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

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

Parasporin-1, a novel cytotoxic protein from Bacillus thuringiensis, induces Ca2+ influx and a sustained elevation of the cytoplasmic Ca2+ concentration in toxin-sensitive cells

Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxin-sensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-1-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-1-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca(2+) concentration; increases in the intracellular Ca(2+) levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca(2+) concentration. The toxin-induced elevation of the intracellular Ca(2+) concentration was markedly decreased in low-Ca(2+) buffer and was not observed in Ca(2+)-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca(2+) buffer and was restored by the addition of Ca(2+) to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca(2+) influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADP-ribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca(2+) level and that the Ca(2+) influx is the first step in the pathway that underlies parasporin-1 toxicity.     

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.     

Fig1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae

During the mating process of yeast cells, two Ca2+ influx pathways become activated. The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described. The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity. Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity. Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor. Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase. Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways. Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS. These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.     

Down-regulating annexin gene GhAnn2 inhibits cotton fiber elongation and decreases Ca2+ influx at the cell apex

Cotton fiber is a single cell that differentiates from the ovule epidermis and undergoes synchronous elongation with high secretion and growth rate. Apart from economic importance, cotton fiber provides an excellent single-celled model for studying mechanisms of cell-growth. Annexins are Ca²⁺- and phospholipid-binding proteins that have been reported to be localized in multiple cellular compartments and involved in control of vesicle secretions. Although several annexins have been found to be highly expressed in elongating cotton fibers, their functional roles in fiber development remain unknown. Here, 14 annexin family members were identified from the fully sequenced diploid G. raimondii (D₅ genome), half of which were expressed in fibers of the cultivated tetraploid species G. hirsutum (cv. YZ1). Among them, GhAnn2 from the D genome of the tetraploid species displayed high expression level in elongating fiber. The expression of GhAnn2 could be induced by some phytohormones that play important roles in fiber elongation, such as IAA and GA₃. RNAi-mediated down-regulation of GhAnn2 inhibited fiber elongation and secondary cell wall synthesis, resulting in shorter and thinner mature fibers in the transgenic plants. Measurement with non-invasive scanning ion-selective electrode revealed that the rate of Ca²⁺ influx from extracellular to intracellular was decreased at the fiber cell apex of GhAnn2 silencing lines, in comparison to that in the wild type. These results indicate that GhAnn2 may regulate fiber development through modulating Ca²⁺ fluxes and signaling.     

On the role of TRPC1 in control of Ca2+ influx, cell volume, and cell cycle

Ca(+) signaling plays a crucial role in control of cell cycle progression, but the understanding of the dynamics of Ca(2+) influx and release of Ca(2+) from intracellular stores during the cell cycle is far from complete. The aim of the present study was to investigate the role of the free extracellular Ca(2+) concentration ([Ca(2+)](o)) in cell proliferation, the pattern of changes in the free intracellular Ca(2+) concentration ([Ca(2+)](i)) during cell cycle progression, and the role of the transient receptor potential (TRP)C1 in these changes as well as in cell cycle progression and cell volume regulation. In Ehrlich Lettré Ascites (ELA) cells, [Ca(2+)](i) decreased significantly, and the thapsigargin-releasable Ca(2+) pool in the intracellular stores increased in G(1) as compared with G(0). Store-depletion-operated Ca(2+) entry (SOCE) and TRPC1 protein expression level were both higher in G(1) than in G(0) and S phase, in parallel with a more effective volume regulation after swelling [regulatory volume decrease (RVD)] in G(1) as compared with S phase. Furthermore, reduction of [Ca(2+)](o), as well as two unspecific SOCE inhibitors, 2-APB (2-aminoethyldiphenyl borinate) and SKF96365 (1-(β-[3-(4-methoxy-phenyl)propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride), inhibited ELA cell proliferation. Finally, Madin-Darby canine kidney cells in which TRPC1 was stably silenced [TRPC1 knockdown (TRPC1-KD) MDCK] exhibited reduced SOCE, slower RVD, and reduced cell proliferation compared with mock controls. In conclusion, in ELA cells, SOCE and TRPC1 both seem to be upregulated in G(1) as compared with S phase, concomitant with an increased rate of RVD. Furthermore, TRPC1-KD MDCK cells exhibit decreased SOCE, decreased RVD, and decreased proliferation, suggesting that, at least in certain cell types, TRPC1 is regulated during cell cycle progression and is involved in SOCE, RVD, and cell proliferation.     

Ca2+ influx through L-type Ca2+ channels controls the trailing tail contraction in growth factor-induced fibroblast cell migration

Growth factor-induced cell migration underlies various physiological and pathological processes. The mechanisms by which growth factors regulate cell migration are not completely understood. Although intracellular elevation of Ca2+ is known to be critical in cell migration, the source of this Ca2+ elevation and the mechanism by which Ca2+ modulates this process in fibroblast cells are not well defined. Here we show that increase of cellular Ca2+ through Ca2+ influx, rather than Ca2+ release from intracellular stores, is essential for growth factor-induced fibroblast cell migration. Voltage-gated L-type Ca2+ channels, previously known to exist in excitable cells such as neurons and muscle cells, are shown here to be present in fibroblasts as well. Furthermore, these channels are responsible for the Ca2+ influx. L-type Ca2+ channel inhibitors block growth factor-induced Ca2+ influx and fibroblast cell migration. One mechanism by which Ca2+ signals control cell migration is to regulate the contraction of the trailing edge of migrating fibroblasts; this process is controlled by the small GTPase Rho in fast migrating cells such as leukocytes. Downstream of Ca2+, both calmodulin and myosin light chain kinase, but not calcineurin, are involved leading to phosphorylation of the myosin light chain at the trailing end. Thus, trailing edge contraction is critically regulated by Ca2+ influx through L-type Ca2+ channels in growth factor-induced fibroblast cell migration.     

Parathyroid hormone secretion in the absence of extracellular free Ca2+ and transmembrane Ca2+ influx

The involvement of extracellular Ca2+ and Ca2+ influx across the plasma membrane in parathyroid hormone (PTH) secretion was investigated in vitro using a new preparation of bovine parathyroid cells. Incubation of these cells in the presence of 25 microM or 2.5 microM free ambient Ca2+ induced a maximal rate of PTH secretion. Low free Ca2+ secretion is not associated with changes in membrane permeability, requires metabolic energy, and is reversible. The Ca2+ channel blocker D600 had no effect on either 45Ca-influx or PTH secretion in these cells. These results, showing that extracellular Ca2+ and Ca2+ influx across the plasma membrane are not required for PTH secretion by parathyroid cells, emphasize the differences in the cellular mechanisms underlying the secretion of PTH vs that of other secretory cells.     

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.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion‐current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage‐clamp technique. All agonists elicited Cl^? currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl^? (F_Cl) currents, while AII activated an oscillatory response; a robust Ca²⁺ influx linked specifically to F_Cl activation elicited an inward current (I_iw,Ca) which was carried mainly by Cl^? ions, through channels with a sequence of permeability of SCN^? > I^? > Br^? > Cl^?. Like F_Cl, I_iw,Ca was not dependent on oocyte [Ca²⁺]_i; instead both were eliminated by preventing [Ca²⁺]_i increase in the follicular cells, and also by U73122 and 2‐APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F_Cl and I_iw,Ca were produced by the expected, PLC‐stimulated Ca²⁺‐release and Ca²⁺‐influx, respectively, and by the opening of I_Cl(Ca) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca²⁺‐influx appeared to be driven through store‐operated, calcium‐like channels. The AII response, which is also known to require PLC activation, did not activate I_iw,Ca and was strictly dependent on oocyte [Ca²⁺]_i increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap‐junction channels. J. Cell. Physiol. 227: 3457–3470, 2012. © 2011 Wiley Periodicals, Inc.     

Quercetin: a novel inhibitor of Ca2+ influx and exocytosis in rat peritoneal mast cells.

The effect of the transport ATPase inhibitor, quercetin on histamine secretion from antigen sensitized mast cells was examined. At micromolar concentrations, quercetin had an immediate inhibitory effect on histamine secretion mediated by antigen, concanavalin A and ATP but it had little effect on release induced by the ionophores A23187 and X537A. Quercetin exerts its effect after the binding of the releasing ligands and the distinction between its effect on ligand induced and A23187 induced secretion suggests that it affects the normal path of Ca2+ entry into the cell. The inhibitory effects of quercetin were compared with those of the structurally related anti-allergic drugs cromoglycate and AH7725.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Polymyxin B, a novel inhibitor of red cell Ca2+-activated K+ channel

Polymyxin B (PXB), a cyclic peptide antibiotic, in concentrations 0.1-3.0 mg/ml (0.08-4.0 mmol/l), inhibited the K+ efflux induced by opening of the Ca2+-activated K+ channel (the Gárdos effect) in intact human red blood cells. The inhibition was observed when the Gárdos effect was elicited by Ca2+ in the presence of vanadate, or propranolol, in ATP-depleted cells, and in A23187-treated cells. The inhibition of the Gárdos effect is caused neither by the inhibition of the anion channel by PXB nor by the inhibition of Ca2+ entry. It can be ascribed to the inhibition of the Ca2+-activated K+ channel. The mechanism of the inhibition remains to be elucidated.     

New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast

The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca(2+) influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca(2+) after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.     

Stimulation of the T3-T cell receptor-associated Ca2+ influx enhances the activity of the Na+/H+ exchanger in a leukemic human T cell line

Three monoclonal antibodies reactive with different structural domains of the T3-T cell receptor complex of the human T cell leukemia line, HPB-ALL, were previously shown to activate a membrane potential-sensitive, La3+-inhibitable Ca2+ influx (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590). OKT3 (anti-T3), WT-31 (anti-receptor constant region), and T40/25 (anti-receptor variable region) also enhance the activity of the Na+/H+ exchanger in these cells. The associated rise in pHi was dependent on the presence of external Ca2+ and Na+, was inhibited by dimethylamiloride and La3+, and was maintained for at least 20 min. Phorbol esters, which are co-mitogenic in T cells and activate protein kinase C, also stimulated the exchanger, but by a mechanism not requiring an elevation in cytoplasmic Ca2+; the rise in pHi rapidly peaked and returned to baseline levels within 20 min. Pretreatment with phorbols prevented an increase in pHi by OKT3 although a transient additive effect was observed when the two were added simultaneously. Receptor function was maintained in the presence of phorbol esters as OKT3 still stimulated a Ca2+ influx. These data demonstrate the existence of two interdependent pathways to activate Na+/H+ exchange in T lymphocytes and suggest a pathway of internal regulation of antigen-activated signal transduction.     

Roles for Ca2+ stores release and two Ca2+ influx pathways in the Fc epsilon R1-activated Ca2+ responses of RBL-2H3 mast cells.

Cross-linking the high affinity IgE receptor, Fc epsilon R1, with multivalent antigen induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent release of intracellular Ca2+ stores, Ca2+ influx, and secretion of inflammatory mediators from RBL-2H3 mast cells. Here, fluorescence ratio imaging microscopy was used to characterize the antigen-induced Ca2+ responses of single fura-2-loaded RBL-2H3 cells in the presence and absence of extracellular Ca2+ (Ca2+o). As antigen concentration increases toward the optimum for secretion, more cells show a Ca2+ spike or an abrupt increase in [Ca2+]i and the lag time to onset of the response decreases both in the presence and the absence of Ca2+o. When Ca2+o is absent, fewer cells respond to low antigen and the lag times to response are longer than those measured in the presence of Ca2+o, indicating that Ca2+o contributes to Ca2+ stores release. Ins(1,4,5)P3 production is not impaired by the removal of Ca2+o, suggesting that extracellular Ca2+ influences Ca2+ stores release via an effect on the Ins(1,4,5)P3 receptor. Stimulation with low concentrations of antigen can lead, only in the presence of Ca2+o, to a small, gradual increase in [Ca2+]i before the abrupt spike response that indicates store release. We propose that this small, initial [Ca2+]i increase is due to receptor-activated Ca2+ influx that precedes and may facilitate Ca2+ stores release. A mechanism for capacitative Ca2+ entry also exists in RBL-2H3 cells. Our data suggest that a previously undescribed response to Fc epsilon R1 cross-linking, inhibition of Ca2+ stores refilling, may be involved in activating capacitative Ca2+ entry in antigen-stimulated RBL-2H3 cells, thus providing the elevated [Ca2+]i required for optimal secretion. The existence of both capacitative entry and Ca2+ influx that can precede Ca2+ release from intracellular stores suggests that at least two mechanisms of stimulated Ca2+ influx are present in RBL-2H3 cells.     

LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca(2+) ([Ca(2+)](i)). Although agonist or ionomycin exposure can raise bulk [Ca(2+)](i) to levels higher than those caused by capacitative Ca(2+) influx, the LAMP assay revealed that only Ca(2+) influx through the plasma membrane store-operated Ca(2+) channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.