The effect of weak low-frequency magnetic fields on the intracellular calcium-dependent proteinases of fish

The in vivo and in vitro effects of weak, low-frequency magnetic fields with resonance parameters for calcium ions upon intracellular calcium-dependent proteinases (calpains) in the crucian carp (Carassius carassius (L.)) and roach (Rutilus rutilus (L.)) were studied. It has been revealed that the impact of a weak lowfrequency magnetic field leads to considerable decrease in the activity of calpains in the fish skeletal muscles and brain.     

Effect of weak combined magnetic fields on the metamorphosis of the mealworm beetle Tenebrio molitor

The effects of weak combined magnetic fields adjusted to the parametric resonance for Ca²⁺ and K⁺ and extremely weak alternating magnetic field on the metamorphosis of the mealworm beetle Tenebrio molitor have been studied. It was shown that the exposure of pupas of insects to all above-indicated types of fields stimulates the metamorphosis. However, after the exposure to weak combined magnetic fields adjusted to the parametric resonance for Ca²⁺ and K⁺, the number of insects with anomalies increases, which is not observed by the action of the weak alternating magnetic field.     

Cell Sensitivity to Magnetic Fields

Attempts to establish extremely low-frequency (ELF) threshold sensitivity limits in biological systems are presently based on estimates of thermal noise in the cell membrane. The Weaver-Astumian (Science 247:459–462, 1990) threshold (8 × 10^?3 V/m) should in principle also apply to electric fields produced by Faraday induction. However, the 60-Hz magnetic field required to induce an electric field of 8 × 10^?3 V/m is improbably large and at variance with the experimental facts, implying either that Faraday induction is not the mode of weak ELF magnetic field biointeractions or that such interactions have nothing to do with the cell membrane, which constitutes only 1 % of the cell volume. We explore the possibility that magnetic field interactions are connected to the periodic changes in free calcium concentration associated with the cellular Ca²⁺ oscillator (CaO). Estimates of the free energy associated with the CaO reveal cyclic voltage changes of the order of 20 mV, suggesting that already existing electric fields within the cytoplasm may be capable of interacting with externally applied magnetic fields. We further hypothesize that CaO frequencies can be reinforced or driven into narrower passbands by weak external ELF signals acting on elements in the Ca²⁺ signaling pathway, e.g., via the calmodulin molecule.     

The role of hydroxyl radicals and calcium ions in the priming of a respiratory burst in neutrophils and the increase in luminol-dependent blood chemiluminescence on exposure to combined magnetic fields with a very weak low-frequency alternating component

The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA AM) used at low concentrations (1.0 and 2.5 μM) was shown to block the priming effect of weak combined static (42 μT) and low-frequency collinear alternating (1.0, 4.4, and 16.5 Hz; 0.86 μT) magnetic fields. This blockage was inferred from a greater increase in chemiluminescence observed for a mouse neutrophil suspension exposed to combined magnetic fields in response to the bacterial peptide N-formyl–Met–Leu–Phe added in the presence of luminol. Similar results were obtained for the effect of BAPTA AM on luminol-dependent chemiluminescence of whole blood. The priming effect of weak combined magnetic fields on the respiratory burst in neutrophils did not depend on the presence of extracellular Ca²⁺ and was not affected by the hydroxyl radical scavenger dimethyl sulfoxide used at 0.025–1.0 mM.     

Intracellular calcium oscillations induced in a T-cell line by a weak 50 Hz magnetic field

Applied weak magnetic fields have been shown to affect cellular activity on several levels, but the mechanisms involved remain elusive. We have decided to study an early signal transduction event in the human T cell line Jurkat; oscillations of free [Ca²⁺]i, of the type seen by crosslinking the CD3 complex. Cells were exposed to a 50 Hz, 0.1 mT, sinusoidal magnetic field while intracellular free calcium was measured in individual cells, using fura-2 as a probe. An acute response was observed with oscillatory increases in [Ca²⁺]i, which subsided when the field was turned off. The effect of the magnetic field on [Ca²⁺]i was comparable to that achieved by an anti-CD3 monoclonal antibody. © 1993 Wiley-Liss, Inc.     

Two cytosolic,Ca2+-dependent,neutral proteinases from rabbit liver: Purification and properties of the proenzymes

Two Ca²⁺-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, M_r = 150,000 and M_r = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca²⁺-requiring proteinases by incubation with Ca²⁺ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca²⁺ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca²⁺ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.     

Magnetic fields at extremely low-frequency (50 Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts

Several studies have been undertaken to elucidate the effects of electromagnetic field (EMF) on intracellular calcium ([Ca²⁺]_i) in the past 20 years. However, still there were controversies of electromagnetic pollution within the scientific community. In this work, we studied the effects of alternative magnetic fields on intracellular calcium. Osteoblastic cells were used as a model both to test the hypothesis that extremely low-frequency (ELF) magnetic fields can alter the concentrations of the intracellular calcium, and to examine the ‘window’ effect predicted by our previous theoretical work. The outcome of this experiment demonstrated that 50 Hz, 0.8 mT magnetic field can induce the uptake of [Ca²⁺]_i in osteoblasts. The empirical evidences of the specified window effects of [Ca²⁺]_i in osteoblastic cells were reported for the first time in this work.     

Proteinases of the calpain family in water invertebrates and fishes

Activity of Ca²⁺-dependent proteinases, or calpains (EC 3.4.22.17), was estimated in a wide range of aquatic invertebrates (Oligochaeta, Hirudinea, Crustacea, Insecta, Gastropoda, Bivalvia) and vertebrates (Osteichthyes). Detected molecular properties of calpains from the tissues of different species allow the consideration of the complications of calpain structural organization and regulatory mechanisms in invertebrates and vertebrates from a comparative-evolutionary perspective. Certain conclusions can be drawn about changes in the functional role of this proteolytic system in cell metabolism.     

The influence of weak magnetic fields on the production of the reactive oxygen species in peritoneal neutrophils of mice

The influence of weak magnetic fields of different types on the rate of the formation of reactive oxygen species in mouse peritoneal neutrophils has been studied. It was found that the exposure of neutrophils activated by phorbol 12-myristate 13-acetate to the magnetic field tuned to the parametric resonance for Ca²⁺ ions leads to a decrease in the rate of the reactive oxygen species (ROS) generation by 23%. Conversely, the generation of ROS in neutrophils exposed to the same field but stimulated by the bacterial peptide FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) increased by about 21%. Pulsed magnetic fields also changed the rate of ROS generation in phorbol-stimulated neutrophils by about 20%, but the sign of the effects observed in this case was opposite to those induced by the magnetic field tuned to the parametric resonance for Ca²⁺ ions.     

Myosin Light Chain Modification Depending on Magnetic Fields: II. Experimental

Recent experiments have revealed that Ca²⁺ -calmodulin dependent myosin light chain phosphorylation in a cell-free preparation exhibits unexpectedly high sensitivity to weak magnetic fields. This enzyme system is a well-studied biochemical system, which appears to depend upon ion binding. A previous article in this journal discussed the theoretical background of myosin phosphorylation and the ion-dependent interactions of EMF with soft tissues. Because of the electromagnetic field (EMF) sensitivity of this cell-free system, experiments were designed to test the effect of a pulsed radio frequency (PRF) field, pulsating magnetic fields (TEMF), gradient magnetic fields (Magnabloc), and homogeneous static magnetic fields (in Helmholtz arrangement) designed for clinical application. It is concluded that these various magnetic fields affect this cell-free enzyme system by modulating ion–protein interactions.     

Effects of Weak Environmental Magnetic Fields on the Spontaneous Bioelectrical Activity of Snail Neurons

We examined the effects of 50-Hz magnetic fields in the range of flux densities relevant to our current environmental exposures on action potential (AP), after-hyperpolarization potential (AHP) and neuronal excitability in neurons of land snails, Helix aspersa. It was shown that when the neurons were exposed to magnetic field at the various flux densities, marked changes in neuronal excitability, AP firing frequency and AHP amplitude were seen. These effects seemed to be related to the intensity, type (single and continuous or repeated and cumulative) and length of exposure (18 or 20 min). The extremely low-frequency (ELF) magnetic field exposures affect the excitability of F1 neuronal cells in a nonmonotonic manner, disrupting their normal characteristic and synchronized firing patterns by interfering with the cell membrane electrophysiological properties. Our results could explain one of the mechanisms and sites of action of ELF magnetic fields. A possible explanation of the inhibitory effects of magnetic fields could be a decrease in Ca²⁺ influx through inhibition of voltage-gated Ca²⁺ channels. The detailed mechanism of effect, however, needs to be further studied under voltage-clamp conditions.     

Transient effect of weak electromagnetic fields on calcium ion concentration in Arabidopsis thaliana

Background  

Weak magnetic and electromagnetic fields can influence physiological processes in animals, plants and microorganisms, but the underlying way of perception is poorly understood. The ion cyclotron resonance is one of the discussed mechanisms, predicting biological effects for definite frequencies and intensities of electromagnetic fields possibly by affecting the physiological availability of small ions. Above all an influence on Calcium, which is crucial for many life processes, is in the focus of interest. We show that in Arabidopsis thaliana, changes in Ca²⁺-concentrations can be induced by combinations of magnetic and electromagnetic fields that match Ca²⁺-ion cyclotron resonance conditions.     

NMR exposure sensitizes tumor cells to apoptosis

NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3–66 mT) static magnetic fields deeply affect apoptosis in a Ca²⁺ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95–102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8–2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca²⁺ overload consequent to NMR-promoted Ca²⁺ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca²⁺ chelation or by inhibition of plasma membrane L-type Ca²⁺ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies.     

Growth of etiolated barley plants in weak static and 50 Hz electromagnetic fields tuned to calcium ion cyclotron resonance

Background

The effects of weak magnetic and electromagnetic fields in biology have been intensively studied on animals, microorganisms and humans, but comparably less on plants. Perception mechanisms were attributed originally to ferrimagnetism, but later discoveries required additional explanations like the "radical pair mechanism" and the "Ion cyclotron resonance" (ICR), primarily considered by Liboff. The latter predicts effects by small ions involved in biological processes, that occur in definite frequency- and intensity ranges ("windows") of simultaneously impacting magnetic and electromagnetic fields related by a linear equation, which meanwhile is proven by a number of in vivo and in vitro experiments.

Methods

Barley seedlings (Hordeum vulgare, L. var. Steffi) were grown in the dark for 5 and 6 days under static magnetic and 50 Hz electromagnetic fields matching the ICR conditions of Ca²⁺. Control cultures were grown under normal geomagnetic conditions, not matching this ICR. Morphology, pigmentation and long-term development of the adult plants were subsequently investigated.

Results

The shoots of plants exposed to Ca²⁺-ICR exposed grew 15–20% shorter compared to the controls, the plant weight was 10–12% lower, and they had longer coleoptiles that were adhering stronger to the primary leaf tissue. The total pigment contents of protochlorophyllide (PChlide) and carotenoids were significantly decreased. The rate of PChlide regeneration after light irradiation was reduced for the Ca²⁺-ICR exposed plants, also the Shibata shift was slightly delayed. Even a longer subsequent natural growing phase without any additional fields could only partially eliminate these effects: the plants initially exposed to Ca²⁺-ICR were still significantly shorter and had a lower chlorophyll (a+b) content compared to the controls. A continued cultivation and observation of the adult plants under natural conditions without any artificial electromagnetic fields showed a retardation of the originally Ca²⁺-ICR exposed plants compared to control cultures lasting several weeks, with an increased tendency for dehydration.

Conclusion

A direct influence of the applied MF and EMF is discussed affecting Ca²⁺ levels via the ICR mechanism. It influences the available Ca²⁺ and thereby regulatory processes. Theoretical considerations on molecular level focus on ionic interactions with water related to models using quantum electrodynamics.

     

Serum plays a critical role in modulating [Ca2+]c of primary culture bone cells exposed to weak ion-resonance magnetic fields

Primary-culture bone cells were exposed to ion-resonance (IR) magnetic fields tuned to Ca²⁺. Cytosolic calcium concentration, [Ca²⁺]_c, was measured by using fura-2 during field exposure. The fields investigated were 20 μT static + 40 μT p-p at either 15.3 or 76.6 Hz, and 0.13 mT static + either 0.5 or 1.0 mT p-p at 100 Hz. Other parameters included field orientation, culture age (2 or 5 days after plating), and the presence of serum (0 or 2%) during exposure. Total experiment time was 29.5 min: The field was applied after 2 min, and bradykinin was added as an agonist control after 22 min. The data were quantified on a single-cell basis during the 2–22 min exposure period in terms of the magnitude of the largest occurring [Ca²⁺]_c spike normalized to local baseline. Field-exposed and control groups were characterized in terms of the percent of cells exhibiting spike magnitudes above thresholds of 100 or 66% over baseline and were compared by using Fisher's exact test. Without serum, there was little evidence that IR magnetic fields altered [Ca²⁺]_c. However, in the presence of 2% serum, 3 of the 16 experiments exhibited significant effects at the 100% threshold. Reducing this threshold to 66% resulted in five experiments exhibiting significant effects. Most strikingly, in all of these cases, the field acted to enhance [Ca²⁺]_c activity as opposed to suppressing [Ca²⁺]_c activity. These findings suggest a role for serum or for constituents within serum in mediating the effects of IR magnetic fields on cells and may provide a resolution pathway to the dilemma imposed by theoretical arguments regarding the possibility of such phenomena. Possible roles of serum and future studies are discussed. Bioelectromagnetics 18:203–214, 1997. © 1997 Wiley-Liss, Inc.     

Thermal dependence of cardiac SR Ca-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca²⁺ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca²⁺ uptake and E–C coupling. Ca²⁺ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca²⁺ uptake as low as 5 °C. As a consequence, the Q₁₀ values in coho salmon were low over a range of different temperature intervals. Maximal Ca²⁺ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca²⁺ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca²⁺ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.     

Calcium efflux of plasma membrane vesicles exposed to ELF magnetic fields-test of a nuclear magnetic resonance interaction model

The question whether very weak, low frequency magnetic fields can affect biological matter is still under debate. The theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely weak 60 Hz magnetic fields on the transport of Ca²⁺ was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested a newly proposed quantum mechanical model postulates that polarization of hydrogen nuclei can elicit a biological effect. Vesicles were exposed for half an hour at 32 °C and the calcium efflux was studied using radioactive ⁴⁵Ca²⁺ as a tracer. A static magnetic field of 26 µT and time‐varying magnetic fields with a frequency of 60 Hz and amplitudes between 0.6 and 6.3 µT were used. The predictions of the model, proposed by Lednev, that at a frequency of 60 Hz the biological effect under investigation would significantly be altered at the amplitudes of 1.3 and 3.9 µT could not be confirmed. Bioelectromagnetics 33:535–542, 2012. © 2012 Wiley Periodicals, Inc.     

Differential contribution of EF-hands to the Ca²⁺-dependent activation in the plant two-pore channel TPC1

Two‐pore channels (TPC) have been established as components of calcium signalling networks in plants and animals. In plants, TPC1 in the vacuolar membrane is gated open upon binding of calcium in a voltage‐dependent manner. Here, we analyzed the molecular mechanism of the Ca²⁺‐dependent activity of TPC1 from Arabidopsis thaliana, using site‐directed mutagenesis of its two canonical EF‐hands. Wild‐type TPC1 and TPC1‐D335A with a mutated first Ca²⁺ ligand in EF‐hand 1 produced channels that retained their voltage‐ and Ca²⁺‐dependent gating characteristics, but were less sensitive at Ca²⁺ concentrations <200 μm . Additional mutation of the first Ca²⁺ ligand in EF‐hand 2 resulted in silent TPC1‐D335A/D376A channels. Similarly, the single mutant TPC1‐D376A could not be activated up to 1 mm Ca²⁺, indicating that the second EF‐hand is essential for the Ca²⁺‐dependent channel gating. Molecular modeling suggests that EF‐hand 1 displays a low‐affinity Ca²⁺/Mg²⁺‐binding site, while EF‐hand 2 represents a high‐affinity Ca²⁺‐binding site. Together, our data prove that EF‐hand 2 is responsible for the Ca²⁺‐receptor characteristics of TPC1, while EF‐hand 1 is a structural site required to enable channel responses at physiological changes in Ca²⁺ concentration.     

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

Ca_V1 and Ca_V2 voltage-gated calcium channels evolved from an ancestral Ca_V1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian Ca_V1 channels are uniquely subject to pronounced, buffer-resistant Ca²⁺/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca²⁺ rises. Early diverging, nonbilaterian invertebrates also possess Ca_V1 and Ca_V2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both Ca_V1 and Ca_V2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of Ca_V1 and Ca_V2 channels. Here, we build upon previous characterization of Trichoplax Ca_V channels by determining the cellular expression and ion-conducting properties of the Ca_V1 channel orthologue, TCa_V1. We show that TCa_V1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca²⁺ currents with kinetics resembling those of rat Ca_V1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCa_V1, but not TCa_V2, exhibits buffer-resistant Ca²⁺/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca²⁺ signaling within various cellular contexts.     

Role of calcium in melanosome aggregation withinLabeo melanophores

In isolated scale melanophores ofLabeo rohita the melanosome aggregating effect of K⁺ was inhibited in Ca²⁺ deprived medium. Moreover, the Ca²⁺-antagonists, verapamil and lanthanum inhibited the action of K⁺ in concentration dependent manner. The elevation of extracellular Ca²⁺ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca²⁺ appeared to be required only for K+ -induced aggregation and not melanosome aggregationper se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca²⁺ free medium