Perturbation Of Intracellular Calcium Homeostasis Research Articles

Effect of PEGylated Magnetic PLGA-PEI Nanoparticles on Primary Hippocampal Neurons: Reduced Nanoneurotoxicity and Enhanced Transfection Efficiency with Magnetofection

Despite broad application of nanotechnology in neuroscience, the nanoneurotoxicity of magnetic nanoparticles in primary hippocampal neurons remains poorly characterized. In particular, understanding how magnetic nanoparticles perturb neuronal calcium homeostasis is critical when considering magnetic nanoparticles as a nonviral vector for effective gene therapy in neuronal diseases. Here, we address the pressing need to systematically investigate the neurotoxicity of magnetic nanoparticles with different surface charges in primary hippocampal neurons. We found that unlike negative and neutral nanoparticles, positively charged magnetic nanoparticles (magnetic poly(lactic-co-glycolic acid) (PLGA)-polyethylenimine (PEI) nanoparticles, MNP-PLGA-PEI NPs) rapidly elevated cytoplasmic calcium levels in primary hippocampal neurons, mainly via extracellular calcium influx regulated by voltage-gated calcium channels. We went on to show that this perturbation of intracellular calcium homeostasis elicited serious cytotoxicity in primary hippocampal neurons. However, our next experiment demonstrated that PEGylation on the surface of MNP-PLGA-PEI NPs shielded the surface charge, thereby preventing the perturbation of intracellular calcium homeostasis. That is, PEGylated MNP-PLGA-PEI NPs reduced nanoneurotoxicity. Importantly, biocompatible PEGylated MNP-PLGA-PEI NPs under an external magnetic field enhanced transfection efficiency (>7%) of plasmid DNA encoding GFP in primary hippocampal neurons compared to NPs without external magnetic field mediation. Moreover, under an external magnetic field, this system achieved gene transfection in the hippocampus of the C57 mouse. Overall, this study is the first to successfully employ biocompatible PEGylated MNP-PLGA-PEI NPs for transfection using a magnetofection strategy in primary hippocampal neurons, thereby providing a nanoplatform as a new perspective for treating neuronal diseases or modulating neuron activities.

Endoplasmic reticulum stress responses in mouse models of Alzheimer's disease: Overexpression paradigm versus knockin paradigm

Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimer's disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused by AD pathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App-knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because of Aβ pathology. Thorough examinations indicated that no ER stress responses arose in App-KI or single APP-Tg mice. These results suggest that PS1 overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology-induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies.

Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach.

MicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses. With the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation. Results of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3'-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress. The finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.

Cell Membrane Injury Induced by Silica Nanoparticles in Mouse Macrophage

Studies on the cytotoxic mechanism of SiO2 nanoparticles is of vital importance for estimating the potential risk of overexposure and cytotoxicity amelioration and safety precautions of SiO2 nanoparticles. The cell membrane injury induced by 20-nm silica nanoparticles was investigated by measuring reactive oxygen species (ROS), membrane fluidity and free Ca2+ content ([Ca2+]i) in cells with laser scanning confocal microscope (LSCM). The 20-nm SiO2 nanoparticles at different concentrations of 31.25, 125, and 500 microg/ml were respectively incubated with RAW264.7 cells for 24 h to determine oxidative stress responses, membrane fluidity, intracellular [Ca2+]i concentration. Exposure to SiO2 nanoparticles at 125 and 500 microg/ml increased ROS levels and intracellular [Ca2+]i, reduced membrane fluidity levels. The decrease of the membrane fluidity indicated membrane damage. Increases of ROS generation and intracellular [Ca2+]i played a role in cytotoxicity. Results showed that mechanisms of cytotoxicity induced by SiO2 nanoparticles was closely correlated to increase oxidative stress, decrease membrane fluidity and perturbation of intracellular calcium homeostasis.

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

Tri- n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri- n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri- n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri- n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri- n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri- n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri- n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl −/HCO 3 − exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri- n-butyltin-induced apoptotic signaling in rat hepatocytes.

Involvement of Calcium in 7β-Hydroxycholesterol and Cholesterol-5β,6β-Epoxide-Induced Apoptosis

Oxidized low-density lipoprotein (oxLDL) is believed to play a central role in the development of atherosclerosis. The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7β-Hydroxycholesterol (7 β-OH) and cholesterol-5 β,6 β-epoxide ( β-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. The exact mechanism through which these oxysterols induce apoptosis remains to be fully elucidated. A perturbation of intra-cellular calcium homeostasis has been found to trigger apoptosis in many experimental systems. The aim of the present study was to determine the involvement of calcium signaling in 7 β-OH and β-epoxide–induced apoptosis. To this end, the authors employed the calcium channel blockers verapamil and nifedipine and inhibitors of calpain activation, ALLM and ALLN. Verapamil protected against the decrease in viability induced by 7 β-OH whereas nifedipine had a protective effect in both 7 β-OH and β-epoxide–treated cells, though these compounds did not restore viability to control levels. Verapamil, nifedipine, and ALLM prevented apoptosis induced by β-epoxide. None of the compounds employed in the current study protected against 7 β-OH–induced apoptosis. Our results implicate calcium signaling in the apoptotic pathway induced by β-epoxide and also highlight differences between apoptosis induced by 7 β-OH and β-epoxide.

Folate deficiency-induced oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhanced activation of NF-κB in human Hep G2 cells

Folate coenzymes are critical for de novo synthesis of purine and thymidine, and for interconversion of amino acids. Folate deficiency inhibits cellular proliferation, disturbs cell cycling, causes genetic damage and eventually results in cell death. Previously, we demonstrated that the demise of human hepatoma Hep G2 cells mediated by folate deficiency proceeded via a p53-independent apoptosis, and the perturbation of intracellular calcium homeostasis was also shown to be involved. To further delineate the mechanism associated with this observed phenomenon, Hep G2 cells were cultivated in the control or folate-deficient media (control media lacking folate, glycine, thymidine and hypoxanthine) for 4 weeks. At the end of this cultivation period, we found that TBARS (an index of lipid peroxidation) concentrations in the folate-deficient cells were drastically increased as compared to the control cells (0.04 vs 0.01 nmole/10 6 cells), indicating that a severe oxidative stress of the former cells had occurred. This phenomenon was also shown to coincide with the ability of these folate-deficient cells to elaborate increased amounts of H 2O 2 as compared to its folate-supplemented cells (2.87 vs 0.98 nmole/10 5 cells/h). Furthermore, the accelerated production of H 2O 2 by the folate-deficient cells was also closely correlated with the elevated homocysteine concentrations released in the culture medium (15.37 ± 2.4 vs 3.58 ± 2.4 μmole/L; P < 0.001). Finally, we demonstrated that folate deficiency was indeed capable of activating a redox-sensitive transcription factor, NF-κB, which is crucial in the control of a reactive oxygen species-mediated apoptosis. In summary, we show that folate deficiency-induced apoptosis is proceeded via the enhanced activation of NF-κB, which is the resulting form of the homocysteine-mediated overproduction of hydrogen peroxide.

Contribution of plasma membrane and endoplasmic reticulum Ca 2+-ATPases to the synaptosomal [Ca 2+] i increase during oxidative stress

In the present study we analyzed the effect of ascorbate (0.8 mM)/Fe 2+ (2.5 μM)-induced membrane lipid peroxidation on the levels of intracellular free calcium, [Ca 2+] i, and on the possible mechanisms involved in the perturbation of intracellular calcium homeostasis during oxidative stress. For this purpose, the influence of the ascorbate/iron oxidant system on the plasma membrane and endoplasmic reticulum Ca 2+-dependent ATPases of brain cortical synaptosomes was studied. In addition, the influence of the peroxidative process on the uptake of calcium ( 45Ca 2+) and on the Na +/Ca 2+ exchange activity at the plasma membrane was evaluated. After ascorbate/Fe 2+-induced membrane lipid peroxidation of the order of 18.05 ± 4.20 nmol TBARS/mg protein, an increase in [Ca 2+] i occured, under basal or depolarizing conditions (30 mM KCI), which was dependent on the extracellular calcium concentration. Thus, for 1 and 3 mM extracellular calcium concentration, an increase of the resting [Ca 2+] i values of 19.8% and 33.7% was observed, while after the K +-depolarization the enhancement of the [Ca 2+] i was 18.4% and 29.5%, respectively. The Na +/Ca 2+ exchange activity and the time-dependent influx of 45 Ca 2+, observed in basal conditions and after the 30 mM K +-depolarization, were not affected under the peroxidative conditions. The Ca 2+-dependent ATPase activity of the synaptosomal plasma membrane was significantly depressed following peroxidation of membrane lipids, decreasing the V max by 48.1%, without significant changes in the affinity of the enzyme for calcium ( K m for Ca 2+ was 0.54 ± 0.04 μM in control conditions and 0.56 ± 0.034 μM in peroxidized conditions). The Ca 2+-ATPase activity of the endoplasmic reticulum was also affected during ascorbate/iron-induced oxidative stress; thus, an inhibition of 45.2% was observed 5 min after adding ATP. These data suggest that the increase in synaptosomal [Ca 2+] i due to oxidative stress may result from the inhibition of the plasma membrane and the endoplasmic reticulum membrane Ca 2+-ATPase activities, probably as a result of the alteration of the lipid environment required for the maximal activity of these membrane enzymes. The consequent increase in [Ca 2+] i may be responsible for the injury of the nervous tissue observed during several pathological conditions in which free radical generation seems to be involved.

Cumene hydroperoxide induced changes in calcium homeostasis in cultured neonatal rat heart cells.

The relationship between oxidative stress induced cell necrosis and perturbation of intracellular calcium homeostasis was investigated in cultured myocytes. Cultured neonatal rat heart cells were loaded with fura-2 AM to measure cytosolic free calcium ([Ca2+]i). Probenecid, an inhibitor of organic anion transport, was present during the experiment to reduce efflux of fura-2 from the cytoplasm. Cells were exposed to cumene hydroperoxide, a toxic organic hydroperoxide that is known to induce oxidative stress in myocytes. The efficacy of the protective agents Trolox C (a vitamin E analogue) and chlorpromazine (a phospholipase inhibitor) on cumene hydroperoxide induced cell injury was determined. [Ca2+]i in control cells was constant (60 nM) during an incubation time of 45 min. Probenecid did not affect [Ca2+]i levels or cell viability under the experimental conditions. Cumene hydroperoxide caused a sustained rise in [Ca2+]i starting after 5-10 min, to a level of 1300 nM at 45 min. After 20-25 min the viability of the heart cells started to decline and after 45 min 44% of the cells were irreversibly injured. The loss of cell viability was expressed as percentage decrease of the fluorescence at 360 nm (the calcium independent wavelength), since the percent release of cellular alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) activity equalled the percent decrease of the fluorescence at 360 nm. Trolox C and chlorpromazine almost completely prevented the cumene hydroperoxide induced alpha-HBDH release. The [Ca2+]i of myocytes incubated with cumene hydroperoxide in combination with Trolox C rose to 1000 nM without affecting cell viability. The cumene hydroperoxide induced rise in [Ca2+]i was markedly reduced by chlorpromazine (at t = 45 min, [Ca2+]i = 360 nM). Addition of Trolox C to untreated cells did not influence [Ca2+]i, whereas chlorpromazine alone induced a slight increase of [Ca2+]i up to 360 nM with complete preservation of cell viability. Trolox C and chlorpromazine are very effective inhibitors of cumene hydroperoxide induced perturbation of calcium homeostasis and subsequent cell death. A role for peroxidation of membrane phospholipids and activation of calcium dependent phospholipase in the cascade of events leading to irreversible injury is suggested.

Silica increases cytosolic free calcium ion concentration of alveolar macrophages in vitro

Rat alveolar macrophages were exposed to silica dust (quartz) suspended in culture medium (SiO 2, dry particle size less than 5 μm in diameter) and fluctuation in their cytosolic free calcium content ([Ca 2+]i) was detected in cell monolayers with a fluorescent calcium probe (Indo-1AM). Cytosolic free calcium content was correlated with lactate dehydrogenase (LDH) release, an index of cell damage. SiO 2 induced a concentration- and time-dependent increase of cytosolic free Ca 2+ ion concentration and LDH release. [Ca 2+]i was increased about fivefold when cells were exposed to 200 μg of SiO 2 per milliliter (3 ml per dish) for 2 hr. [Ca 2+]i changed within 15 min of SiO 2 treatment, whereas LDH release was measurably increased only after 30 min. Chelation of extracellular Ca 2+ by 2 m m ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetate did not prevent SiO 2-induced fluctuation of macrophage [Ca 2+]i, but did partially prevent the SiO 2-induced increase in LDH release ( p < 0.01). We conclude that a very early event in SiO 2-induced damage of alveolar macrophages involves mobilization of intracellular calcium pools to increase [Ca 2+]i. These results suggest that SiO 2-induced macrophage damage, a key event in the development of silicosis, may involve perturbation of intracellular calcium homeostasis.

Perturbation of intracellular calcium homeostasis by oxidative stress in isolated skinned heart cells: K.J.Kako, T.Yanagishita, T.Matsuoka, M.Kato, T.Kaminishi. Department of Physiology, University of Ottawa School of Medicine, Ottawa, Ontario, Canada

Perturbation of intracellular calcium homeostasis by oxidative stress in isolated skinned heart cells: K.J.Kako, T.Yanagishita, T.Matsuoka, M.Kato, T.Kaminishi. Department of Physiology, University of Ottawa School of Medicine, Ottawa, Ontario, Canada