Lack of effect of flurothyl, a non-anesthetic fluorinated ether, on rat brain synaptic plasma membrane calcium-ATPase

Plasma membrane Ca2+-ATPase (PMCA), a regulator of intracellular calcium, is inhibited by volatile anesthetics and by xenon and nitrous oxide. Response of a cellular system to anesthetics, particularly to volatile agents, raises the question of non-specific, even toxic, side effects unrelated to anesthetic action. Compounds with chemical and physical properties similar to halogenated anesthetics, but which lack anesthetic effect, have been used to address this question. We have compared the effects of halothane and flurothyl, a non-anesthetic fluorinated ether, on PMCA Ca2+ transport across isolated brain synaptic plasma membranes (SPM). Flurothyl, at concentrations predicted by the Meyer-Overton curve to range from 0.4 to 2.6 MAC (minimum alveolar concentration), had no significant on PMCA activity. In contrast halothane, 1.3 MAC, reduced Ca2+ transport 30 to 40%. These findings provide further evidence for a specific effect of inhalation anesthetics on neuronal plasma membrane Ca2+-ATPase.     

Participation of the Ca(2+)-calmodulin-activated Kinase II in the control of metaphase-anaphase transition in human cells

Treatment of HeLa S3 cells growing in suspension, and of endothelial cells ECV 304 growing as a monolayer, with the inhibitor of the Ca(2+)-calmodulin activated Kinase II KN-93, blocks cells at metaphase for 15 min (HeLa cells) and 30 min (ECV cells). Thereafter cells resume mitosis and enter anaphase. The inactive isomer KN-92 does not show such effects. The results show the involvement of the CaM K II system in the regulation of the metaphase-anaphase transition whereby the activation of the Kinase II, in particular by calmodulin appears to be affected, the residual autophosphorylation of the CaM K II system apparently sufficing after 15 to 30 min to release the cells into anaphase. The results are compared with the metaphase-blocking effects of the noble gas xenon, where the xenon-induced block can be overcome by small intracellular increases of Ca(2+), thus indicating the CaM K II system as a possible target for xenon.     

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.     

Effects of xenon on intracellular Ca2% release in human endothelial cells

Using human endothelial cells loaded with the Ca^2% indicator Fura2 the effects of xenon on changes in intracellular Ca^2% were studied. The basal level of intracellular Ca^2% is not affected upon incubation of the cells in buffer saturated either with 100% xenon or with 70% xenon/30% air, a concentration which corresponds in humans to the minimum alveolar concentration necessary to induce anesthesia in 50% of patients. A defined cellular response such as the Ca^2% change induced by application of adenosine triphosphate (ATP) makes it possible to study the signalling chain between the stimulus and the various forms of Ca^2% response. ATP induces a typical Ca^2% fingerprint composed of an internal Ca^2% release consisting of several oscillations plus an additional Ca^2%-induced Ca^2% influx from the outside. The latter is absent in Ca^2%-free medium. When cells are incubated with xenon, only the first part of the ATP-induced Ca^2% response is found corresponding to the internal release of Ca^2%; the subsequent Ca^2%-induced Ca^2% influx does not take place. If xenon is removed, a fast recovery is observed and the cells again show both parts of the Ca²⁺ response. Such selective inhibition of Ca²⁺-induced Ca²⁺ influx is not obtained when xenon is replaced by N₂; the ATP response of the cell remains the same as that of untreated cells. Similar effects of xenon treatment can also be observed when the cells are treated with thapsigargin, a specific inhibitor of the SERCA systems. The Ca²⁺-induced Ca²⁺ release is almost completely suppressed in the presence of xenon. We conclude that xenon may act on the cellular level on defined sites of the mechanisms regulating the Ca²⁺-release-activated Ca²⁺ channels of the plasma membrane and that this property may be related to its anesthetic effect.     

Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells.

Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.     

Prevention of postoperative bleeding after complex pediatric cardiac surgery by early administration of fibrinogen,prothrombin complex and platelets: a prospective observational study

Some retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis. Estrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects. Sevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell. Sevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.     

Signal transduction and ligand-receptor dynamics in the neutrophil. Ca2+ modulation and restoration

Intracellular Ca2+ rises when neutrophils are stimulated with formyl peptide ligands. There is enough Ca2+ released to complex approximately 200 microM Quin 2, (220 +/- 90 microM, 7 donors). This result is interpreted in terms of a fixed storage pool of Ca2+ of 44 pmol/10(6) cells. When extracellular Ca2+ is removed from the medium with 5 mM EGTA (final pH 7.4) just prior to cell stimulation, neither the magnitude nor the early time course of the Quin 2 response to formyl peptide is dramatically influenced. This result supports the concept that neither Ca2+ influx nor efflux, which are elevated in stimulated cells, contributes in a major way to the free Ca2+ pool which is monitored by Quin 2 during the early activation phase of cell responses. We have used intracellular Quin 2, and extracellular Ca2+ without the use of EGTA or ionophores to manipulate the levels of intracellular Ca2+. This is accomplished by depleting cells of intracellular Ca2+ by loading with Quin 2 in the absence of Ca2+. Intracellular Ca2+ is modulated by adding back Ca2+ to the medium. Using simultaneous analyses of cell function and Quin 2 fluorescence, we find that at least two aspects of cellular responsiveness (degranulation and O2- production) depend upon the level of available Ca2+. In contrast, the first phase, at least, of a biphasic rapid light scattering response which is related to actin polymerization is independent of Ca2+. We find that the Ca2+- sensitive cell responses can be partially restored in Ca2+-depleted cells if Ca2+ is provided within 30 s, a period which may reflect the putative lifetime of the transiently active ligand-receptor complex.     

Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts

Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.     

Dysregulated ryanodine receptors mediate cellular toxicity: restoration of normal phenotype by FKBP12.6

Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Oscillatory Ca2+ signaling and its cellular function

It is well known that in the cells of many higher eukaryotic organisms Ca2+ ions are used as a signal messenger in the regulation of cellular functions. From recent studies with single cells it was suggested that the intracellular Ca2+ signal comprises repetitive and periodic Ca2+ spikes in a variety of cells. The mechanism by which intracellular Ca2+ oscillates and the biological significance of this oscillation are not well understood. It also remains to be determined how the Ca2+ signaling system sends a message into the cell, intermittently, to amplify the functional response. This review describes and integrates some recent views of oscillatory Ca2+ signaling.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Nifedipine reversibly arrests mitosis in stamen hair cells of tradescantia

Mitotic stamen hair cells of Tradescantia virginiana (cv. Zwanenburg Blue) become arrested in metaphase following a 30-min treatment with 10 to 100 microM nifedipine, a Ca2+-channel entry blocker. The time interval between nuclear envelope breakdown and anaphase onset in untreated cells is approximately 33 min +/- 4 min; nifedipine extends this "metaphase transit time" beyond 70 min. Nifedipine can be photoreversed in situ by exposure to 365 nm light. UV illumination inactivates the drug, its inhibitory effect on Ca2+ is abolished, and cells arrested in metaphase enter anaphase within 3 to 18 min of UV exposure if CaCl2 is present in the medium. The interval between UV illumination and anaphase onset is inversely related to the extracellular concentration of CaCl2. If CaCl2 is not added to the medium, the interval between UV exposure and anaphase onset is usually longer than 18 min. The sole addition of 100 microM CaCl2 to the medium is insufficient to reverse nifedipine inhibition; unless the cells are exposed to UV light, anaphase will not commence. The threshold concentration of free Ca2+ for rapid anaphase onset (less than 10 min after UV photoreversal) is between 1 and 10 microM. These results suggest that an influx of Ca2+ from the extracellular medium to the cytosolic compartment is necessary for normal progression from metaphase to anaphase and that this influx may serve as a trigger for chromosome separation.     

Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nm (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation.     

Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR)

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will focus on the effects of cADPR in different cells and tissues, the mechanisms of cADPR-mediated Ca2+ release and Ca2+ entry, extracellular effects of cADPR, and the role of cADPR in a cell system studied in detail, human T-lymphocytes.     

Removal of the MDCK Cell Primary Cilium Abolishes Flow Sensing

The hypothesis that cell primary cilium is solely responsible for the flow-induced Ca2+ response in MDCK cells was tested by removal of the cilia from mature, responsive cells. Incubation of the cells with 4 mM chloral hydrate for 68 hours resulted in the complete loss of the primary cilia and in disorganization of microtubules, as visualized by immunofluorescence. When intracellular Ca2+ concentration was measured with Fluo-4, the elevation that normally accompanies an increase in fluid flow was abolished after 20 hours exposure to chloral hydrate. At this time, the primary cilia still remained attached to the cells but had become twisted and flexible. Twenty-four hours after return of the deciliated cells to normal medium, intracellular microtubule organization appeared normal, but primary cilia had not yet been expressed. The cells failed to increase intracellular Ca2+ in response to fluid flow until after they had been in normal medium for 120 hours, at which time the primary cilia were 3-4 microm long. Chloral hydrate did not impair the Ca2+ mobilization machinery, as the Ca2+ response to mechanical contact and the spread to neighboring cells was unaffected by the drug. We conclude that the primary cilium is the only sensor for the flow-induced Ca2+ response in MDCK cells and estimate that a single mechanically sensitive channel in the cilium could provide the requisite Ca2+ influx.     

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.     

Induction of calcium influx from extracellular fluid by beauvericin in human leukemia cells

Beauvericin, a cyclic hexadepsipeptide, is a mycotoxin that can induce cell death in human lymphoblastic leukemia CCRF-CEM cells. Our previous data have shown that beauvericin induces cell death in CCRF-CEM cells in a dose- and time-dependent manner, and that this beauvericin-induced cell death can be prevented by administration of intracellular calcium chelator-BAPTA. Therefore, the intracellular Ca2+ concentration ([Ca2+]i) may play an important role in beauvericin-induced cell death in CCRF-CEM cells. In this study, the effect of beauvericin on [Ca2+]i and the possible mechanism responsible for the changes of [Ca2+]i in CCRF-CEM cells were investigated. Beauvericin caused a rapid and sustained [Ca2+]i rise in a dose-dependent manner. Excess extracellular Ca2+ facilitated beauvericin-induced [Ca2+]i rise by adding 1 mM CaCl2 in the bathing medium. On the other hand, beauvericin-induced [Ca2+]i rise was prevented in Ca2+-free Tyrode's solution by 200 microM EGTA. In addition, beauvericin-induced [Ca2+]i rise was also attenuated by intracellular Ca2+ chelator-BAPTA/AM. It is worthy to note that neither the voltage-dependent Ca2+ channel blocker, nimodipine, nor depletion of intracellular Ca2+ with thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, has any effect on beauvericin-induced [Ca2+]i rise. The data from present study indicate that beauvericin acts as a potent Ca2+ mobilizer by stimulating extracellular Ca2+ influx CCRF-CEM cells.     

Listeriolysin of Listeria monocytogenes forms Ca2+-permeable pores leading to intracellular Ca2+ oscillations

Listeriolysin (LLO) is a major virulence factor of Listeria monocytogenes, a Gram-positive bacterium that can cause life-threatening diseases. Various signalling events and cellular effects, including modulation of gene expression, are triggered by LLO through unknown mechanisms. Here, we demonstrate that LLO applied extracellularly at sublytic concentrations causes long-lasting oscillations of the intracellular Ca2+ level of human embryonic kidney cells; resulting from a pulsed influx of extracellular Ca2+ through pores that are formed by LLO in the plasma membrane. Calcium influx does not require the activity of endogenous Ca2+ channels. LLO-formed pores are transient and oscillate between open and closed states. Pore formation and Ca2+ oscillations were also observed after exposure of cells to native Listeria monocytogenes. Our data identify LLO as a tool used by Listeria monocytogenes to manipulate the intracellular Ca2+ level without direct contact of the bacterium with the target cell. As Ca2+ oscillations modulate cellular signalling and gene expression, our findings provide a potential molecular basis for the broad spectrum of Ca2+-dependent cellular responses induced by LLO during Listeria infection.     

Mitogenesis of 3T3 fibroblasts induced by endogenous ganglioside is not mediated by cAMP, protein kinase C, or phosphoinositides turnover

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.