Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.     

Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells.

One of the major biological targets of free radical oxidations, prone, for anatomical reasons, to oxidative challenges, is the cardiovascular system. In the present paper the effect of hydrogen peroxide on intracellular ionized calcium ([Ca2+]i) homeostasis in smooth muscle cells (SMC) is studied, the major aim of the study being a better understanding of the protective effect of antioxidants and Ca2+ channel blockers. The exposure of SMC to 300 microM H2O2 induced a rapid increase of [Ca2+]i, followed by a decrease to a new constant level, higher than the basal before the oxidative challenge. When incubation medium was Ca2+ free, the pattern of [Ca2+]i change was different. The rapid increase was still observed, but it was followed by a rapid decrease to a level only slightly above the basal before the oxidative challenge. The involvement of intracellular Ca2+ stores was tested by using vasopressin, a hormone able to induce discharge of inositol 1,4,5-triphosphate-sensitive Ca2+ stores. When H2O2 was added after vasopressin no [Ca2+]i increase was observed. Treatment of cells, in which the stable increase of [Ca2+]i was induced by H2O2, with disulfide reducing compounds, induced a progressive decrease of [Ca2+]i toward the level observed before the oxidative challenge. Calcium channel blockers and antioxidants, on the other hand, effectively prevented the stabilization of [Ca2+]i at the high steady-state, after the internal Ca2+ release phase. Dihydropyridine Ca2+ channel blockers were by far more active than verapamil and among those the most active was lacidipine. Also the antioxidants trolox and N,N'-diphenyl-1,4-phenylenediamine both prevented the [Ca2+]i unbalance. These results suggest that Ca+ channel blockers and antioxidants, although inactive on oxidative stress-induced Ca2+ release from intracellular stores, prevent the increased influx apparently related to a membrane thiol oxidation.     

An apoplastic Ca2+ sensor regulates internal Ca2+ release in aequorin-transformed tobacco cells

Removal of Ca(2+) from tobacco suspension cell medium has two immediate effects on cytosolic Ca(2+) fluxes: (i) externally derived Ca(2+) influx (occurring in response to cold shock or hypo-osmotic shock) is inhibited, and (ii) organellar Ca(2+) release (induced by a fungally derived defense elicitor, caffeine, or hypo-osmotic shock) is elevated. We show here that the enhanced release of internal Ca(2+) is likely due to increased discharge from a caffeine-sensitive store in response to a signal transduced from an extracellular Ca(2+) sensor. Thus, chelation of extracellular Ca(2+) in the absence of any other stimulus directly activates release of intracellular Ca(2+) into the cytosol. Evidence that this chelator-activated Ca(2+) flux is dependent on a signaling pathway includes its abrogation by prior treatment with caffeine, and its inhibition by protein kinase inhibitors (K252a and staurosporine) and anion channel blockers (niflumate and anthracene-9-carboxylate). An unexpected characteristic of tobacco cell adaptation to low external Ca(2+) was the emergence of a new Ca(2+) compartment that was inaccessible to external EGTA, yet responsive to the usual stimulants of extracellular Ca(2+) entry. Thus, cells that are exposed to EGTA for 20 min lose sensitivity to caffeine and defense elicitors, indicating that their intracellular Ca(2+) pools have been depleted. Surprisingly, these same cells simultaneously regain their ability to respond to stimuli that usually activate extracellular Ca(2+) influx even though all external Ca(2+) is chelated. Because this gradual restoration of Ca(2+) influx can be inhibited by the same kinase inhibitors that block EGTA-activated Ca(2+) release, we propose that chelator-activated Ca(2+) release from internal stores leads to deposition of this Ca(2+) into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca(2+) entry.     

Epidermal growth factor-induced depletion of the intracellular Ca2+ store fails to activate capacitative Ca2+ entry in a human salivary cell line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.     

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.     

Gold activates mast cells via calcium influx through multiple H2O2-sensitive pathways including L-type calcium channels

Heavy metals, including gold, induce severe contact hypersensitivity and autoimmune disorders, which develop through an initial Th2-independent process followed by a Th2-dependent process. It has been shown that mast cell activation plays a role in the Th2-independent process and that gold stimulates histamine release in vitro. However, the mechanisms of the gold-induced mast cell activation remain largely unclear. Here we report that gold directly activates mast cells in a Ca2+-dependent manner. HAuCl4 [Au(III)] at nontoxic concentrations (≤50 μM) induced substantial degranulation and leukotriene C4 secretion in an extracellular Ca2+-dependent manner. Au(III) induced a robust Ca2+ influx but not Ca2+ mobilization from internal stores. Au(III) also stimulated intracellular production of reactive oxygen species, including H2O2, and blockade of the production abolished the mediator release and Ca2+ influx. Au(III) induced Ca2+ influx through multiple store-independent Ca2+ channels, including Cav1.2 L-type Ca2+ channels (LTCCs) and 2-aminoethoxydiphenyl borate (2-APB)-sensitive Ca2+ channels. The 2-APB-sensitive channel seemed to mediate Au(III)-induced degranulation. Our results indicate that gold stimulates Ca2+ influx and mediator release in mast cells through multiple H2O2-sensitive Ca2+ channels including LTCCs and 2-APB-sensitive Ca2+ channels. These findings provide insight into the roles of these Ca2+ channels in the Th2-independent process of gold-induced immunological disorders.     

Genistein inhibits Ca2+ influx and glutamate release from hippocampal synaptosomes: putative non-specific effects

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events.Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.     

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.     

Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.     

Evidence for receptor-mediated calcium entry and refilling of intracellular calcium stores in FRTL-5 rat thyroid cells.

The aim of the present study was to investigate the relationship between agonist-induced changes in intracellular free Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in Fura 2-loaded thyroid FRTL-5 cells. Stimulating the cells with ATP induced a dose-dependent increase in ([Ca2+]i). The ATP-induced increase in [Ca2+]i was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP-induced increase in [Ca2+]i dependent on influx of Ca2+. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]i; this increase was inhibited by Ni2+. In addition, the ATP-induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]i. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]i was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL-5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.     

Inhibition of protein tyrosine phosphatase 1B by reactive oxygen species leads to maintenance of Ca2+ influx following store depletion in HEK 293 cells

Depletion of inositol 1,4,5 trisphosphate-sensitive Ca2+ stores generates a yet unknown signal, which leads to increase in Ca2+ influx in different cell types [J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7 (1986) 1-12]. Here, we describe a mechanism that modulates this store-operated Ca2+ entry (SOC). Ca2+ influx leads to inhibition of protein tyrosine phosphatase 1B (PTP1B) activity in HEK 293 cells [L. Sternfeld, et al., Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells, Cell Signal 17 (2005) 951-960]. Since Ca2+ does not directly inhibit PTP1B, we assumed an intermediate signal, which links the rise in cytosolic Ca2+ concentration and PTP1B inhibition. We now show that Ca2+ influx is followed by generation of reactive oxygen species (ROS) and that it is reduced in cells preincubated with catalase. Furthermore, Ca2+-dependent inhibition of PTP1B can be abolished in the presence of catalase. H2O2 (100 microM) directly added to cells inhibits PTP1B and leads to increase in Ca2+ influx after store depletion. PP1, an inhibitor of the Src family tyrosine kinases, prevents H2O2-induced Ca2+ influx. Our results show that ROS act as fine tuning modulators of Ca2+ entry. We assume that the Ca2+ influx channel or a protein involved in its regulation remains tyrosine phosphorylated as a consequence of PTP1B inhibition by ROS. This leads to maintained Ca2+ influx in the manner of a positive feedback loop.     

Surfactant releases internal calcium stores in neutrophils by G protein-activated pathway

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca(++) stores and that PS+SAP induces depletion through release of Ca(++) stores and through inhibition of Ca(++) influx. Our model predicts that PS+SAP releases Ca(++) stores through insertion of channels, depolarization of neutrophils, and activation of a G protein-dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin-a channel protein with properties similar to those of PS+SAP-is expected to mimic these effects. Human neutrophils were monitored for [Ca(++)] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca(++)] responses were reexamined following preexposure to inhibitors of internal Ca(++) release or the G protein pathway. We observed that (i) 1% PS+SAP-but not PS-SAP-causes transient increase of neutrophil [Ca(++)] within seconds of exposure; (ii) 1% PLG-but not gramicidin alone-closely mimics the effect of PS+SAP on Ca(++) response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca(++) stores releases or of G protein activation suppresses Ca(++) responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca(++) influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca(++) from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca(++) stores and by inhibiting Ca(++) influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.     

VEGF and ATP act by different mechanisms to increase microvascular permeability and endothelial [Ca(2+)](i)

Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.     

The multiple mechanisms of Ca2+ signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes

Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.     

Calcium signalling in squid olfactory receptor neurons

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca(2+) imaging techniques to determine if changes in intracellular Ca(2+) were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca(2+) from intracellular stores while betaine did not change internal Ca(2+) concentrations. Application of 10 mM caffeine also released Ca(2+) from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 microM thapsigargin revealed the presence of a Ca(2+) store depletion-activated Ca(2+) influx. The influx of Ca(2+) through the store-operated channel was reversibly blocked by 10 mM Cd(2+). Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca(2+) release from intracellular stores. Copyright Copyright 1999 S. Karger AG, Basel