Effects of Calmodulin-dependent Protein Kinase Ⅱ Inhibitor,KN-93,on Electrophysiological Features of Rabbit Hypertrophic Cardiac Myocytes

Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.

Involvement of Calcium dependent Protein Kinases in ABA regulation of Stomatal Movement

Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.

MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.

Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.

The calmodulin-dependent protein kinase II inhibitor KN-93 protects rat cerebral cortical neurons from N-methyl-D-aspartic acid-induced injury

In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyI-D-aspartic acid-induced injury. Results showed that, compared with N-methyi-D- aspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca2＋ concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 pM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin- dependent protein kinase II expression.

Ginsenoside Rb1 Pretreatment Attenuates Myocardial Ischemia by Reducing Calcium/Calmodulin-Dependent Protein Kinase Ⅱ-Medicated Calcium Release

Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A myocardial ischemia(IS)rat.model and a myocardial H9 C2 cell hypoxia model were established.MI was induced by occluding the left anterior descending artery for 120 min.Ginsenoside Rb1(10 mg/kg)was administered 30 min before ischemia induction,and the treatment continued for 7 days.Results:In the rat IS injury model,ginsenoside Rb1 reduced myocardial infarct size,mean left ventricular diastolic pressure,incidence of arrhythmia,and levels of serum creatine kinase,lactate dehydrogenase,and malondialdehyde.However,the mean left ventricular systolic pressure,and maximal rising and falling rates of ventricular pressure(±dp/dtmax)increased.In the myocardial H9 C2 cell hypoxia model,ginsenoside Rb1 reduced intracellular calcium concentrations([Ca2+]i)during hypoxia,and markedly reversed the hypoxia-induced decrease in cell survival.Ginsenoside Rb1 was involved in the downregulation of CaMKⅡand the ryanodine receptor,as well as hypoxia-induced H9 C2 cell survival.Conclusion:The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats,partially through the downregulation of CaMKⅡexpression.

微小RNA-34a调控CaMKⅡ/CREB途径改善异氟烷麻醉引起的老年大鼠认知功能障碍

目的探讨微小RNA(miR)-34a对异氟烷麻醉引起的老年大鼠认知功能障碍的影响及其作用机制。方法将40只20月龄大鼠随机分为正常(Con)组、模型(Model)组、miR-34a抑制剂(miR-34a inhibitor)组和miR-34a激动剂(miR-34a mimics)组,每组10只。miR-34a inhibitor组和miR-34a mimics组大鼠尾静脉注射100 nmol/kg对应药物,Con组和Model组大鼠尾静脉注射等剂量生理盐水,1次/d,连续5 d。第6天,除正常组外,其余组大鼠均进行单次异氟烷麻醉6 h,构建术后认知功能障碍(POCD)模型。建模完成后12 h,采用Morris水迷宫实验观察大鼠逃避潜伏期和目标象限停留时间。采用免疫荧光染色观察大鼠海马组织中离子钙结合衔接分子1(Iba-1)阳性表达率。采用实时荧光定量聚合酶链反应(qRT-PCR)检测大鼠海马组织中miR-34a以及B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的mRNA相对表达量。采用酶联免疫吸附实验(ELISA)检测大鼠血清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、活性氧(ROS)和谷胱甘肽过氧化物酶(GSH-Px)水平以及海马组织中谷氨酸(Glu)、Ca^(2+)和N-甲基-D-天冬氨酸受体2B(NMDAR2B)含量。采用蛋白质印迹(Western blotting)检测大鼠海马组织中钙-钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、pCaMKⅡ、环磷腺苷效应元件结合蛋白(CREB)、磷酸化CREB(pCREB)蛋白相对表达量。结果与Con组比较,Model组大鼠逃避潜伏期、血清中IL-6、IL-1β、ROS水平以及海马组织中Iba-1阳性表达率、miR-34a、Bax mRNA相对表达水平和Glu、Ca^(2+)、NMDAR2B含量延长或升高,目标象限停留时间、血清中GSH-Px水平、海马组织中Bcl-2 mRNA相对表达水平、Bcl-2/Bax以及pCaMKⅡ/CaMKⅡ和PCREB/CREB缩短或降低,差异有统计学意义(P<0.05)。下调miR-34a表达可缩短模型大鼠逃避潜伏期,以及降低血清中IL-6、IL-1β、ROS水平和海马组织中Iba-1阳性表达率、miR-34a、Bax mRNA相对表达水平和Glu、Ca^(2+)、NMDAR2B含量(P<0.05),还可延长目标象限停留时间,以及血清中GSH-Px水平、海马组织中Bcl-2 mRNA相对表达水平、Bcl-2/Bax以及pCaMKⅡ/CaMKⅡ和PCREB/CREB(P<0.05)。上调miR-34a表达可通过促进小胶质细胞的异常激活以及炎症、氧化应激和凋亡,抑制CaMKⅡ/CREB信号通路活化,加重模型大鼠认知障碍。结论miR-34a在POCD老年大鼠中高表达,抑制miR-34a表达可通过激活CaMKⅡ/CREB信号通路改善老年大鼠因异氟烷麻醉引起的认知功能障碍。

牡荆素调节环磷酸腺苷激活的交换蛋白1/钙/钙调素依赖性蛋白激酶Ⅱ信号对大鼠心肌梗死后心肌肥大与纤维化的作用

目的探讨牡荆素调控环磷酸腺苷激活的交换蛋白1(Epac1)/钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)信号通路抑制大鼠心肌梗死(MI)后心肌细胞肥大与纤维化的作用。方法24只SD大鼠随机分为:假手术组、MI组、牡荆素组。大鼠进行心脏左前降支结扎建立MI模型,假手术组只穿线不结扎。检测大鼠心功能变化;苏木精-伊红(HE)染色观察心脏病理学变化,天狼星红染色观察心脏纤维化,电镜观察心肌线粒体损伤情况;Western Blot检测Epac1、CaMKⅡ等蛋白变化。结果(1)大鼠MI 4周后,与假手术组比较MI组大鼠左心室短轴缩短率(LVFS)和左心室射血分数(LVEF)均降低(P<0.01),左心室收缩末期内径(LVIDs)、左心室舒张末期内径(LVIDd)和舒张末期左心室后壁厚度(LVPWd)、收缩末期左心室后壁厚度(LVPWs)增加(P<0.01),牡荆素组大鼠心脏LVIDs、LVIDd、LVPWs和LVPWd较MI组降低,LVFS和LVEF均升高,差异有统计学意义(P<0.05);(2)牡荆素可降低大鼠心脏重量指数(P<0.01),并减小MI组大鼠心肌细胞横截面积,同时减轻大鼠左心室壁心肌纤维化程度(P<0.01),牡荆素组大鼠心肌胞浆内线粒体肿胀较MI组减轻,线粒体形态较完整,空泡形成较少;(3)Western Blot结果显示牡荆素抑制大鼠MI后Epac1激活(P<0.01),抑制其下游CaMKⅡ激活与细胞外调节激酶的磷酸化(P<0.01),同时可抑制B淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax)上调(P<0.05),上调Bcl-2(P<0.01),抑制凋亡蛋白裂解半胱天冬酶3(Cleaved-Caspase 3)的活化(P<0.05)。结论牡荆素可通过抑制Epac1/CaMKⅡ通路,改善大鼠MI后心脏功能,抑制心肌肥大和纤维化。

Molecular mechanisms and the role of the protein kinase pathway in rat spatial memory impairment following isoflurane anesthesia

BACKGROUND： Animal experiments have demonstrated that isoflurane exposure alone induces learning and memory deficits for weeks or months. However, the molecular mechanisms of learning and memory remain poorly understood. Hippocampal expression of calcium/phospholipid- dependent protein kinase （PKC） and cAMP-dependent protein kinase （PKA） in rats have been shown to be associated with memory processing. OBJECTIVE： To investigate changes in rat spatial memory and hippocampal CA1 neuronal kinase system following isoflurane anesthesia, and to explore the correlation between molecular changes in cerebral neurons and behavioral manifestations following anesthesia. DESIGN, TIME AND SE＇rFING： A randomized, controlled, animal study. All experiments were performed at the Department of Anesthesia, Beijing Chaoyang Hospital, Capital Medical University from November 2007 to December 2008. MATERIALS： A total of 72 male, 3 month-old （young group）, Sprague Dawley rats, and 36 male, 20 month-old （aged group）, Sprague-Dawley rats were used in the study. Isoflurane was purchased from Baxter, USA. METHODS： Young and aged rats were randomly assigned to control, training （no anesthesia, Morris water maze training）, and isoflurane （1.2% isoflurane, Morris water maze training） groups. The isoflurane group was further subdivided into four groups, which were exposed to anesthesia for 2 or 4 hours, and were subjected to Morris water maze training at 2 days or 2 weeks postanesthesia. Finally, each aged group comprised 6 rats, and the young group comprised 12 rats. MAIN OUTCOME MEASURES： Spatial learning and memory were observed during Morris water maze training. Hippocampal CA1 PKA and PKC expression and activity were detected by immunohistochemistry and enzyme-linked immunosorbent assay （ELISA）. RESULTS： A 4-hour isoflurane exposure induced spatial memory deficits in all rats for 2 days to 2 weeks. In particular, aged rats exhibited more severe spatial memory deficits. Immunohistochemistry and ELISA results showed a significant increase in PKC and PKA expression and activity in the hippocampus CA1 subfield following Morris water maze training （P 〈 0.05）. Moreover, isoflurane anesthesia inhibited PKC and PKA expression and activity, and this inhibition increased with increasing exposure duration and increasing age. CONCLUSION： Results suggested that increased isoflurane exposure and age could extensively inhibit the hippocampal CA1 kinase system. Inhibition of protein kinases could play an important role in the cognitive decline following anesthesia.

A Highly Sensitive Detection Method, Phos-tag^TMAffinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in <i>Arabidopsis</i>

Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABA_A receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and IMUS), respectively in the Mg2+-free external solution containing 1 μmol/L glycine at a holding potential (VH) of -40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 μmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 nmol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 μmol/L) or La3+(30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a cal-cium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.

Stress kinase inhibition modulates acute experimental pancreatitis

AIM: To examine the role of p38 during acute experimental cerulein pancreatitis. METHODS: Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347) and/or a specific p38 inhibitor (SB203580) and pancreatic stress kinase activity was determined. Parameters to assess pancreatitis included trypsin, amylase, lipase, pancreatic weight and histology. RESULTS: JNK inhibition with CEP1347 ameliorated pancreatitis, reducing pancreatic edema. In contrast, p38 inhibition with SB203580 aggravated pancreatitis with higher trypsin levels and, with induction of acinar necrosis not normally found after cerulein hyperstimulation. Simultaneous treatment with both CEP1347 and SB203580 mutually abolished the effects of either compound on cerulein pancreatitis. CONCLUSION: Stress kinases modulate pancreatitis differentially. JNK seems to promote pancreatitis development, possibly by supporting inflammatory reactions such as edema formation while its inhibition ameliorates pancreatitis. In contrast, p38 may help reduce organ destruction while inhibition of p38 during induction of cerulein pancreatitis leads to the occurrence of acinar necrosis.

Salt Stress Triggers Phosphorylation of the Arabidopsis Vacuolar K＋ Channel TPK1 by Calcium-Dependent Protein Kinases （CDPKs）

14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K＋ channel 1 （TPK1） interacts with the 14-3-3 protein GRF6 （GF14-λ）. Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases （CDPKs） can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K＋ homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpkl mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca2＋, Ca2＋-dependent kinases, and 14-3-3 proteins.

Mediation of flowering by a calmodulin-dependent protein kinase

A calmodulin-dependent protein kinase (MCK1) appeared important in regulating flowering in tobacco. The expression of modified MCK1 that lacks the C-terminal including calmodulin-binding domain upsets the flowering developmental program, leading to the abortion of flower primordia initiated on the main axis of the plant and, as well, caused the prolongation of the vegetative phase in axillary buds. The abortion process of flowers began first in the developing anthers and subsequently the entire flower senesces. In axillary buds the prolonged vegetative phase was characterized by atypical elongated, narrow, twisted leaves. These results suggested a role for calmodulin-dependent protein kinase homologs in mediating flowering.

IP3R1调控CaMKⅡ和VDAC1在海洛因致心肌细胞节律异常中的作用

目的:探讨1,4,5-三磷酸肌醇1型受体(inositol 1,4,5-trisphosphate receptor type 1,IP3R1)调控钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)和电压依赖性阴离子通道1(voltage-dependent anion channel 1,VDAC1)在海洛因(heroin,HE)致心肌细胞节律异常中的作用。方法:联合蛋白组学和GEO(Gene Expression Omnibus)数据库分析心律失常芯片数据,寻找关键调控因子。构建IP3R1基因敲减慢病毒并感染原代乳大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs),实验分为对照(control)组、HE组和HE+shIP3R1组。结晶紫染色观察心肌细胞形态;ELISA法检测乳酸脱氢酶(lactate dehydrogenase,LDH)和天冬氨酸转氨酶(aspartate aminotransferase,AST)水平;透射电镜观察线粒体形态学变化;Fluo-4/AM探针法检测细胞内Ca^(2+)浓度;DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS)含量;JC-1染色法检测线粒体膜电位(mitochondrial membrane potential,MMP)水平;ATP检测试剂盒检测细胞内ATP水平;免疫共沉淀(co-immunopre-cipitation,Co-IP)分析IP3R1与CaMKⅡδ和VDAC1蛋白之间的相互作用;Western blot检测IP3R1、CaMKⅡδ、p-CaM-KⅡδ(T287)和VDAC1的蛋白水平。结果:结合蛋白质组学和基因表达谱数据集GSE89410分析,筛选得到80个差异共表达分子,基于基因本体论(Gene Ontology,GO)功能注释和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析结果,最终筛选出关键因子IP3R1,且通过STRING数据库获得IP3R1结合蛋白:CaMKⅡδ和VDAC1。Co-IP结果验证IP3R1与CaMKⅡδ和VDAC1存在相互作用,且HE干预后NRCMs中IP3R1与CaMKⅡδ和VDAC1之间的相互作用增强。体外细胞实验显示,与control组相比,HE组NRCMs数量急剧减少,细胞膜变窄,伪足减少,细胞核结构模糊;LDH和AST水平均显著上升(P<0.05);线粒体超微结构损伤严重,证实HE对NRCMs具有毒性作用并导致线粒体损伤。与control组相比,HE组心肌细胞内Ca^(2+)浓度、ROS水平、MMP以及IP3R1、p-CaMKⅡδ(T287)和VDAC1蛋白水平均显著升高(P<0.05),而HE+shIP3R1组这些指标均显著减低(P<0.05);ATP水平则相反。这证实沉默IP3R1表达可减轻HE干预后NRCMs的钙超载及线粒体损伤。结论:IP3R1通过调控CaMKⅡ和VDAC1引起心肌细胞钙超载和ROS生成增多,参与HE诱导的心肌细胞节律异常。

姜黄素调节NMDAR/Ca^(2+)/CaMKⅡ信号通路对异氟醚诱导的幼龄小鼠术后认知功能障碍的影响

目的探讨姜黄素(Cur)调节N-甲基-D-天冬氨酸受体(NMDAR)/钙离子(Ca^(2+))/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)信号通路对异氟醚(ISO)诱导的幼龄小鼠术后认知功能障碍(POCD)的影响。方法将72只C57BL/6J小鼠分为对照组、ISO组、低剂量Cur组(Cur-L组,50 mg/kg)、中剂量Cur组(Cur-M组,100 mg/kg)、高剂量Cur组(Cur-H组,200 mg/kg)、Cur-H+NMDA(NMDAR激活剂)组(200 mg/kg+8 mg/kg),每组12只。经对应给药处理30 min后,对照组小鼠吸入含30%氧气和空气的混合气体2 h,其余各组小鼠吸入2%ISO 2 h,每天1次,持续14 d。末次给药24 h后,Morris水迷宫实验检测小鼠学习与空间记忆能力;HE染色检测海马CA1区病理学变化;免疫荧光染色检测小鼠海马CA1区神经元特异核蛋白(NeuN)阳性表达;TUNEL染色检测神经细胞凋亡;酶联免疫吸附试验检测海马CA1区组织中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)水平;蛋白印迹法检测海马CA1区组织中NMDAR1和CaMKⅡ蛋白表达;荧光探针检测海马CA1区Ca^(2+)浓度。结果与对照组比较,ISO组小鼠海马CA1区病理损伤严重,逃避潜伏期延长,神经细胞凋亡率升高,海马CA1区组织中IL-1β和TNF-α水平升高,NMDAR1和CaMKⅡ蛋白表达及Ca^(2+)浓度升高(P<0.05),穿越平台次数和NeuN阳性细胞数减少(P<0.05);与ISO组比较,Cur-L组、Cur-M组、Cur-H组小鼠海马CA1区病理损伤减轻,逃避潜伏期缩短,神经细胞凋亡率降低,海马CA1区组织中IL-1β和TNF-α水平降低,NMDAR1和CaMKⅡ蛋白表达及Ca^(2+)浓度降低(P<0.05),穿越平台次数和NeuN阳性细胞数增加(P<0.05),且呈剂量依赖性;NMDA减弱了高剂量Cur对ISO诱导的小鼠POCD的改善作用(P<0.05)。结论Cur可能通过抑制NMDAR/Ca^(2+)/CaMKⅡ信号通路改善ISO诱导的小鼠POCD。

ISO通过CaMKⅡ和PKA激活RyR2诱发心肌细胞凋亡

目的:探讨异丙肾上腺素(ISO)持续激活Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)和蛋白激酶A(PKA)后对心肌细胞凋亡的影响及其作用机制。方法:将H9C2心肌细胞分为Control组、ISO组、ISO+KN93(CaMKⅡ抑制剂)组、ISO+H-89(PKA抑制剂)组,经不同药物干预后,通过CCK-8法检测心肌细胞活力,Annexin V-FITC/PI细胞凋亡试剂盒和流式细胞术检测心肌细胞凋亡,Western blot法检测CaMKⅡ、PKA、兰尼碱受体2(RyR2)及凋亡相关蛋白Cleaved-Caspase3、Bax、Bcl-2的表达情况,荧光显微镜观察细胞形态变化和钙荧光强度。结果:与Control组比较,ISO组细胞活性降低,CaMKⅡ、PKA和RyR2的磷酸化水平增加,凋亡蛋白Cleaved-Caspase3、Bax/Bcl-2表达增加,胞内钙含量增多,细胞凋亡率增多,差异均有统计学意义(P<0.01);与ISO组比较,ISO+KN93和ISO+H-89组细胞活性增高,CaMKⅡ、PKA和RyR2的磷酸化水平降低,凋亡蛋白Cleaved-Caspase3、Bax/Bcl-2表达减少,胞内钙含量减少,细胞凋亡率减少,差异均有统计学意义(P<0.01)。结论:ISO通过CaMKⅡ和PKA共同介导RyR2途径的磷酸化激活,并诱发心肌细胞凋亡。

Cloning and Characterization of a Homologous Ca^(2+)/Calmodulin-Dependent Protein Kinase PSKH1 from Pearl Oyster Pinctada fucata

Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the calcium metabolism in oysters will help elucidate the pearl formation mechanism. This paper describes a full-length PSKH1 cDNA isolated from pearl oyster Pinctada fucata. Oyster PSKH1 shares 65% homology with human PSKH1 and 48% similarity with rat CaM kinase I in the amino acid sequence, and contains a calmodulin-binding domain. The results of semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization revealed that oyster PSKH1 mRNA is highly expressed in the outer epithelial cells of the mantle pallial and in the gill epithelial cells. These studies provide important information describing the complex Ca^2＋ signaling mechanism in oyster calcium metabolism.

山药块茎膨大期DoCDPK1基因生物信息学及低温胁迫下的表达分析

钙依赖性蛋白激酶(CDPK)在植物的生长发育及逆境胁迫方面发挥着重要作用。以大和长芋和毕克齐山药为试验材料,克隆钙依赖性蛋白激酶基因DoCDPK1,并对其进行生物信息学分析,为DoCDPK1基因的功能研究奠定基础。采用生物信息学方法分析DoCDPK1基因结构,构建瞬时表达载体,对DoCDPK1蛋白进行亚细胞定位;采用qRT-PCR分析DoCDPK1基因在不同山药品种的不同发育阶段及低温胁迫处理下的表达模式。结果表明(:1)DoCDPK1基因序列长度为2023 bp,编码521个氨基酸。(2)DoCDPK1与几内亚薯蓣CDPK蛋白序列的一致性为97%,且DoCDPK1存在STKc_CAMK结构域。(3)瞬时表达分析表明,DoCDPK1蛋白定位于细胞核与细胞膜。(4)大和长芋山药中的CDPK活性更高,DoCDPK1可能参与调控山药块茎膨大后期的生长发育。(5)经4℃低温胁迫后,CDPK活性降低,DoCDPK1表达量上调,且表达水平在不同时间处理下存在差异。综上所述,DoCDPK1可能参与调控山药块茎膨大后期的生长发育及低温胁迫响应过程。

生姜提取物对染铝毒大鼠学习记忆功能和神经细胞结构的影响及其可能作用机制

目的分析生姜提取物(GRE)对铝染毒大鼠学习记忆功能及神经细胞结构的影响,并基于钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、神经元型一氧化氮合酶(nNOS)、蛋白激酶C(PKC)探讨其可能的作用机制。方法将36只SD雄性大鼠随机分为空白对照组、铝染毒组、临床药物组、低剂量GRE(L⁃GRE)组、中剂量GRE(M⁃GRE)组、高剂量GRE(H⁃GRE)组,每组6只大鼠。空白对照组大鼠自由饮用不含氯化铝的饮用水,其余组大鼠饮用含10 mg/mL结晶氯化铝的饮用水。3个月后给予空白对照组和铝染毒组大鼠灌胃生理盐水,临床药物组大鼠灌胃盐酸多奈哌齐溶液,分别给予L⁃GRE组、M⁃GRE组、H⁃GRE组大鼠灌胃100 mg/kg、200 mg/kg、400 mg/kg GRE溶液。持续干预4周后,采用Morris水迷宫实验评估大鼠的学习记忆功能,通过HE染色观察大鼠海马组织形态变化,分别采用实时荧光定量PCR和Western blot检测大鼠海马组织CaMKⅡ、nNOS、PKC mRNA和蛋白表达水平。结果(1)与空白对照组相比,铝染毒组大鼠的逃避潜伏期延长且穿越平台次数减少(P<0.05),海马组织的神经细胞皱缩,神经细胞数量明显减少,CaMKⅡ、nNOS、PKC的mRNA表达水平及nNOS、PKC的蛋白表达水平降低(P<0.05)。(2)与铝染毒组比,L⁃GRE组、M⁃GRE组和H⁃GRE组大鼠的逃避潜伏期缩短,临床药物组、M⁃GRE组、H⁃GRE组大鼠的穿越平台次数增加(P<0.05);各给药组大鼠海马组织病理形态有不同程度的改善;临床药物组大鼠的CaMKⅡ、nNOS和PKC mRNA表达水平升高,各剂量GRE组大鼠nNOS和PKC mRNA和蛋白表达水平升高(P<0.05)。(3)与临床药物组相比,M⁃GRE组和H⁃GRE组大鼠的逃避潜伏期,以及各剂量GRE组的穿越平台次数和目标象限停留时间差异无统计学意义(P>0.05);各剂量GRE组大鼠海马组织病理形态改善程度更为明显;M⁃GRE组、H⁃GRE组大鼠的nNOS、PKC蛋白表达水平升高,L⁃GRE组大鼠的nNOS蛋白表达水平升高(P<0.05)。(4)各剂量GRE组组间大鼠逃避潜伏期、穿越平台次数及目标象限停留时间差异无统计意义(P>0.05);随GRE干预剂量增加,大鼠海马组织病理形态的改善效果越明显;H⁃GRE组大鼠的CaMKⅡmRNA表达水平高于L⁃GRE组(P<0.05)。结论GRE能够改善铝染毒大鼠的神经细胞结构和学习记忆功能,其中对神经细胞结构的改善效果优于盐酸多奈哌齐,对学习功能的改善效果与盐酸多奈哌齐相当。GRE的作用机制可能与拮抗铝所致CaMKⅡ、nNOS和PKC表达水平下降有关。