Na^+／Ca^2+交换介导的Nd^3+跨淋巴细胞膜的行为研究

利用Fura-2荧光浓度指示剂法对Na^+／Ca^2+交换介导的Nd^3+跨入外赂血淋巴 细胞膜行为进行了一系列研究。结果表明：当细胞形成向外的Na^+梯度时Nd^3+能 跨膜进入细胞,电压依赖性L-型Ca^2+通道对Nd^3+进入无贡献,提出了Na^+／ Ca^2+交换系统是Nd^3+进入细胞的主要途径;在安全浓度范围内进入胞内的游 Nd^3+浓度成正比,计算表明进入胞内的最大游离Nd^3+浓度为（3.67±0.32）× 10^-14mol·L^-1;当胞外pH值降低时进入胞内的游离Nd^3+浓度减小,胞内游离 Ca^2+浓度减小时进入的游离Nd^3+浓度略微增大,胞外Nd^3+和Ca^2+竞争Na^+／ Ca^2+交换位点;结果进一步推测进入胞内的Nd^3+可被质膜钙泵泵出胞外,初步实 验表明进入胞浆中的Nd^3+会在内质网中进一步累积,而在线粒体中不累积。     

Fura-2探针对希土Y3+跨PC12细胞膜行为研究

使用AR-MIC-CM阳离子测定系统,发展Fura-2荧光测定技术,将其应用于测定细胞内游离希土离子Y³⁺,并以此研究了Y³⁺跨PC12细胞(大鼠嗜铬细胞瘤细胞)膜的行为。结果表明：在模拟细胞内各离子组分,pH=7.05的溶液中,测得表观解离常数为4.5p mol·L^-1。对于PC12细胞,静息条件下Y³⁺不能跨越细胞膜进入胞内。与钙离子通道相关的KCl和去甲肾上腺素均不能刺激希土Y³⁺过膜。用Ouabain(哇巴因)使胞内Na⁺超载后,Y³⁺可过膜进入细胞内,且过膜量与胞外Y³⁺浓度和胞内Na⁺超载程度有一定的浓度依赖关系,提示Y³⁺可以经由Na⁺/Y³⁺交换机制过膜而进入细胞内。     

钙通道关闭及开放状态下稀土离子对淋巴细胞的 跨膜行为

用荧光探针法(以Fura-2为荧光指示剂)通过监测细胞内荧光强度的变化来判断稀土的跨膜行为。在近生理条件下,从单细胞、多细胞两个角度,研究了人外周血淋巴细胞钙离子通道开放、关闭等不同状态下Dy^3^+,La^3^+等稀土离子的跨膜行为。实验得出：微量稀土离子在上述状态下均不能跨膜内流。我们还发现,在钙通道开放的情况下,低剂量稀土离子不影响细胞外钙跨膜内流。     

Fura-2荧光探针法检测细胞内La~(3+)

使用Fura - 2荧光探针技术 ,检测细胞内的镧离子浓度 [La3+]i 变化 ,研究其跨膜行为。结果显示 ,细胞外镧 [La3+]o(0 .1mmol/L)可使细胞内镧离子浓度增加 ,说明镧离子能够跨越小鼠心肌细胞膜 ,讨论了跨膜机理。     

NIH 3T3细胞中钙离子在间期内增加

本文利用钙离子荧光指示剂Fluo-3和结合DNA的荧光染料Hoechst33342对NIH3T3活细胞进行染色,并用显微荧光分光光度计测定单个细胞中DNA含量和细胞内钙离子浓度,测定出细胞从G_1期经S期到G_2期,细胞内钙离子浓度几乎增加了一倍,每个细胞周期时相中的细胞钙离子浓度是非均一的,但S和G_2期细胞中钙离子浓度从不低于G_1和S期细胞中钙离子的一定浓度,G_0期细胞中钙离子浓度也是非均一的,其中一些G_0期细胞低于G_1期细胞中的钙离子浓度,这种钙离子浓度在细胞周期中的变化可能与细胞周期的调控有关。     

人红细胞摄入Cd~(2 )离子的动力学和机理

不同Cd2 离子浓度引起的人红细胞相对溶血率及临界溶血浓度CH0 已经测定 ,CH0 =2 .5× 10 - 4mol·L- 1。Cd2 跨膜进入人红细胞遵从一级动力学过程 ,其一级速率常数为 1.2× 10 - 2 h- 1。阴离子通道抑制剂DIDS及K ,Na ATPase抑制剂均可部分抑制人红细胞摄入Cd2 ,Cl- 浓度和介质pH也影响Cd2 的细胞摄入 ,细胞摄入镉量随胞外Cd2 浓度增加线性增加。这些实验结果证明 ,人红细胞摄入Cd2 是涉及简单被动扩散、阴离子通道转运及主动传送的复杂过程     

大肠杆菌HB101感受态的热化学研究

利用LKB-2277生物活性检测系统,测定了在生理盐水中,采用氯化钙法建立大肠杆菌感受态过程中的代谢热输出。生理盐水中大肠杆菌及其感受态进行内源代谢的热化学方程满足:P=K~c+A,dc/dP~0=K'c^0,氯化钙处理后,K与P~0均增大,说明内源代谢活性增强,这与大肠杆菌感受态建立过程中一系列生理生化活性的调整是一致的,表明大肠杆菌感受态的建立很可能有其内在的调节机制,而不完全是人工诱导的。     

新型钙荧光探针在H2O2对HL-60胞浆钙影响研究中的应用

采用H2O2、黄嘌呤和黄嘌呤氧化酶、H2O2/抗坏血酸/Fe^2＋（或H2O2/Fe^＋）、H2O2和辣根过氧化酶（HRP）等不同活性氧产生体系,对自行合成的胞浆特异性钙荧光探针STDIn的活性氧耐受性进行了考察.以STDIn-AM为探针标记HL-60细胞,激光共聚焦技术检测单细胞内Ca^2＋-STDIn荧光强度的变化;探讨了不同浓度的H2O2对胞内Ca^2＋浓度变化的影响,从膜结构和生物信号传导的角度研究了氧自由基对培养细胞的生物效应.     

高氯酸镱对大鼠背根神经元细胞毒性及膜上钾通道的影响

研究了高氯酸镱(Yb(ClO4)3)诱导大鼠背根神经(DRG)元凋亡、引起胞内钙离子浓度变化以及对膜上钾离子通道的影响.急性分离大鼠DRG细胞,用不同浓度的Yb(ClO4)3处理DRG细胞24和96h,采用流式细胞仪和激光共聚焦法,检测细胞的凋亡和细胞内钙离子荧光强度的变化.利用全细胞膜片钳法,记录Yb(ClO4)3对细胞膜上不同钾通道电流的影响.结果表明,10,100,1000μmol/L Yb(ClO4)3处理DRG神经元24h,细胞基本不表现凋亡;处理96h,细胞出现明显的凋亡(P0.05～0.01),尤其是1000μmol/L Yb(ClO4)3,凋亡率达到了(55.23±3.76)%(P0.01).经Yb(ClO4)3孵育的DRG神经元胞内的Ca2+的荧光强度显著增大;Yb(ClO4)3抑制背根神经节纤维和神经元突起的生长.Yb(ClO4)3抑制DRG神经元膜上的钾电流,胞内和胞外的Yb(ClO4)3作用钾通道的部位不同.细胞外液中的Yb(ClO4)3不同程度地阻断了瞬间外向钾电流IA,对延迟整流钾电流几乎没影响;往电极内液中加入同样浓度的Yb(ClO4)3对IA影响很小,却特异性地阻断了延迟整流钾电流IK.10μmol/L Yb(ClO4)3使IA的激活和失活过程都显著右移,延长了瞬间外向钾电流达到峰值的时间和快速失活时间常数,增加神经元的兴奋性.     

稀土离子跨人血红细胞膜的荧光法研究

采用Fura-2荧光浓度指示剂对红细胞的稀土跨膜作用进行了系列研究.结果表明,稀土离子不能通过完整的红细胞膜进入细胞内.通过与离子载体实验相对照,发现细胞ATP耗竭后,低浓度的稀土离子(5×10^-6mol/L)不能跨膜进入ATP-耗竭红细胞.KCl去极化及加入电压依赖性钙通道刺激剂Bay-K8644对稀土离子的跨膜也没有促进作用.在Ca²⁺内流正常的情况下,低浓度稀土离子(5×10^-6mol/L)对钙离子内流无影响.增大稀土离子浓度到5×10^-4mol/L,用显微镜观察此时红细胞已开始溶血.在模拟胞内离子组分的缓冲液中(pH=7.05),比较了La³⁺,Eu³⁺和Ca²⁺对Fura-2的敏感程度.此条件下Fura-2对La³⁺和Eu³⁺的检测限分别为10-12和10-14mol/L,对Ca²⁺的检测限为10-8mol/L,并测得Fura-2-La³⁺(Eu³⁺)的络合比为1∶1,表观离解常数为1.7×10^-12和4.95×10^-14mol/L,表明用此法检测稀土离子跨膜行为相当灵敏有效.     

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.     

Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes.

Fluorescent dyes have been widely employed as optical indicators of the membrane potential difference in cells, isolated organelles and lipid vesicles that are too small to make microelectrode measurements feasible. We describe here the application of a carbocyanine dye, 3,3'-dipropylthiodicarbocyanine iodide [DiS-C3-(5)], to monitor the transmembrane potential changes induced by a variation of the K+ concentration for the cells of Escherichia (E.) coli and photosynthetic bacterium Rhodospirillum (R.) rubrum. The cells were first incubated in buffers containing DiS-C3-(5) and K+ ions of various concentrations until the fluorescence intensity reached a constant value. Valinomycin was then added to the solution, which caused changes in the fluorescence intensity, depending on the K+ concentrations. The membrane potential is shown to have a linear relationship with the fluorescence intensity of DiS-C3-(5). The results demonstrate that the K+ concentrations inside intact cells are 4.6 mM and 5.3 mM for E. coli and R. rubrum, respectively. The diffusion potentials of K+ ions were determined using the Nernst equation over the range of -1.3 mV to 44 mV, corresponding to K+ concentrations of 5 mM -25 mM outside of the cells.     

Effect of transmembrane Ca2+ gradient on conformation and enzyme activity of reconstituted adenylate cyclase.

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca2+ gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca2+ concentration (approximately 10(-6) mol/L, similar to the physiological condition). If the transmembrane Ca2+ gradient was in the inverse direction (i.e. outside higher Ca2+ concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca2+ ionophore A23187. Proteoliposomes without transmembrane Ca2+ gradient exhibited intermediate activities. The conformation difference of adenylate cyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.     

Hypericin-mediated photodynamic antimicrobial effect on clinically isolated pathogens

The aim of this study was to determine the photodynamic antimicrobial effect of hypericin on clinically isolated Staphylococcus aureus and Escherichia coli cells. Bacterial cells (10(8) cells per mL) were incubated with hypericin (0-40 μM) for 30 min and followed by light irradiation of 600-800 nm at 5-30 J cm(-2). Cell survival was determined by colony counting, cellular hypericin uptake examined by flow cytometer, and cell membrane damage examined by scanning electron microscopy and leakage assay. The effectiveness of hypericin-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake. The combination of hypericin and light irradiation could induce significant killing of Gram positive methicillin-sensitive and -resistant S. aureus cells (>6 log reduction), but was not effective on Gram negative E. coli cells (<0.2 log reduction). The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin.     

Relationship between steps in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence and changes in the energized membrane state and in intracellular and extracellular adenosine 5'-triphosphate (ATP) levels following bacteriophage T5 infection of Escherichia coli.

The addition of bacteriophage T5 to anaerobic, fermenting cells of Escherichia coli B or K-12 in the presence of 8-anilino-1-naphthalene sulfonate (ANS), N-phenylnaphthyl-1-amine (NPN), or dansyl ethylamine causes the fluorescence of these probes to rise in two steps, the first occurring immediately upon addition, the second delayed by 6 min. The conditions necessary for observing this phenomenon are defined (cell density, probe concentration, substrate, absence of an electron acceptor, multiplicity of infection, growth, and harvesting conditions). The magnitudes of the first and second steps in fluorescence are dependent upon the multiplicity of infection; the timing of the steps is not. The first step correlates with a breakdown in the potassium or rubidium permeability barrier of the cells, and it occurs either aerobically or anaerobically, with fermentable or nonfermentable substrates. The second step occurs only with cells that are without an available electron acceptor, are fermenting, and which have a functional membrane-bound, Ca2+-dependent adenosine triphosphatase (ATPase). The results are consistent with disturbance of energization of the cell membrane by the membrane-bound ATPase at the time of the second step in fluorescence. No changes in the intracellular level of adenosine 5'-triphosphate (ATP) was seen, whereas the extracellular level increased sharply, starting 3--6 min after phage addition. The quantity of ATP found in the medium by 30 min after infection amounted to about four times the amount present inside the cells at the time of infection. The quantity and rate of efflux of ATP was similar under aerobic and anaerobic conditions.     

5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

With the imaging fluorescence probe of Ca2+ (fluo-3) and a laser scanning confocal micro-scope, the spontaneous localized calcium release event was first discovered in resting rat cardiac myocytes by Cheng[1] in 1993. A mathematical simulation is developed with computer in order to reveal the effect, which is immediately suggested that these events are likely to reflect the local-ized release of Ca2+ from a small cluster of ryanodine-sensitive Ca2+ release channels in sar-coplasmic reticulum …     

Inhibition of superoxide generation and of increase in intracellular Ca2+ concentration by zinc in rat neutrophils stimulated with zymosan

The effect of Zn2+ on the O2- generation and change in intracellular Ca2+ concentration ([Ca2+]i) of rat peritoneal neutrophils was studied. Zymosan (serum-treated zymosan (STZ))-induced O2- generation was inhibited by Zn2+ at concentrations as low as 10 microM. A large amount of the inhibition was observed in the absence of extracellular Ca2+ but the inhibition could not be restored by increasing the extracellular Ca2+ concentration, indicating that Zn2+ does not necessarily inhibit the O2- generation competitively with extracellular Ca2+. In the absence of extracellular Ca2+, Zn2+ inhibited STZ-induced transient increase in [Ca2+]i in the concentration range that evoked a marked inhibition in the O2- generation. On the other hand, Zn2+ did not inhibit significantly STZ-induced uptake of 45Ca2+ from extracellular medium by the cells. From these results, it is suggested that Zn2+ inhibits STZ-induced release of Ca2+ from intracellular storage sites, resulting in the suppression of the activation mechanism of neutrophils.     

La3+ Transmembrane Research in Guinea Pig Ventricular Cells by Fura-2 Fluorescence

The safety of rare earth fertilizer was paid more attention now, Whether RE' (rare earthion)cross membrane or not is the key problem of rare earth element biological effect. Itwas not solved until now. Ventricular cells which have different type of ions channel andadrenoceptor played very important role in biological functionl. To our knowledge, transventricular cells membrane behavior of rare earth ions has not been researched byFura-2 fluorescent probe technique. Guinea pig ventricular cel…