Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica(prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca²⁺]_i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca²⁺]_i was significantly curtailed in calcium-free buffer (0% Ca²⁺) as compared to that in 100% Ca²⁺ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca²⁺ release-activated Ca²⁺(CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca²⁺]_i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca²⁺]_i in 0% Ca²⁺ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP₃ production, completely abolished increases in [Ca²⁺]_i, induced by OFPC, suggesting that these polyphenols induce the production of IP₃ that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca²⁺]_i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca²⁺]_i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica (prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca2+]i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca2+]i was significantly curtailed in calcium-free buffer (0% Ca2+) as compared to that in 100% Ca2+ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca2+ release-activated Ca2+ (CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca2+]i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca2+]i in 0% Ca2+ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca2+-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP3 production, completely abolished increases in [Ca2+]i, induced by OFPC, suggesting that these polyphenols induce the production of IP3 that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca2+]i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca2+]i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells

Experiments assessed whether long term exposure to 50 Hz pulsed electromagnetic fields with a peak magnetic field of 3 mT can alter the dynamics of intracellular calcium in human astrocytoma U-373 MG cells. Pretreatment of cells with 1.2 microM substance P significantly increased the [Ca(2+)](i). The same effect was also observed when [Ca(2+)](i) was evaluated in the presence of 20 mM caffeine. After exposure to electromagnetic fields the basal [Ca(2+)](i) levels increased significantly from 143 +/- 46 nM to 278 +/- 125 nM. The increase was also evident after caffeine addition, but in cells treated with substance P and substance P + caffeine we observed a [Ca(2+)](i) decrease after exposure. When we substituted calcium-free medium for normal medium immediately before the [Ca(2+)](i) measurements, the [Ca(2+)](i) was similar to that measured in the presence of Ca(2+). In this case, after EMFs exposure of cells treated with substance P, the [Ca(2+)](i), measured without and with addition of caffeine, declined from 824 +/- 425 to 38 +/- 13 nM and from 1369 +/- 700 to 11 +/- 4 nM, respectively, indicating that electromagnetic fields act either on intracellular Ca(2+) stores or on the plasma membrane. Moreover the electromagnetic fields that affected [Ca(2+)](i) did not cause cell proliferation or cell death and the proliferation indexes remained unchanged after exposure.     

Vitamin C decreases intracellular calcium level in human lymphoid cells.

Human lymphocytes have low levels of many antioxidant enzymes however they are know to concentrate vitamin C. Cell injury, including oxidative stress effects, is associated with calcium influx so the influence of vitamin C on the maintenance of calcium levels in leukocytes was studied. Incubation of Molt-3 human lymphoblastoid cells with physiologically relevant concentrations of vitamin C and the calcium ionophore A23187 reversed the calcium influx and increased nuclear protein level associated with the ionophore alone. It is concluded that intracellular vitamin C can inhibit calcium influx into leukocytes so helping to minimise cell damage.     

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Modulation of calcium channel function by drugs

Calcium channel blocking drugs, or "calcium antagonists", have been increasingly used in the last decade, both as valuable cardiovascular drugs, and as tools to investigate the pharmacology of the calcium channels which play a vital role in the excitation-activation coupling of many excitable cells. Three important developments, "patch clamping" to investigate single calcium channels, ligand binding studies to investigate the calcium antagonist "receptor sites", and the introduction of novel calcium channel activators, or "calcium agonists", have recently led to greater understanding of the mechanism of action of drugs on the calcium channel. We show here how the calcium channel modulators interact with the binding sites to increase or decrease calcium flux, and hence to modulate the activity of many excitable tissues. We predict that these new developments will soon result in the isolation of purified calcium channels, and investigation of their subtypes and drug sensitivities. This information could lead to the introduction of novel, more selective calcium antagonists for a variety of indications such as atherosclerosis or neurological disorders. Of particular interest is the potential of tissue-selective calcium agonistic drugs to combat cardiac failure or endocrinological disorders.     

Keratinization of rat vaginal epithelium--V. Modulation of intracellular calcium by estradiol.

Changes in the calcium levels under the influence of estradiol were investigated in rat vaginal epithelial cells (VEC). After single estradiol injection, the immature rats showed 1.5-fold increase in Ca2+ levels within 15 min when compared to control animals. Progesterone priming brought calcium levels well below control values throughout the experimental period (up to 12 h). Ca2+ levels in serum did not show any appreciable change. Localization of calcium in VEC with electron microscopy showed aggregates of calcium oxalate on the inner nuclear membrane, nucleolus, mitochondria and keratohyaline granules. After 15 min of estradiol priming, maximum electron density was seen on all these cell organelles mentioned above, however, by 30 min the electron density was reduced considerably and did not increase during the experimental period (up to 12 h).     

Modulation of platelet-activating-factor-induced calcium influx and intracellular calcium release in platelets by phorbol esters.

1. The rate of 45Ca2+ efflux from prelabelled rat islets of Langerhans was stimulated by carbachol in a dose-dependent manner. 2. Significant stimulation occurred in the presence of 0.2 microM-carbachol; the response was half-maximal at 3-5 microM and was maximal at 20 microM. 3. Stimulation of 45Ca2+ efflux by carbachol was not dependent on the presence of extracellular Ca2+ and was enhanced in Ca2+-depleted medium. 4. Stimulation of 45Ca2+ efflux by 5 microM-carbachol occurred independently of any change in [3H]arachidonic acid release in prelabelled islets, and probably reflected generation of inositol trisphosphate in the cells. 5. The amphipathic peptide melittin failed to increase islet-cell 45Ca2+ efflux at a concentration of 1 microgram/ml, and caused only a modest increase at 10 micrograms/ml. 6. Despite its failure to increase 45Ca2+ efflux, melittin at 1 microgram/ml caused a marked enhancement of 3H release from islets that had been prelabelled with [3H]arachidonic acid. 7. The stimulation of 3H efflux caused by melittin correlated with a dose-dependent increase in the unesterified [3H]arachidonic acid content of prelabelled islets and with a corresponding decrease in the extent of labelling of islet phospholipids. 8. Combined addition of melittin (1 microgram/ml) and 5 microM-carbachol to perifused islets failed to augment 45Ca2+ efflux relative to that elicited by carbachol alone. 9. The data indicate that melittin promotes an increase in arachidonic acid availability in intact rat islets. They do not, however, support the proposal that this can either directly reproduce or subsequently modify the extent of intracellular Ca2+ mobilization induced by agents that cause an increase in inositol trisphosphate.     

CD5 antibodies increase intracellular ionized calcium concentration in T cells

The binding of a variety of monoclonal antibodies to the CD5 (T, gp67) pan T cell differentiation antigen has been shown to potentiate T cell proliferation. In this paper we show that CD5 monoclonal antibodies cause increased intracellular free calcium concentration ([Ca2+]i) in T cells. An increase in [Ca2+]i occurred within 1 min in indo-1-loaded PBMC after the addition of CD5 monoclonal antibodies and cross-linking with a second step anti-mouse kappa light chain antibody. Cross-linking of CD5 was effective when done directly on the cell surface or by the administration of preformed soluble complexes that contained CD5 antibodies. Calcium mobilization induced by suboptimal concentrations of CD3 antibodies was specifically augmented and sustained by CD5 antibodies, although the enhancement was modest in magnitude. When cell surface phenotype was correlated with calcium mobilization, it was found that the CD5 response was restricted to CD5+/CD3+ cells, and that approximately 90% of CD5+ cells had responded. CD5-induced calcium mobilization was found to differ from CD3 stimulation in that EGTA entirely ablated the CD5 response, whereas the CD3 response was resistant to EGTA, indicating that the CD5-induced increased [Ca2+]i is derived primarily or entirely from extracellular calcium. CD5-stimulated calcium mobilization also differed from CD3 in that the CD5 response was inhibited by pretreatment with phorbol myristate acetate, whereas the CD3 response was not, suggesting that depletion of protein kinase C causes an uncoupling of signal transduction between CD5 and calcium channels. Finally, experiments were done with T cells after antigenic modulation of the CD3 or CD5 molecules. Unexpectedly, both the CD5 and the CD3 responses were ablated on CD3-modulated cells, whereas only the CD5 response was ablated on CD5-modulated cells. In addition, several Cd5+/CD3- T cell leukemia lines also failed to respond to CD5 stimulation, providing further evidence which indicates that the CD5 response depends on the cell surface expression of CD3 or a CD3-associated structure. These findings suggest that one mechanism for CD5-induced augmentation of mitogen-stimulated T cell proliferation involves increased [Ca2+]i which is distinct from but interdependent with that induced by stimulation of the CD3 molecule.     

Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene

Background  

Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca²⁺ level ([Ca²⁺]_i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca²⁺/calmodulin-dependent protein kinase.     

Modulation of intracellular glutathione affects adipogenesis in 3T3-L1 cells

Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non‐enzymatic intracellular anti‐oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3‐L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L ‐buthionine‐sulfoximine (BSO), enhances C/EBPβ LAP/LIP ratio and PPARγ expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24 h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti‐adipogenic properties also by increasing GSH content through γ‐glutamyl‐cysteine ligase (GCL) induction. Overall data indicate that in pre‐adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity. J. Cell. Physiol. 226: 2016–2024, 2011. © 2010 Wiley‐Liss, Inc.     

Mobilization of intracellular calcium by endothelin in Swiss 3T3 cells

When intracellular free Ca2+ concentration [( Ca2+]i) was monitored in fura2-loaded Swiss 3T3 cells, endothelin increased [Ca2+]i in a dose-dependent manner; after the addition of endothelin, an initial transient peak was observed immediately and was followed by a sustained increase in [Ca2+]i lasting at least 5 min. 45Ca2+ efflux and influx experiments in endothelin-stimulated Swiss 3T3 cells revealed that the change in [Ca2+]i could be explained by a dual mechanism; an initial transient peak induced mainly by the release of Ca2+ from intracellular stores and the sustained increase by an influx of extracellular Ca2+. Cellular generation of inositol 1,4,5-trisphosphate and cyclic AMP were not induced by endothelin, suggesting that other cellular mediators with the capacity to release Ca2+ from intracellular stores play a significant role in the signal transduction pathway of endothelin in Swiss 3T3 cells.     

EGF receptor—mediated intracellular calcium increase in human hepatoma BEL—7404 cells

Epidermal growth factor(EGF) induced intracellular free calcium ([Ca^2 ]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line.Single cell[Ca^2 ]i analysis and [Ca^2 ]i measurement in cell populations revealed that EGF triggered a rapid[Ca^2 ]i increase in the dose-dependent and time-dependent manner.Pretreatment of cells with an endoplasmic reticulum(ER) Ca^2 -ATPase inhibitor,thapsigargin(TG) at 100nM concentration for 20 min,completely abolished EGF-induced [Ca^2 ]i increase,and chelating extracellular calcium by excess EGTA partially inhibited the increase.Furthermore,the expression of antisense EGF receptor sequence in BEL-7404 cells suppressed the [Ca^2 ]i response to EGF.The results suggest that EGF receptor-mediated [Ca^2 ]i increase in the human hepatoma cells is essentially dependent on the Ca^2 storage in ER.     

Superoxide anion increases intracellular free calcium in human myometrial cells

We investigated the effects of superoxide anion on the intracellular free calcium concentration ([Ca2+]i) in human cultured myometrial cells using a calcium-sensitive fluorescent dye, indo-1, and a digital imaging fluorescence microscopic system. Hypoxanthine (HX) plus xanthine oxidase induced a rise in [Ca2+]i in a manner dose-dependent on xanthine oxidase. The increase in [Ca2+]i in the absence of extracellular calcium ([Ca2+]ex) was 10% of that in the presence of [Ca2+]ex. Nifedipine, which blocks voltage-sensitive calcium channels, also reduced the increase in [Ca2+]i induced by HX-xanthine oxidase. Superoxide dismutase or superoxide dismutase plus catalase, which metabolizes superoxide anion, inhibited the effect of HX-xanthine oxidase on [Ca2+]i. The desensitization of the effect of superoxide anion on [Ca2+]i was investigated by pulsatile administration of HX and xanthine oxidase. Desensitization was observed on pulsatile administration of HX-xanthine oxidase at 2-min intervals. These data suggest that superoxide production may participate in uterine contraction via [Ca2+]i increase.     

Superoxide anion increases intracellular pH, intracellular free calcium, and arachidonate release in human amnion cells.

We determined the effects of superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, on the intracellular pH (pHi) and intracellular free calcium concentration ([Ca2+]i) and release of arachidonate in human cultured amnion cells. Superoxide anion induced a prompt increase of pHi and subsequent increase of [Ca2+]i. The evoked pHi was inhibited by pretreatment with anion channel blockers but not affected by omission of extracellular Na+ or addition of amiloride. The increase of [Ca2+]i was inhibited significantly by the absence of extracellular calcium or by the addition of a calcium channel blocker, cobalt. NH4Cl, which can generally increase pHi, also increased [Ca2+]i of amnion cells. But the increase of [Ca2+]i induced by the NH4Cl was significantly less than that induced by the amount of superoxide anion causing a similar increase in pHi. These results show that superoxide anion, crossed through anion channel in membrane, increased [Ca2+]i at least partially via increase of pHi and that the calcium mobilization was dependent on both extracellular and intracellular sources. Superoxide anion induced the release of arachidonate in a dose-dependent manner and this induction was inhibited by omission of extracellular calcium. These data suggest that the release of arachidonate was dependent on the increase of [Ca2+]i. We also determined the viability of cells in the presence of superoxide anion by flow cytometry. Superoxide anion at the levels used in these experiments did not change the percentage of viable cells. These findings suggested that superoxide anion may regulate biological functions in amnion cells via pHi, [Ca2+]i mobilization, and the release of arachidonate without damaging the cells.     

Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina

A sustained high voltage-activated (HVA), nifedipine- and cadmium- sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na- acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout- dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium- dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.     

Hormonal regulation of free intracellular calcium concentrations in small and large ovine luteal cells

The second messengers mediating hormonal regulation of the corpus luteum are incompletely defined, particularly for the primary luteolytic hormone prostaglandin F2 alpha (PGF2 alpha). In this study, hormonally induced changes in free intracellular calcium concentrations were measured in individual small and large ovine luteal cells by using computer-assisted microscopic imaging of fura-2 fluorescence. This technique could readily detect transient increases in free calcium concentrations within both small and large luteal cells after treatment with 1 microM of the calcium ionophore, A23187. Treatment with PGF2 alpha (1 microM) caused a dramatic increase in free calcium concentrations in large (before = 73 +/- 2 nM; 2 min after PGF2 alpha = 370 +/- 21 nM; n = 33 cells) but not in small (before = 66 +/- 4 nM; 2 min after PGF2 alpha = 69 +/- 8 nM; n = 12 cells) luteal cells. The magnitude and timing of the calcium response was dose- and time-dependent. The PGF2 alpha-induced increase in free intracellular calcium is probably due to influx of extracellular calcium, since inclusion of inorganic calcium channel blockers (100 microM manganese or cobalt) attenuated the response to PGF2 alpha and removal of extracellular calcium eliminated the response. In contrast to PGF2 alpha, luteinizing hormone (LH) (100 ng/ml) caused no change in intracellular levels of free calcium in small or large luteal cells, even though this dose of LH stimulated (p less than 0.01) progesterone production by small luteal cells. Therefore, alterations in free calcium concentrations could be the intracellular second message mediating the luteolytic action of PGF2 alpha in the large ovine luteal cell.