Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.     

Remodelling of Ca2+ homeostasis in type I cortical astrocytes by hypoxia: evidence for association with Alzheimer's disease

Sustained central hypoxia predisposes individuals to dementias such as Alzheimer's disease, in which cells are destroyed in part by disruption of Ca2+ homeostasis. Here, we show that exposure of astrocytes to hypoxia in vitro causes inhibition of plasmalemmal Na+/Ca2+ exchange and excessive mitochondrial Ca2+ loading. Both factors disrupt normal agonist-evoked Ca2+ signalling. Moreover, hypoxia increases the levels of presenilin-1, a major component of a key enzyme involved in Alzheimer's disease. Inhibition of this enzyme partially reverses the effects of hypoxia on Ca2+ signalling. These findings provide an initial cellular basis for understanding the clinical association of hypoxia with Alzheimer's disease.     

Mitochondria as biosensors of calcium microdomains

The notion that the agonist-dependent increases in intracellular Ca2+ concentration, on ubiquitous signalling mechanism, occur with a tightly regulated spatio-temporal pattern has become an established concept in modern cell biology. As a consequence, the concept is emerging that the recruitment of specific intracellular targets and effector system mechanisms depends on exposure to local [Ca2+] that differs substantially from the mean [Ca2+]. A striking example is provided by mitochondria, intracellular organelles that have been overlooked for a long time in the field of calcium signalling due to the low affinity of their Ca(2+)-uptake pathways. We will summarize here some of the evidence indicating that these organelles actively participate in Ca2+ homeostasis in physiological conditions (with consequences not only for the control of their function, but also for the modulation of the complexity of calcium signals) because they have the capability to respond to microdomains of high [Ca2+] transiently generated in their proximity by the opening of Ca2+ channels.     

Noncoenzyme function of vitamin PP and its biologically active derivatives: the current state of the problem

This review is devoted to the current state of investigations of vitamin PP and nicotinamide dinucleotides noncoenzyme functions. Particular attention has been focused on the role of these compounds in post-translation modification of proteins (mono- and poly-ADP-ribosylation), in regulation of gene activity, calcium homeostasis and Ca2+ signalling as well as in modulation of synaptic transmission. Biological significance of these processes in cell function was elicited. The role of deregulation of vitamin PP mediated signalling mechanisms involved in control over the cell function under conditions of different diseases was emphasized.     

The role of sphingosine and its derivatives in the regulation of Ca2+ homeostasis

The role of sphingosine and sphingosine 1-phosphate in the regulation of Ca2+ homeostasis is reviewed. Available data suggest that there are at least three main pathways by which sphingosine and its metabolites affect Ca2+ fluxes in different cell types: (1) indirect action on Ca2+ stores, mediated by other transduction pathways; (2) direct action on the receptors of Ca2+ channels and Ca2+ pumps which are localized at the membranes of Ca2+ stores; (3) indirect action mediated by the regulation of expression of the channel-forming protein Bcl-2, which incorporates into membranes of Ca2+ stores. The effects of sphingosine and its metabolites on Ca2+ homeostasis via mechanisms (1)-(3) are considered. The combination of the mechanisms by which sphingosine affects Ca2+-signalling pathways is tissue-specific. Sphingosine and its metabolites can be physiological modulators of the intracellular Ca2+ signalling pathways.     

A plethora of interacting organellar Ca2+ stores

The endoplasmic reticulum is not the only major agonist-releasable Ca2+ store within cells; it is now clear that virtually all organelles so far studied have the ability to act as mobilizable Ca2+ stores. From recent findings with regard to Ca2+ transportation and Ca2+ homeostasis within a variety of cell organelles such as the mitochondria, nucleus, Golgi and lysosomes, it emerges that many of these organellar Ca2+ stores appear to interact with each other, adding a further level of complexity to Ca2+ signalling events.     

Calcium: Just Another Regulator in the Machinery of Life?

*BACKGROUND: Current hypotheses imply that stimulus-response systems in plants are networks of signal transduction pathways. It is usually assumed that these pathways connect receptors with effectors via chains of molecular events. Diverse intermediate signalling components (transducers) participate in these processes. However, many cellular transducers respond to several stimuli. Hence, there are no discrete chains but rather pathways that interconnect network-modules of different command structure. In particular, the cytosolic free Ca2+ concentration ([Ca2+](cyt)) is thought to perform many different tasks in a wide range of cellular events. However, this range of putative functions is so wide that it is often questioned how Ca2+ can comply with the definition of a second messenger. *THE Ca2+ SIGNATURE HYPOTHESIS: Some authors have suggested the concept of a specific signature of the ([Ca2+](cyt)) response. This implies that characteristics of the time course of changes in ([Ca2+](cyt)) and their localized sites of appearance in cells are used by the plant to identify the type and intensity of the stimulus. This hypothesis has triggered many investigations, which have yielded contradictory results. * THE CURRENT PICTURE: Much evidence suggests that the functions of calcium can be grouped into three classes: Ca2+ as a protective agent, Ca2+ as a chemical switch and Ca2+ as a 'digital' information carrier. Examples of the first two classes are presented here. The third is more controversial; while some investigations seem to support this idea, others call the Ca2+ signature hypothesis into question. Further investigations are needed to shed more light on Ca(2+)-driven signalling cascades.     

Cyclic compression of chondrocytes modulates a purinergic calcium signalling pathway in a strain rate- and frequency-dependent manner

Mechanical loading modulates cartilage homeostasis through the control of matrix synthesis and catabolism. However, the mechanotransduction pathways through which chondrocytes detect different loading conditions remain unclear. The present study investigated the influence of cyclic compression on intracellular Ca2+ signalling using the well-characterised chondrocyte-agarose model. Cells labelled with Fluo4 were visualised using confocal microscopy following a period of 10 cycles of compression between 0% and 10% strain. In unstrained agarose constructs, not subjected to cyclic compression, a subpopulation of approximately 45% of chondrocytes exhibited spontaneous global Ca2+ transients with mean transient rise and fall times of 19.4 and 29.4 sec, respectively. Cyclic compression modulated global Ca2+ signalling by increasing the percentage of cells exhibiting Ca2+ transients (population modulation) and/or reducing the rise and fall times of these transients (transient shape modulation). The frequency and strain rate of compression differentially modulated these Ca2+ signalling characteristics providing a potential mechanism through which chondrocytes may distinguish between different loading conditions. Treatment with apyrase, gadolinium and the P2 receptor blockers, suramin and basilen blue, significantly reduced the percentage of cells exhibiting Ca2+ transients following cyclic compression, such that the mechanically induced upregulation of Ca2+ signalling was completely abolished. Thus cyclic compression appears to activate a purinergic pathway involving the release of ATP followed by the activation of P2 receptors causing a combination of extracellular Ca2+ influx and intracellular Ca2+ release. Knowledge of this fundamental cartilage mechanotransduction pathway may lead to improved therapeutic strategies for the treatment of cartilage damage and disease.     

Synaptic plasticity and Ca2+ signalling in astrocytes

There is a growing body of evidence suggesting a functional relationship between Ca2+ signals generated in astroglia and the functioning of nearby excitatory synapses. Interference with endogenous Ca2+ homeostasis inside individual astrocytes has been shown to affect synaptic transmission and its use-dependent changes. However, establishing the causal link between source-specific, physiologically relevant intracellular Ca2+ signals, the astrocytic release machinery and the consequent effects on synaptic transmission has proved difficult. Improved methods of Ca2+ monitoring in situ will be essential for resolving the ambiguity in understanding the underlying Ca2+ signalling cascades.     

The latest waves in calcium signaling

Ca2+ is a universal second messenger that is a key component of myriad processes in all cell types. Perturbations in normal intracellular Ca2+ concentrations underlie many common pathological conditions, ranging from cardiac hypertrophy to ischemic death of neurons. A recent meeting addressed the contributions of Ca2+ and Ca2+ binding proteins to health and disease. Insights gleaned from mechanistic studies offered the potential for new therapeutic approaches to combat a variety of diseases resulting from alterations in Ca2+ homeostasis.     

Hypoxic remodelling of Ca2+ mobilization in type I cortical astrocytes: involvement of ROS and pro-amyloidogenic APP processing

Chronic hypoxia (CH) alters Ca2+ homeostasis in various cells and may contribute to disturbed Ca2+ homeostasis of Alzheimer's disease. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate the mechanism underlying augmentation of Ca2+ signalling by chronic hypoxia in type I cortical astrocytes. Application of bradykinin evoked significantly larger rises of [Ca2+]i in hypoxic cells as compared with control cells. This augmentation was prevented fully by either melatonin (150 micro m) or ascorbic acid (200 micro m), indicating the involvement of reactive oxygen species. Given the association between hypoxia and increased production of amyloid beta peptides (AbetaPs) of Alzheimer's disease, we performed immunofluorescence studies to show that hypoxia caused a marked and consistent increased staining for AbetaPs and presenilin-1 (PS-1). Western blot experiments also confirmed that hypoxia increased PS-1 protein levels. Hypoxic increases of AbetaP production was prevented with inhibitors of either gamma- or beta-secretase. These inhibitors also partially prevented the augmentation of Ca2+ signalling in astrocytes. Our results indicate that chronic hypoxia enhances agonist-evoked rises of [Ca2+]i in cortical astrocytes, and that this can be prevented by antioxidants and appears to be associated with increased AbetaP formation.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.     

Manipulation of Intracellular Calcium in NCB-20 Cells

A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB-20 cells. However, to date, the regulation of [Ca2+]i homeostasis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2-receptor, elevated [Ca2+]i by a pertussis-toxin-insensitive mechanism. The BK-stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pretreatment with thapsigargin and ionomycin--compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK-sensitive stores; the BK-sensitive Ca2+ pool was a subset of the thapsigargin-sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK-induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.     

Calcium homeostasis and signaling in the blood-stage malaria parasite

The nature of the mechanisms underlying Ca2+ homeostasis in malaria parasites has puzzled investigators for almost two decades. This review summarizes the current knowledge about Ca2+ homeostasis in Plasmodium spp and highlights some key aspects of this process that are specific to this parasite. Plasmodium spp are exposed, during their intracellular stage, not to the usual millimolar concentrations of Ca2+ found in body fluids, but rather to the very low Ca2+ environment of the host cell cytoplasm. Two crucial questions then arise: (1) how is Ca2+ homeostasis achieved by these protozoa; and (2) do they use Ca2+-based signaling pathways? By critically reviewing the recent literature in the field, Célia Garcia here provides at least some partial answers to these questions.     

Rapid spatiotemporal patterning of cytosolic Ca2+ underlies flagellar excision in Chlamydomonas reinhardtii.

Ca(2+)-dependent signalling processes are implicated in many aspects of flagella function in the green alga, Chlamydomonas. In this study, we examine the spatiotemporal dynamics of cytosolic Ca2+ ([Ca2+](cyt)) in single Chlamydomonas cells during the process of flagellar excision, using biolistically loaded calcium-responsive dyes. Acid-induced deflagellation occurred in parallel with a single transient elevation in whole-cell [Ca2+](cyt), which was absent in the acid deflagellation-deficient adf1 mutant. Deflagellation could also be induced by elevated external Ca2+ ([Ca2+](ext)), which promoted very rapid spiking of [Ca2+](cyt) across the whole cell and in the flagella. We also detected very rapid apically localised Ca2+ signalling events with an approximate duration of 500 msec. Ninety-seven per cent of deflagellation events coincided with a rapid elevation in [Ca2+](cyt) in the apical region of the cell, either in the form of a whole cell or an apically localised increase, indicating that [Ca2+](cyt) elevations in the apical region play an underlying role in deflagellation. Our data indicate that elevated [Ca2+](ext) acts to disrupt Ca2+ homeostasis which induces deflagellation by both Adf1-dependent and Adf1-independent mechanisms. Elevated [Ca2+](ext) also results in further [Ca2+](cyt) elevations after the main period of whole cell spiking which are very strongly associated with deflagellation, exhibit a high degree of apical localisation and are largely absent in the adf1 mutant. We propose that these later elevations may act as specific signals for deflagellation.     

Seeing is believing: recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles

The role of Ca2+ in the regulation of the cell cycle has been investigated mostly in studies assessing global cytosolic free Ca2+. Recent studies, however, have used unique techniques to assess Ca2+ in subcellular organelles, such as mitochondria, and in discrete regions of the cytoplasm. These studies have used advanced fluorescence digital imaging techniques and Ca2+-sensitive fluorescence probes, and/or targeting of Ca2+-sensitive proteins to intracellular organelles. The present review describes the results of some of these studies and the techniques used. The novel techniques used to measure Ca2+ in microdomains and intracellular organelles are likely to be of great use in future investigations assessing Ca2+ homeostasis during the cell cycle.     

Participation of mitochondria in calcium signalling in the exocrine pancreas

This minireview is an attempt to put together some of the recent advances regarding the implications of mitochondria in Ca2+ homeostasis. Although the main role of this cytoplasmic organelle is ATP supply to the cell, during the past years strong evidence has been accumulated supporting an active role of these organelles in Ca2+ handling by the cell. The discovery of mitochondrial specific fluorescent dyes has permitted the study of these organelles within living cells. Due to its ubiquitous localisation within the cytosol, mitochondria would play an important role in the modulation of the subcellular patterns of Ca2+ signalling, and therefore would act as modulators of Ca2+-dependent cellular processes.     

The role of calcium in blue-light-dependent chloroplast movement in lemna trisulca L

Chloroplast movements are a normal physiological response to changes in light intensity and provide a good model system to analyse the signal transduction pathways following light perception. Blue-light-dependent chloroplast movements were observed in Lemna trisulca using confocal optical sectioning and 3-D reconstruction or photometric measurements of leaf transmission. Chloroplasts moved away from strong blue light (SBL) towards the anticlinal walls (profile position), and towards the periclinal walls (face position) under weak blue light (WBL) over about 20-40 min. Cytoplasmic calcium ([Ca2 + ]cyt) forms part of the signalling system in response to SBL as movements were associated with small increases in [Ca2 + ]cyt and were blocked by antagonists of calcium homeostasis, including EGTA, nifedipine, verapamil, caffeine, thapsigargin, TFP (trifluoperazine), W7 and compound 48/80. Treatments predicted to affect internal Ca2 + stores gave the most rapid and pronounced effects. In addition, artificially increasing [Ca2 + ]cyt in darkness using the Ca2 + ionophore A23187 and high external Ca2 + (or Sr2 + ), triggered partial movement of chloroplasts to profile position analogous to a SBL response. These data are all consistent with [Ca2 + ]cyt acting as a signal in SBL responses; however, the situation is more complex given that both WBL and SBL responses were inhibited to a similar extent by all the Ca2 + -signalling antagonists used. As the direction of chloroplast movement in WBL is exactly opposite to that in SBL, we conclude that, whilst proper regulation of [Ca2 + ]cyt homeostasis is critical for both SBL and WBL responses, additional factors may be required to specify the direction of chloroplast movement.