Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca²⁺ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m²⁺) on the agonist-induced Ca²⁺ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 μM ATP or by 100 μM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2–100 μM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20–30% decrease in the magnitude of [Ca²⁺]_i elevation induced by a low concentration of ATP (<1 μM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20–40% increase of ATP-induced elevation of [Ca²⁺]_i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, K_m, value in ATP-treated cells and of the maximal [Ca²⁺]_i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca²⁺ and/or ion channels. Bioelectromagnetics 19:366–376, 1998. © 1998 Wiley-Liss, Inc.     

Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field

The effect of exposure of single rat pituitary cells to 50 Hz sine wave magnetic fields of various strengths on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was studied by using dual-emission microfluorimetry, using indo-1 as probe. A 30 min exposure of the cells to vertical 50 μT peak magnetic field triggered a long-lasting increase in [Ca²⁺]_i from a basal value of about 185 ± 4 nM to 326 ± 41 nM (S.E.; n = 150). The vertical and horizontal components of the static magnetic field were 57 and 15 μT, respectively. The 50 Hz ambient magnetic field was always below 0.1 μT rms. The effect was observed both at 25 ± 2 °C and at 37 ± 2 °C. Responsive cells, for which [Ca²⁺]_i rose to values above 309 nM, were identified as lactotrophs and represented 29% of the total pituitaries. [Ca²⁺]_i increase, for the most part, was due to Ca²⁺ influx through voltage-dependent dihydropiridine-sensitive calcium channels inhibited by PN 200-110. However, neither Ca²⁺ channel blockers nor removal of Ca²⁺ from the external medium during exposure completely prevented the field-induced [Ca²⁺]_i increase. Additional experiments using an MTT colorimetric assay showed that alteration of Ca²⁺ homeostasis of lactotrophs was associated with impairment of some mitochondrial processes. © Wiley-Liss, Inc.     

Calcium homeostasis of isolated heart muscle cells exposed to pulsed high-frequency electromagnetic fields

The intracellular calcium concentration ([Ca²⁺]_i) of isolated ventricular cardiac myocytes of the guinea pig was measured during the application of pulsed high-frequency electromagnetic fields. The high-frequency fields were applied in a transverse electromagnetic cell designed to allow microscopic observation of the myocytes during the presence of the high-frequency fields. The [Ca²⁺]_i was measured as fura-2 fluorescence by means of digital image analysis. Both the carrier frequency and the square-wave pulse-modulation pattern were varied during the experiments (carrier frequencies: 900, 1,300, and 1,800 MHz pulse modulated at 217 Hz with 14% duty cycle; pulsation pattern at 900 MHz: continuous wave, 16 Hz, and 50 Hz modulation with 50% duty cycle and 30 kHz modulation with 80% duty cycle). The mean specific absorption rate (SAR) values in the solution were within one order of magnitude of 1 mW/kg. They varied depending on the applied carrier frequency and pulse pattern. The experiments were designed in three phases: 500 s of sham exposure, followed by 500 s of field exposure, then chemical stimulation without field. The chemical stimulation (K⁺-depolarization) indicated the viability of the cells. The K⁺ depolarization yielded a significant increase in [Ca²⁺]_i. Significant differences between sham exposure and high-frequency field exposure were not found except when a very small but statistically significant difference was detected in the case of 900 MHz/50 Hz. However, this small difference was not regarded as a relevant effect of the exposure. © 1996 Wiley-Liss, Inc.     

Structural and physiological properties of leech giant glial cells as studied by confocal microscopy

We have studied the architecture of giant neuropile glial cells of the medicinal leech Hirudo medicinalis L. using confocal laser scanning microscopy. We also measured changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by activation of glutamate receptors or voltage-gated Ca²⁺ channels in different glial cell compartments. Glial cells of isolated segmental ganglia were filled iontophoretically with the Ca²⁺ indicator dye Fluo-3. The three-dimensional structure, calculated from serial sections, showed that numerous fine glial branches extend within the whole neuropile, where most of the synapses between neurones are established. Activation of glial glutamate receptors by glutamate or kainate, or depolarizing the cell membrane by elevating the external K⁺ concentration resulted in a transient increase in [Ca²⁺]_i, as measured by Fluo-3 fluorescence. The comparison of [Ca²⁺]_i changes in glial cell branches with changes in the cell body demonstrated that transients in the branches were 2–3 times larger than those in the cell body. The results suggest that glutamate receptors and voltage-gated Ca²⁺ channels are located in the membrane not only of the glial cell body but also of the cellular branches, which may extend close to synaptic domains.     

Vibration of osteoblastic cells using a novel motion-control platform does not acutely alter cytosolic calcium,but desensitizes subsequent responses to extracellular ATP

Low-magnitude high-frequency mechanical vibration induces biological responses in many tissues. Like many cell types, osteoblasts respond rapidly to certain forms of mechanostimulation, such as fluid shear, with transient elevation in the concentration of cytosolic free calcium ([Ca²⁺]_i). However, it is not known whether vibration of osteoblastic cells also induces acute elevation in [Ca²⁺]_i. To address this question, we built a platform for vibrating live cells that is compatible with microscopy and microspectrofluorometry, enabling us to observe immediate responses of cells to low-magnitude high-frequency vibrations. The horizontal vibration system was mounted on an inverted microscope, and its mechanical performance was evaluated using optical tracking and accelerometry. The platform was driven by a sinusoidal signal at 20–500 Hz, producing peak accelerations from 0.1 to 1 g. Accelerometer-derived displacements matched those observed optically within 10%. We then used this system to investigate the effect of acceleration on [Ca²⁺]_i in rodent osteoblastic cells. Cells were loaded with fura-2, and [Ca²⁺]_i was monitored using microspectrofluorometry and fluorescence ratio imaging. No acute changes in [Ca²⁺]_i or cell morphology were detected in response to vibration over the range of frequencies and accelerations studied. However, vibration did attenuate Ca²⁺ transients generated subsequently by extracellular ATP, which activates P2 purinoceptors and has been implicated in mechanical signaling in bone. In summary, we developed and validated a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Vibration did not elicit acute elevation of [Ca²⁺]_i, but did desensitize responses to later stimulation with ATP.     

Magnesium homeostasis in cardiac cells

Several aspects of Mg²⁺ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg²⁺ indicator, FURAPTRA. The concentration of cytosolic Mg²⁺ ([Mg²⁺]_i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg²⁺]_i below electrochemical equilibrium, we manipulated the Na⁺ gradient and assessed the effects on [Mg²⁺]_i. When extracellular Na⁺ was removed, [Mg²⁺]_i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg²⁺]_i, which was dependent upon extracellular Ca²⁺, was observed when intracellular Na⁺ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca²⁺ can modulate [Mg²⁺]_i. In addition, removing extracellular Na⁺ caused a decrease in intracellular pH (pH_i), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca²⁺ and H⁺ interact intracellularly. Since changes in the Na⁺ gradient can also alter pH_i, we questioned whether pH can modulate [Mg²⁺]_i. pH_i was manipulated by the NH₄Cl prepulse method. NH₄ ⁺-evoked changes in pH_i, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg²⁺]_i; [Mg²⁺]_i changed by –0.16 mM/unit pH. These NH₄ ⁺-evoked changes in [Mg²⁺]_i were not caused by movements of Mg₂₊ or Ca²⁺ across the sarcolemma or by changes in cytosolic Ca²⁺. Additionally, pH_i was manipulated by changing extracellular pH (pH_o). When pH_o was decreased from 7.4 to 6.3, pH_i decreased by 0.64 units and [Mg₂₊]_i increased by 0.12 mM; in contrast, when pH_o was raised from 7.4 to 8.3, pH_i increased by 0.6 units and [Mg²⁺]_i did not change significantly. The results of our investigations suggest that Ca ²⁺ and H⁺ can modulate [Mg²⁺]_i, probably by affecting cytosolic Mg²⁺ binding and/or subcellular Mg²⁺ transport and that such redistribution of intracellular Mg²⁺ may play an important role in Mg²⁺ homeostasis in cardiac cells.     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.     

Calcium Sequestering Ability of Mitochondria Modulates Influx of Calcium through Glutamate Receptor Channel

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca²⁺ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca²⁺ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca²⁺]_i followed by a slow and persistently rising phase. The rapid increase in [Ca²⁺]_i was mainly due to influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca²⁺ in the extracellular medium elicited a small transient rise in [Ca²⁺]_i which can be attributed to the mobilization of Ca²⁺ from IP₃ sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca²⁺ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca²⁺ uniporter. These results imply that mitochondria sequester the Ca²⁺ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca²⁺ caused permeability transition and released the sequestered Ca²⁺ which is manifested as slow rise in [Ca²⁺]_i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca²⁺]_i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca²⁺]_i response. Combined treatment with oligomycin and rotenone further diminished the [Ca²⁺]_i response and also abolished the CCCP mediated rise in [Ca²⁺]_i. However, rotenone alone had no effect on glutamate induced [Ca²⁺]_i response. These changes in glutamate-induced [Ca²⁺]_i response could not be explained on the basis of deficient mitochondrial Ca²⁺ sequestration or ATP dependent Ca²⁺ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca²⁺ buffering processes leading to elevation of [Ca²⁺]_i. In contrast our results showed repression of Ca²⁺ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca²⁺ sequestering ability of mitochondria, the glutamate induced Ca²⁺ influx could be impeded. To validate this, influx of Mn²⁺ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca²⁺ sequestration may desensitize the NMDA receptor operated channel.     

The dynamics of cytosolic calcium in photoreceptor cells

Analysis of the light-induced changes of cytosolic Ca²⁺ ([Ca²⁺]_i) in photoreceptor cells has been taken a step further with two recently published studies^(1,2). In one, changes in [Ca²⁺]_i were measured in single detached rod outer segments from Gecko in response to various light intensities. The advances of the other⁽²⁾ are embodied in its employment of transgenic Drosophila, whose photoreceptors express a visual pigment that is insensitive to the wavelength of light used in the fluorescence imaging of [Ca²⁺]_i. These studies provide a better basis for understanding the regulation of Ca²⁺-mediated events in photoreceptor cells.     

Effects of extremely-law-frequency electromagnetic fields on ion transport in several mammalian cells

We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca²⁺, Na⁺, K⁺, and H⁺) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), the intracellular pH (pH_i), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100–2000 μT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20–1200 μT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12–32% increase of the uptake of ⁴²K within a window at 14.5–15.5 Hz and 100–200 μT (rms), which was found in U937 and Ehrlich cells but not in the other cell types. © 1994 Wiley-Liss, Inc.     

Calcium-activated chloride fluxes in cultured NCL-SG3 sweat gland cells

The dependence of chloride permeability of the human sweat gland cell line NCL-SG3 cell line on cytosolic free calcium ([Ca²⁺]_i) was investigated. X-ray microanalysis, fura-2 fluorescence and patch clamp methodology were used. Carbachol and A23187 decreased cellular Cl and K for cells grown on permeable supports, but carbachol had no effect on cells grown on impermeable supports. In perforated patch experiments with impermeable supports, ATP and calcium ionophores increased the inward current (ic) whereas carbachol had no effect. ic was unaffected by cation channel blockers or removal of extracellular Na⁺ but was blocked by chloride channel blockers. Lowering bath Ca²⁺ decreased ic. On raising bath Ca²⁺ ic and [Ca²⁺]_i responded with a transient rise which was not blocked by La³⁺ or D-600. La³⁺, but not D-600, blocked the entry of Mn²⁺. K⁺-depolarization and Bay-K-8644 had little effect on [Ca²⁺]_i. The rise in [Ca²⁺]_i may be mediated primarily via depletion operated Ca²⁺-channels. Irrespective of substrate NCL-SG3 cells have a chloride permeability which depends on [Ca²⁺]_i.     

Intracellular calcium oscillations in a T-cell line after exposure to extremely-low-frequency magnetic fields with variable frequencies and flux densities

Low-frequency magnetic fields (MF) can increase the cytosolic calcium concentration ([Ca²⁺]_i) in lymphocytes in the same manner as a physiological stimulus such as antibodies directed towards the CD3 complex. In this study, MF with various frequencies and flux densities were used, while [Ca²⁺]_i changes were recorded using microfluorometry with fura-2 as a probe. The applied sinusoidal MF induced oscillatory changes of [Ca²⁺], in the leukemic cell line Jurkat in a manner similar to that seen with stimulation by antibodies. The response at 0.15 mT was over a frequency range from 5 to 100 Hz, with a fairly broad peak having its maximum at 50 Hz. The result of testing increasing flux densities at 50 Hz was a threshold response with no effect below 0.04 mT and a plateau at 0.15 mT. On the basis of the characteristic calcium pattern resulting from an applied MF, we suggest that MF influence molecular events in regular signal transduction pathways of T cells. © 1995 Wiley-Liss, Inc.     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

A comparison of bradykinin,angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca²⁺]_i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca²⁺]_i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca²⁺]_i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca²⁺]_i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca²⁺]_i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.Abbreviations ([Ca²⁺]_i) cytoplasmic free calcium concentration - EGTA ethylene glycol bis (-amino ethyl ether)-N,N,N,N,-tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazinethanesulphonic acid - TPA 12-O-tetradecanoylphorbol-13-acetate - DAG diacyl glycerol - IP₃ inositol-1,4,5-trisphosphate - PIP₂ phosphatidylinositol-4,5-bisphosphate     

Direct inhibitory action of EGTA-Ca complex on reverse-mode Na/Ca exchange inMyxicola giant axons

Summary Giant axons from the marine annelidMyxicola infundibulum were internally dialyzed with solutions containing²²Na ions as tracers of Na efflux. In experiments performed in Li-substituted seawater, Na efflux that is dependent on external Ca ion concentration, [Ca²⁺] _o , was measured using dialysis to maintain [Na⁺] _i at 100mm, which enhances the [Ca²⁺] _o -dependent Na efflux component, (i.e., reverse-mode Na/Ca exchange). When dialysis fluid contained EGTA (1mm) to buffer the internal Ca concentration, [Ca²⁺] _i , to desired levels, Na efflux lost its normal sensitivity to external calcium. The inhibition was not simply due to the Ca-chelating action of EGTA to produce insufficient [Ca²⁺] _i to activate Na/Ca exchange. The addition of EGTA inhibited Ca _o -dependent Na efflux even when a large enough excess of [Ca²⁺] _i was present to saturate the EGTA and still produce elevated values of [Ca²⁺] _i . Control experiments showed that these high values of [Ca²⁺] _i resulted in normal Na/Ca exchange in the absence of EGTA. It is concluded that the presence of EGTA itself interferes with the manifestation of reverse-mode Na/Ca exchange inMyxicola giant axons.     

The role of cytosolic free calcium in the regulation of pyruvate dehydrogenase in synaptosomes

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 (PDH_a) was assessed in isolated nerve terminals (i.e. synaptosomes) under conditions that alter [Ca²⁺]_i. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a and both responses required external calcium. Treatment with KCN, an in vitro model of hypoxia decreased ATP and elevated [Ca²⁺]_i and PDH_a. Furthermore, in the presence of KCN, PDH_a became more sensitive to K⁺ depolarization as indicated by larger changes in PDH_a than in [Ca²⁺]_i. The calcium ionophore Br-A23187 elevated [Ca²⁺]_i, but did not affect PDH_a. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a even if [Ca²⁺]_i was elevated by prior addition of ionophore or KCN. Previous in vivo studies by others show that PDH_a is altered during and after ischemia. The current in vitro results suggest that hypoxia, only one component of ischemia, is sufficient to increase PDH_a. These data also further support the notion that PDH_a is regulated by [Ca²⁺]_i as well as by other factors such as ATP. Our results are consistent with the concept that PDH_a in nerve endings may be affected by [Ca²⁺]_i and that these two processes are clearly linked.Abbreviations (PDH_a) 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     

Ultrafast Ca wave in cultured vascular smooth muscle cells aligned on a micropatterned surface

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which propagates as Ca²⁺ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca²⁺ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca²⁺ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca²⁺]_i dynamics and membrane potential events along the network of cells. The rate of [Ca²⁺]_i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca²⁺ channels (VOCCs) inhibition suppressed the ultrafast Ca²⁺ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca²⁺ entry from the external medium through VOCCs, modeled as an ultrafast Ca²⁺ wave. This wave may trigger and drive slower Ca²⁺ waves observed ex vivo and in vivo.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca²⁺]_i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca²⁺]_i depend on the intensity of external electric field. In the presence of Ca²⁺ chelant EGTA or Ca²⁺ channels blocker La³⁺ in the pulsation solutions, the increase of [Ca²⁺]_i was still observable. It was also observed that [Ca²⁺]_i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca²⁺]_i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Elevated cytosolic calcium levels in human lymphocytes during surface virus infections

Summary Generalised metabolic and electrolyte disturbances are known to accompany both plasma and surface virus infections. We have investigated whether these infections could impair the transport of Ca²⁺ from cells under conditions of controlled concentrations of the energy substrate glucose. Thus, cytosolic calcium levels ([Ca²⁺]_i) were measured in single isolated lymphocytes obtained from healthy volunteers or those suffering from coryza. Before making measurements using a Ca²⁺-sensitive fluorescent dye indo 1, we incubated lymphocytes in buffers containing 0 mM-, 5.6 mM- or 11.2 mM-[glucose]. We found that [Ca²⁺]_i of lymphocytes obtained from the sick were significantly higher than those from healthy controls both at 0 mM and 5.6 mM-[glucose], and that [Ca²⁺]_i was inversely related to the media glucose concentration for both groups. These results suggest a diminished capacity of cation pumping in viral infections, such as coryza, in relationship to the available glucose as energy substrate.