Hydrogen peroxide activates calcium influx in human neutrophils

The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H₂O₂ produced a rapid influx of Mn²⁺ and an increase of intracellular calcium. The H₂O₂ was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca²⁺ to H₂O₂-treated cells suspended in a free Ca²⁺/EGTA buffer resulted in a great increase in [Ca²⁺]_i reflecting influx of Ca²⁺ across the cell membrane. The increase of intracellular calcium was inhibited by Ni²⁺, La³⁺, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H₂O₂ by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.     

Discrimination between receptor- and store-operated Ca influx in human neutrophils

Ca²⁺ and Sr²⁺ entry pathways activated by pro-inflammatory agonists FMLP, LTB₄ and PAF have been compared to thapsigargin in human neutrophils. 2-APB (10 μM) increased Ca²⁺ influx and to a greater extent in agonist than in thapsigargin stimulated neutrophils. This action of 2-APB was specific to Ca²⁺ because 2-APB did not augment Sr²⁺ entry in agonist and thapsigargin stimulated neutrophils. This suggests that Ca²⁺ and Sr²⁺ entry can be used to discriminate between receptor and non-receptor (store)-operated Ca²⁺ influx. Our data show for the first time that Pyr3 whilst partially inhibiting agonist induced Ca²⁺ influx almost completely abolished Ca²⁺ influx after thapsigargin stimulation.     

Fc gammaRIIIb triggers raft-dependent calcium influx in IgG-mediated responses in human neutrophils

Human neutrophils constitutively express a unique combination of FcγRs, namely FcγRIIa and FcγRIIIb. Numerous lines of evidence support the concept that these FcγRs generate only partially characterized intracellular signals. However, despite the fact that both receptors are likely to be engaged simultaneously in a physiological setting, no recent publications have investigated the distinct, although partially convergent, results of their joint activation in IgG-dependent responses. To examine the significance of the co-expression of FcγRIIa and FcγRIIIb on human neutrophils, we analyzed the neutrophil responses to stimuli that engage these FcγRs, namely the phagocytosis of human IgG-opsonized zymosan and the responses to heat-aggregated IgGs. Blocking antibodies to either FcγR significantly decreased the phagocytic index and the stimulated production of superoxide anions. Both receptors are required for optimal IgG-dependent responses by human neutrophils. On the other hand, only blocking antibodies to FcγRIIIb, but not to FcγRIIa, inhibited the mobilization of calcium in response to heat-aggregated IgGs. Furthermore, phagocytosis of IgG-opsonized zymosan by human neutrophils required an extracellular influx of calcium that was blocked only by antibodies against FcγRIIIb. We also observed that this calcium influx as well as the IgG-dependent phagocytosis were dependent on the integrity of the plasma membrane detergent-resistant microdomains to which both isoforms were recruited following stimulation by heat-aggregated IgGs. These data clarify the mechanisms that regulate the FcγRs constitutively expressed on human neutrophils, describe a specific contribution of FcγRIIIb at the level of the mobilization of calcium, and provide evidence for a crucial role of detergent-resistant microdomains in this process.     

An influx of extracelluar calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid

The role of Ca²⁺ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura-2. Previous experiments have shown that a Sephadex G-75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107–121]. Using fura-2, we demonstrated that this Sephadex G-75 fraction also stimulates a rapid, transient increase in intracellular free Ca²⁺. This Ca²⁺ transient is blocked either by chelation of extracellular calcium or by addition of the Ca²⁺ antagonist La³⁺. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G-75 fraction, or ionomycin are all blocked by removal of extracellular Ca²⁺. These results strongly suggest that an influx of extracellular Ca²⁺ is responsible for intiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca²⁺ concomitant with the acrosome reaction.     

Calmodulin inhibits calcium influx current in vascular endothelium

Utilizing the whole-cell configuration of the patch-clamp technique the effect of calmodulin (CaM) on thapsigargin-induced Ca²⁺ current has been studied. Addition of several concentrations of CaM to the patch pipette induced concentration-dependent inhibition of thapsigargin-induced Ca²⁺ current in bovine aortic endothelial cells. The effect of CaM was Ca²⁺ dependent and was not observed when the intracellular Ca²⁺ was buffered to 1 nM with EGTA. CaM produced two major effects on the thapsigargin-induced Ca²⁺ current. First CaM slow down activation of the current by thapsigargin from a control value of 16 ± 5 to 31 ± 6 s with 1 μM CaM in the pipette solution. The second effect of CaM was to reduce the current amplitude in a concentration-dependent manner. The inhibition of Ca²⁺ current was observed at the peak of the current and at the sustained current level. The reduction of current at the sustained level was observed 15–20 s after onset of the thapsigargin response. The half inhibitory concentration determined from these experiments was 0.1 μM. These results indicate that CaM can modulate thapsigargin-induced Ca²⁺ current in this endothelium, suggesting a possible role for CaM in the regulation of store-operated Ca²⁺ influx.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells

OBJECTIVE: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. METHODS: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. RESULTS: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. CONCLUSIONS: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.     

The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.     

Calcium-sensitivity of the plasmalemmal delayed rectifier potassium current suggests that calcium influx in pulvinar protoplasts from Mimosa pudica L. can be revealed by hyperpolarization

Isolated protoplasts from pulvinar motor cells of Mimosa pudica were studied using conventional whole-cell patch clamp techniques. With internal solutions weakly buffered for Ca²⁺ (0.2 mm EGTA), a run-down of the outward delayed rectifier K⁺ current was induced by hyperpolarizing the holding potential, and this effect was strongly promoted by high external Ca²⁺ concentrations. This rundown could be reversed by coming back to less hyperpolarized holding potentials or by lowering the external [Ca²⁺]. Such rundown was absent when pipette internal solutions strongly buffered (10 mm EGTA) for Ca²⁺ were used. Ionomycin induced run-down of the K⁺ current with internal solutions containing 0.2 mm but not 10 mm EGTA. The hyperpolarization-associated rundown was reversibly blocked by Gd³⁺ and La³⁺.We thank Christophe Untereiner and Denis Wagner for expert technical assistance in facilitating the experiments and data acquisition and analysis.     

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca²⁺ channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2_AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2_AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.     

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca²⁺ signalling. Two distinct Ca²⁺ entry pathways amplify and sustain platelet Ca²⁺ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca²⁺ entry (SOCE) and non-capacitative Ca²⁺ entry (NCCE) in thrombin-induced Ca²⁺ signalling and PS exposure by using two Ca²⁺ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca²⁺ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca²⁺ signalling at higher thrombin concentrations. However, despite being involved in Ca²⁺ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca²⁺ entry is an important regulator of thrombin-induced PS exposure in platelets.     

Mitochondrial calcium response in human transformed lymphoblastoid cells

Human lymphoblastoid cell line (LCL) transformed by Epstein-Barr Virus (EBV) is a unique cellular model for the study of human diseases. Although pathophysiological significance of mitochondrial calcium regulation is drawing attention, it is not known whether or not mitochondria in LCLs play a role in intracellular calcium signaling. In this study, role of mitochondria of the lymphoblastoid cell line in calcium signaling was examined. Intra-mitochondrial calcium concentration ([Ca2+]m) was successfully measured using dihydro-Rhod-2, revealed by the decrease of fluorescence after application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and intracellular localization patterns imaged by fluorescent microscope. Platelet activating factor (PAF) concentration-dependently increased cytosolic calcium concentration ([Ca2+]i), while no increase of [Ca2+]m was observed. In contrast, 10 microM thapsigargin increased [Ca2+]i as well as [Ca2+]m. LCLs may be used for the study of possible pathophysiological role of mitochondrial calcium regulation in human diseases.     

Release of calcium from endolysosomes increases calcium influx through N-type calcium channels: Evidence for acidic store-operated calcium entry in neurons

Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such ‘acidic calcium stores’ affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed “acidic store-operated calcium entry (aSOCE)”. aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis.     

Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.     

Growth-plate chondrocytes respond to 17beta-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism

17Beta-estradiol (E(2)) regulates growth-plate chondrocyte differentiation in a gender and cell maturation-dependent manner via classic nuclear receptors ERalpha and ERbeta, and membrane-associated signalling. Here we show that sex-specific effects of E(2) involve changes in intracellular calcium concentration (ICCC). Resting-zone chondrocytes (RC) and growth-zone chondrocytes (GC) were isolated from costochondral cartilage of male and female rats. Confluent cultures were treated with 10(-8)M E(2) or 17alpha-estradiol in the presence of high and low extracellular Ca(2+) concentration. The ICCC was determined using laser scanning confocal microscopy to measure changes in Fluo-4 fluorescence every 5s for a total of 500s. E(2) increased ICCC in the cells from female rats but had no effect on ICCC in male cells. The effect was rapid (peak at 140s) and stereospecific. E(2) increased ICCC in RC and GC chondrocytes but the effect was greater in RC cells. Low Ca(2+) media did not abolish the E(2)-dependent ICCC elevation, nor did inclusion of verapamil, which inhibits Ca(2+) channels on the cell membrane. Thapsigargin reduced the effect of E(2) on ICCC, showing that Ca(2+) pumps on the endoplasmic reticulum were involved. Pre-treatment of the cells with the ER antagonist ICI 182780 did not alter the stimulatory effect of E(2), suggesting that traditional estrogen receptor mechanisms do not play a role. E(2) caused rapid production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) but only in female cells, and the effect was greater in RC chondrocytes. These results indicate that E(2) regulates ICCC in a sex-specific and cell maturation state-dependent manner. The mechanism is membrane-associated and is mediated by PLC-dependent IP3 production and release of Ca(2+) from the endoplasmic reticulum.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells

The effects of Mg²⁺ on the glutamate-, kainate-, N-methyl-d-aspartate- and quisqualate-induced influx of⁴⁵Ca²⁺ were studied in cultured cerebellar granule cells. The N-methyl-d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg²⁺. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg²⁺, whereas N-methyl-d-aspartate was most effective in totally Mg²⁺-free media.d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca²⁺ influx by 1 mM quisqualate in 0.1-mM Mg²⁺ medium. The effect of 10 M quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-d-aspartate, (RS)-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg²⁺ medium. The efficacy of quisqualate in inducing influx of Ca⁺ and the selectivity of antagonists for different receptors are also modified by extracellular Mg²⁺.     

Localised and rapid Ca2+ micro-events in human neutrophils: conventional Ca2+ puffs and global waves without peripheral-restriction or wave cycling

Ultra-localised and peripherally restricted zones of elevated Ca2+ (z-waves) have been reported to cycle around the periphery of neutrophils at low frequency (1/20s) in the absence of conventional localised Ca2+ (puffs) and global Ca2+ (waves) signals. However, we report here that fast confocal laser scanning of human neutrophils loaded with either cytosolic fluo4 or its membrane associated analogue, MOMO reports both "conventional" stationary Ca2+ "puffs" (diameter c.3 microm) and global Ca2+ waves that sweep across the cell. The Ca2+ puff size and frequency of detection suggests that each neutrophil contained only a single release site and that its detection was limited by the location of the confocal plane relative to the event. Both formylated peptide receptor stimulation and cytosolic IP3 uncaging generated Ca2+ puffs (c.6% of cells) and global Ca2+ signals (c.75% of cells). The Ca2+ puffs peaked at approx. 250 nM and had a duration of approx. 235 msec and remained at a single locus. This was similar to other Ca2+ events in other cell types but in direct contrast to the reported z-waves. It was concluded that the micro-events which underlie Ca2+ signalling in neutrophils are conventional and that the existence of novel Ca2+z-waves is doubtful.     

Identification of a novel compound that stimulates intracellular calcium increase and CXCL8 production in human neutrophils from Schisandra chinensis

Schisandra chinensis has been regarded as a useful material in the preventive or treatment of several human diseases. The study of the Schisandra chinensis’ molecular identity has been prioritized because it has been found to regulate cellular responses. Here, we examined the effects from various extracts of Schisandra chinensis to monitor the relative intracellular calcium increase in human neutrophils. We identified an active molecule and structural configuration of a new active compound (α-iso-cubebene), based on the discovery of a cubebene backbone using NMR, MS, and CD spectral data. In terms of its functional aspect, we observed that α-iso-cubebene strongly stimulated CXCL8 production in human neutrophils. Also, α-iso-cubebene-induced CXCL8 production was almost completely inhibited by the calcium chelator, EGTA, thus highlighting the role of calcium signaling in the process. Taken together, our results demonstrate that α-iso-cubebene is a novel natural compound which stimulates intracellular calcium signaling and CXCL8 production. As a result, α-iso-cubebene should be useful for the development of an immune-modulating agent.