Sustained Increase In Intracellular Ca2 Research Articles

Mitochondria control IP3-evoked Ca2+ release triggered by voltage-dependent Ca2+ entry in intact resistance arteries

The contractility of vascular smooth muscle cells (VSMCs) is a major contributor of vascular tone and blood pressure. In VSMCs, intracellular Ca2+ level determines the contractile activity that generates the force to contract arteries. A major source of Ca2+ is via influx through voltage dependent Ca2+ channels (VDCCs). While mitochondria are now recognised as key regulators of intracellular Ca2+ homeostasis in several cell types, their role in modulating Ca2+ signalling mediated by VDCC in VSMCs is unresolved. Given the potential physiological significance, and unclarified interaction between mitochondria and VDCCs in VSMCs, we hypothesize that mitochondria directly regulate Ca2+ signalling mediated by VDCCs. The interplay between mitochondria and VDCCs was investigated by imaging and analysing intracellular Ca2+ signals in smooth muscle cells in intact arteries from rat mesentery. Depolarization of the plasma membrane potential, by high potassium (20 mM) physiological saline solution, triggered Ca2+ influx through VDCCs and a sustained increase in intracellular Ca2+ on which repetitive Ca2+ oscillations occurred. All Ca2+ signals were abolished by removal of external Ca2+ and by dihydropyridine inhibitors of VDCCs. Significantly, the repetitive Ca2+ oscillations, but not the sustained Ca2+ signals, were blocked by the IP3 receptor inhibitor 2-APB and SERCA inhibitor cyclopiazonic acid. Neither the repetitive Ca2+ oscillations or the sustained Ca2+ signals were altered by the ryanodine receptor inhibitors ryanodine and dantrolene. These results suggest that Ca2+ influx via VDCC triggers Ca2+-induced Ca2+ release at IP3 receptors in intact mesenteric arteries. Depolarization of the mitochondrial membrane potential (ψm), with the uncoupler CCCP or complex I inhibitor rotenone, but not ATP deprivation with the ATP synthesis blocker oligomycin, inhibited VDCC evoked IP3 mediated Ca2+ oscillations but not the sustained Ca2+ signals. Furthermore, in intact arteries, depolarization of ψm directly suppressed inositol triphosphate receptors (IP3Rs) mediated Ca2+ release from the internal Ca2+ store evoked by photolysis of caged IP3. These results suggest that mitochondria regulate Ca2+ release via IP3R triggered by Ca2+ entry via VDCC. In return, Ca2+ entry via VDCCs did not alter ψm, but upregulated ROS production. Together, these results suggest that mitochondria regulate Ca2+-induced Ca2+ release at IP3 receptors triggered by Ca2+ influx via VDCCs but do not directly regulate VDCC activity. British Heart Foundation (RG/F/20/110007; PG/20/9/34859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sodium regulates PLC and IP3 R-mediated calcium signaling in invasive breast cancer cells.

Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+ ]i ) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N-methyl-D-glucamine (0Na+ /NMDG) or choline chloride (0Na+ /ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na+ /NMDG and 0Na+ /ChoCl resulted in a slow, sustained depletion in [Na+ ]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+ ]i ). Application of La3+ in nominal Ca2+ -free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca2+ entry channels. Moreover, the Na+ -linked [Ca2+ ]i increase was independent of membrane potential hyperpolarization (NS-1619), but was inhibited by pharmacological blockade of IP3 receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3 -mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+ ]i is perturbed.

In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells.

Several recent studies have demonstrated that the direct precursor of vitamin D3, the calcifediol [25(OH)D3], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D3 by binding the VDR could have a vitamin’s D3 activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca2+ mobilization in response to 25(OH)D3. Our result reveals the ability of 25(OH)D3 to activate rapid, non-genomic pathways, such as an increase of intracellular Ca2+ levels, similar to what observed with the biologically active form of vitamin D3. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca2+ concentration as a result of exposure to 10−5 M of 25(OH)D3. In this work, we show for the first time the in vitro ability of 25(OH)D3 to induce a rapid increase of intracellular Ca2+ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D3 and its role in endocrine system.

Platelet-derived extracellular vesicles regulate cell cycle progression and cell migration in breast cancer cells

Platelets have been extensively implicated in the progression of cancer and platelet-derived extracellular vesicles (PEVs) are gaining growing attention as potential mediators of the platelet-cancer interplay. PEVs are shed from platelet membrane in response to extracellular stimuli and carry important biological signals for intercellular communication. In this study we demonstrate that PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. PEVs were massively internalized by the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not by the MCF-7 cell line. In SKBR3 cells, PEVs decreased mitochondrial dehydrogenase activities and altered cell cycle progression without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the distribution in the different phases of the cell cycle. In all the analyzed breast cancer cells, PEVs triggered a sustained increase of intracellular Ca2+, but only in MDA-MB-231 cells, this was associated to the stimulation of selected signaling proteins implicated in migration, including p38MAPK and myosin light chain. Importantly, inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results demonstrate that PEVs are versatile regulators of cancer cell behavior and elicit a variety of different responses depending on the specific breast cancer cell subtype.

Excitotoxicity and Overnutrition Additively Impair Metabolic Function and Identity of Pancreatic β-Cells.

A sustained increase in intracellular Ca2+ concentration (referred to hereafter as excitotoxicity), brought on by chronic metabolic stress, may contribute to pancreatic β-cell failure. To determine the additive effects of excitotoxicity and overnutrition on β-cell function and gene expression, we analyzed the impact of a high-fat diet (HFD) on Abcc8 knockout mice. Excitotoxicity caused β-cells to be more susceptible to HFD-induced impairment of glucose homeostasis, and these effects were mitigated by verapamil, a Ca2+ channel blocker. Excitotoxicity, overnutrition, and the combination of both stresses caused similar but distinct alterations in the β-cell transcriptome, including additive increases in genes associated with mitochondrial energy metabolism, fatty acid β-oxidation, and mitochondrial biogenesis and their key regulator Ppargc1a. Overnutrition worsened excitotoxicity-induced mitochondrial dysfunction, increasing metabolic inflexibility and mitochondrial damage. In addition, excitotoxicity and overnutrition, individually and together, impaired both β-cell function and identity by reducing expression of genes important for insulin secretion, cell polarity, cell junction, cilia, cytoskeleton, vesicular trafficking, and regulation of β-cell epigenetic and transcriptional program. Sex had an impact on all β-cell responses, with male animals exhibiting greater metabolic stress-induced impairments than females. Together, these findings indicate that a sustained increase in intracellular Ca2+, by altering mitochondrial function and impairing β-cell identity, augments overnutrition-induced β-cell failure.

Ca2+ -dependent down-regulation of human histamine H1 receptors in Chinese hamster ovary cells.

Gq/11 protein-coupled human histamine H1 receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin-dependent endocytosis followed by proteasome/lysosome-mediated down-regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca2+ concentrations induced by a receptor-bypassed stimulation with ionomycin, a Ca2+ ionophore, on the endocytosis and down-regulation of H1 receptors in Chinese hamster ovary cells. All cellular and cell-surface H1 receptors were detected by the binding of [3 H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H1 receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine- and pirdonium-sensitive binding sites of [3 H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca2+ and partially by a ubiquitin-activating enzyme inhibitor (UBEI-41), but were not affected by inhibitors of calmodulin (W-7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin-induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E-64, leupeptin, chloroquine, or NH4 Cl), proteasomes (lactacystin or MG-132), and a Ca2+ -dependent non-lysosomal cysteine protease (calpain) (MDL28170). Since H1 receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H1 receptors, even after the ionomycin treatment, H1 receptors appeared to exist in a form to which [3 H]mepyramine was unable to bind. These results suggest that H1 receptors are apparently down-regulated by a sustained increase in intracellular Ca2+ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

The Role of Vitamin D in Obesity and Diabetes: Ca2+ Signaling, Insulin Secretion, Adipocyte Apoptosis, and Bone Mineralization

Vitamin D has been linked to obesity and type 2 diabetes (T2D) in observational studies, and low vitamin D status is considered a risk factor for these interrelated diseases. However, mechanistic understanding of the role of vitamin D hormone, 1,25(OH)2‐vitamin D3, in obesity and T2D is lacking. We showed that 1,25(OH)2D3‐dependent cellular Ca2+ signals regulate apoptotic death of adipocytes and insulin secretion from pancreatic β‐cells (Sergeev, 2014, 2013, 2009, 2005, 2004). The anti‐obesity effects of 1,25(OH)2D3 in mature adipocytes are determined by its activity to generate, via multiple Ca2+ signaling pathways, a sustained increase in intracellular Ca2+ followed by activation of Ca2+‐dependent initiators and effectors of apoptosis. In pancreatic β‐cells, 1,25(OH)2D3 induces synchronous Ca2+ oscillations, which quantitatively, temporally and spatially pattern oscillatory insulin release from these cells. In recent studies, we examined the effects of increased intake of vitamin D and Ca in a mouse model of high fat diet‐induced obesity (DIO) on development of adiposity and diabetes. DIO mice were characterized by obese phenotype, elevated blood glucose and impaired glucose tolerance, increased insulin and decreased adiponectin concentrations in blood, decreased level of apoptosis in adipose tissue, and decreased Ca and P content in bone. Mice fed high fat diet (DIO) with high vitamin D3 and increased Ca content demonstrated a decreased weight of white adipose tissue depots and improved blood markers related to adiposity, T2D, and vitamin D status. Fasting plasma glucose and insulin concentrations in these mice were significantly decreased, approaching levels found in normal‐weight, non‐obese control, whereas adiponectin concentration demonstrated an increasing trend. DIO was accompanied by low vitamin D status (a decreased level of 25(OH)D) and a decreased plasma concentration of 1,25(OH)2D3. High vitamin D3 intake induced a significant increase in plasma concentrations of 25(OH)D and 1,25(OH)2D3, indicating high vitamin D nutritional status and normal vitamin D hormonal status. High vitamin D3 and Ca intakes were associated with induction of apoptosis (oligonucleosomal fragmentation and ssDNA breaks) and activation of Ca2+‐dependent apoptotic proteases (calpain and caspase‐12) in adipose tissue of DIO mice. High vitamin D3 and Ca intakes were effective in normalization (an increase) of mineral (Ca and P) content in the growing bone of DIO mice via regulatory effects mediated by 1,25(OH)2D3 ‐ PTH axis. The major results obtained demonstrate that increased vitamin D and Ca intake is highly effective in decreasing blood glucose and insulin in DIO and that the hormonal mechanism of this effect can involve 1,25(OH)2D3. Increasing vitamin D and Ca intakes in obesity also activates Ca2+‐mediated apoptotic pathway in adipocytes resulting in a decreased mass of fat tissue and improved markers of adiposity. Moreover, high vitamin D and Ca intakes improve bone status in obesity by increasing bone mineralization via PTH/1,25(OH)2D3‐mediated regulation. The findings obtained strongly imply that increasing vitamin D and Ca intakes may contribute to prevention of obesity, T2D, and bone disorders associated with these diseases.Support or Funding InformationSupported by USDA 2009‐35200‐05008 and SD00H533.

Abstract 4439: Ero1L is a determinant of resistance to bortezomib and collateral sensitivity to MTI-101-induced cell death in myeloma

Abstract Our laboratory previously reported that the novel first-in-class peptidometitc, referred to as MTI-101, binds a CD44/ITGA4-containing complex and induces necrotic cell death in multiple myeloma cells. In this presentation, we worked towards further elucidating the mechanism of action of MTI-101. To this end, we examined changes in gene expression between samples that are either resistant or sensitive to MTI-101 using both an isogenic acquired resistant cell line model and primary myeloma patient specimens. Using cell line models we show that the acquisition of resistance to this class of compounds correlates with changes in genes predicted to regulate Ca2+ homeostasis. Changes included decreased expression of the IP3 receptor(IP3R) isoform 3, phospholipase C (PLC)-β, and SERCA3 channel, as well as decreased expression of the plasma membrane Ca2+ channels TRPC1 and TRPM7. Moreover, we show that MTI-101 treatment evokes a robust and sustained increase in intracellular Ca2+ levels, a phenotype that as predicted was attenuated in the drug-resistant cell line. Blocking Ca2+ release from the IP3R as well as blocking influx of Ca2+ from plasma membrane channels inhibited the MTI-101 induced Ca2+ response. We are also able to inhibit MTI-101 induced cell death by pharmacologically blocking release of Ca2+ from the IP3 receptor and blocking uptake by the mitochondria. Importantly, and consistent with what we previously reported with the linear analog HYD-1, MTI-101 is more potent in specimens obtained from relapsed patients compared to newly diagnosed patients. This differential response to MTI-101 treatment was used to compare baseline GEP in these primary myeloma samples. Using this strategy we show that in primary multiple myeloma specimens, the sensitivity to MTI-101 significantly correlates with increased levels of the thiol oxidase Ero1L. Ero1L has been shown by others to regulate Ca2+ release from the IP3R. Mechanistically, we demonstrate that reducing the expression of Ero1L is sufficient to confer resistance to MTI-101, a finding that correlates with attenuation of MTI-101-induced Ca2+ levels. Moreover, reducing the expression of Ero1L was sufficient to cause increased sensitivity to bortezomib-induced cell death. Importantly, a retrospective analysis of relapsed specimens showed that increased Ero1L levels were found to be a poor prognostic marker for response to bortezomib. Together, our data indicate that Ero1L is a prognostic marker for resistance to bortezomib and may account for the increased sensitivity to MTI-101 in relapsed multiple myeloma specimens. As MTI-101 induces cell death by the deregulation of Ca2+, we speculate that alterations in Ca2+ signaling may play a role in the progression of multiple myeloma. Citation Format: Michael Emmons, Lori Hazlehurst, Steven Escherich, Mark McLaughlin, Javier Cuevas, Daniel Sullivan. Ero1L is a determinant of resistance to bortezomib and collateral sensitivity to MTI-101-induced cell death in myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4439. doi:10.1158/1538-7445.AM2015-4439

Calcium signaling and β2-adrenergic receptors regulate 1-nitropyrene induced CXCL8 responses in BEAS-2B cells

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread environmental pollutants, generated from reactions between PAHs and nitrogen oxides during combustion processes. In the present study we have investigated the mechanisms of CXCL8 (IL-8) responses induced by 1-nitropyrene (1-NP) in human bronchial epithelial BEAS-2B cells, with focus on the possible importance of Ca2+-signaling and activation of β2-adrenergic receptors (β2AR). Ca2+-chelator treatment obliterated 1-NP-induced CXCL8 (IL-8) responses. 1-NP at 10μM (but not 1μM) induced a rapid and sustained increase in intracellular Ca2+-levels ([Ca2+]i). The early but not the later, sustained phase of 1-NP-induced [Ca2+]i was suppressed by beta-blocker treatment (carazolol). Moreover, inhibition of β2AR by blocking-antibody, beta-blocker treatment (ICI 118551) or siRNA transfection attenuated CXCL8 responses induced by 1-NP. The results confirm that PAHs may induce Ca2+-signaling also in BEAS-2B cells, at least partly through activation of β2AR, and suggest that both β2AR- and Ca2+-signaling may be involved in 1-NP-induced CXCL8 responses in bronchial epithelial cells.

Cannabidiol enhances microglial phagocytosis via transient receptor potential (TRP) channel activation

Microglial cells are important mediators of the immune response in the CNS. The phytocannabinoid, cannabidiol (CBD), has been shown to have central anti-inflammatory properties, and the purpose of the present study was to investigate the effects of CBD and other phytocannabinoids on microglial phagocytosis. Phagocytosis was assessed by measuring ingestion of fluorescently labelled latex beads by cultured microglial cells. Drug effects were probed using single-cell Ca²⁺ imaging and expression of mediator proteins by immunoblotting and immunocytochemistry. CBD (10 μM) enhanced bead phagocytosis to 175 ± 7% control. Other phytocannabinoids, synthetic and endogenous cannabinoids were without effect. The enhancement was dependent upon Ca²⁺ influx and was abolished in the presence of EGTA, the Ca²⁺ channel inhibitor SKF96365, the transient receptor potential (TRP) channel blocker ruthenium red, and the TRPV1 antagonists capsazepine and AMG9810. CBD produced a sustained increase in intracellular Ca²⁺ concentration in BV-2 microglia and this was abolished by ruthenium red. CBD rapidly increased the expression of TRPV2 and TRPV1 proteins and caused a translocation of TRPV2 to the cell membrane. Wortmannin blocked CBD enhancement of BV-2 cell phagocytosis, suggesting that it is mediated by PI3K signalling downstream of the Ca²⁺ influx. The TRPV-dependent phagocytosis-enhancing effect of CBD suggests that pharmacological modification of TRPV channel activity could be a rational approach to treating neuroinflammatory disorders involving changes in microglial function and that CBD is a potential starting point for future development of novel therapeutics acting on the TRPV receptor family.

Vitamin D/Ca2+‐dependent calpain and caspases: novel molecular targets for obesity‐related breast cancer drug discovery (261.5)

Removal of breast cancer cells (BCC) via apoptosis results in a decrease in tumor size. Induction of apoptosis in adipocytes decreases adiposity due to long‐term reduction in adipose tissue mass. The hormone 1,25(OH)2‐vitamin D3 (1,25D) triggers apoptosis in both BCC and mature adipocytes via apoptotic Ca2+ signal ‒ a sustained increase in intracellular Ca2+. This signal acts as an initiator of apoptosis that directly recruits Ca2+‐dependent apoptotic effectors, calpain and caspase‐12, in BCC and adipocytes. Normal mammary epithelial cells are resistant to Ca2+‐mediated apoptosis because Ca2+ regulatory mechanisms in these cells do not support the apoptosis‐inducing Ca2+ signal. Remarkably, high vitamin D and Ca intakes activate Ca2+‐mediated apoptotic pathway in adipose tissue and decrease body and fat weight gain in obese mice. Induction of apoptosis with 1,25D in adipocytes, particularly in the tumor‐surrounding adipose tissue involved in tumor progression, contributes to anticancer effects of the hormone. The 1,25D ‒ Ca2+ link between breast cancer and obesity supports rationale to include the vitamin D/Ca2+‐dependent apoptotic proteases as molecular targets for discovery of new therapeutic and preventive agents for breast cancer and obesity as well as to consider increasing vitamin D and Ca intakes as a promising approach to protecting against breast cancer and decreasing adiposity. Supported by USDA 2009‐35200‐05008 and SD00H325.Grant Funding Source: Supported by USDA

Membrane Hyperpolarization Induced by Endoplasmic Reticulum Stress Facilitates Ca2+ Influx to Regulate Cell Cycle Progression in Brain Capillary Endothelial Cells

Upregulation of the Kir2.1 channel during endoplasmic reticulum (ER) stress in t-BBEC117, an immortalized bovine brain endothelial cell line, caused a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)]i) and a facilitation of cell death. Expressions of Ca(2+) influx channels (TRPC, Orai1, STIM1) were unchanged by ER stress. The ER stress-induced [Ca(2+)]i increase was mainly attributed to the deeper resting membrane potential due to Kir2.1 upregulation. ER stress arrested at the G2/M phase and it was attenuated by an inhibitor of Kir2.1. These results indicate that Kir2.1 upregulation by ER stress facilitates cell death via regulation of cell cycle progression in t-BBEC117.

ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.

Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride

Ionic liquids have recently received considerable attention due to their negligible vapor pressure and substitute for conventional organic solvents. However, their solubility in water and a great deal of literature regarding their toxicity on aquatic organisms have caused much concern in recent years. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C8mim][Cl]) on the rat pheochromocytoma (PC12) cell line by cell viability assay and to determine the cytological alterations and damages in PC12 cells after 24h of exposure at the concentrations of 0.07, 0.14, and 0.28mM of [C8mim][Cl]. The results show that [C8mim][Cl] inhibits PC12 cell growth and decreases their viabilities in a remarkable dose-dependent manner, and the 24h EC50 of [C8mim][Cl] for PC12 cells is calculated to be around 0.56mM. Our results also reveal that [C8mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca2+, overproduction of reactive oxygen species, gradually exhausted cellular ATP, mitochondrial permeability transition, and apoptosis in PC12 cells. We suppose that mitochondrial permeability transition and mitochondrial dysfunction maybe the major cytotoxicity mechanism of [C8mim]Cl for PC12 cells.

Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate

In the earlier study, sodium orthovanadate (SOV) has been reported to be a powerful inhibitor of (Na(+), K(+)) adenosine triphosphatase, exhibit widespread actions on the renal and cardiovascular systems, induces smooth muscle contraction by inhibiting the phosphorylation of the protein tyrosine phosphatases. In the current study, we aimed to investigate the cellular mechanisms by which SOV facilitated contractile response of vas deferens smooth muscle and its potential therapeutic advantage. Exogenous application of ATP and NA-caused contraction was strengthened by pretreatment with SOV. This facilitation was inhibited not by bath with the inhibitor of P2 receptor, PPADS, or the inhibitor of α1 receptor, Prazosin, but by bath with the protein tyrosine kinase inhibitor, Genistein. SOV induced a sustained increase in intracellular Ca(2+) of smooth muscle cells, which was abolished by 100 μM Genistein or Ca(2+)-free solution. The facilitation of SOV could also be inhibited by the selective inhibitors of TRP channel, 2-APB and non-selective cation channel, Gd(3+), Ni(+). The in vivo study showed that peritoneal injection of SOV in dystrophic mice (mdx mice) enhanced the contraction of vas deferens smooth muscle stimulated by electrical field stimulation, ATP, noradrenaline, or KCl. The above results suggest that SOV facilitates the concentration of vas deferens smooth muscle through the tyrosine phosphorylation activated the non-selective cation channels, which has potential use in the therapy for muscle dysfunction.

Abstract A30: Inhibition of PMCA4 as a strategy to increase the effectiveness of Bcl-2 inhibitor-mediated cell death in breast cancer cells.

Abstract Intracellular calcium is a vital cell signaling molecule that can influence a myriad of important cellular functions, including those deregulated in the cancer cell. Consequently Ca2+ levels within the cell must be precisely controlled. Amongst the suite of proteins controlling calcium homeostasis are the plasma membrane calcium ATPases (PMCAs), which belong to a family of druggable proteins. PMCAs are pumps that reside on the plasma membrane and are responsible for the efflux of intracellular Ca2+ and thus are important in maintaining basal cellular calcium levels. Sustained increases in intracellular Ca2+ can be a trigger for cell death and agents that induce alterations in calcium signaling could be a strategy to restore the sensitivity of a cancer cell to death stimuli. The aim of this study was to explore the effectiveness of inhibiting two PMCA isoforms, PMCA1 and PMCA4, on the augmentation of cell death initiated by a Ca2+ionophore (ionomycin) and a Bcl-2 inhibitor (ABT-263) in MDA-MB-231 breast cancer cells. Ionomycin represents a caspase-independent mechanism of cell death and ABT-263 enhances cell death via a caspase-dependent mechanism. PMCA1 and PMCA4 were inhibited using siRNA and the effect on cell death was measured by staining with Hoechst 33342 and propidium iodide using high content imaging (ImageXpress). Intracellular Ca2+ was determined using a FLIPRTETRA with cells loaded with the calcium sensor Fluo4-AM. Inhibition of PMCA1 was associated with significant changes in global cytosolic free Ca2+ induced by purinergic receptor activation, whereas the same effect was not seen with PMCA4 inhibition. Consistent with effects on global calcium levels, ionomycin (3 μM)-induced cell death was significantly augmented by PMCA1 but not PMCA4 siRNA. However, ABT-263 (3 μM)-induced cell death was augmented only by inhibition of PMCA4, suggesting that PMCA4 siRNA-mediated inhibition of ABT-263 apoptosis is mediated through effects on local and/or specific calcium signaling processes. PMCA4 can alter the activity of calcium-dependent transcription factors in cardiac myocytes independent of global cytosolic calcium. In MDA-MB-231 cells, the effects of PMCA4 inhibition appear to be associated with alterations in the activity of NFkappaB. These results highlight the specificity of different isoforms of PMCA in the modulation of cell death pathways in MDA-MB-231 breast cancer cells. Inhibition of PMCA4 may be a viable adjuvant therapy when combined with Bcl-2 family inhibitors for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A30.

Annexin A6 suppresses breast cancer cell proliferation by inhibiting excessive receptor‐activated increase in cytosolic calcium

Annexin A6 (AnxA6) is an unusual member of the annexin family of Ca2+-dependent membrane binding proteins that upon cell membrane association inhibits store-operated Ca2+ influx. This suggests that AnxA6 plays a role in a feedback mechanism that regulates receptor-activated rise in cytosolic Ca2+. Here we show that in the AnxA6-expressing BT-549 breast cancer cells AnxA6 translocates from the cytosol to the plasma membrane at high extracellular Ca2+ (2 mM). Its depletion in these cells inhibited the EGF receptor induced Ca2+ influx without affecting store-independent Ca2+ entry. Decreased AnxA6 expression is therefore accompanied by a 2-fold increase in intracellular Ca2+ concentrations and enhanced anchorage-independent cell growth. Moreover, depletion of AnxA6 in BT-549 cells not only led to impaired cell-cell cohesion and cell spreading onto collagen type IV but also inhibited cell motility and invasiveness. Finally, whereas the MAP kinase (ERK1/2) pathway remained constitutively active, Ca2+-dependent activation of focal adhesion kinase and the phosphoinositide-3 (PI3) kinase/Akt pathway were strongly inhibited in the AnxA6-depleted BT-549 cells. These data suggest that loss of AnxA6 promotes breast cancer tumorigenesis by uncoupling the feedback mechanism that prevents abnormal or sustained increases in intracellular Ca2+. Supported by grants: 1 SC1 CA134018-01 and DOD W81XWH-07-1-0254

Kv7-type Channel Currents in Spiral Ganglion Neurons: INVOLVEMENT IN SENSORINEURAL HEARING LOSS

Alterations in K(v)7-mediated currents in excitable cells result in several diseased conditions. A case in DFNA2, an autosomal dominant version of progressive hearing loss, involves degeneration of hair cells and spiral ganglion neurons (SGNs) from basal to apical cochlea, manifesting as high-to-low frequency hearing loss, and has been ascribed to mutations in K(v)7.4 channels. Analyses of the cellular mechanisms of K(v)7.4 mutations and progressive degeneration of SGNs have been hampered by the paucity of functional data on the role K(v)7 channels play in young and adult neurons. To understand the cellular mechanisms of the disease in SGNs, we examined temporal (young, 0.5 months old, and senescent, 17 months old) and spatial (apical and basal) roles of K(v)7-mediated currents. We report that differential contribution of K(v)7 currents in mice SGNs results in distinct and profound variations of the membrane properties of basal versus apical neurons. The current produces a major impact on the resting membrane potential of basal neurons. Inhibition of the current promotes membrane depolarization, resulting in activation of Ca(2+) currents and a sustained rise in intracellular Ca(2+). Using TUNEL assay, we demonstrate that a sustained increase in intracellular Ca(2+) mediated by inhibition of K(v)7 current results in significant SGN apoptotic death. Thus, this study provides evidence of the cellular etiology and mechanisms of SGN degeneration in DFNA2.

Heart-specific Deletion of CnB1 Reveals Multiple Mechanisms Whereby Calcineurin Regulates Cardiac Growth and Function

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca2+ following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca2+ handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca2+-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the α-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca2+-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.

Amyloid-β-Acetylcholinesterase complexes potentiate neurodegenerative changes induced by the Aβ peptide. Implications for the pathogenesis of Alzheimer's disease

The presence of amyloid-β (Aβ) deposits in selected brain regions is a hallmark of Alzheimer's disease (AD). The amyloid deposits have "chaperone molecules" which play critical roles in amyloid formation and toxicity. We report here that treatment of rat hippocampal neurons with Aβ-acetylcholinesterase (Aβ-AChE) complexes induced neurite network dystrophia and apoptosis. Moreover, the Aβ-AChE complexes induced a sustained increase in intracellular Ca2+ as well as a loss of mitochondrial membrane potential. The Aβ-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Aβ-AChE fibrillar complexes. These alterations in calcium homeostasis were reversed when the neurons were treated previously with lithium, a GSK-3β inhibitor; Wnt-7a ligand, an activator for Wnt Pathway; and an N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801), demonstrating protective roles for activation of the Wnt signaling pathway as well as for NMDA-receptor inhibition. Our results indicate that the Aβ-AChE complexes enhance Aβ-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Aβ alone. From a therapeutic point of view, activation of the Wnt signaling pathway, as well as NMDAR inhibition may be important factors to protect neurons under Aβ-AChE attack.