Real-time measurement of cytosolic free calcium concentration in HL-60 cells during static magnetic field exposure and activation by ATP

Calcium ions are involved in a number of important signal transduction pathways in cells. Cytosolic calcium concentration ([Ca(2+)](c)) can be affected by the activation of Ca(2+) channels through the action of ligands such as ATP. The response of [Ca(2+)](c) to ligands may be affected by external factors like magnetic fields. The purpose of this study was to determine if exposure to a static magnetic field (SMF) for 800 s altered the [Ca(2+)](c) response to ATP in undifferentiated HL-60 cells. We sham exposed or field exposed fura-2 loaded HL-60 cells to a SMF of 1, 10, and 100 mT. Cells were activated with ATP 300 s into the exposure. The level of [Ca(2+)](c) was followed before, during, and after field or sham exposure with a ratiometric fluorescence spectroscopy system. It was found that high concentrations of ATP resulted in greater [Ca(2+)](c) responses, but faster recovery to near basal levels. The application of 1, 10, or 100 mT SMF did not affect the [Ca(2+)](c) response to ATP. Future work could examine the effect of a longer SMF exposure on the [Ca(2+)](c) response to ATP. Longer exposures might provide sufficient time for morphological changes in the plasma membrane to occur.     

Real-time measurement of cytosolic free calcium concentration in Jurkat cells during ELF magnetic field exposure and evaluation of the role of cell cycle

Extremely low frequency magnetic fields (ELF MF) have been reported to alter a number of cell signaling pathways, including those involved in proliferation, differentiation and apoptosis where cytosolic free calcium ([Ca(2+)](c)) plays an important role. To better understand the biological conditions under which ELF MF exposure might alter [Ca(2+)](c), we measured [Ca(2+)](c) by ratiometric fluorescence spectrophotometry during exposure to ELF MF in Jurkat E6.1 cells synchronized to different phases of the cell cycle. Suspensions of cells were exposed either to a near zero MF (Null) or a 60 Hz, 100 microT sinusoidal MF superimposed upon a collinear 78.1 microT static MF (AC + DC). An initial series of experiments indicated that the maximum increase in [Ca(2+)](c) above baseline after stimulation with anti-CD3 was significantly higher in samples exposed to AC + DC (n = 30) compared to Null (n = 30) with the largest difference in G2-M enriched samples. However, in a second study with G2-M enriched cells, samples treated with AC + DC (n = 17) were not statistically different from Null-treated samples (n = 27). Detailed analysis revealed that the dynamics in [Ca(2+)](c) before and after stimulation with anti-CD3 were dissimilar between Null samples from each study. From the results, we concluded (i) that the ELF MF increased [Ca(2+)](c) during an antibody-induced signaling event, (ii) that the ELF MF effect did not depend to a large degree on cell cycle, and (iii) that a field-related change in [Ca(2+)](c) signaling appeared to correlate with features in the [Ca(2+)](c) dynamics. Future work could evaluate [Ca(2+)](c) dynamics in relation to the phase of the cell cycle and inter-study variation, which may reveal factors important for the observation of real-time effects of ELF MF on [Ca(2+)](c).     

In situ fiber‐optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence‐lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber‐optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green‐5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Factors confounding cytosolic calcium measurements in Jurkat E6.1 cells during exposure to ELF magnetic fields

Reported changes in the cytosolic calcium concentration ([Ca2+](c)) as a result of exposure to extremely low frequency (ELF) magnetic fields (MF) have been equivocal. In this study, we examine the possibility that some of these differences are attributable to variability associated with the cell cycle, pH of the suspension medium, and response to a calcium agonist. We used a custom designed spectrofluorimeter to measure [Ca2+](c) in Indo 1-AM loaded Jurkat E6.1 cells suspended in conditioned RPMI 1640 medium containing 10% fetal bovine serum. Four exposures were examined: zero static MF (Null), 60 Hz 100 microT(peak) sinusoidal MF (AC), 78 microT static MF (DC), and the combination of the 60 Hz and the 78 microT static MF (AD + DC). A significant decrease in normalized [Ca2+](c) values between 375-495 s for the DC and AC + DC groups was found in comparison to the Null group. However, statistical analysis indicated that cell cycle and quality of the alpha-CD3 monoclonal antibody response were significant covariates, while pH was not a significant covariate. When the effect of these covariates was taken into account, all exposure groups were significantly different from the control. Our results suggest that ELF MF effects may not be seen unless correction is made for biological variability of each cell preparation with respect to cell cycle and [Ca2+](c) response to antigen stimulation.     

Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation

In guard cells, activation of anion channels (I_anion) is an early event leading to stomatal closure. Activation of I_anion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the dynamics of the action of [Ca²⁺]_i on I_anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca²⁺]_i dynamics of I_anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca²⁺]_i using Fura‐2 fluorescence imaging. We found that I_anion rises with [Ca²⁺]_i only at concentrations substantially above the mean resting value of 125 ± 13 nm , yielding an apparent K_d of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca²⁺ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I_anion activity, but without a dependence of the current on [Ca²⁺]_i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca²⁺]_i sensitivity of I_anion, displacing the apparent K_d for [Ca²⁺]_i to 573 ± 38 nm . These findings support previous evidence for different modes of regulation for I_anion, only one of which depends on [Ca²⁺]_i, and they underscore an independence of [Ca²⁺]_i from protein (de‐)phosphorylation in controlling I_anion. Most importantly, our results demonstrate a significant displacement of I_anion sensitivity to higher [Ca²⁺]_i compared with that of the guard cell K⁺ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.     

ATP-induced calcium oscillations and change of P2Y subtypes with culture conditions in HeLa cells

ATP, UTP, ADP and UDP induced intracellular Ca(2+) responses and oscillations in HeLa cells that sometimes lasted over 1 h. The response is due to the activation of P2Ys, G-protein coupled ATP receptors, because the oscillations persisted for several minutes even in Ca(2+)-free solution, and suramin and PPADS, antagonists of ATP receptors, partially inhibited the response. The potency of these nucleotides varied with the culture or cell conditions, i.e. UTP was generally most potent but in some cases UDP was more potent; responses to UDP were variable while those to ATP were constant. In addition, Ca(2+) responses to ATP and UDP were additive. These findings suggested the existence of two or more subtypes of P2Ys in HeLa cells. RT-PCR experiments revealed the existence of P2Y(2), P2Y(4) and P2Y(6). Recovery from starvation (culture in FBS-free medium overnight and re-addition of FBS) increased the responses to UTP and UDP but not to ATP, suggesting that the number or activity of P2Y(6) and/or P2Y(4) receptors may increase with cell proliferation in HeLa cells.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Label‐free in vivo cellular‐level detection and imaging of apoptosis

Cell death plays a critical role in health and homeostasis as well as in the pathogenesis and treatment of a broad spectrum of diseases and can be broadly divided into two main categories: apoptosis, or programmed cell death, and necrosis, or acute cell death. While these processes have been characterized extensively in vitro, label‐free detection of apoptosis and necrosis at the cellular level in vivo has yet to be shown. In this study, for the first time, fluorescence lifetime imaging microscopy (FLIM) of intracellular reduced nicotinamide adenine dinucleotide (NADH) was utilized to assess the metabolic response of in vivo mouse epidermal keratinocytes following induction of apoptosis and necrosis. Results show significantly elevated levels of both the mean lifetime of NADH and the intracellular ratio of protein bound‐to‐free NADH in the apoptotic compared to the necrotic tissue. In addition, the longitudinal profiles of these two cell death processes show remarkable differences. By identifying and extracting these temporal metabolic signatures, apoptosis in single cells can be studied in native tissue environments within the living organism.

     

Intracellular calcium activity in isolated bovine adrenal chromaffin cells in the presence and absence of 60 Hz magnetic fields

This study examined whether 60 Hz magnetic field (MF) exposure alters intracellular calcium levels ([Ca(2+)](i)) in isolated bovine adrenal chromaffin cells, a classic model of neural responses. [Ca(2+)](i) was monitored by fluorescence video imaging of cells loaded with the calcium indicator fluo-4 during exposures to magnetic flux densities of 0.01, 0.1, 1.0, 1.4, or 2.0 mT. MFs generated by Helmholtz coils constructed from bifilar wire allowed both 60 Hz field and sham exposures. Following a 5 min monitoring period to establish baseline patterns, cells were subjected for 10 min to a 60 Hz MF, sham field or no field. Reference calcium responses and assessment of cell excitability were obtained by the sequential addition of the nicotinic cholinergic receptor agonist dimethylphenylpiperazinium (DMPP) and a depolarizing concentration of KCl. Throughout an 8 day culture period, cells exhibited spontaneous fluctuations in [Ca(2+)](i). Comparisons of the number of cells exhibiting transients, the number and types of calcium transients, as well as the time during monitoring when transients occurred showed no significant differences between MF exposed cells and either sham exposed or nonexposed cells. With respect to the percentage of cells responding to DMPP, differences between 1 and 2 mT exposed cells and both nonexposed and sham exposed cells reached statistical significance during the first day in culture. No statistically significant differences were observed for responses to KCl. In summary, our data indicate that [Ca(2+)](i) in chromaffin cells is unaffected by the specific 60 Hz MF intensities used in this study. On the other hand, plasma membrane nicotinic receptors may be affected in a manner that is important for ligand-receptor interactions.     

Effect of ultra‐strong static magnetic field on bacteria: Application of Fourier‐transform infrared spectroscopy combined with cluster analysis and deconvolution

A new method based on Fourier‐transform infrared (FTIR) spectroscopy combined with cluster analysis and deconvolution was established to investigate the biological effect of an ultra‐strong static magnetic field (SMF) of 10.0 T on Escherichia coli and Staphylococcus aureus. FTIR spectroscopy was applied to characterize the spectroscopic fingerprints of these bacterial cells with or without the treatment of the SMF. After the calculation, the results of cluster analysis indicated that the SMF had significant effects on E. coli compared with S. aureus, and the effects were reflected by the changes of spectral region of 1500–1200 cm^?1. The deconvolution results of this major indication region showed that the composition and conformation of nucleic acid, protein, and fatty acid of E. coli were altered under the magnetic conditions. Bioelectromagnetics 30:500–507, 2009. © 2009 Wiley‐Liss, Inc.     

Development of chromatographic and free radical scavenging activity fingerprints by thin‐layer chromatography for selected Salvia species

Introduction – Plant‐derived free radical scavengers have become the subject of intensive scientific interest. Recently, the concept of coupling chromatographic fingerprints with biological fingerprinting analysis has gained much attention for the quality control of plant extracts. However, identification of free radical scavenging activity of each single compound in a complex mixture is a difficult task. Thin‐layer chromatography with post‐chromatographic derivatisation with the methanol solution of DPPH can be a valuable tool in such analyses. Objective – Development of chromatographic and free radical scavenging fingerprints of nineteen Salvia species grown and cultivated in Poland. Methodology – Chromatography was performed on the silica gel layers with use of two eluents, one for the resolution of the less polar compounds, and the other one for the resolution of the medium and highly polar ones. The plates were sprayed with the vanillin–sulfuric acid reagent to produce chemical fingerprints, and with DPPH solution to generate free radical scavenging fingerprints. Results – With four Salvia species, it was revealed that their strong free radical scavenging properties are not only due to the presence of polar flavonoids and phenolic acids, but also due to the presence of several free radical scavengers in the less polar fraction. Because of the similarities in both the chromatographic and the free radical scavenging fingerprints, S. triloba can be introduced as a possible equivalent of the pharmacopoeial species, S. officinalis. Conclusion – Fingerprints developed in the experiments proved useful for the analysis of complex extracts of the different Salvia species. Copyright © 2010 John Wiley & Sons, Ltd.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

Alzheimer mouse brain tissue measured by time resolved fluorescence spectroscopy using single‐ and multi‐photon excitation of label free native molecules

Time resolved spectroscopic measurements with single‐photon and multi‐photon excitation of native molecules were performed ex vivo on brain tissues from an Alzheimer's disease (AD) and a wild type (WT) mouse model using a streak camera. The fluorescence decay times of native NADH and FAD show a longer relaxation time in AD than in WT tissue, suggesting less non‐radiative processes in AD. The longer emission time of AD may be attributed to the coupling of the key native building block molecules to the amyloid‐tau and/or to the caging of the native fluorophores by the deposition of amyloid‐beta or tau plaques and neurofibrillary tangles that affect the local non‐radiative interactions.

     

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA

By using a FURA2 ratio imaging method, the intracellular free calcium concentration was investigated in cultured mice neuroblastoma cells under the influence of an amplitude-modulated (AM) field (5 kHz sine wave AM 16 Hz sinusoidal 800 V/m and 80 V/m), as well as of electric field pulses (300-ms unipolar pulses of 1000 V/m and 800 V/m, 5 pulses during 10 s and 50 pulses during 100 s). An increase in free intracellular calcium was found in about 50% of cells after field application, whereas in control experiments only about 20% of the cells showed similar increases. However, this effect depended on the amount of UV irradiation used for excitation of FURA2 fluorescence. Experiments with 1/30 to former total illumination no longer demonstrated an increase in control cells or in cells treated with AM fields. The number of cells showing calcium increase after the application of pulsed fields was reduced significantly. Therefore, the UV light itself, applied as double flashes for the fluorescence measurement, activates the cellular calcium regulation. These findings offer a possible explanation for the low reproducibility of field effects found in different laboratories, in which investigations were performed with different equipment using different intensities of UV excitation. Bioelectromagnetics 18:595–597, 1997. © 1997 Wiley-Liss, Inc.     

Characterization and applications of serum‐free induced adhesion in jurkat suspension cells

In this study, we demonstrate that the presence of serum in different media plays an important role in inducing transient and reversible adhesion in Jurkat suspension cells. Attachment of Jurkat cells in two distinct media formulations (serum‐fortified and serum‐free) to untreated polystyrene (PS), plasma‐treated PS, and fibronectin‐coated PS was compared. Additional analysis characterized the occurrence of this transient cell adhesion, including attachment rate, reversibility of attachment, and viability and preservation of phenotype in cells during and after attachment. As a demonstration of the utility of this technique, a few applications of transiently adhering Jurkat cells are shown which would be otherwise difficult with freely suspended cells, such as increased gene delivery, confocal‐based apoptosis detection, and real‐time electric‐field effect monitoring in Jurkat cells. Biotechnol. Bioeng. 2010;106: 784–793. © 2010 Wiley Periodicals, Inc.     

Eupatilin inhibits T‐cell activation by modulation of intracellular calcium flux and NF‐κB and NF‐AT activity

Eupatilin, one of the pharmacologically active ingredients of Artemisia princeps, exhibits a potent anti‐ulcer activity, but its effects on T‐cell immunity have not been investigated. Here, we show that eupatilin has a profound inhibitory effect on IL‐2 production in Jurkat T cells as well as in human peripheral blood leukocytes. Eupatilin neither influenced clustering of CD3 and LFA‐1 to the immunological synapse nor inhibited conjugate formation between T cells and B cells in the presence or absence of superantigen (SEE). Eupatilin also failed to inhibit T‐cell receptor (TCR) internalization, thereby, suggesting that eupatilin does not interfere with TCR‐mediated signals on the membrane proximal region. In unstimulated T cells, eupatilin significantly induced apoptotic cell death, as evidenced by an increased population of annexin V⁺/PI⁺ cells and cleavage of caspase‐3 and PARP. To our surprise, however, once cells were activated, eupatilin had little effect on apoptosis, and instead slightly protected cells from activation‐induced cell death, suggesting that apoptosis also is not a mechanism for eupatilin‐induced T‐cell suppression. On the contrary, eupatilin dramatically inhibited I‐κBα degradation and NF‐AT dephosphorylation and, consequently, inhibited NF‐κB and NF‐AT promoter activities in PMA/A23187‐stimulated T cells. Interestingly, intracellular calcium flux was significantly perturbed in cells pre‐treated with eupatilin, suggesting that calcium‐dependent cascades might be targets for eupatilin action. Collectively, our results provide evidence for dual regulatory functions of eupatilin: (1) a pro‐apoptotic effect on resting T cells and (2) an immunosuppressive effect on activated T cells, presumably through modulation of Ca²⁺ flux. J. Cell. Biochem. 108: 225–236, 2009. © 2009 Wiley‐Liss, Inc.