General properties of the interaction between animals and ELF electric fields

An analysis is given of the interaction between extremely low-frequency (ELF) electric fields and animals of arbitrary body shape. This analysis is based on three approximations which are valid in the ELF range: In living tissues, capacitive (displacement) currents are negligible compared to conduction currents; effects resulting from the finite velocity of propagation of electromagnetic fields are negligible; skin effect in living tissues is negligible. Major conclusions of the analysis are: (a) The electric field outside the body, the induced charge on the surface of the body, and the total current crossing any section through the body (eg, through the neck or limbs) are completely determined by the characteristics of the applied ELF electric field, the shape of the body, its location relative to ground and other conductors, and any conduction currents from the body to ground or other conductors. (b) All of the quantities in (a) can be measured using conducting animal models. (c) The magnitudes of the electric field outside the body and the induced charge density on the surface of the body are independent of frequency, in the ELF range, when the body is either insulated from or shorted to ground (and any other conductors in the system). (d) The only quantities affected by the electrical properties of the tissues comprising the body are the current density and electric field inside the body. (e) The electric field outside and inside a body will be unchanged by a scaled change in its size.     

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2–0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^?5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^?5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.     

Short-circuit currents,surface electric fields,and axial current densities for guinea pigs exposed to ELF electric fields.

Short-circuit currents, surface electric fields, and axial current densities were measured in electrically grounded guinea pigs exposed to a uniform, vertical, ELF electric field. These data are 70–110% of corresponding values obtained in grounded rats exposed to the same electric field.     

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.     

A fluorometric approach to local electric field measurements in a voltage-gated ion channel

Site-specific electrostatic measurements have been limited to soluble proteins purified for in vitro spectroscopic characterization or proteins of known structure; however, comparable measurements have not been made for functional membrane bound proteins. Here, using an electrochromic fluorophore, we describe a method to monitor localized electric field changes in a voltage-gated potassium channel. By coupling the novel probe Di-1-ANEPIA to cysteines in Shaker and tracking field-induced optical changes, in vivo electrostatic measurements were recorded with submillisecond resolution. This technique reports dynamic changes in the electric field during the gating process and elucidates the electric field profile within Shaker. The extension of this method to other membrane bound proteins, including transporters, will yield insight into the role of electrical forces on protein function.     

The recent evolution of a symbiotic ion channel in the legume family altered ion conductance and improved functionality in calcium signaling

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.     

Biologic effects of prolonged exposure to ELF electromagnetic fields in rats. I. 50 Hz electric fields

A three-year investigation was conducted on the biological effects of high-intensity electric field exposures of rats for up to 18% of their life span. Two hundred and forty adult male rats, divided into groups of 20 animals each, were exposed at ground potential for 8 h/ day at 25-kV/m and 100-kV/m 50-Hz electric fields or were sham exposed for 280, 440, and 1240 h. The corresponding ages at sacrifice were 140, 164, and 315 days. An additional group of 40 rats was investigated under similar experimental conditions after 440 h of exposure at floating potential. Independent of exposure duration, mode of grounding, and field strength, no statistical differences in body weight, morphology, and histology of the liver, heart, mesenteric lymph nodes, and blood variables (hematology and serum chemistry) were found in comparison with sham-exposed animals. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (TS)at sacrifice varied widely among experimental animals in the same group but did not differ in exposed compared with sham-exposed rats. A nonsignificant tendency toward a decrease in the testes/body weight ratio was found after 1240 h of exposure. Microscopic examination of a large number of specimens showed no quantitative or qualitative statistical differences in testes alterations either among exposed animals or between exposed and their corresponding sham-exposed groups. We conclude that 50-Hz electric field exposure, even of long duration at very high field strengths, does not induce harmful effects on tissues with high cellular turnover rates and does not impair the reproductive function of rats. Moreover, after exposure, all variables investigated were well within the normal physiological range. © 1993 Wiley-Liss. Inc.     

Pulsed magnetic field effects on calcium signaling in lymphocytes: dependence on cell status and field intensity.

The effect of 3-Hz, monopolar, quasi-rectangular magnetic field pulses on 45Ca2+ uptake in resting and mitogen-treated rat thymic lymphocytes was evaluated. A 30-min, non-thermal exposure to the pulsed magnetic field (Bpeak = 6.5 mT, Emax = 0.69 mV/cm, Jmax = 2.6 microA/cm2) reduced Concanavalin A-induced 45Ca2+ uptake by 45%. It was observed that (i) the induction of the 3-Hz field response depended on Ca2+ signal transduction activation; (ii) the response direction (stimulation or inhibition) depended on the level of lymphocyte mitogen responsiveness, and (iii) the field response magnitude increased with increasing magnetic field flux densities (Bpeak = 0, 1.6, 6.5 and 28 mT). Our results demonstrate field effects at Bmax nearly 10(4) greater than that of the average human environment for low-frequency magnetic fields and they are consistent with the independent results from other 3-Hz pulsed magnetic field studies with lymphocytes.     

ELF in vitro exposure systems for inducing uniform electric and magnetic fields in cell culture media.

Many in vitro experiments on the biological effects of extremely low frequency (ELF) electromagnetic fields utilize a uniform external magnetic flux density (B) to expose biological materials. A significant number of researchers do not measure or estimate the resulting electric field strength (E) or current density (J) in the sample medium. The magnitude and spatial distribution of the induced E field are highly dependent on the sample geometry and its relative orientation with respect to the magnetic field. We have studied the E fields induced in several of the most frequently used laboratory culture dishes and flasks under various exposure conditions. Measurements and calculations of the E field distributions in the aqueous sample volume in the containers were performed, and a set of simple, quantitative tables was developed. These tables allow a biological researcher to determine, in a straightforward fashion, the magnitudes and distributions of the electric fields that are induced in the aqueous sample when it is subjected to a uniform, sinusoidal magnetic field of known strength and frequency. In addition, we present a novel exposure technique based on a standard organ culture dish containing two circular, concentric annular rings. Exposure of the organ culture dish to a uniform magnetic field induces different average electric fields in the liquid medium in the inner and outer rings. Results of experiments with this system, which were reported in a separate paper, have shown the dominant role of the magnetically induced E field in producing specific biological effects on cells, in vitro. These results emphasize the need to report data about the induced E field in ELF in-vitro studies, involving magnetic field exposures. Our data tables on E and J in standard containers provide simple means to enable determination of these parameters.     

Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels

To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.     

Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulation in tobacco BY-2 cells

Trivalent cations such as those of Al, La, and Gd are phytotoxic. Our previous works showed that addition of LaCl(3) or GdCl(3) to tobacco cells triggers the generation of superoxide (O(2)*-). Here, we show that AlCl(3) at normal physiological pH (5.8) induces much greater production of O(2)*- (detected with a specific chemiluminescence probe), indicating that these trivalent cations similarly induce the oxidative bursts. It was shown that NADPH oxidase is involved in the generation of O(2)*- and the yield of O(2)*- was dose-dependent (ca. 6mM Al, optimal). Following the acute spike of O(2)*-, a gradual increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) was detected with the luminescence of recombinant aequorin over-expressed in the cytosol. Interestingly, a O(2)*- scavenger and a Ca(2+) chelator significantly lowered the level of [Ca(2+)](c) increase, indicating that the Al-induced O(2)*- stimulates the influx of Ca(2+). Compared to the induction of O(2)*- generation, the [Ca(2+)](c) elevation was shown to be maximal (340 nM) at relatively lower Al concentrations (ca. 1.25 mM). Thus, the Al concentration optimal for O(2)*- is too much (inhibitory) for [Ca(2+)](c). In addition, high concentrations of Al were shown to be inhibitory to the H(2)O(2)-induced Ca(2+) influx. This explains the ineffectiveness of high Al concentration in the oxidative burst-mediated induction of [Ca(2+)](c) increase. It is likely that Al-induced [Ca(2+)](c) elevation is manifested from the finely geared balance between the O(2)*- -mediated driving force and the channel inhibition-mediated brake. Furthermore, it is note-worthy that Al (< or =10mM) showed no inhibitory effect on the hypo-osmolarity-induced Ca(2+) influx, implying that Al may be a selective inhibitor of redox-responsive Ca(2+) channels. Possible target channels of Al actions are discussed.     

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

Ca²⁺ entry through store-operated Ca²⁺ channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca²⁺ entry through SOCs but rendered SOCs susceptible to inhibition by H₂O₂. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.     

Basolateral potassium channel in turtle colon. Evidence for single-file ion flow

Treatment of the apical surface of the isolated, ouabain-inhibited turtle colon with the polyene antibiotic amphotericin B permitted the properties of a barium-sensitive potassium conductance in the basolateral membrane to be discerned from the measurements of transepithelial fluxes and electrical currents. Simultaneous measurements of potassium currents and 42K fluxes showed that the movement of potassium was not in accord with simple diffusion. Two other cations, thallium and rubidium, were also permeable and, in addition, exhibited strong interactions with the potassium tracer fluxes. The results indicate that permeant cations exhibit positive coupling, which is consistent with a single-file mechanism of ion translocation through a membrane channel.     

Calcium ions inhibit the allosteric interaction between the dihydropyridine and phenylalkylamine binding site on the voltage-gated calcium channel in heart sarcolemma but not in skeletal muscle transverse tubules

Calcium channel blockers bind with high affinity to sites on the voltage-sensitive Ca2+ channel. Radioligand binding studies with various Ca2+ channel blockers have facilitated identification and characterization of binding sites on the channel structure. In the present study we evaluated the relationship between the binding sites for the Ca2+ channel blockers on the voltage-sensitive Ca2+ channel from rabbit heart sarcolemma and rabbit skeletal muscle transverse tubules. [3H]PN200-110 binds with high affinity to a single population of sites on the voltage-sensitive Ca2+ channel in both rabbit heart sarcolemma and skeletal muscle transverse tubules. [3H]PN200-110 binding was not affected by added Ca2+ whereas EGTA and EDTA noncompetitively inhibited binding in both types of membrane preparations. EDTA was a more potent inhibitor of [3H]PN200-110 binding than EGTA. Diltiazem stimulates the binding of [3H]PN200-110 in a temperature-sensitive manner. Verapamil inhibited binding of [3H]PN200-110 to both types of membrane preparations in a negative manner, although this effect was of a complex nature in skeletal muscle transverse tubules. The negative effect of verapamil on [3H]PN200-110 binding in cardiac muscle was completely reversed by Ca2+. On the other hand, Ca2+ was without effect on the negative cooperativity seen between verapamil and [3H]PN200-110 binding in skeletal muscle transverse tubules. Since Ca2+ did not affect [3H]PN200-110 binding to membranes, we would like to suggest that Ca2+ is modulating the negative effect of verapamil on [3H]PN200-110 binding through a distinct Ca2+ binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Current density in a model of a human body with a conductive implant exposed to ELF electric and magnetic fields

A numerical model of a human body with an intramedullary nail in the femur was built to evaluate the effects of the implant on the current density distribution in extremely low frequency electric and magnetic fields. The intramedullary nail was chosen because it is one of the longest high conductive implants used in the human body. As such it is expected to alter the electric and magnetic fields significantly. The exposure was a simultaneous combination of inferior to superior electric field and posterior to anterior magnetic field both alternating at 50 Hz with the values corresponding to the ICNIRP reference levels: 5000 V m^?1 for electric field and 100 µT for magnetic flux density. The calculated current density distribution inside the model was compared to the ICNIRP basic restrictions for general public (2 mA m^?2). The results show that the implant significantly increases the current density up to 9.5 mA m^?2 in the region where it is in contact with soft tissue in the model with the implant in comparison to 0.9 mA m^?2 in the model without the implant. As demonstrated the ICNIRP basic restrictions are exceeded in a limited volume of the tissue in spite of the compliance with the ICNIRP reference levels for general public, meaning that the existing safety limits do not necessarily protect implanted persons to the same extent as they protect people without implants. Bioelectromagnetics 30:591–599, 2009. © 2009 Wiley‐Liss, Inc.     

Review of in vivo static and ELF electric fields studies performed at Gazi Biophysics Department

In vivo effects of Static Electric and ELF Magnetic and Electric fields have been carried out for more than 20 years in the Bioelectromagnetic Laboratory at the Biophysics Department of the Medical Faculty of Gazi University. In this article, the results of in vivo ELF Electric field studies are presented as a review. Static and 50 Hz ELF (Extremely Low Frequency) Electric (E) fields effects on free radical synthesis, antioxidant enzyme level, and collagen synthesis were analyzed on tissues of guinea pigs, such as brain, liver, lung, kidney, spleen, testis, and plasma. Animals were exposed to static and ELF electric fields with intensities ranging from 0.3 kV/m to 1.9 kV/m in vertical and horizontal directions. Exposure periods were 1, 3, 5, 7, and 10 days. Electric fields were generated from a specially designed parallel plate capacitor system. The results indicate that the effects of electric fields on the tissues studied depend significantly on the type and magnitude of electric field and exposure period.     

Frequency-dependent effects of ELF magnetic field on chromatin conformation in Escherichia coli cells and human lymphocytes.

The effects of magnetic fields of extremely low frequency (ELF, 21 microT r.m.s.) on cells of different Escherichia coli K12 strains and human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD). Within the frequency range of 6-24 Hz, two resonance-type frequency windows with maximal effects at 9 Hz and 16 Hz were observed in response of GE499 strain. Only one frequency window with maximum effect at 8.5 Hz was found for GE500 cells. These data along with previously obtained for two other E. coli strains, AB1157 and EMG2, indicate that frequency windows are dependent on genotype of cells exposed to ELF. Resonance-type effects of ELF were also observed in human lymphocytes in frequency windows around 8 and 58 Hz. These ELF effects differed significantly between studied donors, but were well reproducible in independent experiments with lymphocytes from the same donors. The frequency windows in response of E. coli strains and human lymphocytes to ELF significantly overlapped suggesting that the same targets may be involved in this response. We compared the frequency windows with predictions based on the ion cyclotron resonance (ICR) model and the magnetic parametric resonance model. These models predicted effects of ELF magnetic fields at the 'cyclotron' frequencies of some ions of biological relevance. According to the ICR model, ELF effects should be also observed at harmonics of cyclotron frequencies and, contrary, parametric resonance model predicted effects at subharmonics. While we observed coincidence of each experimental resonance frequency with predictions of one of these two models, all experimentally defined effective frequency windows were in good agreement with relatively narrow frequency ranges of both harmonics and subharmonics for natural isotopes of Na, K, Ca, Mg, and Zn ions. The experimental data support idea that both harmonics and subharmonics of several biologically important ions are involved in frequency-dependent ELF effects in cells of different types.