细胞外Ca^2＋内流入胞质的机制

细胞外Ｃａ＾２＋主要是通过塌压依赖性Ｃａ＾２＋通道和钙池耗竭依赖性Ｃａ＾２＋通道而内流的。前者主要见于电兴奋细胞,这一过程比较清楚;后者主要见非兴奋细胞,情况远较复杂：外来信号激活内贮钙池,钙池在释放Ｃａ＾２＋同时通过目前尚不清楚的途径将直接或间接传至质膜Ｃａ＾２＋通道,而诱发Ｃａ＾２＋内流。     

小鼠卵激活过程中胞质游离Ca^2＋的变化及孤雌发育研究

乙醇和电刺激均可使小鼠ＭⅡ期卵母细胞激活并在体外孤雌发育至囊胚。小鼠卵对乙醇十分敏感。用７％－８％乙醇处理５ｍｉｎ后９５％以上的卵母细胞（卵龄为ＨＣＧ注射后１８－１９ｈ）内形成原核。３－４次电刺激后卵的激活率为６３．６３％。乙醇刺激可诱导卵内游离Ｃａ＾２＋浓度出现多次升高;单一电刺激仅能诱导卵内游离Ｃａ＾２＋浓度出现１次升高;多次电刺激可诱导卵内游离Ｃａ＾２＋浓度多次升高,而且电刺激次数与Ｃａ＾２     

小鼠卵受精和人工激活过程中胞质游离Ca^2＋变化及其在卵激活中的作用

休止于第二次成熟分裂中期（ＭＩ）的小鼠卵母细胞分别乙醇,钙离子载体Ａ２３１８７、电刺激或精子激活并用Ｃａ＾２＋特异荧光探针－Ｆｕｒａ２／ＡＭ测定细胞内游离Ｃａ＾２＋的变化。结果表明,受精诱导ＭⅡ卵内游离Ｃａ＾２＋浓度多次跃升（ｏｓｃｉｌｌａｔｉｏｎ）乙醇,钙离子载体及１次电刺激仅诱导胞内Ｃａ＾２＋１次升高,人工诱导激活的卵可象正常受精卵一样卵裂并发育至囊胚,用ＥＧＴＡ阻止受精和人工激活过程中卵内游     

介导心肌肥大的一条新的信号通路--Calcineurin通路

心肌肥大是心肌细胞对外界刺激,如工作负荷、神经体液因子及内在心肌蛋白遗传突变一种基本应答。已知胞内Ｃａ＾２＋浓度升高在各种刺激诱导心肌肥大的信号传递中起重要作用,但对Ｃａ＾２＋信号下游的传递机制一直不甚清楚。新近研究证实,由Ｃａ＾２＋活化的钙调神经磷酸酶（ＣａＮ）在心肌肥大的信号传递中起重要作用,基可能是Ｃａ＾２＋信号致肥大基因活化的偶联环节。抑制ＣａＮ活性可阻滞各种因素诱导的心肌肥大发生与发展,     

若干蝙蝠葛碱衍生物对人红细胞膜Ca^2＋—Mg^2＋——ATPase活力的影响

本文测定了数种蝙蝠葛碱衍生物对钙调素激活的人红细胞膜Ｃａ＾２＋－Ｍｇ＾２＋－ＡＴＰａｓｅ和的影响,结果表明,这些化合物对该酶都有不同程度的抑制作用,其机制表现为性抑制,过量的ＣａＭ能完全逆转这些化合物所引起的抑制。当Ｃａ＾２＋－Ｍｇ＾２＋－ＡＴＰａｓｅ被胰蛋白酶限制性酶解完全活化后,其活力不再受ＣａＭ激活,但仍被这些化合物所抑制。     

GM1激活环核苷酸磷酸二酯酶的作用

神经节苷脂ＧＭ１能激活ＣａＭ依赖性环核苷酸磷酸二酯酶,其ＡＣ为５０为１．５６μｇ／ｍｌ,这种作用并不一定依赖Ｃａ＾２＋的存在,在有Ｃａ＾２＋存在时,ＧＭ１对ＰＤＥ的最大激活活性低于ＣａＭ,仅为ＣａＭ的８０．４％;没有Ｃａ＾２＋存在时,ＧＭ１与ＣａＭ对ＰＤＥ有同样的最大激活活性。     

钙在植物乙烯生成及信号传递中的生理作用

Ｃａ＾２＋对植物乙烯生成的调节与作用位点有关,胞外Ｃａ＾２＋在维持质膜功能的同时,抑制乙烯生成,延缓衰老;过量Ｃａ＾２＋进入胞质,胞内Ｃａ＾２＋促进乙烯生成和衰老。内源ＣａＭ与乙烯生成关系密切,介入了乙烯代谢和外源激素对乙烯的调控。此外,Ｃａ＾２＋是乙烯信号传递所必需的。     

花生幼苗下胚轴质膜Ca2+-ATP酶及其对低温胁迫的反应

经６％－１２％ＤｅｘｔｒａｎＴ７０密度梯度离心,获得了纯度较高的７ｄ龄花生幼苗下胚轴质膜制剂,质膜Ｃａ＾２＋－ＡＴＰａｓｅ在反应系统不存在Ｍｇ＾２＋时,可正常表现水解ＡＴＰ的活性,但此活性明显低于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,Ｃａ＾２＋－ＡＴＰａｓｅ不受Ｎａ３ＶＯ４抑制,不被Ｋ＾＋激活,而被Ｃｌ＾－抑制,Ｃａ＾２＋－ＡＴＰａｓｅ的最适,ｐＨ不同于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,低温胁迫显著提高质膜Ｃ     

神经节苷脂（Gangliosides）对人红细胞膜Ca^2＋—Mg^2＋ATPase的影响

神经节苷脂（Ｇａｎｇｌｉｏｓｉｄｅｓ）是红细胞膜Ｃａ＾２＋－Ｍｇ２＋ＡＴＰａｓｅ的一种激活剂,这种激活作用也是依赖于Ｃａ＾２＋存在。在２００μｍｏｌ／Ｌ Ｃａ＾２＋存在的反应体系中,１００μｇ／ｍＬ Ｇａｎｇｌｉｏｓｉｄｅｓ对Ｃａ＾２＋－Ｍｇ＾２＋ＡＴＰａｓｅ的激活作用最大,为基本酶活性的１５０％以上。实验还发现ＣａＭ拮抗剂三氟嗪（ＴＥＰ）、粉防已碱（Ｔｅｔ）等也同样抑制Ｇａｎｇｌｉｏｓｉｄｅｓ的     

苹果果肉质膜微囊主动动输Ca^2＋的Ca^2＋—ATP酶特性

应用＾４５Ｃａ＾２＋示踪法研究了苹果果肉质膜微囊依赖于Ｃａ＾２＋的ＡＴＰ酶（Ｃａ＾２＋－ＡＴＰ酶）活性与Ｃａ＾２运输之间的关系及激素对该酶活性的影响。结果表明：Ｃａ＾２＋－ＡＴＰ酶存在于质膜上并受载体Ａ２３８７刺激而活性增加,该酶活性与依赖于ＡＴＰ的Ｃａ＾２＋运输依抑制剂ＥＢ,游离Ｃａ＾２＋和ＡＴＰ浓度的变化并呈极为相似的饱和动力学特征,而其ＥＢ半抑制浓度,Ｃａ＾２＋和ＡＴＰ半饱和浓度分别为０．１     

Local Ca2+ signals in cellular signalling

Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions.     

Calcium, mitochondria and apoptosis studied by fluorescence measurements

Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years. Evidence has been presented that calcium oscillations can effectively trigger apoptosis under certain conditions and that dysregulation of calcium signalling is a common cause of cell death. These effects are regularly mediated through calcium signal propagation to the mitochondria and the ensuing mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria to the cytoplasm. The progress in this area depended on the development of (1) fluorescent/luminescent probes, including fluorescent proteins that can be genetically targeted to different intracellular locations and (2) the digital imaging technology, fluorescence-activated cell sorting and fluorescent high throughput approaches, which allowed dynamic measurements of both [Ca2+] in the intracellular compartments of interest and the downstream processes. Fluorescence single cell imaging has been the only possible approach to resolve the cell-to-cell heterogeneity and the complex subcellular spatiotemporal organization of the cytoplasmic and mitochondrial calcium signals and downstream events. We outline here fluorometric and fluorescence imaging protocols that we set up for the study of calcium in the context of apoptosis.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Cytosolic calcium microdomains by arachidonic acid and nitric oxide in endothelial cells

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plasmamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors.     

Cholinergic signal transduction cascades in rat pinealocytes: functional and ontogenetic aspects.

In adult rat pinealocytes, acetylcholine activates nicotinic receptors whose stimulation causes a depolarization of the cells, opening of voltage-gated cation channels of the L-type and subsequent increase in the intracellular calcium ion concentration. These events trigger a release of glutamate that, by its action on metabotropic glutamate type 3 receptors, activates an inhibitory cyclic AMP cascade and suppresses norepinephrine-induced melatonin biosynthesis. The nicotinic response is fully developed in the third postnatal week. Prior to this timepoint, rat pinealocytes possess functional muscarinic receptors whose activation causes a rise in the intracellular calcium ion concentration through a calcium release from thapsigargin-sensitive intracellular calcium stores and an opening of store-operated calcium channels. This cascade may influence tissue differentiation and maturation of the melatonin pathway. The demonstration of functional cholinoreceptors and the ontogenetic switch from muscarinic to nicotinic signalling in rat pinealocytes supports the concept that pineal functions in mammals are influenced by neuronal inputs other than the sympathetic innervation which serves as the major regulatory system.     

Hydrogen peroxide signalling

Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis.     

Modulation of Elementary Calcium Release Mediates a Transition from Puffs to Waves in an IP3R Cluster Model

The oscillating concentration of intracellular calcium is one of the most important examples for collective dynamics in cell biology. Localized releases of calcium through clusters of inositol 1,4,5-trisphosphate receptor channels constitute elementary signals called calcium puffs. Coupling by diffusing calcium leads to global releases and waves, but the exact mechanism of inter-cluster coupling and triggering of waves is unknown. To elucidate the relation of puffs and waves, we here model a cluster of IP₃R channels using a gating scheme with variable non-equilibrium IP₃ binding. Hybrid stochastic and deterministic simulations show that puffs are not stereotyped events of constant duration but are sensitive to stimulation strength and residual calcium. For increasing IP₃ concentration, the release events become modulated at a timescale of minutes, with repetitive wave-like releases interspersed with several puffs. This modulation is consistent with experimental observations we present, including refractoriness and increase of puff frequency during the inter-wave interval. Our results suggest that waves are established by a random but time-modulated appearance of sustained release events, which have a high potential to trigger and synchronize activity throughout the cell.     

Cytochalasin D abolishes the schistosomicidal activity of praziquantel

To test the hypothesis that calcium channels of schistosomes are the targets for the action of praziquantel, we subjected schistosomes in vitro to pharmacological agents capable of interfering with the functioning of calcium channels. After 1-h exposure to these agents, praziquantel was added and incubation continued overnight. Worms were then washed, resuspended in drug-free medium and observed during the following 7-10 days. About 50% of schistosomes pre-exposed to the calcium channel blockers nicardipine and nifedipine were able to survive a praziquantel concentration (3 microM) that normally killed the majority of adult male worms. Since the organization of the actin cytoskeleton controls the activity of calcium channels in a number of different systems, we also pre-exposed schistosomes to the actin depolymerizing agent cytochalasin D. This treatment rendered the parasites completely refractory to the effects of very high praziquantel levels (up to 36 microM). These results are consistent with the hypothesis that schistosome calcium channels are involved in the mechanism of action of praziquantel.