Single pulses of cytoplasmic calcium associated with phagocytosis of individual zymosan particles by macrophages

We have measured cytosolic free calcium levels ([Ca++]i) in individual macrophages during the phagocytosis of single zymosan particles. We report here that the contact of a macrophage with zymosan results in a rapid transient elevation of [Ca++]i. Each [Ca++]i pulse is symmetrical lasting for up to 30 seconds. In contrast, macrophage spreading is associated with repetitive [Ca++]i spiking occurring in salvos of up to four smaller spikes, each lasting for between 8 and 18 seconds. These qualitative and kinetic differences might suggest that the role of [Ca++]i in phagocytosis is distinct from its role in spreading.     

A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 fibroblasts. Estimated [Ca++]i of serum-deprived, subconfluent fibroblasts was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells growing in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal growth factor (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit growth factor-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent fibroblasts, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate growth factor-induced increases in [Ca++]i.     

Arachidonic acid induces an increase in the cytosolic calcium concentration in single pancreatic islet beta cells.

The insulin secretagogue D-glucose induces both accumulation of nonesterified arachidonic acid (35 microM) in pancreatic islets and a rise in beta cell cytosolic [Ca++]i. Arachidonate amplifies both voltage-dependent Ca++ entry in secretory cells and depolarization-induced insulin secretion. Here, arachidonate induced a biphasic rise in [Ca++]i of Fura-2AM loaded beta cells which increased with arachidonate concentration (5-30 microM), was reversed upon washout, and was unaffected by the arachidonate oxygenase inhibitor BW755C. The sustained phase of the rise was abolished by removal of extracellular Ca++ and amplified by depolarization with KCl. The accumulation of nonesterified arachidonate in islets stimulated by D-glucose may therefore promote the D-glucose-induced rise in beta cell [Ca++]i.     

Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells

Effects on Ca++ translocation and [Ca++]i were studied in dog thyro?d cell monolayers using both 45Ca++ efflux and the indicator quin-2. Carbamylcholine, a non hydrolysable analog of acetylcholine, through muscarinic receptors, and to a lesser extent TRH and PGF2 alpha increased both these parameters. [Ca++]i increased by 171, 100 and 75% respectively over a basal level of 66 +/- 17 nM (mean +/- SD). The response to carbamylcholine was biphasic. A transient increase in [Ca++]i was followed by a more sustained phase where the [Ca++]i was slightly higher than the basal level. Only the first phase was insensitive to extracellular Ca++ depletion. This phase is probably due to a release of Ca++ from an intracellular store. NaF also induced a sustained rise in [Ca++]i dependent on extracellular Ca++ and affected 45Ca++ efflux. Our data provide direct evidence of an implication of intracellular Ca++ in the response of dog thyro?d cells to all these agents.     

Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels

Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i.     

A comparative study of three methods for intracellular loading of the calcium indicator aequorin in ferret papillary muscles

We compared the results of loading the bioluminescent Ca++ indicator aequorin by standard microinjection techniques to those obtained with two new chemical approaches to loading that utilize low concentrations of Ca++ chelator; i.e., 1) Immersion and 2) Macroinjection. After loading with the immersion and macroinjection methods, twitch tension returned to pre-load values indicating lack of damage to the muscles. The aequorin signals obtained with all three methods were similar and converted to similar quantitative values for [Ca++]i. Our data suggest that chemical loading (in particular macroinjection) may be preferable to microinjection, particularly in muscles with increased connective tissue content.     

Leukotriene B4 induced steady state calcium rise and superoxide anion generation in guinea pig eosinophils are not related events.

We have examined the nature of the leukotriene B4 (LTB4) induced steady state intracellular calcium rise [Ca++]i in guinea pig eosinophils and the relationship between LTB4 induced [Ca++]i and superoxide anion (O2-). LTB4 induced a rise in intracellular Ca++ (following a Ca(++)- transient) in a dose dependent manner with an optimal increase around 50-100 nM. Depolarizing concentrations of K+ did not induce [Ca++]i in eosinophils nor did the voltage operated calcium channel inhibitor, nifedipine, inhibit the LTB4 induced Ca++ entry. In contrast, SK&F 96365 a purported receptor operated calcium channel (ROCC) inhibitor, and its parent compound SC 32849, attenuated LTB4 induced [Ca++]i. Five reference anti-asthmatics (ketotifen, formoterol, disodium-cromoglycate, theophylline and budesonide) had no influence on LTB4 induced [Ca++]i. LTB4 also induced O2- generation (a functional response) in a dose dependent manner with optimal effect around 100 nM. However, in contrast to Ca(++)- influx, LTB4 induced O2- generation was not affected by either SK&F 96365 or its analogues or reference anti-asthmatics. The results of this study suggest a) the presence of a non-voltage gated, receptor operated, calcium sequestration process in guinea pig eosinophils, b) that LTB4 induced [Ca++]i and O2- generation are apparently unrelated events in these cells, and c) that standard anti-asthmatics do not have an influence on either LTB4 induced [Ca++]i or O2- generation in these cells.     

小鼠巨噬细胞内游离钙离子变化介导的吞噬作用

为评估小鼠巨噬细胞吞噬死亡细胞时胞质内游离钙离子的变化。实验使用F luo-3标记巨噬细胞内钙离子和碘化丙碇对死亡细胞核染色,观察吞噬过程中细胞内钙离子的变化和显示巨噬细胞的吞噬功能,检测含死亡细胞的巨噬细胞内荧光密度图像。利用激光扫描共聚焦显微镜检测钙离子的释放。在缺钙的溶液中,可见巨噬细胞接触死细胞时细胞内钙离子快速地聚集和增高。在吞噬体形成时,巨噬细胞内钙离子上升到较高的水平。快速上升后,当吞噬小泡消化时,细胞内游离钙下降,随后钙离子恢复到低水平。研究显示伴随着吞噬小泡中红色荧光的死细胞的出现和消失,巨噬细胞内出现一系列钙离子变化的图像。提示巨噬细胞内钙离子改变在细胞吞噬作用中具有一定的作用。     

Evaluation of cellular mechanisms for modulation of calcium transients using a mathematical model of fura-2 Ca2+ imaging in Aplysia sensory neurons.

A theoretical model of [Ca++]i diffusion, buffering, and extrusion was developed for Aplysia sensory neurons, and integrated with the measured optical transfer function of our fura-2 microscopic recording system, in order to fully simulate fura-2 video or photomultiplier tube measurements of [Ca++]i. This allowed an analysis of the spatial and temporal distortions introduced during each step of fura-2 measurements of [Ca++]i in cells. In addition, the model was used to evaluate the plausibility of several possible mechanisms for modulating [Ca++]i transients evoked by action potentials. The results of the model support prior experimental work (Blumenfeld, Spira, Kandel, and Siegelbaum, 1990. Neuron. 5: 487-499), suggesting that 5-HT and FMRFamide modulate action potential-induced [Ca++]i transients in Aplysia sensory neurons through changes in Ca++ influx, and not through changes in [Ca++]i homeostasis or release from internal stores.     

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]i is high, but there is a region of [Ca]i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]i change.     

Extracellular calcium ion depletion in frog cardiac ventricular muscle.

The extracellular free [Ca++] in frog ventricular muscle strips was monitored using single-barrel calcium ion-selective microelectrodes. During trains of repetitive stimulation, a heart rate-dependent, sustained fall (depletion) of the extracellular free [Ca++] occurs, which is most likely a consequence of net Ca++ influx into ventricular cells. The magnitude of the [Ca++]0 depletion increases for higher Ringer's solution [Ca++], and is reversibly blocked by manganese ion. Prolonged repetitive field stimulation (20-30 min) activates additional cellular Ca++ efflux, which can balance the additional Ca++ influx caused by stimulation, resulting in abolition of extratrabecular [Ca++]0 depletion in 20-30 min, and hence zero net transmembrane Ca++ flux at steady state. In the poststimulation period of quiescence, cellular Ca++ efflux persists and causes an elevation (accumulation) of the extracellular free [Ca++]. From these [Ca++]0 depletions, quantitative estimates for the net transmembrane Ca++ flux were derived using an analytical solution to the diffusion equation. In the highest Ringer's solution [Ca++] used (1 mM) the calculated net increase of the total intracellular calcium per beat was 6.5 +/- 1.4 mumol/l of intracellular space. This corresponds to an average net transmembrane Ca++ influx of 0.81 +/- 0.17 pmol/cm2/s during the 800-ms action potential. In lower bath [Ca++] the net transmembrane [Ca++] flux was proportionately reduced.     

BAY-K-8644, a calcium channel agonist, induces a rise in cytoplasmic free calcium and iodide discharge in thyroid cells

BAY-K-8644, a calcium channel agonist, induces a rise in cytoplasmic free calcium and iodide discharge in cultured porcine thyroid cells. The cytoplasmic free calcium concentration, [Ca2+]i, was measured using aequorin, a calcium-sensitive photoprotein. BAY-K-8644, a dihydropyridine derivative, acts as a Ca channel agonist and induces a rise in [Ca2+]i and iodide discharge; 0.5 nM BAY-K-8644 is a minimal dose to effect a rise in [Ca2+]i and iodide discharge and 50 nM BAY-K-8644 produces the maximal effect. The data indicate that BAY-K-8644-induced iodide discharge is mediated by a rise in [Ca2+]i.     

Biphasic increasing effect of angiotensin-II on intracellular free calcium in isolated rat early proximal tubule

In the freshly isolated early proximal tubule (S1), the effect of angiotensin II (ANG II) on cytosolic Ca++ concentration ([Ca++]i) was determined using the fluorescent indicator fura-2. In order to establish an adequate experimental system, we investigated firstly the relationship between cellular ATP and [Ca++]i under various conditions in late proximal tubule, the most fragile nephron segment, and cortical collecting tubule, a relatively stable one. We found out that cellular ATP depletion caused [Ca++]i to rise, and ANG II response to [Ca++]i under high ATP condition was higher than that under low ATP condition. ANG II-induced [Ca++]i rise in S1 was biphasic, demonstrating the two peaks corresponding to the 10(-11) and 10(-7) M ANG II. This study suggests for the first time 1) the necessity of high intracellular ATP to evaluate a high affinity ANG II actions and 2) the biphasic characteristics of [Ca++]i increase by ANG II in intact S1.     

Ca(2+)-independent F-actin assembly and disassembly during Fc receptor- mediated phagocytosis in mouse macrophages

Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F-actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F-actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i.     

The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium

A monoclonal antibody to the antigen-receptor on the T cell line Jurkat induces substantial increases in [Ca++]i. Ca++ ionophores can substitute for this antibody in activation by increasing [Ca++]i to levels comparable with those seen with the antigen-receptor antibody. Stimulation with either the antigen-receptor antibody or a Ca++ ionophore leads to the appearance of the same phosphoproteins. These results suggest that the antigen-receptor initiates T cell activation by increasing [Ca++]i.     

Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells

The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator "Quin 2." [Ca++]i rose from approximately 150 nM to greater than 2 microM over a time course that was strongly dependent on the concentration of H2O2 used (5 X 10(-5) to 5 X 10(-3) M). After exposure of P388D1 cells to 5 X 10(-3) M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points greater than 45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 X 10(-4) M H2O2 to 14.0 min at 5.0 X 10(-3) M. When the total [Ca++]i-buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68-fold (1.1 X 10(2) - 7.6 X 10(3) amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6-fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that "surface blebs" appeared during this phase of injury. Both the rise in [Ca++]i and "blebbing" were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; the rise in [Ca++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; the rate of rise of [Ca++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; during the early phase (less than 30 min) of rise of [Ca++]i, only intracellular calcium is involved in the response; these events occur concomitantly with gross morphological changes to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevated cytosolic calcium in cerebrocortical nerve terminals of rats during prolonged ethanol ingestion

Increases in cytosolic free calcium concentrations ([Ca++]i) may underlie acute neuronal degeneration during ischemic or anoxic episodes, seizures and excitotoxin treatment. With quin-2 and fura-2 fluorescent probes, we have obtained evidence for elevated [Ca++]i in cerebrocortical terminals of adult rats following chronic consumption of ethanol-containing liquid diets for "neurotoxic" durations. Compared to isocaloric carbohydrate-fed controls, ethanol-fed rats had significantly higher [Ca++]i in P2 synaptosomal fractions after 4 months of diet intake, and in purified cerebrocortical synaptosomes after diet ingestion for 10 months. In addition, [Ca++]i in the synaptosomal fractions of ethanol-fed rats from either exposure time were markedly resistant to K(+)-dependent potentiation. Persistently increased synaptic [Ca++]i and a blunted response to K+ depolarization following chronic ethanol ingestion lead us to associate impaired Ca++ homeostasis in the neurodegenerative processes of alcoholism.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Human recombinant interleukin 1 beta has no effect on intracellular calcium or on functional responses of human neutrophils

The effect of human recombinant interleukin 1 beta (rIL 1 beta) on human neutrophils was examined. rIL 1 beta, even at concentrations of 100 ng/ml (100 half-maximal T cell stimulating U/ml) did not change significantly the intracellular free calcium concentration, [Ca++]i, whereas the control stimulus, fmet-leu-phe, significantly elevated [Ca++]i. rIL 1 beta also failed to stimulate production of superoxide, degranulation of lysosomal enzymes, phagocytosis of bacterial particles, chemotaxis, or chemokinesis of human neutrophils. This is substantial evidence that superphysiologic concentrations of interleukin 1 have no direct effect on [Ca++]i, as well as on functional responses of neutrophils.     

Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line.

Digital imaging microscopy revealed that human platelets show periodic intracellular Ca++ elevation in response to 0.01 U/ml thrombin. MEG-01, a megakaryoblastic leukemia cell line, also responded with oscillatory intracellular Ca++ elevation (0.7-1 times/min) to thrombin (0.001-0.003U/ml). Ca++ transients appears to be fused with higher thrombin doses. With extracellular Ca++ concentrations of 0.1 mM or less, Ca++ oscillation could not be elicited, or even when present, it disappeared after a few spikings of [Ca++]i. Extracellular Ca++ concentrations of 0.3 mM or more were required to facilitate ongoing Ca++ oscillation, suggesting an important role of Ca++ influx for Ca++ oscillation.