内皮素对培养心肌细胞内游离钙浓度的作用

实验用培养新生ＳＤ大鼠心室肌细胞,以Ｆｕｒａ－２／ＡＭ荧光指示剂负载检测收肌细胞内游离钙浓度（「Ｃａ＾２＋」）的变化,探讨内皮素－１（ＥＴ－１）对「Ｃａ＾２＋」ｉ的作用及其机制。结果显示：ＥＴ－１引起心肌细胞「Ｃａ＾２＋」ｉ升高有两个时相,瞬时相持续相。ＥＴ－１诱导的瞬时相「Ｃａ＾２＋」ｉ升高呈浓度依赖性,预先用ＥＴＡ特异性受阻断剂ＢＱ１２３处理,可阻断ＥＴ－１引起的「Ｃａ＾２＋」ｉ升高,揭示上述     

α1-肾上腺素受体亚型介导细胞内游离Ca2+增高的信号转导途径

本文探讨了α１ａ,α１ｂ,α１ｄ三种亚型肾上腺素受体激动时细胞内Ｃａ６２＋浓度升高的信号转导途径。在稳定表达三亚型α１－ＡＲ的ＨＥＫ２９３细胞２系中,用ｆｕｒａ－２方法细胞内Ｃａ＾２＋信号强弱的变化。结果显示,百日咳毒素对去甲肾上腺素激动三亚型α１－ＡＲ而引起的「Ｃａ＾２＋」ｉ升高无影响,Ｕ－７３１２２和ＰＭＡ明显抑制「Ｃａ＾２＋」ｉ升高．     

血小板激活时胞质游离钙浓度与两种颗粒数变化的相关性

用电镜形态计量法检测血小板α颗粒（αＧ）和致密颗粒（ｄＧ）的数密度,用钙荧光指示剂Ｆｕｒａ２检测血小板胞质游离Ｃａ＾２＋浓度（「Ｃａ＾２＋」ｉ）,观察到在钙离子导体Ａ２３１８７作用下,血小板「Ｃａ＾２＋」ｉ明显升高。凝血酶与ＡＤＰ也都分别引起「Ｃａ＾２＋」ｉ升高,且有浓度依赖性,选用三种激动剂的不同量以反映血小板不同程度激活时,测定「Ｃａ＾２＋」与颗粒数密度,分析两者间的相关性,发现αＧ和ｄＧ的数     

M型受体在大鼠肾上腺嗜铬细胞内钙库释放和激素分泌中的作用

在单个大鼠肾上腺嗜铬细胞上,用显微荧光测量和碳纤电极记录方法,测量可激活毒蕈碱（ｍｕｓｃａｒｉｎｅ,Ｍ）受体的激动剂乙酰甲胆碱（ｍｅｔｈａｃｈｏｌｉｎｅ,ＭＣｈ）对胞内游离钙浓度「Ｃａ＾２＋」ｉ和儿茶酚胺激素分泌的影响。在细胞外液含２ｍｍｏｌ／Ｌ Ｃａ＾２＋时,用含钙或不含钙的ＭＣｈ（１ｍｍｏｌ／Ｌ）刺激细胞,均引起「Ｃａ＾２＋」ｉ的升高或钙振荡,并诱发激素的分泌。     

中枢神经系统钙稳态失调和老龄脑功能

脑的老化表现为记忆力的减退。脑老化的钙假说认为脑的老化与中枢神经系统「Ｃａ＾２＋」ｉ的调节机制紊乱有关,衰老可以通过多种因素导致「Ｃａ＾２＋」ｉ升高,影响突触传导,神经递质释放,信号转导等导致记忆障碍,本文综述了近年来的进展。     

牛磺酸对血管紧张素Ⅱ改变培养心肌细胞内游离Ca^2＋深度作？…

目的和方法：用Ｆｕｒａ－２／ＡＭ荧光显示测定细胞内游离Ｃａ＾２＋浓度（〖Ｃａ＾２＋〗ｉ）的方法,我们研究了牛磺酸（Ｔａｕ）对血管紧张素Ⅱ（ＡｎｇⅡ）引起的培养心肌细胞（〖Ｃａ＾２＋〗ｉ）变化的影响。结果：在有Ｃａ＾２＋和无Ｃａ＾２＋的缓冲液中,ＡｎｇⅡ（１,１０,１００,１０００ｎｍｏｌ／Ｌ）引起的〖Ｃａ＾２＋）ｉ和蔼同。在含Ｃａ＾２＋的缓冲液中,Ｔａｕ（１０,２０ｍｏｌ／Ｌ）可隽浓度地抑制Ａｎｇ     

用Fura－2双波长荧光法测定神经细胞内游离钙

采用新型Ｃａ＾２＋荧光指示剂Ｆｕｒａ－２建立双波长荧光法测定分离的大鼠神经细胞内游离钙浓度（［Ｃａ＾２＋］ｉ）。结果显示,在静息状态下,其［Ｃａ＾２＋］ｉ为１０９±１２ｎｍｏｌ／Ｌ．３０ｍｍｏｌ／ＬＫＣｌ可显增加［Ｃａ＾２＋］ｉ,并且ＫＣｌ的这种效应呈一定的剂量依赖关系,提示该法灵敏、可靠。     

应用流式细胞仪研究抗IL——8单抗对IL——8激活人颗粒细胞活力的中和作用

以ＩＬ－８免疫的ＢＡＬＢ／Ｃ小鼠脾细胞与Ｓｐ２／０或６５３小鼠骨髓瘤细胞融合构建了淋巴细胞杂交瘤克隆Ｉ８－Ｓ２和Ｉ８－６３。ＥＬＩＳＡ叠加试验（ＥＬＩＳＡ Ａｄｄｉｔｉｖｉｔｙ Ｔｅｓｔ）表明这两杂交瘤克隆分泌的单抗分别识别ＩＬ－８分子的不同表位。ＩＬ－８能激活人颗粒细胞,引起细胞内Ｃａ＾２＋浓度（「Ｃａ＾２＋」）上升。通过流式细胞仪分析「Ｃａ＾２＋」的变化,发现两个克隆单抗对ＩＬ－８激活细胞的活     

新疆呼图壁种牛场地区13种优势植物水溶性盐分的含量特点及数量分析

通过对新疆呼图壁种牛场地区１３种耐盐植物水溶性盐分含量的分析,结果阐述了盐分分布和积累的特点：１）大部分植物在盐分含量水平上是：Ｎａ＾＋〉Ｋ＾＋〉Ｍｇ＾２＋〉Ｃａ＾２＋,Ｃｌ＾－〉ＳＯ４＾２－〉ＨＣＯ３＾－〉ＣＯ３＾２－,Ｎａ＾＋超过２００００μｇ／ｇ,Ｃａ＾２＋不足８００μｇ／ｇ;Ｃｌ＾－达４．１６％,ＣＯ３＾２－仅为０．１１％。全盐量平均为２５．８１％;２）ＣＯ３＾２－、ＨＣＯ３＾－和Ｃａ＾２     

腺苷对缺氧时海马神经元内游离Ca^2＋的影响

实验用Ｆｌｕｏ－３负载细胞,在激光扫描共聚焦显微镜下直接监测缺氧后分散培养的大鼠海马ＣＡ１区神经元内游离Ｃａ＾２＋浓度（［Ｃａ＾２＋］ｉ）的变化,观察腺苷对这种变化的影响并初步探讨其作用机制。结果发现,急性缺氧使海马神经元［Ｃａ＾２＋］ｉ显著升高;腺苷（１００μｍｏｌ／Ｌ）明显抑制缺氧引起的［Ｃａ＾２＋］ｉ增高,腺苷Ａ１受体拮抗剂ＣＰＴ以及Ｋ＾＋通道阻断剂４－ＡＰ和ＡＴＰ敏感性Ｋ＾＋通道阻断剂ｇｌ     

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Characterization of pressure-induced calcium response in neuronal cell lines

BACKGROUND: Cation channels that respond to mechanical stress have been described in neuronal and nonneuronal cells. These nonselective cation ([C+(SA)]) channels are believed to regulate volume and osmolarity of cells in the central nervous system and are therefore believed to be involved in brain injury, resulting in intracellular calcium accumulation and cell death. METHODS: Activation of pressure-sensitive channels was monitored as an increase in [Ca2+](i) by flow cytometry using indo-1. Several neuronal cell lines including NH15-CA2 neuroblastoma x glioma cells were stimulated by rectangular pressure increase. RESULTS: Neuronal cell lines showed a pressure-sensitive increase in [Ca2+](i) but no pressure sensitivity was found in fibroblasts and embryonic P19 cells. [C+(SA)] channels in NH15-CA2 cells were not blocked by inhibitors of voltage-dependent calcium channels and G-proteins. Depletion of extracellular calcium and of internal Ca2+ stores inhibited pressure-induced [Ca2+](i) increase. Elevated [C+(SA)] channel activity was also observed in confluent NH15-CA2 thus accumulated in the G(0)/G(1)-phase of the cell cycle. P19 cells showed occurrence of [C+(SA)] channel activity only after neuronal differentiation. CONCLUSION: Pressure-sensitive channel activity is present in cells of neuronal origin. This activity depends on neuronal differentiation and might have a pivotal role in neuronal development and differentiation.     

药物诱导后的人大肠癌细胞的[Ca^2＋]i的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１,２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化,并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响,从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１,２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下,ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流,ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外,Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池,亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

pH改变对心肌细胞内Ca~(2 )浓度和细胞长度的影响

目的 :探讨细胞内 pH(pHi)改变对心肌细胞内Ca2 浓度 ([Ca2 ]i)和细胞长度的影响。方法 :心肌细胞内分别灌注 2 0mmol/L丙酸钠和 15mmol/LNH4Cl,建立细胞内酸碱中毒模型。荧光指示剂indo 1和SNARF 1载入大鼠心肌细胞内 ,用荧光显微镜同时测定心肌 [Ca2 ]i、pHi 和细胞长度。结果 :细胞内酸中毒早期 ,收缩期和舒张期[Ca2 ]i 轻度增加 ,细胞缩短 (CS)降低 ,细胞长度增加 ,心肌纤维对Ca2 的敏感性和CS/ [Ca2 ]i 降低 (P <0 .0 1) ;碱中毒时 ,收缩期和舒张期 [Ca2 ]i 均较对照组降低 ,CS增加 ,细胞长度变短 ,心肌纤维对Ca2 的敏感性和CS/[Ca2 ]i 增加 (P <0 .0 1)。结论 :酸中毒早期 [Ca2 ]i 和细胞长度增加 ,碱中毒时 [Ca2 ]i和细胞长度降低。酸、碱中毒对Ca2 敏感性的影响并非线性关系 ,即单位 pHi变化时酸中毒对敏感性的影响较碱中毒小     

胞内钙平衡在培养的牛主动脉内皮细胞低氧和复氧损伤中的作用

本文通过建立培养的牛主动脉内皮细胞体外低氧复氧模型,观察低氧和复氧时胞内钙浓度的改变与存活率的关系。结果显示,随低氧时间延长内皮细胞存活率下降,低氧后再复氧存活率进一步降低。低氧时无钙溶液孵育降低细胞的存活率,但复氧时无钙溶液孵育则增加细胞的存活率。低氧２ｈ胞内钙浓度从９９ｎｍｏｌ／Ｌ降对６９ｎｍｏｌ／Ｌ,无钙时胞内钙进一步降低,低氧４ｈ再复氧４０ｍｉｎ,胞内钙浓度恢复至正常。提示细胞内钙浓度平衡     

丙二醛破坏大鼠海马神经元胞质钙离子稳态的信号机制

目的研究丙二醛（MDA）对原代培养的海马神经元胞质中钙离子稳态的破坏作用及可能的信号机制。方法以Fur2／AM为荧光指示剂,采用荧光分光光度法定量测定原代培养海马神经元胞质游离钙浓度变化。结果随着MDA浓度的升高和作用时间的延长,导致胞质中游离钙水平显著升高,破坏其钙稳态。MDA所导致的海马神经元胞质游离钙水平升高包括两个过程：100μmol／L的MDA可使胞质[Ca2＋]i水平在0—10min内的早期渐进升高过程,经历中间大约5min的平台期后,接下来15—30min的晚期显著升高。以细胞膜电压依赖的Ca2＋通道抑制剂nimodipine抑制外钙内流后,可显著抑制晚期胞质[Ca2＋]i水平的升高,以PLC的抑制剂U73122作用后,则可抑制早期胞质[Ca2＋]i水平的升高。结论100μmol／L的MDA作用下,海马神经元胞质中早期钙离子水平的升高和晚期钙离子水平的升高可能分别由不同的信号机制所介导。     

低氧条件下大脑皮层微血管平滑肌细胞内皮细胞中[Ca~(2 )]_i变化的研究

目的和方法：本研究采用离子探针Ｆｕｒａ２／ＡＭ 结合计算机图象分析技术,并通过施加ＮＯ合酶抑制剂ＬＮＮＡ和ＮＯ的作用靶———鸟苷酸环化酶（ＧＣ）的抑制剂美兰（Ｍｅｔｈｙｌｅｎｅ Ｂｌｕｅ;ＭＢ）,观察经培养的大鼠大脑皮层微血管内皮细胞和平滑肌细胞中的［Ｃａ２＋］ｉ 在低氧作用后的变化以及与有关血管舒张因子ＮＯ和ｃＧＭＰ之间的关系。结果：低氧时大脑微血管内皮细胞和平滑肌细胞内的Ｃａ２＋ 浓度有所下降,变化幅度的大小与低氧的程度及低氧作用的时间有关,且可以被ＬＮＮＡ和ＭＢ所抑制。结论：低氧时大脑微血管的舒张反应与ＮＯ的产生有关,ＮＯ通过细胞内的多种机制,最终使得胞内Ｃａ２＋ 下降而导致血管舒张