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1.
The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N−'tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca2+]i, we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 μM BAPTA/AM reduced the number of cells showing deregulation of [Ca2+]i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCI were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3–30 μM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 μM BAPTA/AM, the time constant of decline of [Ca2+]i was increased by a factor of 4.1 ± 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 μM BAPTA/AM, the K+-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca2+]i-induced cell damage, however, at the expense of a disturbed calcium signalling. 相似文献
2.
Ronny Fransson Pierluigi Nicotera Lars Wärng»ardi Ulf G. Ahlborg 《Cell biology and toxicology》1990,6(2):235-244
Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca2+ concentration ([Ca2+]i). In the present study, the role of[Ca2+]i in DDT-induced loss of GJIC was investigated in WB-F344 rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca2+ fourescent indicator, furà-2. Our results show that DDT at non-cytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca2+]i, and was not prevented by loading cells with the intracellular Ca2+ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2+3 fold sustained increase in [Ca2+]i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca2+]i are not responsible for DDT-induced loss of communication and that, in general an increase in [Ca2+]i, within physiological levels is not sufficient to abolish GJIC. However, Ca2+-dependent processes that are active at normal resting [Ca2+
i appear to be required for the maintenance of GJIC.Abbreviations [Ca2+]
cytosolic free Ca2+ concentration
- GJIC
gap junctional intercellular communication
- SLDT
scrape-loading/dye transfer assay
- DDT
1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane
- tBuBHQ
2,5-di(tert-butyl)-1,4-benzohydroquinone
- LDH
lactate dehydrogenase
- ER
endoplasmic reticulum
- Fura-2
1-[2-(5carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid
- BAPTA
bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraaceticacid
- Fura-2/AM and BAPTA/AM
are the cell permeant acetoxymethyl ester forms of fura-2 and BAPTA, respectively 相似文献
3.
BAPTA‐AM dramatically improves maturation and development of bovine oocytes from grade‐3 cumulus‐oocyte complexes
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Hongmei Hu Xianhong Mo Xue Li Xiangwei Fu Yunpeng Hou 《Molecular reproduction and development》2018,85(1):38-45
Intracellular free calcium ([Ca2+]i) is essential for oocyte maturation and early embryonic development. Here, we investigated the role of [Ca2+]i in oocytes from cumulus‐oocyte complexes (COCs) with respect to maturation and early embryonic development, using the calcium‐buffering agent BAPTA‐AM (1,2‐bis[2‐aminophenoxy]ethane‐N,N,N′,N′‐tetraacetic acid tetrakis [acetoxymethyl ester]). COCs were graded based on compactness of the cumulus mass and appearance of the cytoplasm, with Grade 1 indicating higher quality and developmental potential than Grade 3. Results showed that: (i) [Ca2+]i in metaphase‐II (MII) oocytes from Grade‐3 COCs was significantly higher than those from Grade‐1 COCs, and was significantly reduced by BAPTA‐AM; (ii) nuclear maturation of oocytes from Grade‐3 COCs treated with BAPTA‐AM was enhanced compared to untreated COCs; (iii) protein abundance of Cyclin B and oocyte‐specific Histone 1 (H1FOO) was improved in MII oocytes from Grade‐3 COCs treated with BAPTA‐AM; (iv) Ca2+ transients were triggered in each group upon fertilization, and the amplitude of [Ca2+]i oscillations increased in the Grade‐3 group upon treatment with BAPTA‐AM, with the magnitude approaching that of the Grade‐1 group; and (v) cleavage rates and blastocyst‐formation rates were improved in the Grade‐3 group treated with BAPTA‐AM compared to untreated controls following in vitro fertilization and parthenogenetic activation. Therefore, BAPTA‐AM dramatically improved oocyte maturation, oocyte quality, and embryonic development of oocytes from Grade‐3 COCs. 相似文献
4.
《Neurochemistry international》1987,10(1):55-64
The utility of the acetoxymethyl esters of two tetracarboxylic acids, fura-2 and quin-2, in the determination of ionic calcium levels within synaptosomes and mitochondria was compared. Synaptosomes and isolated mitochondria both accumulated the esters but mitochondria had a much more limited capacity to hydrolyze them. Dye-loaded synaptosomes maintain their external membrane potential of magnitude similar to values for unloaded controls and do not accumulate radioactive Ca2+ in excess with time. Both fluorescent compounds yielded similar values (about 300–400 nM) for free intrasynaptosomal calcium [Ca2+]i. Mitochondrial Ca2+ could be measured only with fura-2. Isolated mitochondria contained 0.9–1 μM free Ca2+ in a similar extrasynaptosomal medium. Fura-2 tended to overestimate [Ca2+]i while quin-2 tended to underestimate it due to chelation of these dyes with intrasynaptosomal trace elements. Fura-2 requiring the use of two excitation wavelengths was significantly superior to the single wavelength method using quin-2. Advantages included reduced danger of erroneous readings due to (i) synaptosomal sedimentation, (ii) photobleaching of the dye, (iii) underestimation of intrasynaptosomal calcium during correction for dye leakage by manganese entry into synaptosomes. Fura-2 interfered less with synaptosomal Ca2+ transients than quin-2, probably due to lower intrasynaptosomal concentration of dye needed. Both unstimulated and K+-stimulated 45Ca2+ uptake were increased in quin-2-loaded synaptosomes but only K+-stimulated uptake in fura-2 loaded ones. This series of advantages makes fura-2 of superior utility in the determination of free intrasynaptosomal calcium. 相似文献
5.
《Biochemical and biophysical research communications》2014,443(3):888-893
Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca2+]c) and nuclear calcium ([Ca2+]n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca2+]n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca2+]n and [Ca2+]c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells. 相似文献
6.
Calcium homeostasis and mitochondrial oxidative metabolism interact closely in brain and both processes are impaired during hypoxia. Since the regulation of the pyruvate dehydrogenase complex (PDHC) may link these two processes, the relation of cytosolic free calcium ([Ca2+]i) to the activation state of PDHC (PDHa) was assessed in isolated nerve terminals (i.e. synaptosomes) under conditions that alter [Ca2+]i. K+ depolarization elevated [Ca2+]i and PDHa and both responses required external calcium. Treatment with KCN, an in vitro model of hypoxia decreased ATP and elevated [Ca2+]i and PDHa. Furthermore, in the presence of KCN, PDHa became more sensitive to K+ depolarization as indicated by larger changes in PDHa than in [Ca2+]i. The calcium ionophore Br-A23187 elevated [Ca2+]i, but did not affect PDHa. K+ depolarization elevated [Ca2+]i and PDHa even if [Ca2+]i was elevated by prior addition of ionophore or KCN. Previous in vivo studies by others show that PDHa is altered during and after ischemia. The current in vitro results suggest that hypoxia, only one component of ischemia, is sufficient to increase PDHa. These data also further support the notion that PDHa is regulated by [Ca2+]i as well as by other factors such as ATP. Our results are consistent with the concept that PDHa in nerve endings may be affected by [Ca2+]i and that these two processes are clearly linked.Abbreviations (PDHa)
Activation state of the pyruvate dehydrogenase complex
- ([Ca2+]i)
cytosolic free calcium concentrations
- (MOPS)
3(N-morpholino)propanesulfonic acid
- (fura-2AM)
fura-2 acetoxymethyl ester
- (AABS)
p-(p-aminophenylazo)benzene sulfonic acid
- (PDHC)
pyruvate dehydrogenase complex
- (TES)
N-tris{[hydroxymethyl]methyl}-2-amino-ethanesulfonic acid
- (SNK-test)
Student-Newman-Keuls test 相似文献
7.
ATP receptor-mediated changes in the Ca2+ concentration were recorded from neurons of the sensorimotor cortex in brain slices from 3-week-old rats. To measure the
cytoplasmic concentration of Ca2+, slices were incubated with Fura-2/AM, and a microfluorimetry system was focused on an individual cell. Possible glutamatergic
signals resulting from ATP-evoked glutamate release were excluded. After elimination of calcium from the extracellular solution,
the first ATP-induced [Ca2+]
i
transient decreased to 62±9% of a similar response in the normal solution, suggesting the participation of metabotropic purinoreceptor-triggered
Ca release in transient generation. Depletion of the caffeine-sensitive calcium store results in diminution of ATP-induced
[Ca2+]
i
transient in the Ca2+-free solution by 31.4±7.0% (P<0.01). This may indicate that in pyramidal neurons of the sensorimotor cortex InsP3- and Ca-induced Ca-releases demonstrate noticeable functional interaction. Nevertheless, there is no single compartment in
the endoplasmic reticulum bearing both IICR and CICR channels. 相似文献
8.
Jiuhan Zhao Biao Wang Xiaohong Wang Xiuli Shang 《Molecular and cellular biochemistry》2018,448(1-2):71-76
The purpose of the study was to investigate the changes of Ca2+/calmodulin-dependent protein kinases II (CaMKII)/cAMP response element-binding protein (CREB) signaling pathway in a rat tinnitus model. Eighteen Wistar rats were randomly divided into three groups: normal control (NC), normal saline (NS), and tinnitus model (TM) groups. Tinnitus model was induced by intraperitoneal injection of salicylate. The concentration of intracellular calcium level in auditory cortex cells was determined using Fura-2 acetoxymethyl ester (Fura-2 AM) method with fluorospectrophotometer. Expressions of calmodulin (CaM), N-methyl-d-aspartate receptor 2B subunit (NR2B), calcium-calmodulin kinase II (CaMKII), and cAMP response element-binding protein (CREB) were detected with Western blot. Tinnitus model was successfully established by the intraperitoneal administration of salicylate in rats. Compared with rats in NC and NS groups, salicylate administration significantly elevated CaM, NR2B, phospho-CaMKII and phospho-CREB expression in auditory cortex from tinnitus model group (p?<?0.05), and the free intracellular Ca2+ concentrations (p?<?0.05). Our data reveal that salicylate administration causes tinnitus symptoms and elevates Ca2+/CaMKII/CREB signaling pathway in auditory cortex cells. Our study likely provides a new understanding of the development of tinnitus. 相似文献
9.
We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar
neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular
concentration of free Ca2+ ([Ca2+]
і
) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons
of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca2+, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation
of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition,
the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments
are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells. 相似文献
10.
Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5mol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca2+]in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 mol/L, and overall production of neurites was reduced at 2 mol/L. The length of axons and the number of axons and dendrites were reduced at 1 mol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 mol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca2+]in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 mol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca2+]in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.Abbreviations BAPTA-AM
1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester
- [Ca2+]in
intracellular free calcium ion concentration
- DMSO
dimethyl sulfoxide
- E18
embryonic day 18
- FBS
fetal bovine serum
- fura-2AM
fura-2 acetoxymethyl ester
- HBSS
Hanks' Balanced Salt Solution
- MEM
Eagle's Minimum Essential Medium 相似文献
11.
WenHao Zhang Zdenko Rengel John Kuo 《The Plant journal : for cell and molecular biology》1998,15(1):147-151
Loading of Ca2+-sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca2+ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca2+-sensitive dyes is the presence of cell-wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane-impermeable. A novel non-invasive loading protocol was described in this paper to load the Ca2+-sensitive fluorescent probe Fluo-3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo-3/AM ester solution at 4°C for 2 h followed by 2-h incubation in the dye-free solution at 20°C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, because hydrolysis of Fluo-3/AM ester by extracellular esterases is a chemical process (high Q10), while diffusion of Fluo-3/AM across the plasma membrane is a physical process (low Q10). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20°C, releasing the membrane-impermeable Ca2+-sensitive Fluo-3 in the cytoplasm. The root cells loaded with Fluo-3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca2+ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca2+ ions. 相似文献
12.
Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells 总被引:1,自引:0,他引:1
The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca(2+)](i) and pH(i) were determined by loading the cells with the fluorescent dyes, Fura-2 acetoxymethyl ester (Fura-2/AM) and 2',7'-bis(2-carboxyethyl)-5-(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [(3)H]-thymidine. The results indicated that (1) LPS (10 microg/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca(2+)](i) and [(3)H]-thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [(3)H]-thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca(2+)](i) elevation. 相似文献
13.
肺腺癌A549/DDP细胞周期变化及其多药耐药性 总被引:3,自引:2,他引:1
用Fura-2/AM标记药物敏感的肺腺癌细胞A549和抗顺铂药物的肺腺癌细胞A549/DDP两种细胞胞内游离Ca2+,用碘化丙锭(PI)标记细胞DNA,检测其胞内Ca2+的变化及两种细胞增殖能力和细胞周期.实验结果表明,抗药性细胞株A549/DDP胞浆内游离Ca2+的浓度仅为药物敏感细胞株A549的1/3左右,同时前者的细胞增殖能力较后者明显增强,而且细胞周期也明显缩短.当用BAPTA-AM和EGTA或A23187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2+浓度时可改变细胞的生长周期,二者也呈现明显差别.这些结果表明,对顺铂产生耐药性的人肺腺癌A549/DDP细胞胞内Ca2+浓度的降低,可能影响细胞的增殖,缩短细胞的生长周期,特别是影响起决定作用的G1期,从而有利于肿瘤细胞多药耐药特性的维持. 相似文献
14.
A method is described for monitoring intracellular ionized calcium (Ca2+) and determining kinetic and thermodynamic parameters of Ca+-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca2+ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca2– that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca2+ with a rate constant of 0.031 ± 0.003 sec–1, maximal rate of 23 ± 7 nM/sec, dissociation constant of 366 ± 63 nM, Hill coefficient of 2.3 ± 0.4, Q10 of 2.58 ± 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca2+-extrusion system of lymphocytes and may be applicable to other blood cell types.Abbreviations DMSO
dimethyl sulfoxide
- HEPES
[N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid],sodium salt
- Indo-1/AM
acetoxymethyl ester of indo-1
- IP3
inositol 1,4,5-triphosphate
- IP4
inositol 1,3,4,5-tetrakisphosphate
- RPMI
Roswell Park Memorial Institute — 1640 culture medium
- TPEN
tetrakis-[2-pyridylmethyl]-ethylenediamine 相似文献
15.
Quantitative analysis of calcium gradients and activity in growing pollen tubes ofLilium longiflorum
Summary Pollen tubes ofLilium longiflorum were loaded with quin-2 to determine the cytoplasmic free calcium. Quin-2-fluorescence was detected at 500 nm with alternating excitation at 340 nm and 360 nm. The calcium2+-concentration was obtained using the intensity ratio R=I340/I360. The analysis exhibits a [Ca2+] of nearly 10–7mol·l–1 in the tip region and about 2·10–8 mol·l–1at the tube base, near the pollen grain. The data give evidence for the existence of a continuous gradient of free calcium within growing pollen tubes of various length. 相似文献
16.
Summary Intracellular free calcium ion concentrations ([Ca2+]i) in the anterior prestalk and posterior prespore cells of theDictyostelium discoideum slug were determined, using the highly selective Ca2+ indicators, quin-2/AM and fura-2/AM. Temporal changes in [Ca2+]i in response to chemotactic stimulation with cAMP were also monitored at the single-cell level and compared between the two types of cells. The results obtained showed that resting [Ca2+]i in the prestalk cells is considerably higher than that in the prespore cells. Moreover, transient increase in [Ca2+]i upon stimulation with a low concentration of cAMP (20 nM) was noticed only in the prestalk cells, but not in the prespore cells. These facts are discussed in relation to the polarized movement and cellular differentiation in the migrating slug.Abbreviations cAMP
3,5-cyclic adenosine monophosphate
- DIF
differentiation-inducing factors
- IP3
inositol 1,4,5-triphosphate 相似文献
17.
《Life sciences》1993,53(22):PL359-PL364
This study examines the mechanism of action of ketamine, a dissociative anesthetic, with a specific focus on its ability to inhibit changes in the concentration of intracellular free calcium, [Ca2+]i, in PC-12 cells. The resting [Ca2+]i as measured with the fluorescent probe Fura-2 AM in control cells is 184.8±8.6 nM (mean±SEM, n = 15). Changes in [Ca2+]i via influx through voltage-gated calcium channels after membrane depolarization with potassium chloride were monitored in the absence and presence of various concentrations of ketamine. Potassium-depolarization caused a dose-dependent rapid increase in [Ca2+]i, averaging 62±5%, 33±2% and 18±3% (n = 10 each) above control levels for 70 mM, 50 mM and 35 mM KCl, respectively. Ketamine, in the dosage range studied (5 – 500 μM), inhibited the increase in [Ca2+]i stimulated by potassium-depolarization in a dose-dependent manner. The computer-fitted dose-response curve of the pooled data yielded a half maximal suppression concentration, ED50, of 33 μM. In conclusion, this study demonstrates that ketamine inhibits Ca2+ influx through voltage-gated Ca2+ channels in PC-12 cells at clinically relevant doses, and may play a role in ketamine's action as a general anesthetic agent. 相似文献
18.
Assessment of Fura-2 for measurements of cytosolic free calcium 总被引:21,自引:0,他引:21
Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
19.
C. Bandeira-Duarte C. A. M. Carvalho E. J. Cragoe Jr A. P. Carvalho 《Neurochemical research》1990,15(3):313-320
Preparations of synaptosomes isolated in sucrose or in Na+-rich media were compared with respect to internal pH (pH1), internal Ca2+ concentration ([Ca2+]i), membrane potential and45Ca2+ uptake due to K+ depolarization and Na+/Ca2+ exchange. We found that synaptosomes isolated in sucrose media have a pHi of 6.77±0.04 and a [Ca2+]i of about 260 nM, whereas synaptosomes isolated in Na+-rich ionic media have a pHi of 6.96±0.07 and a [Ca2+]i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca2+ only by voltage sensitive calcium channels (VSCC'S) when K+-depolarized, while the Na+-rich synaptosomes take up45Ca2+ both by VSCC'S and by Na+/Ca2+ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca2+ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca2+ uptake by VSCC'S, but not by Na+/Ca2+ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca2+ flux through channels and through Na+/Ca2+ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca2+ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF
2,7-Biscarboxyethyl-5(6)-carboxyfluorescein
- BCECF/AM
acetoxymethyl ester of BCECF
- [Ca2+]i
Internal free calcium ion concentration
- CBZ-DMB
5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil
- DMB
2, 4-dimethylbenzamil
- DMSO
dimethyl sulfoxide
- Indo-1/AM
acetoxymethyl ester of Indo-1
- MES
2-|N-Morpholino|ethanesulfonic acid
- NMG
N-methyl-D-glucamine
- pHi
internal pH
- TPP+
tetraphenylphosphonium
- p
plasma membrane potential 相似文献
20.
《Electromagnetic biology and medicine》2013,32(1):77-84
AbstractCalcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15?Hz, 50?Hz, 75?Hz and 100?Hz) and at a flux density of 2?mT. Intracellular calcium concentration ([Ca2+]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10?mM) was carried out to verify the function of the cardiac Na+/Ca2+ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca2+]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes. 相似文献