Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Calcium signal induced by mechanical perturbation of osteoclasts

Multinucleated osteoclasts from rabbit long bone, 1–6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca²⁺]_i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca²⁺]_i responses decreases with decreasing bathing [Ca²⁺]. Following mechanical perturbation, a thapsigargin-induced [Ca²⁺]_i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca²⁺]_i is larger and longer than in control cells. Ni²⁺ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd³⁺ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca²⁺]_i signal via both a calcium influx and an internal calcium release. © 1995 Wiley-Liss, Inc.     

Signal percolation through plants and the shape of the calcium signature

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

Calcium signalling in glial cells

Various electrical, mechanical, and chemical stimuli, including the influences of neurotrasmitters, neuromodulators, and hormones, trigger complex changes in [Ca²⁺] _i in all types of glial cells. Glial [Ca²⁺] _i responses are controlled by coordinated activity of several molecular cascades. The initiation of [Ca²⁺] _i signal in glial cells results from activation of either plasmalemmal, or intracellular Ca²⁺ permeable channels. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide the means for intracellular and intercellular information coding. Furthermore, glial [Ca²⁺] _i signals can travel with no decrement within glial networks. These intercellular Ca²⁺ waves can be regarded as a substrate for information exchange between the glial cells. Neuronal activity can trigger [Ca²⁺] _i signals in neighboring glial cells and, moreover, there is some evidence that glial [Ca²⁺] _i waves can activate neuronal electrical and/or [Ca²⁺] _i , responses. Glial Ca²⁺ signalling can be regarded as a form of glial excitability.     

Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca²⁺]_cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca²⁺]_cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca²⁺]_cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Mitochondrial Calcium Signaling Driven by the IP3 Receptor

Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca²⁺]([Ca²⁺]_c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP₃). Imaging studiesof single cells have demonstrated that [Ca²⁺]_c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP₃ receptor Ca²⁺ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP₃ receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca²⁺-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca²⁺ transport appears to modulate the spatiotemporal organizationof [Ca²⁺]_c responses evoked by IP₃ and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP₃-dependent mitochondrial calcium signaling.     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

Background

Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca²⁺ concentration ([Ca²⁺]i) elevation induced by infection can cause cell death, but [Ca²⁺]i changes and high [Ca²⁺]i-induced death of macrophages due to infection of Leptospira have not been previously reported.

Methodology/Principal Findings

We first used a Ca²⁺-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca²⁺]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca²⁺]i elevation was caused by both extracellular Ca²⁺ influx through the purinergic receptor, P₂X₇, and Ca²⁺ release from the endoplasmic reticulum, as seen by suppression of [Ca²⁺]i elevation when receptor-gated calcium channels were blocked or P₂X₇ was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP₂) into inositol-1,4,5-trisphosphate (IP₃), which in turn induces intracellular Ca²⁺ release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S^-1. Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca²⁺]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca²⁺]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca²⁺]i increases induced apoptosis and necrosis of macrophages, while mild [Ca²⁺]i elevation only caused apoptosis.

Conclusions/Significance

This study demonstrated that L. interrogans infection induced [Ca²⁺]i elevation through extracellular Ca²⁺ influx and intracellular Ca²⁺ release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca²⁺]i elevation.     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.     

Okadaic acid induces the release of Ca from intracellular stores in ECV304 endothelial cells

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca²⁺ ([Ca²⁺]_c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca²⁺]_c both in the presence and absence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ stores with either ATP (1 μM) or thapsigargin (100 nM) prevented any further release of Ca²⁺ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca²⁺), a prior release of Ca²⁺ induced by okadaic acid reduced the magnitude of the response to ATP (1 μM). Taken together these observations indicate that okadaic acid induces Ca²⁺ release from the agonist-sensitive pool. The okadaic acid-induced Ca²⁺ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca²⁺]_c oscillations, which at their peak resulted in 40–100% cells, at any one time, having an elevated [Ca²⁺]_c. The response appeared to propagate between adjacent cells and the elevation of [Ca²⁺]_c. appeared initially in the cell periphery. In adherent cells, the release of Ca²⁺ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca²⁺ from the agonist- and thapsigargin-sensitive intracellular stores.     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

Calcium ions as second messengers in guard cell signal transduction

Ca²⁺ is a ubiquitous second messenger in plant cell signalling. In this review we consider the role of Ca²⁺-based signal transduction in stomatal guard cells focusing on three important areas: (1) the regulation of guard cell turgor relations and the control of gene expression in guard cells, (2) the control of specificity in Ca²⁺ signalling, (3) emerging technologies and new approaches for studying intracellular signalling. Stomatal apertures alter in response to a wide array of environmental stimuli as a result of changes in guard cell turgor. For example, the plant hormone abscisic acid (ABA) stimulates a reduction in stomatal aperture through a decrease in guard cell turgor. Furthermore, guard cells have been shown to be competent to relay an ABA signal from its site of perception to the nucleus. An increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺]₁) is central to the mechanisms underlying ABA-induced changes in guard cell turgor. We describe a possible model of Ca²⁺-based ABA signal transduction during stomatal closure and discuss recent evidence which suggests that Ca²⁺ is also involved in ABA nuclear signal transduction. Many other environmental stimuli which affect stomatal apertures, in addition to ABA, induce an increase in guard cell [Ca²⁺]₁) This raises questions regarding how increases in [Ca²⁺]₁) can be a common component in the signal transduction pathways by which stimuli cause both stomatal opening and closure. We discuss several mechanisms of increasing the amount of information contained within the Ca²⁺ signal, including encoding information in a stimulus-specific Ca²⁺ signal or Ca²⁺ signature', the concept of the ‘physiological address’ of the cell, and the use of other second messengers. We conclude by addressing the emerging technologies and new approaches which can be used in conjunction with guard cells to dissect further the molecular mechanisms of Ca²⁺-mediated signalling in plants.     

Calcium channels and signal transduction in plant cells

An increasing number of studies indicate that changes in cytosolic free Ca²⁺ ([Ca²⁺]_c) mediate specific types of signal transduction in plant cells. Modulation of [Ca²⁺]_c is likely to be achieved through changes in the activity of Ca²⁺ channels, which catalyse passive influx of Ca²⁺ to the cytosol from extracellular and intracellular compartments. Voltage-sensitive Ca²⁺ channels have been detected in the plasma membranes of algae, where they control membrane electrical properties and cell turgor. These channels are sensitive to 1,4-dihydropyridines, which in animal cells specifically affect one class of voltage-regulated plasma membrane Ca²⁺ channel. Ca²⁺-permeable channels with different pharmacological properties have been found in the plasma membrane of higher plants. Recent evidence suggests the existence of two discrete classes of Ca²⁺ channel co-resident in the vacuolar membrane (tonoplast) of higher plants. The first is gated by inositol 1,4,5-trisphosphate, and bears a number of similarities to its animal counterpart which is located in the endoplasmic reticulum (ER). The second tonoplast Ca²⁺ channel is voltage-operated. However, the specific roles of these tonoplast channels in signal transduction have yet to be elucidated.     

Voltage-Induced Ca2+ Release in Postganglionic Sympathetic Neurons in Adult Mice

Recent studies have provided evidence that depolarization in the absence of extracellular Ca²⁺ can trigger Ca²⁺ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca²⁺ from intracellular stores in response to depolarization, independent of Ca²⁺ influx. We measured changes in cytosolic ΔF/F₀ in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K⁺ depolarization in the presence (2 mM) and absence of extracellular Ca²⁺ ([Ca²⁺]_e). Progressive elevations in extracellular [K⁺]_e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca²⁺]_e. Peak amplitude of ΔF/F₀ transients in 2 mM [Ca²⁺]_e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F₀ in 0 mM [Ca²⁺]_e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca²⁺]_e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F₀ transients in 0 mM [Ca²⁺]_e were slower than those of ΔF/F₀ transients evoked in 2 mM [Ca²⁺]_e. Rises in ΔF/F₀ evoked by high [K⁺]_e in the absence of extracellular Ca²⁺ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ ATPase, or the inositol 1,4,5-triphosphate (IP₃) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd²⁺, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca²⁺ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca²⁺ from IP₃-sensitive internal stores in response to membrane depolarization, independent of Ca²⁺ influx.     

Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L.

 The inward K⁺ channels (I_Kin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I_Kin inhibition requires an elevation in cytosolic free Ca²⁺ levels ([Ca²⁺]_c) because: (i) experimental increases in [Ca²⁺] _c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca²⁺]_c in guard cells. However, not all guard cells respond to ABA with a [Ca²⁺]_c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca²⁺ in the regulation of downstream effectors of ABA response, such as the I_Kin channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca²⁺ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca²⁺]_c and I_Kin inhibition. In the presence of extracellular Ca²⁺, the extent of [Ca²⁺]_c elevation correlated with the extent of I_Kin inhibition. However, upon chelation of either extracellular Ca²⁺, [Ca²⁺]_c, or both, extracellular Ca²⁺ and [Ca²⁺]_c, [Ca²⁺]_c elevation did not occur in response to ABA yet I_Kin currents were still strongly inhibited. These data illustrate that Ca²⁺-independent regulation is involved in ABA-inhibition of stomatal opening processes. Received: 17 September 1999 / Accepted: 26 October 1999     

Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes

Abstract: Rilmenidine, a ligand for imidazoline and α₂-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca²⁺]_i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca²⁺]_i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca²⁺-free medium, indicating that rilmenidine evokes release of Ca²⁺ from intracellular stores. However, the sustained elevation of Ca²⁺ was completely dependent on extracellular Ca²⁺, consistent with rilmenidine activating Ca²⁺ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca²⁺ influx pathway. The α₂-adrenergic antagonist rauwolscine attenuated the increase in [Ca²⁺]_i induced by clonidine (a selective α₂ agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca²⁺ release from intracellular stores and Ca²⁺ influx by a mechanism independent of α₂-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca²⁺ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims

The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.

Methods

Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.

Results

Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, respectively, but not affected by Cav channels antagonists.

Conclusions

Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.     

Extracellular Nucleotides Elicit Cytosolic Free Calcium Oscillations in Arabidopsis

Extracellular ATP induces a rise in the level of cytosolic free calcium ([Ca²⁺]_cyt) in plant cells. To expand our knowledge about the function of extracellular nucleotides in plants, the effects of several nucleotide analogs and pharmacological agents on [Ca²⁺]_cyt changes were studied using transgenic Arabidopsis (Arabidopsis thaliana) expressing aequorin or the fluorescence resonance energy transfer-based Ca²⁺ sensor Yellow Cameleon 3.6. Exogenously applied CTP caused elevations in [Ca²⁺]_cyt that displayed distinct time- and dose-dependent kinetics compared with the purine nucleotides ATP and GTP. The inhibitory effects of antagonists of mammalian P2 receptors and calcium influx inhibitors on nucleotide-induced [Ca²⁺]_cyt elevations were distinct between CTP and purine nucleotides. These results suggest that distinct recognition systems may exist for the respective types of nucleotides. Interestingly, a mutant lacking the heterotrimeric G protein Gβ-subunit exhibited a remarkably higher [Ca²⁺]_cyt elevation in response to all tested nucleotides in comparison with the wild type. These data suggest a role for Gβ in negatively regulating extracellular nucleotide signaling and point to an important role for heterotrimeric G proteins in modulating the cellular effects of extracellular nucleotides. The addition of extracellular nucleotides induced multiple temporal [Ca²⁺]_cyt oscillations, which could be localized to specific root cells. The oscillations were attenuated by a vesicle-trafficking inhibitor, indicating that the oscillations likely require ATP release via exocytotic secretion. The results reveal new molecular details concerning extracellular nucleotide signaling in plants and the importance of fine control of extracellular nucleotide levels to mediate specific plant cell responses.The calcium ion, Ca²⁺, is a ubiquitous second messenger that is used to regulate a wide range of cellular processes (Clapham, 2007). A number of plant environmental and developmental responses are encoded to distinct Ca²⁺ signal patterns with specific frequencies and amplitudes of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). These signal patterns can take the form of pulsating [Ca²⁺]_cyt spiking/oscillations (Berridge et al., 2003). In plants, such [Ca²⁺]_cyt oscillations occur in various cell types (e.g. stomatal guard cells, pollen tubes, and legume root hairs) and play a critical role in responding to environmental signals (Evans et al., 2001; Oldroyd and Downie, 2008; McAinsh and Pittman, 2009).ATP is a ubiquitous compound in all living cells; it not only provides the energy to drive many biochemical reactions but also functions in signal transduction as a substrate for kinases, adenylate cyclases, etc. However, ATP was also shown to be an essential signaling agent outside of cells in animals, where it is referred to as extracellular ATP. Extracellular ATP is involved in numerous cellular processes, including neurotransmission, immune responses, cell growth, and cell death (Khakh and Burnstock, 2009). In mammalian cells, plasma membrane-localized receptors, purinoceptors of the P2X and P2Y classes, bind ATP as well as other nucleotides at the cell surface to activate intracellular signaling cascades via second messengers. Binding of extracellular ATP to P2X receptors gates calcium influx, whereas activation of P2Y receptors stimulates the recruitment of heterotrimeric G proteins to trigger cytoplasmic signaling and gene expression. As a common phenomenon, the activated receptors induce the elevation of [Ca²⁺]_cyt, which in turn activates the production of downstream messengers such as nitric oxide and reactive oxygen species (ROS; Shen et al., 2005; Fields and Burnstock, 2006).A possible physiological role for extracellular ATP in plants was first reported in studies in which exogenously applied ATP was found to stimulate closure of the Venus flytrap (Dionaea muscipula; Jaffe, 1973), to induce the formation of nucleases in excised Avena leaves (Udvardy and Farkas, 1973), and to induce potassium ion uptake into cells of maize (Zea mays) leaf slices (Lüttge et al., 1974). Over the past several years, extracellular ATP was found to be an important signaling compound in plants that induces various plant responses, including root-hair growth (Lew and Dearnaley, 2000; Kim et al., 2006), stress responses (Thomas et al., 2000; Jeter et al., 2004; Song et al., 2006), gravitropism (Tang et al., 2003), cell viability (Chivasa et al., 2005), pathogen responses (Chivasa et al., 2009), and thigmotropism (Weerasinghe et al., 2009). The release of extracellular ATP from root cells was directly imaged by Kim et al. (2006) using a luciferase construct engineered to bind to plant cell wall cellulose. Recently, using this reporter, Weerasinghe et al. (2009) measured the release of ATP from root cells in response to touch. This documentation of the presence of extracellular ATP in plants at levels sufficient to induce cellular responses suggests that extracellular ATP likely plays an important role throughout plant growth and development. However, no P2 receptor homologs have been identified in plants, despite the fact that plants share a number of cellular responses to ATP with animal cells. For example, the addition of exogenous ATP or ADP triggers an increase in [Ca²⁺]_cyt levels in whole seedlings, dissected root tissues, and root epidermal protoplasts of Arabidopsis (Arabidopsis thaliana; Demidchik et al., 2003, 2009; Jeter et al., 2004). The production of ROS in response to ATP addition was detected in various plant tissues (Kim et al., 2006; Song et al., 2006; Wu et al., 2008; Demidchik et al., 2009). More recently, the plasma membrane NADPH oxidase RBOHC (for respiratory burst oxidase homolog C) was shown to be required for extracellular ATP-induced ROS production in Arabidopsis primary roots (Demidchik et al., 2009). Extracellular ATP also stimulates the production of nitric oxide in tomato (Solanum lycopersicum) culture cells and in Salvia miltiorrhiza hairy roots (Foresi et al., 2007; Wu and Wu, 2008). These reports suggest that extracellular ATP signals across the plasma membrane by triggering elevation in [Ca²⁺]_cyt, which activates the production of downstream messengers. Ultimately, these cell responses induce the expression of various genes, such as MAPKs, LOX, and ACS6 (Jeter et al., 2004; Song et al., 2006), and cause physiological responses, as described above.In animal cells, extracellular ATP-evoked elevations in [Ca²⁺]_cyt are often observed in the form of oscillations that result from the transient opening of Ca²⁺ channels located either in the plasma membrane or in cytosolic Ca²⁺ stores. Intracellular calcium release is often mediated through phospholipase C (PLC)-mediated signaling coupled to heterotrimeric G proteins (Mahoney et al., 1992; Visegrady et al., 2000; Hanley et al., 2004). In plants, plasma membrane Ca²⁺-permeable channels are known to contribute to extracellular ATP-induced [Ca²⁺]_cyt elevation (Demidchik et al., 2009). However, neither the mechanisms underlying extracellular ATP-evoked Ca²⁺ signaling nor the possible involvement of heterotrimeric G proteins has been characterized in plants.In order to explore their roles as possible ligands of putative nucleotide receptors, the plant [Ca²⁺]_cyt response to six different nucleotides (Fig. 1A) was measured using Arabidopsis seedlings expressing one of two [Ca²⁺]_cyt sensors, either aequorin or the fluorescence resonance energy transfer (FRET)-based Ca²⁺ sensor Yellow Cameleon 3.6 (YC3.6). The pyrimidine nucleotide CTP as well as the purine nucleotides ATP and GTP induced a strong elevation of [Ca²⁺]_cyt in seedlings. Interestingly, the effects of all the nucleotides on Ca²⁺ signaling were negatively regulated by a heterotrimeric G protein β-subunit, AGB1. The addition of ATP to aequorin-expressing seedlings induced distinct [Ca²⁺]_cyt oscillations in the presence of the apyrase inhibitor NGXT191. However, in the absence of this inhibitor, such [Ca²⁺]_cyt oscillations could be localized to specific root cell layers using YC3.6 fluorescence. Given the importance of [Ca²⁺]_cyt oscillations in intracellular signaling, the data suggest an important, unexplored role of extracellular ATP in the plant signaling pathways.Open in a separate windowFigure 1.NTPs increase bioluminescence in aequorin-expressing transgenic Arabidopsis seedlings. A, Chemical structures of purine and pyrimidine derivatives. B, Individual 5-d-old aequorin seedlings were transferred to individual wells of a 96-well microplate and incubated overnight in reconstitution buffer containing coelenterazine. Each NTP was then applied at a final concentration of 100 μm. The line graph shows time-dependent changes in photon counts from representative wells of each treatment (bin size = 50 frames, 1 s, 20 bin smoothing). The inset shows a pseudocolored photon-counting image integrated over 400 s after nucleotide treatment calibrated to the inset scale.