A comparative study of the calcium system in memory T cells and naive T cells

The comparative analysis of responses of memory and naive T lymphocytes to Ca2+-mobilizing agents, namely Con A, thimerosal, thapsigargin and ionomycin, was carried out. The effect of these agents on both types of T cells differed qualitatively and quantitatively. The lack of intracellular Ca2+ stores in memory T cells was shown. Ca2+-mobilizing agents did not induce influx of Ca2+ in memory T cells from outside and this was the reason for their stability to Ca2+ ionophores. It was also shown that memory T cells were resistant to the 'Ca2+ paradox'.     

Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin. Intracellular Ca2+ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca2+, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca2+ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca2+ into internal stores and a delayed decrease that correlates with the inhibition of a Mn2+-permeable Ca2+ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca2+ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca2+ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn2+, and (iii) identical rates of Ca2+ influx are observed when Ca2+ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca2+. Taken together, these data suggest that the sustained influx of extracellular Ca2+ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn2+-permeable, Ca2+ store-operated Ca2+ channel.     

Membrane potential changes associated with calcium signals in human lymphocytes and rat mast cells

Human lymphocytes and rat mast cells, two non-excitable cellular models, were used to investigate membrane potential changes accompanying Ca2+ signals. Cells were stimulated with agents known to induce both Ca2+ release from internal stores and influx of extracellular Ca2+, namely thapsigargin, ionomycin and compound 48/80. Thapsigargin and ionomycin were used to activate lymphocytes, while compound 48/80 was used to stimulate mast cells. Membrane potential changes and Ca2+ concentration were monitored with the fluorescent dyes bis-oxonol and fura-2, respectively. In lymphocytes, thapsigargin induced a hyperpolarization temporally correlated with the increase in intracellular Ca2+ concentration. This hyperpolarization is due to activation of a K+ conductance which consists of two phases, a first phase independent on external Ca2+ and a second one blocked in a Ca2+-free medium. Ionomycin induced a Ca2+-dependent depolarization attributed to a massive influx of external Ca2+. On the other hand, stimulation of mast cells with compound 48/80 produced a fast hyperpolarization and an increase in intracellular Ca2+ levels. Besides different time-courses, this hyperpolarization differs from that induced by thapsigargin in lymphocytes in two aspects, it is mainly due to a Cl(-)-entry current and exit of K+ and it is completely inhibited in the absence of extracellular Ca2+. Compound 48/80-induced histamine release is not related to membrane potential changes.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Calcium fluxes in T lymphocytes.

Mechanisms controlling Ca2+ fluxes through the plasma membrane of lymphocytes have been characterized in a human T-cell clone and in the Jurkat T-cell line. Due to endogenous buffers, about 1/125 of the Ca2+ ions that enter the cell are free. Ca2+ fluxes were estimated from the variations in intracellular Ca2+ concentration ([Ca2+]i) elicited by concentration jumps in extracellular Ca2+ ([Ca2+]o). Thapsigargin was used to inhibit Ca2+ uptake into intracellular stores and to stimulate Ca2+ entry. Ca2+ extrusion was strictly due to the activity of plasma membrane Ca(2+)-ATPases since there was no detectable Na+/Ca2+ exchange activity in these cells. The rate of Ca2+ extrusion was mainly influenced by [Ca2+]i and less by [Ca2+]o but was insensitive to cell depolarization. In depolarized cells, thapsigargin-induced Ca2+ influx was reduced to 10% of the value measured in normally polarized cells, suggesting that depolarization not only reduces the electrochemical gradient for Ca2+ ions, but also inhibits Ca2+ permeation. When Ca2+ ions enter the cell, they bind to a site inside the channel, with a Kd of 3.3 mM. Stimulation of clonal T-cells with low concentrations of either anti-CD3 antibodies or thapsigargin elicited Ca2+ oscillations. Both the amplitude and the frequency of CD3-induced Ca2+ oscillations were sensitive to [Ca2+]o. These oscillations were immediately interrupted when extracellular Ca2+ was removed. The properties of Ca2+ oscillations in T lymphocytes suggest that they are mainly due to variations of Ca2+ influx, modulated by variations in [Ca2+]i.     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis.

Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2+ ([Ca2+]i) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2+]i results from two sources: an initial release of Ca2+ from intracellular stores followed by an influx of extracellular Ca2+. These two phases, release from intracellular stores and Ca2+ influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2+ from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2+]i response in interphase HeLa cells: a rapid rise in [Ca2+]i followed by a sustained elevation, the latter dependent entirely on extracellular Ca2+. In mitotic cells only the initial elevation, derived by Ca2+ release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2+ influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2+, being replenished by reuptake of Ca2+ that is retained within the cell. To ensure the depletion of Ca2+ stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2+ from intracellular stores by inhibition of a specific Ca(2+)-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2+ stores, stimulates Ca2+ influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+]i that is dependent on extracellular Ca2+ and is associated with a strong stimulation of 45Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+]i elevation that is initially comparable in magnitude and largely independent of extracellular Ca2+. However, unlike interphase cells, in mitotic cells the elevation of [Ca2+]i is not sustained and 45Ca2+ influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2+ influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2+ influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2+ influx and sustained elevations of [Ca2+]i.     

Mechanisms of miconazole-induced rise in cytoplasmic calcium concentrations in Madin Darby canine kidney (MDCK) cells

The effect of miconazole on intracellular calcium levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ indicator. Miconazole increased [Ca2+]i dose-dependently at concentrations of 5-100 microM. The [Ca2+]i transient consisted of an initial rise, a gradual decay and an elevated plateau (220 s after addition of the drug). Removal of extracellular Ca2+ partly reduced the miconazole response. Mn2+ quench of fura-2 fluorescence confirmed that miconazole induced Ca2+ influx. The miconazole-sensitive intracellular Ca2+ store overlapped with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, because 20 microM miconazole depleted the thapsigargin (1 microM)-sensitive store, and conversely, thapsigargin abolished miconazole-induced internal Ca2+ release. Miconazole (20-50 microM) partly inhibited the capacitative Ca2+ entry induced by 1 microM thapsigargin, measured by depleting intracellular Ca2+ store in Ca(2+)-free medium followed by addition of 10 mM CaCl2. Miconazole induced capacitative Ca2+ entry on its own. Pretreatment with 0.1 mM La3+ partly inhibited 20 microM miconazole-induced Mn2+ quench of fura-2 fluorescence and [Ca2+]i rise, suggesting that miconazole induced Ca2+ influx via two pathways separable by 0.1 mM La3+. Miconazole-induced internal Ca2+ release was not altered when the cytosolic level of inositol 1,4,5-trisphosphate (IP3) was substantially inhibited by the phospholipase C inhibitor U73122.     

Mechanism of action of calcium ionophores on intact cells: ionophore-resistant cells

Calcium ionophores are generally assumed to directly facilitate the transport of Ca2+ across the plasma membrane. The ability of Ca2+ ionophores ionomycin and A23187 to increase Ca2+ concentration in the cytosol ([Ca2+]i) in different cells was analyzed in detail using fluorescent Ca2+ probes. In fura-2-loaded cells, the dependence of the level of [Ca2+]i on ionomycin and A23187 concentrations had a complex character and could not be explained by ionophoric properties only. The Ca2+ signal induced by the Ca2+ ionophores consisted of three components. The first component was due to the activation of Ca2+ influx through native Ca2+ channels and was sensitive to drugs which inhibited the receptor-operated Ca2+ influx. The second component originated from phospholipase C-dependent mobilization of Ca2+ from intracellular stores. An additional influx of Ca2+ into the cells was activated in this case by a store-regulated mechanism. The third ionophoric component was very small at low concentrations of the ionophores. The effect of the ionophores on Ca2+ influx and Ca2+ mobilization was demonstrated on different cells such as Ehrlich ascites tumour cells, murine peritoneal neutrophils, macrophages, and T-lymphocytes. Thymocytes, neutrophils, and Ehrlich ascites tumour cells were more sensitive to the Ca2+ ionophores. Memory T-cells and brown preadipocytes were ionophore-resistant. The insensitivity to Ca2+ ionophores correlated with the absence of Ca2+ in the intracellular Ca2+ stores and the low activity of plasma membrane store-regulated Ca2+ channels.     

Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotrophs.

In rat pituitary somatotrophs, the stimulation of growth hormone secretion by growth hormone-releasing hormone (GHRH) is a Ca(2+)-dependent event involving Ca2+ influx. The presence of calcium-induced calcium release (CICR) Ca2+ stores has been suggested in these cells. The aim of our study was to demonstrate the presence of CICR stores in rat somatotrophs and to determine their function in GHRH Ca2+ signalling. To this end we measured cytosolic free Ca2+ concentration ([Ca2+]i), using indo-1 in purified rat somatotrophs in primary culture, while altering intracellular Ca2+ stores. Ionomycin (10 ttM) or 4-bromo-A23187 (10 ItM), used to mobilise organelle-bound Ca2+, raised [Ca2+]i in the absence of extracellular Ca2+. Caffeine (5 to 50 mM), used to mobilise Ca2+ from CICR stores, transiently raised [Ca2+]i in 65% of cells tested. The response to 40 mM caffeine was abolished when Ca2+ stores were depleted, with 1 microM thapsigargin or with 10 microM ryanodine. All cells that responded to 40 mM caffeine responded to 10 nM GHRH. The [Ca2+]i response to 10 nM GHRH was reversible and repeatable. However, the second response was 38% smaller than the first. Ryanodine treatment abolished the reduction in the second [Ca2+]i response, while thapsigargin increased the reduction by 67%. We conclude that rat somatotrophs possess CICR Ca2+ stores and that they account for 30-35% of the GHRH-induced increase in [Ca2+]i, and that their partial depletion is involved in somatotroph desensitization.     

Reduction of the Ca2+ transport systems in mouse young brown fat cells

Principal differences in the kinetics and amplitude of Ca2+ response to norepinephrine were found between freshly isolated young differentiated brown fat cells. An increase in the Ca2+ concentration in the cytoplasm ([Ca2+]i) in the young cells was unusually slow (A[Ca2+]i = 0.03 nM/s) in comparison with that in the differentiated cells, and the Ca2+ influx from the outside was not induced by Ca2+ mobilization agents, such as thapsigargin and ionomycin. Ionomycin increased [Ca2+]i up to 150 nM in a Ca2+-free medium and up to 270 nM in the normal medium. This results in that the intracellular Ca2+ stores in freshly isolated young cells are rather poor, and the mechanism of capacitive calcium entry does not virtually function. The data on chemical modification of Ca2+ channels in the plasma membrane by thimerosal suggest that the conductance of these channels is low and/or their number in young brown fat cells is insignificant.     

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.     

Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.     

Stimulation of leukotriene production and membrane translocation of 5-lipoxygenase by cross-linking of the IgE receptors in RBL-2H3 cells.

Recent studies in rat basophilic leukemia cells (RBL-2H3) have shown that two pharmacological agents, ionomycin and thapsigargin, induce leukotriene C4 production and translocation of 5-lipoxygenase from cytosol to membrane, primarily by causing an influx of extracellular calcium. In the present study, we investigate the induction of these events by receptor activation. Cross-linking of high-affinity IgE receptors (Fc epsilon RI) by antigen in RBL-2H3 cells leads to leukotriene C4 production and membrane translocation of 5-lipoxygenase. As in the ionomycin-stimulated cells, leukotriene C4 production in antigen-stimulated cells is calcium-dependent since the amount of leukotriene C4 produced correlates quantitatively with the increase in intracellular free calcium concentration ([Ca2+]i). However, the increase in [Ca2+]i required for equivalent leukotriene C4 production by antigen is not as high as it is using ionomycin. In addition, no threshold [Ca2+]i level is required for leukotriene production by antigen, which is in contrast to the ionomycin stimulation that a [Ca2+]i level of 300-400 nM is required. Furthermore, antigen causes an additive increase in leukotriene C4 production in cells stimulated by the ionomycin. These results suggest that another as yet unidentified intracellular pathway acts in conjunction with Ca2+ for leukotriene synthesis in antigen-stimulated cells. Antigen stimulation causes 20-30% of the total cell 5-lipoxygenase to associate with membranes (compared with 10% in unstimulated cells) as demonstrated by enzyme activity assay and by Western Blot using antibodies to 5-lipoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clomiphene, an ovulation-inducing agent, mobilizes intracellular Ca2+ and causes extracellular Ca2+ influx in bladder female transitional carcinoma cells

BACKGROUND/METHODS: The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of BFTC human bladder cancer cells was explored by using fura-2 as a Ca2+ indicator. RESULTS: Clomiphene at concentrations between 10 and 75 microM increased [Ca2+]i in a concentration-dependent manner and the signal saturated at 50 microM. The [Ca2+]i signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by about 40-50% in maximum [Ca2+]i. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 50 microM clomiphene in Ca2+-free medium, suggesting that clomiphene induced capacitative Ca2+ entry. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and CCCP (to uncouple mitochondria) inhibited 85% of clomiphene-induced intracellular Ca2+ release. Conversely, pretreatment with 50 microM clomiphene in Ca2+-free medium abolished the [Ca2+]i increase induced by brefeldin, thapsigargin or CCCP. The intracellular Ca2+ release was unaltered by inhibiting formation of inositol-1,4,5-trisphosphate (IP3) with 2 mM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; a phospholipase C inhibitor). CONCLUSION: The [Ca2+]i increase induced by 50 microM clomiphene was not affected by 10 microM of nifedipine, verapamil or diltiazem. Collectively, the results suggest that clomiphene releases intracellular Ca2+ in an IP3-independent manner and also activates extracellular Ca2+ influx.     

Activation of calcium oscillations by thapsigargin in parotid acinar cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.     

Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy.

High resolution digital video imaging has been employed to monitor the spatial and temporal development of agonist-induced cytosolic Ca2+ signals in fura 2-loaded exocrine acinar cells. Enzymatically isolated mouse pancreatic and lacrimal acinar cells or small acinar cell clusters were used. These retain their morphological polarity so that the secretory granules in individual cells are located at one pole, the secretory pole. In acinar cell clusters the granules are located centrally, oriented to surround what would be in situ referred to as the lumen. In pancreatic and lacrimal acinar cells inositol-1,4,5-triphosphate-generating agonists [acetylcholine (ACh) and cholecystokinin octapeptide (CCK) for the pancreas and ACh in the lacrimal gland] give rise to a rapidly spreading Ca2+ signal that is initiated at the secretory pole of the cells. The initial increase in [Ca2+]i in the luminal pole is independent of extracellular Ca2+ indicating that the earliest detectable intracellular Ca2+ release is specifically located at the secretory pole. In lacrimal acinar cells ATP acts as an extracellular agonist, independent of phosphoinositide metabolism to activate a receptor-operated calcium influx pathway which, as for ACh, gives rise firstly to an increase in intracellular Ca2+ concentration in the secretory pole. We propose that this polar rise in intracellular Ca2+ concentration is due to Ca(2+)-induced Ca2+ release. By contrast, when Ca2+ release and Ca2+ influx are induced in the absence of receptor activation by thapsigargin and ionomycin, the Ca2+ signal develops diffusely and slowly with no localization to the secretory pole.(ABSTRACT TRUNCATED AT 250 WORDS)