Increase in intracellular Zn concentration by thimerosal in rat thymocytes: Intracellular Zn release induced by oxidative stress

Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn²⁺ concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn²⁺. To reveal the property of TMR-induced increase in intracellular Zn²⁺ concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn²⁺, of rat thymocytes was examined. TMR at concentrations ranging from 0.3 μM to 10 μM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca²⁺- and Zn²⁺-free condition. The threshold concentration was 0.3–1 μM. The increase in the intensity was significant when TMR concentration was 1 μM or more. N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn²⁺, completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H₂O₂) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was −0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H₂O₂ and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn²⁺ concentration via decreasing cellular thiol content.     

金属离子Cu2+和Zn2+诱导β-淀粉样蛋白的聚集

为探讨金属离子Cu²⁺和Zn²⁺对β-淀粉样蛋白 (Αβ) 聚集的影响, 用紫外光谱、荧光光谱和透射电镜等方法, 从Aβ40聚集体的形态、大小和细胞毒性等多个角度研究了Cu²⁺和Zn²⁺对Aβ40聚集的作用。结果表明, Cu²⁺和Zn²⁺都能促进Aβ40的聚集, 并且两者能改变Aβ40聚集体的形态和大小。Zn²⁺诱导Aβ40聚集产生纤维状聚集体, 而Cu²⁺诱导Αβ40聚集产生纤维状和无定形聚集体。此外, Aβ40还原Cu²⁺产生了H₂O₂。本研究结果分析了金属离子与Αβ40聚集的关系, 阐明了金属离子在阿尔兹海默病 (AD) 中的作用。     

Protective effects of β-dihydroagarofuran-type sesquiterpene against Aβ25-35-induced neuronal apoptosis and oxidative damage

Excessive beta-amyloid (Aβ) plays a detrimental role in the pathogenesis of Alzheimer’s disease (AD), which is closely associated with apoptosis and oxidative stress in neurons. Therefore, identification of active small molecules with potent effects on neutralizing Aβ-induced neurotoxicity would be a promising strategy for delaying or preventing AD progression. In the present study, we discovered that pretreatment with CF-1 ((1R,2S,4R,5S,7R,9S,10S)-1,15-diacetoxy-2-benzoyloxy-9-cinnamoyloxy-β-di-hydroagarofuran), a sesquiterpene isolated from the seeds of Celastrus flagellaris, attenuated Aβ_25-35-induced reduction in cell viability in a concentration-dependent manner, as evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Above neuroprotective effect of CF-1 was associated with a significant decrease of apoptotic cells as measured by 4,6-diamidino-2-phenylindole (DAPI) staining, which concurrently happened with marked inhibition in the level of cleaved Caspase-3, an apoptotic executive protein. CF-1 pretreatment also markedly reduced the intracellular accumulation of reactive oxygen species (ROS) following Aβ exposure in SH-SY5Y neuroblastoma cells, but such pretreatment had no notable influence on 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging. In conclusion, we demonstrated that a novel natural product, CF-1, possessed promising effects against Aβ-induced neuronal apoptosis and oxidative stress, which could be a potential drug lead or candidate for the treatment of Aβ-associated neurotoxicity.     

Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons

The study of ischemic tolerance is critical in the development of strategies for the treatment of ischemic stroke. We used the oxygen and glucose deprivation (OGD) paradigm in cultured cortical neurons as an in vitro approach to elucidate the mechanism of protection conferred by glutamate preconditioning. Pretreatment of neurons with N-methyl-d-aspartate (NMDA) receptor antagonists prevented OGD-induced cell death whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and voltage-dependent Ca(++) channel (VDCC) blockers were without effect. Neurons preconditioned with glutamate exhibited resistant to damage induced by OGD. The ischemic tolerance depended on the duration of preconditioning exposure and the interval between preconditioning exposure and test challenge. Protective efficacy was blocked by the NMDA or AMPA receptor antagonists but not by the VDCC blocker. Furthermore, neuroprotective effect was not seen if extracellular Ca(++) was omitted or removed with EGTA. Pretreatment with staurosporin and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93) but not 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002) or 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio] butadiene (U0126) significantly reduced ischemic tolerance. Preconditioning increased phosphorylated levels of cAMP responsive element binding protein (CREB) and pretreatment with CRE-decoy oligonucleotide completely blocked preconditioning-induced increase in cell viability. Importantly, glutamate preconditioning increased Bcl-2 expression that was blocked by KN93, staurosporin and CRE-decoy oligonucleotide. These results suggest that preconditioning with glutamate conferred neuroprotection against subsequent OGD by inducing p-CREB-mediated Bcl-2 expression.     

Royal jelly can diminish secondary neuronal damage after experimental spinal cord injury in rabbits

The aim of this experimental study was to investigate the neuroprotective effect of Royal jelly (RJ) on traumatic spinal cord injury (SCI). Twenty-one New Zealand male rabbits, weighing between 2.5 and 3.0 kg were divided into three groups: Sham (no drug or operation, n = 7), Control (laminectomy + single dose of 1 ml/kg saline orally, after trauma; n = 7) and RJ (laminectomy + 100 mg/kg RJ, orally, after trauma, n = 7). Laminectomy was performed at T10 and balloon catheter was applied extradurally for traumatic SCI. Four and 24 h after surgery, rabbits were evaluated according to the Tarlov scoring system. Blood, cerebrospinal fluid and tissue sample from spinal cord were taken for measurements of antioxidant status or detection of apoptosis. Four hours after SCI, all animals in control or RJ treated groups became paraparesic. Significant improvement was observed in RJ treated group, 24 h after SCI, with respect to control. Traumatic SCI led to increase in the lipid peroxidation and decrease enzymic or non-enzymic endogenous antioxidative defense systems, and increase in apoptotic cell numbers. RJ treatment mostly prevented lipid peroxidation and also augmented endogenous enzymic or non-enzymic antioxidative defense systems. Again, RJ treatment significantly decreased the apoptotic cell number induced by SCI.     

Pramipexole protects against H2O2-induced PC12 cell death

Pramipexole, a novel non-ergot dopamine (DA) agonist, has been successfully applied to the treatment of Parkinson’s disease (PD). Although the specific cause of PD remains unknown, recent studies have provided evidence that oxidative stress plays a role in the parthenogenesis of the disease. In the present study, we examined the effect of pramipexole on hydrogen peroxide (H₂O₂, 100 μM)-induced PC12 cell death, and the intracellular mechanism of this effect. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay revealed that pretreatment of PC12 cells with pramipexole (1–100 μM) resulted in significant protection against H₂O₂-induced cell death in a concentration-dependent manner. The protective effect of pramipexole was not affected by pretreatment with the DA receptor antagonists sulpiride, spiperone or domperidone, suggesting that the effect of pramipexole is not mediated by DA receptors. In PC12 cells, pramipexole inhibited H₂O₂-induced lactate dehydrogenase (LDH) leakage, as well as H₂O₂-induced cytochrome c release and caspase-3 activation with the resultant apoptosis. It was also observed in PC12 cells that H₂O₂ stimulated phosphorylation of mitogen-activated protein (MAP) kinases, i.e., extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH₂-terminal kinase (JNK) and p38 MAP kinase. Pramipexole inhibited H₂O₂-induced JNK and p38 MAP kinase, but not ERK1/2 phosphorylation. Furthermore, in these cells experiments with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, revealed that pramipexole, the JNK inhibitor SP600125 and the p38 MAP kinase inhibitor SB203580 inhibited the generation of H₂O₂-induced reactive oxygen species. Caspase inhibitors Z-DEVD-FMK and Z-IETD-FMK, as well as SP600125 and SB203580, inhibited H₂O₂-induced PC12 cell death to a similar extent as pramipexole. These results suggest that pramipexole exerts a protective effect against oxidative stress-induced PC12 cell death in part through an inhibition of JNK and p38 MAP kinase.     

In vivo treatment with the K+ channel blocker 4-aminopyridine protects against kainate-induced neuronal cell death through activation of NMDA receptors in murine hippocampus

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured neurons in vitro. To elucidate in vivo neuroprotective role of NMDA receptors, we investigated the effects of activation of NMDA receptors by endogenous glutamate on kainate-induced neuronal damage to the mouse hippocampus in vivo. The systemic administration of the K+ channel blocker 4-aminopyridine (4-AP, 5 mg/kg, i.p.) induced expression of c-Fos in the hippocampal neuronal cell layer, which expression was completely abolished by the noncompetitive NMDA receptor antagonist MK-801, thus indicating that the administration of 4-AP would activate NMDA receptors in the hippocampal neurons. The prior administration of 4-AP at 1 h to 1 day before significantly prevented kainate-induced pyramidal cell death in the hippocampus and expression of pyramidal cells immunoreactive with an antibody against single-stranded DNA. Further immunohistochemical study on deoxyribonuclease II revealed that the pretreatment with 4-AP led to complete abolition of deoxyribonuclease II expression induced by kainate in the CA1 and CA3 pyramidal cells. The neuroprotection mediated by 4-AP was blocked by MK-801 and by the adenosine A1 antagonist 8-cyclopenthyltheophylline. Taken together, in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation induced by deoxyribonuclease II in the murine hippocampus.     

EGCG inhibits Cd2+-induced apoptosis through scavenging ROS rather than chelating Cd2+ in HL-7702 cells

Context and objective: Epigallocatechin-3-gallat (EGCG), the major catechin in green tea, shows a potential protective effect against heavy metal toxicity to humans. Apoptosis is one of the key events in cadmium (Cd²⁺)-induced cytotoxicity. Nevertheless, the study of EGCG on Cd²⁺-induced apoptosis is rarely reported. The objective of this study was to clarify the effect and detailed mechanism of EGCG on Cd²⁺-induced apoptosis.

Methods: Normal human liver cells (HL-7702) were treated with Cd²⁺ for 21?h, and then co-treated with EGCG for 3?h. Cell viability, apoptosis, intracellular reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP) and caspase-3 activity were detected. On the other hand, the chelation of Cd²⁺ with EGCG was tested by UV-Vis spectroscopy analysis and Nuclear Magnetic Resonance (¹H NMR) spectroscopy under neutral condition (pH 7.2).

Results and conclusion: Cd²⁺ significantly decreased the cell viability and induced apoptosis in HL-7702 cells. Conversely, EGCG co-treatment resulted in significant inhibition of Cd²⁺-induced reduction of cell viability and apoptosis, implying a rescue effect of EGCG against Cd²⁺ poisoning. The protective effect most likely arises from scavenging ROS and maintaining redox homeostasis, as the generation of intracellular ROS and MDA is significantly reduced by EGCG, which further prevents MMP collapse and suppresses caspase-3 activity. However, no evidence is observed for the chelation of EGCG with Cd²⁺ under neutral condition. Therefore, a clear conclusion from this work can be made that EGCG could inhibit Cd²⁺-induced apoptosis by acting as a ROS scavenger rather than a metal chelating agent.     

Calcium and reactive oxygen species mediated Zn2+ -induced apoptosis in PC12 cells

The release of excessive Zn(2+) from presynaptic boutons into extracellular regions contributes to neuronal apoptotic events, which result in neuronal cell death. However, the mechanisms of Zn(2+)-induced neuronal cell death are still unclear. Therefore, we investigated the dynamics of intracellular Zn(2+), calcium, and reactive oxygen species in PC12 cells. The addition of Zn(2+) produced cell death in a concentration- and time-dependent manner. (45)Ca(2+) influx occurred just after the treatment with Zn(2+), although subsequent hydroxyl radical ((*)OH) production did not begin until 3 h after Zn(2+) exposure. (*)OH production was significantly attenuated in Ca(2+)-free medium or by L-type Ca(2+) channel antagonist treatment, but it was independent of the intracellular Zn(2+) content. Dantrolene treatment had no protective effects against Zn(2+)-induced cell death. Treatment with N-acetyl-L-cysteine blocked (*)OH generation and subsequent cell death. These data indicate that Ca(2+) influx and subsequent (*)OH production are critical events in Zn(2+)-induced toxicity in PC12 cells.     

Involvement of Ca(2+)-induced Ca2+ releasing system in interleukin-1beta-associated adenosine release

Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.     

Role of intracellular Ca2+ signal in the ascorbate-induced apoptosis in a human hepatoma cell line

Although ascorbate (vitamin C) has been shown to have anti-cancer actions, its effect on human hepatoma cells has not yet been investigated, and thus, the exact mechanism of this action is not fully understood. In this study, the mechanism by which ascorbate induces apoptosis using HepG2 human hepatoblastoma cells is investigated. Ascorbate induced apoptotic cell death in a dose-dependent manner in the cells, was assessed through flow cytometric analysis. Contrary to expectation, ascorbate did not alter the cellular redox status, and treatment with antioxidants (N-acetyl cysteine and N,N-diphenyl-p-phenylenediamine) had no influence on the ascorbate-induced apoptosis. However, ascorbate induced a rapid and sustained increase in intracellular Ca2+ concentration. EGTA, an extracellular Ca2+ chelator did not significantly alter the ascorbate-induced intracellular Ca2+ increase and apoptosis, whereas dantrolene, an intracellular Ca2+ release blocker, completely blocked these actions of ascorbate. In addition, phospholipase C (PLC) inhibitors (U-73122 and manoalide) significantly suppressed the intracellular Ca2+ release and apoptosis induced by ascorbate. Collectively, these results suggest that ascorbate induced apoptosis without changes in the cellular redox status in HepG2 cells, and that the PLC-coupled intracellular Ca2+ release mechanism may mediate ascorbate-induced apoptosis.     

Discovery of HDAC Inhibitors That Lack an Active Site Zn2+-Binding Functional Group

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Loss of endoplasmic reticulum Ca2+ homeostasis: contribution to neuronal cell death during cerebral ischemia

The loss of Ca²⁺ homeostasis during cerebral ischemia is a hallmark of impending neuronal demise. Accordingly, considerable cellular resources are expended in maintaining low resting cytosolic levels of Ca²⁺. These include contributions by a host of proteins involved in the sequestration and transport of Ca²⁺, many of which are expressed within intracellular organelles, including lysosomes, mitochondria as well as the endoplasmic reticulum (ER). Ca²⁺ sequestration by the ER contributes to cytosolic Ca²⁺ dynamics and homeostasis. Furthermore, within the ER Ca²⁺ plays a central role in regulating a host of physiological processes. Conversely, impaired ER Ca²⁺ homeostasis is an important trigger of pathological processes. Here we review a growing body of evidence suggesting that ER dysfunction is an important factor contributing to neuronal injury and loss post-ischemia. Specifically, the contribution of the ER to cytosolic Ca²⁺ elevations during ischemia will be considered, as will the signalling cascades recruited as a consequence of disrupting ER homeostasis and function.     

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+ -induced neuronal death in cultured hippocampal neurons

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb(2+) causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb(2+). Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb(2+)-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb(2+) treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 microM) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb(2+). And that Pb(2+)-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb(2+) and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.     

Effect of Zn^2＋ ions on ryanodine binding to sarcoplasmic reticulum of striated muscles in the presence of pyrithione

AIM: To explore whether the differential effects of Zn^2 on ryanodine binding to the sarcoplasmic reticulum (SR)of skeletal and cardiac muscles resulted from different permeability of the SR to Zn^2 . METHODS: [^3H]ryanodinebinding assays were performed to examine the effect of Zn^2 on ryanodine binding to the SR in the presence ofpyrithione sodium (PyNa), a specific Zn^2 ionophore. RESULTS: As a control, PyNa up to 50 μmol/L did notinduce any effect on ryanodine binding to the SR of cardiac muscle. But PyNa 1-100 μnol/L increased ryanodinebinding in skeletal muscle with maximum binding (222.2 % 20.9 % of the control) and inhibited ryanodine bindingto 50 % of the control at about 500 μmol/L. In the presence of PyNa 10 and 50 μnol/L the dose-dependence of theeffect of Zn^2 in cardiac muscle was still monophasic and not changed by PyNa, while the biphasic effect of Zn^2 in skeletal muscle became monophasic. CONCLUSION: Different permeability of the SR to Zn^2 may account forthe differential effects of Zn^2 on ryanodine binding in skeletal and cardiac muscles. PyNa is not a strictly specific Zn^2 ionophore.     

The influence of humic acid on the toxicity of nano‐ZnO and Zn2+ to the Anabaena sp.

This study explored the effects of humic acid (HA) on the toxicity of ZnO nanoparticles (nano‐ZnO) and Zn²⁺ to Anabaena sp. Typical chlorophyll fluorescence parameters, including effective quantum yield, photosynthetic efficiency and maximal electron transport rate, were measured by a pulse‐amplitude modulated fluorometer. Results showed that nano‐ZnO and Zn²⁺ could inhibit Anabaena sp. growth with the EC₅₀ (concentration for 50% of maximal effect) of 0.74 ± 0.01 and 0.3 ± 0.01 mg/L, respectively. In the presence of 3.0 mg/L of HA, EC₅₀ of nano‐ZnO increased to 1.15 ± 0.04 mg/L and EC₅₀ of Zn²⁺ was still 0.3 ± 0.01 mg/L. Scanning electron microscopy observation revealed that HA prevented the adhesion of nano‐ZnO on the algae cells due to the increased electrostatic repulsion. The generation of intracellular reactive oxygen species and cellular lipid peroxidation were significantly limited by HA. Nano‐ZnO had more damage to the cell membrane than Zn²⁺ did, which could be proven by the malondialdehyde content in Anabaena sp. cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 895–903, 2015.     

Natural iron chelators: Protective role in A549 cells of flavonoids-rich extracts of Citrus juices in Fe3+-induced oxidative stress

Exogenous iron in particulate matter and imbalanced iron homeostasis cause deleterious effects on health. Natural and synthetic iron chelators may be of therapeutic benefit, therefore we evaluated the protective effect of Citrus flavonoids-rich extracts from bergamot and orange juices in iron overloaded human lung epithelial cells.Cytofluorimetric, biochemical and genotoxic analyses were performed in Fe₂(SO₄)₃ exposed A549, pretreated with each extract whose chemical composition was previously detected. Chelating activity was assessed in cells by a calcein ester.Both extracts reduced the generation of reactive oxygen species and membrane lipid peroxidation, improved mitochondrial functionality, and prevented DNA-oxidative damage in iron-exposed cells. Antioxidant effects were attributed to the chelating property, blocking upstream the redox activity of iron. Flavonoid-rich extracts also induced antioxidant catalase.The bergamot and orange juice extracts had a broad-spectrum protective effect. Their use prevents iron oxidative injury and these natural iron chelators could be used as therapeutic agents.     

S-2474, a novel nonsteroidal anti-inflammatory drug, rescues cortical neurons from human group IIA secretory phospholipase A(2)-induced apoptosis

The elevated level of group IIA secretory phospholipase A(2) (sPLA(2)-IIA) activity contributes to neurodegeneration in the cerebral cortex after ischemia. The up-regulation of cyclooxygenase-2 (COX-2) is also relevant to cerebral ischemia in humans. Studies of ischemia with COX-2 inhibitors suggest a clinical benefit. In the present study, we investigated effects of S-2474 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons, which was established as an in vitro model of brain ischemia. S-2474 is a novel nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 and contains the di-tert-butylphenol antioxidant moiety. S-2474 significantly prevented neurons from undergoing sPLA(2)-IIA-induced cell death. S-2474 completely ameliorated sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. sPLA(2) also generated neurotoxic prostaglandin D(2) (PGD(2)) and free radicals from neurons before cell death. S-2474 significantly inhibited the sPLA(2)-IIA-induced generation of PGD(2). The present cortical cultures contained few non-neuronal cells, indicating that S-2474 affected neuronal survival directly, but not indirectly via non-neuronal cells. The inhibitory effect of S-2474 on COX-2 might contribute to its neuroprotective effect. In conclusion, S-2474 exhibits neuroprotective effects against sPLA(2)-IIA. Furthermore, the present study suggests that S-2474 may possess therapeutic potential for stroke via ameliorating neurodegeneration.     

Ca2+-induced Ca2+ Release from Internal Stores in INS-1 Rat Insulinoma Cells

The secretion of insulin from pancreatic β-cells is triggered by the influx of Ca²⁺ through voltage-dependent Ca²⁺ channels. The resulting elevation of intracellular calcium ([Ca²⁺]_i) triggers additional Ca²⁺ release from internal stores. Less well understood are the mechanisms involved in Ca²⁺ mobilization from internal stores after activation of Ca²⁺ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic β-cell line, INS-1 cells. To measure cytosolic and stored Ca²⁺, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. [Ca²⁺]_i was repetitively increased by caffeine stimulation in normal Ca²⁺ buffer. However, peak [Ca²⁺]_i was only observed after the first caffeine stimulation in Ca²⁺ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in [Ca²⁺]_i were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca²⁺ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca²⁺ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca²⁺ release from internal stores was activated by caffeine, Ca²⁺, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic β-cells.     

Modulation of intracellular Ca2+ signalling in HeLa cells by the apoptotic cell death enhancer PK11195

1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a proven enhancer of apoptotic cell death in a variety of cellular models. This effect is independent of its established cellular target, the mitochondrial benzodiazepine receptor (mBzR), since it is able to promote cell death also in mBzR knockout cells. Thus recently it was suggested that PK11195 might exert its effect by modulating the expression and function of the oncogene Bcl-2. We have previously demonstrated that Bcl-2 modulates cellular Ca²⁺ homeostasis as its overexpression reduces the Ca²⁺ concentration in the endoplasmic reticulum (ER) ([Ca²⁺]_er), impairing mitochondrial and cytosolic Ca²⁺ overload during cellular stress and therefore inhibiting the induction of the apoptotic cascade. Here, using ER, mitochondria and cytosolic targeted aequorin probes, we show that cellular treatment with PK11195 induces opposite changes in cellular Ca²⁺ homeostasis, increasing the [Ca²⁺]_er and amplifying IP₃ induced Ca²⁺ transients in mitochondria ([Ca²⁺]_m) and cytosol ([Ca²⁺]_c). This work provides evidence for a novel pharmacological effect of PK11195 on Ca²⁺ signalling which may be linked to its effect on Bcl-2 and account for its role in apoptotic cell death.