Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans

It has been shown that the intracellular concentrations of Na+, K+, and Cl- ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na' ions. An increase in the extracellular concentration of Na+ results in the accumulation of K+ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCI added to the medium was increased to 4%, the concentration gradient of Cl- ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase--periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na+, K+, and Li+ salts and pH. The enzymes were found to be resistant to high concentrations of Na+ and K+ chlorides and Na+ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na+ and Li+ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na+ concentrations, close to those typical of cells growing at optimal salt concentrations.     

Effects of sinusoidal magnetic field observed on cell proliferation, ion concentration, and osmolarity in two human cancer cell lines

Low frequency magnetic fields have previously been shown to affect cell functions. In this article, the effects of 20 mT, 50 Hz sinusoidal magnetic field on cell proliferation, ion concentration, and osmolarity in two human cancer cell lines (HL-60 and SK-Hep-1) were investigated. Inhibition of cell growth was observed. On the other hand, the exposure also increased the Na+, K+ ion concentration and osmolarity in cell supernatant compared to the control group. To our knowledge, this is the first study on cancer cells where magnetic fields affect osmolarity in cell supernatant. In addition, a model of cells exposed to the oscillating magnetic field is described as well as the characteristics of ions in and out of cells. The experimental data appears to be consistent with the theoretical analysis. The results are also discussed in terms of the relationships among cell growth, ion concentration, and osmolarity. Magnetic field inhibitions of cell growth in vitro may relate to changes in cell ion concentration and osmolarity.     

Copper induced alterations of biochemical parameters in the gill and plasma of Oreochromis niloticus

The main objective of this study was to determine the effects of copper exposure on copper accumulated in branchial tissue, gill Na+/K+-ATPase activity and plasma Na+, Cl-, osmolality, protein, glucose and cortisol, in Oreochromis niloticus. Fish were experimentally exposed to 40 and 400 microg L(-1) of waterborne copper and sacrified after 0, 3, 7, 14 and 21 days. Copper accumulation and Na+/K+-ATPase activity were determined in branchial tissue, whereas osmolality, Na+, Cl-, protein, glucose and cortisol concentrations were measured in plasma samples. Gill copper accumulation increased linearly with exposure time and concentration, whereas gill Na+/K+-ATPase activity was maximally inhibited after 3 days of exposure and showed a significant negative correlation with copper tissue levels. Plasma Cl- values decreased with time of exposure but only at 400 microg L(-1) of copper. Plasma Na+, protein and osmolality decreased with exposure time at the highest copper concentration tested, whereas at 40 microg L(-1) of copper this effect was only observed after 21 days of exposure. Plasma glucose and cortisol levels increased in a dose and time dependent manner, while showing complex fluctuations during the intermediate exposure times. In conclusion, copper induces an early maximum inhibition of gill Na+/K+-ATPase activity in O. niloticus. The subsequent slow decrease in ion plasma levels was related to compensatory mechanisms involving a non-specific stress response that appeared overcome at long-term exposures.     

Effect of spironolactone on the renin-aldosterone system in rats

PRA, PRC and the plasma concentration of aldosterone (Aldo) were measured in rats (Sp-rats) receiving a daily sc injection of Spironolactone, (Sp, 20 mg in olive oil) and in control rats (C-rats) receiving olive oil only. Animals were studied one day after starting treatment, 5 days on treatment or after 5 weeks on the study. PRA, PRC and Aldo were significantly increased in Sp-rats as compared to C-rats throughout all the study. In additional Sp-rats and C-rats, the urine volume, serum Na+ and K+ concentration, Na+ and K+ intake and the urinary excretion of Na+, K+ and aldosterone-18-glucuronide (UAldV) were serially measured during 5 weeks. The total radioactivity plasma clearance after an i.v. bolus injection of 3H-aldosterone was subsequently measured in (5 Sp-rats and 5 C-rats). No significant differences in serum Na+ and K+ concentration and in Na+ and K+ balance were observed between Sp-rats and C-rats. UAldV was significantly higher in Sp-rats than in C-rats during all the study. After 5 weeks on treatment the total radioactivity plasma clearance was significantly higher in Sp-rats than in C-rats. These results indicate that Sp, at high dosage, stimulates renin release and aldosterone secretion by a mechanism unrelated to alterations in Na+ and K+ balance.     

Effect of weak combined magnetic fields on the metamorphosis of the meal-worm 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 meal-worm 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.     

Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump.

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.     

On the effect of unstirred layers on K+-activated electrogenic Na+ pumping in cardiac Purkinje strands.

Many studies of electrogenic Na+ pumping in Purkinje strands have involved intracellular Na+ loading by exposure to 0 mM K+, followed by reexposure to K+. For sheep Purkinje strands the K+ concentration for half-maximal stimulation (K0.5) in such studies is higher than K0.5 of canine Purkinje strands. A model was developed to determine if gradients in the K+ concentration of extracellular fluid layers during enhanced pump activity can account for the discrepancy. Pump activity was assumed linearly dependent on [Na+]i and dependent on [K+]o, according to Michaelis-Menten kinetics. The model simulated diffusion of K+ across unstirred layers and both depletion and accumulation of K+ in extracellular clefts of Purkinje strands during changes in the K+ concentration of the tissue bath. Errors in estimates of K0.5 occurred when delay in achieving a steady state extracellular K+ concentration was simulated. The simulations suggested that a linear relationship between pump current and intracellular Na+, a monoexponential decay of pump current, independence of the rate constants for the current decay on the initial Na+ load and holding potential, and apparent Michaelis-Menten K+ kinetics is not sufficient evidence against pump-induced interstitial K+ depletion having introduced errors in determination of K0.5. It is concluded that interstitial K+ depletion may account for the difference between determinations of K0.5 in sheep and canine Purkinje strands.     

Involvement of Na+/K+pump in fine modulation of bursting activity of the snail Br neuron by 10?mT static magnetic field

The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na(+)/K(+) pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15?min. Biochemical data showed that Na(+)/K(+)-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, (31)P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na(+)/H(+) exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na(+)/K(+) pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na(+)/K(+) pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field.     

Effects of Static Magnetic Field Exposure on Plasma Element Levels in Rat

The interaction of static magnetic fields (SMFs) with living organisms is a rapidly growing field of investigation. The magnetic fields (MFs) effect observed with radical pair recombination is one of the well-known mechanisms by which MFs interact with biological systems. SMF influenced cellular antioxidant defense mechanisms by affecting antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). However, there were insufficient reports about the effects of SMF on macro and trace elements in serum, and the results were contradictory until now. In the current study, 12 rats were divided into two groups, namely as control and exposure group (128 mT and 1 h/day during five consecutive days). The macro and trace element concentrations in serum were examined. No significant difference was observed in the sodium (Na), potassium (K), calcium (Ca), phosphorus (P), and selenium (Se) levels in rat compared to control. By contrast, exposure to SMF showed an increase in the zinc (Zn) level and a decrease in iron (Fe) concentration. Under our experimental conditions, SMF exposure cannot affect the plasma levels of macroelements, while it can disrupt Zn and Fe concentrations in rat.     

Inhibition of Na+,K+-ATPase in Penaeus indicus postlarvae by lead

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg2+-dependent ATPase, Na+,K+-stimulated ATPase, Na+-stimulated ATPase and K+-stimulated ATPase. The Na+,K+-activated, Mg2+-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC50 value of 204.4 microM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC50 (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na+,K+-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.     

Nuclear magnetic resonance studies of sodium/calcium exchange in frog perfused, beating hearts

This study explores the effect of extracellular Ca2+ concentration ([Ca2+]o), on the intracellular Na+ concentration ([Na+]i), in frog intact hearts using nuclear magnetic resonance spectroscopy, which allows for the measurement of [Na+]i in perfused, beating hearts. Decreases in [Ca2+]o yielded marked increases in [Na+]i. A similar effect was seen during inhibition of the Na+/K+ pump and was fully reversible. This sensitivity of [Na+]i to [Ca2+]o, previously observed using microelectrodes, supports a crucial physiological role for Na+/Ca2+ exchange in frog intact, beating hearts.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM-500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase activity was studied. The response of enzymatic activity in the presence of various K+ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na+ / K+-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K+ ions below 20 mM in the medium assay. The IC50 values for high/low isoforms 2.77 x 10(-6) M / 8.56 x 10(-5) M and 7.06 x 10(-7) M / 1.87 x 10(-5) M were obtained in the presence of optimal (20 mM) and 2 mM K+, respectively. However, preincubation in the presence of elevated K+ concentration (50-500 mM) in the medium assay prior to Na+ / K+-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC50 values for both isoforms was the same as in the presence of the optimal K+ concentration. On the contrary, addition of 200 mM K+ into the medium assay after 10 minutes exposure of Na+ / K+-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na+ / K+-ATPase by reducing maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction.     

Effects of extremely low-frequency magnetic field on growth and differentiation of human mesenchymal stem cells

Human Mesenchymal Stem Cells (hMSCs) were exposed to a developed extremely low-frequency (ELF) magnetic fields (50?Hz ,20?mT ELF) system to evaluate whether exposure to (ELF) magnetic fields affects growth, metabolism, and differentiation of hMSCs. MTT method was used to determine the growth and metabolism of hMSCs following exposure to ELF magnetic fields. Na(+)/K(+) concentration and osmolality of extracellular were measured after exposured culture. Alkaline phosphatase (ALP) assay and Calcium assay, ALP staining, and Alizarin red staining were performed to evaluate the osteogenic differentiation of hMSCs under the ELF magnetic field exposure. In these experiments, the cells were exposed to ELF for up to 23 days. The results showed that exposure to ELF magnetic field could inhibit the growth and metabolism of hMSC, but have no significant effect on differentiation of hMSCs. These results suggested that ELF magnetic field may influence the early development of hMSCs related adult cells.     

Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions

Changes in the intracellular concentrations of Na+ and K+ of fetal human fibroblasts have been followed after replacement of serum-containing growth media with unsupplemented and serum-supplemented saline solution (Earle's balanced salt solution). Incubation in unsupplemented salt solution was followed by a progressive increase of the internal Na+ counterbalanced by a decrease of internal K+, without major alterations of the internal osmolarity. After 3 h incubation the intracellular Na+ and K+ concentrations were 120 mM and 50 mM, respectively. These intracellular ion derangements were not associated with a failure of the (Na+ + K+)-ATPase pump, whose activity actually increased with enhanced intracellular Na+ concentration. Ion changes did not take place when serum (in excess of 0.5%, final concentration) was present in the saline solution and a complete restoration to normal of the Na+ and K+ gradients occurred upon addition of serum to cells previously incubated in plain saline solution. The effects of serum were mimicked by furosemide, thus suggesting that channels sensitive to this diuretic are involved in the movement of Na+ and K+ following fibroblast incubation in unsupplemented saline solution.     

Na+ modulates the K+ permeability and the membrane potential of alkalophilic Bacillus

In the absence of Na+ in the medium, the membrane potential of obligately alkalophilic Bacillus cells was found to be decreased by the addition of K+ to the medium, whereas K+ addition in the presence of Na+ had no effect. Rb+ showed essentially the same effect as K+. The decreased membrane potential was quickly restored by lowering the K+ concentration in the medium or by adding Na+ or Li+ to the medium. Thus, in the absence of Na+, the membrane potential of alkalophilic Bacillus seems to be affected by the concentration difference of K+ between inside and outside of the cell, and Na+ or Li+ in the medium suppresses the K+ effect. An exchange between extracellular Rb+ and intracellular K+ was observed in the absence of Na+. However, the exchange was greatly suppressed by the addition of Na+ or Li+ to the medium, indicating that Na+ in the medium modulates the K+ permeability of the alkalophilic Bacillus cell membrane. The K+-induced decrease in the membrane potential of alkalophilic Bacillus in the absence of Na+ is accounted for by the increased K+-permeability of the cell membrane.     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.     

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.     

Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells

Rapid effects of serum stimulation on electrical and ionic membrane properties and their relationship to the initiation of DNA synthesis and cell division have been investigated in mouse N1E-115 neuroblastoma cells. Addition of 10% fetal calf serum to serum-deprived N1E-115 cells results in the initiation of DNA synthesis after a lag of approximately 10 hr. The earliest events following serum addition include: transient membrane potential and resistance changes, detectable within seconds and lasting 5--10 min; a persistent increase in the initial rate of 22Na+ influx, the major part of which is not of electrodiffusional origin, and which is potentiated by weak acid anions; and an external Na+-dependent increase in the rate of the Na+, K+ pump. In the absence of serum the stimulation of the Na+, K+ pump can be mimicked by increasing net Na+ influx with monensin or neurotoxins. Growth-depleted serum fails to induce any of the electrical and ionic events. The diuretic amiloride (0.4 mM) inhibits serum-induced Na+ influx, Na+, K+ pump stimulation and DNA synthesis, but does not affect the electrical response or the basal influx rates. The results suggest that serum growth factors act, at least in part, by stimulating an electroneutral, amiloride-sensitive Na+/H+ exchange mechanism. The enhanced Na+ influx then results in the observed stimulation of the Na+, K+ pump, while the simultaneous efflux of protons may raise the intracellular pH.     

The lack of the effect of a strong constant magnetic field on isolated membrane preparations of Na,K-dependent ATPase

Effect of constant magnetic field (CMF) with induction 10 T on membrane preparations of Na,K-dependent ATPase of bovine brain (lipoproteid vesicules with 300-500 A diameter) were studied. No CMF effect on the activity of Na,K-dependent ATPase was observed under different experimental conditions (three temperature points 15, 20 and 37 degrees C and great variation of Na+,K+ concentrations ratio). CMF also produced no effect on the preparations of Na,K-dependent ATPase immobilized by adsorption on millipore filters.