U-73122 inhibits carbachol-induced increases in [Ca2+]i,IP3, and insulin release in β-TC3 cells

We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca²⁺]_i, and IP₃ in β-TC3 cells. Carbachol (0.1–100 μM) increased [Ca²⁺]_i and carbachol (0.1–1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP₃) production. U-73122 (2–12 νM) inhibited the effects of carbachol on [Ca²⁺]_i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca²⁺]_i by 20% and did not inhibit the effect of carbachol on insulin release. Since carbachol increased IP₃, [Ca²⁺]_i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-depenent processes in β-TC3 cells.     

定量研究菲啶对酵母朊病毒[PSI+]的治愈作用

为系统研究菲啶对酵母朊病毒的治愈效果,借助表达融合蛋白GFP-Sup35p的酵母朊病毒模型(NGMC),引入半变性琼脂糖凝胶电泳技术和荧光漂白后恢复技术在蛋白和细胞水平定量分析了菲啶对酵母朊病毒的治愈效果。结果表明,蛋白和细胞水平采用的定量分析方法能够精确定量菲啶对酵母朊病毒的治愈作用,菲啶作用酵母朊病毒[PSI+]1~5 d的治愈率分别为0%、0%、51.7%、87.5%和94.4%。另外,菲啶作用酵母朊病毒[PSI+]细胞1~2 d后出现的粉色菌落中朊病毒的聚集状态与[PSI+]相似,而3~5 d后出现的粉色菌落中朊病毒的状态与[psi-]相似。     

NaF、NaBr、NaI对酵母朊病毒[PSI~+]形成的影响

为研究NaF、NaBr、NaI对酵母朊病毒[PSI+]形成的影响,利用表达融合蛋白GFP-Sup35p酵母朊病毒模型,借助半变性琼脂糖凝胶电泳技术,结合免疫印迹方法在蛋白水平上定量分析NaF、NaBr、NaI对酵母朊病毒[PSI+]形成的影响。结果显示,在细胞表型方面NaF、NaBr诱导出酵母朊病毒[PSI+],且所需浓度分别为0.02、1.0 mol/L;NaI在浓度为0.25 mol/L可以诱导出[PSI+]经盐酸胍治愈后的酵母朊病毒[psi-]。在蛋白水平,NaF、NaBr、NaI盐作用后的[psi-]细胞内Sup35p并没有形成聚集体。     

光电响应对紫膜外表面[k~+]依赖性与质子泵效率相关

本文研究发现,不锈钢／电沉积紫膜薄膜／含水胶（电介质）／铜型菌紫质光电池的光电响应对紫膜外表面的钾离子浓度的依赖性与质子泵效率的这种依赖性成相关性。这种相关性说明：１．膜中菌紫质的光电响应主要来自质子泵运而非钾离子直接的贡献;２．存在一合适的离子浓度使菌紫质的光电响应有极大值;３．作用在外表面的离子主要影响质子释放结构域     

大豆黄酮对衰老小鼠不同脑区突触内[Ca2+]i的影响

目的: 检测大豆黄酮对衰老小鼠不同脑区突触内[Ca2 ]i影响.方法: 灌喂衰老模型小鼠大豆黄酮,利用荧光指示剂Fura-2/AM测定[Ca2 ]i.结果: 大豆黄酮使衰老小鼠大脑皮质、海马、间脑突触内游离[Ca2 ]i分别下降0.18倍(P<0.05)、0.34倍(P<0.01)、0.21倍(P<0.05).结论: 大豆黄酮可部分抑制由于突触[Ca2 ]i代谢失调引发的脑老化.     

Zn[2+]对中华绒螯蟹zao状幼体肝胰腺超微结构的影响

研究了不同Zn[2+]浓度对中华绒螯蟹(Eriocheir sinensis)zao状幼体肝胰腺细胞超微结构的影响。当Z[2+]浓度超过200 μg/L时，与对照组相比，肝胰腺结构受到了显著的影响。成熟和正在形成的B细胞的空泡中有很多含有金属的电子致密颗粒(EDG，可能为金属蛋白复合体)，随着B细胞的成熟和B细胞从肝胰腺管壁上脱落，这些EDG也被释放到管腔中，因此肝胰腺的管腔中常有许多此类颗粒存在。B细胞空泡或管腔中的EDG与环境中Zn[2+]浓度高低呈正相关。E细胞质中也出现了很多空泡，与正常E细胞的结构明显不同。R细胞的细胞质常解体形成大的空泡，被破坏的程度较为严重。在Zn[2+]浓度>1000 μg/L时，肝胰腺遭到严重破坏，细胞结构几乎不存在。研究结果表明，肝胰腺的B细胞在Zn[2+]的解毒方面起重要作用，但当Zn[2+]浓度超过了肝胰腺B细胞的解毒能力时，就会引起肝胰腺细胞(如R和E细胞)结构异常，甚至破坏整个肝胰腺细胞结构，从而影响幼体的生长，甚至造成死亡。     

β-Adrenergic-agonist stimulated taurine release from astroglial cells is modulated by extracellular [K+] and osmolarity

Astroglial cells are known to release taurine in response to stimulation by a variety of stimuli including -adrenergic receptor agonists such as isoproterenol (IPR). The effects of changing osmolarity and extracellular [K⁺] on IPR-stimulated taurine release were studied with LRM55 cells, a continuous astroglial cell line. IPR-stimulated taurine release decreased almost 8% for each 1% increase in osmolarity, indicating that IPR-stimulated release is highly regulated by the osmolarity of the medium. IPR-stimulated taurine release was greatly enhanced when external [K⁺] was increased isosmotically by substituting KCl for NaCl but was strongly suppressed when external [K⁺] was increased hyperosmotically by adding KCl to the medium. Both IPR-stimulated and K⁺-stimulated taurine release depended on external [Cl^–]; IPR-stimulated release declined approximately in parallel to K⁺-stimulated release as [Cl^–] in the medium was reduced. The high sensitivity of IPR-stimulated release to factors that change cell volume (osmolarity, external [K⁺], external [Cl^–]) is consistent with the idea that IPR, elevated [K⁺], and reduced osmolarity all elicit taurine release via a single tension-controlled mechanism.Special issue dedicated to Dr. Claude Baxter.     

小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca2+]cyt的关系

以小麦叶肉细胞原生质体为材料,通过免疫荧光标记和Ca~(2 )荧光染料的装载并结合药物学试验,借助激光共聚焦扫描显微镜观察,探讨微管骨架和Ca~(2 )之间的内在联系。试验结果表明,[Ca~(2 )]_(cyt)的升高能够诱发微管骨架的解聚;而微管骨架的解聚也会促使胞外Ca~(2 )内流,进而造成[Ca~(2 )]_(cyt)的升高。     

激发子诱发小麦叶肉细胞原生质体微管骨架排列格局与[Ca2+]cyt的变化

以小麦叶肉细胞原生质体-激发子互作为研究体系,通过免疫荧光标记和Ca~(2 )荧光染料的装载并结合药理学试验,借助激光共聚焦扫描显微镜观察,探讨小麦抵抗叶锈菌侵染过程中微管骨架和Ca~(2 )之间的内在联系。试验结果表明,激发子处理可引起抗性品种原生质体[Ca~(2 )]_(cyt)的升高并诱发微管骨架的解聚,预解聚微管骨架,再用激发子处理,可使抗性品种原生质体[Ca~(2 )]_(cyt)的升高幅度增加。     

激发子诱发的小麦叶肉细胞原生质体[Ca2+]cyt升高主要源于胞外钙离子内流

以小麦叶肉细胞原生质体-激发子互作为研究体系,借助共聚焦激光扫描显微镜观察结合药物学试验,对激发子刺激后不同抗叶锈性小麦品种原生质体[Ca2+]cyt的动态变化和[Ca2+]cyt升高的钙来源进行了研究。结果表明：抗叶锈小麦品种‘洛夫林10’的原生质体在激发子处理后,[Ca2+]cyt明显升高,随后有所下降,但在试验检测时间范围内仍保持较高浓度水平;而感病品种‘郑州5389’经激发子处理后,[Ca2+]cyt只发生轻微的波动。使用质膜钙通道抑制剂抑制胞外钙离子流入胞内,再经激发子处理,原生质体[Ca2+]cyt虽也有升高,但升高幅度大大降低。这一结果表明,激发子刺激诱发的[Ca2+]cyt升高主要源于胞外钙离子内流。     

Mechanism of [Ca2+]i rise induced by angiotensin 1–7 in MDCK renal tubular cells

The effect of angiotensin 1–7 (Ang 1–7) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Ang 1–7 at concentrations of 10–50 µM induced a [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Ang 1–7 evoked store operated Ca²⁺ entry that was inhibited by La³⁺ and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin prevented Ang 1–7 from releasing more Ca²⁺. Inhibition of phospholipase C with U73122 abolished Ang 1–7-induced [Ca²⁺]_i rise. Ang 1–7-induced [Ca²⁺]_i rise was abolished by the angiotensin type 1 receptor antagonist losartan, but was not affected by the angiotensin type 2 receptor antagonist PD 123,319. In sum, in MDCK cells, Ang 1–7 stimulated angiotensin type 1 receptors leading to a [Ca²⁺]_i rise that was composed of phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A2-sensitive store-operated Ca²⁺ channels.     

Determination of [Mg2+]i – an update on the use of Mg2+-selective electrodes

Since their invention, ion-selective microelectrodes have become an indispensable tool for investigations of intracellular ion regulation and transport. While highly selective sensors for all major intracellular monovalent ions have been available for decades, the development of sensors for divalent cations seems to have presented more difficulties. As ion-selective microelectrodes typically have time-constants in the range of 0.5 to several seconds they turned out to be inapt for the investigation of intracellular Ca²⁺. The development of sensors for Mg²⁺-selective electrodes has made its most striking progress only over the past few years. While the first Mg²⁺ sensor, ETH 1117, was barely able to detect physiological Mg²⁺ concentrations in the presence of other mono- and divalent cations, the newest sensors allow measurements in the micromolar range. When used in macroelectrodes, the most recent developments, ETH 5506 and ETH 5504, have even been reported to do so in the presence of millimolar Ca²⁺ concentrations. Although there is still room for improvement to make these sensors applicable in microelectrodes, some preliminary data look extremely promising and indicate that a new era for Mg²⁺-selective microelectrodes is about to start.     

超氧阴离子通过提高[Ca2+]cyt调节保卫细胞K+通道和气孔运动

氧化信号参与了许多生理过程的调控。用膜片钳和激光共聚焦显微镜,采用可以产生O2^ 的甲基紫精处理蚕豆(Vicia faba L)保卫细胞,测定了O2^ 对气孔运动调节过程中胞质Ca^2 离子浓度和细胞质膜K^ 通道活性的变化,结果表明甲基紫精可以促进气孔的关闭,乙二醇四乙酸酯(Ethylene glycol bis(2-aminoethyl)tetra-acetic acid,EGTA)、抗坏血酸(Ascorbic acid,AsA)和过氧化物酶(Catalase,CAT)可以消除小于10^-5mol／L甲基紫精对气孔运动的影响;10^-2和10^-5mol/L的甲基紫精可使保卫细胞胞质Ca^2 浓度有不同程度提高,并伴随有钙震荡。蚕豆气孔保卫细胞质膜内向K^ 通道可被咆外甲基紫精抑制,而这种抑制和[Ca^2 ]cyt有关。推测甲基紫精产生的O2^-对蚕豆气孔运动的调节,主要是通过O2^ 诱导的胞内游离Ca^2 浓度的升高,从而抑制了通过保卫细胞质膜K^ 内向电流。     

EFFECTS OF ELEVATED [K+]0 ON THE RELEASE OF NEUROTRANSMITTERS FROM CORTICAL SYNAPTOSOMES: EFFLUX OR SECRETION?

Abstract— Elevated K⁺₀ elicited a substantial Ca-independent efflux of accumulated GABA from cortical synaptosomal fractions. Efflux from tissue labelled with either NE or choline was affected considerably less by elevated K⁺ pulses in the absence of calcium. K-facilitated Ca-dependent efflux was large for all three of the accumulated substances. K-dependent (Ca-independent) efflux of accumulated GABA was associated with all subcellular fractions exhibiting GABA accumulation whereas K-facilitated Ca-dependent efflux was restricted to fractions containing synaptosomes. Eighty per cent of both GABA accumulation and K-dependent efflux was, however, recovered in a purified synaptosomal fraction. Alanine slightly decreased GABA accumulation, but % K-dependent efflux was not affected.
Elevated K⁺, in the absence of calcium, released GABA from accumulated pools in preference to endogenous pools, whereas the Ca-dependent efflux, facilitated by K⁺, was similar for both accumulated and endogenous GABA.
The Ca-independent efflux of accumulated GABA increased linearly with log [K⁺]₀ between 10 and 70 mM-K⁺ in sodium-containing media. Prior treatment with veratridine or Na-free medium substantially decreased the Ca-independent but not the Ca-dependent GABA efflux produced by elevated K⁺ pulses.
The Ca-dependent and Ca-independent efflux of accumulated GABA in response to elevated K⁺ pulses is suggested to arise not only via different flux mechanisms but also from different GABA pools. The Ca-dependent efflux is interpreted to reflect stimulus-secretion coupling processes whereas the Ca-independent efflux may reflect membrane transport processes.     

Single-cell screening of cytosolic [Ca2+] reveals cell-selective action by the Alzheimer’s Aβ peptide ion channel

Interaction of the Alzheimer’s Aβ peptides with the plasma membrane of cells in culture results in chronic increases in cytosolic [Ca²⁺]. Such increases can cause a variety of secondary effects leading to impaired cell growth or cell degeneration. In this investigation, we made a comprehensive study of the changes in cytosolic [Ca²⁺] in single PC12 cells and human neurons stressed by continuous exposure to a medium containing Aβ42 for several days. The differential timing and magnitude of the Aβ42-induced increase in [Ca²⁺] reveal subpopulations of cells with differential sensitivity to Aβ42. These results suggest that the effect produced by Aβ on the level of cytosolic [Ca²⁺] depends on the type of cell being monitored. Moreover, the results obtained of using potent inhibitors of Aβ cation channels such as Zn²⁺ and the small peptide NA7 add further proof to the suggestion that the long-term increases in cytosolic [Ca²⁺] in cells stressed by continuous exposure to Aβ is the result of Aβ ion channel activity.     

血管性痴呆小鼠海马神经细胞静息态[Ca2+]i及CaMmRNA、CaMPKⅡmRNA表达的变化

目的:观察血管性痴呆小鼠海马神经细胞内静息态游离Ca2 浓度([Ca2 ]i)和钙调素(CaM)、CaM依赖性蛋白激酶Ⅱ(CaMPKⅡ)mRNA表达水平的变化,探讨上述因素在血管性痴呆发病中的作用.方法:采用双侧颈总动脉线结、缺血/再灌注的方法,制备小鼠血管性痴呆模型,并设假手术组作为对照.分别于术后第29 d、第30 d进行学习、记忆成绩测试,然后快速取海马制备海马活细胞,以Fluo-3/AM为荧光探针,在激光扫描共聚焦显微镜下观察两组海马神经细胞静息态[Ca2 ]i变化,并应用RT-PCR技术检测海马神经细胞内CaMmRNA、CaMPKⅡmRNA的表达水平.结果:①模型组的学习和记忆成绩均低于假手术组(P＜0.05);②模型组神经细胞静息态[Ca2 ]i显著高于假手术组(P＜0.05);而CaMmRNA、CaMPKⅡmRNA表达水平低于假手术组,具有极其显著性差异(P＜0.01).结论:海马神经细胞静息态[Ca2 ]i增高、CaMmRNA和CaMPKⅡmRNA表达减少是导致小鼠血管性痴呆发生的机制之一.