Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells.     

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.     

Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy.

We describe here the use of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca2+]i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca2+]i in optically sectioned unit volumes of the cell. Images of the calcium wave obtained during propagation between paired cells revealed that the wavefront is constant in shape and propagates at constant velocity without any delay at the cell-to-cell junction. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living cells.     

激光扫描共聚焦显微镜观察胰蛋白酶对肺上皮细胞游离钙离子的影响

目的：研究PAR-2激动剂SLIGKV和tc-LIGRLO、胰蛋白酶及其抑制剂对H292肺上皮细胞[Ca^2＋]i的影响.方法：应用Fluo-3/AM 荧光标记技术和激光扫描共聚焦显微镜（LSCM） 检测不同因素处理的H292肺上皮细胞[Ca^2＋]i.结果：胰蛋白酶、SLIGKV、tc-LIGRLO均能引发H292细胞[Ca^2＋]i的增加,平均荧光强度分别比加入药物前增加267%,60%和37%.胰蛋白酶抑制剂大豆胰蛋白酶抑制剂（SBTI）和α1-抗胰蛋白酶（α1-AT）可以抑制胰蛋白酶诱导的细胞[Ca^2＋]i的增加.结论：PAR-2可以介导H292肺上皮细胞[Ca^2＋]i的释放增加,胰蛋白酶抑制剂可以抑制胰蛋白酶诱导的细胞[Ca^2＋]i的增加.     

Improved method for measuring intracellular Ca++ with fluo-3

The accuracy of flow cytometric measurement of intracellular calcium with fluo-3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo-3 and SNARF-1. When SNARF-1 fluorescence is collected at approximately 600 nm, its intensity does not change upon cell activation. Furthermore, fluo-3 and SNARF-1 fluorescence signals exhibit a linear relationship. The ratio of fluo-3 to SNARF-1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo-3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.     

双重荧光染色监测听毛细胞游离钙

Calcium distribution and mobilization during mechanical stimulation in outer hair cells of the guinea pig were monitored using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Spatial calcium gradients were revealed among various subcellular areas. The ratios of the fluorescence intensity of fluo-3 and fura-red were 1.71 +/- 0.85, 1.61 +/- 0.75, 1.47 +/- 0.65 and 1.39 +/- 0.66 for the cytoplasm, the cytoplasmic membrane, the cuticular plate and the nucleus respectively, indicating that free calcium ion concentrations are the highest in the cytoplasm and the lowest in the nucleus. While the calcium concentration remained relatively constant under resting conditions, it increased during mechanical stimulation. The results show that confocal ratio imaging of fluo-3 and fura-red enables us to determine more accurately the subcellular calcium distribution and that the calcium ions make a contribution to the mechanic-electrical transduction in hair cells.     

Differential regulation of intracellular Ca2+ signalling induced by high K+ and endothelin-1 in single smooth muscle cells of intact canine basilar artery: detection by means of confocal laser microscopy

Changes in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells play the key role in regulation of vascular smooth muscle tone and pathogenesis of cerebral vasospasm. In this study, we adopted the confocal laser microscopy to detect the fluorescence signals arising from the individual smooth muscle cells of canine basilar artery. Ring preparations were made, loaded with fluo-3 and changes in fluorescence induced by high K+ and endothelin-1 (ET-1) were measured by confocal laser microscopy. In some unstimulated smooth muscle cells Ca2+ waves arising from discrete region of the cell propagated to the whole cell with a velocity of approximately 10 microm/s. High K+ (80 mmol/L) induced a rapid rise in [Ca2+]i, the peak level being consistently reached approximately 10 s after stimulation. In contrast, the time to peak level of [Ca2+]i induced by ET-1 (0.3 micromol/L) varied widely between 13 and 26 s among individual cells, an indication that the extent of nonuniform coordination of increases in [Ca2+]i in individual cells may be partly responsible for the different time courses of tension development of vascular smooth muscle in response to the vasoactive stimulants. The increase in [Ca2+]i induced by ET-1 was transient but a pronounced and sustained contraction developed further in response to ET-1. Thus ET-1 has a biological property as a potential candidate to elicit cerebral vasospasm. Confocal laser microscopy could be a useful tool to measure the changes in [Ca2+]i in individual smooth muscle cells of cerebral artery.     

Spatial non-uniformities in [Ca2+]i during excitation-contraction coupling in cardiac myocytes.

The intracellular calcium ([Ca2+]i) transient in adult rat heart cells was examined using the fluorescent calcium indicator fluo-3 and a laser scanning confocal microscope. We find that the electrically evoked [Ca2+]i transient does not rise at a uniform rate at all points within the cell during the [Ca2+]i transient. These spatial non-uniformities in [Ca2+]i are observed immediately upon depolarization and largely disappear by the time the peak of the [Ca2+]i transient occurs. Importantly, some of the spatial non-uniformity in [Ca2+]i varies randomly in location from beat to beat. Analysis of the spatial character of the non-uniformities suggests that they arise from the stochastic nature of the activation of SR calcium-release channels. The non-uniformities in [Ca2+]i are markedly enhanced by low concentrations of Cd2+, suggesting that activation of L-type calcium channels is the primary source of activator calcium for the calcium transient. In addition, the pattern of calcium release in these conditions was very similar to the spontaneous calcium sparks that are observed under resting conditions and which are due to spontaneous calcium release from the SR. The spatial non-uniformity in the evoked [Ca2+]i transient under normal conditions can be explained by the temporal and spatial summation of a large number of calcium sparks whose activation is a stochastic process. The results are discussed with respect to a stochastic local control model for excitation-contraction (E-C) coupling, and it is proposed that the fundamental unit of E-C coupling consists of one dihydropyridine receptor activating a small group of ryanodine receptors (possibly four) in a square packing model.     

Role of calcium in differentiation of murine erythroleu-kemia cells

Calcium plays a crucial role in the normal and abnomal cell metabolism.The role of calcium in the differentiation process of murine erythroleukemia cells(MELC)remains controversial.Here,based upon quantitative measurement of fluorescence in single cells,a method was developed to investigate the intracellular free calcium[Ca^2 ]i concentration and DNA contents simultaneously,by employing the fluorescent probe,fluo-3 acetoxymethyl ester and DNA dye Hoechst 33342.During MELC differentiation.[Ca^2 ]i concentration incresed.We also demonstrated that calcium ionophore,A23187,enhanced the HMB-induced MELC differentiation,while verapamil,an inhibitor of calcuim uptake,slightly reduced differentiation.These results suggested that an increase in the [Ca^2 ]i level was an essential step in HMBA-induced MELC differentiation.     

Real-time confocal microscopy and calcium measurements in heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution

Preliminary experiments and characterization of a modified confocal fluorescence microscope have been carried out and are presented in this article. We have made use of commercially available hardware and software (having modified and extended the system) and are continuing the process of making modifications to this system to enable us to carry out investigations in living and mobile cells. We report on identified problems in measuring intracellular calcium in myocardial cells, important lessons that have been learned and new findings regarding myocardial cells. Specifically, we have found that myocardial cellular organelles (e.g. nuclei and mitochondria) are sharply defined unlike images obtained with standard epifluorescence microscopes using intracellular indicators. Furthermore, we show that the organelles can accumulate or largely exclude certain indicators relative to the concentration in the cytosol. Additionally, we have examined the use of the new calcium indicator fluo-3 in contracting heart cells and have more clearly defined certain limitations in its use in this preparation. We are working on modifications of the present equipment to enable the system to work with an ultraviolet laser as a light source. The confocal microscope offers the prospect of extraordinarily good spatial and temporal resolution under specific conditions in heart cells.     

A single cell observation of staurosporine effect on the Ca signals in rat basophilic leukemia cells

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen-dependent calcium signals in an individual rat basophilic leukemia cell (RBL-2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL-2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.     

增强UV-B辐照对小麦幼苗叶肉细胞内Ca2＋水平的研究

以小麦（Triticum aestivum）幼苗叶片为材料,利用提取原生质体方法在小麦幼苗叶肉细胞中成功地装载了钙离子荧光指示剂fluo-3/AM,采用激光共聚焦显微技术检测了增强UV-B辐射后小麦幼苗叶肉细胞内游离钙离子荧光强度的分布,并对[Ca2＋];进行了测定.结果显示,对照组细胞内钙离子荧光分布较均匀,主要分布于紧贴质膜处和核周围,UV-B辐射组钙离子荧光与对照组分布相似,但其原生质体表面不如对照组平滑;同时发现增强UV-B辐射组细胞内钙离子荧光强度值较对照组高,说明增强UV-B辐射组小麦幼苗叶肉细胞维持较高浓度的钙离子水平.这些变化表明Ca2＋信号有可能以一定的方式参与了小麦响应UV-B辐射胁迫的过程.     

活细胞内5-羟色胺可见荧光的多光子激发成像

应用多光子激发激光扫描显微镜对5-羟色胺（5-hydroxytryptamine, 5-HT）孵育的大鼠粘膜型肥大细胞进行自发荧光成像,首次观察到了活细胞内5-HT相关的可见荧光,并对其产生机理进行了初步探讨.实现了对活细胞内5-HT空间分布的高分辨成像,为研究活组织或细胞内5-HT的空间分布和含量与细胞功能状态的关系提供了新的实验方法.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system.The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field.The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field.In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions,the increase of [Ca2+]i was still observable.It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.     

High concentration of calcium ions in Golgi apparatus

The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6-NBD-ceramide,and then the single cells were examined by laser scanning confocal microscopy(LSCFM) for subcellular distributions of Ca^2 and the location of Golgi apparatus.In these cells,the intracellular Ca^2 were found to be highly concentrated in the Golgi apparatus.The changes of distribution of cytosolic high Ca^2 region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium,when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin,the fluorescence of the Golgi region decreased far less than that of the cytosol.Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.     

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.     

大鼠肺动脉平滑肌培养细胞内Ca~(2+)反应的多样性

用Ｃａ２＋荧光色素Ｆｌｏｕ－３／ＡＭ负荷原代培养的大鼠肺动脉平滑肌细胞,在共聚焦激光显微镜下观察细胞内Ｃａ２＋对各种缩血管物质反应的非均一性。实验结果提示：各种Ｃａ２＋通道的反应与细胞培养时间相关。８０％以上的Ｃａ２＋贮库具有ＣＩＣＲＣａ２＋通道,在肺血管管平滑肌细胞可能存在一种只具有ＣＩＣＲ通道的Ｃａ２＋贮库,ＣＩＣＲ的Ｃａ２＋释放作用强于ＩＣＲＣａ２＋通道