Intracellular Calcium Ion Levels Research Articles

Centchroman regulates breast cancer angiogenesis via inhibition of HIF-1α/VEGFR2 signalling axis

AimsAngiogenesis is a recognized hallmark of cancer which promotes cancer cell progression and metastasis. Inhibition of angiogenesis to attenuate cancer growth is becoming desirable strategy for breast cancer management. The present study is aimed to investigate the antiangiogenic efficacy of a novel selective estrogen receptor modulator Centchroman (CC) on human breast cancer cells. Main methodsEffect of CC on cell viability was evaluated using Sulforhodamine B assay. Endothelial cell proliferation, wound healing, Boyden chamber cell invasion, tube formation and chorioallantoic membrane (CAM) assays were performed to assess the effect of CC on migration, invasion and angiogenesis. Apoptosis, reactive oxygen species generation, caspase-3/7 and intracellular calcium ion level were measured through flow cytometry. Expression levels of HIF-1α, VEGF, VEGFR2, AKT and ERK were assessed by western blot analysis. Key findingsCC selectively induces apoptosis in human breast cancer cells without affecting non-tumorigenic breast epithelial cells MCF-10A. Moreover, it inhibits migratory, invasive and mammosphere forming potential of breast cancer. Furthermore, CC also inhibited VEGF-induced migration, invasion and tube formation of HUVECs in vitro. CC effectively inhibited neovasculature formation in chicken CAM. Western blot analysis demonstrated that CC inhibited expression of HIF-1α and its downstream target VEGF. Interestingly, CC also suppressed VEGFR2 phosphorylation and consequently attenuated AKT and ERK phosphorylation. SignificanceOur findings suggest that CC downregulates VEGF-induced angiogenesis by modulating HIF-1α/VEGFR2 pathway and recommend it (CC) as a potential therapeutic drug for breast cancer treatment.

The PMR1 pump in alpha-synuclein toxicity and neurodegeneration.

Proteinopathies constitute a diverse group of devastating neurodegenerative disorders, characterized by aberrant aggregation of specific proteins within neurons and in the brain parenchyma. Parkinson's disease (PD) is among the most common proteinopathies, caused by the accumulation of different species of α-synuclein and the formation of protein inclusions known as Lewy bodies. Although several mutations in the α-synuclein gene have been linked to PD, the mechanisms mediating the aggregation and toxicity of α-synuclein are not fully understood. Here, we review recent evidence that highlight an intricate interplay between α-synuclein and ionostasis, focusing on the PMR1 pump, a Golgi resident Ca2+/Mn2+ P-type ATPase, which plays a pivotal role in regulating the intracellular levels of calcium and manganese ions.

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100mM EtOH (in culture medium) for 2days. For interaction studies, cells were treated with memantine (4μM), EDTA (1mM), or BAPTA-AM (10μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Involvement of oxidative stress and calcium signaling in airborne particulate matter - induced damages in human pulmonary artery endothelial cells

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H2DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O2−) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM2.5 leads to an intrinsic free radical production (ROS, O2−) and a 4h-exposure to PM2.5 (5–15μg/cm2), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2− levels. The basal intracellular calcium ion level [Ca2+]i was also increased after 4h-exposure to PM2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM2.5 is closely correlated to an increase of oxidative stress.

Monocrotophos, an organophosphorus insecticide, disrupts the expression of HpNetrin and its receptor neogenin during early development in the sea urchin (Hemicentrotus pulcherrimus)

Netrins, chemotropic guidance cues, can guide the extension of serotonergic axons by binding to netrin receptors during neural development. However, little is known about whether disruption of netrin signaling is involved in the mechanisms by which organophosphorus pesticides affect serotonergic nervous system (SNS) development. In this study, we evaluated the effects of the pesticide monocrotophos (MCP) on the expression patterns of HpNetrin and its receptor neogenin as well as on the intracellular calcium ion (Ca2+) levels in Hemicentrotus pulcherrimus (sea urchin) by exposing fertilized embryos to 0, 0.01, 0.10, and 1.00mg/L MCP. The results showed that MCP disrupted HpNetrin and neogenin expression at different developmental stages in H. pulcherrimus and that Ca2+ appeared to be involved in the MCP-induced developmental neurotoxicity. Specifically, the lower concentrations of MCP elevated HpNetrin and neogenin transcription, resulting in higher intracellular Ca2+ levels during the early developmental stages in the sea urchin; this may affect netrin-directed cell migration/axon extension and subsequently disrupt serotonergic axon branching and synapse formation. In contrast, 1.00mg/L MCP exhibited an inhibitory effect on HpNetrin and neogenin transcription. This finding implies that the regulatory roles of these factors may be diminished during early development, thereby causing developmental defects in the sea urchin. Collectively, our results provide a basis for exploring the involvement of netrin and neogenin in the organophosphate-induced disruption of the SNS during development.

Verapamil augments carmustine- and irradiation-induced senescence in glioma cells by reducing intracellular reactive oxygen species and calcium ion levels.

Resistance to conventional therapies and frequent recurrence are the major obstacles to the treatment of high-grade gliomas, including glioblastoma. Thus, the development of new therapeutic strategies to overcome these obstacles is necessary to improve the treatment outcomes. In this study, we found that verapamil, a pan-adenosine triphosphate-binding cassette transporter and L-type voltage-dependent calcium channel inhibitor, sensitized U87MG glioma cells to carmustine- and irradiation-induced senescence. Furthermore, our results indicated that verapamil treatment, in combination with carmustine and irradiation, rendered U87MG glioma cells and several patient-derived glioma stem cells more sensitive to therapy-induced senescence than individual or dual-combination treatments. When investigating the underlying mechanism, we found that verapamil treatment markedly decreased intracellular reactive oxygen species and calcium ion levels. Reactive oxygen species reduction with N-acetylcysteine, a reactive oxygen species scavenger, rendered U87MG glioma cells more sensitive to carmustine and irradiation whereas the protein kinase C agonist, phorbol 12-myristate 13-acetate, mitigated the effects of carmustine and irradiation. Taken together, our results indicate that verapamil may be a potent therapeutic sensitizer for increasing the effectiveness of glioblastoma treatment.

Improvement of dexamethasone sensitivity by chelation of intracellular Ca2+ in pediatric acute lymphoblastic leukemia cells through the prosurvival kinase ERK1/2 deactivation

Previous studies have demonstrated that glucocorticoid hormones, including dexamethasone, induced alterations in intracellular calcium homeostasis in acute lymphoblastic leukemia (ALL) cells. However, the mechanism by which intracellular calcium homeostasis participates in dexamethasone sensitivity and resistance on ALL cells remains elusive. Here, we found that treatment of cells with dexamethasone resulted in increased intracellular calcium concentrations through store-operated calcium entry stimulation, which was curtailed by store-operated calcium channel blockers. We show that BAPTA-AM, an intracellular Ca2+ chelator, synergistically enhances dexamethasone lethality in two human ALL cell lines and in three primary specimens. This effect correlated with the inhibition of the prosurvival kinase ERK1/2 signaling pathway. Chelating intracellular calcium with Bapta-AM or inhibiting ERK1/2 with PD98059 significantly potentiated dexamethasone-induced mitochondrial membrane potential collapse, reactive oxygen species production, cytochrome c release, caspase-3 activity, and cell death. Moreover, we show that thapsigargin elevates intracellular free calcium ion level, and activates ERK1/2 signaling, resulting in the inhibition of dexamethasone-induced ALL cells apoptosis. Together, these results indicate that calcium-related ERK1/2 signaling pathway contributes to protect cells from dexamethasone sensitivity by limiting mitochondrial apoptotic pathway. This report provides a novel resistance pathway underlying the regulatory effect of dexamethasone on ALL cells.

Chemical Shift Assignment of the Regulatory Domain of Calcineurin Using NMR Spectroscopy

Calcineurin (CaN) is a serine/threonine phosphatase that is universally expressed in eukaryotic cells. In humans, CaN participates in cardiovascular and central nervous system development, and is known for its role in T-cell activation. Because of its signaling activity, regulatory problems that occur with CaN have been associated with several human diseases, including Alzheimer's disease and cardiac hypertrophy. CaN is activated by increased levels of intracellular calcium ions (Ca2+). These ions bind to a regulator protein, Calmodulin (CaM), which activates CaN by binding to CaN's calmodulin binding region (CaMBR). The binding site is located in the regulatory domain (RD) of CaN. The RD is intrinsically disordered until Ca2+-bound CaN binds, whereupon the RD folds and releases the previously bound autoinhibitory domain (AID). Our research is centered on understanding the molecular interactions that facilitate binding of CaN to the RD. To accomplish this, we used 2D and 3D NMR techniques to examine the structural determinants of binding and completed the protein's backbone and sidechain assignments in the solution phase. Our protein production methods included expressing protein from transformed E. coli and purifying the cell lysate on a Ni-NTA column followed by a CaM-Sepharose column. Purity was affirmed using SDS-PAGE and LC-MS.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in Molt-4 cells

Purpose: Transforming growth factor-β-activated kinase1 (TAK1) plays an anti-apoptotic role in response to multiple stresses. TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) has been studied for its apoptotic effects. However, the combined effect of OZ with physical stresses remains to be elusive. Therefore, in this study we focussed to determine the combined effects of OZ with hyperthermia (HT) using Molt-4 cell line.Materials and methods: Molt-4 cells were pre-treated with OZ for 1 h followed by heat exposure (44 °C, 10 min) and harvested 24 h after incubation at 37 °C, apoptosis was measured by Annexin V-FITC/PI double staining assay using flow cytometry and cell growth was observed by cell counting assay. Further mechanism involved in the combination was investigated by measuring mitochondrial membrane potential (MMP), intracellular ROS generation, expression of apoptosis related protein, intracellular calcium ion level and Fas activity.Results: Combination of OZ with HT significantly enhances MMP loss and superoxide generation. Furthermore, OZ pre-treatment promotes caspase-8 cleavage, Fas externalisation, caspase 3 activity and intracellular calcium ion levels. OZ pre-treatment decreased the expression of HT-induced Bcl-2 and increased the expression of pro-apoptotic Bax, while markedly suppressed the phosphorylation of JNK and p38. In addition, increased expression of CHOP following combined treatment indicates that ER stress may also involve in the enhancement of HT-induced apoptosis.Conclusion: Our data showed for the first time that OZ sensitizes Molt-4 cells to HT-induced apoptosis via extrinsic and intrinsic apoptotic pathways. Furthermore, ROS and ER stress may also play role in the enhancement of HT-induced apoptosis by OZ.

Role of orexin receptor subtypes in the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from dorsal root ganglion of carrageenan-treated rats

We analysed the roles of orexin receptors in the effects of orexin-A on KCl-induced increases in intracellular calcium ion levels ([Ca2+]i) in C-fiber-like small neurons of rats with inflammation induced by intraplantar injection of carrageenan into the hind paw. Controls were treated with saline. Paw withdrawal and threshold forces in response to tactile stimuli were determined using von Frey filaments. [Ca2+]i in C-fiber-like neurons derived from dorsal root ganglia was visualised using a calcium fluorescence probe. Changes in neuronal [Ca2+]i were assessed as relative fluorescence intensity (F/F0). One day after carrageenan injection, the paw withdrawal response to tactile stimuli and the paw withdrawal threshold were increased and reduced, respectively. KCl loading of neurons from either carrageenan-treated or control rats increased F/F0 to about 2.0. KCl-induced increases in F/F0 of carrageenan-treated, but not control, rats were inhibited by orexin-A. The OX1 and OX2 receptor antagonist MK-4305, but not the OX1 receptor antagonist SB334867, counteracted the effects of orexin-A on the KCl-induced increase in F/F0. These results suggest that OX2, but not OX1 receptors mediate the inhibitory effect of orexin-A on KCl-induced increases in [Ca2+]i in C-fiber-like neurons of rats with inflammation.

Usefulness of cardiotoxicity assessment using calcium transient in human induced pluripotent stem cell-derived cardiomyocytes

Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be an attractive strategy for assessing compounds on cardiac contractility. In addition, as arrhythmogenic compounds are known to induce characteristic waveform changes in hiPSC-CMs, it is expected that calcium transient would allow evaluation of not only compound-induced effects on cardiac contractility, but also compound arrhythmogenic potential. Using a combination of calcium transient in hiPSC-CMs and a fast kinetic fluorescence imaging detection system, we examined in this study changes in calcium transient waveforms induced by a series of 17 compounds that include positive/negative inotropic agents as well as cardiac ion channel activators/inhibitors. We found that all positive inotropic compounds induced an increase in peak frequency and/or peak amplitude. The effects of a negative inotropic compound could clearly be detected in the presence of a β-adrenergic receptor agonist. Furthermore, most arrhythmogenic compounds raised the ratio of peak decay time to peak rise time (D/R ratio) in calcium transient waveforms. Compound concentrations at which these parameters exceeded cutoff values correlated well with systemic exposure levels at which arrhythmias were reported to be evoked. In conclusion, we believe that peak analysis of calcium transient and determination of D/R ratio are reliable methods for assessing compounds' cardiac contractility and arrhythmogenic potential, respectively. Using these approaches would allow selection of compounds with low cardiotoxic potential at the early stage of drug discovery.

Potential Protective Effects of Bioactive Constituents from Chinese Propolis against Acute Oxidative Stress Induced by Hydrogen Peroxide in Cardiac H9c2 Cells.

Chinese propolis (CP) is known as a health food but its beneficial effects in protecting cardiomyocytes remain elusive. Here, we investigated the effects of CP and its active compounds on hydrogen peroxide (H2O2) induced rats cardiomyocytes (H9c2) oxidative injury. Cell viability decreases induced by H2O2 were mitigated by different CP extracts using various solvents. From these active fractions, six active compounds were separated and identified. Among tested isolated compound, the cytoprotective activities of three caffeates, caffeic acid phenethyl ester (CAPE), benzyl caffeate (BZC), and cinnamyl caffeate (CNC), exerted stronger effects than chrysin, pinobanksin, and 3,4-dimethoxycinnamic acid (DMCA). These three caffeates also increased H9c2 cellular antioxidant potential, decreased intracellular calcium ion ([Ca2+]i) level, and prevented cell apoptosis. Overall, the cardiovascular protective effects of the CP might be attributed to its caffeates constituents (CAPE, BZC, and CNC) and provide evidence for its usage in complementary and alternative medicine.

Graphene oxide induces apoptotic cell death in endothelial cells by activating autophagy via calcium-dependent phosphorylation of c-Jun N-terminal kinases.

Graphene oxide (GO) have received considerable attention with respect to their utilization in biomedical applications. However, GO-related safety issues concerning human vasculature are very limited. In this manuscript, we report for the first time the differential size-related biological effects of GOs on endothelial cells (ECs). Notably, Subnanometer- and nanometersized GOs induce apoptotic death in ECs via autophagy activation. We propose a molecular mechanism for the GO-induced autophagic cell death through the PLCβ3/IP3/Ca2+/JNK signaling axis. Our findings could be provide a better understanding of the GO sizedependent cytotoxicity in vasculature and facilitate the future development of safer biomedical applications of GOs.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

LicochalconeA (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER)stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamineB assay. Apoptosis was detected by flow cytometry with AnnexinV/propidium iodide staining, and by fluorescent microscopy with Hoechst33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be considered an important mechanism by which LCA induces apoptosis of T24 bladder cancer cells.

Salivary Peptides Regulate Intracellular Calcium in Rat Pancreatic Beta Cells

ObjectivesProline Rich Proteins (PRPs), secreted by parotid gland, have a variety of functions including anti‐caries and anti‐bacterial properties, but less is known about their actions outside the oral cavity. One of these proteins, called Parotid Hormone (PH) in Sus scrofa, is believed to play a significant role in the maintenance of healthy dentin and have effects on other organs including pancreas by having regulatory effects on maintaining the sensitivity of beta cells to circulating glucose. Basic Salivary Proline Rich Protein 2 and 3 (PRB2 & PRB3), in human, have the highest sequence homology with porcine Parotid Hormone. Changes in cytosolic calcium ion concentration constitute an important element of signal transduction that may lead to insulin release. We hypothesized that PH, PRB2 and PRB3 are involved in insulin stimulation by regulating the intracellular calcium ion levels in pancreatic beta cells.MethodsCultured RIN‐m beta cells from Rattus Norvegicus were used as an in vitro model for measuring intra‐cellular calcium levels using the fluorescent dye Fura2‐AM. Cells in calcium‐rich or calcium‐free medium were treated with PH (34nmoles/200 μL), PRB2 (11nmoles/200μL) or PRB3 (11nmoles/200μL). In some experiments cells were pretreated with the L‐type calcium channel blocker diltiazem (10μM) or with thapsigargin (2μM) to deplete internal calcium stores, before treatment with the peptides.ResultsPH, PRB2 and PRB3 increased intracellular calcium levels in cultured beta cells by 3‐, 10‐ and 3‐fold respectively. In calcium‐free medium, no change was observed in intracellular calcium level after treatment with PH or PRB2; however treatment with PRB3 caused an increase in intracellular calcium level. Interestingly, calcium uptake by cells following PH and PRB2 treatment was partially blocked by diltiazem (10μM), whereas thapsigargin (2μM) treatment blocked the response caused by PRB3.ConclusionSalivary peptides PH, PRB2 and PRB3 show differential regulation of intracellular calcium in RIN‐m beta cells. Our data indicates that PH and PRB2 regulate an influx of external calcium partially through L‐type calcium channels, while PRB3 potentially regulates an increase of calcium from internal stores. In summary, our results suggest that PH, PRB2 and PRB3 may have regulatory functions beyond those that have been described in the literature, and may play a role in a parotid‐pancreatic axis.Support or Funding InformationDepartmental funding from the Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry.

Inhibitory gene expression of the Cav3.1 T-type calcium channel to improve neuronal injury induced by lidocaine hydrochloride

Cav3.1 is a low-voltage-activated (LVA) calcium channel that plays a key role in regulating intracellular calcium ion levels. In this study, we observed the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y cells that silenced Cav3.1 mRNA by RNA interference, and investigated the roles of p38 MAPK in these effects. We constructed the pNC-puro-CACNA1G-SH-SY5Y cells and pshRNA-CACNA1G –SH-SY5Y cells by the RNA interference. All the cells were cultured with or without 10mM lidocaine hydrochloride for 24h. The cell morphology, cell viability, Cav3.1 and p38 protein expression, cell apoptosis rate and intracellular calcium ion concentration were detected. We found that all cells treated with 10mM lidocaine hydrochloride for 24h showed cellular rounding, axonal regression, and cellular floating. Compared with the cells in SH-SY5Y+Lido group and NC+Lido group, those in the RNAi+Lido group showed similar changes, but of smaller magnitude. Additionally, following lidocaine hydrochloride all cells displayed increased Cav3.1 and p38 MAPK protein, apoptosis rate, and intracellular calcium ion levels; however,these changes in the RNAi+Lido group were less pronounced than in the SH-SY5Y+Lido and NC+Lido groups. The cell viability decreased following lidocaine hydrochloride treatment, but viability of the cells in the RNAi+Lido group was higher than in the SH-SY5Y+Lido and NC+Lido groups. The results showed that Cav3.1 may be involved in neuronal injury induced by lidocaine hydrochloride and that p38 MAPK phosphorylation was reduced upon Cav3.1 gene silencing.

Crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis.

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.

Tweeters, Woofers and Horns: The Complex Orchestration of Calcium Currents in T Lymphocytes.

Elevation of intracellular calcium ion (Ca2+) levels is a vital event that regulates T lymphocyte homeostasis, activation, proliferation, differentiation, and apoptosis. The mechanisms that regulate intracellular Ca2+ signaling in lymphocytes involve tightly controlled concinnity of multiple ion channels, membrane receptors, and signaling molecules. T cell receptor (TCR) engagement results in depletion of endoplasmic reticulum (ER) Ca2+ stores and subsequent sustained influx of extracellular Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane. This process termed store-operated Ca2+ entry (SOCE) involves the ER Ca2+ sensing molecule, STIM1, and a pore-forming plasma membrane protein, ORAI1. However, several other important Ca2+ channels that are instrumental in T cell function also exist. In this review, we discuss the role of additional Ca2+ channel families expressed on the plasma membrane of T cells that likely contribute to Ca2+ influx following TCR engagement, which include the TRP channels, the NMDA receptors, the P2X receptors, and the IP3 receptors, with a focus on the voltage-dependent Ca2+ (CaV) channels.

G-CSF protects human brain vascular endothelial cells injury induced by high glucose, free fatty acids and hypoxia through MAPK and Akt signaling.

Granulocyte-colony stimulating factor (G-CSF) has been shown to play a neuroprotective role in ischemic stroke by mobilizing bone marrow (BM)-derived endothelial progenitor cells (EPCs), promoting angiogenesis, and inhibiting apoptosis. Impairments in mobilization and function of the BM-derived EPCs have previously been reported in animal and human studies of diabetes where there is both reduction in the levels of the BM-derived EPCs and its ability to promote angiogenesis. This is hypothesized to account for the pathogenesis of diabetic vascular complications such as stroke. Here, we sought to investigate the effects of G-CSF on diabetes-associated cerebral vascular defect. We observed that pretreatment of the cultured human brain vascular endothelial cells (HBVECs) with G-CSF largely prevented cell death induced by the combination stimulus with high glucose, free fatty acids (FFA) and hypoxia by increasing cell viability, decreasing apoptosis and caspase-3 activity. Cell ultrastructure measured by transmission electron microscope (TEM) revealed that G-CSF treatment nicely reduced combination stimulus-induced cell apoptosis. The results from fluorescent probe Fluo-3/AM showed that G-CSF greatly suppressed the levels of intracellular calcium ions under combination stimulus. We also found that G-CSF enhanced the expression of cell cycle proteins such as human cell division cycle protein 14A (hCdc14A), cyclinB and cyclinE, inhibited p53 activity, and facilitated cell cycle progression following combination stimulus. In addition, activation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt, and deactivation of c-Jun N terminal kinase (JNK) and p38 were proved to be required for the pro-survival effects of G-CSF on HBVECs exposed to combination stimulus. Overall, G-CSF is capable of alleviating HBVECs injury triggered by the combination administration with high glucose, FFA and hypoxia involving the mitogen-activated protein kinases (MAPK) and Akt signaling cascades. G-CSF may represent a promising therapeutic agent for diabetic stroke.