Fifty-Hertz low frequency magnetic field modifies sarcoplasmic reticulum function

Among the putative mechanisms, by which extremely low frequency magnetic field (ELF-MF) modify calcium metabolism is that of affecting Ca2+ fluxes across cell membrane or internal Ca2+ stores. To test this hypothesis, whether ELF MF can modulate Ca2+ fluxes of isolated skeletal muscle sarcoplasmic reticulum vesicles (SR) was investigated in the present study. The Ca2+ transport and Ca2+-Mg2+-ATPase activity were observed by means of dynamic Ca2+ dye spectrum, the function of ryanodine receptor (RyR1) was monitored by [3H]-ryanodine binding assay. The membrane fluidity of SR was measured by fluorescence polarization. 50 Hz 0.4 mT MF exposure for 30 min led to a significant decrease in the Ca2+ uptake initial rate and Ca2+-Mg2+-ATPase activity compared to sham exposed SR. These results strongly suggest that prevention of SR Ca2+ uptake by ELF MF exposure was due to the inhibition of Ca2+-Mg2+- ATPase activity, and the increase of SR Ca2+ release was due to the activation of RyR1.


INTRODUCTION
With increasing use of electric appliances, high voltage power transmission and magnetic resonance imaging, it has resulted in the increased exposure to a complex mix of artificially elevated extremely low frequency (ELF) magnetic field (MF). Epidemiological data suggest that there is an association between ELF MF exposure and the incidence of a few types of cancers, particularly in childhood leukemia, brain and breast cancers [1], but these results have not been confirmed in other studies [2][3]. However, any possible harmful effects could not be excluded because of many positive results which were independently confirmed.
In vitro studies, ELF MF have been reported to affect several basic cellular processes, such as cell proliferation [4], apoptosis [5], DNA and RNA synthesis [4,6], and gene transcription [7]. Since all these effects can be related more or less directly to the signal transduction pathways, Ca2+ as an intracellular critical second message has been of focus to scientists for many years [8][9][10][11][12]. In many types of cell the signal transduction from plasmalemma to intracellular targets is linked to the oscillatory behaviour of intracellular Ca2+. Experimental investigations of ELF MF on living systems seem to indicate that calcium signaling pathways, and a cytosolic Ca2+ oscillator in particular, may be the processes which are affected by external ELF MF. A few scattered studies have not detected an effect [8] [5], erythrocytes [8], T-lymphocytes (Jurkat E 6.1) [9,11,12], pituitary cells et al [10]. Experiments with different cells have the disadvantage that they represent a system with different Ca2+ stores, Ca2+ transporters and Ca2+ regulating receptor sides. In addition, it has been reported in the literature that ELF MF penetrate through cells or tissues without significant attenuation [13], if ELF MF could influence internal Ca2+ stores on cellular level, it is very likely to induce some effects on isolated Ca2+ stores models. Furthermore, contraction of skeletal muscle provides one of the best studied examples of calcium signalling. Muscle can be homogenized to give a preparation of sealed vesicles derived from the Sarcoplasmic reticulum (SR) that can accumulate Ca2+ from the external medium in the presence of ATP, following uptake of Ca2+, SR vesicles will spontaneously release a fraction of the accumulated Ca2+ [15]. For all these reasons, using isolated internal Ca2+ stores (SR vesicles) while not cells to investigate the effects of ELF MF on the basic mechanisms of Ca2+ regulation system in the present study. SR is the storage site for the bulk of Ca2+ and its transport across the SR membrane is managed by two molecules: the ryanodine receptor (RyR1) and Ca2+-Mg2+-ATPase.
To understand how and at which point ELF MF exposure alter intracellular calcium homeostasis, we observed the effects of ELF MF on the Ca2+ transport with isolated SR vesicles.

Isolation of rabbit skeletal muscle SR vesicles
SR vesicles were isolated from hind leg and back skeletal muscle from New Zealand White rabbits by the method of MacLennan with small modifications [15]. Fifty micromolar dithiothreitol and 0.2 µg/ml leupeptin were added to all buffers except for the final SR resuspension buffer. The final samples were flash-frozen in liquid nitrogen and stored at -80 0C until use. The protein concentration was determined by absorption spectroscopy [16].

Magnetic field exposure
The MF exposure system is composed of three major parts: a pair of circular horizontal Helmholz coils with 150 turns of a copper wiring (20 cm in height and 20 cm in radius), a signal generator and a power amplifier. The whole equipment is placed in a cell culture incubator, and is shielded from external field contamination.
Measurements of the intensity of the magnetic field with a Gauss-meter (HT 22, shanghai) showed that it was fairly constant for the center area (10 cm in height and 5 cm in radius) between the coils. Our preliminary experiments indicated that the field intensity of 0.1 mT had no obvious effects on SR Ca2+ transport, whereas a higher intensity (0.4 mT) would lead to the sharp decrease in Ca2+ transport. In the present study, a 0.4 mT 50 Hz magnetic field was used. The uniformity of the field over the experimental region is (0.400±0.012) mT, calculated from the values measured in the middle of the field and at the eight corners of cube box (5cm× 5cm×5cm).
Purified SR (10 mg/ml) in 0.5ml-Eppendorf tubes were randomly divided into two groups: the shamexposed group (Control), the MF-exposed group (+MF). Each group contained three samples. The +MF groups were exposed to a 0.4 mT 50 Hz magnetic field for 30 min. The Control groups were sham exposed for the same time in a MF exposure system with power off which was shielded from external field contamination.

A-23187-stimulated Ca2+-Mg2+-ATPase activity
A-23187-stimulated Ca2+-Mg2+-ATPase activity was determined according to the method of Ref [17]. The standard assay buffer contained 100 mM KCl, 20 mM Hepes, 3 mM NADH, 1 mM EGTA, 1 mM MgCl2, 0.5 mM Mg2+-ATP, 1 mM phosphoenol pyruvate, 5 units of lactate dehydrogenase, 5 units of pyruvate kinase, at pH 7.0 in a 1.5-ml volume. Background ATPase activity was initiated by the addition of SR (0.15 mg) to the cuvette in the absence of Ca2+, and the absorbance changes of the oxidation of NADH at 340 nm were recorded for 2 min (25 0C). Maximal Ca2+-stimulated activity was determined in the presence of 8 µM free Ca2+ (the free Ca2+ concentrations in the presence of 1 mM EGTA were calculated by WinMaxc) and 3 µM A-23187 (A-23187 is a Ca2+ ionophore), and absorbance changes were monitored for 5 min until the reaction was run to completion. A-23187 was added to prevent back inhibition that results from overloading Ca2+ of vesicles with. Ca2+-Mg2+-ATPase activity was calculated as the differences between maximal ATPase and background ATPase activities. Enzyme activity was expressed as µmol NADH. mg-1. min-1. The data are the average of four independent experiments.

Measurement of Ca2+ uptake
Ca2+ fluxes across SR vesicles were monitored by using anti-pyrylazo III Ca2+ chelometric dye [17]. The standard procedure was as follows, Ca2+ uptake into SR vesicles (0.2 mg/ml) was carried out in a buffer containing 100 mM KCl, 20 mM Hepes, 1 mM MgCl2, 200 µM APIII, 20 µM free Ca2+, pH 7.0. Ca2+ uptake was initiated by the addition of 0.5-1 mM Mg2+-ATP and followed continuously on a Cary 100 spectrophotometer operating at a measuring wavelength of 710 nm and a reference wavelength of 790 nm. In all experiments, the free extravesicular Ca2+ concentration was recorded as a function of time and stored in the computer. The Ca2+ uptake capacity and the initial Ca2+ uptake rate was determined from the uptake curve of extravesicular Ca2+ concentration versus time.
Considering that net Ca2+ uptake is an equilibrium between Ca2+ entry into the vesicles and Ca2+ efflux through the release channel, 10 µM ruthenium red (RyR specific inhibitor) was added to the assay buffer in order to observe the effects of MF on single aspect of Ca2+-ATPase uptake. That is sufficient to inhibit Ca2+ efflux through the release channel. The independent experiment was repeated more than eight times.

Measurements of Ca2+ release
Assays of Ca2+ release were performed with two different methods from actively loaded and passively loaded SR vesicles. During the actively loaded SR assay in the absence of ruthenium red, upon completion of Ca2+ uptake, at which time the Ca2+ concentration had reached a steady state, Ca2+ release was initiated by the addition of RyR modulator (1 mM NADH). At the end of Ca2+ release, 3 µM A-23187 was used to release the rest Ca2+ in SR lumen.
In order to obtain the effects of MF on the RyR initial Ca2+ release rate under conditions of the same SR Ca2+ uptake capacity, we also carried out the passively loaded SR Ca2+ release assays. The procedure was as follows: SR vesicles (10 mg/ml) were passively loaded for 90 min in a medium containing 1.0 mM CaCl2, 100 mM KCl, 20 mM Hepes, pH 7.0 at 25 ℃. Then divided the sample into MF group and sham group, MF group was exposed to 50 Hz、0.4 mT MF for 30 min at 25 ℃, and sham group was placed the same time at the same temperature. Passively loaded SR vesicles were diluted 50-fold into the assay buffer (200 µM APIII, 100 mM KCl, 20 mM Hepes, pH 7.0). The free extravesicular Ca2+ concentration was recorded as a function of time and the Ca2+ initial release rate was determined from the initial slope of extravesicular Ca2+ concentration versus time.

Statistical analysis
Each independent experiment was repeated certain times as indicated in corresponding sections. The results were expressed as means ± standard deviation (SD). of n repeating times. Statistical comparisons between groups were performed with Student's t test. Differences were considered statistically significant when p<0.05.

Effects of MF exposure on SR Ca2+-ATPase activity.
The ATPase studies were conducted in the presence of A-23187, Ca2+-ionophore to ensure that maximal catalytical activity was not prone to "back inhibition" of the pump or stimulation of ATPase activity via opening of the RyR1. SR vesicles were exposed to 50 Hz 0.4 mT MF for 30 min caused the hydrophobic activity of Ca2+-Mg2+-ATPase to decrease significantly, which reduced to (0.91±0.13) from that of sham exposure SR vesicles (1.25±0.16), as shown in Fig.1. Thapsigargin (TG) was the Ca2+-Mg2+-ATPase specific inhibitor, the addition of 1.5 µM TG resulted in both MF exposure (0.22±0.05) and sham exposure (0.37±0.08) decreasing to nearly their baseline activity. Ca2+-Mg2+-ATPase activity was determined by subtracting the rate of oxidation NADH in the presence of TG from the rate of oxidation NADH in the absence of TG, namely the Ca2+-Mg2+-ATPase activity of MF group (0.69±0.12) reduced to 78% from sham exposure group (0.88±0.10).

Effects of MF exposure on SR Ca2+ uptake.
When cytosolic [Ca2+]i is elevated, the Ca2+-ATPase will actively transports Ca2+ back into the SR lumen. Ca2+-Mg2+-ATPase is the major SR protein that transports 2 mol of Ca2+ across the SR bilayer membrane with hydrolysis of 1 mol ATP.
Inhibition of SR Ca2+-Mg2+-ATPase activity resulted in a parallel reduction in the Ca2+ uptake capacity (CUC) and the initial Ca2+ uptake rate (IUR). The CUC and the IUR of MF group decreased to 80% and 69% of sham exposure SR, respectively. Because Ca2+ uptake was a dynamic process between Ca2+ entry into the vesicles and Ca2+ effluxes through RyR1, the inhibition of Ca2+ uptake on MF exposure is caused by either the Ca2+-Mg2+-ATPase influxes decrease or the RyR1 effluxes increase. To investigate the effects of MF on SR Ca2+-Mg2+-ATPase net Ca2+ uptake, the addition of 10 µM RR in assay buffer to inhibit RyR1 Ca2+ release. Results shown that the net CUC and IUR of MF group only recovered to 86% and 84% that of control. These results demonstrated that MF exposure decreased CUC and IUR of Ca2+-Mg2+-ATPase by 14% and 16%.
The decrease percentage caused by MF exposure are only partially recovered from 80% to 86% for CUC and from 69% to 84% for IUR, in the presence of RyR1 inhibitor, which indicated that MF exposure may also activated SR RyR1 Ca2+ release activity.

Effects of MF on SR Ca2+ release.
The analysis from above results indicate, that the original decreases of MF group CUC (20%) and IUR (31%) was due to the decreases in Ca2+-Mg2+-ATPase influx (CUC 14%, IUR 16%) and the increase in RyR1 Ca2+ release (CUC 6%, IUR 14%). In order to investigate the effects of 50 Hz 4 G MF on RyR1 Ca2+ release, we carried out Ca2+ effluxes assays from actively loaded SR and passively loaded SR.
MF activates the SR Ca2+ release channel/RyR1 and induces rapid release of Ca2+ from actively loaded vesicles. What's more, the effects of MF on the RyR1 Ca2+ release obviously magnified by 1 mM NADH, which caused the initial Ca2+ release rate (IRR) to increase 17% and 23% in the absence or presence of 1 mM NADH. Considering the inhibitor effects of MF on SR Ca2+-Mg2+-ATPase may be affected the RyR1 Ca2+ release, we also carried out the passively loaded SR Ca2+ release assay.
Data from passively loaded SR further demonstrated that MF markedly increase the IRR, from control group (8.58±0.70) increase to (10.81±1.61)(n=5). If the whole process of passively loaded SR 2h were exposed to MF, the IRR increased to (10.13±0.97) (n=5). Moreover, passively loaded SR with exposing to MF 0.5 h, if not measured immediately and placed at room temperature for another 2 h, MF activating effect on RyR is significantly weakened (data not shown). These results implicated that MF increased RyR Ca2+ release may be time-dependent and can be reversible.

Discussions and conclusions
Cellular studies have demonstrated that ELF MF can influence processes, such as DNA, RNA or protein synthesis in various cell types [6][7]. The cellular and molecular mechanisms by which these fields trigger biological effects are still unknown. Data reported in the literature suggested that cell membrane mediated signal transduction pathway, especially those involving calcium transport might be candidates for ELF field interactions.
In this study, we evaluated the effects of 5o Hz MF exposure at field intensity of 0.4 mT on isolated internal Ca2+ stores (SR) activity. Results of Ca2+ uptake experiments showed that 50 Hz sinusoidal MF reduced CUC and IUR to 80% and 69% of control respectively. the addition of ruthenium red could only partially restore the decreased Ca2+ uptake by MF exposure. These finding provide insight into the role Since Ca2+ is an integral part of various cellular signaling pathways, the effects of ELF MF on various cells intracellular [Ca2+]i or calcium signal transduction have been widely carried out. Studies suggest that ELF MF exposure influences intracellular Ca2+ movement and signal transduction pathways. The exposure to ELF MF causes oscillation in fibroblasts and lymphocytes. Changes in Ca2+ influx across the cell membrane of lymphocytes and decreases in cytoplasmic Ca2+ oscillation are also reported. But for all this, neither the effect itself, nor the corresponding mechanism is clarified.
Our results showed that ELF MF significantly decreased SR Ca2+ loading, leading to a 16% decrease of the initial Ca2+ loading rate. Decreased the initial Ca2+ loading rate was paralleled by a significant decrease of the percentage of Ca2+ -ATPase activity.