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Y Eilam

Publications and source records attributed to Y Eilam.

At least 55 records · Page 3Linked to original sources
PubMed1983

Membrane effects of phenothiazines in yeasts. I. Stimulation of calcium and potassium fluxes.

Application of trifluoperazine (10-50 microM) to suspensions of the yeast Saccharomyces cerevisiae induces the following effects. (1) A marked increase in the initial rate of 45Ca2+ influx into the cells, accompanied by an increase in the cellular content of calcium. This stimulation in 45Ca2+ influx (10-20-fold) is observed only in the presence of a metabolic substrate and is completely inhibited by LaCl3. The dose-response curves of the cellular accumulation of 45Ca2+ are of a bell shape, indicating a biphasic response. The concentration of the drug yielding maximal accumulation depends on the density of the cells in the suspensions. The results indicate that the stimulation of 45Ca2+ influx is mediated by an energy-dependent carrier-mediated process and not by the increase in the passive membrane permeability to Ca2+. (2) Efflux of K+ from the cells is induced. Removal of metabolic substrate abolishes the effect at concentrations of up to 35 microM and reduces it at higher concentrations. Addition of high concentrations of cations (K+, Na+, Mg2+) to the medium abolishes the stimulation of both K+ efflux and Ca2+ influx. Chloropromazine, thioridazine and chlorprothixene display similar effects, but at higher concentrations. The results are discussed in terms of two possible alternative mechanisms; (1) calmodulin-independent effects of trifluoperazine on cell membranes, or (2) inhibition of some calmodulin-dependent processes by low concentrations of trifluoperazine.

Antipsychotic Agents↗
PubMed1982

Nystatin effects on cellular calcium in Saccharomyces cerevisiae.

The primary effects of nystatin, a polyene antibiotic, on the yeast Saccharomyces cerevisiae were investigated. Though K+ leakage was observed shortly after the addition of nystatin, Ca2+ leakage was delayed 2-3 h after its application and it occurred only at an acidic pH and in the absence of K+, Na+ or Mg2+ from the medium. However, within 4 min after application nystatin induced a passive influx of Ca2+ into the cells even at a concentration of 1 microM in the medium. These results led to the conclusion that the primary membranal lesion induced by nystatin is not restricted to monovalent cations but is also manifested by increased permeability to Ca2+. The delayed leakage of Ca2+ is explained by the assumption that the bulk of cellular calcium is sequestered so that the concentration of free Ca2+ in the cytoplasm is very low. The sequestered calcium may be liberated 2-3 h after the addition of nystatin as a consequence of secondary damage to the cells such as intracellular acidification and loss of cations.

2,4-Dinitrophenol↗
PubMed1982

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. Herpes 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, inhibited 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.

Calcium↗
PubMed1982

Effect of insulin on folic-acid transport in cultured fibroblasts.

Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of 3H-folic acid was linear with time during 15 seconds and reached a plateau level at 2-3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of experiments (up to 10 minutes). Insulin caused a two-fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 microM insulin. A lag period of 7-10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentrations (above 5 X 10(-6) M) a linear component was observed. Insulin increased the slope of the linear component and the Vmax of the saturable component while the Km remained unaltered.

Animals↗
PubMed1982

Effects of estradiol on uptake of folate in isolated intestinal epithelial cells.

Estradiol 17 beta caused significant changes in the time course of 3H-folate uptake into isolated chick-intestinal epithelial cells. The radioactivity within the cells initially increased reaching a level of 1.5-2 times that of the control thereafter decreased to somewhat below the control levels. These changes were prevented by sodium azide but not by inhibitors of protein synthesis.

Animals↗
PubMed1982

Studies on calcium efflux in the yeast Saccharomyces cerevisiae.

The properties of the 45Ca efflux systems in Saccharomyces cerevisiae were investigated in yeast cells grown overnight in medium containing 45Ca. Efflux was measured in medium containing glucose and Tris-Hepes buffer adjusted to the required pH. In the absence of permeable cations in the medium, at pH 5.2, 20% of the cellular Ca was extruded from the cells during the first 2 h. There was no further decrease in the amount of cellular Ca during an additional 24 h of incubation. The initial rate of Ca efflux was markedly reduced with the increase in the pH of the medium. On the other hand the efflux during the second phase (2-24 h) increased with the increase in medium-pH up to pH 7.5. It is suggested that the initial rapid phase of Ca efflux, in the absence of permeable cations, represents transport across the plasma membrane and is mediated via a Ca2+/H+ antiport. The second phase represents the release of Ca sequestered in some cellular organelles, probably the vacuoles, and is mediated via a different mechanism. Addition of Ca or Mg to the medium markedly stimulated the rate of Ca efflux from both cellular compartments. At the same time a predominant influx of divalent cations was observed. This exchange between intracellular Ca and extracellular divalent cations was not affected by the pH of the medium between pH 5.2 and 7.0. Both processes, Ca efflux and Ca-Mg exchange, required cellular energy; they were almost completely inhibited in the absence of glucose and the presence of antimycin A, a respiratory inhibitor.

Antimycin A↗
PubMed1982

Effect of phenytoin on folic acid uptake in isolated intestinal epithelial cells.

The anticonvulsant drug phenytoin was found to inhibit the uptake of folic acid into isolated chick intestinal epithelial cells. At a concentration of 100 micrograms/ml the drug inhibited the cellular accumulation of folic acid by 60%. The efflux of folic acid from preloaded cells was not affected by the drug. The inhibition was observed at an acidic pH of 5.8 and at a neutral pH of 7.4. Both the Na+-dependent and the Na+-independent components of folate uptake were inhibited. Phenytoin had no effect on the uptake of the glucose analog 3-O-methylglucose. Thus it was concluded that the effect of phenytoin on folate uptake was not due to changes in Na+ fluxes, concentration gradients or energy metabolism, and suggested instead a direct effect on the folic acid uptake system.

Animals↗
PubMed1981

Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells.

Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.

4-Chloromercuribenzenesulfonate↗
PubMed1981

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts.

Primary cultures of bone cells and skin fibroblasts were examined for their Ca++ content, intracellular distribution and Ca++ fluxes. Kinetic analysis of 45Ca++ efflux curves indicated the presence of three exchangeable Ca++ compartments which turned over at different rates: a "very fast turnover" (S1), a "fast turnover" (S2, and a "slow turnover" Ca++ pool (S3). S1 was taken to represent extracellular membrane-bound Ca++, S2 represented cytosolic Ca++, and S3 was taken to represent Ca++ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca++ as compared with cultured fibroblasts. Most of this extra Ca++ was localized in the "slow turnover" intracellular Ca++ pool (S3). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca++: (1) In both types of cells serum caused an increase in the amount of Ca++ in the "very fast turnover" Ca++ pool, and an increase in the rate constant of 45Ca++ efflux from this pool, indicating a decrease in the strength of Ca++ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca++ in the "slow turnover" Ca++ pool (S3), an increase in the rates of Ca++ efflux from the cells to the medium, and from S3 to S2, as well as a decrease in the rate of influx into S3. (3) In bone cells the amount of Ca++ in S3 remained high in "serum activated" cells, the rate of efflux from S3 to S2 increased, and the rate of influx into S3 also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca++ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca++ in bone cells and fibroblasts, in parallel with measurements of 3H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca++ which was followed by a biphasic increase lasting until cell division.

Animals↗
PubMed1981

On the mechanism of folate transport in isolated intestinal epithelial cells.

The mechanism of folic acid (FA) uptake was studied in isolated intestinal epithelial cells prepared from 2- to 6-wk-old chicks. The cells accumulated FA, reaching a level of three- to fivefold that at equilibrium. In the presence of the metabolic inhibitors, NaN3 or KCN, FA was taken up only until equilibration while accumulation of FA was inhibited. Addition of these inhibitors at a steady state of FA accumulation caused a release of intracellular FA. The kinetics of FA uptake were found to be saturable (Km = 3.5 x 10(-5) M), indicating a carrier-mediated mechanism. The steady-state level of FA accumulation was higher as the concentration of NA+ in the medium increased from 0 to 120 mM. This stimulation of FA uptake by Na+ was not due to the stimulation of glucose uptake, because in experiments carried out in the presence of phlorizin, a glucose-transport inhibitor, FA accumulation was not diminished. It is suggested that FA is taken up by a Na+-coupled transport system.

Animals↗
PubMed1980

Effects of vitamin D metabolites on cellular Ca2+ and on Ca transport in primary cultures of bone cells.

Both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) exerted direct effects on Ca2+ transport and accumulation in primary cultures of bone cells. The following changes were recorded. (1) A significant decrease in the amount of intracellular exchangeable Ca2+. (2) A marked increase in the rate constants of efflux from the 'slow'-turnover intracellular Ca pool. (3) A marked increase in the 'initial rate' of Ca influx into the cells. Thus, vitamin D metabolites caused an increase in the turnover of Ca2+ in bone cells and altered the steady-stae level of intracellular exchangeable Ca2+. Whereas the changes in the rate of efflux were abolished in the presence of inhibitors of protein synthesis, the increase in the rate of influx was not sensitive to these inhibitors. It is suggested that the changes in the two fluxes were mediated by different mechanisms and that the changes in influx were due to a direct effect of vitamin D metabolites on the cellular membranes.

24,25-Dihydroxyvitamin D 3↗
PubMed1980

Effects of calcitonin on transport and intracellular distribution of exchangeable Ca2+ in primary culture of bone cells.

The effects of calcitonin (CT) on the cellular transport and accumulation of Ca were studied in bone cells grown in primary culture. The following sequence of events was detected: (1) After 1--2 h of incubation with CT the initial rate of Ca influx into bone cells transiently increased. A longer incubation with CT (3--24 h), caused a gradual decrease in the initial rate of Ca influx, reaching a level below the control at 24 h. (2) After 2 h incubation with CT, efflux studies revealer Ca pool and (b) an increase in the accumulation of Ca in the slow-turnover Ca pool. (3) After 30 min of incubation with CT the amount of Ca in bone cells gradually increased and remained at a higher level for up to 24 h incubation with CT. It is suggested that the transient increase in the initial rate of Ca influx into bone cells, caused by CT, may provide the signal for the accumulation of Ca in the slow-turnover Ca pool, which is probably the mitochondria.

Animals↗
PubMed1980

Combined effect of calcitonin and glucocorticoid hormone on calcium content of cultured bone cells.

The combined effect of calcitonin and triamcinolone acetonide on Ca2+ content in cultured bone cells was studied. Cultures were first preincubated with 45Ca for 24 hrs thus achieving a steady state between the extracellular and intracellular 45Ca2+. Calcitonin was added for 24 hrs and subsequently, triamcinolone acetonide was added for time intervals ranging from 1 to 24 hrs. Calcitonin induced a significant increase in the content of cellular exchangeable Ca2+. When triamcinolone was added to cultures pretreated with calcitonin a marked efflux activity was noted. Concomitant ultrastructural histochemical examinations, using the K-pyroenthymonate fixation method, further substantiated the above finding: calcitonin treated cells revealed increased number of Ca-pyroenthymonate precipitates, whereas the combined treatment led to the disappearance of such Ca-aggregates.

Animals↗
PubMed1978

Nucleoside transport in mammalian cell membranes. IV. Organomercurials and organomercurial-mercaptonucleoside complexes as probes for nucleoside transport systems in hamster cells.

Organomercurials form stable stoichiometric complexes with thiolated nucleosides. The complexes inhibited uptake of ribonucleosides and cytosine arabinoside (CAR) in various types of normal and transformed cells. The inhibition was competitive and reversible (Ki = 3--6 micrometer). The interaction between complexes and transport system displayed a 1:1 stoichiometry. Chemical factors which contributed to the inhibitory power were evaluated with a series of S-alkylated derivatives and S--Hg--R complexes of mercaptonucleosides. The inhibitory potency was not determined exclusively by the hydrophobic nature of either the S-alkylated or the S--Hg--R moieties. Chemical modification of cells with penetrating and nonpenetrating organomercurials lead to stimulation of nucleoside uptake and to an increase in its susceptibility to inhibition by S--Hg--R complexes or S-aklylated derivatives of mercaptopurine ribosides. The kinetic and chemical data obtained with nucleoside analogs and with chemical modifiers suggested complex features of nucleoside transport systems. Four distinct classes of sites were implied: (i) a substrate binding site susceptible directly to competitive inhibition by organomercurial-mercaptonucleoside complexes, (ii) an additional site susceptible either to S-arylalkylated or S-mercuriated derivatives of 6-mercaptopurine ribosides, (iii) SH-containing modifier sites which stimulate uridine uptake upon binding of organomercurials, and (iv) SH-containing modifier sites which inhibit the function upon binding of organomercurials. From the observation that only SH sites related to stimulation were susceptible to modification by macromolecular-SH modifier probes, some conclusions can be drawn regarding the disposition of the various sites in the cell membrane in general and among membrane components in particular.

Binding, Competitive↗
PubMed1977

Distinct properties of uridine transport systems in growing, quiescent and serum-stimulated hamster embryo cells.

The kinetics of uridine uptake in growing, quiescent and serum-activated hamster embryo cells are investigated. The maximum velocity of uridine uptake in growing hamster embryo cells, is lower than in the methylcholanthrene transformed hamster cell line (MCT). This kinetic constant is further reduced in quiescent cells. The Km values in growing and in quiescent hamster embryo cells, as well as in MCT cells are of the same magnitude. Distinct alterations in the pattern of inhibition by nitrobenzyl 6-mercaptoinosin (NBMI) are detected as growing hamster embryo cells become quiescent. In quiescent cells the maximum level of inhibition is lower and the apparent Ki value for the inhibition is much higher. These changes are due to the lower apparent K'm values of NBMI-bound carriers and to the slower rate of formation of the carrier-inhibitor complex. The changes in the kinetic properties of the carriers are partly reversed by serum-activation. The number of inhibitor binding sites (i.e. nucleoside carriers) does not increase by serum-stimulation of quiescent cells (0.36 and 0.34-10(5) sites/cell in quiescent and serum-stimulated cells, respectively). It is implied that the reduction in uridine transport in quiescent cells is probably due to changes in the turnover of the carriers. These changes may be connected with the observed alterations in the properties of carriers or their immediate environment in quiescent cells.

Animals↗