NK cell-induced cytotoxicity is dependent on a Ca2+ increase in the target

In previous work we showed that programmed cell death (PCD) in thymocytes is mediated by a sustained increase in cytosolic Ca2+ concentration, resulting in the activation of an endogenous endonuclease, DNA fragmentation, and cell death. In this study we investigated the roles of Ca2+ and DNA fragmentation in target cell killing by natural killer (NK) cells. The effector cells induced a rapid, sustained increase in cytosolic Ca2+ concentration in Jurkat target cells. Buffering the target cell cytosolic Ca2+ with the Ca2(+)-selective dye, quin-2, prevented target cell killing. Extensive DNA fragmentation was associated with killing in every target tested, and this response was also blocked by quin-2. The endonuclease inhibitor, aurintricarboxylic acid, inhibited both DNA fragmentation and killing without influencing the Ca2+ increase in target cells. Thus, it is concluded that NK cell killing depends on a Ca2+ increase and appears to involve endogenous endonuclease activation in target cells.     

PGE2-induced apoptotic cell death in K562 human leukaemia cells.

Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells (apoptosis) and enucleated erythrocytes (eryptosis). In erythrocytes PGE2 induced suicidal cell death involves activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. The present study was performed to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from Fluo3 fluorescence, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 human leukaemia cells with PGE2 (50 microM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin binding, led to caspase activation and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels, which are activated by PGE2 and participate in the triggering of apoptosis.     

Calcium binding of ARC mediates regulation of caspase 8 and cell death

Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca(2+)-dependent regulator of caspase 8 and cell death. We found that in Ca(2+) overlay and Stains-all assays, ARC protein bound to Ca(2+) through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca(2+) transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca(2+)-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca(2+)-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca(2+) concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.     

Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca(2+)](c) leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca(2+)](c) and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca(2+) mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca(2+)](c) which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca(2+)-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca(2+)](c) was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.     

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.     

Ca2+ signaling, mitochondria and cell death

In the complex interplay that allows different signals to be decoded into activation of cell death, calcium (Ca2+) plays a significant role. In all eukaryotic cells, the cytosolic concentration of Ca2+ ions ([Ca2+]c) is tightly controlled by interactions among transporters, pumps, channels and binding proteins. Finely tuned changes in [Ca2+]c modulate a variety of intracellular functions ranging from muscular contraction to secretion, and disruption of Ca2+ handling leads to cell death. In this context, Ca2+ signals have been shown to affect important checkpoints of the cell death process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the evidence supporting the involvement of Ca2+ in the three major process of cell death: apoptosis, necrosis and autophagy (ii) the complex signaling interplay that allows cell death signals to be decoded into mitochondria as messages controlling cell fate.     

Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases.

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.     

Ca(2+)-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor-1 (APAF-1)

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro-apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease-activating factor-1 (APAF-1). This complex activates procaspase-9 in the presence of dATP, resulting in caspase-mediated execution of apoptotic neuron death. Many forms of Ca(2+)-mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca(2+) induced prominent degradation of APAF-1 in human SH-SY5Y neuroblastoma cells and in a neuronal cell-free apoptosis system. Loss of APAF-1 correlated with a reduced ability of cytochrome c to activate caspase-3-like proteases. Ca(2+) induced the activation of calpains, monitored by the cleavage of full-length alpha-spectrin into a calpain-specific 150-kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF-1 degradation also occurred via calpain-independent pathways. Our data suggest that Ca(2+) inhibits caspase activation during Ca(2+)-mediated neuron death by triggering the degradation of the cytochrome c-binding protein APAF-1.     

Early-phase occurrence of K+ and Cl? efflux in addition to Ca2+ mobilization is a prerequisite to apoptosis in HeLa cells

Sustained rise in cytosolic Ca(2+) and cell shrinkage mainly caused by K(+) and Cl(-) efflux are known to be prerequisites to apoptotic cell death. Here, we investigated how the efflux of K(+) and Cl(-) as well as the rise in cytosolic Ca(2+) occur prior to caspase activation and are coupled to each other in apoptotic human epithelial HeLa cells. Caspase-3 activation and DNA laddering induced by staurosporine were abolished by blockers of K(+) and Cl(-) channels or cytosolic Ca(2+) chelation. Staurosporine induced decreases in the intracellular free K(+) and Cl(-) concentrations ([K(+)](i) and [Cl(-)](i)) in an early stage prior to caspase-3 activation. Staurosporine also induced a long-lasting rise in the cytosolic free Ca(2+) concentration. The early-phase decreases in [K(+)](i) and [Cl(-)](i) were completely prevented by a blocker of K(+) or Cl(-) channel, but were not affected by cytosolic Ca(2+) chelation. By contrast, the Ca(2+) response was abolished by a blocker of K(+) or Cl(-) channel. Strong hypertonic stress promptly induced a cytosolic Ca(2+) increase lasting >50 min together with sustained shrinkage and thereafter caspase-3 activation after 4 h. The hypertonic stress induced slight increases in [K(+)](i) and [Cl(-)](i) in the first 50 min, but these increases were much less than the effect of shrinkage-induced condensation, indicating that K(+) and Cl(-) efflux took place. Hypertonicity induced caspase-3 activation that was prevented not only by cytosolic Ca(2+) chelation but also by K(+) and Cl(-) channel blockers. Thus, it is concluded that not only Ca(2+) mobilization but early-phase efflux of K(+) and Cl(-) are required for caspase activation, and Ca(2+) mobilization is a downstream and resultant event of cell shrinkage in both staurosporine- and hypertonicity-induced apoptosis.     

Arachidonic acid released by phospholipase A(2) activation triggers Ca(2+)-dependent apoptosis through the mitochondrial pathway

We studied the effects of the divalent cation ionophore A23187 on apoptotic signaling in MH1C1 cells. Addition of A23187 caused a fast rise of cytosolic Ca(2+) ([Ca(2+)](c)), which returned close to the resting level within about 40 s. The [Ca(2+)](c) rise was immediately followed by phospholipid hydrolysis, which could be inhibited by aristolochic acid or by pretreatment with thapsigargin in Ca(2+)-free medium, indicating that the Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) was involved. These early events were followed by opening of the mitochondrial permeability transition pore (PTP) and by apoptosis in about 30% of the cell population. In keeping with a cause-effect relationship between addition of A23187, activation of cPLA(2), PTP opening, and cell death, all events but the [Ca(2+)](c) rise were prevented by aristolochic acid. The number of cells killed by A23187 was doubled by treatment with 0.5 microm MK886 and 5 microm indomethacin, which inhibit arachidonic acid metabolism through the 5-lipoxygenase and cyclooxygenase pathway, respectively. Consistent with the key role of free arachidonic acid, its levels increased within minutes of treatment with A23187; the increase being more pronounced in the presence of MK886 plus indomethacin. Cell death was preceded by cytochrome c release and cleavage of caspase 9 and 3, but not of caspase 8. All these events were prevented by aristolochic acid and by the PTP inhibitor cyclosporin A. Thus, A23187 triggers the apoptotic cascade through the release of arachidonic acid by cPLA(2) in a process that is amplified when transformation of arachidonic acid into prostaglandins and leukotrienes is inhibited. These findings identify arachidonic acid as the causal link between A23187-dependent perturbation of Ca(2+) homeostasis and the effector mechanisms of cell death.     

Mitochondrial modulation of calcium signaling at the initiation of development

Fertilization triggers cytosolic Ca(2+) oscillations that activate mammalian eggs and initiate development. Extensive evidence demonstrates that Ca(2+) is released from endoplasmic reticulum stores; however, less is known about how the increased Ca(2+) is restored to its resting level, forming the Ca(2+) oscillations. We investigated whether mitochondria also play a role in activation-associated Ca(2+) signaling. Mitochondrial dysfunction induced by the mitochondrial uncoupler FCCP or antimycin A disrupted cytosolic Ca(2+) oscillations, resulting in sustained increase in cytosolic Ca(2+), followed by apoptotic cell death. This suggests that functional mitochondria may participate in sequestering the released Ca(2+), contributing to cytosolic Ca(2+) oscillations and preventing cell death. By centrifugation, mouse eggs were stratified and separated into fractions containing both endoplasmic reticulum and mitochondria and fractions containing endoplasmic reticulum with no mitochondria. The former showed Ca(2+) oscillations by activation, whereas the latter exhibited sustained elevation in cytosolic Ca(2+) but no Ca(2+) oscillations, suggesting that mitochondria take up released cytosolic Ca(2+). Further, using Rhod-2 for detection of mitochondrial Ca(2+), we found that mitochondria exhibited Ca(2+) oscillations, the frequency of which was not different from that of cytosolic Ca(2+) oscillations, indicating that mitochondria are involved in Ca(2+) signaling during egg activation. Therefore, we propose that mitochondria play a crucial role in Ca(2+) signaling that mediates egg activation and development, and apoptotic cell death.     

Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C

Glucocorticoid hormones and Ca2+ ionophores stimulate a suicide process in immature thymocytes, known as apoptosis or programmed cell death, that involves extensive DNA fragmentation. We have recently shown that a sustained increase in cytosolic Ca2+ concentration stimulates DNA fragmentation and cell killing in glucocorticoid- or ionophore-treated thymocytes. However, a sustained increase in the cytosolic Ca2+ level also mediates lymphocyte proliferation, suggesting that apoptosis is blocked in proliferating thymocytes. In this study we report that phorbol esters, which selectively stimulate protein kinase C (PKC), blocked DNA fragmentation and cell death in thymocytes exposed to Ca2+ ionophore or glucocorticoid hormone. The T cell mitogen, concanavalin A, which stimulates thymocytes by a mechanism that involves PKC activation, caused concentration-dependent increases in the cytosolic Ca2+ level that did not result in DNA fragmentation, but incubation with concanavalin A and the PKC inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) resulted in both DNA fragmentation and cell death. Phorbol ester directly inhibited Ca2+-dependent DNA fragmentation in isolated thymocyte nuclei. Our results strongly suggest that PKC activation blocks thymocyte apoptosis by preventing Ca2+-stimulated endonuclease activation.     

Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.     

Hypochlorous acid induces apoptosis of cultured cortical neurons through activation of calpains and rupture of lysosomes

3-Chlorotyrosine, a bio-marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimer's disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic-necrotic continuum of concentration-dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 microm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa alpha-fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of alpha-fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl-induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca²⁺ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca²⁺ as well as PG synthesis. The increase of cytosolic Ca²⁺ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca²⁺ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGIA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca²⁺, whereas TMB-8 (10 μM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca²⁺, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca²⁺.These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca²⁺ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca²⁺ and on the other hand, PG synthesis.     

A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis.

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.     

Ca2+ store depletion and endoplasmic reticulum stress are involved in P2X7 receptor-mediated neurotoxicity in differentiated NG108-15 cells

P2X7 receptor (P2X7R) activation by extracellular ATP triggers influx of Na(+) and Ca(2+), cytosolic Ca(2+) overload and consequently cytotoxicity. Whether disturbances in endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress are involved in P2X7R-mediated cell death is unknown. In this study, a P2X7R agonist (BzATP) was used to activate P2X7R in differentiated NG108-15 neuronal cells. In a concentration-dependent manner, application of BzATP (10-100 μM) immediately raised cytosolic Ca(2+) concentration ([Ca(2+)]i) and caused cell death after a 24-h incubation. P2X7R activation for 2 h did not cause cell death but resulted in a sustained reduction in ER Ca2+ pool size, as evidenced by a diminished cyclopiazonic acid-induced Ca(2+) discharge (fura 2 assay) and a lower fluorescent signal in cells loaded with Mag-fura 2 (ER-specific Ca(2+)-fluorescent dye). Furthermore, P2X7R activation (2 h) led to the appearance of markers of ER stress [phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF2α) and C/EBP homologous protein (CHOP)] and apoptosis (cleaved caspase 3). Xestospongin C (XeC), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor (IP3R), strongly inhibited BzATP-triggered [Ca(2+)]i elevation, suggesting that the latter involved Ca(2+) release via IP3R. XeC pretreatment not only attenuated the reduction in Ca(2+) pool size in BzATP-treated cells, but also rescued cell death and prevented BzATP-induced appearance of ER stress and apoptotic markers. These novel observations suggest that P2X7R activation caused not only Ca(2+) overload, but also Ca(2+) release via IP3R, sustained Ca(2+) store depletion, ER stress and eventually apoptotic cell death.     

Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells

Abnormal T cell activation and cell death underlie the pathology of systemic lupus erythematosus. Although mitochondrial hyperpolarization (MHP) represents an early and reversible checkpoint of T cell activation and apoptosis, lupus T cells exhibit persistent MHP. NO has recently been recognized as a key signal of mitochondrial biogenesis and mediator of MHP in human T lymphocytes. In this study, we show that persistent MHP was associated with increased mitochondrial mass (+47.7 +/- 2.8%; p = 0.00017) and increased mitochondrial (+21.8 +/- 4.1%; p = 0.016) and cytoplasmic Ca2+ content in T cells from 19 systemic lupus erythematosus patients with respect to 11 control donors (+38.0 +/- 6.4%; p = 0.0023). Electron microscopy revealed that lupus lymphocytes contained 8.76 +/- 1.0 mitochondria, while control donors contained 3.18 +/- 0.28 mitochondria per cell (p = 0.0009). Increased mitochondrial mass in T cells was associated with 2.08 +/- 0.09-fold enhanced NO production by lupus monocytes (p = 0.0023). Activation of T cells through the TCR initiates a biphasic elevation in cytosolic free Ca2+ concentration, a rapid initial peak observed within minutes, and a plateau phase lasting up to 48 h. In response to CD3/CD28 costimulation, rapid Ca2+ fluxing was enhanced while the plateau phase was diminished in lupus T cells. NO-induced mitochondrial biogenesis in normal T cells enhanced the rapid phase and reduced the plateau of Ca2+ influx upon CD3/CD28 costimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Mitochondria constitute major Ca2+ stores and NO-dependent mitochondrial biogenesis may account for altered Ca2+ handling by lupus T cells.