that ISO-treated (i.p., daily 3 mg/kg, 2 weeks) rats developed an obvious cardiac hypertrophy as expressed by increases of HW/BW and myocyte cross-section area. Cardiac mitochondrial ROS level was significantly enhanced in ISO group as compared to CTRL group (P < 0.05). The expressions of NOX(4) and p-CaMKII in ISO group were also up-regulated as compared to CTRL group (1.4 and 1.6 times of CTRL, respectively, P < 0.05). NAC supplement significantly suppressed the hypertrophic development of heart in ISO-stimulated rats. The cardiac mitochondrial ROS level showed a significant decrease in rats of ISO+NAC group (P < 0.05 vs ISO). In accordance with this, ISO+NAC group rats also showed marked reductions in the expressions of NOX(4) and p-CaMKII/CaMKII compared to ISO group rats (P < 0.05). There were no significant differences of the detected indices between the rats from CTRL+NAC and CTRL groups. SBP showed no differences among four groups. These results suggest that both oxidative stress and CaMKII play important roles in sustained βAR-stimulated cardiac hypertrophy. NAC may suppress ISO-induced cardiac hypertrophy by down-regulating the expression of activated-CaMKII, and by reducing the level of oxidative stress originated from mitochondria and NADPH oxidase pathways.
Acetylcysteine ; pharmacology ; Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cardiomegaly ; physiopathology ; Isoproterenol ; pharmacology ; Male ; Mitochondria, Heart ; metabolism ; Myocardium ; pathology ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Receptors, Adrenergic, beta ; metabolism

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