Endoplasmic Reticulum Calcium Release Research Articles

Neuregulin β1 enhances peak glutamate-induced intracellular calcium levels through endoplasmic reticulum calcium release in cultured hippocampal neuronsThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Modulation of intracellular free calcium levels is the primary second messenger system of the neuronal glutamatergic system, playing a role in regulation of all major cellular processes. The protein neuregulin (NRG) beta1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic glutamate receptors. The effect NRG may have on glutamate-induced changes in intracellular free calcium concentrations has not been examined, however. In this study, cultured embryonic rat hippocampal neurons were treated with NRGbeta1 to determine a possible effect on glutamate-induced intracellular calcium levels. Long-term (24 h), but not short-term (1 h), incubation with NRGbeta1 resulted in a significantly greater glutamate-mediated acute peak elevation of intracellular calcium levels than occurred in vehicle-treated neurons. Long-term NRGbeta1 incubation significantly enhanced calcium increase induced by specific stimulation of metabotropic glutamate receptors, but did not significantly alter the N-methyl D-aspartate (NMDA)- or KCl-induced calcium increase and paradoxically decreased the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) treatment on intracellular calcium. Metabotropic glutamate receptors cause increased intracellular free calcium via release of calcium from intracellular stores; thus this system was examined in more detail. NRGbeta1 treatment significantly (greater than 2-fold) enhanced calcium release from endoplasmic reticulum stores after stimulation of ryanodine receptors with caffeine, but did not significantly increase calcium release from endoplasmic reticulum mediated by inositol trisphosphate (IP3) receptors. In addition, ryanodine receptor inhibition with ruthenium red prevented the glutamate-induced increase in intracellular calcium levels in NRGbeta1-treated neurons. These data show that long-term NRGbeta1 treatment can enhance glutamate-induced peak intracellular calcium levels through metabotropic glutamate receptor activation by increasing endoplasmic reticulum calcium release through ryanodine receptors.

Role of ERO1-α–mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress–induced apoptosis

Endoplasmic reticulum (ER) stress-induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-alpha (ER oxidase 1 alpha). In ER-stressed cells, ERO1-alpha is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-alpha suppresses apoptosis. IP3-induced calcium release (IICR) is increased during ER stress, and this response is blocked by siRNA-mediated silencing of ERO1-alpha or IP3R1 and by loss-of-function mutations in Ero1a or Chop. Reconstitution of ERO1-alpha in Chop(-/-) macrophages restores ER stress-induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not Chop(-/-) mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ERO1-alpha-IP3R pathway.

Death by Committee: Organellar Trafficking and Communication in Apoptosis

Apoptosis proceeds through a set of evolutionarily conserved processes that co-ordinate the elimination of damaged or unneeded cells. This program of cell death is carried out by organelle-directed regulators, including the Bcl-2 proteins, and ultimately executed by proteases of the caspase family. Although the biochemical mechanisms of apoptosis are increasingly understood, the underlying cell biology orchestrating programmed cell death remains enigmatic. In this review, we summarize the current understanding of Bcl-2 protein regulation and caspase activation while examining cell biological mechanisms and consequences of apoptotic induction. Organellar contributions to apoptotic induction include death receptor endocytosis, mitochondrial and lysosomal permeabilization, endoplasmic reticulum calcium release and fragmentation of the Golgi apparatus. These early apoptotic events are accompanied by stabilization of the microtubule cytoskeleton and translocation of organelles to the microtubule organizing center. Together, these phenomena establish a model of apoptotic induction whereby a cytoskeletal-dependent coalescence and 'scrambling' of organelles in the paranuclear region co-ordinates apoptotic communication, caspase activation and cell death.

The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor

Although the presence of a BH4 domain distinguishes the antiapoptotic protein Bcl-2 from its proapoptotic relatives, little is known about its function. BH4 deletion converts Bcl-2 into a proapoptotic protein, whereas a TAT-BH4 fusion peptide inhibits apoptosis and improves survival in models of disease due to accelerated apoptosis. Thus, the BH4 domain has antiapoptotic activity independent of full-length Bcl-2. Here we report that the BH4 domain mediates interaction of Bcl-2 with the inositol 1,4,5-trisphosphate (IP3) receptor, an IP3-gated Ca(2+) channel on the endoplasmic reticulum (ER). BH4 peptide binds to the regulatory and coupling domain of the IP3 receptor and inhibits IP3-dependent channel opening, Ca(2+) release from the ER, and Ca(2+)-mediated apoptosis. A peptide inhibitor of Bcl-2-IP3 receptor interaction prevents these BH4-mediated effects. By inhibiting proapoptotic Ca(2+) signals at their point of origin, the Bcl-2 BH4 domain has the facility to block diverse pathways through which Ca(2+) induces apoptosis.

Deviant Ryanodine Receptor-Mediated Calcium Release Resets Synaptic Homeostasis in Presymptomatic 3xTg-AD Mice

Presenilin mutations result in exaggerated endoplasmic reticulum (ER) calcium release in cellular and animal models of Alzheimer's disease (AD). In this study, we examined whether dysregulated ER calcium release in young 3xTg-AD neurons alters synaptic transmission and plasticity mechanisms before the onset of histopathology and cognitive deficits. Using electrophysiological recordings and two-photon calcium imaging in young (6-8 weeks old) 3xTg-AD and non-transgenic (NonTg) hippocampal slices, we show a marked increase in ryanodine receptor (RyR)-evoked calcium release within synapse-dense regions of CA1 pyramidal neurons. In addition, we uncovered a deviant contribution of presynaptic and postsynaptic ryanodine receptor-sensitive calcium stores to synaptic transmission and plasticity in 3xTg-AD mice that is not present in NonTg mice. As a possible underlying mechanism, the RyR2 isoform was found to be selectively increased more than fivefold in the hippocampus of 3xTg-AD mice relative to the NonTg controls. These novel findings demonstrate that 3xTg-AD CA1 neurons at presymptomatic ages operate under an aberrant, yet seemingly functional, calcium signaling and synaptic transmission system long before AD histopathology onset. These early signaling alterations may underlie the later synaptic breakdown and cognitive deficits characteristic of later stage AD.

Dual Mechanisms of sHA 14-1 in Inducing Cell Death through Endoplasmic Reticulum and Mitochondria

HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

Dysregulated Endoplasmic Reticulum calcium release in Alzheimer's disease: A paradigm for high throughput drug/siRNA screens

Dysregulated Endoplasmic Reticulum calcium release in Alzheimer's disease: A paradigm for high throughput drug/siRNA screens

Anesthesia, Calcium Homeostasis and Alzheimers Disease

While anesthetics are indispensable clinical tools generally safe and effective, in some situations there is grown concern about selective neurotoxicity of these agents; the clinical significance is unclear as of yet. The mechanisms for inhalational anesthetics mediated cell damage are still not clear, although a role for calcium dysregulation has been suggested. For example, the inhaled anesthetic isoflurane decreases endoplasmic reticulum (ER) calcium concentration and increases that in the cytosol and mitochondria. Inhibition of ER calcium release, via either IP(3) or ryanodine receptors, significantly inhibited isoflurane neurotoxicity. Neurons made vulnerable to calcium dysregulation by overexpression of mutated presenilin-1 (PS1) or huntingtin (Q-111) proteins showed enhanced apoptosis upon isoflurane exposure. Sevoflurane and desflurane were less potent than isoflurane in altering intracellular calcium, and produced less apoptosis. Short exposures to inhalational anesthetics may provide neuroprotection by preconditioning via a sublethal stress, while prolonged exposures to inhalational anesthetics may induce cell damage by apoptosis through direct cytotoxic effects.

Novel Function of PERK as a Mediator of Force-induced Apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.

Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1-42: Involvement of endoplasmic reticulum calcium release in oligomer-induced cell death

The nature of the toxic form of amyloid-beta peptide (Aβ) involved in early Alzheimer's disease (AD) pathology and whether it is the fibrillar or the oligomeric peptide that is the most deleterious to neurons remain controversial. This work aimed to compare the neurotoxicity of different amyloid-beta peptide 1-42 (Aβ1-42) assemblies, using fresh and aged samples enriched in oligomeric and fibrillar species, respectively, and also isolated oligomers and fibrils. The results obtained with fresh and aged Aβ1-42 preparations suggested that oligomeric species are more toxic to cortical neurons in culture than fibrillar forms, which was confirmed by using isolated oligomers and fibrils. In order to further elucidate the mechanisms involved in soluble Aβ toxicity, the involvement of endoplasmic reticulum (ER) calcium (Ca 2+) release in oligomer-induced apoptosis was evaluated. We observed that oligomeric Aβ1-42 depletes ER Ca 2+ levels leading to intracellular Ca 2+ dyshomeostasis involving phospholipase C activation. Moreover, in the presence of dantrolene, an inhibitor of ER Ca 2+ release through ryanodine receptors, the oligomer-induced apoptosis was prevented demonstrating the involvement of ER Ca 2+ release.

Mefloquine-Induced Disruption of Calcium Homeostasis in Mammalian Cells Is Similar to That Induced by Ionomycin

In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.

NF-κB activated by ER calcium release inhibits Aβ-mediated expression of CHOP protein: Enhancement by AD-linked mutant presenilin 1

Mutations in presenilin which result in early-onset Alzheimer disease (AD) cause both increased calcium release from intracellular stores, primarily endoplasmic reticulum (ER), and changes in NF-κB activation. Some studies have also reported that neurons containing AD-linked mutant presenilins (mPS1) show increased vulnerability to various stresses, while others report no differences in neuronal death. The majority of these reports center on potential changes in ER stress, because of the enhanced ER calcium release seen in mPS1 neurons. One of the primary death effectors of ER stress is CHOP, also termed GADD153, which acts to transcriptionally inhibit protective cellular molecules such as Bcl-2 and glutathione. Because both CHOP and NF-κB are activated by increased intracellular calcium and stress, yet have diametrically opposite effects on neuronal vulnerability, we sought to examine this interaction in greater detail. We observed that IP3-mediated calcium release from ER, stimulated by Aβ exposure, mediated both CHOP expression and NF-κB DNA binding activity. Further, specific inhibition of NF-κB resulted in greater expression of CHOP, while activation of NF-κB inhibited CHOP expression. The enhanced release of calcium from IP3-mediated ER stores in mPS1 neurons stimulated increased NF-κB compared to normal neurons, which inhibited CHOP expression. Upon blockage of NF-κB, exposure to Aβ caused significantly greater Aβ-mediated CHOP expression and death in mPS1 neurons compared to normal neurons. Thus, AD-linked PS1 mutations disrupt the balance between stress-induced NF-κB and CHOP, resulting in greater dependence on stress-induced NF-κB activation in mPS1 neurons.

Transient Receptor Potential Vanilloid 1 Agonists Cause Endoplasmic Reticulum Stress and Cell Death in Human Lung Cells

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest- and DNA damage-inducible transcript 3 (GADD153), GADD45alpha, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N'-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2alpha) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2alphaK3/EIF2alpha pathway, or stable overexpression of the EIF2alpha-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2alphaK3/EIF2alpha pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.

The immunophilin ligand GPI1046 protects neurons from the lethal effects of the HIV‐1 proteins gp120 and Tat by modulating endoplasmic reticulum calcium load

The dysfunction and death of neuronal cells is thought to underlie the cognitive manifestations of human immunodeficiency virus (HIV)-associated neurological disorders. Although HIV-infected patients are living longer owing to the effectiveness of anti-retroviral therapies, the number of patients developing neurological disorders is on the rise. Thus, there is an escalating need for effective therapies to preserve cognitive function in HIV-infected patients. Using HIV-protein-induced neurotoxicity as a model system, we tested the effectiveness of a non-immunosuppressive immunophilin ligand to attenuate gp120 and Tat-induced modification of neuronal function. The immunophilin ligand GPI1046 attenuated endoplasmic reticulum (ER) calcium release induced by gp120 and Tat and protected neurons from the lethal effect of these neurotoxic HIV proteins. Both inositol 1,4,5 trisphosphate (IP(3)) and ryanodine-sensitive ER calcium release was attenuated by pre-incubation with GPI1046. Using the sarco/endoplasmic reticulum calcium pump inhibitor thapsigargin to release ER calcium, we determined that GPI1046 reduced the total ER calcium load. These findings suggest that non-immunosuppressive immunophilin ligands may be useful neuroprotective drugs in HIV dementia.

3,3′-Diindolylmethane (DIM) and its derivatives induce apoptosis in pancreatic cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5

3,3'-Diindolylmethane (DIM), ring-substituted DIMs and 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) inhibit growth of Panc-1 and Panc-28 pancreatic cancer cells. Although DIMs (diarylmethanes) and selected C-DIMs (triarylmethanes), such as the p-t-butyl derivative (DIM-C-pPhtBu), activate the aryl hydrocarbon receptor and peroxisome proliferator-activated receptor gamma, respectively, this study shows that both DIM and DIM-C-pPhtBu induce common receptor-independent pathways. Both DIM and DIM-C-pPhtBu increased endoplasmic reticulum (ER) staining and ER calcium release in Panc-1 cells, and this was accompanied by increased expression of glucose related protein 78 and C/EBP homologous transcription factor (CHOP/GADD153) proteins. Similar results were observed after treatment with thapsigargin (Tg), a prototypical inducer of ER stress. The subsequent downstream effects of DIM/DIM-C-pPhtBu- and Tg-induced ER stress included CHOP-dependent induction of death receptor DR5 and subsequent cleavage of caspase 8, caspase 3, Bid and PARP. Activation of both receptor-dependent and receptor-independent (ER stress) pathways by DIM and DIM-C-pPhtBu in pancreatic cancer cells enhances the efficacy and potential clinical importance of these compounds for cancer chemotherapeutic applications.

The effects of ruthenium red, dantrolene and nimodipine, alone or in combination, in NMDA induced neurotoxicity of cerebellar granular cell culture of rats

In the present study, we used the L-type calcium channel blocker nimodipine, the endoplasmic reticulum (ER) calcium release blocker dantrolene and the calcium transport blocker in mitochondria ruthenium red (RuR), in NMDA induced neurotoxicity, to observe the most suitable combination for neuroprotection in cerebellar granular cell culture of rat pups. In addition we tested the effect of RuR on intact neuronal cells without adding NMDA into the flask, in order to compare the effects. As was expected, NMDA induced neuronal cell death. In NMDA induced neurotoxicity, RuR was the most neuroprotective agent of all three compounds tested, but interestingly, RuR alone was found to be neurotoxic in non-NMDA treated cultures. RuR showed neuroprotectivity in a dose dependent manner in NMDA toxicity. Dantrolene and nimodipine were also found to have neuroprotective properties in NMDA induced cell death in rat cerebellum. On the other hand, the combined application of the compounds was not found as protective as ruthenium red applications alone. The present study revealed a neurotoxic feature of ruthenium red, however we also demonstrated a neuroprotective role for it in NMDA-mediated neuron culture. This could be interpreted as a result of the partial agonistic effect of ruthenium red.

Reactive Blue Prevented Caffeine-Induced Neurotoxicity by an Independent Mechanism from Intracellular Calcium Currents in Cell Culture from Auditory Cortex of Rats

Neurotoxicity induced by caffeine in auditory-neuron cultures was studied in rat pups. For possible protective effect, reactive blue (RB) alone and in combination with dantrolene were tested in subsequent doses. RB was found to have a U-shape neuroprotective effect in caffeine neurotoxicity. Dantrolene was also tested in combined application in caffeine neurotoxicity. Despite the existing neuroprotection, no additional protection was obtained with various doses of dantrolene. In conclusion, RB may exert neuroprotective effect by increasing intracellular ATP levels in caffeine toxicity. High ATP levels may postpone the toxic cascade. Dantrolene as an endoplasmic reticulum calcium release blocker had no additional protective effect, suggesting that the increased intracellular calcium levels may be involved in later states of the toxic cascade, occurring after the compensatory phase of the cell death.

Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca 2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca 2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca 2+-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca 2+ channels, and the Ca 2+-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential ΔΨ m appeared to precede a rise in cytosolic free Ca 2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (ΔΨ m), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.

ATP- and Adenosine-Mediated Signaling in the Central Nervous System: Adenosine Stimulates Glutamate Release From Astrocytes via A2a Adenosine Receptors

Adenosine enhanced intracellular Ca2+ concentrations in astrocytes via A2a adenosine receptors involving protein kinase A (PKA) activation. The Ca2+ rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP3-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A2a adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A2a adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca2+ release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes.

Cytochrome c and IP 3 R: A Deadly Handshake

The mitochondria and endoplasmic reticulum (ER) are both involved in the apoptosis pathway, and release of calcium from the ER contributes to this process of cell death (see Mattson and Chan). ER calcium release is also involved in many nonpathological signaling processes, and Boehning et al. show that the interaction of cytochrome c (cyt c) released from mitochondria with the inositol trisphosphate receptors (IP 3 Rs) of the ER may be how the ER calcium release can reach pathologic levels. An interaction between cyt c and the IP 3 R was identified in a yeast two-hybrid screen, and these two proteins coimmunoprecipitated from cells treated with the broad-spectrum kinase inhibitor STS, which induces apoptosis. Furthermore, cell fractionation studies of cells treated with STS showed that cyt c accumulated in the ER fraction and was depleted from the mitochondrial fraction. Movement of cyt c to the ER fraction was not inhibited by exposure of the cells to caspase inhibitors, which suggests that translocation of cyt c occurred before caspase activation during apoptosis. Fluorescence resonance energy transfer assays indicated that cyt c and IP 3 Rs come into close association after induction of apoptosis with STS. IP 3 Rs are sensitive to calcium such that at concentrations in the nanomolar range (resting levels in a cell), calcium stimulates IP 3 R calcium release. In contrast, at higher micromolar concentrations of calcium, IP 3 R-induced calcium release is inhibited by calcium. Microsomal preparations exposed to cyt c and micromolar concentrations of calcium did not exhibit calcium-induced inhibition of IP 3 R calcium release. In vivo calcium imaging suggested that a spike in calcium precedes cyt c release from the mitochondria and then calcium levels remain elevated. Expression of the cyt c-binding domain of the IP 3 R acted as a dominant negative, preventing calcium oscillations and cyt c release in response to STS. Thus, release of small amounts of cyt c leads to an interaction with the IP 3 R on the ER, preventing calcium inhibition of ER calcium release. The resulting global increase in calcium triggers massive release of cyt c, which then acts in the positive feedback loop to maintain ER calcium release through the IP 3 Rs. M. P. Mattson, S. L. Chan, Calcium orchestrates apoptosis. Nat. Cell Biol. 5 , 1041-1043 (2003). [Online Journal] D. Boehning, L. Patterson, L. Sadaghat, N. O. Blebova, T. Kurosaki, S. H. Snyder, Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat. Cell Biol. 5 , 1051-1061 (2003). [Online Journal]