Microwave radiation-induced calcium ion efflux from human neuroblastoma cells in culture

Monolayer cultures of human neuroblastoma cells were exposed to 915-MHz radiation, with or without sinusoidal amplitude modulation (80%) at 16 Hz, at specific absorption rates (SAR) for the culture medium and cells of 0.00, 0.01, 0.05, 0.075, 0.1, 0.5, 0.75, 1.0, 1.5, 2, or 5 mW/g. A significant increase in the efflux of calcium ions (⁴⁵Ca²⁺) as compared to unexposed control cultures occurred at two SAR values: 0.05 and 1 mW/g. Increased efflux at 0.05 mW/g was dependent on the presence of amplitude modulation at 16 Hz but at the higher value it was not. These results indicate that human neuroblastoma cells are sensitive to extremely low levels of microwave radiation at certain narrow ranges of SAR.     

Calcium-ion efflux from brain tissue: Power-density versus internal field-intensity dependencies at 50-MHz RF radiation

In previous experiments changes were found in calcium-ion efflux from chickbrain tissue that had been exposed in vitro to 147-MHz radiation across a specific range of power densities when the field was amplitude modulated at 16 Hz. In the present study, 50-MHz radiation, similarly modulated as a sinusoid, was found to produce changes in calcium-ion efflux from chick brains exposed in vitro in a Crawford cell. Exposure conditions were optimized to broaden any power-density window and to enhance the opportunity to detect changes in the calcium-ion efflux. The results of a power-density series demonstrated two effective ranges: One spanning a range from 1.44 to 1.67 mW/cm², and the other including 3.64 mW/cm², which were bracketed by no-effect results at 0.72, 2.17, and 4.32 mW/cm². Peaks of positive findings are associated with near-identical rates of energy absorption: 1.4 μW/g at 147 MHz, and 1.3 μW/g at 50 MHz, which indicates that the enhanced-efflux phenomenon is more dependent on the intensity of fields in the brain than on the power density of incident radiation. In addition, the phenomenon appears to occur at multiples of some, as yet unknown, rate of radiofrequency (RF) energy absorption. Because of the extremely small increments of temperature associated with positive findings (< 4 × 10^?4°C), and the existence of more than one productive absorption rate, a solely thermal explanation appears extremely unlikely.     

Effect of amplitude-modulated 147 MHz radiofrequency radiation on calcium ion efflux from avian brain tissue

Cerebral cortex tissue slices and cerebral hemispheres prepared from Gallus domesticus chicks were exposed to 147 MHz radiofrequency radiation, amplitude modulated at 16 Hz and applied at a power density of 0.75 mW/cm2, to determine the effect of such exposure of 45Ca2+ efflux from the avian brain tissue. Statistical analysis of these data demonstrates that such exposure has no significant effect on 45Ca2+ efflux.     

Low frequency amplitude modulated microwave fields change calcium efflux rates from synaptosomes

Calcium(⁴⁵Ca^{2 +}) efflux from preloaded synaptosomes was studied with a continuous perfusion technique and the rate constants of a two-phase efflux process calculated. When 16-Hz sinusoidally amplitude modulated 450-MHz microwave field (maximal incident intensity 0.5 mW/ cm², modulation depth 75%) was applied during the second phase, the rate constant increased by 38%. Unmodulated or 60-Hz modulated signals were not effective. This microwave fieldinduced change can be distinguished from CaCl₂-stimulated ⁴⁵Ca^{2 +} efflux which is most probably derived intracellularly. These data suggest that the microwave-field-induced change in calcium efflux probably did not involve intracellular calcium. Also, this change in the dynamic property of synaptosomes did not require gross anatomically intact tissue as a substrate for field tissue interaction.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane

The characteristics of Ca²⁺ transport across the excitable membrane of Paramecium aurelia were studied by measuring ⁴⁵Ca²⁺ influx and efflux. The intracellular concentration of free Ca²⁺ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca²⁺ influx was easily measurable at 0°C, but not at 23°C. The influx of ⁴⁵Ca²⁺ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na⁺ and K⁺ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca²⁺ influx. An externally applied, pulsed, electric field (1–2 mA/cm² of electrode surface), caused the rate of Ca²⁺ influx to increase 3–5 times, with the extent of stimulation dependent on the current density and the pulse width Ca²⁺ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca²⁺ “gating” mechanism, which has been studied electrophysiologically. In contrast, Ca²⁺ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0°C with ⁴⁵Ca²⁺, Ca²⁺ efflux was rapid at 23°C, but did not occur at 0°C. This active Ca²⁺ efflux mechanism is probably responsible for maintaining the low internal Ca²⁺ levels in unstimulated cells.     

Acidosis and Ca2+ distribution in myocardial tissue of flounder and rat

Summary The effect of acidosis on the myocardial Ca²⁺ distribution was examined at 15°C in ventricular strips of the flounder (Platichthys flesus) and at 30°C in atrial strips of the rat (Rattus norvegicus).Lowering the Ringer pH from 7.6 to 6.9 by increasing its CO₂ (flounder 2% to 12%, rat 4% to 14%), resulted in an elevated Ca²⁺ efflux in resting strips as well as in strips stimulated (12/min) to contraction. A decrease in pH of the Ringer used for the flounder myocardium by a lowering of bicarbonate (30 mM to 5 mM) also resulted in an elevation of the Ca²⁺ efflux, but the effect was smaller than that produced by an increased CO₂.With 11 mM Ca²⁺ and 10 mM EGTA added to the Ringer to reduce the amount of⁴⁵Ca²⁺ bound to extracellular sites, an increased CO₂ with a concomitant drop in Ringer pH resulted in an increased Ca²⁺ efflux in both myocardia. The Ca²⁺ efflux was only marginally elevated in the flounder myocardium and unchanged in that of rat when the same drop in Ringer pH was produced with a lowering in bicarbonate.In a nominally Ca²⁺-free Ringer with 0.1 mM EGTA the⁴⁵Ca²⁺ efflux was stimulated for both myocardia by an increase in CO₂.The flounder myocardium was exposed to high CO₂ in a nominally Na⁺, Ca²⁺-free Ringer and again the⁴⁵Ca²⁺ efflux increased. After a return to Na, Ca and low CO₂ in the Ringer, a higher efflux persisted in the strips being subjected to a high CO₂ than in the controls.The Ca²⁺ uptake rate was about the same at high and low CO₂ for both myocardia.Based on these results the measured increase in Ca efflux following an increase in CO₂ or a decrease in bicarbonate probably results from an elevated cytoplasmatic Ca²⁺ activity. It seems unlikely that an increased uptake rate of Ca²⁺ or a direct stimulation of Ca²⁺ transporting mechanisms in the cell membrane are responsible for the change.     

Effect of extracellular adenosine 5′-triphosphate on Ca2+ efflux from freshly isolated adult rat cardiomyocytes

The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration-dependent manner (0.01–1 mM). The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated ⁴⁵Ca²⁺ efflux was not affected by deprivation of the extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. These results indicate that ATP stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P₂ purinoceptors which may couple to Na⁺/Ca²⁺ exchange.     

Attempts to alter 45Ca2+ binding to brain tissue with pulse-modulated microwave energy

Rat brain tissue, loaded with ⁴⁵Ca²⁺ by intraventricular injection was exposed in vitro to pulsemodulated 1-GHz (SAR of 0.29 or 2.9 W/kg) or 2.45-GHz radiation (SAR = 0.3 W/kg), and in vivo to 2.06-GHz radiation (SAR of 0.12 to 2.4 W/kg). There were no significant differences in efflux of ⁴⁵Ca²⁺ between the microwave- and sham-irradiated groups.     

Concanavalin a binding and Ca2+ fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca²⁺ content of the cells. ⁴⁵Ca²⁺ fluxes were measured under conditions where artifacts due to Ca²⁺ binding to concanavalin A could be excluded. Both ⁴⁵Ca²⁺ influx into and efflux from these cells are significantly activated by the lectin. If ⁴⁵Ca²⁺ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in ⁴⁵Ca²⁺ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 μg/ml) no significant increase in ⁴⁵Ca²⁺ influx occurs but an increase in ⁴⁵Ca²⁺ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca²⁺ influx into the cells and that activation of Ca²⁺ efflux occurs as a response to an increase in the cytosolic Ca²⁺ activity. Thus, Ca²⁺ may well play a role in triggering lymphocyte activation.     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000     

Biochemical Changes in Rat Brain Exposed to Low Intensity 9.9 GHz Microwave Radiation

Present study concerns with various biochemical changes in the developing rat brain exposed to 9.9 GHz (square wave modulated, 1 kHz) at power density 0.125 mW/cm² (specific absorption rate 1.0 W/kg) for 2 h/day for 35 days. Thirty days old male wistar rats were used for this present study. Each group consists of eight animals. After the exposure, biochemical assays such as calcium ion efflux, calcium-dependent protein kinase (PKC), and ornithine decarboxylase (ODC) were performed on the brain tissue. Results of this study reveal that chronic exposure of rat to microwave radiation alter the activity of certain enzymes. There was a significant increase in calcium ion efflux and the activity of ODC. On the other hand, there is a significant decrease in PKC activity. Since these enzymes are related to growth, any alteration may lead to affect functioning of the brain and its development.     

Tetrahydropapaveroline and salsolinol alter45Ca2+ efflux within perfused hippocampus of unrestrained rats

The kinetics of⁴⁵Ca²⁺ efflux were examined at circumscribed sites in the perfused hippocampus of the freely moving rat with either one of two tetrahydroisoquinoline (TIQ) products, tetrahydropapaveroline (THP) or salsolinol. Guide tubes for unilateral push-pull perfusion were implanted stereotaxically to rest just above sites within the dorsal hippocampus. Upon recovery from surgery, a tissue site in the hippocampus was prelabeled with 1.0 l of⁴⁵Ca²⁺ (2.0 Ci) injected through the indwelling guide tube. After 16–20 hr had elapsed, successive push-pull perfusions of the site were carried out with an artificial cerebrospinal fluid (CSF), at 5.0 min intervals and at a rate of 20 l/min, in order to obtain a control washout curve of declining radioactivity. On the fifth of a series of 5.0 min perfusions, either THP or salsolinol was added to the perfusion medium in a concentration of 10 or 100 ng/l. Then the hippocampal site was perfused again with control CSF for the collection of an additional three samples. Although THP in both of the test concentrations generally augmented the efflux of⁴⁵Ca²⁺, the temporal course and magnitude of the enhancement depended on the anatomical site of the perfusion. In the more rostral hippocampal planes of AP 3.0 and AP 4.0, THP caused a delayed efflux of the cation, after the perfusion of THP had been discontinued, in nearly half of the loci reactive to the TIQ. Similarly, salsolinol enhanced significantly the efflux of⁴⁵Ca²⁺ in a concentration-dependent manner during the interval of its perfusion within the hippocampal plane of AP 3.0. These results suggest that both THP and salsolinol can affect differentially the kinetics of⁴⁵Ca²⁺ efflux and that these differences are contingent on the circumscribed anatomical site of push-pull perfusion. It is envisaged that a part of the neurochemical effects of the two TIQs when acting centrally are mediated by membrane mechanisms involving Ca²⁺ transport, metabolism or other cellular activity of the cation.     

Cation-induced efflux of calcium from the mitochondria of Hymenolepis diminuta

We have previously reported that Ca²⁺ influx into the mitochondria of Hymenolepis diminuta, a rat intestinal eestode, takes place through an electrophoretic uniport system. Sodium and lithium were found to induce efflux of ⁴⁵Ca²⁺ from the mitochondria of H. diminuta. The two cations induced the efflux in a hyperbolic and linear fashion, respectively. The efflux as well as an exchange of external Ca²⁺ with internal ⁴⁵Ca²⁺ was inhibited by lanthanum. The type of Ca²⁺ transport system in the cestode organelle has been discussed and compared with that of the host (mammalian) counterpart.     

Concanavalin a binding and Ca fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca²⁺ content of the cells. ⁴⁵Ca²⁺ fluxes were measured under conditions where artifacts due to Ca²⁺ binding to concanavalin A could be excluded. Both ⁴⁵Ca²⁺ influx into and efflux from these cells are significantly activated by the lectin. If ⁴⁵Ca²⁺ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in ⁴⁵Ca²⁺ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 μg/ml) no significant increase in ⁴⁵Ca²⁺ influx occurs but an increase in ⁴⁵Ca²⁺ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca²⁺ influx into the cells and that activation of Ca²⁺ efflux occurs as a response to an increase in the cytosolic Ca²⁺ activity. Thus, Ca²⁺ may well play a role in triggering lymphocyte activation.     

Effects of noradrenaline on45Ca2+ efflux from isolated rat islets of Langerhans

Noradrenaline caused a prompt but transient increase in the rate of⁴⁵Ca²⁺ efflux from isolated rat islets of Langerhans perifused in Ca²⁺ depleted medium. The response was modest in size and was unaffected by isosmotic replacement of NaCl with choline chloride or by inclusion of 0.5 mM dibutyryl cAMP in the perifusion medium, suggesting that it was not mediated by Na+: Ca²⁺ exchange nor by lowered cAMP. Despite its effect on⁴⁵Ca²⁺ efflux, noradrenaline treatment did not alter the kinetics of⁴⁵Ca²⁺ efflux in response to the muscarinic agonist, carbamylcholine, nor did it change the magnitude of the response to this agent. Simultaneous introduction of 20 mM glucose with noradrenaline prevented a rise in⁴⁵Ca²⁺ efflux and indeed resulted in inhibition of⁴⁵Ca²⁺ efflux. The data suggest that noradrenaline does not directly activate the mechanisms which regulate Ca²⁺ extrusion from islets cells, and they do not support a primary role for the Ca²⁺ efflux response in mediating adrenergic inhibition of insulin secretion.     

The stimulus-secretion coupling of glucose-induced insulin release: effects of valinomycin upon pancreatic islet function.

In isolated rat pancreatic islets, valinomycin (0.01 to 1.0 μm) caused a dose-related facilitation of ⁸⁶Rb⁺ outflow and a dose-related inhibition of the glucose-induced changes in both outflow and net uptake of ⁸⁶Rb⁺. At high concentrations (0.1–1.0 μm), the ionophore also inhibited the oxidation of glucose and endogenous nutrients, decreased the adenylate charge, and lowered the concentration of reduced pyridine nucleotides in the islet cells. However, as little at 1.0 to 10.0 nm valinomycin caused anomalies in the handling of ⁴⁵Ca²⁺ (suppression of the early inhibitory effect of glucose upon ⁴⁵Ca²⁺ efflux, and reduction in the amount of ⁴⁵Ca²⁺ recovered in the islets after an extensive washing procedure) and inhibition of insulin release. Moreover, when the effect of glucose upon K⁺ conductance was abolished by high concentrations of valinomycin (0.1–1.0 μm), the glucose-induced secondary rise in ⁴⁵Ca²⁺ efflux was still observed. These findings suggest that the effects of glucose upon ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ handling, respectively, although normally concomitant with one another, can be dissociated, in part at least, from one another. It is concluded that the glucose-induced reduction in K⁺ outflow may be unnecessary for the sugar to cause a partial remodeling of Ca²⁺ fluxes in the islet cells.     

The calcium requirement for stimulation of rubidium efflux from isolated rat parotid acinar cells by carbachol.

Stimulation of ⁸⁶Rb⁺ efflux from isolated parotid acinar cells by carbchol was biphasic. The phases of stimulated ⁸⁶Rb⁺ efflux were separated on the basis of their relative requirements for extracellular Ca²⁺. If the isolated cells were incubated in Ca²⁺ free buffer containing 1.0 mM ethylene glycol bis (β-aminoethyl ether) N, N¹ - tetra acetic acid (EGTA) for 30 min. before adding carbachol an initial phase of ⁸⁶Rb⁺ efflux was observed. A second phase of ⁸⁶Rb⁺ efflux was obtained upon addition of 2.0 mM Ca²⁺. However when cells were incubated for 60 min. in Ca²⁺ free buffer containing 1.0 mM EGTA the initial phase of release caused by carbachol was inhibited by 95 percent. If the EGTA was titrated with Ca²⁺ to give 1.0 mM Ca²⁺, following the 60 min. depletion regimen, the second phase was observed. Although 60 min. of Ca²⁺-depletion in EGTA buffer was required for complete inhibition of the effect of carbachol on the initial phase of ⁸⁶Rb⁺ efflux, the response was fully restored within 4 min. after the readdition of Ca²⁺.     

Amylase release from rat parotid glands II. Calcium kinetics

The kinetics of ⁴⁵Ca²⁺ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM ⁴⁵Ca²⁺ medium increased the total tissue ⁴⁵calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on ⁴⁵Ca²⁺ kinetics, or total ⁴⁵calcium content. Carbamylcholine increased the initial rate of ⁴⁵Ca²⁺ uptake, but had no effect on total uptake.Elevation of the extracellular Ca²⁺ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40–50 min to reach the maximal response.The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca²⁺ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.     

THE EFFECT OF LOW LEVEL CONTINUOUS 2.45 GHz WAVES ON ENZYMES OF DEVELOPING RAT BRAIN

The present work describes the effect of low level continuous microwaves (2.45 GHz) on developing rat brain. Some 35-day-old Wistar rats were used for this study. The animals were exposed 2 hr/day for 35 days at a power density of 0.34 mW/cm² [specific absorption rate (SAR), 0.1 W/kg] in a specially made anechoic chamber. After the exposure, the rats were sacrificed and the brain tissue was dissected out and used for various biochemical assays. A significant increase in calcium ion efflux and ornithine decarboxylase (ODC) activity was observed in the exposed group as compared to the control. Correspondingly, a significant decrease in the calcium-dependent protein kinase activity was observed. These results indicate that this type of radiation affects the membrane bound enzymes, which are associated with cell proliferation and differentiation, thereby pointing out its possible role as a tumor promoter.     

Relationship between efflux of ionic calcium and phosphorus during excitation of pancreatic islets with glucose

Simultaneous rates of [³²P]orthophosphate and ⁴⁵Ca²⁺ efflux from prelabeled rat pancreatic islets have been evaluated to assess whether these ions move in concert throughout all phases of “stimulus-secretion coupling”. Perifusion with stimulatory concentrations of glucose elicited immediate but transitory increases in ³²P outflow accompanied by initial retardations and subsequent augmentations in net ⁴⁵Ca²⁺ outflows. These monophasic ³²P and biphasic ⁴⁵Ca²⁺ responses to secretory stimulation were abolished completely by membrane stabilization with tetracaine. However, certain manipulations enabled individual components to be modified separately. During stimulation with glucose, inhibition of insulin release by Ni²⁺ abolished the late increases in ⁴⁵Ca²⁺ outflow without affecting the initial retentions of ⁴⁵Ca²⁺ or the increased releases of ³²P. Under basal conditions, the ionophore A23187 “triggered” increased releases of ⁴⁵Ca²⁺ and insulin without prior retentions of ⁴⁵Ca²⁺ or enhancements of ³²P efflux. Thus, the immediate retardations of ⁴⁵Ca²⁺ outflow and heightened efflux of ³²P may reflect early events in stimulus-secretion coupling which can be dissociated from the augmented release of ⁴⁵Ca²⁺ accompanying activated emiocytosis.