Calcium spiking activity and baseline calcium levels in ROS 17/2.8 cells exposed to extremely low frequency electromagnetic fields (ELF EMF).

PURPOSE: To determine whether extremely low frequency electromagnetic fields can alter average free cytosolic calcium ion concentrations [Ca2+]i and transient increases in [Ca2+]i in populations of ROS 17/2.8 cells. MATERIALS AND METHODS: Cells loaded with the calcium-selective luminescent photoprotein, aequorin, were placed in the bottom of a sample chamber, which was inserted into the gap of a previously described air gap reactor system where they were exposed either to sinusoidal magnetic fields at a variety of frequencies and flux densities or to sham conditions. Real-time recordings of photon counts due to aequorin luminescence were obtained and data were analysed with the use of probit plots. RESULTS: Probit plots of data obtained from cells exposed to the various magnetic fields were virtually superimposable over the data obtained for the same cultures during pre- and post-exposure sham or no-field periods. CONCLUSION: These experiments provided no evidence for any effects of ELF EMF, either positive or negative, on either average [Ca2+]i or on transient increases in [Ca2+]i.

Mechanisms underlying spontaneous calcium spiking in aequorin-loaded ROS 17/2.8 cells.

In earlier studies, McLeod and coworkers reported the detection of spontaneous calcium spiking in ROS 17/2.8 cells which they suggested was derived from individual cells progressing through mitosis or the cell cycle. They also indicated that the degree of spiking could be modulated by exposure of the cells to time-varying extremely low frequency electric fields. Given the implications of such observations for our understanding of the effects of electromagnetic fields on biological systems, it appeared important for mechanistic reasons to understand the basis of this spiking. In this study, we were able to confirm that spontaneous calcium spiking activity could be detected in ROS 17/2.8 cells and that this appeared to emanate from individual cells. We found this spiking to be completely dependent on extracellular calcium ions and to be independent of the inositol 1,4,5-trisphosphate-sensitive intracellular calcium store. This spiking is not reduced by treatments which slow down or block the passage of cells through the cell cycle. Further, we found that spiking was only detectable in the most highly aequorin-loaded subpopulation of cells whose growth rate is reduced and whose morphological appearance is abnormal. In conjunction with what is known about calcium spiking in other, nonexcitable mammalian cells in culture, the data presented strongly argue that the spontaneous calcium spiking observed in ROS 17/2.8 cells is unrelated to normal events of the cell cycle and most likely result from the damaging effects of excessive loading with aequorin.

Power-frequency electromagnetic fields and the capacitative calcium entry system in SV40-transformed Swiss 3T3 cells.

The present study was designed to test the hypothesis that a 60 Hz electromagnetic field could affect the influx of calcium ions across the plasma membrane through the so-called capacitative calcium entry system. Recordings of cytosolic calcium-ion concentrations in SV40-transformed Swiss 3T3 cells were obtained in real time during exposure to magnetic fields ranging from 0.3-50 mT or to sham conditions using the calcium-sensitive photoprotein aequorin. This was done for cell populations whose capacitative entry system was activated by either bradykinin or thapsigargin under a variety of experimental conditions. No effects of the magnetic field were observed on bradykinin-induced calcium transients and, with the exception of a small but statistically significant increase observed in experiments performed at 50 mT, no effects of the fields were observed on baseline calcium levels prior to or after such transients. The magnetic fields also had no effects on the size or kinetics of any of the thapsigargin-induced calcium transients. Overall, the data fail to support the hypothesis tested in this work.

Stimulus-secretion coupling in porcine adrenal chromaffin cells: effect of dexamethasone.

Recent studies from this laboratory established that dexamethasone (DEX) potentiates Ca2+ current via voltage-gated Ca2+ channels (VGCC), and as a consequence potentiates agonist-induced cytosolic Ca2+ transients in rat adrenal chromaffin cells. The present study examined whether DEX can also modulate VGCC activity and agonist-induced cytosolic Ca2+ transients in porcine adrenal medullary chromaffin (PAMC) cells, and if so whether this results in alterations in catecholamine secretion. Forty-eight-hr exposure to 1 microM DEX significantly increased peak Ca2+ current (delta + 138%; n = 6; P < 0.05) in PAMC cells. DEX treatment also significantly potentiated the increase in cytosolic Ca2+ in response to membrane depolarization with KCl (delta + 20%; n = 29; P < 0.05), but did not affect the amplitude of Ca2+ transients elicited by nicotine or acetylcholine. Despite the potentiation of intracellular Ca2+, DEX treatment had no effect on KCl-induced secretion of either norepinephrine or epinephrine. These data demonstrate that as in the rat chromaffin cell, DEX can also increase VGCC activity in PAMC cells. However, the subsequent potentiation of selected agonist-induced increases in intracellular Ca2+ does not appear to be sufficient to alter catecholamine secretion.

Blockade of plasma membrane calcium pumping ATPase isoform I impairs nerve growth factor-induced neurite extension in pheochromocytoma cells.

Numerous lines of evidence indicate that calcium signaling is essential for nerve growth factor (NGF)-directed neuronal cell differentiation. We report here that blocking production of the plasma membrane Ca(2+)-ATPase isoform 1 (PMCA1) in PC6 cells with antisense RNA impairs their ability to extend normal neurites in response to NGF. This result does not appear to be due to loss in NGF signaling as NGF-dependent tyrosine phosphorylation of erk1 and erk2, as well as expression of the NGF-inducible immediate early gene, NGFI-A, was observed in these cells. Resting cytosolic calcium levels did not appear to be altered in the antisense transfectants and release of calcium from internal bradykinin-sensitive calcium pools was unchanged. However, the rate of removal of free cytosolic calcium following this release was reduced in the antisense-transfected cells compared with controls. It is concluded that PMCA1 is involved in neurite extension and/or stabilization either through moderation of local calcium levels, or by some other mechanism.

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts.

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.

Altered calcium regulation in SV40-transformed Swiss 3T3 fibroblasts.

Calcium homeostasis has long been thought to be altered in transformed cells but mechanisms have not been established. In this study, the photoprotein, aequorin, was used to examine calcium regulation in 3T3 and SV40-transformed 3T3 cells. It was found that calcium transients induced by bradykinin or serum in serum-starved cells are lower and delayed in the transformed cells and decay kinetics are altered. These changes are not related to differences in cell cycle distribution. Though the serum transient is insensitive to nifedipine, verapamil, or lanthanum, removal of extracellular calcium accelerates transient decay in both cell types. Treatment of unstimulated cells with the ER Ca(2+)-ATPase inhibitor, thapsigargin, causes a 4-5-fold greater increase in [Ca2+]i in the transformed than in the nontransformed cells. Following serum stimulation, transformed cells still exhibit a large thapsigargin-induced increase in [Ca2+]i whereas the response in nontransformed cells is nearly abolished. When the 3T3 or SV3T3 cells are exposed to serum or thapsigargin in the absence of extracellular calcium and subsequently exposed to 11.8 mM Ca2+, a much greater influx of calcium again occurs in the SV3T3 cells. The observed changes in SV3T3 cells are most likely due to an alteration in a capacitative mechanism which regulates influx of calcium through the plasma membrane.

Design and characterization of a system for exposure of cultured cells to extremely low frequency electric and magnetic fields over a wide range of field strengths.

A system is described that is capable of producing extremely low frequency (ELF) magnetic fields for relatively short-term exposure of cultured mammalian cells. The system utilizes a ferromagnetic core to contain and direct the magnetic field of a 1,000 turn solenoidal coil and can produce a range of flux densities and induced electric fields much higher than those produced by Helmholtz coils. The system can generate magnetic fields from the microtesla (microT) range up to 0.14 T with induced electric field strengths on the order of 1.0 V/m. The induced electric field can be accurately varied by changing the sample chamber configuration without changing the exposure magnetic field. This gives the system the ability to separate the bioeffects of magnetic and induced electric fields. In the frequency range of 4-100 Hz and magnetic flux density range of 0.005-0.14 T, the maximum total harmonic distortion of the induced electric field is typically less than 1.0%. The temperature of the samples is held constant to within 0.4 degrees C by constant perfusion of warmed culture medium through the sample chamber.

Cytokinesis is more rapid in Ha-T24-ras transfected rat embryo fibroblasts than in non-transfected control cells.

It has long been known that neoplastic cells are characterized by increases in cell motility. Earlier studies from this laboratory indicated that mitotic events were also altered in many tumor and experimentally transformed cells and that this included increases in metaphase duration and a reduction in the duration of cytokinesis. The studies presented in this paper were done to determine whether or not transfection of normal rat embryo fibroblasts by the Ha-T24-ras oncogene could also produce such alterations in mitotic events. The results obtained with the use of time lapse video microscopy indicate that neither the duration of metaphase nor the rate of chromosome movement during anaphase was altered but that the rate of furrow progression during cytokinesis occurred at a significantly more rapid rate. Thus, the cellular alterations induced by transfection with Ha-T24-ras accelerate microfilament-dependent cytokinetic furrowing without significant effects on microtubule-dependent mitotic events. One of several possible mechanisms that could account for these observations involves a down regulation of protein kinase C which has been reported to occur in many neoplastic cells including those transformed by ras. Such a hypothesis could also have broader implications because it may be applicable to the increase in motility and metastatic activity generally observed in transformed cells.

Sensitivity of cultured human embryonic cerebral cortical neurons to excitatory amino acid-induced calcium influx and neurotoxicity.

Although there has been a large body of literature from animal studies concerning neuronal excitatory amino acid (EAA) receptors and their possible roles in brain development, function, and pathology, essentially no direct information on actions of EAAs in humans has previously been available. We now report on experiments in cell cultured human embryonic cerebral cortical neurons which directly addressed the actions of EAAs in the developing human brain. In cultures established from 14-week fetuses, neurons were insensitive to glutamate neurotoxicity during the first 30 days in culture. After 30 days in culture increasingly more neurons became vulnerable to glutamate acting at the N-methyl-D-aspartate and kainate type receptors. The development of calcium responses to glutamate (as measured with the calcium indicator dye fura-2) preceded sensitivity to excitotoxicity by several weeks in the human neurons. Glutamate-induced rises in intracellular calcium and neurotoxicity developed much more rapidly in rat cortical neurons. Studies of dynamic aspects of calcium responses to calcium ionophore A23187 in human and rat cortical neurons demonstrated a direct relation between calcium buffering ability and resistance to EAA neurotoxicity. Interestingly, the human neurons were better able to buffer a calcium load than were rat neurons, suggesting that species-specific and/or developmental stage-specific differences in calcium-buffering systems are likely to play roles in determining neuronal vulnerability to EAAs. These initial observations indicate that human cortical neurons become sensitive to EAAs during the prenatal period, and suggest that EAAs may play important roles in both normal human brain development and neurodegenerative processes.

Roberts syndrome with normal cell division.

Roberts-SC phocomelia syndrome (RS) is an autosomal recessive disorder of symmetric limb defects, craniofacial abnormalities, pre- and postnatal growth retardation, and mental retardation. Patients with RS have been reported to have premature separation of heterochromatin of many chromosomes and abnormalities in the cell-division cycle. We report an infant whose clinical and radiologic findings resemble those of RS but who lacks the cytogenetic and cell division abnormalities reported in RS. This patient may represent a variant of RS or a new syndrome.

Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.

Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection of mammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to utilize the salvage pathway of pyrimidine biosynthesis. These loading methods can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and stably. Optimal transfection is observed when the DNA is added to cells in physiological saline lacking divalent cations and containing K+ in place of Na+. DNA molecules 7.1 to 30 kilobases long have been introduced by the scrape loading procedure. In addition, the scrape loading procedure has been employed for cotransfection and subsequent expression of nonselectable genes encoded on DNA molecules added in a mixture with DNA molecules whose expression is selected. Cell lines expressing oncogenes or proteins that are important for regulation of cell growth and division have been obtained by this procedure. The scrape loading procedure is also useful for studies of the cellular changes that occur upon expression of an exogenous gene. As many as 80% of cells scrape loaded with the plasmid pC6, which encodes the simian virus 40 large tumor antigen, contained this protein in the nucleus between 1 and 5 days after transfection. Thus, scrape loading and sonication loading are simple, economical, and reproducible methods for introduction of DNA molecules into adherent and nonadherent cells, and these methods may be useful in the future for experimentation at both fundamental and applied levels.

The study of fluorescent probes by quantitative video intensification microscopy (QVIM).

In this paper we describe a system for the quantitation and display of fluorescence at the cellular level. It uses a low light level video camera which is interfaced to a fluorescence microscope and to a microprocessor-controlled video digitizing system. With the use of a light pen entry system one can specify areas of the field for measurement. The data obtainable are the area and perimeter of the delimited zone, the distribution of pixel intensities within this zone over a 16-level gray scale, and a value for total fluorescence intensity. Statistical outputs for repeated measurements are also obtained. The system responds linearly to light input, has a high degree of reproducibility, and provides good spatial resolution. Using the DNA-specific dye, Hoechst 33248, in diploid fibroblasts as test material, the system is shown to be able to reproduce expected distributions for amounts of DNA per cell. The capabilities and advantages of pseudocolor display are also demonstrated. We conclude that, in conjunction with appropriate fluorescent probes, systems such as the one described make it possible to do quantitative histochemistry of living cells and to measure substances not previously amenable to study.

Differential sensitivity of metaphase to diamide and ouabain in HeLa cells.

We have examined the effects of diamide, an oxidizer of glutathione, on the progress of HeLa cells through the cell cycle. At concentrations which do not significantly alter generation time, anaphase or cytokinesis, diamide causes a two-fold increase in the duration of metaphase. At 3 X 10(-8) M, ouabain also prolongs metaphase without effect on anaphase or cytokinesis, though with a different time course. The data suggest that the metaphase stage of mitosis is particularly sensitive to alterations in both sulfhydryl groups and Na+ levels but that the effects of diamide are probably not primarily due to the oxidation of sulfhydryl groups of the Na+/K+-ATPase.

The alteration of mitotic events by ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone.

A large quantity of published work indicates that calcium ions may be involved in the regulation of mitotic events and recent reports suggest that the onset of chromosome movement is dependent upon a transient increase in free cytosolic calcium ions. In this paper we examine the effects of two agents known to perturb intracellular calcium pools on mitosis in HeLa cells. These were the calcium-selective ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone (CCCP), which is a protonophoric inhibitor of oxidative phosphorylation. Owing to a stimulation of glycolysis, the latter agent does not decrease intracellular ATP in HeLa but does cause mitochondria to release calcium ions. Our data show that, at low concentrations, both agents prolong metaphase but differ in their effects on anaphase and cytokinesis. Studies with chlorotetracycline, a commonly used probe for membrane-associated calcium, verify that these agents do affect calcium pools under the conditions of our experiments. The data presented are consistent with the idea that increased cytosolic calcium levels can directly or indirectly affect mitotic events but, contrary to other suggestions, cause a prolongation of metaphase, i.e. they delay the onset of chromosome movement.

Alterations in metaphase durations in cells derived from human tumours.

With the use of time-lapse cinemicrography, we previously found that metaphase durations were significantly prolonged in SV40-transformed human fibroblasts when compared to untransformed controls. This was consistent with some earlier reports and suggested that prolonged metaphases could account for high metaphase/prophase ratios and possibly, in part, for increased mitotic indices seen in advanced tumours. However, there are inconsistencies in the literature and no comparable data available from malignant carcinomas. Presented in this paper are data from two cervical dysplasias, two cases of carcinoma in situ, nine malignant carcinomas and several other types of human cells. The results show that mean metaphase durations were prolonged in cells derived from most of the carcinomas but not from the other cell types. On the other hand, cytokinesis appears to progress more rapidly than normal in most of the tumour-derived cells. These and other findings indicate that the changes are a result of some metabolic alteration common to many but not all tumour cells. For reasons presented, we suggest as a working hypothesis that the alterations may be due to changes in calcium regulation, possibly resulting from alterations in mitochondrial metabolism.

Studies on the role of Ca++ in cell division with the use of fluorescent probes and quantitative video intensification microscopy.

It is clear that QVIM systems, when combined with appropriate fluorescent probes can be utilized to perform quantitative cytochemical studies on living and fixed cells. They also have the potential to facilitate studies of substances which like Ca++ are not easily studied by other means. The preliminary studies we have described support the idea that calcium ions and calcium transport enzymes may indeed play important roles in cell division and indicate that the tools we have at hand should help us further our understanding of the mitotic process.

Abnormalities in the cell-division cycle in Roberts syndrome fibroblasts: a cellular basis for the phenotypic characteristics?

Roberts-SC phocomelia syndrome (RS) is a recessively inherited developmental disorder characterized by profound pre- and postnatal growth reduction, symmetrical limb reductions of varying severity, and craniofacial abnormalities. Many patients with RS exhibit a striking chromosomal abnormality involving the heterochromatic, C-banding regions of most chromosomes. Dermal fibroblast strains from three such patients were used to investigate in vitro cellular growth characteristics. Plating efficiency, colony-forming ability, and cell density at confluence in RS were compared with dermal fibroblast strains from pediatric patients without RS and fetal lung fibroblast strains. Time-lapse cinematography was used to study mitotic duration and cytokinesis in RS and various control fibroblast strains. Clearly, cell from affected individuals had deficiencies that led to a multitude of abnormalities at the cellular level. These included: abnormal mitosis and cytokinesis, reduced cell growth, atypical cell morphology, and altered chromosomal morphology at peri- and paracentromeric and nucleolar-organizing regions. These findings suggest that the basis for at least some of the phenotypic abnormalities characteristic of this trait may reside in the reduced growth rates of the cells during the course of development. This could account for the reduced pre- and postnatal growth rates as well as for the developmental abnormalities, since deficiencies of cells in developing anlagen could well lead to alterations in developmental patterns.

Measurement of fluorescence using digital integration of video images.

The authors describe a system for the quantitation of fluorescence light output by individual cells using the signal obtained from a silicon intensifier target video camera. The video image is digitized to 4 bits (16 levels), and a 512 X 512 matrix is constructed and stored in 128K of video memory. Areas to be measured are user-specified by means of a light pen entry system. Recorded intensities are integrated under microprocessor control to provide a measure of total fluorescence in the selected areas. The distribution of light intensities per pixel over the 16-level gray scale as well as morphometric data are also obtainable. Linear response to transmission and fluorescence standards was verified. Reproducibility of the system was evaluated using fluorescent beads and glutaraldehyde-fixed chick red blood cells, which gave coefficients of variation comparable to those obtainable from other systems. Measurements of DNA per nucleus of human diploid fibroblasts using Hoechst dye 33258 yielded the two sharp peaks corresponding to the 2C and 4C values of DNA expected from such cells. We conclude that digital video measurement of fluorescence provides meaningful data and has considerable promise as a sensitive tool for the quantitation of fluorescence at the cellular level.