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Recent evidence suggests that multiple calcium-releasing messengers might be activated simultaneously to regulate patterns of intracellular calcium signalling. In this way, agonists might use different messenger cocktails to encode specific signals and target selected processes.
Calcium signalling was studied in porcine aortic endothelial cells stably transfected with wild type or mutants of the human platelet-derived growth factor (PDGF) beta-receptor and fibroblast growth factor (FGF) receptor-1 (FGFR1). Phospholipase C-gamma (PLC-gamma) has a consensus binding site at phosphorylated Tyr1021 in the PDGF beta-receptor. The phosphorylated tyrosine at 1009 is a binding site for Syp/PTP1D, an adaptor molecule mediating Grb2/RAS signalling. Also, Tyr1009 has been shown to be a minor binding site for PLC-gamma; however previous data have indicated that it does not have any functional significance in PLC-gamma signalling. The concentration of cytoplasmic calcium ([Ca2+]i) was measured by microfluorometry and digital imaging. About 72% of the cells transfected with wild type PDGF beta-receptor responded to a challenge with PDGF-BB. Mutants in which both Tyr1009 and Tyr1021 in the PDGF beta-receptor were exchanged for phenylalanine totally lacked [Ca2+]i responses. However, in those with a single mutation at Tyr1009 or Tyr1021, 36% and 12% of the cells responded, respectively. In cells transfected with FGFR1 or FGFchim, with the kinase insert of FGFR1 replaced by the insert of the PDGF beta-receptor, a [Ca2+]i increases was observed in similar proportions of cells. The amplitudes of the growth factor-induced [Ca2+]i responses was comparable in the different transfectants. Thrombin, activating a G-protein coupled receptor, triggered [Ca2+]i peaks more rapidly, and in a higher proportion of cells compared to the growth factors. The present data indicate that both Tyr1009 and Tyr1021 alone and in cooperation mediate PDGF-BB triggered calcium signalling.
Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus.
Calcium ion is a universal second messenger in numerous cell physiological processes. The paper describes the structure and the activation mechanisms of the bioluminescent (aequorin) and fluorescent based GFP calcium sensitive probes (Cameleon) and the data obtained with such probes in genetically transformed animal and vegetal organisms. The importance of these in vivo Ca2+ imaging molecules in the understanding of calcium signalling is discussed.
Calcium is an ubiquitous second messenger that is involved in the regulation of a number of cell functions. The mechanism by which the specificity of calcium signaling is achieved is not well understood. We suggest that calcium release from the ER can occur selectively at different spatial locations in response to different extracellular stimuli. We discuss a possible mechanism for such selectivity and present a model based on this mechanism. The suggested mechanism is based on the regulation of local Ca2+ release by cyclic AMP-dependent protein kinase (PKA) and relies upon two experimental observations: first, some G-protein coupled signaling pathways activate PLC and regulate adenylate cyclase at the same time, leading to IP3 production and altering PKA activity via changes in cAMP level; second, phosphorylation by PKA alters the properties of IP3 receptor (IP3R). In our model we consider allosteric regulation of IP3Rs by IP3 and cAMP-dependent phosphorylation. The differences in IP3Rs and PKA densities at different spatial locations within the cell allow the release of calcium selectively at each location in response to certain combination of IP3 and cAMP concentration. Specificity of agonist-response coupling is achieved if different combinations in the levels of these second messengers are specific for different extracellular stimuli.
Calcium entry into neuronal cells through N-methyl-D-aspartate (NMDA)-type glutamate receptors or L-type voltage-gated calcium channels is a key event in the control of gene expression following electrical activation. Calcium acts both in the cytoplasm and the nucleus to activate signalling pathways that stimulate gene expression through different DNA regulatory elements. Differential control of transcription by spatially distinct calcium signals provides a mechanism by which a single second messenger can generate diverse transcriptional responses. This may allow for stimulation-specific modulation of gene expression critical for adaptive changes in the nervous system.
Understanding of intracellular calcium signal and its modulations allows a better comprehension of mechanisms of action of calcium antagonists. This paper deals with one possible functional classification of calcium antagonists based on the actual concepts on cellular calcium regulation.
Calcium is a second messenger responsible for regulating a wide range of cellular processes. It is normally presented as brief spikes even in non-excitable cells. The necessity of limiting the period of calcium stimulation to brief bursts may depend upon the fact that prolonged elevation of calcium can be toxic. It can act on endonucleases in the nucleus to trigger programmed cell death. It will be argued that non-lethal effects of elevated calcium can lead to a variety of pathological conditions including hypertension, atherosclerosis, transformation, malignant hyperthermia and possible neural disorders such as spreading depression and manic-depressive illness.
Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e. between 20 and 100 nmol l-1) in order to avoid the cytotoxic effects of a prolonged elevation of [Ca2+]. Cells have evolved a sophisticated signalling system based on the generation of brief pulses of Ca2+ which enables this ion to be used as a messenger, thus avoiding its toxic effects. Such Ca2+ spikes usually result from the coordinated release of Ca2+ from internal stores using either inositol 1,4,5-trisphosphate or ryanodine receptors. Using Ca2+ imaging techniques, the opening of individual channels has now been visualized and models have been proposed to explain how these elementary events are coordinated to generate the global Ca2+ signals that regulate cellular activity.
Calcium regulates three different aspects of cardiac contraction. It drives pacemaker activity, excitation-contraction coupling and the transcriptional events that remodel the Ca(2+) signalling system in both health and disease.
Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.
We have recently reported that acetylcholinesterase expression was induced during apoptosis in various cell types. In the current study we provide evidence to suggest that the induction of acetylcholinesterase expression during apoptosis is regulated by the mobilization of intracellular Ca(2+). During apoptosis, treatment of HeLa and MDA-MB-435s cells with the calcium ionophore A23187 resulted in a significant increase in acetylcholinesterase mRNA and protein levels. Chelation of intracellular Ca(2+) by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester), an intracellular Ca(2+) chelator, inhibited acetylcholinesterase expression. A23187 also enhanced the stability of acetylcholinesterase mRNA and increased the activity of acetylcholinesterase promoter, effects that were blocked by BAPTA-AM. Perturbations of cellular Ca(2+) homeostasis by thapsigargin resulted in the increase of acetylcholinesterase expression as well as acetylcholinesterase promoter activity during thapsigargin induced apoptosis in HeLa and MDA-MB-435s cells, effects that were also inhibited by BAPTA-AM. We further demonstrated that the transactivation of the human acetylcholinesterase promoter by A23187 and thapsigargin was partially mediated by a CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter. This motif was able to bind to CCAAT binding factor (CBF/NF-Y). These results strongly suggest that cytosolic Ca(2+) plays a key role in acetylcholinesterase regulation during apoptosis induced by A23187 and thapsigargin.
After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.
Two kinds of lymphocyte populations were prepared from human resected tonsils: one was a mixed population consisting of T cells, B cells, other lymphocytes, and a few macrophages, and the other was a T-enriched population obtained by removing adherent cells from the mixed population with a nylon wool column. A transient rise in cytosolic free calcium ion concentration ([Ca2+]i) was observed in both the populations within a few minutes of stimulation with concanavalin A (Con A). However, emergence of cells which had high [Ca2+]i after 4 min of Con A stimulation was observed practically only in the mixed population. The [Ca2+]i elevation occurring within a few minutes of stimulation in both populations was interpreted as being due to a release from the intracellular Ca-storing organelles, whereas the high [Ca2+]i in a group of cells after 4 min of Con A stimulation in the mixed population was caused by an influx of extracellular calcium that probably corresponds to a transient enhancement of Ca2+ uptake observed only in the mixed population during early stimulation. Con A induced both interleukin 2 receptor (IL-2R) expression and interleukin 2 (IL-2) production. A nonmitogenic lectin, wheat germ agglutinin (WGA), also induced a rise in the [Ca2+]i within a few minutes. WGA induced IL-2 production, but did not induce IL-2R expression. Chelation of extracellular calcium with EGTA at the time of Con A addition resulted in a decrease in IL-2R expression, IL-2 production, and DNA synthesis, but not when CaCl2 equimolar to EGTA was present in the culture medium. Chelation of calcium 12 hr after Con A stimulation decreased IL-2R expression, but had no effect on IL-2 production. These results indicate that IL-2 production required Ca2+ only in the early (G0) stage and that IL-2R expression was dependent on Ca2+ in both the G0 and the G1 stages.
Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.
Wherever you travel through the cytoplasm of the cells you will find organelles with internal [Ca(2+)] levels higher than in the surrounding cytosol. This is particularly true of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells); such organelles serve as the main sources of releasable Ca(2+) for cytosolic cellular signalling. Calcium pumps of the SERCA family (sarcoplasmic and endoplasmic reticulum calcium ATP-ases) import calcium into the organelle lumen. The other mechanism that is responsible for the steady state calcium level within the lumen of ER or SR is a calcium leak that balances the influx created by the pumps. The leak remains the most enigmatic of the processes involved in calcium regulation. The molecular nature of the leak mechanism is not known. The basal leak is a relatively slow process, which is difficult to investigate and which is easily outmatched (both in the amplitude of calcium responses and in attractiveness to experimenters) by substantially faster second messenger-induced release. Nevertheless, information on the properties of the calcium leak, although thinly scattered through the pages of PubMed, has been slowly accumulating. In this review we will discuss the properties of the calcium leak and speculate about possible mechanisms, which could mediate this process.
There have been extensive efforts to characterize the mechanism of action of volatile anesthetics, but their molecular and cellular actions are still a matter of debate. Volatile anesthetics act primarily on synaptic transmission in the central nervous system but proof of this as the predominant mechanism of action remains elusive. Changes in neurotransmitter release may relate to direct interaction of the anesthetic molecule with an ion channel protein or synaptic protein, but can also be a consequence of alterations in intracellular signaling. Calcium is one of the most important messengers in cells and its intracellular concentration may be modified by several agents including volatile anesthetics. Neuronal excitability is in part determined by calcium availability that is controlled by several mechanisms. Because voltage-gated calcium channels (VGCC) play a key role in controlling Ca2+ entry and in initiating cellular responses to stimulation through an elevation of intracellular calcium concentration ([Ca2+](i)), they are thought to be one of the targets for volatile anesthetics. However, [Ca2+](i) can also be altered without the participation of VGCC through receptor-mediated pathways. Indeed, calcium homeostasis is also controlled by plasma membrane Ca2+ -adenosine triphosphatase, sarcoplasmic-endoplasmic reticular Ca2+ -ATPase, the Na+ -Ca2+ exchanger, and mitochondrial Ca2+ sequestration. Alteration of any of those mechanisms that control [Ca2+](i) may lead to a change in presynaptic transmission or postsynaptic excitability. Here we will review some of the recent progress in identifying putative actions of volatile anesthetics, specifically the effect on intracellular calcium homeostasis in neurons.
Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.