Nuclear Calcium Research Articles

An update on nuclear calcium signalling.

Over the past 15 years or so, numerous studies have sought to characterise how nuclear calcium (Ca2+) signals are generated and reversed, and to understand how events that occur in the nucleoplasm influence cellular Ca2+ activity, and vice versa. In this Commentary, we describe mechanisms of nuclear Ca2+ signalling and discuss what is known about the origin and physiological significance of nuclear Ca2+ transients. In particular, we focus on the idea that the nucleus has an autonomous Ca2+ signalling system that can generate its own Ca2+ transients that modulate processes such as gene transcription. We also discuss the role of nuclear pores and the nuclear envelope in controlling ion flux into the nucleoplasm.

Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum

The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca(2+) to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca(2+) dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca(2+) indicators genetically targeted to NE/endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca(2+)](ne/er) and [Ca(2+)](n) respectively. Cytosolic Ca(2+) ([Ca(2+)](cyt)) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca(2+)](ne/er), which was rapidly replaced on addition of extrinsic Ca(2+) to those cells containing fully functional NCX/GM1: differentiated NG108-15 and C6 cells. Reduced elevation of [Ca(2+)](ne/er) following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108-15 and NG-CR72 cells. No change in [Ca(2+)](ne/er) due to applied Ca(2+) was seen in Jurkat cells, which entirely lack NCX. Ca(2+) entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca(2+)](n) and [Ca(2+)](cyt) were elevated independent of [Ca(2+)](ne/er) and remained in approximate equilibrium with each other. Ca(2+) rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca(2+) transfer from cytosol to ER, an alternate route to the sarcoplasmic/endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca(2+).

Alterations In Binding Properties Of Myocardial Nuclear Membrane Receptors Induce Nuclear Calcium Overload In Rat Ischemia-reperfusion

Cell nuclei possess an independent calcium regulatory system consisting of nuclear Ca2+-ATPases (NCA), IP3 receptors (IP3R), IP4 receptors (IP4R), ryanodine receptors(RyR), and nuclear pore complexes(NPC). We studied the changes in Ca2+n and its regulatory system in rat model of myocardial ischemia-reperfusion injury(IRI) induced by 30 min coronary occlusion followed by 180 min reperfusion. The Ca2+n content in isolated nuclei was measured with atomic absorption spectrophotometer. NPC permeability was assessed through the amount of calmodulin conjugated Alexa FluoTM 488 as fluorescent probes. NCA activity was evaluated by phosphate group released from ATP in enzymatic reaction. The maximum binding capacity(Bmax) and dissociation constant(Kd) of IP3R, IP4R and RyR were determined by radioligand binding of [3H]IP4, [3H]IP4 and [3H]-ryanodine to isolated cardiomyocytic nuclei. All results are shown in the table. Our findings suggest that in vivo rat myocardial IRI is characterized by Ca2+n overload, upregulations of nuclear IP3R and IP4R, downregulations of NCA activity and RyR, and an increase in permeability of NPC. The upregulation of nuclear IP3R and IP4R may be responsible for the nuclear calcium overload.

The Morphology of Cell Nuclei Regulates Calcium Coding in Hippocampal Neurons

Event Abstract Back to Event The Morphology of Cell Nuclei Regulates Calcium Coding in Hippocampal Neurons Gillian Queisser1* 1 University of Heidelberg, Interdisziplinares Zentrum fur Wissenschaftliches Rechnen, Germany Calcium acts as a key regulator in the nucleus for biochemical events that trigger gene transcription and is involved in processes such as memory formation and information storage. Recent research shows that the morphology of hippocampal neuron nuclei is regulated by NMDA receptors, which led us to investigate the morphological influence in a modeling environment. We introduce novel concepts of neuron nuclei and their morphology as a regulator for nuclear calcium signaling. In a model study we developed a three-dimensional mathematical model for nuclear calcium signaling based on experimental data and three-dimensionally reconstructed cell nuclei. When investigating the influence of the nuclear morphology on calcium signals, we find two main types of nuclei, infolded and spherical. While spherical nuclei are observed to be "signal-integrators", infolded nuclei are adept at resolving high-frequency signals, an area not yet explored in detail until this point. Downstream of calcium, the morphology of nuclei might affect biochemical processes that are involved in gene transcription. Conference: Bernstein Conference on Computational Neuroscience, Frankfurt am Main, Germany, 30 Sep - 2 Oct, 2009. Presentation Type: Poster Presentation Topic: Information processing in neurons and networks Citation: Queisser G (2009). The Morphology of Cell Nuclei Regulates Calcium Coding in Hippocampal Neurons. Front. Comput. Neurosci. Conference Abstract: Bernstein Conference on Computational Neuroscience. doi: 10.3389/conf.neuro.10.2009.14.066 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Aug 2009; Published Online: 26 Aug 2009. * Correspondence: Gillian Queisser, University of Heidelberg, Interdisziplinares Zentrum fur Wissenschaftliches Rechnen, Heidelberg, Germany, gillian.queisser@bioquant.uni-heidelberg.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gillian Queisser Google Gillian Queisser Google Scholar Gillian Queisser PubMed Gillian Queisser Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspects

Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca(2+) store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P(3) receptors [Ins(1,4,5)P(3)Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P(3)Rs by Ins(1,4,5)P(3). We have also investigated the possible consequences of nuclear calcium signals: the role of Ca(2+) in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca(2+)-mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca(2+): the IEG expression level was lowest in Ca(2+)-free solution, increased at the physiological level of 1 mm [Ca(2+)](o) and was maximal at 10 mm [Ca(2+)](o), i.e.: 102 +/- 22% and 163 +/- 15% for c-fos; c-myc -73 +/- 13% and 106 +/- 24%; c-jun -49 +/- 8% and 59 +/- 9% at 1 and 10 mm of extracellular Ca(2+) respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca(2+) together with nuclear Ca(2+) signals induced by CCK play important roles in the induction of IEG expression.

Nonpeptidic antagonists of ETA and ETB receptors reverse the ET-1-induced sustained increase of cytosolic and nuclear calcium in human aortic vascular smooth muscle cellsThis article is one of a selection of papers published in the special issue (part 2 of 2) on Forefronts in Endothelin.

Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic Ca2+ ([Ca]c) and nuclear Ca2+ ([Ca]n) in human aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional confocal microscopy coupled to the Ca2+ fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10(-6) mol/L) and BQ-788 (10(-7) mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10(-8)-10(-6) mol/L) and A-192621 (10(-7) mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore, activation of the ETB receptor alone using the specific agonist IRL-1620 (10(-9) mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10(-7) mol/L) further increased nuclear Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by extracellular application of the Ca2+ chelator EGTA. Pretreatment with the G protein inhibitors pertussis toxin (PTX) and cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane depolarization with KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of intracellular Ca2+. These results suggest that both ETA- and ETB-receptor activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better pharmacological tools for blocking ET-1-induced sustained increase of intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via activation of PKC, but via a PTX- and CTX-sensitive G protein calcium influx through the R-type Ca2+ channel.

Role of Yersinia enterocolitica heat-stable enterotoxin (Y-STa) on differential regulation of nuclear and cytosolic calcium signaling in rat intestinal epithelial cells

The heat-stable enterotoxin (Y-STa) produced by the pathogenic strains of Yersinia enterocolitica is a causative agent of secretory diarrhea. We have reported earlier that Y-STa-induced inositol trisphosphate-mediated cytosolic calcium rise occurs in rat intestinal epithelial cells. In the present communication, the involvement of a nuclear calcium store in the action mechanism of Y-STa in rat intestinal epithelial cells has been shown. Calcium imaging with time series confocal microscopy shows that Y-STa stimulates both the nuclear and cytosolic calcium levels in rat intestinal epithelial cells where a rise in nuclear calcium precedes the cytosolic events. Moreover, Y-STa stimulates both cytosolic and nuclear inositol trisphosphate (IP(3)) levels in a time-dependent manner. Western blot and immunocytochemical analysis reveal a higher density of IP(3) receptor type II in the nuclear membrane compared to the cytosol, which may be the cause of an early rise of the nuclear calcium level. Therefore, it is suggested that Y-STa regulates the nuclear and cytosolic calcium signals in a distinct temporal manner in rat intestinal epithelial cells.

G Protein-Coupled Receptor Signaling in the Nucleus

G protein-coupled receptors (GPCRs) are best known for their role as plasma membrane-localized receptors that respond to extracellular ligands. However, evidence is building that there may be complete GPCR signaling systems in the nucleus as well. Kumar et al . show that a particular type of metabotropic glutamate receptor, mGluR5, can couple to G q/11 and stimulate phosphatidylinositol-phospholipase C (PI-PLC) to generate inositol trisphosphate (IP 3 ) and oscillatory calcium signals in nuclei from transfected human embryonic kidney (HEK) 293 cells or cultured dissociated striatal neurons. Hemagglutinin-tagged mGluR5 expressed in HEK293 cells exhibited both plasma membrane and nuclear membrane localization. To analyze the nuclear mGluR5 signaling, nuclei were isolated from cells either expressing the hemagglutinin-tagged wild-type mGluR5 or a point mutant that could not couple to G proteins (F767S mutant). Bath application of glutamate to the isolated nuclei showed that only those from cells expressing wild-type mGluR5 produced oscillations in calcium concentration (measured with a fluorescent calcium indicator). Glutamate-induced calcium oscillations required PLC activity and were inhibited by the pan-PLC inhibitor U73122 and the PI-PLC-specific inhibitor ET-18-OCH 3 . Although mGluR5 frequently exhibits constitutive activity in heterologous systems, a modest glutamate-induced increase in IP 3 concentration was detected in nuclei from cells expressing mGluR5. Transfection of a biosensor that binds to membranes through an interaction with phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and then is released from membranes when IP 3 is produced and PIP 2 concentrations decrease revealed that the isolated nuclei from the mGluR5-expressing cells had glutamate-stimulated oscillatory changes in the PIP 2 to IP 3 ratio, leading to repeated release from and recruitment to the nuclear membrane of the biosensor. Pharmacological inhibitors of IP 3 receptors and ryanodine receptors inhibited glutamate-induced calcium oscillations in nuclei isolated from cells expressing mGluR5. Several types of glutamate receptors are present in striatal neurons. To isolate the mGluR5 response, the authors added the agonist quisqualate in the presence of inhibitors of both AMPA-type and mGluR1 receptors and found that the neurons exhibited both cytosolic and nuclear calcium oscillations. The quisqualate-stimulated calcium signals were blocked if the neurons were transfected with a dominant-negative Gα q or if PLCβ1 was knocked down with siRNA. Application of pharmacological inhibitors to isolated striatal neuron nuclei suggested that these cells also exhibited PI-PLC-dependent calcium signaling in response to quisqualate. Thus, there appears to be a complete GPCR signaling system in the nucleus. How these receptors are targeted to the nucleus and how they are activated when their ligand-binding domains are in the nuclear lumen remain to be investigated. V. Kumar, Y.-J. I. Jong, K. L. O’Malley, Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear G q/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca 2+ release. J. Biol. Chem. 283 , 14072-14083 (2008). [Abstract] [Full Text]

CRF Facilitates Calcium Release from Intracellular Stores in Midbrain Dopamine Neurons

Changes in cytosolic calcium are crucial for numerous processes including neuronal plasticity. This study investigates the regulation of cytosolic calcium by corticotropin-releasing factor (CRF) in midbrain dopamine neurons. The results demonstrate that CRF stimulates the release of intracellular calcium from stores through activation of adenylyl cyclase and PKA. Imaging and photolysis experiments showed that the calcium originated from dendrites and required both functional IP3 and ryanodine receptor channels. The elevation in cytosolic calcium potentiated calcium-sensitive potassium channels (sK) activated by action potentials and metabotropic Gq-coupled receptors for glutamate and acetylcholine. This increase in cytosolic calcium activated by postsynaptic Gs-coupled CRF receptors may represent a fundamental mechanism by which stress peptides and hormones can shape Gq-coupled receptor-mediated regulation of neuronal excitability and synaptic plasticity in dopamine neurons.

A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity

BackgroundExtrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic) may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death.ResultsHere we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours) periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6%) reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting.ConclusionThe slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity-dependent neuroprotection. Other factors, in particular calcium signaling to the nucleus and the induction of survival promoting genes are more likely to mediate acquired neuroprotection.

Nucleoplasmic reticulum is not essential in nuclear calcium signalling mediated by cyclic ADPribose in primary neurons

Nuclear calcium regulation is essential for controlling nuclear processes such as gene expression. Recent studies, mostly performed on immortalized or transformed cell lines, reported the presence of a nucleoplasmic reticulum (NR). It has been suggested that NR acts as a storage organelle having an important role in nuclear Ca2+ signalling. However, whether NR is present and necessary in primary neurons for generation of nuclear Ca2+ signalling has never been investigated. Here, we show, by confocal microscopy and by electronic microscopy, that nuclei in intact neurons or isolated nuclei are not endowed with NR. Finally, our experiments performed on isolated nuclei from Aplysia giant neurons show that the nuclear envelope acts as a functional Ca2+ store which can be mobilized by the second messenger cyclic ADPribose to elicit a nucleoplasmic Ca2+ elevation. Our study provides evidence that nuclear Ca2+ signals can be independent of the presence of NR in neurons.

Mechanism of nuclear calcium signaling by inositol 1,4,5-trisphosphate produced in the nucleus, nuclear located protein kinase C and cyclic AMP-dependent protein kinase

Nuclear phospholipase C-gamma 1 can be phosphorylated by nuclear membrane located epidermal growth factor receptor sequel to epidermal growth factor-mediated signaling to the nucleus. The function of mouse liver phospholipase C-gamma 1 is attributed to a 120 kDa protein fragment which has been found to be a proteolytic product of the 150 kDa native nuclear enzyme. The tyrosine-phosphorylated 120 kDa protein band interacts with activated EGFR, binds phosphatidyl-3-OH kinase enhancer, and activates nuclear phosphatidylinositol-3-OH-kinase, and is capable of generating diacylglycerol in response to the epidermal growth factor signal to the nucleus in vivo. Thus a mechanism for nuclear production of inositol-1,4,5-trisphophate is unraveled. Nuclear generated inositol-1,4,5-trisphophate interacts with the inner membrane located inositol-1,4,5-trisphophate receptor and sequesters calcium into the nucleoplasm. Nuclear inositol-1,4,5-trisphophate receptor is phosphorylated by native nuclear protein kinase C which enhances the receptor-ligand interaction. Nuclear calcium-ATPase and inositol-1,3,4,5-tetrakisphophate receptor are located on the outer nuclear membrane, thus facilitating calcium transport into the nuclear envelope lumen either by ATP or inositol-1,3,4,5-tetrakisphophate depending upon the external free calcium concentrations. Nuclear calcium ATPase is phosphorylated by cyclic AMP-dependent protein kinase with enhanced calcium pumping activity. A holistic picture emerges here where tyrosine phosphorylation compliments serine phosphorylation of key moieties regulating nuclear calcium signaling. Evidence are forwarded in favor of proteolysis having a profound implications in nuclear calcium homeostasis in particular and signal transduction in general.

Filtering, reconstruction, and measurement of the geometry of nuclei from hippocampal neurons based on confocal microscopy data

The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium.

GM1 in the nuclear envelope regulates nuclear calcium through association with a nuclear sodium‐calcium exchanger

The inner membrane of the nuclear envelope (NE) of neurons and other cells has been shown to contain GM1 tightly associated with a Na(+)/Ca(2+) exchanger (NCX) whose activity it potentiates in mediating transfer of Ca(2+) from nucleoplasm to the NE lumen. This is consistent with localization of the NCX/GM1 complex in the inner membrane of the NE. NCXs of the plasma membrane, in contrast, appear to bind GM1 much less avidly. This is believed due to different isoforms of NCX in the two membranes, and a difference in topology of NCX relative to GM1. A cytoprotective function for nuclear NCX/GM1 was suggested in the observation that cultured neurons from mice lacking GM1 (GM2/GD2 synthase knockout) were vulnerable to Ca(2+)-induced apoptosis. These neurons in culture were rescued to some extent by GM1 but more effectively by LIGA-20, a membrane-permeant derivative of GM1 that entered the NE. Further indication came from a study of the mutant mice, which were highly susceptible to kainate-induced seizures and could be rescued by LIGA-20. This correlated with the ability of LIGA-20 to cross the blood-brain barrier, enter brain cells, insert into the NE, and potentiate nuclear NCX.

Regulation of nuclear calcium concentration.

Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.

Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients

BackgroundIn hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types.ResultsUsing laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border.ConclusionThus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.

Nuclear calcium signaling by inositol trisphosphate in GH3 pituitary cells

It has been proposed that nuclear and cytosolic Ca2+ ([Ca2+]N and [Ca2+]C) may be regulated independently. We address here the issue of whether inositol trisphosphate (IP3) can, bypassing changes of [Ca2+]C, produce direct release of Ca2+ into the nucleoplasm. We have used targeted aequorins to selectively measure and compare the changes in [Ca2+]C and [Ca2+]N induced by IP3 in GH3 pituitary cells. Heparin, an IP3 inhibitor that does not permeate the nuclear pores, abolished the [Ca2+]C peaks but inhibited only partly the [Ca2+]N peaks. The permeant inhibitor 2-aminoethoxy-diphenyl-borate (2-APB) blocked both responses. Removal of ATP also inhibited more strongly the [Ca2+]C than [Ca2+]N peak. The [Ca2+]N and [Ca2+]C responses differed also in their sensitivity to IP3, the nuclear response showing higher affinity. Among IP3 receptors, type 2 (IP3R2) has a higher affinity for IP3 and is not inactivated by ATP removal. We find that IP3R2 immunoreactivity is present inside the nucleus whereas the other IP3R subtypes are detected only in the cytoplasm. The nuclear envelope (NE) of GH3 cells showed deep invaginations into the nucleoplasm, with cytosol and cytoplasmic organella inside. These results indicate that GH3 pituitary cells possess mechanisms able to produce selective increases of [Ca2+]N.

Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco

Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Δ4-unsaturated LCB d- erythro-sphingosine and the saturated LCB d- ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-, N-Dimethyl- d- erythro-sphingosine, a structural analogue of d- erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.

Light-induced Apoptosis Involves a Defined Sequence of Cytoplasmic and Nuclear Calcium Release in AlPcS4-photosensitized Rat Bladder RR 1022 Epithelial Cells¶

Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose–dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate–labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-l-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.

Arraying the Pro-Life and Neurotoxic Responses to NMDA Receptor Signaling

Glutamate receptors of the N -methyl-D-asparate (NMDA) type are implicated in physiological neuronal signaling (for example, neuroprotection and synaptic plasticity) as well as in pathological neuronal processes (for example, excitotoxicity and neurodegenerative disease). Zhang et al . performed microarray analysis to monitor gene expression in cultured hippocampal neurons in response to two different paradigms that stimulated NMDA receptors, which are calcium-conducting ion channels. In one, the NMDA receptors were activated by synaptic activity through the pharmacological blockade of inhibitory receptors, and in the other, both synaptic and extrasynaptic receptors were activated by glutamate added to the bath. The NMDA-specific transcriptional profile was assessed by comparing the genes stimulated or repressed in the presence or absence of NMDA-specific inhibitors. The activity-dependent profile resulted in a total of 478 induced genes, with 115 of those specific to NMDA receptor activation and 108 of those unique to the synaptic activation profile--that is, they were not also activated by bath-applied glutamate. Similar analysis showed that of 106 total genes activated by bath-applied glutamate, only 11 were specific to the extrasynaptic activation of the receptors. Only a single gene was specifically repressed by extrasynaptic NMDA receptors, whereas 34 genes were specifically repressed by synaptic activation of NMDA receptors. Using gene ontology to classify the regulated genes, the authors noted that synaptic receptor activation seemed to stimulate a prosurvival, antiapoptotic profile by enhancing genes associated with survival and repressing genes associated with cell death. The functions of two genes up-regulated by the synaptic activity, Bcl6 and Btg2 , were further assessed by overexpression studies and RNA inhibition studies. Bcl6 encodes a transcriptional repressor that has been implicated in B cell lymphomas, where it has been reported to repress p53 expression and prevent cell death due to DNA damage. The exact function of the protein encoded by Btg2 is unknown, but it has been implicated in protection of PC12 cells from cell death. Overexpression of either of these proteins in the hippocampal cultures protected the neurons from death induced by growth factor withdrawal or by staurosporine, and coexpression was most effective. Knockdown of Btg2 by RNA interference prevented activity-dependent protection from staurosporine, and knockdown of Bcl6 resulted in enhanced cell death in the absence of any stressful stimuli. Both of these genes required a nuclear calcium signal for transcriptional activation, and their activity-dependent transcription was blocked by overexpression of nuclear calcium-calmodulin binding protein. Analysis of the consequence of overexpression or knockdown of the Clca1 gene, which showed increased transcription in response to extrasynaptic glutamate and encodes a calcium-activated chloride channel, revealed that overexpression promoted cell death but knockdown did not promote survival. Altogether, these results help explain how a single receptor can promote opposing responses and emphasize the importance of spatially restricted signaling. S.-J. Zhang, M. N. Steijaert, D. Lau, G. Schütz, C. Delucinge-Vivier, P. Descombes, H. Bading, Decoding NMDA receptor signaling: Identification of genomic programs specifying neuronal survival and death. Neuron 53 , 549-562 (2007). [PubMed]