卵细胞受精相关胞质钙离子波、钙离子震荡信号特征及其形成机制

胞质[Ca2 ]i震荡的动力学变化在哺乳动物早期胚胎发育中发挥重要作用。卵母细胞的成熟伴随间断的、快速的[Ca2 ]i震荡的时空表达;在受精过程中精子因子诱导的反复[Ca2 ]i震荡的振幅和持续时间是卵细胞最有效的激活信号,这种信号形成自然连续的受精[Ca2 ]i波,并以长时持续[Ca2 ]i震荡形式在受精卵空间传递并持续数小时,直至受精完成;受精卵内源性的Ca2 释放所引起的[Ca2 ]i震荡形成第一次卵裂信号,启动早期胚胎的发育。精子PLCζ和cPKCs是形成受精卵[Ca2 ]波、[Ca2 ]震荡的重要因素。     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.     

谷氨酸促进大鼠海马神经元的内钙升高

谷氨酸能影响大鼠海马神经元胞内钙信号的变化,进而影响海马神经元神经冲动的发放和学习记忆过程。运用荧光测钙技术实时监测了大鼠海马神经元内钙信号的动态变化,同时分析了谷氨酸对其胞内钙信号的影响。试验表明：谷氨酸能够显著提高胞内游离钙离子的浓度;细胞外钙离子的存在、谷氨酸刺激时间及刺激频率的增加都能引起胞内钙信号不同程度的升高;但谷氨酸的过度刺激会引起钙离子浓度的超负荷,从而导致神经元结构和功能的损坏。     

An insulin-sensitive cation channel controls [Na+]i via [Ca2+]o-regulated Na+ and Ca2+ entry.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.     

Depolarization decreases the [Ca2+]i sensitivity of myosin light-chain kinase in arterial smooth muscle: comparison of aequorin and fura 2 [Ca2+]i estimates

Histamine stimulation of swine arterial smooth muscle is associated with a high [Ca2+]i sensitivity for increases in myosin light-chain phosphorylation. In contrast, KCl depolarization produces a relatively lower [Ca2+]i sensitivity (i.e., similar increases in [Ca2+]i induce less myosin phosphorylation). We evaluated whether 1) artifacts in the methodology for measuring [Ca2+]i or 2) true alterations in the [Ca2+]i sensitivity of myosin light-chain kinase were responsible for these apparent changes in the [Ca2+]i sensitivity of phosphorylation. The [Ca2+]i sensitivity of phosphorylation was higher with histamine stimulation regardless of whether the [Ca2+]i indicator was aequorin (which was loaded intracellularly by reversible hyperpermeabilization) or Fura 2 (which was loaded intracellularly by incubation of the tissues in Fura 2 AM). Aequorin and Fura 2 appeared to detect qualitatively similar stimulus-induced changes in [Ca2+]i with the exception that the initial response to histamine stimulation was different (histamine initially induced a large aequorin light transient and a relatively smaller increase in Fura 2 fluorescence). The [Ca2+]i sensitivity of myosin light-chain kinase extracted from KCl depolarized tissues was lower than the [Ca2+]i sensitivity of myosin light-chain kinase extracted from unstimulated or histamine stimulated tissues. These results suggest that depolarization specifically modifies myosin light-chain kinase to decrease its [Ca2+]i sensitivity. Changes in the [Ca2+]i sensitivity of myosin light-chain phosphorylation are not an artifact of the [Ca2+]i measurement technique.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Interplay between Ca2+ release and Ca2+ influx underlies localized hyperpolarization-induced [Ca2+]i waves in prostatic cells.

Calcium seems to be a major second messenger involved in the regulation of prostatic cell functions, but the mechanisms underlying its control are poorly understood. We investigated spatiotemporal aspects of Ca2+ signals in the LNCaP cell line, a model of androgen-dependent prostatic cells, by using non-invasive external electric field pulses that hyperpolarize the anode facing membrane and depolarize the membrane facing the cathode. Using high-speed fluo-3 confocal imaging, we found that an electric field pulse (10-15 V/cm, 1-5 mA, 5 ms) initiated rapidly, at the hyperpolarized end of the cell, a propagated [Ca2+]i wave which spread through the cell with a constant amplitude and an average velocity of about 20 microns/s. As evidenced by the total wave inhibition either by the block of Ca2+ entry or the depletion of Ca2+ stores by thapsigargin, a specific Ca(2+)-ATPase inhibitor, the [Ca2+]i wave initiation may imply a localized Ca2+ influx linked to a focal auto-regenerative process of Ca2+ release. Using different external Ca2+ and Ca2+ entry blockers concentrations, Mn2+ quenching of fluo-3 and fura-2 fluorescence and inhibitors of InsP3 production, we found evidence that the [Ca2+]i wave progression required, in the presence of basal levels of InsP3, an interplay between Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ influx through channels possibly activated by the [Ca2+]i rise.     

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.     

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca²⁺ level ([Ca²⁺]_i) and viability in human glioblastoma cells.Main methodsThe Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca²⁺]_i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive, non store-operated Ca²⁺ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.     

2-hydroxyoleic acid affects cardiomyocyte [Ca2+]i transient and contractility in a region-dependent manner

Monounsaturated fatty acids such as oleic acid are cardioprotective, modify the physicochemical properties of cardiomyocyte membranes, and affect the electrical stability of these cells by regulating the conductance of ion channels. We have designed a nonhydrolysable oleic acid derivative, 2-hydroxyoleic acid (2-OHOA), which regulates membrane lipid structure and cell signaling, resulting in beneficial cardiovascular effects. We previously demonstrated that 2-OHOA induces PKA activation and PKCalpha translocation to the membrane; both pathways are thought to regulate transient outward K(+) current (I(to)) depending on the stimulus and the species used. This study was designed to investigate the effect of 2-OHOA on isolated cardiomyocytes. We examined the dose- and time-dependent effect of 2-OHOA on cytosolic Ca(2+) concentration ([Ca(2+)](i)) transient and contraction of myocytes isolated from different parts of the rat ventricular myocardium. Although this drug had no effect on [Ca(2+)](i) transient and cell shortening in myocytes isolated from the septum, it increased (up to 95%) [Ca(2+)](i) transient and cell shortening in subpopulations of myocytes from the right and left ventricles. The pattern of the effects of 2-OHOA was similar to that observed following the application of the I(to) blocker 4-aminopyridine, suggesting that the drug may act on this channel. Unlike the effect of 2-OHOA on [Ca(2+)](i) transient and cell shortening, PKCalpha translocation to membranes was not region specific. Thus 2-OHOA-induced effects on [Ca(2+)](i) transients and cell shortening are likely related to reductions in I(to) function, but PKCalpha translocation does not seem to play a role. The present results indicate that 2-OHOA selectively increases myocyte inotropic responsiveness, which could underlie its beneficial cardiovascular effects.     

钙离子与缺氧性神经干细胞凋亡的相关性研究

目的检测钙离子浓度在缺氧性神经干细胞凋亡过程中的变化,以探讨缺氧性神经干细胞凋亡的发生机制。方法从大鼠胚胎神经管获取神经干细胞,经无血清悬浮培养技术获得神经球。对所获神经球行干细胞克隆试验、Br-dU掺入标记试验、nestin、NSE和GFAP免疫荧光染色,以确认神经干细胞的生物学特性。三气培养箱予以缺氧干预,按缺氧程度分为5%O2组、10%O2组和正常对照组(20%O2),每个实验组又依缺氧干预时间的不同,分为24h、48h、72h、96h、120h5个亚组。用激光扫描共聚焦显微镜和Fluo-3荧光探针标记技术检测神经干细胞内钙离子浓度;采用An-nexinⅤ-FITC/PI检测细胞凋亡率。结果5%O2120h组和10%O2120h组中神经干细胞凋亡率显著高于正常对照组和其他缺氧干预组,并且伴随有胞内钙超载。结论细胞内钙超载可能是缺氧性神经干细胞凋亡机制中的一个重要环节。     

Cysteinyl leukotriene-dependent [Ca2+]i responses to angiotensin II in cardiomyocytes

With the use of fura 2 measurements in multiple and single cells, we examined whether cysteinyl leukotrienes (CysLT) mediate angiotensin II (ANG II)-evoked increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in neonatal rat cardiomyocytes. ANG II-evoked CysLT release peaked at 1 min. The angiotensin type 1 (AT(1)) antagonist losartan, but not the AT(2) antagonist PD-123319, attenuated the elevations in [Ca(2+)](i) and CysLT levels evoked by ANG II. Vasopressin and endothelin-1 increased [Ca(2+)](i) but not CysLT levels. The 5-lipoxygenase (5-LO) inhibitor AA-861 and the CysLT(1)-selective antagonist MK-571 reduced the maximal [Ca(2+)](i) responses to ANG II but not to vasopressin and endothelin-1. While MK-571 reduced the responses to leukotriene D(4) (LTD(4)), the dual CysLT antagonist BAY-u9773 completely blocked the [Ca(2+)](i) elevation to both LTD(4) and LTC(4). These data confirm that ANG II-evoked increases, but not vasopressin- and endothelin-1-evoked increases, in [Ca(2+)](i) involve generation of the 5-lipoxygenase metabolite CysLT. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] antagonist 2-aminoethoxydiphenyl borate attenuated the [Ca(2+)](i) responses to ANG II and LTD(4). Thus AT(1) receptor activation by ANG II is linked to CysLT-mediated Ca(2+) release from Ins(1,4,5)P(3)-sensitive intracellular stores to augment direct ANG II-evoked Ca(2+) mobilization in rat cardiomyocytes.