Paracrine Ca2+ signaling in vitro: Serotonin—mediated cell—cell communication in mast cell/smooth muscle cocultures

Mast cells are tissue-resident immune cells that are capable of signaling many different cell types in vascularized tissue including epithelia and smooth muscle. We have developed an in vitro coculture system in which secretion of serotonin by a mucosal mast cell line (RBL-2H3) can be studied at a single cell level by measuring Ca²⁺ transients in fura-2 loaded mast cells and serotonin-sensitive A7r5 smooth muscle cells using fluorescence video microscopy and digital image processing. A7r5 cells elevate intracellular Ca²⁺ via 5HT₂ receptors in response to bath-applied serotonin with an ED₅₀ for serotonin of 550nM. Crosslinking lgE receptors with antigen caused Ca²⁺ transients in the mucosal mast cells. Ca²⁺ responses in the smooth muscle were detected ≈? 30–240 sec after the initiation of the mast cell Ca²⁺ responses. Smooth muscle Ca²⁺ responses were dependent on preloading mast cells with serotonin and were blocked by the 5HT₂ antagonist ketanserin. The timing and magnitude of the smooth muscle responses indicated that secretion from mast cells can lead to local concentrations of serotonin in the range of 300 nM within 1 min of antigen stimulation. This coculture technique has allowed the first direct demonstration of serotonin-mediated signaling between immune cells and vascular elements. © 1994 Wiley-Liss, Inc.     

Protein kinase C activator PMA reduces the Ca2+ response to antigen stimulation of adherent RBL-2H3 mucosal mast cells by inhibiting depletion of intracellular Ca2+ stores

Activation of protein kinase C has been shown to reduce the Ca²⁺ responses of a variety of cell types. In most cases, the reduction is due to inhibition of Ca²⁺ influx, but acceleration of Ca²⁺ efflux and inhibition of Ca²⁺ store depletion by protein kinase C activation have also been described. For adherent RBL-2H3 mucosal mast cells, results from whole-cell patch clamp experiments suggest that protein kinase C activation reduces Ca²⁺ influx, while experiments with intact, fura-2-loaded cells suggest that Ca²⁺ influx is not affected. Here we present single-cell data from Ca²⁺ imaging experiments with adherent RBL-2H3 cells, showing that antigen-stimulated Ca²⁺ responses of phorbol 12-myristate 13-acetate (PMA)-treated cells are more transient than those of control cells. PMA also reduced the response to antigen in the absence of extracellular Ca²⁺, indicating that depletion of intracellular Ca²⁺ stores is inhibited. If PMA was added after stores had been depleted by thapsigargin, a small decrease in [Ca²⁺]_i was observed, consistent with a slight inhibition of Ca²⁺ influx. However, the major effect of PMA on the antigen-stimulated Ca²⁺ response is to inhibit depletion of intracellular Ca²⁺ stores. We also show that inhibition of protein kinase C did not enhance the Ca²⁺ response to antigen, suggesting that inhibition of the Ca²⁺ response by activation of protein kinase C does not contribute to the physiological response to antigen. J. Cell. Physiol. 181:113–123, 1999. © 1999 Wiley-Liss, Inc.     

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca²⁺ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca²⁺ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca²⁺ levels, respectively. Histamine release and Ca²⁺ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca²⁺ increase. FcεRI-induced Ca²⁺ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca²⁺ release was both necessary and sufficient for mast cell degranulation, extracellular Ca²⁺ influx enhanced the process. Furthermore, cytosolic Ca²⁺ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca²⁺ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca²⁺ signaling in regulating histamine degranulation.     

Protective effect of chitosan oligosaccharides against FcɛRI-mediated RBL-2H3 mast cell activation

The activation of mast cells by immunoglobulin E-mediated stimuli is considered as a central event in allergic responses. In this regard, chitosan oligosaccharides (COS) of two different molecular weight ranges (1–3 kDa and 3–5 kDa) were investigated for their capabilities against the activation of RBL-2H3 mast cell sensitized with dinitrophenyl-specific immunoglobulin E antibody and stimulated by antigen dinitrophenyl-bovine serum albumin. It was found that COS significantly inhibited RBL-2H3 cell degranulation via attenuating the releases of histamine and β-hexosaminidase. Moreover, the inhibitory activity of COS was accompanied by a reduction in intracellular Ca²⁺ elevation. Notably, the expression of immunoglobulin Fc epsilon receptor I (Fc?RI) in RBL-2H3 cells was down-regulated by COS treatment in a dose-dependent manner. The suppressive effect of COS on RBL-2H3 cell activation suggested that COS may be potential candidates of novel inhibitors against allergic reactions.     

Orai-2 is localized on secretory granules and regulates antigen-evoked Ca mobilization and exocytosis in mast cells

The increase in intracellular Ca²⁺ through the Ca²⁺ channel is an indispensable step for the secretion of inflammatory mediators by mast cells. It was recently reported that Orai-1 is responsible for the Ca²⁺ influx that is activated by depletion of stored Ca²⁺. There are three isoforms of Orai: Orai-1, Orai-2, and Orai-3; however, isoforms other than Orai-1 are poorly understood. We found that Orai-2 is expressed and localized on secretory granules in RBL-2H3. Ca²⁺ release from Ca²⁺ store, induced by antigen stimulation, was significantly attenuated by knockdown of Orai-2, while that induced by thapsigargin was not affected. Furthermore, exocytotic release induced by antigen stimulation was inhibited in knockdown cells. This observation suggests a new role of Orai isoforms in secretory cells.     

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca²⁺ level ([Ca²⁺]_i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca²⁺]_i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca²⁺]_i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca²⁺]_i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca²⁺]_i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca²⁺ signaling and degranulation. mast cell; immunoglobulin E; cytochalasin D; Y-27632; wortmannin     

The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses

Ionizing radiation has different biological effects according to dose and dose rate. In particular, the biological effect of low-dose radiation is unclear. Low-dose whole-body gamma irradiation activates immune responses in several ways. However, the effects and mechanism of low-dose radiation on allergic responses remain poorly understood. Previously, we reported that low-dose ionizing radiation inhibits mediator release in IgE-mediated RBL-2H3 mast cell activation. In this study, to have any physiological relevance, we investigated whether low-dose radiation inhibits allergic responses in activated human mast cells (HMC-1(5C6) and LAD2 cells), mouse models of passive cutaneous anaphylaxis and the late-phase cutaneous response. High-dose radiation induced cell death, but low-dose ionizing radiation of <0.5 Gy did not induce mast cell death. Low-dose ionizing radiation that did not induce cell death significantly suppressed mediator release from human mast cells (HMC-1(5C6) and LAD2 cells) that were activated by antigen-antibody reaction. To determine the inhibitory mechanism of mediator released by low-dose ionizing radiation, we examined the phosphorylation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, and protein kinase C, as well as the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). The phosphorylation of signaling molecules and [Ca²⁺]_i following stimulation of FcεRI receptors was inhibited by low dose ionizing radiation. In agreement with its in vitro effect, ionizing radiation also significantly inhibited inflammatory cells infiltration, cytokine mRNA expression (TNF-α, IL-4, IL-13), and symptoms of passive cutaneous anaphylaxis reaction and the late-phase cutaneous response in anti-dinitrophenyl IgE-sensitized mice. These results indicate that ionizing radiation inhibits both mast cell-mediated immediate- and delayed-type allergic reactions in vivo and in vitro.     

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca²⁺ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP₃) concentration changes using a fluorescent IP₃ sensor in single HeLa cells showing different patterns of histamine-induced Ca²⁺ oscillations in terms of the time constant of Ca²⁺ spike amplitude decay and the Ca²⁺ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca²⁺ signals and we found that there were cell-specific IP₃ dynamics depending on the patterns of Ca²⁺ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP₃ increases in HeLa cells. Modulation of IP₃ dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca²⁺ oscillations, such as the time constant of the temporal changes in the Ca²⁺ spike amplitude and the Ca²⁺ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP₃ production, rather than IP₃-induced Ca²⁺ release, can cause cell-to-cell variability in the patterns of Ca²⁺ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca²⁺ signals evoked by G protein-coupled receptor stimulation.     

Ca2+-Atpase inhibitor,cyclopiazonic acid,releases Ca2+ from intracellular stores in rbl-2h3 mast cells and activates a Ca2+ influx pathway that is permeable to sodium and manganese

Cyclopiazonic acid has been reported to inhibit the Ca²⁺-ATPase of intracellular calcium stores in some nonexcitable cell types, such as myeloid cells and lymphocytes. The present study examines the effects of cyclopizonic acid on rat basophilic leukemia (RBL) cells, a mucosal mast cell line. Addition of cyclopiazonic acid to fura-2-loaded RBL cells evoked a biphasic increase in free ionized intracellular calcium. Release of stored calcium accounted for the first phase of this response. The second phase was determined to be calcium entering through an influx pathway activated by cyclopiazonic acid. The influx pathway was selective for calcium, But was somewhat permeable to manganese. However, in a Ca²⁺-free solution containing EGTA, sodium ions permeated freely. This influx pathway appears to be identical to that which is activated by antigen, the physiological stimulus to the cells. Cyclopiazonic acid also induced secretion when combined with the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate, which activates protein kinae C. © 1995 Wiley-Liss, Inc.     

Ca2+ signals and T lymphocytes; "New mechanisms and functions in Ca2+ signalling"

Abstract Interaction between a T cell and an antigen‐presenting cell leads to the rapid formation of an immunological synapse allowing antigen detection by the T cell and the development of an immune response. Antigen detection triggers various cellular responses including a modest but sustained T cell Ca²⁺ increase. In this review are discussed a series of related questions. What are the various molecular events by which a T cell Ca²⁺ response can be triggered in the immunological synapse by a very small amount of antigen ? How is Ca²⁺ released from intracellular stores and how can these stores remain empty for hours ? Through which channels does Ca²⁺ influx takes place, and how is Ca²⁺ influx coupled to Ca²⁺ release from intracellular stores ? What are the main immediate and indirect cellular targets of the Ca²⁺ increase ?     

The characterization and quantification of antigen-induced Caoscillations in a rat basophilic leukaemia cell line (RBL–2H3)

Using1999 Harcourt Publishers Ltdthe ratiometric Ca²⁺indicator, indo-1, the antigen-induced increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) was measured in individual RBL–2H3 cells which had been passively sensitized with monoclonal antibody to the dintrophenyl (DNP) haptenic group. Antigenic stimulation using DNP-human serum albumin conjugate (DNP-HSA) induced concentration-dependent asynchronous Ca²⁺oscillations, or irregular spikes. To achieve a quantitative comparison of the effects of different concentrations of antigen on changes in [Ca²⁺]_i, the area under the curve (AUC) of Ca²⁺oscillations in each cell was calculated.The dose–response curve of the calculated AUC is consistent with the bell-shaped dose–response curve for antigen-induced mediator release, depolarization and⁸⁶Rb⁺-efflux. Ca²⁺oscillations induced by antigenic stimulation were abolished by removal of external Ca²⁺and the subsequent reintroduction of external Ca²⁺caused their resumption.To investigate the role of Ca²⁺oscillations in the secretory response, changes in [Ca²⁺]_iinduced by concanavalin A (Con-A), A23187, thapsigargin and NECA were also monitored. Con-A mimicked the response induced by antigen, whilst A23187 and thapsigargin induced a large transient non-oscillatory response. NECA, an adenosine receptor agonist, induced only a small transient rise in [Ca²⁺]_iwithout oscillatory behaviour. Since all these stimuli accept NECA-induced degranulation in these cells, it is suggested that, although Ca²⁺oscillations are not essential for the initiation of secretion, they probably underlie the in-vivo physiological response of mast cells and basophils to an antigenic challenge. They also seem to enhance the efficacy of the Ca²⁺signal.     

Signal percolation through plants and the shape of the calcium signature

Plants respond to almost any kind of external stimulus with transients in their cytoplasmic free calcium concentration ([Ca²⁺]_c). A huge variety of kinetics recorded by optical techniques has been reported in the past. This variety has been credited the specificity needed to explain how information about incoming stimuli is evaluated by the organism and turned into the right physiological responses which provide advantages for survival and reproduction. A physiological response often takes place away from the site of stimulation. This requires cell-to-cell communication. Hence, responding cells are not necessarily directly stimulated but rather receive an indirect stimulus via cell-to-cell communication. It appears unlikely that the ‘[Ca²⁺]_c signature’ in the primarily stimulated cell is conveyed over long distances via cell-to-cell communication from the ‘receptor cells’ to the ‘effector cells’. Here, a novel aspect is highlighted to explain the variety of [Ca²⁺] kinetics seen by integrating methods of [Ca²⁺]_c recording. Plants can generally be seen as cellular automata with specific morphology and capable for cell-to-cell communication. Just a few rules are needed to demonstrate how waves of [Ca²⁺]_c-increases percolate through the organism and thereby deliver a broad variety of ‘signatures’. Modelling intercellular signalling may be a possible way to find explanations for different kinds of signal transmission, signal amplification, wave formation, oscillations and stimulus-response coupling. The basic examples presented here show that care has to be taken when interpreting cellular ‘[Ca²⁺]_c signatures’ recorded by optical techniques which integrate over a big number of cells or even whole plants.Key words: cellular automata, cell-to-cell communication, cytoplasmic calcium, modelling, percolation, signature     

Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis

All three members of the Orai family of cation channels–Orai1, Orai2 and Orai3–are integral membrane proteins that can form store-operated Ca²⁺ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca²⁺ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca²⁺-dependent exocytosis of inflammatory mediators. We show that the Ca²⁺ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca²⁺ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2^−/− PMCs at high (2 mM) extracellular Ca²⁺ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1 mM) Ca²⁺ concentration. Likewise, the density of CRAC currents, Ca²⁺-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca²⁺ transients, store-operated Ca²⁺ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca²⁺ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.     

Role of Janus kinase-2 in IgE receptor-mediated leukotriene C4 production by mast cells

We have previously shown that Janus kinase 3, a member of the family of non-receptor protein tyrosine kinases, plays a critical role in the regulation of FcεRI-mediated mast cell responses. In the current study, we investigated the role of another JAK family member, JAK2, in these responses. Our results show that the treatment of IgE-sensitized mouse mast cells with an inhibitor of JAK2 (AG490) blocked the release of leukotriene C₄ in a dose-dependent fashion after antigen challenge. However, prostaglandin PG D₂ production and degranulation were not affected under identical experimental conditions. Transfection of RBL-2H3 mast cells with JAK-2 specific small interfering RNA resulted in a 50% reduction of LTC₄ release in response to FcεRI crosslinking, but did not inhibit mast cell degranulation or calcium ionophore-induced LTC₄ release, indicating involvement of JAK2 in IgE receptor-mediated leukotriene release. Taken together, these data suggest that JAK2 is a critical regulator of IgE/antigen-induced production of LTC₄ in mast cells.     

Ca2+-Mg2+-activated adenosine triphosphatase in plasma and granule membranes in non-secreting and secreting mast cells

An adenosine triphosphatase (ATP) activated by Ca²⁺ or Mg²⁺ is shown morphologically on the outer surface of non-secreting and secreting rat peritoneal mast cells. ATPase having the same properties is also seen on the external surface of the other peritoneal cells, i.e. macrophages, mononuclear cells and lymphocytes. When histamine release from the mast cells was induced by exposing them to antigen (anaphylactic reaction) or compound 48/80, ATPase activated by Ca²⁺ or Mg²⁺ could in addition be demonstrated in the granule membranes. Granule membrane ATPase is also shown in non-secreting mast cells after freezing and thawing. ATPase on the outer surface of the plasma membrane is seen in the secreting mast cells as in the non-secreting cells except in the areas where the plasma membrane fuses with the granule membrane. The role of ATPase in granule secretion process has been discussed.     

Distinct Stages of Stimulated FcεRI Receptor Clustering and Immobilization Are Identified through Superresolution Imaging

Recent advances in fluorescence localization microscopy have made it possible to image chemically fixed and living cells at 20 nm lateral resolution. We apply this methodology to simultaneously record receptor organization and dynamics on the ventral surface of live RBL-2H3 mast cells undergoing antigen-mediated signaling. Cross-linking of IgE bound to FcεRI by multivalent antigen initiates mast cell activation, which leads to inflammatory responses physiologically. We quantify receptor organization and dynamics as cells are stimulated at room temperature (22°C). Within 2 min of antigen addition, receptor diffusion coefficients decrease by an order of magnitude, and single-particle trajectories are confined. Within 5 min of antigen addition, receptors organize into clusters containing ∼100 receptors with average radii of ∼70 nm. By comparing simultaneous measurements of clustering and mobility, we determine that there are two distinct stages of receptor clustering. In the first stage, which precedes stimulated Ca²⁺ mobilization, receptors slow dramatically but are not tightly clustered. In the second stage, receptors are tightly packed and confined. We find that stimulation-dependent changes in both receptor clustering and mobility can be reversed by displacing multivalent antigen with monovalent ligands, and that these changes can be modulated through enrichment or reduction in cellular cholesterol levels.     

Vacuolin-1-modulated exocytosis and cell resealing in mast cells

The small chemical vacuolin-1 induces rapid formation of large vacuoles in various cell types. In epithelial cells, vacuolin-1 has been shown to inhibit Ca²⁺ ionophore-induced exocytosis depending on experimental conditions used but had no effect on repair of damaged membranes. However, it is not known whether vacuolin-1 could inhibit exocytosis induced by immunoreceptor triggering in professional secretory cells and whether there is any correlation between effect of vacuolin-1 on exocytosis and membrane repair in such cells. Here we show that in rat basophilic leukemia (RBL-2H3) cells activated by the high-affinity IgE receptor (FcεRI) triggering vacuolin-1 enhanced exocytosis. Under identical conditions of activation, vacuolin-1 inhibited exocytosis in mouse bone marrow-derived mast cells (BMMCs). This inhibition was not reflected by decreased phosphorylation of the FcεRI α and β subunits, linker for activation of T cells, non-T cell activation linker, Akt and MAP kinase Erk, and uptake of extracellular Ca²⁺, indicating that early activation events are not affected. In both cell types vacuolin-1 led to formation of numerous vacuoles, a process which was inhibited by bafilomycin A1, an inhibitor of vacuolar H⁺-ATPase. Thapsigargin- or Ca²⁺ ionophore A23187-induced exocytosis also showed different sensitivity to the inhibitory effect of vacuolin-1. Pretreatment of the cells with vacuolin-1 followed by permeabilization with bacterial toxin streptolysin O enhanced Ca²⁺-dependent repair of plasma membrane lesions in RBL-2H3 cells but inhibited it in BMMCs. Our data indicate that lysosomal exocytosis exhibits different sensitivity to vacuolin-1 depending on the cell type analyzed and mode of activation. Furthermore, our results support the concept that lysosomal exocytosis is involved in the repair of injured plasma membranes.     

Glutamate Mediated Astrocytic Filtering of Neuronal Activity

Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca²⁺ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca²⁺ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca²⁺ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP₃ and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca²⁺ activity as a nonlinear integrator of glutamate-dependent neuronal activity.     

A Ca2+ Current Activated by Release of Intracellular Ca2+ Stores in Rat Basophilic Leukemia Cells (RBL-1)

We have characterized a Ca²⁺ current activated by depletion of intracellular Ca²⁺ stores (capacitative Ca²⁺ entry current) as a first step to investigate the mechanisms underlying communication between the intracellular Ca²⁺ stores and the plasma membrane Ca²⁺ permeability. Whole cell currents in response to voltage ramps from −125 to +60 mV from a holding potential of −40 mV were recorded in rat basophilic leukemia cells (RBL-1 cells) in solutions designed to optimize detection of a Ca²⁺ current. An inwardly rectifying current could be activated upon dialysis of the cell interior with pipette solutions devoid of Ca²⁺ and containing 20 mm BAPTA, a procedure expected to passively deplete intracellular Ca²⁺ stores. The current was maximally activated within 2 min, was sensitive to extracellular Ca²⁺ concentration and was abolished by removal of extracellular Ca²⁺. The current was markedly reduced in the presence of Ni²⁺ or La³⁺. The pathway activated by this protocol was permeant to Ba²⁺, displaying complex permeability characteristics at negative potentials. A small inward Mn²⁺ current consistent with a finite permeability of the pathway to Mn²⁺ was detected. In contrast Ni²⁺ displayed no detectable current carrying ability. Extracellular Na⁺ permeated the pathway in the absence of extracellular Ca²⁺. Under conditions designed to reduce passive depletion of intracellular Ca²⁺ stores, a Ca²⁺ current indistinguishable from that described above was activated by addition of ionomycin. This observation is consistent with the activation of the Ca²⁺ influx pathway occurring as a result of events associated with depletion of intracellular Ca²⁺ stores. Importantly, application of extracellular Ni²⁺ in the presence of ionomycin irreversibly inhibited the current. The presence of an inwardly rectifying K⁺ current in RBL cells could confound studies of the capacitative Ca²⁺ entry current when recorded using pipette solutions devoid of K⁺ since this current would be inward over the voltage range used to investigate the capacitative Ca²⁺ entry current. This study compares an inward rectifying K⁺ current and the capacitative Ca²⁺ entry current in RBL cells and highlights some similarities and differences between the two currents. The results demonstrate that caution should be exercised in interpreting recordings made using extracellular solutions containing even modest amounts of K⁺ when studying the capacitative Ca²⁺ entry current in RBL cells. Received: 12 September 1995/Revised: 18 June 1996