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PubMed1996

Alcohol inhibits the depolarization-induced stimulation of oxidative phosphorylation in synaptosomes.

The effects of alcohol and Ca2+ transport inhibitors on depolarization-induced stimulation of oxidative phosphorylation and free-Ca2+ concentrations in rat synaptosomes were investigated. Glucose oxidation was stimulated by depolarization with K+ or veratridine and by the Ca2+ ionophore ionomycin. The stimulation by K+, veratridine, and ionomycin was correlated with elevation of synaptosomal free Ca2+. Depolarization-stimulated respiration was inhibited by verapamil, Cd2+, and ruthenium red but not by diltiazem. Synaptosomal Ca2+ elevation was inhibited by verapamil but not by ruthenium red. These results indicate that the stimulation depends on elevation of mitochondrial free Ca2+. Ethanol, at pharmacological concentrations (50-200 mM), inhibited the Ca2+-dependent stimulation of oxidative phosphorylation. This inhibition resulted, in part, from the inhibition of voltage-gated Ca2+ channels, which inhibited the elevation of synaptosomal free Ca2+, and, in part, from the stimulation of the mitochondrial Ca2+/Na+ antiporter, which inhibited the elevation of the mitochondrial matrix free Ca2+. The inhibition by ethanol of the excitation-induced stimulation of oxidative phosphorylation in the synapse may contribute to the depressant and narcotic effects of alcohol and enhance excitotoxicity.

Adenosine Triphosphate↗
PubMed1997

Calcium-activated neutral protease activity is decreased in PC12 cells after ethanol exposure.

Calcium-activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence-based assay with N-succinyl-Leu-Tyr 7-amido-4-methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 microM) or low (140 microM) Ca2+ concentrations in the presence of 20 microM ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F(t) = F(final)*(1 - e(-k(obs)t). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k(obs) beginning at 20 mM, to 27.8% of control at 1,400 microM Ca2+ and 29.2% of control at 140 microM Ca2+ in the presence of 20 microM ionomycin. No changes in mu-calpain or m-calpain activities were found in cell extracts from cells exposed to 20 mM ethanol, whereas at 40 and 80 mM ethanol, significant decreases in both mu-calpain and m-calpain activities were discovered.

Animals↗
PubMed2002

Localization of calcineurin/NFAT in human skin and psoriasis and inhibition of calcineurin/NFAT activation in human keratinocytes by cyclosporin A.

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

Biological Transport↗
PubMedDate not supplied

Na+-Ca2+ exchange and Ca2+ efflux in transfected Chinese hamster ovary cells.

The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.

Animals↗
PubMed2000

Cytosolic [Ca(2+)] modulates basal GLUT1 activity and plays a permissive role in its activation by metabolic stress and insulin in rat epithelial cells.

The aim of this work was to investigate the role of cytosolic free calcium ([Ca(2+)]c) in the stimulation of GLUT1 by metabolic stress and insulin. Chelation of [Ca(2+)]c with bapta, introduced in rat liver epithelial Clone 9 cells in the acetoxymethyl (AM) form, decreased their basal rate of 2-deoxyglucose uptake in a dose-dependent fashion. Maximal inhibition at 75 microM bapta was by 38 +/- 8% (n = 8). The effect was partially reversed by ionomycin. Basal sugar uptake was also decreased by lowering extracellular [Ca(2+)] in ionomycin-permeabilized cells. Increasing [Ca(2+)]c over its resting level of 168 +/- 32 (n = 27) had no affect on sugar uptake. Chelation of [Ca(2+)]c did not change the abundance of surface GLUT1 and had no significant effect on the affinity of GLUT1 for sugars. In addition, calcium chelation abolished the activation of GLUT1 by azide, arsenate, 2,4-dinitrophenol and insulin. However, [Ca(2+)]c did not increase in the presence of azide. We conclude that [Ca(2+)]c, near or below its resting level, modulates GLUT1 activity over a considerable range and plays a permissive role in the activation of the carrier by metabolic stress and insulin.

2,4-Dinitrophenol↗
PubMed2003

Ligation of intercellular adhesion molecule 3 inhibits GM-CSF production by human eosinophils.

BACKGROUND: Intercellular adhesion molecule 3 (ICAM-3) has recently been identified on the surface of eosinophils. OBJECTIVE: The purpose of this study was to characterize ICAM-3 expression on eosinophils in response to cytokines and to determine whether ligand binding of ICAM-3 modulates inflammatory responses of eosinophils, as it does in other leukocytes. METHODS: To determine effects of ICAM-3 on eosinophil function, we isolated human eosinophils and used a monoclonal antibody directed against the epitope of ICAM-3 that binds to leukocyte-function antigen-1 to mimic binding of ICAM-3 and this natural ligand. We measured granulocyte-macrophage colony stimulating factor (GM-CSF) production by unstimulated eosinophils and eosinophils stimulated with ionomycin (1 micromol/L), both in the presence and absence of this anti-ICAM-3 antibody. RESULTS: We found that 99% of eosinophils expressed ICAM-3, regardless of whether allergic symptoms were present or absent. Expression of ICAM-3 was not enhanced by proinflammatory cytokines. Expression of ICAM-3 was reduced in apoptotic cells and in cells incubated with the combination of GM-CSF and tumor necrosis factor-alpha (n = 3). Antibody binding of ICAM-3, which mimics leukocyte-function antigen-1 binding, had no effect on baseline GM-CSF production but reduced by 80% the production of GM-CSF stimulated by ionomycin (control 1969 pg/mL +/- 1259 SD versus anti-ICAM-3 396 pg/mL +/- 207 SD, n = 8) and reduced GM-CSF mRNA content. CONCLUSIONS: ICAM-3 is highly expressed on the surface of human eosinophils, and downregulation of GM-CSF production by anti-ICAM-3 mAb suggests that ICAM-3 ligation may inhibit eosinophil inflammatory responses and survival.

Adult↗
PubMed1985

Activation of tracheal smooth muscle contraction: synergism between Ca2+ and activators of protein kinase C.

The effects of divalent ionophores (A23187 and ionomycin), Ca2+ channel agonist (BAY K 8644), and protein kinase C (C-kinase) activators [phorbol 12-myristate 13-acetate (PMA), mezerein] on bovine tracheal smooth muscle contraction were investigated. A23187 (5 microM) and ionomycin (0.5 microM) produced a prompt but transient contraction. C-kinase activators either produced no effect--e.g., PMA at 200 nM--or produced a rise in tension that was slow in onset but then gradually increased--e.g., mezerein at 400 nM. In contrast, ionophores and C-kinase activators, in combination, acted synergistically to produce a prompt and sustained contractile response that is reminiscent of that observed in response to carbachol, a cholinergic agonist. In addition, BAY K 8644 (20 nM), which has a minimal effect on tension by itself, could significantly enhance contraction induced by C-kinase activators. The contraction induced by all of these agents was quickly reversed either by removal of extracellular Ca2+ or upon addition of forskolin, an activator of adenylate cyclase. A similar reversal of carbachol-induced contraction by forskolin was observed with carbachol-induced contraction. These findings strongly suggest that C-kinase plays an important role in mediating tracheal smooth muscle contraction.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation.

This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or the calcium ionophore ionomycin. The antibody directed against HLA-A, -B, and -C locus gene products (W6/32) and the antibody directed against HLA-B locus gene products (4E) inhibited proliferation induced by either mitogen by 70-90%. The HLA-A locus-specific antibody (131), though inhibiting ionomycin-induced proliferation by 80-90%, was much less effective when OKT3 was the stimulus. The inhibition affected T4+ and T8+ cells and was not mediated by DR+ accessory cells. The inhibitory effect of these antibodies was associated with a decrease in the level of interleukin 2 activity present in culture supernatants, decreased interleukin 2 receptor expression, and decreased transferrin receptor expression and was not overcome by the addition of exogenous interleukin 2. Our results suggest that HLA class I molecules are directly involved in the early critical events of human lymphocyte activation and proliferation.

Antibodies, Monoclonal↗
PubMed1992

Platelet activation by simultaneous actions of diacylglycerol and unsaturated fatty acids.

Several cis-unsaturated fatty acids such as oleic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids added directly to intact human platelets greatly enhance protein kinase C activation as judged by the phosphorylation of its specific endogenous substrate, a 47-kDa protein. This enhancement absolutely requires the presence of a membrane-permeant diacylglycerol, 1,2-dioctanoylglycerol, or a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. In the presence of ionomycin and either 1,2-dioctanoylglycerol or phorbol 12-myristate 13-acetate, the release of serotonin from the platelets is also remarkably increased by cis-unsaturated fatty acids. The effect of these fatty acids is observed at concentrations less than 50 microM. Saturated fatty acids and trans-unsaturated fatty acids are inactive. Titration of ionomycin to induce a release reaction and measurement of the intracellular Ca2+ level by the fura-2 procedure indicate that cis-unsaturated fatty acids increase an apparent sensitivity of the platelet response to Ca2+. The results suggest that cis-unsaturated fatty acids, which are presumably produced from phosphatidylcholine by signal-dependent activation of phospholipase A2, may take part directly in cell signaling through the protein kinase C pathway.

Diglycerides↗
PubMed1994

Calcium oscillations in pituitary gonadotrophs: comparison of experiment and theory.

We have developed a mathematical model that describes several aspects of agonist-induced Ca2+ signaling in single pituitary gonadotrophs. Our model is based on fast activation of the inositol 1,4,5-trisphosphate (InsP3) receptor Ca2+ channels at low free cytosolic Ca2+ concentration ([Ca2+]i) and slow inactivation at high [Ca2+]i. Previous work has shown that these gating properties, when combined with a Ca(2+)-ATPase, are sufficient to generate simulated Ca2+ oscillations. The Hodgkin-Huxley-like description we formulate here incorporates these different gating properties explicitly and renders their effects transparent and easy to modulate. We introduce regulatory mechanisms of channel opening which enable the model, both in the absence and in the presence of Ca2+ entry, to give responses to a wide range of agonist doses that are in good agreement with experimental findings, including subthreshold responses, superthreshold oscillations with frequency determined by [InsP3], and nonoscillatory "biphasic" responses followed occasionally by small-amplitude oscillations. A particular added feature of our model, enhanced channel opening by reduced concentration of Ca2+ in the lumen of the endoplasmic reticulum, allows oscillations to continue during pool depletion. The model predicts that ionomycin and thapsigargin can induce oscillations with basal [InsP3] and zero Ca2+ entry, while Ca2+ injection cannot. Responses to specific pairings of sub- or superthreshold stimuli of agonist, ionomycin, and thapsigargin are also correctly predicted. Since this model encompasses a wide range of observed dynamic behaviors within a single framework, based on well-established mechanisms, its relevance should not be restricted to gonadotrophs.

Calcium↗
PubMed1995

Monocytes are required to prime peripheral blood T cells to undergo apoptosis.

Freshly isolated, human peripheral blood T (PBT) cells are largely resistant to the apoptotic effects of anti-CD3 monoclonal antibody, ionomycin, or phorbol 12-myristate 13-acetate (PMA). We demonstrate here, however, that PBT cells, including both CD4+ and CD8+ cell populations, can be readily induced to undergo apoptosis when cocultured with either autologous or allogeneic monocytes (Mo) in PMA-containing medium. Incubation of PBT cells with Mo at a ratio of 1:1 for 18 hr resulted in maximal levels (80%) of apoptotic cell death. The mechanism whereby Mo enable PBT cells to undergo apoptosis in PMA-containing medium appeared to depend on cell-cell contact or close proximity between Mo and PBT cells rather than solely via soluble mediators. It was demonstrated that Mo acquire the ability to prime PBT cells for apoptosis after treatment with PMA and that treated Mo maintain this ability even after fixation with formaldehyde. It was also found that once PBT cells became primed for apoptosis by incubation with PMA-pretreated Mo, the primed PBT cells were susceptible to apoptosis triggered not only by PMA but also by either ionomycin or by monoclonal antibody crosslinking of T-cell surface molecules such as CD4 and CD3. Interestingly, the degree of apoptosis of CD4+ T cells by crosslinking of CD4 molecules via a combination of gp120, anti-gp120, and goat anti-mouse IgG was significantly greater for T cells primed with PMA-treated Mo than for unprimed T cells. Together, these findings reveal an important role for accessory cells in priming resting PBT cells for apoptosis and suggest a possible Mo-dependent mechanism by which T cells may become primed for apoptosis in human immunodeficiency virus-infected asymptomatic individuals.

Apoptosis↗
PubMed1996

Inhibition of cytokines and JAK-STAT activation by distinct signaling pathways.

An important component of cytokine regulation of cell growth and differentiation is rapid transcriptional activation of genes by the JAK-STAT (signal transducer and activator of transcription) signaling pathway. Ligation of cytokine receptors results in tyrosine phosphorylation and activation of receptor-associated Jak protein tyrosine kinases and cytoplasmic STAT transcription factors, which then translocate to the nucleus. We describe the interruption of cytokine triggered JAK-STAT signals by cAMP, the calcium ionophore ionomycin, and granulocyte/macrophage colony-stimulating factor. Jak1 kinase activity, interleukin 6-induced gene activation, Stat3 tyrosine phosphorylation, and DNA-binding were inhibited, as was activation of Jak1 and Stat1 by interferon gamma. The kinetics and requirement for new RNA and protein synthesis for inhibition of interleukin 6 by ionomycin and GM-CSF differed, but both agents increased the association of Jak1 with protein tyrosine phosphatase ID (SH2-containing phosphatase 2). Our results demonstrate that crosstalk with distinct signaling pathways can inhibit JAK-STAT signal transduction, and suggest approaches for modulating cytokine activity during immune responses and inflammatory processes.

Blotting, Northern↗
PubMed1996

Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes.

Despite intensive efforts, the intracellular signaling pathways that mediate apoptosis remain unclear. The human B lymphoma cell line, B104, possesses characteristics that make it an attractive model for analysis of receptor-mediated apoptosis. Although these cells express both membrane IgM (mIgM) and membrane IgD (mIgD) crosslinking mIgM results in significant apoptosis while crosslinking mIgD does not. Our results show that crosslinking mIgM but not mIgD induced a delayed and sustained activation of the mitogen-activated protein kinase (MAPK) family members stress-activated protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin, which also induces apoptosis in B104 cells, stimulated a similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor of apoptosis induced by either mIgM or ionomycin, inhibited activation of both SAPK and p38 MAPK, suggesting that stimulation of these kinases may be required for induction of apoptosis. Collectively, our results indicate that SAPK and p38 MAPK may be downstream targets during mIgM-induced, calcium-mediated, apoptosis in human B lymphocytes.

Apoptosis↗
PubMed1996

Contribution of sustained Ca2+ elevation for nitric oxide production in endothelial cells and subsequent modulation of Ca2+ transient in vascular smooth muscle cells in coculture.

To elucidate the intracellular Ca2+ (Ca2+i ) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca2+i reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca2+i-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca2+i of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO2-. The order of potency of the agonists in terms of the peak Ca2+i in ECs was bradykinin (100 nM) > ATP (10 microM) > ionomycin (50 nM) > thapsigargin (1 microM). In contrast, the order in reference to both the extent of Ca2+i reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by NG-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca2+i response in cocultured VSMCs to the isoculture level. In ECs, when the Ca2+ influx was blocked by Ni2+ or by chelating extracellular Ca2+, all four agonists markedly decreased NO production, the half decay time of the Ca2+i degenerating phase, and the area under the Ca2+i curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca2+i curve but not to the Ca2+i peak level. Thus, the sustained elevation of Ca2+i in ECs, mainly a result of Ca2+ influx, determines the active NO production and subsequent Ca2+i reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca2+i in ECs, and markedly decreased the Ca2+i in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T. (1992) J. Biol. Chem. 267, 20377-20382).

Animals↗
PubMed1996

Desensitization of gonadotropin-releasing hormone action in alphaT3-1 cells due to uncoupling of inositol 1,4,5-trisphosphate generation and Ca2+ mobilization.

Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool.

Calcium↗
PubMed1996

Intracellular calcium stores are not required for Bcl-2-mediated protection from apoptosis.

The ability of Bcl-2 to inhibit cell death is well documented but its mechanism of action remains elusive. Recent reports have suggested that Bcl-2 prevents apoptosis by inhibiting the release of Ca2+ from the thapsigargin-sensitive Ca2+ store. The mobilization of Ca2+ from this store has been implicated as a signal regulating apoptotic cell death induced by glucocorticoids and by interleukin-3 withdrawal. The present study was designed to determine if Bcl-2 would still inhibit apoptosis after depletion of intracellular Ca2+ stores. We compared the response of two Chinese hamster ovary cell lines (5AHSmyc and 5A300bcl-2.2) following incubation with the calcium ionophore ionomycin to deplete intracellular Ca2+ stores. Continued incubation of 5AHSmyc cells in calcium-free media induced substantial apoptotic DNA fragmentation within 4 h and >95% loss of viability within 48 h. However, 5A300bcl-2.2 cells showed no evidence of DNA fragmentation or loss of viability over the same time period. Intracellular Ca2+ was analyzed with the Ca2+-sensitive fluorescent dye INDO-1 and confirmed that ionomycin was capable of releasing Ca2+ from intracellular stores in both cell lines. These results show that depletion of intracellular Ca2+ stores induces apoptosis and that these Ca2+ stores are not required for the protection afforded by Bcl-2.

Animals↗
PubMed1996

Differential regulation of nuclear and cytosolic Ca2+ in HeLa cells.

The results reported in this study address the controversial issue that nuclear free Ca2+ ([Ca2+]n) may be regulated independently of cytosolic free Ca2+ ([Ca2+]c). We have measured [Ca2+]n and [Ca2+]c with recombinant aequorin targeted to the nucleus and cytosol in HeLa cells. We found that histamine, ATP, and ionomycin increased [Ca2+]c quantitatively more than [Ca2+]n, although the time course of these changes was similar. The difference between [Ca2+]c and [Ca2+]n depended on the stimulus, and the relative difference between [Ca2+]n and [Ca2+]c was less with ionomycin than with histamine or ATP. After depletion of the internal Ca2+ store, restoration of extracellular Ca2+ resulted in only increased [Ca2+]c without a significant increase in [Ca2+]n. Treatment with cyclopiazonic acid resulted in a delayed increases in [Ca2+]n compared to [Ca2+]c. These differences in both timing and magnitude of nuclear Ca2+ signals confirm that the cell can limit or delay increases in nuclear free Ca2+. Taken with the fact that an inositol phosphate signaling system resides in the nucleus and its envelope, our data support the hypothesis that [Ca2+]n may be independently regulated.

Adenosine Triphosphate↗
PubMed1997

Modulation of endoplasmic reticulum calcium pump expression during T lymphocyte activation.

Calcium mobilization from intracellular storage organelles is a key component of the second messenger system inducing cell activation. Calcium transport ATPases associated with intracellular calcium storage organelles play a major role in controlling this process by accumulating calcium from the cytosol into intracellular calcium pools. In this study the modulation of the expression of the sarco-endoplasmic reticulum calcium transport ATPase (SERCA) isoenzymes has been studied in lymphocytes undergoing phorbol myristate acetate and ionomycin-induced activation. In several T lymphocyte cell lines a combined treatment by the two drugs resulted in an approximately 90% decrease of the expression of the calcium pump isoform recognized by the PLIM430 isoform-specific antibody, whereas the expression of the SERCA 2b isoform was increased approximately 2-fold. Phorbol ester or ionomycin applied separately was ineffective. In Jurkat T cells the down-modulation of expression of the SERCA isoform recognized by the PLIM430 antibody appeared concomitantly with the induction of interleukin-2 expression and could be inhibited by the immunosuppressant drug cyclosporine-A. These data indicate that T cell activation induces a selective and cyclosporine-A-sensitive modulation of the expression of the SERCA calcium pump isoforms. This reflects a profound reorganization of the calcium homeostasis of T cells undergoing activation and may open new avenues in the understanding of the plasticity of the calcium homeostasis of differentiating cells and in the pharmacological modulation of lymphocyte function.

Calcium-Transporting ATPases↗