茉莉酸甲酯诱导人神经母细胞瘤细胞株BE(2)-C凋亡作用机制

探讨茉莉酸甲酯对人神经母细胞瘤细胞株BE(2)-C的生长抑制作用及其对凋亡抑制蛋白家族成员XIAP和survivin表达的影响。1～2 mmol·L^-1茉莉酸甲酯处理BE(2)-C细胞6～24 h后， MTT法检测瘤细胞生长活性， 克隆形成实验检测细胞增殖能力， PI单染流式细胞术检测细胞周期， AO/EB荧光染色法、 Hoechst 33258荧光染色法、 Annexin V-FITC/PI双染流式细胞术检测细胞凋亡， 半定量逆转录聚合酶链反应(RT-PCR)、 实时荧光定量逆转录聚合酶链反应(real-time RT-PCR)检测细胞周期素D1(cyclin D1)、 X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein， XIAP)及survivin表达。结果表明， 各浓度茉莉酸甲酯作用后， BE(2)-C细胞呈时间、 浓度依赖性生长抑制， 1， 1.5和2 mmol·L^-1茉莉酸甲酯作用24 h后， 细胞生长抑制率为20.6%～85.5%(P<0.01)， IC₅₀为1.35 mmol·L^-1； S期细胞比率增高， 部分瘤细胞发生典型的凋亡形态学改变， 早期凋亡细胞(FITC⁺ PI^-)分别占13.51%、 17.32%和24.59%(均P<0.01)，细胞死亡率分别为29.36%、 54.73%和75.52%(均P<0.01)； XIAP和survivin表达分别下调18.5%～68.9%和22.4%～48.7%(均P<0.05)， 而cyclin D1表达无显著变化(P>0.05)。可见， 茉莉酸甲酯能通过诱导细胞周期阻滞和凋亡抑制人神经母细胞瘤细胞株BE(2)-C的生长活性， 下调XIAP和survivin基因表达可能是其作用机制之一。     

银杏叶提取物对低氧复氧、H2O2和谷氨酸损伤时谷氨酸引起的大鼠星形胶质细胞［Ca2+］i变化的影响

目的研究银杏叶提取物对低氧复氧、H₂O₂和L-谷氨酸损伤时谷氨酸升高大鼠星形胶质细胞［Ca²⁺］_i的影响。方法钙荧光探针Fluo-3/AM标记胞浆内游离钙离子，激光扫描共聚焦显微镜测定［Ca²⁺］_i的变化。结果 在低氧复氧、H₂O₂以及高浓度的L-谷氨酸损伤后，外源性谷氨酸（27 μmol·L^-1）均不能引起培养乳大鼠星形胶质细胞正常的［Ca²⁺］_i升高，反而使［Ca²⁺］_i分别降低(3.3±1.6)%，(81±11)%和(81±7)%；损伤前预先给予GbE（10 mg·L^-1）不能明显改善星形胶质细胞的谷氨酸反应，但预先给予GbE（100 mg·L^-1）后，27 μmol·L^-1谷氨酸可使损伤的星形胶质细胞［Ca²⁺］_i分别升高(135±98)%，(117±93)%和(89±36)%。结论低氧复氧、H₂O₂以及高浓度的L-谷氨酸均能损伤星形胶质细胞的谷氨酸反应，影响神经细胞与胶质细胞的双向交流。GbE能明显逆转不同损伤后谷氨酸诱导星形胶质细胞［Ca²⁺］_i的异常变化，使星形胶质细胞在不同损伤时能维持正常功能，该作用可能与GbE的脑保护作用有关。     

糖皮质激素促进β-淀粉样蛋白对海马神经元损伤的研究

目的在体外实验条件下,观察糖皮质激素是否促进β-淀粉样蛋白的神经元毒性作用。方法通过LDH释放率法检测细胞活力、TUNEL法测细胞凋亡以及LSCM测胞内Ca²⁺浓度的变化。结果单独的地塞米松(10^-8,10^-7,10^-6mol·L^-1)组或Aβ25-35(0.5μmol·L^-1)组分别作用海马神经元后,细胞活力变化不明显(P>0.05);但与同剂量的DEX、Aβ_25-35组相比,DEX+Aβ_25-35组的细胞活力明显降低(P<0.05),同时胞内Ca²⁺显著上升(P<0.05),神经元的凋亡率增加明显,细胞内出现凋亡的典型形态学特征。结论糖皮质激素可能通过促进Aβ引起海马神经元胞内Ca²⁺升高增加神经元毒性作用。     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

钙拮抗剂TMB-8抑制BHQ,NE和KCl引起的培养乳牛基底动脉单个平滑肌细胞内游离钙的升高

用AR CM MIC阳离子测定系统,测量单个细胞内游离钙浓度([Ca²⁺]i),研究8-(N,N-二乙胺)-n-辛基 3,4,5-三甲氧基苯甲酸酯(TMB-8)对培养乳牛基底动脉平滑肌[Ca²⁺]i的作用。在细胞外钙浓度为1.3mmol·L^-1时,TMB-8(30μmol·L^-1)可明显抑制BHQ,NE及KCl引起[Ca²⁺]i的升高。在细胞外钙为零+EGTA 0.1mmol·L^-1时,TMB-8(10,30及100μmol·L^-1)可浓度依赖性地降低静息[Ca²⁺]i,TMB-8(30μmol·L^-1)可几乎完全阻断BHQ及NE引起[Ca²⁺]i的增加。研究表明TMB-8降低培养乳牛基底动脉平滑肌[Ca²⁺]i的机制,主要是抑制肌浆网Ca²⁺的释放,或增加肌浆网对Ca²⁺的摄入,并由此间接地抑制细胞外钙的内流。     

芍药苷对醋酸铅诱导海马神经元凋亡及Bcl-2/Bax蛋白表达的影响

目的 探讨芍药苷(paeoniflorin,PF)对醋酸铅诱导海马神经元凋亡及Bcl-2/Bax蛋白表达的影响。方法 分离培养胎鼠海马神经元细胞,细胞免疫荧光染色鉴定纯度。MTT测定海马神经元细胞活力以确定醋酸铅最适造模浓度及时间,同时筛选合适剂量PF干预海马神经元凋亡。依据MTT测定结果,分为空白组、模型组和20,40,80 μmol·L^-1 PF组干预海马神经元细胞,作用24 h后,加醋酸铅染毒,检测细胞色素C (cytochrome C,Cyt-C)含量、线粒体膜电位及细胞内Ca²⁺浓度。流式细胞仪检测细胞凋亡情况,Western blotting测定海马神经元细胞中caspase-3、cleaved-caspase-3、caspase-8、cleaved-caspase-8、caspase-9、cleaved-caspase-9、Bax、Bcl-2蛋白表达水平。结果 细胞免疫荧光染色鉴定结果显示分离培养的细胞为海马神经元细胞,且纯度较高。MTT测定结果显示醋酸铅最适造模浓度及时间为25 μmol·L^-1染毒24 h;PF剂量为20,40,80 μmol·L-1可显著改善海马神经元细胞活性,呈剂量依赖性。与空白组相比,模型组Cyt-C含量、凋亡率、细胞内Ca²⁺浓度显著升高(P<0.01),线粒体膜电位显著降低(P<0.01)。与模型组相比,40 μmol·L^-1 PF组和80 μmol·L^-1 PF组可降低Cyt-C含量、凋亡率、细胞内Ca²⁺浓度(P<0.05或P<0.01),升高线粒体膜电位(P<0.01),20 μmol·L^-1PF组可显著升高线粒体膜电位(P<0.05)。此外,一定剂量的PF可下调cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9、Bax蛋白表达,上调Bcl-2蛋白表达。结论 PF可抑制醋酸铅诱导的海马神经元凋亡,可通过调控Bcl-2/Bax蛋白表达发挥神经保护作用。     

(-)-S·R-蝙蝠葛苏林碱对谷氨酸引起的大鼠大脑皮质神经元损伤的保护作用

用细胞培养方法,在原代培养的大鼠皮质神经细胞上,观察了(-)-S·R-蝙蝠葛苏林碱对谷氨酸引起的神经元损伤的保护作用。以Fura-2/AM为Ca²⁺的荧光指示剂,用AR-CM-MIC阳离子测定系统观察(-)-S·R-蝙蝠葛苏林碱对谷氨酸诱发大鼠脑皮质神经细胞内Ca²⁺升高的影响。结果表明(-)-S·R-蝙蝠葛苏林碱能剂量依赖性地抑制谷氨酸的神经毒作用,对谷氨酸诱发的神经细胞内游离Ca²⁺升高有明显的抑制作用。提示蝙蝠葛苏林碱对缺血性脑损伤有保护作用。     

蚓激酶的心肌保护作用及机制

目的研究蚓激酶对心肌缺血的保护作用，并进一步探讨其可能机制。方法采用结扎大鼠左冠状动脉前降支制备急性心肌缺血模型，观察蚓激酶对心肌缺血的保护作用；应用全细胞膜片钳和激光扫描共聚焦技术，研究蚓激酶对L-型钙电流(I_Ca-L)和细胞内游离钙离子浓度的影响。结果蚓激酶80，40和20 mg·kg^-1剂量组均可缩小心肌梗死面积。膜片钳研究结果表明，当刺激电压为+10 mV时，10和50 μmol·L^-1蚓激酶使I_Ca-L降低共聚焦结果显示，在静息状态下，10 μmol·L^-1蚓激酶对［Ca²⁺］_i无明显影响；但10 μmol·L^-1蚓激酶对60 mmol·L^-1 KCl诱导的［Ca²⁺］_i升高却有明显抑制作用，并且在整个实验过程中(240 s)并未出现明显的峰值。结论蚓激酶对大鼠心肌缺血具有保护作用，其机制可能与抑制I_Ca-L及下调［Ca²⁺］_i有关。     

钙离子对4-氨基吡啶诱发肥大细胞释放组胺作用的影响

4-Aminopyridine(4-AP) has been shown to induce histamine release fromisolated peritoneal mast cells(PMC)in mice and rats. In the presence of extracellular calcium atnormal level(0.9 mmol·L^-1)histamine release induced by 4-AP(13. 6 mmol·L^-1) from mice PMCwas 33.0%±4.6%,while at low calcium concentration (0.5 mmol·L^-1) and in calcium-freemedium this parameter decreased to 25.5%±4.2%and 16.3%±3.7%respectively.Histaminerelease in response to 4-AP(10 mmol·L^-1)from rat PMC was 39.1%±6.7%(0.9 mmol·L^-1calcium),while at low calcium concentration(0. 5 mmol·L^-1) and in calcium-free medium thisparameter decreased to 29.3%±4.7%and 20.2%±2.9% respectively. Results of statisticalanalysis indicate that 4-AP induced histamine release is related to Ca²⁺ concentration. When rat PMCwere preincubated in calcium-free medium with EDTA 0.1 mmol·L^-1 for 180 min　4-AP inducedhistamine release was 13.8%±1.6%.This shows that 4-AP also elicited mobilization of endogenous calcium stores in mast cells.The mechanism of 4-AP　induced histamine release was discussed.     

毒毛旋花子苷元对豚鼠心室肌细胞内钙浓度的影响

观察毒毛旋花子苷元(strophanthidin, Str)对分离豚鼠心室肌细胞内游离钙浓度(［Ca²⁺］_i)的影响。酶解分离豚鼠心室肌细胞, 用Fluo 3-AM负载, 激光共聚焦显微镜法测定单个豚鼠心室肌细胞［Ca²⁺］_i的荧光密度。Str可浓度依赖性地升高［Ca²⁺］_i, Str (10 μmol·L^-1)在［Ca²⁺］_i升高达峰值时, 可使细胞挛缩, 而Str (1和10 nmol·L^-1)对细胞形态无影响。TTX、 尼索地平或升高细胞外钙可影响Str (1和100 nmol·L^-1)对［Ca²⁺］_i的升高作用,而对Str (10 μmol·L^-1)无明显影响。在外液中加入ryanodine或去除细胞外钙, 则3个检测浓度的Str升高［Ca²⁺］_i作用均被明显抑制。在无K⁺、 无Na⁺液中, 10 μmol·L^-1 Str升高［Ca²⁺］_i的作用减弱, 而Str (1和100 nmol·L^-1)升高［Ca²⁺］_i的作用无明显影响。加入TTX、 尼索地平或增加细胞外的钙离子浓度, 则3个检测浓度Str的作用均受到影响。提示低浓度Str对［Ca²⁺］_i的升高作用与抑制Na⁺、K⁺-ATP酶活性无关, 而与促进L-型钙通道和TTX敏感性钠通道的“slip-mode”钙电导有关; 高浓度Str升高［Ca²⁺］_i的作用则是抑制Na⁺、K⁺-ATP酶的结果。此外, Str对［Ca²⁺］_i的升高作用还与直接作用于ryanodine受体促进内钙释放有关。     

埃他卡林增强星形胶质细胞摄取谷氨酸的作用

目的研究新型ATP敏感性钾通道开放剂(KATPCO)埃他卡林(iptakalim,Ipt)对星形胶质细胞摄取谷氨酸的影响。方法取新生大鼠脑星形胶质细胞作原代培养，将Ipt直接作用于细胞，观察它对正常和6-羟基多巴胺(6-OHDA)损伤模型细胞摄取谷氨酸的影响；分别加入阳性对照药吡那地尔和非特异性钾通道阻断药格列本脲分析Ipt的作用机制。根据［³H］标记的D,L-谷氨酸摄入量判断细胞谷氨酸摄取作用强度。结果Ipt和吡那地尔都能增强星形胶质细胞的谷氨酸摄取作用、逆转6-OHDA引起的谷氨酸摄取抑制效应，预先加入格列本脲后，上述作用均被取消。结论埃他卡林可能通过促进钾通道开放增强星形胶质细胞摄取谷氨酸的作用。     

Cellular mechanism for spontaneous calcium oscillations in astrocytes

AIM: To determine the Ca2+ source and cellular mechanisms of spontaneous Ca2+ oscillations in hippocampal astrocytes. METHODS: The cultured cells were loaded with Fluo-4 AM, the indicator of intracellular Ca2+, and the dynamic Ca2+ transients were visualized with confocal laser-scanning microscopy. RESULTS: The spontaneous Ca2+ oscillations in astrocytes were observed first in co-cultured hippocampal neurons and astrocytes. These oscillations were not affected by tetrodotoxin (TTX) treatment and kept up in purity cultured astrocytes. The spontaneous Ca2+ oscillations were not impacted after blocking the voltage-gated Ca2+ channels or ethylenediamine tetraacetic acid (EDTA) bathing, indicating that intracellular Ca2+ elevation was not the result of extracellular Ca2+ influx. Furthermore, the correlation between the spontaneous Ca2+ oscillations and the Ca2+ store in endoplasmic reticulum (ER) were investigated with pharmacological experiments. The oscillations were: 1) enhanced when cells were exposed to both low Na+ (70 mmol/L) and high Ca2+ (5 mmol/L) solution, and eliminated completely by 2 micromol/L thapsigargin, a blocker of sarcoplasmic reticulum Ca2+-ATPase; and 2) still robust after the application with either 50 micromol/L ryanodine or 400 micromol/L tetracaine, two specific antagonists of ryanodine receptors, but depressed in a dose-dependent manner by 2-APB, an InsP3 receptors (InsP3R) blocker. CONCLUSION: InsP3R-induced ER Ca2+ release is an important cellular mechanism for the initiation of spontaneous Ca2+ oscillation in hippocampal astrocytes.     

A study of the cardioprotective effect of breviscapine during hypoxia of cardiomyocytes

Breviscapine is a flavonoid extracted from Erigeron breviscapus. Hand.-Mazz, and it has been reported that breviscapine can activate K+ channels and block Ca2+ channels. In this paper, we studied the cardioprotective effects of breviscapine on electrocardiogram (ECG) changes (ST-segment elevation), infarction size in dog heart subjected to myocardial infarction caused by left coronary artery ligation and lactate dehydrogenase (LDH) leakage, changes of intracellular free Ca2+ levels, apoptosis and necrosis in cultured neonatal rat cardiomyocytes subjected to hypoxia. Additionally, the effect of breviscapine on myocardial oxygen consumption was detected in dog myocardium in vitro. The results showed that breviscapine treatment (1 mg/kg, 2 mg/kg and 4 mg/kg) significantly reduced ST-segment elevation and infarction size in hearts subjected to myocardial infarction, that breviscapine treatment (14.29 microg/mL, 28.57 microg/mL and 57.14 microg/mL) significantly decreased oxygen consumption in myocardium, and that breviscapine treatment (5 microg/mL, 10 microg/mL and 20 microg/mL) significantly reduced LDH leakage, intracellular free Ca2+ levels, apoptosis and necrosis in cardiomyocytes subjected to hypoxia. In conclusion, the present study indicates that breviscapine is in favor of myocardial protection.     

Neuroprotective effects of stearic acid against toxicity of oxygen/glucose deprivation or glutamate on rat cortical or hippocampal slices

AIM: To observe the effects of stearic acid, a long-chain saturated fatty acid consisting of 18 carbon atoms, on brain (cortical or hippocampal) slices insulted by oxygen-glucose deprivation (OGD), glutamate or sodium azide (NaN3) in vitro. METHODS: The activities of hippocampal slices were monitored by population spikes recorded in the CA1 region. In vitro injury models of brain slice were induced by 10 min of OGD, 1 mmol/L glutamate or 10 mmol/L NaN3. After 30 min of pre-incubation with stearic acid (3-30 micromol/L), brain slices (cortical or hippocampal) were subjected to OGD, glutamate or NaN3, and the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride method. MK886 [5 mmol/L; a noncompetitive inhibitor of proliferator-activated receptor (PPAR-alpha)] or BADGE (bisphenol A diglycidyl ether; 100 micromol/L; an antagonist of PPAR-gamma) were tested for their effects on the neuroprotection afforded by stearic acid. RESULTS: Viability of brain slices was not changed significantly after direct incubation with stearic acid. OGD, glutamate and NaN3 injury significantly decreased the viability of brain slices. Stearic acid (3-30 micromol/L) dose-dependently protected brain slices from OGD and glutamate injury but not from NaN3 injury, and its neuroprotective effect was completely abolished by BADGE. CONCLUSION: Stearic acid can protect brain slices (cortical or hippocampal) against injury induced by OGD or glutamate. Its neuroprotective effect may be mainly mediated by the activation of PPAR-gamma.     

Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus.

1. We studied the release of [3H]-dopamine and [3H]-noradrenaline (NA) from hippocampal synaptosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo-1 as a probe for Ca2+. 2. Presynaptic glutamate receptor activation induced Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA from rat hippocampal synaptosomes. Thus, L-glutamate induced the release of both neurotransmitters in a dose-dependent manner (EC50 = 5.62 microM), and the effect of 100 microM L-glutamate was inhibited by 83.8% in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX), but was not affected by 1 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801). 3. Other glutamate receptor agonists also stimulated the Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA as follows: N-methyl-D-aspartate (NMDA), at 200 microM, released 3.65 +/- 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 microM MK-801; quisqualate (50 microM), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (100 microM) or kainate (100 microM) released 1.57 +/- 0.26%, 1.93 +/- 0.17% and 2.09 +/- 0.22%, of the total 3H catecholamines, respectively. 4. The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tonic potentiation and attenuation produced by membrane depolarization in guinea-pig trachealis

1. We studied how membrane depolarization directly affected intracellular Ca2+ signalling when voltage-operated Ca2+ channels (VOCC) were not available in guinea-pig tracheal smooth muscle. To block VOCC, we used 3 micromol/L verapamil, which completely abolished high K+ (20-60 mmol/L)-induced contraction, and elevation of fura-2 signal. 2. Muscle tone was generated by adding Ca2+ to the extracellular Ca2+-free solution containing prostaglandin (PG)E2 (100 nmol/L) after abolishing basal tone with indomethacin (1 micromol/L). 3. In the absence of verapamil, high K+ (20-60 mmol/L) solution potentiated 2.4 mmol/l Ca2+-induced sustained contractions. Even in the presence of 3 micromol/L verapamil, replacement with 20 and 40 mmol/L K+ solution induced tonic potentiation, which was changed to attenuation with a higher K+ solution (60 mmol/L), lower extracellular Ca2+ concentration ([Ca2+]o) and pretreatment with cyclopiazonic acid (10 micromol/L), a Ca2+ sequestration inhibitor. 4. These results indicate that the balance between depolarization-dependent Ca2+ release and receptor-operated cation channel inhibition may determine whether tonic potentiation or attenuation is manifested, depending on the availability of VOCC, the magnitude of the depolarization, [Ca2+]o and Ca2+ content in the sarcoplasmic reticulum.     

Fendiline mobilizes intracellular Ca2+ in Chang liver cells

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.