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).     

Lack of adverse effects in pregnant/lactating female rats and their offspring following pre- and postnatal exposure to ELF magnetic fields

We have recently reported that exposure of pregnant rats to 60 Hz at field strengths up to 0.5 mT during the entire period of pregnancy did not induce any biologically significant effects on both pregnant dams and embryo-fetal development. The present study was carried out to investigate the potential effects of gestational and lactational MF exposure on pregnancy, delivery, and lactation of dams and growth, behavior, and mating performance of their offspring in rats. Timed-pregnant female Sprague-Dawley (SD) rats (24/group) received continuous exposure to 60 Hz magnetic field (MF) at field strengths of 0 (sham control), 5 microT, 83.3 microT, or 0.5 mT. Dams received MF or sham exposures for 21 h/day from gestational day 6 through lactational day 21. Experimentally generated MF was monitored continuously throughout the study. No exposure-related changes in clinical signs, body weight, food consumption, pregnancy length, and necropsy findings were observed in dams. Parameters of growth, behavior, and reproductive performance of offspring showed no changes related to MF exposure. There were no adverse effects on embryo-fetal development of F2 offspring from dams exposed to MF. In conclusion, exposure of pregnant SD rats to 60 Hz at field strengths up to 0.5 mT from gestational day 6 to lactational day 21 did not produce biologically significant effects in dams, F1 offspring, or F2 fetuses.     

An extremely low frequency magnetic field attenuates insulin secretion from the insulinoma cell line, RIN-m

In this study, we investigated the effects of exposure to an extremely low frequency magnetic field (ELFMF) on hormone secretion from an islet derived insulinoma cell line, RIN-m. We stimulated RIN-m cells to secrete insulin under exposure to an ELFMF, using our established system for the exposure of cultured cells to an ELFMF at 5 mT and 60 Hz, or under sham exposure conditions for 1 h and observed the effects. In the presence of a depolarizing concentration of potassium (45 mM KCl), exposure to ELFMF significantly attenuated insulin release from RIN-m cells, compared to sham exposed cells. Treatment with nifedipine reduced the difference in insulin secretion between cells exposed to an ELFMF and sham exposed cells. The expression of mRNA encoding synaptosomal associated protein of 25 kDa (SNAP-25) and synaptotagmin 1, which play a role in exocytosis in hormone secretion and influx of calcium ions, decreased with exposure to an ELFMF in the presence of 45 mM KCl. These results suggest that exposure to ELFMF attenuates insulin secretion from RIN-m cells by affecting calcium influx through calcium channels.     

Temperature sensitivity of catecholamine secretion and ion fluxes in bovine adrenal chromaffin cells

The effects of temperature on ion fluxes and catecholamine secretion that are mediated by nicotinic acetylcholine receptors (nAChRs), voltage-sensitive calcium channels (VSCCs), and voltage-sensitive sodium channels (VSSCs) were investigated using bovine adrenal chromaffin cells. When the chromaffin cells were stimulated with DMPP, a nicotinic cholinergic agonist, or 50 mM K+, the intracellular calcium ([Ca2+]i) elevation reached a peak and decreased more slowly at lower temperatures. The DMPP-induced responses were more sensitive to temperature changes compared to high K+-induced ones. In the measurement of intracellular sodium concentrations ([Na+]i), it was found that nicotinic stimulation required a longer time to attain the maximal level of [Na+]i at lower temperatures. In addition, the VSSCs-mediated [Na+]i increase evoked by veratridine was also reduced as the temperature decreased. The measurement of [3H]norepinephrine (NE) secretion showed that the secretion within the first 3 min evoked by DMPP or high K+ was greatest at 37 degrees C. However, at 25 degrees C, the secretion evoked by DMPP, but not that by the 50 mM K+, was greater after 10 min of stimulation. This data suggest that temperature differentially affects the activity of nAChRs, VSCCs, and VSSCs, resulting in differential [Na+]i and [Ca2+]i elevation, and in the [3H]NE secretion by adrenal chromaffin cells.     

Cytosolic organelles shape calcium signals and exo-endocytotic responses of chromaffin cells

The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis.     

Developmental toxicity evaluation of ELF magnetic fields in Sprague-Dawley rats

To identify possible effects of horizontally polarized magnetic field (MF) exposure on maintenance of pregnancy and embryo-fetal development, an MF exposure system was designed and constructed and 96 time-mated female Sprague-Dawley (SD) rats (24/group) received continuous exposure to 60 Hz MF at field strengths of 0 (sham control) and 5, 83.3, or 500 microT (50, 833, or 5000 mG). Dams received MF or sham exposures for 22 h/day on gestational day 6-20. MF was monitored continuously throughout the study. There were no evidences of maternal toxicity or developmental toxicity in any MF exposed groups. Mean maternal body weight, organ weights, and hematological and serum biochemical parameters in groups exposed to MF did not differ from those in sham control. No exposure related differences in fetal deaths, fetal body weight, and placental weight were observed between MF exposed groups and sham control. External, visceral, and skeletal examination of fetuses demonstrated no significant differences in the incidence of fetal malformations between MF exposed and sham control groups. In conclusion, exposure of pregnant rats to 60 Hz at MF strengths up to 500 microT during gestation day 6-20 did not produce any biologically significant effect in either dams or fetuses.     

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.     

Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells

Experiments assessed whether long term exposure to 50 Hz pulsed electromagnetic fields with a peak magnetic field of 3 mT can alter the dynamics of intracellular calcium in human astrocytoma U-373 MG cells. Pretreatment of cells with 1.2 microM substance P significantly increased the [Ca(2+)](i). The same effect was also observed when [Ca(2+)](i) was evaluated in the presence of 20 mM caffeine. After exposure to electromagnetic fields the basal [Ca(2+)](i) levels increased significantly from 143 +/- 46 nM to 278 +/- 125 nM. The increase was also evident after caffeine addition, but in cells treated with substance P and substance P + caffeine we observed a [Ca(2+)](i) decrease after exposure. When we substituted calcium-free medium for normal medium immediately before the [Ca(2+)](i) measurements, the [Ca(2+)](i) was similar to that measured in the presence of Ca(2+). In this case, after EMFs exposure of cells treated with substance P, the [Ca(2+)](i), measured without and with addition of caffeine, declined from 824 +/- 425 to 38 +/- 13 nM and from 1369 +/- 700 to 11 +/- 4 nM, respectively, indicating that electromagnetic fields act either on intracellular Ca(2+) stores or on the plasma membrane. Moreover the electromagnetic fields that affected [Ca(2+)](i) did not cause cell proliferation or cell death and the proliferation indexes remained unchanged after exposure.     

Prolonged Ca2+ transients in ATP-stimulated endothelial cells exposed to 50 Hz electric fields

Human umbilical vein endothelial cells were exposed to sinusoidal electric fields of 0.3 or 30 kV/m, 50 Hz, for 24 h. Changes in intracellular calcium concentration ([Ca(2+)](i)) induced by ATP-stimulation in the absence of extracellular Ca(2+) were observed in individual cells. No differences were observed between the exposure and sham-exposure groups in [Ca(2+)](i) resting level before ATP-stimulation, or in the [Ca(2+)](i) peak levels induced by stimulation. However, the duration of the initial transients in [Ca(2+)](i) following an ATP stimulus was significantly prolonged by exposure to a 30 kV/m field. The inositol trisphosphate receptor inhibitor, xestospongin C, inhibited the ATP-induced elevation in [Ca(2+)](i) in both the exposure and sham-exposure groups. The ATP-receptor P2Y appeared to play an important role in the increase of [Ca(2+)](i). The present results suggest that an extremely low-frequency electric field affects the function of vascular endothelial cells by a mechanism involving activation of P2Y.     

Effects of 50 Hz electric currents and magnetic fields on the prokaryote Propionibacterium acnes

The effects of 50 Hz sinusoidal electric currents and magnetic fields on the Gram-positive skin bacterium Propionibacterium acnes were investigated. Intracellular free calcium ([Ca(2+)](i)), intracellular pH (pH(i)), and cell viability were examined, based on their relevance to ELF field studies and on previous studies conducted on P. acnes (UVA irradiation, photosensitization using porphyrin-based sensitizers, and broad-band red light). The [Ca(2+)](i) and the pH(i) were measured spectrofluorimetrically using the fluorescent probes fura-2 and BCECF, respectively. Sham-exposed controls were used to assess the field exposed samples. Cell suspensions were exposed to 50 Hz, 0.2 mT sinusoidal magnetic fields generated by using Helmholtz coils for up to 30 min. The estimated maximum induced electric field was 0.2 mV/m. Changes in [Ca(2+)](i) and cell viability were not detected. Ag/AgCl electrodes were used to expose cell suspensions to 50 Hz sinusoidal electric currents. The current densities were in the range 0.015-1500 A/m(2) (corresponding electric fields congruent with0.01-1000 V/m). Changes in [Ca(2+)](i) were not observed after current exposure. Current densities of 800 A/m(2) (electric field E congruent with550 V/m) were required for a 50% reduction in cell viability. Current densities greater than 800 A/m(2) were required for a reduction in pH(i). However, a pH gradient across the cell membrane (inside alkaline) was maintained even when exposure resulted in less than 0. 2% survival (1400 A/m(2), E congruent with950 V/m). Thus, dissipation of the pH gradient across the cell membrane and changes in [Ca(2+)](i) were not a consequence of cell inactivation by 50 Hz electric currents. This is in contrast to inactivation of P. acnes by UVA irradiation or photosensitization, where such changes have been obtained.     

Nifedipine reveals the existence of two discrete components of the progesterone-induced [Ca2+]i transient in human spermatozoa

The steroid progesterone, an agonist of acrosome reaction, induces a biphasic [Ca(2+)](i)-signal in human sperm comprising an initial transient [Ca(2+)](i) elevation, and a subsequent ramp or plateau. Nifedipine, an inhibitor of voltage-operated Ca(2+) channels, inhibits progesterone-induced acrosome reaction in human sperm, but fluorimetric studies have detected no effect of this compound on the progesterone-induced [Ca(2+)](i) signal. We have used single-cell imaging to study the effects of nifedipine on [Ca(2+)](i) signalling in human sperm. Analysis of mean responses from large numbers of cells showed that treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced [Ca(2+)](i) transient. In control cells, the latency of the transient peak (maximum fluorescence) fell within the range of 30-105 s. In the presence of nifedipine, very few cells peaked "late" (>60 s after application of progesterone). Analysis of transient responses in control cells revealed characteristic "early" and "late" responses, most cells showing both "early" and "late" transients, whereas "late" transients were rare and smaller in the presence of nifedipine. Sustained responses showed strong association with late transients, and occurrence and amplitude of sustained responses were significantly reduced in nifedipine pretreated cells.These findings are consistent with the occurrence of a discrete, nifedipine-sensitive component of the progesterone-induced [Ca(2+)](i) transient that peaks 1-2 min after exposure to the hormone and is crucial for the induction of the sustained [Ca(2+)](i) signal.     

No Evidence of Cellular Alterations by MilliTesla-Level Static and 50 Hz Magnetic Fields on S. cerevisiae

In an attempt to determine whether magnetic field (MF) exposures might induce cellular alterations, S. cerevisiae yeast cells were exposed to static or sinusoidal 50?Hz homogeneous MF (0.35?mT, 1.4?mT, and 2.45?mT) for 1?h and 72?h. Unsynchronized cells grown exponentially while exposed to MF, containing cells in all stages of the mitotic cell cycle. MF was generated by a pair of Helmholtz coils (40?cm in diameter, coaxial, separated by 20?cm). Survival, cell cycle distribution, colony forming ability, and mutation frequency were assayed. No differences in the above-mentioned parameters were observed in MF exposed samples in relation to unexposed controls, suggesting that homogeneous MF at these intensities do not produce appreciable cellular alterations in this organism under typical in vitro growth conditions.     

Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells

In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca(2+) influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca(2+)](i) could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.     

No evidence of cellular alterations by MilliTesla-level static and 50 Hz magnetic fields on S. cerevisiae

In an attempt to determine whether magnetic field (MF) exposures might induce cellular alterations, S. cerevisiae yeast cells were exposed to static or sinusoidal 50?Hz homogeneous MF (0.35?mT, 1.4?mT, and 2.45?mT) for 1?h and 72?h. Unsynchronized cells grown exponentially while exposed to MF, containing cells in all stages of the mitotic cell cycle. MF was generated by a pair of Helmholtz coils (40?cm in diameter, coaxial, separated by 20?cm). Survival, cell cycle distribution, colony forming ability, and mutation frequency were assayed. No differences in the above-mentioned parameters were observed in MF exposed samples in relation to unexposed controls, suggesting that homogeneous MF at these intensities do not produce appreciable cellular alterations in this organism under typical in vitro growth conditions.     

Modes of secretagogue-induced [Ca]i responses in individual chromaffin cells of the perfusedrat adrenal medulla

Chromaffin cells in the perfused rat adrenal medulla were loaded with indo-1 for confocal image analyses. Resting levels of [Ca(2+)](i) in chromaffin cells were similar and were stable with time. This is in contrast to the situation in isolated rat chromaffin cells, in which spontaneous oscillations of [Ca(2+)](i) are known to occur. When chromaffin cells were stimulated for 3-4 min by high K(+) or nicotine, [Ca(2+)](i) increased to a peak in 20-30 s and then declined rather smoothly. In contrast, chromaffin cells stimulated by muscarine or low pH (6.5) commonly exhibited irregular oscillations in [Ca(2+)](i). This provides additional evidence supporting the previous claim that muscarine and low pH evoke catecholamine secretion using partly shared mechanisms. Although muscarine and low pH were speculated to produce weaker responses in noradrenaline-secreting cells due to their selective stimulation of adrenaline secretion, no clear indications for segregation of cell types from [Ca(2+)](i) responses to these stimulants were found. The perfused adrenal medulla loaded with Indo-1 was also employed for simultaneously monitoring integrated changes in [Ca(2+)](i)(Ca responses) by conventional microfluorometry and in catecholamine secretion from a whole medulla (secretory responses). When the profiles of secretory responses were approximated by the kth power of the profiles of Ca responses, the k-values were estimated to be 2.2 and 2.3 for high-K(+)- and nicotine-elicited responses, respectively, whereas a k-value of 1.4 was obtained for both muscarine- and low-pH-elicited responses. An analysis showed that the significant difference in the k-value with these two classes of stimulants is accounted for by the stimulant-dependent patterns of [Ca(2+)](i) responses found in confocal image analysis.     

The effect of oscillating low intensity magnetic field on the Na+, K+, Ca++, and Mg++ concentrations in the maternal and fetal circulation of the dually perfused human placental cotyledon

Dual-sided perfusions of the human placental cotyledon in vitro were used to study effects of low intensity magnetic fields (MFs) of 2 mT, 50 Hz (E1, 10 perfusions) and 5 mT, 50 Hz (E2, 10 perfusions). In the control group C (10 experiments) no field was used. Perfusions lasted 180 min each. Increased release of calcium ions from the placental cotyledon was found in the fetal circulation during perfusion when the 2 mT, 50 Hz MF was used. No changes in the release of sodium and magnesium ions were observed compared to the control group. The 5 mT, 50 Hz oscillating MF intensified the release of sodium ions from the perfused cotyledon both to the fetal and maternal circulation up to the 150th min of the experiment. Increased release of magnesium ions was observed only to the fetal circulation between 120 and 180 min and of calcium ions to the fetal circulation between 60 and 180 min. No significant differences in K concentrations were found between the control and MF exposed cotyledons under conditions of these experiments.     

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.     

Effects of docosahexaenoic acid on calcium pathway in adult rat cardiomyocytes

In this study we examined the effect of polyunsaturated fatty acids (PUFAs), in particular of docosahexaenoic acid (DHA), on calcium homeostasis in isolated adult rat cardiomyocytes exposed to KCl, ET-1 and anoxia. Free [Ca(2+)](i) in rat cardiomyocytes was 135.7 +/- 0.5 nM. Exposure to 50 mM KCl or 100 nM ET-1 resulted in a rise in free [Ca(2+)](i) in freshly isolated cells (465.4 +/- 15.6 nM and 311.3 +/- 12.6 nM, respectively) and in cultured cells (450.8 +/- 14.8 nM and 323.5 +/- 14.8 nM respectively). An acute treatment (20 minutes) with 10 microM DHA significantly reduced the KCl- and ET-1-induced [Ca(2+)](i) increase (300.9 +/- 18.1 nM and 232.08 +/- 11.8 nM, respectively). This reduction was greater after chronic treatment with DHA (72 h; 257.7 +/- 13.08 nM and 192.18 +/- 9.8 nM, respectively). Rat cardiomyocytes exposed to a 20 minute superfusion with anoxic solution, obtained by replacing O(2) with N(2) in gas mixture, showed a massive increase in cytosolic calcium (1200.2 +/- 50.2 nM). Longer exposure to anoxia induced hypercontraction and later death of rat cardiomyocytes. Preincubation with DHA reduced the anoxic effect on [Ca(2+)](i) (498.4 +/- 7.3 nM in acute and 200.2 +/- 12.2 nM in chronic treatment). In anoxic conditions 50 mM KCl and 100 nM ET-1 produced extreme and unmeasurable increases of [Ca(2+)](i.) Preincubation for 20 minutes with DHA reduced this phenomenon (856.1 +/- 20.3 nM and 782.3 +/- 7.6 nM, respectively). This reduction is more evident after a chronic treatment with DHA (257.7 +/- 10.6 nM and 232.2 +/- 12.5 nM, respectively). We conclude that in rat cardiomyocytes KCl, ET-1 and anoxia interfered with intracellular calcium concentrations by either modifying calcium levels or impairing calcium homeostasis. Acute, and especially chronic, DHA administration markedly reduced the damage induced by calcium overload in those cells.     

Extremely low frequency magnetic field effects on premorbid behaviors produced by cocaine in the mouse

We investigated the premorbid behavioral changes produced by the administration of cocaine and acute exposure to extremely low frequency (ELF) magnetic field (MF) in the mouse. ICR mice received intraperitoneal injections of cocaine at two doses (65 and 70 mg/kg) and were subsequently exposed to one of eight ELF-MF fields (2, 3, 4, 8, 10, 15, 25, or 60 Hz) of about 20 G (2 mT) intensity immediately after injection. Twelve mice were used for each of applied cocaine dose and ELF-MF level. For a given dose of cocaine, the applied MF frequencies were randomly ordered, and blind tests were carried out in which the behavior observer did not know the frequencies of MF. The premorbid behaviors were defined in the ICR mice and their changes were observed over the exposure of various ELF-MFs. Our data show that the onset times of stop rearing and tonic-clonic seizure in the 4 Hz MF exposure group are significantly different from those of the sham group.