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     

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     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通     

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．     

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。     

衰老性记忆障碍与脑突触体内游离[Ca^2＋]i的相关性

采用行为观察和生化检测相结合的方法,在过去工作的基础上,研究了１２月龄和１８月龄小鼠学习记忆能力的变化和１８月龄小鼠四个脑区（海马、大脑皮层、四叠体和小脑）交触体内「Ｃａ＾２＋」ｉ的水平,同时还比较了老年记忆保持良好组与记忆障碍组小鼠的脑钙水平。结果表明,随着年龄的增长,小鼠的学习记忆能力显著下降,上述脑区（除大脑皮层外）突触内「Ｃａ＾２＋」ｉ均明显升高,其中老年记忆障碍小鼠脑钙水平升高最为显著。     

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     

压力超负荷心肌肥大晚期和心衰大鼠心肌力学及Ca^2＋反？…

目的：观测比较肥大心肌和衰竭心肌在基础力学性能、「Ｃａ＾２＋」。正性变力作用和Ｃａ＾２＋通道阻滞剂负性变力作用三方面的变化和差别。方法：大鼠升主动脉缩窄法建立左心室肥大（ＬＶＨ）和充血性心衰（ＣＨＦ）模型,记录离体灌流乳头肌的和长度－张力曲线,比较基础状态和ｎｉｒｉｄｉｐｉｎｅ处理后「Ｃａ＾２＋」。与峰张力的量效关系。结果：①ＣＨＦ组长度－张力曲线料ＬＶＨ组和假手术对照组（Ｓｈａｍ）明显下移;基础     

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     

ACTH4-9类似物对小鼠海马突触体Ca2+水平的调节作用

本工作观察了Ｏｒｇ２７６６（ＡＣＴＨ４－９的类似物）对海马突触体内游离Ｃａ＾２＋水平及对＾４５Ｃａ＾２＋摄取的影响。采用细胞内钙离子荧光探针Ｆｕｒａ－２,通过Ｓｐｅｘ－阳离子测定系统检测突触体内［Ｃａ＾２＋］ｉ的动态变化,并用液闪光谱仪测定突触体对＾４５Ｃａ＾２＋的摄取。结果表明：较低剂量的ＯＲＧ２７６６对突触体内［Ｃａ＾２＋］ｉ的影响不明显,却显著降低突触体对＾４５Ｃａ＾２＋的摄取;高剂量的Ｏｒ     

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     

衰老性记忆障碍与脑突触体内游离[Ca~(2 )]_i的相关性

采用行为观察和生化检测相结合的方法 ,在过去工作的基础上 ,研究了 12月龄和 18月龄小鼠学习记忆能力的变化和 18月龄小鼠四个脑区 (海马、大脑皮层、四叠体和小脑 )突触体内 [Ca2 ]i 的水平 ,同时还比较了老年记忆保持良好组与记忆障碍组小鼠的脑钙水平。结果表明 ,随着年龄的增长 ,小鼠的学习记忆能力显著下降 ,上述脑区 (除大脑皮层外 )突触内 [Ca2 ]i 均明显升高 ,其中老年记忆障碍小鼠脑钙水平升高最为显著。提示 ,小鼠衰老性记忆障碍可能与其脑突触体内 [Ca2 ]i 的超载有关。     

保卫细胞钙信号的研究进展

钙(Ca^2 )是多种信号途径的第二信使。Ca^2 成像技术的成熟和发展为显示保卫细胞胞质Ca^2 浓度([Ca^2 ]cyt)的分布及外界刺激引起[Ca^2 ]cyt的变化模式提供了很好的研究工具,关于细胞内外Ca^2 库释放Ca^2 的机制也有了较清楚的认识。拟南芥突变体的研究使Ca^2 信号上游分子及其排序更加明确,[Ca^2 ]cyt增加下游的磷酸化和去磷酸化过程也是气孔关闭必需的生理过程。     

Evaluation of the Ca2+ Concentration in Purified Nerve Terminals: Relationship Between Ca2+ Homeostasis and Synaptosomal Preparation

The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin-2 and fura-2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 +/- 2 nM for Percoll (C-band) synaptosomes, loaded at 30 degrees C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37 degrees C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30 degrees or 37 degrees C). Loading quin-2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin-2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura-2 is preferable in synaptosomes, although above a critical fura-2-AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura-2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37 degrees C. This study shows the applicability of measuring steady-state [Ca]i and dynamic [Ca]i changes quantitatively in fura-2-loaded synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

Sigma-1 receptor as regulator of neuronal intracellular Ca2+: clinical and therapeutic relevance

Preserving brain function and cognitive faculties during aging and psychiatric diseases (e.g. psychotic, anxiety and affective disorders, dementia) is essential for the self-reliance and quality of life of patients. Cognitive loss involves not only memory, but also motor function. The decrease of catecholaminergic and excitatory neurotransmissions, as well as of protein phosphorylation, have currently been identified as prominent biological markers of the above-mentioned diseases. Such deleterious biological events are well known to occur downstream of a progressive decline of intracellular Ca2+ signalling. This latter constitutes a key target for the neuronal plasticity that has also been reported during aging and psychiatric disorders. Most of the medicines used in psychiatry are active on the sigma-1 receptor. This membrane bound receptor is widely distributed in memory-associated cortical and motor-related brainstem areas, prompting the hypothesis that it might contribute to the pathophysiology of these behavioural brain diseases. The sigma-1 receptor is characterized by a unique mode of action by regulating both Ca2+ entry at the plasma membrane level (i.e. via potassium channels, voltage-sensitive Ca2+ channels) and Ca2+ mobilization from endoplasmic stores [i.e. via Ins(1,4,5)P3 receptors]. This review presents recent data supporting the notion that drugs acting via the endoplasmic reticulum-coupled sigma-1 receptor might reverse these deleterious events by restoring both extra- and intra-cellular Ca(2+)-dependent neuronal responses.     

Expansion of the calcium hypothesis of brain aging and Alzheimer's disease: minding the store

Evidence accumulated over more than two decades has implicated Ca2+ dysregulation in brain aging and Alzheimer's disease (AD), giving rise to the Ca2+ hypothesis of brain aging and dementia. Electrophysiological, imaging, and behavioral studies in hippocampal or cortical neurons of rodents and rabbits have revealed aging-related increases in the slow afterhyperpolarization, Ca2+ spikes and currents, Ca2+transients, and L-type voltage-gated Ca2+ channel (L-VGCC) activity. Several of these changes have been associated with age-related deficits in learning or memory. Consequently, one version of the Ca2+ hypothesis has been that increased L-VGCC activity drives many of the other Ca2+-related biomarkers of hippocampal aging. In addition, other studies have reported aging- or AD model-related alterations in Ca2+ release from ryanodine receptors (RyR) on intracellular stores. The Ca2+-sensitive RyR channels amplify plasmalemmal Ca2+ influx by the mechanism of Ca2+-induced Ca2+ release (CICR). Considerable evidence indicates that a preferred functional link is present between L-VGCCs and RyRs which operate in series in heart and some brain cells. Here, we review studies implicating RyRs in altered Ca+ regulation in cell toxicity, aging, and AD. A recent study from our laboratory showed that increased CICR plays a necessary role in the emergence of Ca2+-related biomarkers of aging. Consequently, we propose an expanded L-VGCC/Ca2+ hypothesis, in which aging/pathological changes occur in both L-type Ca2+ channels and RyRs, and interact to abnormally amplify Ca2+ transients. In turn, the increased transients result in dysregulation of multiple Ca2+-dependent processes and, through somewhat different pathways, in accelerated functional decline during aging and AD.     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

本文对fMLP诱导的嗜中性白细胞胞内钙浓度变化与凋亡的关系进行了研究。用膜受体激动剂fMLP和钙离子载体A23187诱导细胞内钙浓度升高,BAPTA螯合胞质钙。运用荧光显微镜,流式细胞仪,电泳等方法对培养细胞的凋亡百分率及细胞骨架变化进行了研究。结果表明:fMLP和A23187均有效地抑制了凋亡,而BAPTA促进了凋亡。对骨架测定表明随细胞凋亡微丝解聚明显,胞内钙升高抑制骨架解聚,胞内钙降低促进其解聚。故嗜中性白细胞凋亡过程中伴随有微丝的解聚,胞内钙浓度升高时凋亡被有效抑制,胞内钙浓度降低时促进了凋亡。     

适当浓度的过氧化氢诱导人支气管上皮细胞发生钙振荡

包括过氧化氢（Hzoz）在内的活性氧通过引起细胞内钙的变化而造成细胞损伤。然而,不同浓度的H202可以导致细胞内不同的钙变化,并激活不同的信号通路。细胞内钙振荡是其中的一种钙信号变化形式,钙振荡可以调控转录因子NF—KB的活性。该研究探讨可以诱导支气管上皮细胞内钙振苏发生的H2o2浓度。体外培养人支气管上皮细胞,采取钙离子荧光探针Fura_2标记细胞。并使用离子成像系统,观测不同浓度的H：0：（0～1000μmol／L）作用下细胞内钙浓度的变化。结果发现,低于50μmol／L的H202仅仅引起“钙火花”;50～500μmol／L的H202导致细胞内钙振荡的发生;而1000μmol／L的H202引起细胞内持续的高钙;同时也证实150μmol／L的H202诱发明显的钙振荡,而钙振荡随后引起了NF—KB活性的升高。该研究提示,适当浓度的H：0：可以诱发支气管上皮细胞内钙振荡的发生,推测可能是活性氧导致慢性气道炎症损伤的一个机制。