Disturbance Of Intracellular Calcium Homeostasis Research Articles

Blockage of Orai1-Nucleolin interaction meditated calcium influx attenuates breast cancer cells growth

As an important second messenger, calcium (Ca2+) regulates a wide variety of physiological processes. Disturbance of intracellular calcium homeostasis implicated in the occurrence of multiple types of diseases. Orai1 is the major player in mediating store-operated calcium entry (SOCE) and regulates calcium homeostasis in non-excitable cells. Over-expression and activation of Orai1 have been reported in breast cancer. However, its molecular mechanisms are still not very clear. Here, we demonstrated that Nucleolin (NCL) was a novel interacting partner of Orai1. NCL is a multifunctional nucleocytoplasmic protein and is upregulated in human breast tumors. The binding of C-termini of NCL (NCL-CT) to N-termini of Orai1 (Orai1-NT) is critical for mediating calcium influx and proliferation of breast cancer cells. Blocking the NCL-Orai1 interaction by synthesized Orai1 peptide can effectively reduce the intracellular calcium influx and suppress the proliferation of breast cancer cells in vitro and in vivo. Our findings reveal a novel activation mechanism of Orai1 via direct interaction with NCL, which may lead to calcium homeostasis imbalance and promote the proliferation of breast cancer cells. Blocking NCL-Orai1 interaction might be an effective treatment of breast cancer.

DNA Methylation-Dependent Gene Expression Regulation of Glutamate Transporters in Cultured Radial Glial Cells.

Exposure to xenobiotics has a significant impact in brain physiology that could be liked to an excitotoxic process induced by a massive release of the main excitatory neurotransmitter, L-glutamate. Overstimulation of extra-synaptic glutamate receptors, mainly of the N-methyl-D-aspartate subtype leads to a disturbance of intracellular calcium homeostasis that is critically involved in neuronal death. Hence, glutamate extracellular levels are tightly regulated through its uptake by glial glutamate transporters. It has been observed that glutamate regulates its own removal, both in the short-time frame via a transporter-mediated decrease in the uptake, and in the long-term through the transcriptional control of its gene expression, a process mediated by glutamate receptors that involves the Ca2+/diacylglycerol-dependent protein kinase and the transcription factor Ying Yang 1. Taking into consideration that this transcription factor is a member of the Polycomb complex and thus, part of repressive and activating chromatin remodeling factors, it might direct the interaction of DNA methyltransferases or dioxygenases of methylated cytosines to their target sequences. Here we explored the role of dynamic DNA methylation in the expression and function of glial glutamate transporters. To this end, we used the well-characterized models of primary cultures of chick cerebellar Bergmann glia cells and a human retina-derived Müller glia cell line. A time and dose-dependent increase in global DNA methylation was evident upon glutamate exposure. Under hypomethylation conditions, the glial glutamate transporter protein levels and uptake activity were increased. These results favor the notion that a dynamic DNA methylation program triggered by glutamate in glial cells modulates one of its major functions: glutamate removal.

Bisphenol-A induces neurodegeneration through disturbance of intracellular calcium homeostasis in human embryonic stem cells-derived cortical neurons

Bisphenol-A (BPA) is a representative exogenous endocrine disruptor, which is extensively composed in plastic products. Due to the capability of passing through the blood-brain barrier, evidence has linked BPA exposure with multiple neuropsychological dysfunctions, neurobehavioral disorders and neurodegenerative diseases. However, the underlying mechanism by which BPA induces neurodegeneration still remains unclear. Our study used human embryonic stem cells-derived human cortical neurons (hCNs) as a cellular model to investigate the adverse neurotoxic effects of BPA. hCNs were treated with 0, 0.1, 1 and 10 μM BPA for 14 days. Impacts of BPA exposure on cell morphology, cell viability and neural marker (MAP2) were measured for evaluating the neurodegeneration. The intracellular calcium homeostasis, reactive oxygen species (ROS) generation and organelle functions were also taken into consideration. Results revealed that chronic exposure of BPA damaged the neural morphology, induced neuronal apoptosis and decreased MAP2 expression at the level of both transcription and translation. The intracellular calcium levels were elevated in hCNs after BPA exposure through NMDARs-nNOS-PSD-95 mediating. Meanwhile, BPA led to oxidative stress by raising the ROS generation and attenuating the antioxidant defense in hCNs. Furthermore, BPA triggered ER stress and increased cytochrome c release by impairing the mitochondrial function. Ultimately, BPA triggered the cell apoptosis by regulating Bcl-2 family and caspase-dependent signaling pathway. Taken together, BPA exerted neurotoxic effects on hCNs by eliciting apoptosis, which might due to the intracellular calcium homeostasis perturbation and cell organellar dysfunction.

Nanoparticles and neurotoxicity: Dual response of glutamatergic receptors.

Although the use of nanoparticles for neuro-diagnostic and neurotherapeutic purposes provides superior benefits than the conventional approaches, it may be potentially toxic in central nervous system. In this respect, nanotechnological research focuses on nanoneurotoxicity-nanoneurosafety concepts. Despite these efforts, nanoparticles (NPs) may cause neurotoxicity, neuroinflammation, and neurodegeneration by penetrating the brain-olfactory route and blood-brain barrier (BBB). Indeed, due to their unique structures nanomaterials can easily cross biological barriers, thus avoid drug delivery problems. Despite the advancement of nanotechnology for designing therapeutic agents, toxicity of these nanomaterials is still a concern. Activation of neurons by astrocytic glutamate is a result of NPs-mediated astrocyte-neuron crosstalk. Increased extracellular glutamate levels due to enhanced synthesis and reduced reuptake may induce neuronal damage by abnormal activation of extrasynaptic N-methyl d-aspartate receptor (NMDAR) subunits. NMDAR is the key factor that mediates the disturbances in intracellular calcium homeostasis, mitochondrial dysfunction and generation of reactive oxygen species in NPs exposed neurons. While some NPs cause neuronal death by inducing NMDARs, others may be neurotoxic through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors or protect the neurons via blocking NMDARs. However, mechanisms of dual effects of NPs, neurotoxicity or neuroprotection are not precisely known. Some NPs present neuroprotective effect either by selectively inhibiting extrasynaptic subunit of NMDARs or by attenuating oxidative stress. NPs-related proinflammatory activation of microglia contributes to the dysfunction and cytotoxicity in neurons. Therefore, investigation of the interaction of NPs with the neuronal signaling molecules and neuronal receptors is necessary for the better understanding of the neurotoxicity or neurosafety of nanomaterials.

Antioxidant supplementation upregulates calbindin expression in cerebellar Purkinje cells of rat pups subjected to post natal exposure to sodium arsenite

Optimal cytoplasmic calcium (Ca2+) levels have been associated with adequate cell functioning and neuronal survival. Altered intracellular Ca2+ levels following impaired Ca2+ homeostasis could induce neuronal degeneration or even cell death. There are reports of arsenite induced oxidative stress and the associated disturbances in intracellular calcium homeostasis. The present study focused on determining the strategies that would modulate tissue redox status and calcium binding protein (CaBP) (Calbindin D28k-CB) expression affected adversely by sodium arsenite (NaAsO2) exposure (postnatal) of rat pups.NaAsO2 alone or along with antioxidants (AOXs) (alpha lipoic acid or curcumin) was administered by intraperitoneal (i.p.) route from postnatal day (PND) 1–21 (covering rapid brain growth period – RBGP) to experimental groups and animals receiving sterile water by the same route served as the controls. At the end of the experimental period, the animals were subjected to euthanasia and the cerebellar tissue obtained therefrom was processed for immunohistochemical localization and western blot analysis of CB protein. CB was diffusely expressed in cell body as well as dendritic processes of Purkinje cells (PCs) along the PC Layer (PCL) in all cerebellar folia of the control and the experimental animals. The multilayered pattern of CB +ve cells along with their downregulated expression and low packing density was significantly evident in the arsenic (iAs) alone exposed group as against the controls and AOX supplemented groups.The observations are suggestive of AOX induced restoration of CaBP expression in rat cerebellum following early postnatal exposure to NaAsO2.

Anesthesia-induced neurotoxicity in an animal model of the developing brain: mechanism and therapies.

Children are being exposed to an increasingly greater variety of anesthetics with advances in pediatric and obstetric surgery. Recent animal and retrospective human data suggest that the general anesthetics commonly used in pediatric medicine could be damaging to the developing brain when used at clinical concentrations. In vivo primate and rodent models have shown that neonatal exposure to clinical concentrations of anesthetics causes neural apoptosis and long-term cognitive impairment. Many general anesthetics, such as isoflurane, sevoflurane, barbiturates, benzodiazepines, ketamine, propofol, and nitrous oxide, cause adverse changes in the neonatal rodent and primate brain. Animal and human data suggest an association between general anesthesia during the neonatal period and long-term cognitive impairment. Cohort studies involving humans have recently been started. The window of vulnerability to these neurotoxic effects of anesthetics is restricted to the period of synaptogenesis, also known as the “brain growth spurt” (BGS) period. To minimize the risk of neurodegeneration, it is necessary to study both the mechanism of neurotoxicity and preventative medicine. Neonatal anesthetic exposure affects many mechanisms of neurotoxicity. Mechanisms of anesthetic-induced neurotoxicity seem to involve altered expression of ligand-gated ion channels, disturbance of intracellular calcium homeostasis, and the mitochondria-mediated apoptotic pathway. Several agents reportedly help to prevent anesthesia-induced neurotoxicity, including hydrogen, melatonin, apocynin, and ketorolac, and should thus be co-administered with anesthetics. After anesthesia, only environmental enrichment can improve learning deficits due to anesthesia-induced neurotoxicity. Further studies of environmental enrichment (Wu et al., 2016) after anesthesia are necessary to develop preventative and therapeutic strategies for anesthesia-induced neurotoxicity.

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague–Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.

Ang-(1–7) Offers Cytoprotection Against Ischemia–Reperfusion Injury by Restoring Intracellular Calcium Homeostasis

Accumulating evidence indicates that angiotensin-(1-7) [Ang-(1-7)] offers protective effects against ischemia-reperfusion (I/R) induced arrhythmias and contractile dysfunction, which are related to disturbances of intracellular calcium homeostasis. However, whether or not Ang-(1-7) regulates intracellular calcium in I/R is not clear. To shed light on this issue, we carried out studies with a cellular model of simulated I/R in isolated rat ventricular myocytes and measured calcium transients using laser scanning confocal microscopy. Our results showed that Ang-(1-7) had no effects on the calcium transient in myocytes superfused with normal solution; however, in myocytes of simulated I/R, Ang-(1-7) significantly attenuated the increased diastolic intracellular Ca during reperfusion, restored the decreased peak Ca of calcium transient during ischemia, and reversed the decreased amplitude of calcium transient throughout the I/R periods. Additionally, Ang-(1-7) significantly suppressed the reactive oxygen species production in I/R, especially during the ischemic phase. These data indicated that Ang-(1-7) affords significant cytoprotective effects through directly improving calcium homeostasis independent of its anti-oxidative action. Most notably, the effects of Ang-(1-7) on intracellular Ca dynamics manifests only in the diseased states, that is, I/R. This unique property suggests that upregulation of Ang-(1-7) expression and/or activation of the Ang-(1-7)/Mas signaling cascade is a highly desirable strategy for the treatment of myocardial impairment induced by I/R.

Disturbance of Intracellular Calcium Homeostasis and CaMKII/CREB Signaling is Associated with Learning and Memory Impairments Induced by Chronic Aluminum Exposure

Aluminum-induced neuronal injury has been implicated in various neurodegenerative disorders. However, the underlying mechanism involved in this pathogenesis still remains unknown. Our present findings demonstrated that chronic aluminum exposure resulted in spatial learning impairment and significantly increased intracellular calcium level in the hippocampus of rats. Examination of the associated protein molecules essential for induction and maintenance of long-term potentiation revealed that aluminum exposure could increase the expression level of calmodulin (CaM), but the expression levels of CaM-dependent protein kinase II (CaMKII), and phosphorylated cAMP-responsive element binding protein (CREB) were significantly reduced, whereas the total protein levels of CaMKII and CREB did not change in the aluminum-treated hippocampus. Thus, we provide a previously unrecognized mechanism whereby chronic aluminum exposure impairs hippocampal learning and memory, at least in part, through disruption of intracellular calcium homeostasis and CaM/CaMKII/CREB signaling pathway.

Role of Intracellular Calcium in Proteasome Inhibitor-Induced Endoplasmic Reticulum Stress, Autophagy, and Cell Death

Proteasome inhibition induces endoplasmic reticulum (ER) stress and compensatory autophagy to relieve ER stress. Disturbance of intracellular calcium homeostasis can lead to ER stress and alter the autophagy process. It has been suggested that inhibition of the proteasome disrupts intracellular calcium homeostasis. However, it is unknown if intracellular calcium affects proteasome inhibitor-induced ER stress and autophagy. Human colon cancer HCT116 Bax positive and negative cell lines were treated with MG132, a proteasome inhibitor. BAPTA-AM, a cell permeable free calcium chelator, was used to modulate intracellular calcium levels. Autophagy and cell death were determined by fluorescence microscopy and immunoblot analysis. MG132 increased intracellular calcium levels in HCT116 cells, which was suppressed by BAPTA-AM. MG132 suppressed proteasome activity independent of Bax and intracellular calcium levels in HCT116 cells. BAPTA-AM inhibited MG132-induced cellular vacuolization and ER stress, but not apoptosis. MG132 induced autophagy with normal autophagosome-lysosome fusion. BAPTA-AM seemed not to affect autophagosome-lysosome fusion in MG132-treated cells but further enhanced MG132-induced LC3-II levels and GFP-LC3 puncta formation, which was likely via impaired lysosome function. Blocking intracellular calcium by BAPTA-AM relieved MG132-induced ER stress, but it was unable to rescue MG132-induced apoptosis, which was likely due to impaired autophagic degradation.

Nephroprotective effect of calcium channel blockers against toxicity of lead exposure in mice

Exposure to lead (Pb) can induce kidney damage, which is related to induction of oxidative damage and disturbance of intracellular calcium homeostasis. Pb can readily permeate through dihydropyridine-sensitive L-type calcium channels and accumulate within cells. The objective of this study was to investigate protective effects of calcium channel blockers (CCBs) verapamil and nimodipine on nephrotoxicity induced by Pb acetate in mice. One hundred and twenty male mice were randomly divided into 6 groups: control, Pb, low-dose verapamil, high-dose verapamil, low-dose nimodipine and high-dose nimodipine (n=20 per group). Pb acetate was injected intraperitoneally (i.p.) at 40mg/kg body weight/day for 10 days to establish the Pb toxicity model. While control mice received saline, mice of the treated groups simultaneously received i.p. injections of verapamil or nimodipine daily for 10 days. Both verapamil and nimodipine showed protection against Pb-induced kidney injury, including alleviation of renal pathological damage and decreasing the level of Pb in kidney homogenate and extent of apoptosis in nephrocytes. Moreover, verapamil and nimodipine significantly down-regulated levels of blood urea nitrogen and creatinine in the serum. In addition, verapamil and nimodipine administration decreased malondialdehyde content and increased activities of super oxide dismutase activity and glutathione peroxidase in the kidney homogenate. The findings in the present study implicate the therapeutic potential of CCBs for Pb-induced nephrotoxicity, which were at least partly due to the decrease of Pb uptake and inhibition of lipid peroxidation.

Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty-five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage-gated, type IV alpha subunit (SCN4A), calcium channel voltage-dependent, L type, alpha 1S subunit (CACNA1S), and L-type voltage-gated calcium channel alpha 2/delta-subunit (CACNL2A). Twenty-nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational 'hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia-nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.

Sonodynamic effects of hematoporphyrin monomethyl ether on CNE‐2 cells detected by atomic force microscopy

Hematoporphyrin monomethyl ether (HMME) has been effectively used to treat solid tumors of some types. However, its application in nasopharyngeal carcinoma has not been studied yet. In this paper, the detailed sonodynamic effects of HMME-SDT (sonodynamic therapy) on CNE-2 cells including cell growth inhibition, apoptosis induction, and membrane toxicity were investigated. It was found that HMME alone had less cytotoxicity whereas HMME-SDT could suppress the cell proliferation in a dose-dependent manner as detected by MTT assay. The annexin V-based flow cytometric data indicated that upon SDT, different concentrations of HMME induce distinct types of cell death, apoptosis by low concentration (60 µg/ml) of HMME and necrosis by higher concentration (120 µg/ml). The immunofluorescence of cytoskeleton and nuclei morphology showed that upon HMME-SDT, the cells became rounding and the cytoskeletal network disappeared, and, the nuclei represented a total fragmented morphology of nuclear bodies. These alternations showed the apoptosis induction by HMME-SDT. Further AFM study showed that the cell membrane structure and cytoskeleton networks were destroyed, and, the Young's modulus, tip-cell-surface adhesion force decreased to 0.22 ± 0.11 Mpa, 35.4 ± 12.8 pN of cells with 120 µg/ml HMME-SDT from 0.48 ± 0.21 Mpa, 69.6 ± 22.3 pN of native cells, respectively. These membrane changes caused the collapse of mitochondrial transmembrane potential and disturbance of intracellular calcium homeostasis, which was consistent with the results detected by flow cytometry. Therefore, membrane toxicity and cytoskeleton disrupture induced by HMME-SDT maybe important factors to induce cell apoptosis, and, the disturbance of mitochondrial transmembrane potential and calcium channels might be the apoptosis mechanisms.

FAD mutations in amyloid precursor protein do not directly perturb intracellular calcium homeostasis.

Disturbances in intracellular calcium homeostasis are likely prominent and causative factors leading to neuronal cell death in Alzheimer's disease (AD). Familial AD (FAD) is early-onset and exhibits autosomal dominant inheritance. FAD-linked mutations have been found in the genes encoding the presenilins and amyloid precursor protein (APP). Several studies have shown that mutated presenilin proteins can directly affect calcium release from intracellular stores independently of Aβ production. Although less well established, there is also evidence that APP may directly modulate intracellular calcium homeostasis. Here, we directly examined whether overexpression of FAD-linked APP mutants alters intracellular calcium dynamics. In contrast to previous studies, we found that overexpression of mutant APP has no effects on basal cytosolic calcium, ER calcium store size or agonist-induced calcium release and subsequent entry. Thus, we conclude that mutated APP associated with FAD has no direct effect on intracellular calcium homeostasis independently of Aβ production.

Mutation screening of the RYR1-cDNA from peripheral B-lymphocytes in 15 Swedish malignant hyperthermia index cases

Malignant hyperthermia (MH), linked to the ryanodine receptor 1 gene (RYR1) on chromosome 19, is a potentially lethal pharmacogenetic disorder which may lead to a disturbance of intracellular calcium homeostasis when susceptible individuals are exposed to halogenated anaesthetics, suxamethonium, or both. Central core disease (CCD) is a rare dominantly inherited congenital myopathy allelic to MH-susceptibility. In this study, 14 unrelated MH-susceptible probands and one CCD patient from Sweden were screened for mutations in the RYR1. Since the RYR1 is also expressed in B-lymphocytes, RYR1-cDNA was transcribed from total RNA extracted from white blood cells. We detected two known RYR1 mutations and two previously described unclassified sequence variants. In addition, six novel sequence variants were detected. All mutations or sequence variants were verified on genomic DNA. Seven of the probands did not show any candidate mutation, although the total coding region of RYR1 was sequenced. Segregation data in in vitro contracture tested family members of three probands support a causative role of three of the novel sequence variants. Our study contributes to the genetic aetiology of MH in Sweden, but also raises questions about the involvement of genes other than RYR1 since nearly half of the probands did not show any sequence variants in the total coding region of the RYR1.

P1-381: Anionic interactions are involved in transthyretin (TTR) aggregation and disturbance of intracellular calcium homeostasis as an initial event in TTR-induced cytotoxicity

P1-381: Anionic interactions are involved in transthyretin (TTR) aggregation and disturbance of intracellular calcium homeostasis as an initial event in TTR-induced cytotoxicity

Calcium dysregulation in Alzheimer's disease: Recent advances gained from genetically modified animals

Alzheimer's disease is a progressive and irreversible neurodegenerative disorder that leads to cognitive, memory and behavioural impairments. Two decades of research have implicated disturbances of intracellular calcium homeostasis as playing a proximal pathological role in the neurodegeneration associated with Alzheimer's disease. A large preponderance of evidence has been gained from the use of a diverse range of cell lines. Whilst useful in understanding the principal mechanism of neurotoxicity associated with Alzheimer's disease, technical differences, such as cell type or even the form of amyloid-beta used often underlie conflicting results. In this review, we discuss recent contributions that transgenic technology has brought to this field. For example, the triple transgenic mouse model of Alzheimer's disease has implicated intraneuronal accumulation of the amyloid-beta peptide as an initiating factor in synaptic dysfunction and behavioural deficits. Importantly, this synaptic dysfunction occurs prior to cell loss or extracellular amyloid plaque accumulation. The cause of synaptic dysfunction is unknown but it is likely that amyloid-beta and its ability to disrupt intracellular calcium homeostasis plays a key role in this process.

On the mechanism of callose synthesis induction by metal ions in onion epidermal cells.

Metal ions induce the synthesis of callose in Allium cepa epidermal cells. Callose is deposited as single knoblike local accumulations, aggregates of knobs, or furrowed clusters tightly attached to the cell wall. The most effective metal is copper, it induces callose formation at micromolar concentrations. Agents acting on inositolphosphate metabolism, phospholipase inhibitors, calcium channel inhibitors, modulators of cytoplasmic calcium, or receptor antagonists influence callose synthesis. It is concluded that metal ions, especially Cu(2+), initiate a signal transduction chain by activation of phospholipases and generation of inositol 1,4,5-trisphosphate, and that callose synthesis is a cellular defence reaction caused by the disturbance of intracellular calcium homeostasis.

Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis.

Glutamate toxicity on PC12 cells is mediated by oxidative stress as a consequence of the inhibition of a cystine uptake system with depletion of GSH. In this study we report that glutamate decreases PC12 cell viability, inhibiting the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This decrease was prevented by the antioxidants vitamin E, idebenone and L-deprenyl, which were also shown to be effective in reducing the accumulation of reactive oxygen species (ROS) in cells exposed to glutamate, decreasing the fluorescence of 2',7'-dichlorofluorescein (DCF). Incubation of PC12 cells with high glutamate concentrations induced mitochondrial dysfunction, leading to the loss of mitochondrial transmembrane potential, evaluated as a decrease in rhodamine 123 (Rh123) retention by mitochondria, and to the decrease of intracellular ATP levels. The mitochondrial dysfunction, induced by glutamate, can be involved in the observed increase of [Ca2+]i. The elevation of [Ca2+]i occurred after GSH depletion, suggesting that oxidative stress is involved in the disturbances of intracellular calcium homeostasis. In conclusion, our data indicate that glutamate, at concentrations which block cystine uptake in PC12 cells leading to GSH depletion and inducing oxidative stress, increases ROS accumulation and decreases cell survival by a mechanism involving mitochondrial dysfunction and impairment of Ca2+ homeostasis.

Repair of wounded monolayers of cultured vascular endothelial cells after simultaneous exposure to lead and zinc

We investigated the interaction between lead and zinc on the repair of wounded monolayers of cultured bovine aortic endothelial cells. A half area of the monolayers was wounded and then incubated in the presence of lead (5.0 and 10 μM) and/or zinc (10 μM). It was morphologically observed that the appearance of the cells in the wounded area was strongly decreased by lead alone but considerably increased by zinc alone. The repair of wounded area after simultaneous exposure to lead and zinc showed that lead inhibits not only spontaneous but also zinc-promoted repair of endothelial cell layers without a change of the leakage of lactate dehydrogenase. Interaction between lead and zinc on the DNA synthesis of growing endothelial cells was similar to that on the repair, suggesting that the repair reflected the proliferation. In growing endothelial cells, the intracellular accumulation of lead was significantly increased by zinc; that of zinc was unaffected by lead; and that of metallothionein was slightly increased by zinc and lead but the effect of zinc was suppressed in the presence of lead. Although zinc significantly decreased the intracellular accumulation of radioactive calcium, lead increased it in the presence or absence of zinc. It was therefore concluded that lead inhibits not only spontaneous but also zinc-promoted repair of the damaged endothelial cell layers through an inhibition of the proliferation mediated by the calcium-mediated signalling pathways and/or a disturbance of intracellular calcium homeostasis.