The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Accelerating effect of 2,4,6-trinitrophenol on the glycolytic rate of human red cells

A number of instantaneous changes occurred when picrate was added to a suspension of human red cells in steady state with respect to glycolysis and ion distribution across the membrane at pH 7.40. The rate of glycolysis increased, without change in glycolytic quotient, to a new steady-state value, the effect reaching a maximum of 1.75 times the rate of the control at 0.5 mM picrate. Inorganic phosphate (P(i)) was released at a relatively constant rate, increasing with picrate concentration to 1.0 mmol P(i)/liter cells x h at 5-6 mM picrate. The steady- state concentrations of ATP and 1,3-diphosphoglycerate (1,3-DPG) decreased to new stable values within 15-45 min after the addition of picrate. The ATP level was affected only at picrate concentrations of 1 mM or more, and the level of ATP stabilized at 75 percent of the control values at 4 mM of picrate. In contrast, 1,3-DPG concentrations decreased to 40 percent of the control value of 0.5 mM picrate. Higher concentrations of picrate resulted in only a small additional decrease in the stationary concentration of 1,3-DGP. A net efflux of cellular potassium at constant rate took place. This net efflux was an almost linear function of picrate concentration in the range of 0.1-3 mM. At the latter concentration the net efflux amounted to about 2.7 meq/liter cells x h and a further increase in picrate concentration caused only a minor increase in the potassium efflux. Possible mechanisms for the effects of picrate on human red cell glycolysis are discussed.     

The effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro

Human leucocytes incubated in tissue culture fluid of low-sodium concentration (2 mM; iso-osmolarity maintained with choline chloride) reached a new equlibrium within 1 hour and lost approximately 25% of intracellular potassium and 70% of intracellular sodium. The rate constant for ouabainsensitive sodium efflux fell by more than 50% and the ouabain-insensitive rate constant increased nearly threefold in the low-sodium medium. Total sodium efflux fell in proportion to internal sodium whereas ouabain-insensitive sodium efflux remained unchanged. A reduction in external sodium from 140 to 2 mM was associated with a 75% fall in sodium influx. In the low-sodium medium ouabainsensitive potassium influx exceeded ouabain-sensitive sodium efflux and no ouabain-sensitive potassium efflux could be demonstrated. Ouabain-insensitive potassium influx and that portion of potassium efflux which is dependent on external potassium fell in parallel in low-sodium cells, suggesting reduced activity of a ouabain-insensitive K:K exchange system.     

Vitamin B6 compounds prevent the death of yeast cells due to menadione, a reactive oxygen generator

The antioxidant effects of natural vitamin B(6) compounds on Schizosaccharomyces pombe cells treated with menadione sodium bisulfite (water-soluble menadione and a generator of superoxide, MSB) and the mechanism underlying the function were examined with the yeast cells treated with pyridoxal 5'-phosphate. Vitamin B(6) compounds showed no ex vivo reactivity toward MBS at pH 5.5 or 7.0. The yeast cells showed no growth in the medium containing 1.0 mM MSB. The coexistence of 1.0 mM of each vitamin B(6) compound supported the growth of the yeast cells. The efficacy order was pyridoxal 5'-phosphate>/=pyridoxamine 5'-phosphate>pyridoxamine>pyridoxal>/=pyridoxine. The first three compounds showed higher antioxidant activity than vitamin C did. Pyridoxal 5'-phosphate prevented the reduction of the glutathione content in the MSB-treated cells and, in turn, suppressed the increases in peroxide and thiobarbituric acid reactive substances in the yeast cells and increased the viability of the yeast cells under oxidative stress. The antioxidant function of pyridoxal 5'-phosphate was not dependent on the phosphorelay pathway, which finally triggers the expression of the catalase gene.     

Activation of the calcium permeability of erythrocyte membranes by perfringolysin O

Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1 mM, but not the hemolysis by digitonin at 10 mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1 mM CaCl2 containing 45CaCl2, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.     

Interaction of Saccharomyces cerevisiae with gold: toxicity and accumulation

This paper examines the effects of ionic gold on Saccharomyces cerevisiae, as determined by long-term (growth in gold-containing media) and short-term interactions (H⁺ efflux activity). An increasing gold concentration inhibited growth and at <0.2 mM Au, growth was not observed. Transmission electron microscopy revealed no differences in ultrastructure but fine electron dense particles were observed in unstained preparations from gold-containing medium. After glucose addition (to 10mM) to starved suspensions of S. cerevisiae, glucose-dependent reduction of external pH occurred as the cells extruded protons. In the presence of increasing gold concentrations, the lag time before proton extrusion did not change but the rate and duration decreased significantly with a marked influence on proton efflux rate being observed at 10 M. Extension of preincubation time of yeast cells in gold-containing medium resulted in a decreasing proton efflux rate and colloidal phase formation in the cell suspensions, the time between gold addition and the beginning of colloidal phase formation depending on the gold concentration used. Both Ca and Mg enhanced the inhibitory effect of gold on the yeast cells with Ca showing a stronger inhibitory effect than Mg.     

Characteristics of copper tolerance in Yarrowia lipolytica

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20–25 μmol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.     

Characterization of a BALB/c 3T3 preadipose cell mutant with altered Na+K+Cl- cotransport activity

A BALB/c 3T3 cell mutant (3T3-E12) was isolated by its ability to survive at a low extracellular K+ concentration (0.14 mM). The growth rate of mutant cells was less dependent on external K+ than parental cells. Analysis of potassium transport revealed that 3T3-E12 cells have a decreased activity of the furosemide-sensitive Na+K+Cl- cotransport system, both in the efflux and influx modes. This is shown to be a result of a decrease in the apparent affinity of the transport system for K+ and Na+, but not Cl-. Upon exposure to the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA), BALB/c 3T3 cells exhibited a maximal volume decrease of 20%, while mutant cells shrunk by only 7%, suggesting that regulation of cell volume, at least four exposure to a tumor promoter, is impaired in mutant cells compared to parental 3T3 cells.     

The amiloride-sensitive Na+/H+ exchange system in skeletal muscle cells in culture

Chick skeletal muscle cells in culture have an amiloride-sensitive Na+-transporting system that has the following properties. Na+ uptake is dependent on the extracellular Na+ concentration. The Km value for Na+ is 25 mM and remains constant between pH 7.5 and 8.5. The maximal rate of Na+ transport is higher at alkaline pH. An ionizable group with a pK of 7.6 is essential for the system to be functional. The activity of the amiloride-sensitive Na+ uptake system is controlled by internal Na+ and H+ concentrations. Amiloride inhibition of Na+ uptake is competitively antagonized by increasing Na+ concentration. The dissociation constant for amiloride is 5 microM in Na+-free conditions and is constant between pH 7.5 and 8.5. The Km value for Na+ found from competition experiments is 13 mM. The amiloride-sensitive Na+ influx occurs in parallel with an amiloride-sensitive H+ efflux. This H+ efflux is stimulated by increasing external Na+ concentrations, the Km for Na+ being 15 mM. It is inhibited by amiloride with the same concentration dependence as Na+ influx.     

Transport and cytotoxicity of the anticancer drug 3-bromopyruvate in the yeast Saccharomyces cerevisiae

We have investigated the cytotoxicity in Saccharomyces cerevisiae of the novel antitumor agent 3-bromopyruvate (3-BP). 3-BP enters the yeast cells through the lactate/pyruvate H⁺ symporter Jen1p and inhibits cell growth at minimal inhibitory concentration of 1.8 mM when grown on non-glucose conditions. It is not submitted to the efflux pumps conferring Pleiotropic Drug Resistance in yeast. Yeast growth is more sensitive to 3-BP than Gleevec (Imatinib methanesulfonate) which in contrast to 3-BP is submitted to the PDR network of efflux pumps. The sensitivity of yeast to 3-BP is increased considerably by mutations or chemical treatment by buthionine sulfoximine that decrease the intracellular concentration of glutathione.     

Ammonia accumulation in acetate-growing yeast

During growth on acetate, the pH of yeast cultures rises from 5.8 to around 7-8 in the stationary phase. This was found to result from acetic acid uptake and accompanying H+ loss. In addition, acetate-growing yeast were found to accumulate ammonia. The influence of pH on ammonia transport and accumulation was studied with the analogue [14C]methylamine with the following results. (a) Methylamine uptake kinetics from 0.1-50 mM were consistent with a single-component uptake system (NH+4 permease) at pH values more acidic than 6.5, and with a two-component system (NH+4 permease and NH3 diffusion) above pH 7.5. (b) Equilibrium accumulation of methylamine was found to increase with increasing pH. (c) Methylamine efflux from methylamine-loaded cells increased as the external pH decreased. It was concluded from measurements of the internal pH under various culture conditions that the accumulation of ammonia in acetate-growing alkaline cultures resulted from the sum of two processes: (1) an energy-driven NH+4 transport; and (2) NH3 diffusion dependent on the delta pH.     

Furosemide and Ca2+ affect 86Rb+ efflux from pancreatic beta-cells by different mechanisms

The interaction between furosemide, calcium and D-glucose on the 86Rb+ efflux from beta-cell-rich mouse pancreatic islets was investigated in a perifusion system with high temporal resolution. Raising the glucose concentration from 4 to 20 mM induced an initial decrease in 86Rb+ efflux, which was followed by a steep increase and then a secondary decrease. Removal of extracellular calcium increased the 86Rb+ efflux at 4 mM D-glucose but reduced it at 20 mM. The initial biphasic changes in 86Rb+ efflux induced by 20 mM D-glucose were inhibited by calcium deficiency. Furosemide (100 microM) reduced the 86Rb+ efflux rate both at 4 and 20 mM D-glucose and the magnitudes appeared to be similar at either glucose concentration. Furosemide (100 microM) reduced the glucose-induced (10 mM) 45Ca+ uptake but did not affect the basal (3 mM D-glucose) 45Ca+ uptake. However, the ability of furosemide (100 microM) to reduce the 86Rb+ efflux at a high glucose concentration (20 mM) was independent of extracellular calcium. The inhibitory effects of furosemide and calcium deficiency on the 86Rb+ efflux rate appeared to be additive. It is concluded that the effect of furosemide on 86Rb+ efflux is not secondary to reduced calcium uptake and that the effects of furosemide and calcium deficiency are mediated by different mechanisms. The effect of furosemide is compatible with inhibition of loop diuretic-sensitive co-transport of Na+, K+ and Cl- and the effect of calcium deficiency with reduced activity of calcium-regulated potassium channels.     

Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts.

The multiplication rate of sparse cultures of chick embryo cells is only slightly lower at pH 6.9 than at pH 7.4. There is, however, a marked reduction in the multiplication rate of the pH 6.9 cultures before they reach confluency. Cultures at pH 7.4 continue to multiply beyond confluency with only a slight decrease in the multiplication rate. Eighty to ninety percent of the glucose taken up by the cells growing at each pH is converted to lactic acid which is released into the medium. Metabolic reduction in pH of the medium is almost entirely accounted for by the amount of lactic acid produced by the cells. Neither the intracellular nor extracellular accumulation of lactic acid nor the accompanying reduction in pH is sufficient to explain density dependent inhibition of the rate of multiplication of chick cells. The rate of lactic acid production and the multiplication rate of chick cells are independent of glucose concentration in the range of 2--16 mM. In view of the kinetic parameters for the uptake of glucose, this shows that glycolysis is not limited by the rate of glucose uptake and that depletion of glucose from the medium cannot account for the onset of density dependent inhibition of multiplication. However, when cells reach very high population densities, conventional glucose concentrations of 5 mM can be depleted overnight by chick cells. Since the multiplication rate of cells is dependent on glucose concentration when it falls below 2 mM, depletion of glucose may cause some growth inhibition in crowded cultures supplied with conventional medium.     

Release of glutamate and gamma-aminobutyric acid in the ovine fetal hippocampus: ontogeny and effect of hypoxia.

The effects of increased potassium ion concentration (50 mM) and hypoxia on the efflux of glutamate and gamma-aminobutyric acid (GABA) were studied in ovine fetal hippocampal slices using the static-pool-interface superfusion method at three selected gestational ages (85 days, 105 days, 135 days; term, about 147 days). There was no difference in spontaneous efflux of either amino acid across the three gestational ages. Potassium ion stimulated the efflux of glutamate in the hippocampus of the 85-days-old fetus only, and this efflux of glutamate was not calcium-ion dependent. Potassium ion stimulated the efflux of GABA in the ovine fetal hippocampus at days 85 and 105 only; this efflux was calcium-ion dependent. A ten-minute period of hypoxia did not enhance the efflux of either glutamate or GABA. The data indicate that both glutamate and GABA are present in the ovine fetal hippocampus, and can be released by depolarizing concentrations of potassium ion in the immature fetus. The lack of potassium ion-evoked efflux of glutamate and GABA in the mature fetal hippocampus may reflect a toxic response to this stimulus. The lack of calcium ion regulation of glutamate efflux compared with GABA efflux indicates either a difference in maturation of glutamatergic synaptic mechanisms compared with GABAergic mechanisms, or is indicative of glial release of glutamate. Prolonged, severe hypoxia (greater than 10 min) may be required to evoke efflux of glutamate in the developing fetal hippocampus.     

Kinetics of sulfate uptake by yeast.

Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H+ and an efflux of 1 K+ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but Km is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.     

The influence of cytoplasmic sodium concentration on the stoichiometry of the sodium pump

Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).     

Dose-Dependent, K+-Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+-Dependent

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.     

Characterization of anion permeability in AH-66 hepatoma ascites cells

The sulfate transport in AH-66 hepatoma ascites cells was examined under various controlled conditions using 35SO42- as a tracer. The sulfate efflux rate was dependent on temperature, pH and anion species of the cell suspending medium. The efflux rate became saturated as the concentration of extracellular anions was increased. The efflux of anion was inhibited by some chemical reagents specifically reactive with amino or sulfhydryl groups. The results obtained in this study suggest that sulfate anions were transported by a facilitated transport system(s), and that some membrane protein(s) is involved in the anion transport system(s) of AH-66 cells. Both amino and sulfhydryl groups are thought to play a determinant role at the sulfate transport site in AH-66 cells.     

The interaction of chloride with the sulfate transport system of Ehrlich ascites tumor cells.

The effect of Cl- on SO4(-2) efflux was studied in both Cl--containing and Cl--free ascites tumor cells loaded with 35SO4(-2) to test the hypothesis that Cl--SO4(-2) exchange is mediated by the same mechanism responsible for SO4(-2)-self exchange. The addition of Cl--free, 35SO4(-2) loaded cells to a SO4(-2)-free, Cl- medium results in: (1) SO4(-2) efflux that is dependent on the extracellular Cl- concentration (Km = 4.85 mM; ke = 0.048 min-1 at 50 mM Cl-) and (2) net Cl--uptake that exceeds SO4(-2) loss. Both SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) and ANS (1-anilino-8-napthalene sulfonate) inhibit S04(-2) efflux but are without effect on Cl- uptake. The addition of Cl--containing, 35SO4(-2) loaded cells to a SO4(-2)-free, Cl- medium results in: (1) a slight gain in cellular Cl- and (2) ke for SO4(-2) efflux identical to that for Cl--free cells.