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.

Aluminum neurotoxicity effects on intracellular Ca^(2+) homeostasis in the rat cerebral cortex

Studies have suggested that aluminum, a neurotoxic metal, is involved in the progression of neurodegenerative diseases. Previous studies have confirmed that aluminum influences intracellular Ca^2＋ homeostasis. However, it remains unclear whether aluminum increases or decreases intracellular Ca^2＋ concentrations. The present study demonstrated that Al^3＋ competitively binds to calmodulin （CAM）, together with Ca^2＋, which resulted in loss of capacity of CaM to bind to Ca^2＋, leading to increased [Ca^2＋]i. Al^3＋ stimulated voltage-gated calcium channels on cell membranes, which allowed a small quantity of Ca^2＋ into the cells. Al^3＋ also promoted calcium release from organelles by stimulating L-Ca^2＋αlc to trigger calcium-induced calcium release. Although Al^3＋ upregulated expression of Na＋/Ca^2＋exchanger mRNA, increased levels of Ca^2＋ and Na＋/Ca^2＋ exchanger did not maintain a normal Ca^2＋ balance. Al^3＋ resulted in disordered intracellular calcium homeostasis by affecting calcium channels, calcium buffering, and calcium expulsion.

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.

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。

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。

羟墓自由基引起神经细胞［Ca^（2＋）］i增高的机制和Ebselen的抑制作用

用Ｆｕｒａ－２显微荧光测量技术研究了羟基自由基对单个皮层神经细胞内游离钙离子浓度［Ｃａ２＋］ｉ影响和硒化合物Ｅｂｅｌｅｎ对［Ｃａ２＋］ｉ的抑制作用。结果表明羟基自由基的作用首先引起胞内［Ｃａ２＋］ｉ以时间常数τ＝３８９５．４±５０７．２Ｓ速度缓慢增加，然后加入了以τ＝４２０．６±１２２．０Ｓ的外钙大量涌入。钙通道阻断剂、疏基还原剂、疏基还原制和自由基清除剂对羟基自由基损伤作用的影响提示外钙的大量涌入部分与通道的开放有关，疏基损伤在羟基自由基引起的［Ｃａ２＋］ｉ升高中起着重要的作用。具有类谷胱甘肽过氧化酶活性的小分子硒化合物Ｅｂｓｅｌｅｎ（１０－５ｍｏｌ／Ｌ和１０－６ｍｏｌ／Ｌ）抑制羟基自由基引起的［Ｃａ２＋］ｉ升高，推测它可以抑制钙库的释放或促进内钙的外排以及抑制外钙的流入。

实时监测GHRP对心肌细胞内[Ca^(2+)]i和NO调控的双标记方法研究

目的探索实时监测GHRP对心肌细胞内[Ca2+]i和NO调控的双标记方法,为研究GHRP对心脏直接保护效应机制提供新方法。方法用改良的Langendorff恒流灌注仪系统和酶解法急性分离SD成年大鼠心肌细胞,在LSCM下分别以Ca2+探针Rhod-2/AM和NO探针DAF-FM/DA对心肌细胞内Ca2+和NO进行双标记并观察GHRP即时刺激对两者的调控影响。结果在LSCM下心肌细胞内Ca2+呈红色荧光,NO呈绿色荧光,两者双标记后呈现黄绿色荧光,GHRP使红色荧光出现瞬时增强后又迟缓地回降,绿色荧光强度没有明显变化,双标记下GHRP对两者的调控同单标记结果。结论LSCM下实验中设计的双标记方法能实时监测成年大鼠心肌细胞内Ca2+和NO存在及GHRP对两者同一时相调控作用,引起[Ca2+]i两个时相的变化,而对NO信号系统未见明显影响。

镉对人体外周血淋巴细胞内游离Ca^(2+)及钙调素影响的研究

采用体外实验方法，观察CdCl2对培养24h的人外周血淋巴细胞内游离Ga2+浓度及CaM活性的影响。结果表明：细胞内游离Ca2+浓度与镉浓度（≤100μmol／L）及染毒时间（≤60min）存在明显的剂量及时相相关，在≤50μmol／LCdCl2作用下，CaM活性随染毒剂量增加而升高，50μmol／LCdCl2使CaM活性增至0μmol／L组的190．22％（P＜0．05），但100μmol／LCaCl2则对CaM无激活作用。在一定镉浓度（0～50μmol／LCdCl2）及染毒时间（0～40min）内，细胞内游离Ca2+浓度与CaM活性呈*一致性变化，提示镉的免疫毒作用机制与Ca2+、CaM系统有关。

Effect of Ca^(2+) on CO_2 corrosion properties of X65 pipeline steel

The effect of Ca^2＋ on CO2 corrosion to X65 pipeline steel was investigated in the simulated stratum water of an oil field containing different concentrations of Ca^2＋. It is found that Ca^2＋ can enhance the corrosion rate, especially in the Ca^2＋ concentration from 256 to 512 mg/L, which can be attributed to the growing grain size and loosing structure of corrosion scales with increasing Ca^2＋ concentration. X-ray diffraction （XRD） and X-ray photoelectron spectroscopy （XPS） investigations reveal that a complex carbonate （Fe, Ca）CO3 forms at high Ca^2＋ concentration due to the gradual replacement of Fe^2＋ in FeCO3 by Ca^2＋.

Trpm7下调对大鼠心肌细胞游离Ca^(2+)浓度影响的研究

目的 TRPM7是心肌细胞内一种阳离子通道,对Mg^(2+)和Ca^(2+)都具有通透性。为了深入研究TRPM7基因在心肌细胞的功能,本试验试图研究下调TRPM7后大鼠心肌细胞内Ca^(2+)浓度的变化。方法大鼠心肌细胞的原代培养,心肌细胞存活度的测定,小干扰RNA(siRNA)技术,大鼠心肌细胞胞内游离Ca^(2+)荧光强度的检测。结果经过胞内游离Ca^(2+)荧光强度的检测发现下调TRPM7后大鼠心肌细胞内Ca^(2+)浓度无显著变化。结论调TRPM7后对心肌细胞钙的代谢没有显著影响。

用Fura 2－AM检测血小板胞浆游离钙浓度

本文对应用荧光指示剂Ｆｕｒａ２－ＡＭ（国产）测定血小板胞浆游离钙浓度的某些实验条件进行了探讨。结果表明：实验最适ｐＨ范围为７．３５～７．４５。实验不受溶血、黄疸及高脂血样品的干扰。批内ＣＶ４．９８％，批间ＣＶ５．６％。正常成人参考值为１６７．７５±２０．６６ｎｍｏｌ／Ｌ。与使用进口Ｆｕｒａ２－ＡＭ（Ｓｉｇｍａ产品）检测结果比较，结果无统计学差异，相关系数：γ＝０．９８。

小鼠神经细胞中胱硫醚β合成酶催化生成的H_(2)S对脑微血管内皮细胞增殖、迁移的影响及与VEGFR _(2)的关系

目的探讨神经细胞中的胱硫醚β合成酶(CBS)催化生成的H_(2)S对小鼠脑血管内皮细胞增殖、迁移的影响及其与VEGFR 2的关系。方法用CCK-8法检测细胞增殖,细胞划痕法和Transwell法分别检测细胞迁移,甲基蓝法检测H_(2)S含量和钙荧光成像法检测内皮细胞的细胞内游离Ca^(2+)浓度,HT22细胞和bEnd.3细胞进行Transwell细胞共培养。结果H_(2)S供体NaHS(1×10^(-8)—1×10^(-3.5) mol·L^(-1))可明显增加HUVEC细胞和bEnd.3细胞的增殖、迁移面积和迁移的细胞数,而VEGFR 2阻断剂SU5416(10μmol·L^(-1))可明显抑制NaHS促进增殖和迁移的作用;在共培养体系中,H_(2)S合酶底物L-cys(100μmol·L^(-1))可明显促进bEnd.3细胞增殖和迁移及细胞内Ca^(2+)荧光强度,并提高体系中的H_(2)S含量,但CBS抑制剂AOAA(1 mmol·L^(-1))和SU5416则明显减弱L-cys的增殖和迁移,促进作用及细胞内Ca^(2+)荧光强度的增高。结论神经细胞中CBS催化生成的H_(2)S可促进脑血管内皮细胞增殖和迁移,其作用可能与激活VEGFR 2,导致内皮细胞的胞内游离Ca^(2+)浓度升高有关。

问号钩端螺旋体内化过程中细胞内游离Ca^(2+)水平变化及细胞凋亡

目的 建立观察问号钩端螺旋体 (简称钩体 )黏附的双荧光染色法 ,探讨不同毒力钩体对细胞内游离Ca2 + 水平及细胞凋亡的影响。方法 以问号钩体黄疸出血群赖型 5 6 6 0 1株和双曲钩体三堡垄群patoc型PatocⅠ株抗血清为一抗、羊抗兔IgG荧光素F(ab) 2 和罗丹明F(ab) 2 片段为二抗的双荧光染色法 ,分别检测 5 6 6 0 1株和PatocⅠ株钩体对Vero、J774A .1细胞的黏附作用。采用fluo 3 AM胞内Ca2 + 特异荧光标记激光共聚焦技术 ,检测问号钩体 5 6 6 0 1株和波摩那群波摩那型 5 6 6 0 8株、双曲钩体PatocⅠ株作用的J774A .1细胞胞内游离Ca2 + 水平的变化。采用FITC annexinⅤ PI荧光标记流式细胞术 ,检测紫外线灭活前后的 5 6 6 0 1株钩体诱导Vero和J774A .1细胞凋亡的情况。结果 所建立的双荧光染色法能清晰地观察到强毒力的 5 6 6 0 1株钩体对Vero和J774A .1细胞的黏附 ,无毒力的PatocⅠ株钩体则否。正常J774A .1细胞胞内游离Ca2 + 基础值为 (1 0 5 .0± 7.0 ) %,PatocⅠ株钩体作用细胞的荧光强度变化百分数一直波动于 (1 0 2 .2± 5 .2 ) %。 5 6 6 0 1株钩体感染J774A .1细胞胞内游离Ca2 + 浓度迅速增高 ,呈现为双峰型曲线 ,其荧光强度变化百分数分别为 (74 7.5± 35 .7) %和 (80 4 .6±4 0 .8) %。 5 6

SH－SY5Y细胞胞内钙库特性研究

运用单细胞显微荧光测量技术测量了单个ＳＨ—ＳＹ５Ｙ细胞内游离钙离子浓度的变化。首次报道了ＳＨ—ＳＹ５Ｙ细胞内存在毒蕈碱敏感而非咖啡因敏感的钙库，并研究了它的动力学特征。Ｎ￣￣Ｃ￣ｈｅｔｗＭａｄＵｇｈ＊ＴＯｗｈｏｍＣｂ￣ｐｏｎｄｅｎｅｅｓｈｏｕｌｄｂｅ￣．

莪术抑制晶状体上皮细胞增殖的信号转导机制

目的探讨天然药物莪术(rhizoma curcumae,RC)对体外培养的晶状体上皮细胞(lens ep-ithelial cell,LEC)增殖的抑制作用,并从信号转导方面研究其抑制LEC增殖的作用机制,为寻求防治后发性白内障(after cataract)的确切有效药物提供实验依据。方法用牛LEC进行原代和传代培养,取第三代细胞。用10ng/ml重组人碱性成纤维细胞生长因子(recombinant human basic fibroblast growthfactor,rhbFGF)诱导LEC增殖。同时分别将5mg/ml、10mg/ml、20mg/ml的莪术作用于增殖状态下的LEC72小时。(1)通过四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)比色测定法检测莪术对LEC增殖的抑制程度;(2)用钙荧光指示剂Fura-2/AM测定莪术抑制LEC增殖时细胞内游离钙离子浓度(Ca2+i)变化;(3)用放射免疫分析法测定莪术抑制LEC增殖时细胞内环化腺苷酸(cyclic adenosine monophosphate,cAMP)和环化鸟苷酸(cyclicguanosine monophosphate,cGMP)浓度变化。结果莪术可明显抑制LEC增殖。抑制LEC增殖时细胞内游离钙离子浓度(Ca2+i)升高,细胞内cAMP浓度显著升高,细胞内cGMP浓度显著降低,cAMP/cGMP比值升高,并具有剂量依赖关系。结论诱导Ca2+i升高,激活细胞内Ca2+信号转导途径;上调细胞内cAMP水平,下调细胞内cGMP水平,使cAMP/cGMP比值升高,激活细胞内cAMP-PKA、cGMP-PKG信号转导途径可能是莪术抑制LEC增殖的作用机制之一。莪术能够明显抑制LEC增殖的作用,有望成为防治后发性白内障的有效药物。

血小板衍生生长因子和钙拮抗剂对大鼠肝细胞胶原合成及细胞内游离钙浓度的影响

目的：观察血小板衍生生长因子（Ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｄｅｇｒｏｗｔｈｆａｃｔｏｒ，ＰＤＧＦ）和钙拮抗剂（（Ｃａｌｃｉｕｍａｎｔａｇｏｎｉｓｔ，Ｃａ－Ａ）对原代培养大鼠肝细胞胶原合成以及细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响。方法：应用３Ｈ－脯氨酸掺入率法和Ｆｕｒａ－２／ＡＭ荧光示踪法分别测定细胞内胶原含量及［Ｃａ２＋］ｉ。结果：ＰＤＧＦ能促进细胞内胶原合成，提高肝内［Ｃａ２＋］ｉ，且与浓度呈正相关，而Ｃａ－Ａ具有反作用。结论：ＰＤＧＦ参与肝纤维化的形成。

Studies on the Effects of Epimedium Extract on Erythrocytes

The effect of Epimedium extract （EE） on erythrocytes was investigated by atomic force microscopy （AFM）. The images of the surface structures showed clear concave and progressive increase of surface roughness of erythrocyte after incubation with EE at concentration of 0.2 or 0.05 g/L, far below its critical hemolytic levels. The AFM results also indicated that the granules of the fine surface structure increased, which caused by aggregation of membrane protein. Further study showed that the change in surface topography of erythrocyte membrane might be connected with the increase of intracellular free Ca^2＋ induced by EE.

血小板衍生生长因子对肝细胞游离钙浓度影响

目的观察血小板衍生生长因子（PDGF）对SD大鼠肝细胞内游离钙浓度（[Ca2+]i）的影响，同时观察钙拮抗剂（Ca－A）对PDGF作用的影响。方法应用Fura－2／AM荧光示踪法检测肝细胞（n=15）内[Ca2+]i的变化；及PDGF存在下和PDGF、Ca－A同时存在下细胞内[Ca2+]i变化。结果PDGF可明显升高细胞内[Ca2+]i，Ca－A具有抑制PDGF的增钙作用。结论PDGF参与SD大鼠肝细胞内Ca2+浓度的转运，Ca－A对肝细胞具有保护作用。

大鼠感染性脑损伤中神经细胞内游离钙的变化

采用荧光法测定负载Ｆｕｒａ－２／ＡＭ大脑皮层突触体内游离钙［Ｃａ２＋］ｉ浓度在大鼠感染性脑损伤（感脑）中的变化及其意义。结果显示：百日咳菌液组（０．５ｈ，４ｈ，２４ｈ）的［Ｃａ２＋］ｉ，含水量（ＷＣ）及伊文思蓝（ＥＢ）含量均高于对应盐水对照组（Ｐ＜０．０５或＜０．０１）；菌液组［Ｃａ２＋］ｉ增高呈时效关系，以２４ｈ组含量最高（４ｈｖｓ０．５ｈ，Ｐ＜０．０５；２４ｈｖｓ４ｈ，Ｐ＜０．０１），并分别与ＷＣ及ＥＢ含量呈高度正相关（０．５ｈ，４ｈ，Ｐ＜０．０１）及无相关（２４ｈ，Ｐ＞０．０５）。结果表明。

Spontaneous Neurotransmitter Release Depends on Intracellular Rather than ER Calcium Stores in Cultured Xenopus NMJ

Calcium ions are important in many vital neuron processes, including spontaneous neurotransmitter release. Extracellular calcium has long been known to be related to spontaneous neurotransmitter release, but the detailed mechanism for the effect of intracellular Ca^2＋ on synaptic release has not yet been understood. In this research, 1,2-bis-（o-aminophenoxy）-ethane-N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester （BAPTA-AM） was used to combine with cytosolic free Ca^2＋ in a calcium free medium of cultured Xenopus neuromuscular junctions （NMJ）, The spontaneous synaptic current （SSC） frequency was obviously reduced. Then, drugs were applied to interrupt and activate the Ca2＋ release channels in the endoplasmic reticulum （ER） membrane, but the SSC frequency was not affected. The results show that spontaneous neurotransmitter release depends on intracellular rather than ER calcium in cultured Xenopus NMJ without extracellular calcium.