NITRIC OXIDE INHIBITS A RISE OF ATP-INTRODUCED CYTOSOLIC FREE Ca^(2+) CONCENTRATION AND RELEASE FROM INTRACELLULAR STORED Ca^(2+)

Object. The effects of ATP-introduced a rise in cytosolic free Ca2+ concentration and inhibition of nitric oxide were investigated. Method. Measurement of free Ca2+([Ca2+] i)of cultured rat tail arterial smooth muscle cells using Fura-2/AM dual excitation wavelength spectrofluorometer. Results. There are two components of [Ca2+] i can be evoked by ATP. One part is Ca2+ entry from Ca2+ channel and formed a plateau. The another part is a peak that released from Ca2+ store. Both of them can be inhibited by NO. Conclusion. The ATP induced [Ca2+] i rise that release Ca2+ from both Insp 3 and ryanochine receptors and Ca2+ entry through calcium channels. The inhibition of NO on ATP induced [Ca2+] i rise that was mediated by cGMP.

18β-glycyrrhetinic Acid-induced Apoptosis and Relation with Intracellular Ca^2＋ Release in Human Breast Carcinoma Cells

Objective:To study the effects of 18β-glycyrrhetinic acid (GA) on proliferation inhibition, apop totic induction, and the relationship between GA-induced apoptosis and intracellular Ca2+ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 μmol/L to 250 μmol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 μmol/L GA for 24 h, intracellular Ca2+ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 μmol/L to 250 μmol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose dependent fashion. IC50 of the proliferation inhibition was 234.33 μmol/L. Treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca2+ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18β-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca2+ level may be depended on apoptosis induced by GA in MCF-7 cells.

Ca^(2+) Entry Through TRPC1 Channels Contributes to Intracellular Ca^(2+) Dynamics and Consequent Glutamate Release from Rat Astrocytes

各种不同的刺激作用于星型胶质细胞,可以导致胞浆内Ca2+浓度增加,进而释放更多谷氨酸作用于周边的神经元。大部分Ca2+来源于细胞内,小部分来源于细胞外。Ca2+内流是通过钙池操纵Ca2+通道(SOC)实现的。因此,作者观察在星型胶质细胞内Ca2+激活与谷氨酸释放过程中钙池操纵Ca2+通道(SOC)发挥了什么样的作用。已有研究显示星型胶质细胞所表达的TRPC通道(Ca2+通过瞬时受体电位通道相关蛋白)介导了钙池操纵Ca2+的内流。本文发现培养的星形胶质细胞以及从视皮质中新分离的星形胶质细胞表达TRPC1,TRPC4,和TRPC5。间接免疫组化显示这些蛋白存在于整个细胞中,然而机能检测TRPC1主要表达在质膜上。在新分离的星形胶质细胞中做标记,显示了在细胞发育过程中TRPC表达的改变。应用抗TRPC1的抗体,可以阻断TRPC1通道并且可以测定它们在培养的星形细胞的机械性和激动剂触发的钙离子内流过程中的作用。阻断TRPC1可以减少机械诱导的钙离子依赖性的谷氨酸盐的释放。这些实验数据表明,钙离子通过TRPC1通道的内流有助于钙离子在星形细胞中的信号传导以及由此引起的谷氨酸盐的释放。

Relationship of Intracellular Free Ca^(2+) Concentration and Calcium-activated Chloride Channels of Pulmonary Artery Smooth Muscle Cells in Rats under Hypoxic Conditions

To investigate the relationship between intracellular free Ca^2＋ concentration （[Ca^2＋ ]i ） and calcium-activated chloride （Clca） channels of pulmonary artery smooth muscle cells （PASMCs） in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca^2＋ indicator Fura-2/AM was used to observe [Ca^2＋ ]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay. The Clca channel blockers niflumic acid （NFA） and indaryloxyacetic acid （IAA-94） exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca^2＋ ]i was increased. Under normoxic condition, [Ca^2＋ If was （123.634-18.98） nmol/ L, and in hypoxic condition, [Ca^2＋]i wag （281. 754-16.48） nmol/L （P〈0. 01）. Under normoxic condition, [Ca^2＋ ]i showed no significant change and no effect on Clca channels was observed （P〉 0. 05）. Chronic hypoxia increased [Ca^2＋ ]i which opened Clca channels. The NFA and IAA-94 blocked the channels and decreased [Ca^2＋ ]i from （281.75± 16.48） nmot/L to （117.66 ±15.36） nmol/L （P〈0.01）. MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency （A value） from 0. 459±0. 058 to 0. 224±0. 025 （P〈0. 01）. Hypoxia increased [Ca^2＋ ]i which opened Cl~ channels and had a positive-feedback in [Ca^2＋ ]i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Clca channel may play a part in the regulation of proliferation of PASMCs.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

Influence of Panax quinquefolium saponins on increased intracellular Ca^(2+) in PC12 cells

BACKGROUND： Previous studies have demonstrated that intracellular Ca^2＋ （[Ca^2＋]） overload, excitotoxicity, free radical injury, and nitric oxide toxicity are involved in mechanisms of neuronal death in the ischemic brain. OBJECTIVE： To investigate the influence of Panax quinquefo/ium saponins （PQS） on multiple factors-induced Ca^2＋ overload in the rat pheochromocytoma （PC12） cell line. DESIGN, TIME AND SETTING： Intergroup comparison, in vitro study. The experiment was performed at the Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University between November 2007 and April 2008. MATERIALS- In vitro cultured PC12 cells in the logarithmic phase were assigned into blank control, model, and drug treatment groups （10 μmol/L nimodipine; 40 μg/L, 100 μg/L, and 250 μg/L PQS）. Nimodipine was purchased from Jiangsu Yangtze River Pharmacy Group Co., China; PQS （purity 〉 95%, HLPC grade） was provided by School of Basic Medical Sciences, Jilin University. Caffeine, Na2S2O4, L-glutamic acid （Glu）, Fura-2/AM, and calcium ionophore A23187 were purchased from Sigma, USA. METHODS： PC12 cells in the model and drug treatment groups were separately incubated in glucose-free Hank＇s buffered saline solution ＋ Na2S2O4 （2 mmol/L） for 6 hours, Glu （200 μmot/L） plus A23187 （0.05 μmol/L） for 6 hours, KCI （50 mmol/L） for 1 hour, and caffeine （5 mmol/L） for 3 hours to establish models of intracellular Ca^2＋ overload induced by oxygen and glucose deprivation, Glu, A23187, high K＋, or caffeine. In addition, control cells were incubated in high-glucose DMEM culture medium. MAIN OUTCOME MEASURES： [Ca^2＋]i changes in PC12 cells exposed to oxygen-glucose deprivation, Glu, A23187, high K^＋, or caffeine were detected using spectrofluorometer. RESULTS： PQS blocked the [Ca^2＋]i increase induced by oxygen-glucose deprivation, Glu, A23187, high K＋, or caffeine. In particular, high-dose PQS was most effective （P 〈 0.01）. PQS significantly inhibited Glu- or caffeine-induced [Ca^2＋]i increases in the absence of extracellular Ca^2＋, but nimodipine did not. CONCLUSION： PQS blocked intracellular Ca^2＋ overload induced by oxygen-glucose deprivation, Glu, A23187, high K^＋, or caffeine. This mechanism might be involved in the attenuation of neuronal apoptosis following ischemic brain injury.

Ca^2＋信号参与铝诱导黑麦根系分泌有机酸的调控

【目的】揭示铝诱导根系分泌有机酸的机制,探讨胞质钙信号对铝诱导黑麦根系分泌有机酸的调控作用。【方法】采用药理学研究方法和激光共聚焦扫描显微技术探讨铝胁迫下根尖胞质游离钙离子浓度([Ca2+]cyt)及其与有机酸分泌的关系。【结果】铝不仅诱导黑麦根系分泌柠檬酸和苹果酸,而且使根尖细胞Ca2+的荧光强度增强、波动加剧。铝引起的根尖细胞Ca2+荧光强度的变化在受Ca2+通道抑制剂异搏定、胞内Ca2+通道阻断剂辽红干扰后,显著减少了铝诱导的有机酸分泌。铝诱导的根尖[Ca2+]cyt的升高及有机酸分泌还受CaM阻断剂三氟拉嗪的干扰和抑制。钙螯合剂EGTA处理不仅减弱了铝诱导的Ca2+荧光信号、加大了Ca2+信号的波动幅度,而且显著抑制铝诱导的柠檬酸分泌。此外,在铝胁迫下,根系有机酸分泌受阴离子通道抑制剂尼氟灭酸的抑制。【结论】根尖[Ca2+]c可能是介导铝诱导黑麦根系分泌有机酸的胞内信号因子,而质膜上的阴离子通道可能是铝诱导的钙信号传导途径中的下游效应器。

广枣总黄酮对大鼠心室肌细胞IC_a、I_(to)和细胞[Ca^(2+)]_i的影响

目的 观察广枣总黄酮对大鼠心室肌细胞L 型钙通道电流 (ICa)和瞬时外向钾通道电流 (Ito)以及对心肌细胞内游离钙浓度 ([Ca2 + ]i)的影响 ,探讨其抗心律失常作用机制。方法 全细胞膜片钳记录大鼠心室肌细胞ICa、Ito,激光共聚焦显微镜观察细胞 [Ca2 + ]i 的变化。结果 在钳制电压- 4 0mV ,实验电压 - 4 0～ +5 0mV时 ,广枣总黄酮 10 0mg·L-1对心室肌细胞ICa无显著影响 ;在钳制电压 - 6 0mV ,实验电压 - 4 0～ +5 0mV时显著抑制瞬时外向钾通道Ito(P <0 0 5 ) ;而激光共聚焦显微镜结果显示广枣总黄酮在 5 0、10 0、2 0 0mg·L-1却降低缺氧复氧心肌细胞收缩期和静息期[Ca2 + ]i 的浓度。结论 广枣总黄酮对心肌细胞ICa无显著影响 ,可显著抑制瞬时外向钾通道Ito,并可明显降低心肌细胞收缩期和静息期细胞 [Ca2 + ]i 浓度。这可能是其抗心律失常和保护缺血心肌的主要作用机制。

小檗碱对培养大鼠神经细胞内游离Ca^(2+)的影响

以Ｆｕｒａ２／ＡＭ为细胞内钙离子的荧光指示剂，用ＡＲＣＭＭＩＣ阳离子测定系统，直接测定了体外培养的新生大鼠神经细胞内游离钙（［Ｃａ２＋］ｉ）值，并观察了小檗碱（Ｂｅｒ）的影响。结果表明，Ｂｅｒ对神经细胞静息［Ｃａ２＋］ｉ无明显影响，Ｂｅｒ１～１００μｍｏｌ·Ｌ－１能剂量依赖地抑制去甲肾上腺素和Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高，其ＩＣ５０分别为３９．９和１７．９μｍｏｌ·Ｌ－１。高剂量Ｂｅｒ（１０～１００μｍｏｌ·Ｌ－１）能抑制高Ｋ＋引起的［Ｃａ２＋］ｉ升高。姐果提示，Ｂｅｒ对去甲肾上腺素，高Ｋ＋及Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高的抑制作用可能是其抗脑缺血作用机制之一。

18β-甘草次酸诱导人乳腺癌细胞凋亡及其细胞内Ca^(2+)水平的变化

背景与目的:18-甘草次酸是甘草的重要成分,近年来的研究发现18-甘草次酸具有抑制人淋巴细胞性白血病、肝癌和肺癌的细胞增殖。本研究探讨18-甘草次酸对人乳腺癌MCF-7细胞诱导凋亡的作用。目前研究已证实细胞内游离Ca2+浓度([Ca2+]i)的动态变化在诱发细胞凋亡过程的多个环节中起重要作用,因此本研究也探讨由18-甘草次酸诱导的MCF-7细胞凋亡发生与[Ca2+]i变化的关系。方法:用50~250μmol/L浓度梯度的18β-甘草次酸处理MCF-7细胞24h,用MTT比色法测定细胞增殖能力。100μmol/L和150μmol/L 18β-甘草次酸处理细胞24 h,用末端脱氧核苷酸转移酶介导dUTP末端标记法、Annexin V流式细胞仪法和单细胞凝胶电泳法检测凋亡细胞。150μmol/L 18β-甘草次酸处理细胞24 h,用Fura-2荧光负载方法测定[Ca2+]i的变化。分别用100μmol/L BAPTA-AM和0.5 mmol/L EGTA与150μmol/L 18β-甘草次酸联合处理MCF-7细胞24 h,用单细胞凝胶电泳法检测凋亡细胞。结果:从100μmol/L 18β-甘草次酸浓度起对MCF-7细胞的增殖抑制率显著升高(P<0.01和P<0.05),呈剂量依赖性,半增殖抑制浓度(IC 50)为234.33μmol/L。100μmol/L和150μmol/L 18β-甘草次酸使细胞凋亡率显著升高(P<0.01和P<0.05);18β-甘草次酸处理组的[Ca2+]i也明显高于对照组(P<0.05)。18β-甘草次酸与BAPTA-AM联合处理组和18β-甘草次酸与EGTA联合处理组的凋亡率均明显低于单纯的150μmol/L18β-甘草次酸处理组(P<0.05和P<0.01)。结论:18-甘草次酸具有抑制MCF-7细胞增殖和诱导其凋亡的作用,而18-甘草次酸诱导MCF-7细胞凋亡的作用在抑制该细胞增殖方面起主要作用。18-甘草次酸诱导MCF-7细胞凋亡依赖于细胞内Ca2+水平上调,而细胞外Ca2+内流是导致细胞内Ca2+水平上调的原因之一。

巯亚硝基卡托普利对环匹阿尼酸引起的平滑肌细胞胞浆游离Ca^(2+)浓度升高作用的影响

目的 探讨巯亚硝基卡托普利 (S nitrosocaptopril,CapNO)对Ca2 + 池操纵性Ca2 + 内流信号转导过程的影响。方法 以Fura 2荧光探针测定胞浆游离Ca2 + 浓度([Ca2 + ]i)。结果 ①CapNO(2 0～ 12 0 μmol·L-1)呈浓度依赖性抑制环匹阿尼酸 (cyclopiazonicacid ,CPA)引起的[Ca2 + ]i 升高 ,80 μmol·L-1CapNO为最大效应浓度。相同浓度的captopril对CPA升高 [Ca2 + ]i 无明显抑制作用。②在 80 μmol·L-1CapNO抑制CPA引起 [Ca2 + ]i 升高作用的基础上 (31%± 11% ) ;随后加入 1μmol·L-1硝苯地平不能降低 [Ca2 + ]i,再加入 2 0 μmol·L-1SK&F96 36 5 (最大效应浓度 )可进一步降低 [Ca2 + ]i(5 4%± 18% ) ,其中SK&F96 36 5净抑制率为 2 4%± 10 % ,与SK&F96 36 5单独作用抑制率(5 4%± 11% )比较差异有显著性。③不同顺序给予最大效应浓度CapNO (80 μmol·L-1)和tyrphostinAG490 (2 μmol·L-1)对CPA引起 [Ca2 + ]i 升高存在交叉抑制作用。结论 CapNO可通过阻断电压依赖性Ca2 + 通道和Ca2 + 池操纵性Ca2 + 通道抑制CPA引起的 [Ca2 + ]i 升高 ,CapNO对CPA引起 [Ca2 + ]i 升高的阻断作用存在酪氨酸激酶 (Janus2亚型 )

慢性染铅对大鼠海马区神经细胞Ca^(2+)浓度及Ca^(2+)-ATP酶活性的影响

目的：观察慢性染铅对大鼠海马区神经细胞Ｃａ２＋浓度及Ｃａ２＋－ＡＴＰ酶活性的影响。方法：用０．１５％醋酸铅饲养大鼠建立慢性染铅动物模型，参照Ｄｉｌｄｙ法和徐友涵法测定海马神经细胞Ｃａ２＋浓度及Ｃａ２＋－ＡＴＰ酶活性。结果发现：细胞内Ｃａ２＋浓度，染铅组（２０３．８３±３０．５０）ｎｍｏｌ／Ｌ，对照组（９７．６２±１９．８３）ｎｍｏｌ／Ｌ，ｔ＝８．３１Ｐ＜０．００５；Ｃａ２＋－ＡＴＰ酶活性，染铅组（３２６．４２±４０．０６）ｎｍｏｌ（Ｐｉ）·ｍｇ－１·ｍｉｎ－１，对照组（２５３．０７±２５．４０）ｎｍｏｌ（Ｐｉ）·ｍｇ－１·ｍｉｎ－１，ｔ＝３．５４，Ｐ＜０．０１。结论：慢性染铅可使大鼠海马区神经细胞内Ｃａ２＋浓度升高。

环维黄杨星D对分离大鼠心室肌细胞内Ca^(2+)和L型钙电流的影响

目的 研究环维黄杨星D(CD)对大鼠心室肌细胞内Ca2 + 动员和L型钙电流 (ICa L)的影响。方法 采用全细胞膜片钳和激光扫描共聚焦显微术研究CD对心肌细胞ICa L以及氯化钾、咖啡因诱发心肌细胞内Ca2 + 动员的影响。结果 CD浓度依赖性抑制ICa L。指令电压为 10mV时 ,1和 10 μmol·L- 1 CD分别使ICa L 电流密度从 (- 9 9±1 8)pA pF降至 (- 6 4± 1 4 )pA pF和 (- 4 2± 0 6 )pA pF。共聚焦实验显示 1和 10 μmol·L- 1 CD不影响静息心肌细胞 [Ca2 + ]i,对氯化钾诱发 [Ca2 + ]i升高水平无明显抑制作用 ;咖啡因引起的细胞内Ca2 + 动员可被CD进一步增强。结论 CD浓度依赖性抑制大鼠心室肌细胞ICa L,并有促进咖啡因诱发心肌细胞内Ca2 +

外源Ca^(2+)对糙米发芽力及贮藏物动员的影响

以水稻南粳 39为材料 ,研究了外源Ca2 + 对糙米萌发和有关贮藏物动员的影响。在 0～ 15mmol·L-1Ca2 + 浓度范围内 ,低浓度Ca2 + 能提高糙米的发芽率、发芽指数、活力指数、呼吸速率和淀粉酶活力及淀粉等贮藏物质的降解 ;随着Ca2 + 浓度的升高 ,上述指标下降 ;最适浓度为 0 5mmol·L-1。

库容性Ca^(2+)内流参与ACh诱导的大鼠远端结肠平滑肌收缩

应用生物换能技术和Ca2+通道特异性阻断剂观察并记录大鼠离体远端结肠平滑肌收缩张力的变化,分析库容性 Ca2+内流(capacitative Ca2+ entry,CCE)是否与ACh诱导的离体远端结肠平滑肌收缩反应有关。结果表明,以无钙的Krebs 液灌流或应用EGTA螯合细胞外Ca2+后,高K+及ACh引起的远端结肠平滑肌收缩几乎完全消失。电压操纵性Ca2+通道阻断剂veiapamil也能减弱高K+及ACh引起的远端结肠平滑肌收缩,其减弱的程度分别为74％和41％。在无钙的Krebs液中, 5 μmol／L ACh可引起离体肠管瞬时性收缩,这是由肌质网(sarcoplasmic reticulum,SR)释放钙所致;然后加入10 μmol／L阿托品(atropine),并在此基础上恢复细胞外Ca2+(2．5 mmol／L),结肠平滑肌则出现持续性收缩,待收缩反应达峰值时,加入5μmol／L verapamil,收缩无明显变化,且该收缩反应对钙库操纵性通道(store-operated Ca2+ channel,SOCC)阻断剂La3+ 敏感,20,50和100 μmol／L的La3+使上述收缩张力分别降低15％,23％和36％,且呈浓度依赖性,但对Cd2+不敏感。研究结果提示,细胞外Ca2+内流对高K+及ACh介导的离体远端结肠平滑肌持续性收缩是必需的,由ACh诱导的远端结肠平滑肌收缩至少包括SR释放钙引起的短暂性收缩及受体操纵性Ca2+通道(receptor-operated Ca2+ channel,ROCC)、电压操纵性Ca2+通道(voltage-operated Ca2+ channel,VOCC)和CCE介导的胞外Ca2+内流等途径。这将从通道水平进一步分析消化管平滑肌收缩的机制和特征,亦将为预防和控制因胃肠动力紊乱所致的消化管疾病寻求有针对性的药物干预和治疗提供理论依据。

大黄酸对IL-2诱发T淋巴细胞增殖和细胞[Ca^(2＋)]_i变化的作用

研究大黄酸对IL-2引起大鼠T淋巴细胞增殖和细胞内[Ca^(2+)]_i变化的影响.采用MTT法检测细胞增殖,用单细胞钙成像系统记录胞内游离Ca^(2+)浓度([Ca^(2+)]_i).结果表明,大黄酸对IL-2引起的细胞增殖有显著抑制作用,且抑制效果与剂量有关;IL-2引起[Ca^(2+)]_i升高,并持续维持高[Ca^(2+)]_i水平,[Ca^(2+)]_i升高的机制包括胞内Ca^(2+)释放和胞外Ca^(2+)内流;大黄酸显著抑制IL-2引起的[Ca^(2+)]_i升高.结果提示大黄酸可抑制T淋巴细胞增殖,作用机制可能与抑制细胞内[Ca^(2+)]_i升高相关.

绞股蓝总黄酮对缺氧心肌细胞损伤标记物及细胞内游离Ca^2＋浓度的影响

探讨绞股蓝总黄酮(TFG)对缺氧心肌细胞损伤标记物及细胞内游离Ca2+浓度的影响.在离体乳鼠心肌细胞原代培养基础上制备心肌细胞缺氧模型,随机分为正常对照组、缺氧损伤组和绞股监总黄酮干预缺氧损伤组.分别测定各组心肌培养基中乳酸脱氢酶(LDH)、肌钙蛋白I(cTn I)的含量,心肌细胞内游离Ca2+的浓度(荧光探针法).缺氧损伤组心肌培养基中LDH和cTnI的含量及心肌细胞内游离Ca2+浓度较正常对照组明显增加(P<0.05或0.01);经TFG干预后细胞培养基中LDH利cTnI的含量及心肌细胞内游离Ca2+浓度较缺氧损伤组明显降低(P<0.05或0.01).TFG对缺氧心肌细胞具有保护作用,其机理可能与减轻心肌细胞内钙超负荷有关.

高钾离子引起PC12细胞内游离Ca^(2+)浓度升高的机制

目的：分析高钾离子（Ｋ＋）引起ＰＣ１２细胞内游离钙离子浓度（［Ｃａ２＋］ｉ）升高的可能机制。方法：利用ＭｉｒａＣａｌＩｍａｇｅＳｙｓｔｅｍ检测［Ｃａ２＋］ｉ，Ｃａ２＋荧光探针为Ｆｕｒａ－２／ＡＭ。结果：（１）ＫＣｌ浓度为３０～１００ｍｍｏｌ／Ｌ时，可剂量依赖性地诱导ＰＣ１２细胞［Ｃａ２＋］ｉ升高；（２）在细胞外无Ｃａ２＋时，高Ｋ＋对ＰＣ１２细胞［Ｃａ２＋］ｉ无影响；（３）Ｌ－型电压门控钙通道阻滞剂维拉帕米、地尔硫和硝苯地平的浓度分别为５×１０－５，１×１０－４和５×１０－３ｍｏｌ／Ｌ时，可完全阻断高Ｋ＋诱导的ＰＣ１２细胞［Ｃａ２＋］ｉ升高，但Ｎ－型电压门控钙通道阻滞剂ω－ｃｏｎｏｔｏｘｉｎＧＶＩＡ在浓度为１×１０－６ｍｏｌ／Ｌ时，对高Ｋ＋诱导的ＰＣ１２细胞［Ｃａ２＋］ｉ升高没有影响；（４）５×１０－５ｍｏｌ／Ｌ维拉帕米对细胞外由无Ｃａ２＋到有Ｃａ２＋过程引起的［Ｃａ２＋］ｉ升高无抑制作用。结论：高Ｋ＋引起ＰＣ１２细胞［Ｃａ２＋］ｉ升高以细胞质膜上Ｌ－型电压门控Ｃａ２＋通道开放为基础。

单细胞内Ca^（2＋）时空变化的激光共聚焦显微测定

应用激光扫描共聚焦显微系统（ＬＳＣＭ）和Ｆｌｕｏ－３／ＡＭ荧光探剂标记技术，测定了单个活细胞胞内游离Ｃａ２＋的动态变化与立体分布影像．结果显示，在３７℃，Ｆｌｕｏ－３／ＡＭ终浓度为６μｍｏｌ／Ｌ的条件下，Ｃ５７ＢＬ／６Ｊ小鼠巨噬细胞负载１ｈ左右即可获得良好的标记效果．相反，若探剂浓度太高或负载时间太长，胞内荧光强度太强，影响在共聚焦显微镜镜下分辨细胞内结构．因此用ＬＳＣＭ研究细胞内游离Ｃａ２＋变化时，荧光探剂的负载应以获得最适荧光信号而不是以最大荧光强度为标准．上述方法在其他如平滑肌细胞、卵母细胞中的测定亦获得满意的结果，这对进一步研究各种生理和病理条件下细胞内Ｃａ２＋信号的动态变化、与跨膜Ｃａ２＋梯差的关系及对活细胞功能活动的调节提供了一种可行的、直观的研究手段。

神经肽Y经Ca^2＋/CaM依赖的钙调神经磷酸酶途径诱导心肌细胞肥大

目的观察神经肽Y(NPY)诱导心肌细胞肥大效应,及Ca2+/CaM依赖的钙调神经磷酸酶(CaN)途径在其中起的作用。方法用NPY(10、100nmol)刺激心肌细胞,用环胞素A(CsA)特异性抑制CaN活性。测定心肌细胞蛋白合成速率(3H-Leu掺入量)、CaN-蛋白表达、c-junmRNA表达、CaN酶活性、细胞内游离钙含量。结果(1)在100nmolNPY作用下,心肌细胞3H-Leu掺入量及c-junmRNA表达量均较对照组显著增高(P<0.05,P<0.001)。NPY+CsA组和对照组相比无显著差别。(2)在100nmolNPY作用下,NPY组心肌细胞内CaN酶比活力、CaN-α表达、胞浆及核内钙含量较对照组显著增高(P<0.05,P<0.05,P<0.001,P<0.001)。结论NPY可刺激心肌细胞肥大,CaN信号途径在其中起重要作用。