三羟异黄酮对豚鼠心室肌细胞内游离钙浓度的影响

用激光共聚焦显微镜观察研究三羟异黄酮(genistein,GST)对豚鼠心室肌细胞内游离钙浓度([Ca^2 ]i)的影响。结果用相对荧光强度(FI-F0／FX0,％)表示。实验结果显示,在正常台氏液、无钙台氏液和正常台氏液中加入3mmol／L EGTA后,GST(10～40μmol／L)浓度依赖性地降低细胞内钙浓度。蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate)和L-型Ca^2 通道激动剂Bay K8644可部分抑制正常台氏液时GST的效应。当细胞外液钙浓度由1mmol／L增加到10mmol／L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,GST(40μmol／L)可降低钙波的传播速度和持续时间,最终阻断钙波。以上结果提示,GST降低心室肌细胞内游离钙浓度,此作用与其抑制电压依赖性Ca^2 通道、减弱酪氨酸激酶抑制和豚鼠心室肌细胞肌浆网内钙释放有关。     

代谢型谷氨酸受体激动引起星形神经胶质细胞肿胀

用[~3H]-3-氧-甲基-D-葡萄糖摄取的方法测定细胞水含量,观察谷氨酸受体激动剂、拮抗剂对培养的星形神经胶质细胞水含量的影响,并观察细胞内、外钙的作用.结果发现:0.5,1,10 mmol/L的谷氨酸和1mmol/L的trans-ACPD(代谢型谷氨酸受体激动剂)1h均可以引起细胞的水含量增加,1mmol/L的 AMPA(离子型谷氨酸受体激动剂)不影响细胞的水含量,1mmol/L的L-AP3(代谢型谷氨酸受体拮抗剂)可以拮抗1mmol/L谷氨酸和trans-ACPD的作用;撤除细胞外液的钙,谷氨酸不再引起细胞的水含量增加, 20 μmol/ L 的 CdCl₂不能减轻谷氨酸的作用,而300μmol/L的CdCl₂及30μmol/L的胆罗啉(Dantrolene)均可以减轻谷氨酸的作用,提示代谢型谷氨酸受体激动引起星形细胞肿胀,细胞内、外Ca²⁺在谷氨酸引起的星形细胞肿胀中起一定的作用.     

白藜芦醇降低大鼠心室肌细胞内游离钙浓度

实验旨在研究白藜芦醇(resveratrol)对大鼠心室肌细胞内钙浓度(intracellular calcium concentratoin,[Ca2+]i)的影响.应用激光共聚焦显微镜技术记录心室肌细胞内的钙荧光强度.结果表明在正常台氏液和无钙台氏液中,白藜芦醇(15～60μmol/L)呈浓度依赖性地降低[Ca2+]i.蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate,1.0 mmol/L)和L型Ca2+通道激动剂Bay K8644(10 μmol/L)可部分抑制正常台氏液中白藜芦醇的效应.但NO合酶阻断剂L-NAME(1.0 mmol/L)对白藜芦醇的作用无影响.白藜芦醇也能明显抑制无钙台氏液中由低浓度ryanodine(1.0 nmol/L)引起的[Ca2+]i增加.当细胞外液钙浓度由1 mmol/L增加到10 mmol/L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,白藜芦醇(60 μmol/L)可降低钙波的传播速度和持续时间,最终阻断钙波.结果提示,白藜芦醇能够降低心室肌细胞内游离钙浓度,此作用可能与其抑制电压依赖性Ca2+通道、酩氨酸激酶和肌浆网内钙释放有关.     

谷氨酸、NMDA 和吗啡对培养大鼠星形胶质细胞内钙振荡的影响

目的:研究谷氨酸、NMDA、吗啡对原代培养的大鼠星形胶质细胞的胞内钙信号的影响及受体作用机制.方法:利用Leica AF6000活细胞工作站,检测谷氨酸、NMDA、吗啡分别灌流前后Fura-2/AM加载的星形胶质细胞内钙信号的动态变化,进一步观 察分别阻断代谢性谷氨酸受体5 (mGluR5)、NMDA受体(NMDA receptor,NMDAR)和阿片μ受体对诱导的胞内钙振荡的影响.结果:谷氨酸、NMDA、吗啡均可明显升高胞内游离钙的浓度([Ca2+]i),而将其相应受体拮抗后,星形胶质细胞[Ca2+]i升高的现象可以被显著抑制.结论:离体培养的星形胶质细胞膜上存在mGluR5、NMDAR和阿片μ受体,这些受体的激活可以升高星影胶质细胞的[Ca2+]i,且这些受体依赖的[Ca2+]i的调控机制可能是星形胶质细胞与神经元交互作用的重要途径之一.     

Bay K8644及nifedipine对大鼠下丘脑神经元L—型Ca^2＋通道特性的影响

采用神经元急性分离和膜片箍技术以及细胞贴附式方式记录通道活动,探讨DHP类Ca^2 通道激动剂Bay K8644及拮抗剂nifedipine对下丘脑神经元L－型Ca^2 通道的影响,结果显示,在Bay K8644作用下,通道开放形式发生变化,明显可见多级开放;通道平均开放时间,平均开放概况显著增加,但单通道电导无明显变化。nifedipine的作用与Bay K8644相反。结果提示,Bay K8644对下丘脑神经元L－型Ca^2 通道有明显激动作用 nifedipine有显著抑制作用。     

植物多肽PA1b打开胰腺β 细胞L型钙通道

利用海藻酸在pH2.7的条件下对小分子多肽的吸附作用, 从豌豆种子中分离并纯化出含37个氨基酸的小分子肽PA1b (pea albumin 1b), 它的肽链内具有6个半胱氨酸并形成一个胱氨酸结构模体. 采用荧光显微技术和膜片钳技术, 发现胞外施加PA1b在胞外钙离子存在的情况下使胰腺β细胞内钙离浓度增加, 该效应被特异性的L型钙通道的阻断剂尼莫地平(nimodipine)阻断, 在零钙外液中PA1b对胞内钙离子浓度无影响; 此外, PA1b使β细胞膜去极化并使膜电容增加. 因此推断PA1b使原代β细胞上去极化细胞膜, 使L型钙离子通道开放, 细胞外钙离子内流并促发细胞分泌.     

植物多肽PA1b打开胰腺β细胞L型钙通道促使钙内流和细胞分泌

利用海藻酸在pH2.7的条件下对小分子多肽的吸附作用,从豌豆种子中分离并纯化出含37个氨基酸的小分子肽PA1b(pea albumin 1b),它的肽链内具有6个半胱氨酸并形成一个胱氨酸结构模体.采用荧光显微技术和膜片钳技术,发现胞外施加PA1b在胞外钙离子存在的情况下使胰腺β细胞内钙离浓度增加,该效应被特异性的L型钙通道的阻断剂尼莫地平(nimodipine)阻断,在零钙外液中PA1b对胞内钙离子浓度无影响;此外,PA1b使β细胞膜去极化并使膜电容增加.因此推断PA1b使原代β细胞上去极化细胞膜,使L型钙离子通道开放,细胞外钙离子内流并促发细胞分泌.     

烟碱对脑细胞内钙离子浓度的调控及其机制分析

神经肌肉接头及神经节N受体均可引起细胞外Ca~(2 )内流和细胞内Ca~(2 )释放,增加细胞内Ca~(2 )浓度。尚无资料证明脑N受体是否影响细胞内Ca~(2-)浓度。本实验观察烟碱对脑细胞内Ca~(2 )浓度的影响并探讨其可能的机理。 烟碱对大鼠脑突触体主动摄取~(45)Ca~(2 )的影响 本实验条件下钙通道激动剂Bay-k-8644(10~(-7)～10~(-4)～mol/L)浓度依赖性地增加突触体~(45)Ca~(2 )主动摄取量;钙通道拮抗剂异搏定(10~(-9)～10~(-5)mol/L)浓度依赖性地抑制~(45)Ca~(2 )摄取     

细胞内高镁对豚鼠心室肌细胞L-型钙通道活性的影响

本文旨在研究细胞内高镁对离体豚鼠心室肌细胞L-型钙通道的单通道门控特性的影响。急性酶消化法分离豚鼠心室肌细胞,应用膜片钳膜内向外单通道记录模式观察不同浓度细胞内镁([Mg2+]i)对心肌细胞L-型钙通道单通道电流的影响。结果显示,对照组([Mg2+]i 0.9 mmol/L)的心肌细胞L-型钙通道相对活性为(176.5±34.1)%,而高镁组([Mg2+]i 8.1 mmol/L)钙通道相对活性显著降低至(64.8±18.1)%(P0.05)。另外,8.1 mmol/L[Mg2+]i使心肌细胞L-型钙通道的平均开放时间缩短至对照组的约25%(P0.05),但对通道平均关闭时间无明显影响。以上结果提示,细胞内高镁主要通过缩短通道平均开放时间抑制豚鼠心室肌细胞L-型钙通道的活性。     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

钙指示剂的发展及其研究现状

钙指示剂常被用于细胞及细胞器钙信号的检测,是钙信号转导研究中必不可少的工具.目前的钙指示剂分两大类,包括化学钙指示剂,如Fura-2、Indo-1、Fluo-4等,和基因编码的钙指示蛋白如D1ER、GCaMP、CEPIAler等.随着技术的发展及研究需求的不断提升,各版本的钙指示剂也在不断更新.本文对已有的钙指示剂进行...     

Ganglioside Function in Calcium Homeostasis and Signaling

Ganglioside function in eukaryotic cells encompasses a variety of modulatory interactions related to both development and mature cellular behavior. In relation to the nervous system this includes induction of neurite outgrowth and trophic/neuroprotective phenomena; more generally this applies to ganglioside effects on receptor function, adhesion reactions, and signal transduction mechanisms in neural and extraneural systems. Underlying many of these trophic effects are ganglioside-induced changes in cellular calcium, accomplished through modulation of Ca²⁺ influx channels, Ca²⁺ exchange proteins, and various Ca²⁺-dependent enzymes that are altered through association with gangliosides. A clear distinction needs to be drawn between intrinsic functions of gangliosides as naturally expressed by the cell and activities created by application of exogenous ganglioside(s) that may or may not reflect natural function. This review attempts to summarize findings in this area and point to possible future directions of research.     

Heparin inhibits the reconstituted plasma membrane Ca2+-ATPase from porcine brain synaptosome

Heparin has been shown to be involved in the regulation of cellular Ca(2+) by binding to many proteins with high affinity. Here we examined the effects of heparin on the plasma membrane Ca(2+)-ATPase from porcine brain synaptosome. Our results showed that heparin dramatically inhibited the ATP hydrolysis and Ca(2+) uptake in the presence and absence of calmodulin. Together with controlled proteolysis by trypsin, we concluded that the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase was less important for the heparin inhibition. Excess phosphatidylserine was able to eliminate the heparin inhibition. We observed that Ca(2+) affinity kept no obvious changes, but the ATP affinity of plasma membrane Ca(2+)-ATPase was apparently decreased in the presence of heparin. Our results indicated that heparin had little effects on ATP or Ca(2+) binding sites of the enzyme.     

Aging-related changes in calcium oscillations in fertilized mouse oocytes

Aging of oocytes, being not fertilized after ovulation for a prolonged time, considerably affects normal development of the fertilized oocyte. We examined effects of the aging on a series of highly repetitive Ca²⁺ transients commonly seen in fertilized mouse oocytes (Ca²⁺ oscillations). Frequency of Ca²⁺ oscillations in the aged oocyte [20 hrs after induction of superovulation by i.p. human chorionic gonadotropin (hCG)] was significantly higher (34.1 ± 5.8 1/hr) than the fresh oocyte (14 hr post-hCG, 21.8 ± 7.9 1/hr). Rates of rise and fall of individual Ca²⁺ transient in the aged oocyte were significantly slower than the fresh oocyte, whereas durations of individual Ca²⁺ transients were similar. When extracellular Ca²⁺ was raised from 2.04 mM to 5.00 mM, aged oocytes showed significant prolongation of the duration of individual Ca²⁺ transient, that resulted in a sustained elevation of intracellular Ca²⁺ ([Ca²⁺]_i) in 33% of the aged oocyte. Transient increase in [Ca²⁺]_i by photolysis of a caged Ca²⁺, Nitr-5, injected into cytoplasm was completely restored in the fresh oocyte [fluorescence intensity of [Ca²⁺]_i indicator dye Fluo-3 (F₄₈₀) returned to 97 ± 2% of the control level, time constant = 37 ± 9 sec]. In contrast, in the aged oocyte, restoration of F₄₈₀ following Nitr-5 photolysis was incomplete (115 ± 12% of the control) and slow (time constant = 64 ± 23 sec). Because inhibition of the Ca²⁺ pump of the endoplasmic reticulum (ER) by 5 μM thapsigargin almost completely inhibited restoration of F₄₈₀ following Nitr-5 photolysis in the fresh oocyte, we conclude that the aging-related changes in Ca²⁺ oscillations may be accounted for by dysfunction of intracellular Ca²⁺ regulation, presumably of the Ca²⁺ pump of the ER. Mol. Reprod. Dev. 48:383–390, 1997. © 1997 Wiley-Liss, Inc.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

Mode of action of proctolin on locust visceral muscle

Proctolin increases the frequency and amplitude of myogenic contractions and results in a sustained contraction of the oviducts of Locusta migratoria. The possible mode of action of proctolin receptors on this visceral muscle has been investigated. Calcium-free saline, containing either 20 mM magnesium ions or 100 μM EGTA, inhibited myogenic contractions, lowered basal tension, and abolished all the effects of proctolin following a 20 min incubation. These effects were reversible upon washing with normal saline. Similar results were obtained with normal saline containing 10 mM cobalt ions. Nifedipine at 50 μM lowered basal tension, abolished myogenic contractions, and reduced the proctolin-induced sustained contraction by 42-62% at 0.5 nM proctolin and by 33-37% at 5 nM proctolin. Similar results were obtained with 100 μM verapamil. Proctolin was still capable of eliciting considerable contractions (25-67% of controls) in preparations depolarized with 100 mM potassium saline. The removal of calcium from the high-potassium saline reversibly abolished the potassium-induced contraction and reversibly blocked the action of proctolin. Nifedipine was ineffective in blocking the action of proctolin in high-potassium saline. Neither cyclic AMP levels nor cyclic GMP levels of the lateral oviducts were elevated by proctolin in the presence of a phosphodiesterase inhibitor. The results indicate that proctolin mediates its effects via an influx of external calcium ions. This calcium appears to enter through two channels, a voltage-dependent channel and a receptor-operated channel. Cyclic nucleotides do not appear to be involved in the action of proctolin in this visceral muscle.     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

Plasma membrane calcium pump and sodium-calcium exchanger in maintenance and control of calcium concentrations in platelets

The purpose of this research was to elucidate the activity of the mechanisms responsible for control of cytosolic calcium concentration in platelets by modeling the time-course of the concentration changing in response to discharge of the intracellular stores or store-operated calcium entry (SOCE). The parameters estimated as a result of model fitting to experimental data are related to physiological or pathological state of the cells. It has been shown that: (a) the time-course is determined by the passive calcium fluxes and activities of the corresponding mechanisms; (b) the decline in the concentration (after its rise) develops due to activity of plasma membrane calcium ATPase (PMCA) both in the case of discharge of the stores of platelets contained in calcium-free medium and in the case of SOCE; (c) impulsive extrusion of calcium in response to its sudden influx, presumably, is the main function of PMCA; (d) the function of sodium-calcium exchanger (NCX) is to extrude calcium excess by permanent counteracting its influx.     

Calcium in plant defence-signalling pathways

In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.     

Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca²⁺ concentration. To better understand this process, we measured the cytosolic Ca²⁺ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca²⁺ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca²⁺-sensitive dyes that possess different affinities for Ca²⁺ allowed the quantitative determination of the cytosolic Ca²⁺ concentration in the steady state. Determining the cytosolic Ca²⁺ concentration as a function of the adapting light intensity shows that the Ca²⁺ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca²⁺ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca²⁺ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca²⁺ concentration, we conclude that the Ca²⁺-buffering capacity is limited. The percentage of the Ca²⁺ influx that is buffered gradually decreases with increasing Ca²⁺ concentrations; at cytosolic Ca²⁺ concentration levels above 10 μM, buffering becomes minimal.