Endogenous regulation of 2-deoxyglucose uptake in C6 glioma cells correlates with cytoskeleton-mediated changes of surface morphology

The cellular basis of the membrane-limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time-dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2-deoxyglucose (2-DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F-actin content. Because the changes of 2-DG uptake rates occurred independent of the intracellular 2-DG concentration, the bulk of this 2-DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2-DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton-mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. II: intracellular metabolite and enzyme assays at steady state chemostat cultures

Intracellular metabolite concentration and enzyme activity measurements were made to explain the new metabolic and growth phenomena seen in the micro-aerobic, continuous yeast cultures described in Part I. The results of these assays suggested mechanisms for the observed maximum in the specific ethanol productivity as a function of the oxygen feed rate, changing ATP yields, the effects of antifoam, and the sharp changes in the biomass concentration with small changes in the oxygenation. Measured were the intracellular concentrations of ATP, NADH, glucose 6-phosphate, pyruvate, glycerol, and ethanol, and the activities of hexokinase and alcohol dehydrogenase. Rate-limiting steps were identified by the accumulation of metabolites upstream and the depletion of metabolites downstream of the step.A potential mechanism for the stimulation of fermentation with decreasing oxygenation was an activation of glucose transport by an accumulating intracellular ATP concentration. The inhibition of fermentation at yet lower oxygenation rates may have been caused by the continued accumulation of ATP to the point that the glycolytic kineses were inhibited. A mechanism for the changing ATP yields and intracellular ATP concentration proposed the existence of ATPases or ATP waste reactions stimulated by both oxygen and ATP. Antifoam had the effect of decreasing the resistance for glycerol transport out of the cell. The resulting stimulation of glycerol production and inhibition of ethanol production decreased the intracellular ATP content. Finally, intracellular ethanol was found not to accumulate to levels of higher than the extracellular concentration.     

Intracellular ATP in a glucosephosphate isomerase mutant ofSaccharomyces cerevisiae

The degree of ATP depletion caused by glucose in a glucosephosphate isomerase-deficient strain of Saccharomyces cerevisiae was determined. Even in the presence of a sugar normally fermentable by the mutant, the addition of glucose can decrease the intracellular ATP, depending on the competition of the sugars for transport and subsequent phosphorylation. For both parent and mutant cells, a correlation exists between the calculated velocity of ATP formation or ATP consumption during the utilization of different concentrations of sugars and the experimental intracellular ATP level. For initially resting yeast cells, a rate increase of 35 mumol per min per g ATP was calculated to increase the intracellular level of this nucleotide by 1 mumol per g cell mass.     

Caloric restriction mimetic 2-deoxyglucose antagonizes doxorubicin-induced cardiomyocyte death by multiple mechanisms

Caloric restriction (CR) is a dietary intervention known to enhance cardiovascular health. The glucose analog 2-deoxy-D-glucose (2-DG) mimics CR effects in several animal models. However, whether 2-DG is beneficial to the heart remains obscure. Here, we tested the ability of 2-DG to reduce cardiomyocyte death triggered by doxorubicin (DOX, 1 μm), an antitumor drug that can cause heart failure. Treatment of neonatal rat cardiomyocytes with 0.5 mm 2-DG dramatically suppressed DOX cytotoxicity as indicated by a decreased number of cells that stained positive for propidium iodide and reduced apoptotic markers. 2-DG decreased intracellular ATP levels by 17.9%, but it prevented DOX-induced severe depletion of ATP, which may contribute to 2-DG-mediated cytoprotection. Also, 2-DG increased the activity of AMP-activated protein kinase (AMPK). Blocking AMPK signaling with compound C or small interfering RNA-mediated knockdown of the catalytic subunit markedly attenuated the protective effects of 2-DG. Conversely, AMPK activation by pharmacological or genetic approach reduced DOX cardiotoxicity but did not produce additive effects when used together with 2-DG. In addition, 2-DG induced autophagy, a cellular degradation pathway whose activation could be either protective or detrimental depending on the context. Paradoxically, despite its ability to activate autophagy, 2-DG prevented DOX-induced detrimental autophagy. Together, these results suggest that the CR mimetic 2-DG can antagonize DOX-induced cardiomyocyte death, which is mediated through multiple mechanisms, including the preservation of ATP content, the activation of AMPK, and the inhibition of autophagy.     

ADP Compartmentation Analysis Reveals Coupling between Pyruvate Kinase and ATPases in Heart Muscle

Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.     

Effect of 2-deoxyglucose on cell wall formation in Saccharomyces cerevisiae and its relation to cell growth inhibition

The growth inhibition and the lysis of Saccharomyces cerevisiae caused by 2-deoxy-d-glucose (2-DG) were shown to be a consequence of unbalanced cellular growth and division. The lysis, but not the repression of growth and osmotic fragility of cells, could be suppressed by the addition of mannitol as an osmotic stabilizer. This result, as well as the morphological changes observed in the cells and changes in the chemical composition of the cell walls, showed that S. cerevisiae grown in the presence of 2-DG formed weakened cell walls responsible for the osmotic fragility. Evidence is presented for the first time demonstrating the incorporation of 2-DG into yeast cell wall material. Other data suggest that the inhibition of yeast growth by 2-DG results from an interference of phosphorylated metabolites of 2-DG with metabolic processes of glucose and mannose involved in the synthesis of structural cell wall polysaccharides.     

The Ability of 2-Deoxyglucose to Promote the Lysis of Streptococcus bovis JB1 via a Mechanism Involving Cell Wall Stability

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (<5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (>85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture superntant. Received: 11 April 1997 / Accepted: 10 June 1997     

The proton electrochemical gradient across the plasma membrane of yeast is necessary for phospholipid flip

Recently, two members of the P4 family of P-type ATPases, Dnf1p and Dnf2p, were shown to be necessary for the internalization (flip) of fluorescent, 7-nitrobenz-2-oxa-1,3-diazol-4-yl(NBD)-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae. In the current study, we have demonstrated that ATP hydrolysis is not sufficient for phospholipid flip in the absence of the proton electrochemical gradient across the plasma membrane. This requirement was demonstrated by two independent means. First, collapse of the plasma membrane proton electrochemical gradient by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) almost completely blocked NBD-phospholipid flip while only moderately reducing the cytosolic ATP concentration. Second, strains with point mutations in PMA1, which encodes the plasma membrane proton pump that generates the proton electrochemical gradient, are defective in NBD-PC flip, whereas their cytosolic ATP content is actually increased. These results establish that the proton electrochemical gradient is required for NBD-phospholipid flip across the plasma membrane of yeast and raise the question whether it contributes an additional required driving force or whether it functions as a regulatory signal.     

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.     

Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance

The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.     

Glucose transport and catabolite repression in Endomycopsis fibuligera yeasts

The role of systems for glucose transport in the manifestation of carbon catabolite repression of glucoamylase synthesis was studied in the yeast Endomycopsis fibuligera. Experimentas were conducted with its mutant AB-192 defective in the system of transport universal for glucose and 2-deoxy-D-glucose (2-DG). The nature of the mutation was established from the following data: (1) transport of labeled glucose into the mutant cells was twice as low in comparison with the parent culture 20-9; (2) transport of labeled 2-DG was suppressed almost entirely; (3) no competition was found between glucose and 2-DG for penetration into the mutant cells. Glucoamylase synthesis in the mutant AB-192 was not sensitive to catabolite repression by glucose. This was confirmed by the resistance of the AB-192 cells to the inhibition by glucose and their complete resistance to the repression by 2-DG. Moreover, an addition of cAMP did not stimulate glucoamylase synthesis by the mutant culture in the presence of glucose and 2-DG. It can be concluded therefore that the resistance of the yeast to catabolite repression by the glucose is caused by the mutation in the system for carbohydrate transport. The results suggest that the system of glucose transport plays an important role in the manifestation of carbon catabolite repression in the yeast Endomycopsis fibuligera.     

Purinergic modulation of glucose uptake into cultured rat podocytes: effect of diabetic milieu

Extracellular purines act via P1 and P2 receptors on podocytes and may influence on their function. This action may be modified under various (patho)physiological conditions leading to development of podocytopathy. Aim of study was to investigate effects of diabetic milieu, represented by high glucose concentration (HG, 30 mM glucose) on purinergic-induced changes of 2-deoxy-d-glucose (2-DG) uptake and on extracellular purines metabolism in cultured rat podocytes. Basal 2-DG uptake was 2.7-fold enhanced in HG compared to normal glucose concentration, NG (1271 ± 86 vs. 477 ± 37 nmol/h/mg protein, P < 0.001). ATP stimulated 2-DG uptake by 44 ± 4% and 29 ± 5% in NG and HG, respectively. ATP analogues, β, γ-methylene ATP and 2-methylthio ATP stimulated 2-DG uptake in range of 18–34% in NG and 16–17% in HG. Benzoylbenzoyl ATP increased 2-DG uptake about 24 ± 2% in NG however, its effect in HG reached 50 ± 1%. The antagonists of P2 receptors (suramin, reactive blue 2, PPADS) decreased basal 2-DG uptake in NG and HG; suramin and reactive blue 2 at average of 15 ± 4% in NG but in HG the effect was in following order: suramin 28 ± 3%; PPADS 20 ± 3% and RB-2 9 ± 0.9%. Extracellular adenosine concentration was higher in HG than in NG (0.48 ± 0.01 vs. 5.05 ± 0.39 μM, P < 0.05), however intracellular ATP content and extracellular ATP concentration were not affected. Neither ecto-ATPase nor ecto-5′-nucleotidase activities were affected in HG. In conclusion, diabetic milieu affects purinergic modulation of glucose transport into podocytes which may play a role in development of diabetic podocytopathy.     

CD1 stimulation of human T cell lines induces a rapid increase in the intracellular free Ca2+ concentration and the production of IL-2.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.     

On the mechanism of the Crabtree effect in mouse ascites tumor cells

The addition of glucose to ELD and ELT/B1 mouse ascites tumor cell suspensions caused a 2.3-fold increase in the phosphorylation state ratio, (ATP)/(ADP) (Pi), because of a decrease in the intracellular Pi concentration. The addition of glucose to these cell suspensions has been reported by Chance and Hess ('59) to cause an increase in the study state reduction of cytochrome b and an increase in the steady state oxidation of cytochrome c. On a quantitative basis these two independent measurements suggest that a near equilibrium exists between the oxidation-reduction state of the mitochondrial electron carriers and the reactions of ATP synthesis (as expressed by the phosphorylation state ratio) both before and after glucose addition. We conclude that the mechanism of the inhibition of respiration by glycolysis (the Crabtree effect) is a decrease in the rate of electron transport caused by the mass action effect of the elevated phosphorylation state ratio.     

Inhibition of protein phosphorylation by chloroquine

The rapid phase of fructose-1,6-bisphosphatase (FBPase) inactivation following glucose addition to starved yeast cells [reported previously] is inhibited on addition of 10 mM chloroquine (CQ) at about pH 8. This inhibition of inactivation was shown to be due to the prevention of phosphorylation of the enzyme. CQ was also found to inhibit general protein phosphorylation in the yeast cells. Glycolysis, as observed by changes in intracellular glucose-6-phosphate and extracellular glucose and ethanol concentrations, was shown to be significantly inhibited in cells treated with CQ. Similarly, a decrease in ATP concentrations was observed. However, during the early stages of phosphorylation of FBPase, levels of ATP were similar in cells containing CQ as in those without CQ. Thus, decrease in ATP levels is not thought to be significantly responsible for the inhibition of protein phosphorylation. However, the phosphorylating activity of cyclic AMP-dependent protein kinases is inhibited in vitro by relatively low concentrations of CQ. Thus, prevention of protein phosphorylation by CQ is believed to be due to inhibition of protein kinases in yeast cells.Abbreviations FBPase fructose-1,6-bisphosphatase - CQ chloroquine - SDS sodium dodecyl sulfate - G6P glucose-6-phosphate - TCA trichloroacetic acid     

2-Deoxyglucose Enhances 1-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine-Induced ATP Loss in the Mouse Brain

Abstract: The effects of 2-deoxyglucose (2-DG), an inhibitor of the uptake and use of glucose, on ATP loss caused by the neurotoxicant 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) were determined in the mouse brain. 2-DG alone had no effect on brain ATP levels, but when administered 30 min before MPTP exposure, 2-DG significantly enhanced MPTP-induced ATP reduction. This was reflected as an increase in ATP loss in the striatum (from 15 to 27%) as well as a significant decrease in ATP in the cerebellar cortex, an area of the brain that was not affected after exposure to MPTP alone. In mice pretreated with 2-DG, striatal ATP levels remained significantly decreased for >8 h after MPTP administration. In contrast, ATP levels in the cerebellar cortex returned to normal values within 4 h from MPTP exposure. Mazindol, a catecholamine uptake blocker, completely protected against MPTP-induced loss of striatal ATP in the absence of 2-DG, but it only partially prevented striatal ATP decrease after administration of both 2-DG and MPTP; mazindol was also ineffective in protecting against ATP loss caused by 2-DG and MPTP in the cerebellar cortex. 2-DG/MPTP-induced ATP loss appeared to be associated with the presence of the 1 -methyl-4-phenylpyridinium (MPP⁺) metabolite because (1) the pattern of ATP recovery in the striatum and cerebellar cortex appeared to reflect the pattern of MPP⁺clearance from these areas of the brain (i.e., significant MPP⁺ levels persisted longer in the striatum than in the cerebellar cortex), and (2) ATP decrease was completely prevented by blocking the conversion of MPTP to MPP⁺with the monoamine oxidase B inhibitor deprenyl. Data indicate that impairment of glucose metabolism dramatically enhances the effects of MPTP/MPP⁺ on cerebral energy supplies, making these effects relatively nonselective for dopaminergic neurons of the nigrostriatal pathway.     

The role of metabolic memory in the ATP paradox and energy homeostasis

In yeast, a sudden transition from glucose limitation to glucose excess leads to a new steady state at increased metabolic fluxes with a sustained decrease in the ATP concentration. Although this behaviour has been rationalized as an adaptive metabolic strategy, the mechanism behind it remains unclear. Nevertheless, it is thought that, on glucose addition, a metabolite derived from glycolysis may up-regulate ATP-consuming reactions. The adenine nucleotides themselves have been ruled out as the signals that mediate this regulation. This is mainly because, in that case, it would be expected that the new steady state at increased fluxes would be accompanied by an increased stationary ATP concentration. In this study, we present a core model consisting of a monocyclic interconvertible enzyme system. Using a supply-demand approach, we demonstrate that this system can account for the empirical observations without involving metabolites other than the adenine nucleotides as effectors. Moreover, memory is an emerging property of such a system, which may allow the cell to sense both the current energy status and the direction of the changes.     

Phosphorus-31 and carbon-13 nuclear magnetic resonance study of glucose and xylose metabolism in agarose-immobilized Candida tropicalis.

Candida tropicalis can ferment both hexose and pentose sugars. Here, we have used 31P and 13C nuclear magnetic resonance spectroscopy to study the capacity of this yeast species to metabolize glucose or xylose when immobilized in small (< 1-mm-diameter) agarose beads. Immobilized C. tropicalis metabolizing glucose showed rapid initial growth within the beads. A corresponding drop in the intracellular pH (from 7.8 to 7.25) and hydrolysis of intracellular polyphosphate stores were observed. Although the initial rate of glucose metabolism with immobilized C. tropicalis was similar to the rate observed previously in cell suspensions, a decrease by a factor of 2.5 occurred over 24 h. In addition to ethanol, a significant amount of glycerol was also produced. When immobilized C. tropicalis consumed xylose, cell growth within the beads was minimal. The intracellular pH dropped rapidly by 1.05 pH units to 6.4. Intracellular ATP levels were lower and intracellular Pi levels were higher than observed with glucose-perfused cells. Consumption of xylose by immobilized C. tropicalis was slower than was previously observed for oxygen-limited cell suspensions, and xylitol was the only fermentation product.     

Molecular analysis of the dissociation between IL-2 production and proliferation in a response of a T cell clone to the antigen presented by B cells

Using B cells as APC, antigen specific responses of two murine T cell clones, 34-7F and 35-8H, were analyzed. 34-7F cells produced IL-2 but failed to proliferate, whereas 35-8H cells both produced IL-2 and proliferate. The antigenic stimulation increased intracellular free Ca2+ concentration in both clones, but enhanced inositol phospholipid metabolism only in 35-8H cells. The treatment of 34-7F cells with PMA, an activator of protein kinase C, synergized with the antigenic stimulation to induce the proliferation of the T cells. Thus, the failure of 34-7F cells to proliferate in the Ag response appears to result from the absence of an increase in inositol phospholipid metabolism. The absence is likely due to the defect in B cells as APC, inasmuch as the antigenic stimulation of 34-7F cells with whole spleen cells induced increases in inositol phospholipid metabolism and proliferation. The PMA treatment synergized with the Ag on B cells to enhance IL-2R expression, which was not inhibited by the addition of nifedipine, a calcium channel blocker. The agent inhibited the IL-2 production. Taken together, the results in the present experiments suggest the association of IL-2 production with increases in intracellular free Ca2+ concentration but not in inositol phospholipid metabolism, and that of IL-2R expression with increases in the metabolism but not in intracellular free Ca2+ concentration.