The influence of calcium on the deformability of human granulocytes

Experiments were carried out to determine the importance of extra- and intracellular calcium for the deformability of granulocytes during filtration tests. At low calcium concentration (0.1 mM), granulocytes are more deformable than at the physiological free-calcium concentration of 1.25 mM. Increasing calcium concentrations up to 10 mM do not further impair the deformability. Parallel measurements of the intracellular calcium concentration by means of the fura fluorescence method were performed to explain this. Extracellular calcium concentrations between 1.25 mM and 10 mM had no influence on the intracellular calcium level. A lower extracellular calcium concentration (0.1 mM), however, decreased the intracellular calcium level. Therefore, the measurements of the intracellular calcium concentrations are consistent with the deformability results. Studies with the calcium entry blocker nifedipine suggested that a low intracellular calcium improves the deformability of granulocytes. It is concluded; (i) the physiological calcium concentration of 1.25 mM is stressful for isolated granulocytes, and (ii) the intracellular calcium level plays a crucial role in granulocyte deformability, i.e. the lower the intracellular calcium concentration the greater the deformability.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.     

The nonstructural glycoprotein of rotavirus affects intracellular calcium levels.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.     

Cytosolic calcium oscillators

Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.     

Mechanism of intracellular calcium transients.

Calcium ions mediate extracellular signals on intracellular processes. The signalling system based on transient rises or oscillations of the cytoplasmic calcium concentration has potential advantages. The relevant mechanisms of intracellular concentration changes include calcium-induced calcium release and calcium dependent inactivation of calcium release. A model has been devised based on these processes to generate repetitive transients of the cytoplasmic calcium concentration.     

Pharmacology of capacitative calcium entry

The versatility of Ca(2+) as a messenger in multiple signaling events requires that the concentration of calcium ions within the cytoplasm be highly regulated. In particular, the release of calcium from intracellular stores must often be linked to calcium influx across the cell membrane. Capacitative calcium entry, whereby the depletion of intracellular Ca(2+) stores induces the influx of extracellular calcium, is a crucial element of concerted calcium signaling. Investigations into the phenomenon are contributing to a new appreciation for the organized cytoplasmic framework that supports calcium signaling.     

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.     

Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.     

Membrane depolarization inhibits thrombin-induced calcium influx and aggregation in human platelets.

The relationship between thrombin-evoked changes in intracellular calcium concentration [( Ca2+]i) and aggregation was examined in Indo-1-loaded human platelets. The stimulus-induced intracellular calcium release and external calcium influx, as well as platelet aggregation, were studied in the same cell preparation. A close correlation between the sustained high [Ca2+]i level, depending on calcium entry, and the aggregation response was found. Gramicidin, at a concentration high enough to induce membrane depolarization, strongly inhibited the calcium influx and aggregation, but did not influence the thrombin-induced intracellular calcium release. We conclude that calcium influx through depolarization-inhibited calcium channels is a prerequisite of thrombin-induced platelet aggregation.     

Revisiting intracellular calcium signaling semantics

Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.     

Monitoring cytosolic calcium in the dinoflagellate Crypthecodinium cohnii with calcium orange-AM

Calcium plays several important roles in the signal transduction pathways of dinoflagellates. We describe here the development of calcium orange-AM as an intracellular calcium reporter for the heterotrophic dinoflagellate Crypthecodinium cohnii. We demonstrated with confocal microscopy that by restricting the incubation period to 30-45 min, no compartmentalization of the dye occurs in the mitochondria or endoplasmic reticulum. The dye fluorescence responded well to the effects of calcium ionophores and calcium chelators. By calibrating the dye with known calcium concentrations, we determined the intracellular calcium concentration of C. cohnii to be 158 +/- 56 nM, which rose to about 550 nM upon mechanical stimulation.     

Effects of nanomolar concentration dihydroouabain on calcium current and intracellular calcium in guinea pig ventricular myocytes

The effects of nanomolar concentration of dihydroouabain (DHO) on L-type calcium current (ICa-L), TTX-sensitive calcium current (ICa(TTX)), and intracellular calcium concentration ([Ca2+]i) were investigated in guinea pig ventricular myocytes. The whole-cell patch-clamp technique was used to record ICa-L and ICa(TTX); [Ca2+]i was detected and recorded with the confocal microscopy. The nanomolar concentration of DHO increased the ICa-L, ICa(TTX), and [Ca2+]i, which could be partially inhibited by nisoldipine or TTX, but still appeared in the absence of extracellular K+ and Na+. These data suggest that DHO could increase [Ca2+]i in non-beating myocytes via stimulating the ICa-L and ICa(TTX), or perhaps triggering directly a release of intracellular calcium.     

Arachidonic acid induces mobilization of calcium stores and c-jun gene expression: evidence that intracellular calcium release is associated with c-jun activation.

Arachidonic acid (AA) plays a signaling role in the induction of several genes. We previously demonstrated that AA induces c-jun gene expression in the stromal cell line +/+.1 LDA 11 by a signaling pathway involving activation of the c-jun amino-terminal kinase (JNK). This study investigated the role of calcium in AA signaling of c-jun activation in +/+.1 LDA 11 cells. AA (10-50 microM) caused a rapid dose-dependent rise in cytosolic calcium. AA-induced calcium mobilization involved both influx of extracellular calcium and the release of intracellular calcium. The importance of calcium was investigated by variation of the extracellular calcium concentration, chelation of intracellular calcium and by calcium ionophore-induced influx of extracellular calcium. AA-induced c-jun gene expression and increased luciferase activity of a construct containing the high affinity AP-1 binding site was decreased in cells preincubated with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)-eThane-N,N,N',N',-tetraacetic acid tetra(aceToxymethyl-esTer) (BAPTA-AM, 10 microM) prior to stimulation with AA. Similarly, chelation of intracellular calcium decreased AA-induced JNK activation. On the contrary, changes in the extracellular calcium concentration had no effect. Also, ionophore A23187 failed to induce c-jun and JNK activation either alone than in combination with AA. These results suggested that calcium was required for AA-dependent activation of c-jun, but that calcium alone was insufficient to induce activation of c-jun. Thus, release of calcium from intracellular stores is implicated in the signaling pathway of AA-induced c-jun activation in stromal cells.     

Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism

Current knowledge suggests that normal parathyroid glands and parathyroid adenomas have different sensitivities to environmental calcium. In search for morphological equivalents, 5 normal human and 10 porcine parathyroid glands, as well as 10 human parathyroid adenomas were investigated with regard to intracellular and extracellular calcium distribution. The glands were incubated for 2, 4, 6 and 20 h in tissue cultures using HAM's F10 medium with various calcium concentrations. For visualization of the calcium distribution in the tissue the method of pyroantimonate precipitation was applied. Specificity of the reaction was controlled by X-ray microanalysis. Shifts of the calcium pyroantimonate precipitates were quantitated by morphometry using an area-counting system. The results demonstrate that in normal parathyroid glands calcium precipitates are distributed randomly. Incubation of normal glands in medium with low calcium concentration (0.6 mM) provoked reduced amounts of intracellular and extracellular calcium complexes. When the incubations were performed in medium with high calcium content (2.6 mM), calcium accumulated inside parathyroid and stroma cells. In contrast to normal parathyroid glands, parathyroid adenomas fixed immediately after surgery showed an atypical calcium distribution with low amounts of intracellular and high amounts of extracellular calcium grains. The data suggest that in normal parathyroid glands the intracellular calcium concentration follows the extracellular environmental calcium concentration. Thus, calcium modulates parathyroid hormone (PTH) secretion via intracellular regulatory mechanisms. In parathyroid adenomas the calcium transport via the tumor cell membrane appears to be disturbed resulting in lowered intracellular calcium levels. This is remarkable since the environmental calcium concentration is elevated due to the hypercalcemia of primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of external calcium and calcium sensors on ginsenoside Rb1 biosynthesis by Panax notoginseng cells

The effects of external calcium concentrations on biosynthesis of ginsenoside Rb1 and several calcium signal sensors were quantitatively investigated in suspension cultures of Panax notoginseng cells. It was observed that the synthesis of intracellular ginsenoside Rb1 in 3-day incubation was dependent on the medium Ca2+ concentration (0-13 mM). At an optimal Ca2+ concentration of 8 mM, a maximal ginsenoside Rb1 content of 1.88 +/- 0.03 mg g(-1) dry weight was reached, which was about 60% and 25% higher than that at Ca2+ concentrations of 0 and 3 mM, respectively. Ca2+ feeding experiments confirmed the Ca2+ concentration-dependent Rb1 biosynthesis. In order to understand the mechanism of the signal transduction from external Ca2+ to ginsenoside biosynthesis, the intracellular content of calcium and calmodulin (CaM), activities of calcium/calmodulin-dependent NAD kinase (CCDNK) and calcium-dependent protein kinase (CDPK), and activity of a new biosynthetic enzyme of ginsenoside Rb1, i.e., UDPG:ginsenoside Rd glucosyltransferase (UGRdGT), in the cultured cells were all analyzed. The intracellular calcium content and CCDNK activity were increased with an increase of external Ca2+ concentration within 0-13 mM. In contrast, the CaM content and activities of CDPK and UGRdGT reached their highest levels at 8 mM of initial Ca2+ concentration, which was also optimal to the ginsenoside Rb1 synthesis. A similar Ca2+ concentration-dependency of the intracellular contents of calcium and CaM and activities of CCDNK, CDPK, and UGRdGT was confirmed in Ca2+ feeding experiments. Finally, a possible model on the effect of external calcium on ginsenoside Rb1 biosynthesis via the signal transduction pathway of CaM, CDPK, and UGRdGT is proposed. Regulation of external Ca2+ concentration is considered a useful strategy for manipulating ginsenoside Rb1 biosynthesis by P. notoginseng cells.     

Cell calcium of ejaculated rabbit spermatozoa before and following in vitro capacitation

Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2. Measurements of trypan blue exclusion indicated that cell viability was not affected by quin-2 loading. The concentration of intracellular free calcium of quin-2 loaded sperm was calculated to be 144 +/- 14 nM. Spermatozoa capacitated in vitro either before or following quin-2 loading had intracellular free calcium levels similar to that of non-capacitated sperm. These studies indicate that the concentration of intracellular free calcium of ejaculated rabbit spermatozoa does not change as a result of in vitro capacitation.     

Role of intracellular calcium ions in the physiopathology of fibromyalgia syndrome.

Calcium ions have a key role in the physiology of muscular contraction: changes in calcium ion concentration may be involved in the pathogenesis of fibromyalgia. Although, since the plasmatic level of calcium in fibromyalgia patients is always in the normal range, it seemed interesting to evaluate the intracellular calcium concentration. The study was carried out on two groups of subjects: 70 affected by fibromyalgia and 40 healthy controls. The results obtained show that in fibromyalgia patients the intracellular calcium concentration is significantly reduced in comparison to that of healthy controls: the reduced intracellular calcium concentration seems to be a peculiar characteristic of fibromyalgia patients and may be potentially responsible for muscular hypertonus. The effective role of this anomaly in the physiopathology of fibromyalgia and the potential role of drugs active on the calcium homeostasis are still to be confirmed.     

Mannitol enhances intracellular calcium diffusion in the rat ileum--a hypothesis

The addition of 92 or 136 mM mannitol to a modified saline solution that contained 1.25 mM Ca2+ led to a mannitol concentration-dependent increase in the amount of calcium absorbed in 1 h from 8 cm long ileal loops prepared from fasted male Sprague-Dawley rats, with body weights of 190 +/- 10 g. It is argued that this mannitol-enhanced movement of calcium out of the loop cannot have utilized the paracellular pathway, inasmuch as the luminal calcium concentration of the mannitol instillate decreased during the experiment, with a negative calcium gradient between luminal and body fluids. Instead it is proposed that uncomplexed mannitol and the uncharged calcium complex of mannitol entered the ileal cells. The uncomplexed intracellular mannitol would bind additional calcium that had crossed the brush border down its gradient. The increase in total intracellular calcium will raise the effective intracellular gradient and thereby amplify intracellular calcium diffusion. This in turn increases calcium absorption.     

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.