Stimulation Of Calcium Release Research Articles

Cow milk and its dairy products ameliorate bone toxicity in the Coragen-induced rat model

Abstract Coragen is an insecticide that stimulates calcium release from intracellular stores of muscle cells causing death to sensitive species. The present study aimed to evaluate the bone toxic effect of Coragen and the potential therapeutic effect of cow milk, yogurt, and soft cheese in rats. Toxicity was induced by Coragen administration with different doses of 1/20 or 1/40 LD50 in rats. Groups of rats (n = 6) were treated with either 5 g milk, 5 g yogurt, or 1.5 g cheese. Coragen administration elevated alkaline and acid phosphatases activity and reduced the calcium and phosphorus level in urine and serum of rats administered with Coragen. Femur and tibia length, thickness, weight, and breaking force were decreased by Coragen administration and femur Ca and P contents as well. Bone mineral area (BMA), bone mineral content (BMC), bone mineral density (BMD), protein profile (total, albumin, and globulin), and antioxidant system (TAC, GSH, GPX, GST, and SOD) were decreased by Coragen. All these parameters were improved on the treatment with milk and milk products. The results showed that yogurt treatment was significantly superior to the other treatments in increasing BMD (27%), breaking force (9%), femur Ca (41%), serum Ca (14%), and serum P (16%) and in reducing acid phosphatases (14%) and urine Ca and P by 8 and 10%, respectively. It can be concluded that the treatment with milk and milk products may provide treatment against osteoporosis and toxicity caused by Coragen.

Роль рианодиновых и IP 3 -рецепторов в генерации кальциевых ответов, вызываемых трициклическими антидепрессантами в нейронах неокортекса крысы

Tricyclic antidepressants, in particular amitriptyline (ATL) and desipramine (DES), are currently used to treat depression and chronic pain of various origins, in which NMDA receptor dysfunctions play an important role. The effect of therapeutic concentrations of ATL on calcium-dependent desensitization of NMDA receptors, driven by the level of free calcium in the cytoplasm, is well-known. In addition, in cardiomyocytes, ATL and DES can cause the release of calcium into the cytoplasm from intracellular stores by opening inositol-3-phosphate (IP3R) and / or ryanodine receptors (RyR) channels. The aspect of the effect of these drugs on neurons remains poorly understood. We studied the dependence of the calcium response to DES and ATL on the activation of IP3R and RyR of the endoplasmic reticulum and mitochondria using rat neocortex neurons in primary culture. Short-term (30 s) paired (5 min interval) applications of 200 μM DES or 200 μM ATL induce similar magnitude calcium responses in cortical neurons. The use of RyR and IP3R antagonists showed that responses to ATL are blocked by the IP3R antagonist 2-APB (100 μM), while responses to DES are blocked by ryanodine, the RyR antagonist (100 nM). Since intracellular distribution of RyR and IP3R is not homogenous, it can be assumed that DES and ATL stimulate calcium release from different calcium depots, representing segments of the reticulum or mitochondria. In addition, ATL and DES, being channel blockers of NMDA receptors, inhibited calcium entry from outside the cell via activated NMDA receptors. Considering the high concentrations of DES and ATL (more than 100 μM) required to stimulate the release of deposited calcium in neurons, it seems unlikely that such effects participate their therapeutic action. However, the found specificity of DES and ATL for RyR and IP3R, respectively, can be used as a tool for experimental purposes.

The Parkinson's disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Pathway to liver disease

Signal Transduction Nonalcoholic steatohepatitis (NASH) is a prominent cause of chronic liver disease in humans but how it develops is not fully understood. Wang et al. traced a signaling pathway through which increased cholesterol in the liver promoted NASH. In a mouse model of NASH, cholesterol internalization led to activation of soluble adenylyl cyclase and production of cyclic adenosine monophosphate–activated protein kinase A. This in turn stimulated calcium release, activation of the small guanosine triphosphatase RhoA, and the stabilization of TAZ, the transcriptional regulator. This pathway may provide targets for therapeutic intervention in NASH and enhance understanding of cholesterol signaling. Cell Metab. 10.1016/j.cmet.2020.03.010 (2020).

STIM1 a Calcium Sensor Promotes the Assembly of an ECM that Contains Extracellular Vesicles and Factors that Modulate Mineralization

Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. Several proteins work in coordination and contribute to this tightly regulated process. STIM1 (Stromal interaction molecule 1) is one such protein that has been recently identified to function in bone and enamel mineralization. STIM1 is a calcium sensor localized on the ER membrane and is well recognized for its physiological role in maintaining calcium homeostasis. We have demonstrated earlier that DMP1 stimulation of preosteoblasts and preodontoblasts stimulate calcium release from internal Ca2+ stores and this store depletion is sensed by STIM1. Store-operated calcium entry (SOCE) is one of the major Ca2+ influx mechanisms following store depletion in the ER. To demonstrate a role for STIM1 in dentin matrix mineralization, we generated transgenic DPSCs in which STIM1 was either overexpressed or silenced. These cells were characterized for their differentiation potential by gene expression analysis, Alizarin Red staining and the topology of the matrix examined by Field Emission Scanning Electron Microscopy (FESEM). Results suggest upregulation of genes involved in mineralization and increased calcium deposition with STIM1 overexpression. FESEM results demonstrate that STIM1 overexpression resulted in release of large amount of extracellular microvesicles and promoted matrix mineralization. Interestingly, knockdown of STIM1 resulted in release of fewer microvesicles and less mineral deposits in the ECM. Reduced dentin thickness, malformed and highly porous alveolar bone of STIM-1 null mice confirmed the role of STIM1 in the formation of calcified tissues. Overall, STIM1 is a crucial molecule in biomineralization as STIM1 can influence intracellular Ca2+ oscillations and thus provide a signal for activation of upstream and downstream effectors to promote precursor cell differentiation and matrix mineralization.

The Role of NAADP-Mediated Endo-Lysosomal Calcium Release in the Cardiac Atria

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a highly potent calcium-mobilising second messenger which stimulates calcium release from endo-lysosomes via the opening of TPC2 channels. In cardiac ventricular myocytes, lysosomes form nano-junctions with the sarcoplasmic reticulum (SR) and mitochondria and exposure to NAADP (synthesised by CD38 upon beta-adrenergic stimulation), leads to an augmented calcium transient and SR calcium content, and contributes to the hypertrophy and arrhythmogenesis associated with chronic beta-adrenergic stress. We utilised transmission electron microscopy (TEM) and optical mapping in lapine and murine models to investigate endo-lysosomal calcium signalling in the cardiac atria. TEM of fixed, imbedded rabbit atrial myocytes or tissue (n=3 animals) confirmed that lysosomes form nano-junctions with both the SR (median distance 17nm, IQR 13-21nm, n=15 lysosomes) and mitochondria (median distance 13nm, IQR 10-57nm, n=16 lysosomes). These are close enough to have the potential for localised calcium signalling. In intact murine left atria, 10nM isoprenaline caused a 59.3±4.8% increase in calcium transient amplitude (n=6, fluorophore rhod-2). Abolishing endo-lysosomal calcium storage using 5µM Bafilomycin A1 (Baf) significantly reduced the calcium transient response to isoprenaline (to 35.3±2.7%, n=6, P<0.01). In CD38KO mice, left atrial responses were significantly reduced (35.9±2.6%, n=6) in comparison to wild-type controls (59.3±4.9%, n=6, P<0.01) and there was no longer an effect of Baf on isoprenaline response (35.9±2.6% and 33.6±4.0% in CD38KO control and Baf respectively, P>0.05, both n=6). Our data suggest that, endo-lysosomes can form nanojunctions for calcium signalling within cardiac atrial myocytes and that the endo-lysosomal NAADP pathway is functionally relevant during beta-adrenergic stimulation. The changes to and effects of this stereotyped architecture and pathway in atrial disease states are unknown and will be a vital avenue for future research.

Fine-Tuning of GPCR-Chemokine Interactions. Design and Identification of Chemokine Analogues as Receptor Agonists, Biased Agonists, and Antagonists.

Chemokines play important roles in immune defense by directing migration of leukocytes and serve as key promoters of tumorigenesis and metastasis. This study explores the molecular mechanisms of recognition and activation of two homologous chemokine receptors, CXCR1 and CXCR2, using CXCL8 analogues with residue substitutions in the conserved Glu4Leu5Arg6 (ELR) triad. Analysis of the binding of CXCL8 analogues to CXCR1 is consistent with the two-site model for signal recognition of CXCR1, whereas analysis of the binding of CXCL8 analogues to CXCR2 supported a single-site model for signal recognition of CXCR2. The CXCL8-Arg6His analogue stimulated calcium release, phosphorylation of ERK1/2, and chemotaxis in cells expressing CXCR1. However, CXCL8-Arg6His failed to stimulate calcium release and chemotaxis in cells expressing CXCR2, although it stimulated phosphorylation of ERK1/2, indicating that CXCL8-Arg6His operated as a classical CXCR2 biased agonist. The CXCL8-Glu4AlaLeu5AlaArg6His analogue was inactive in cells expressing CXCR1 and CXCR2. These findings suggest that the Glu4Leu5 motif in CXCL8 is essential for activation of CXCR1 and CXCR2. Importantly, CXCL8-Glu4AlaLeu5AlaArg6His blocked specifically the calcium release and chemotaxis of cells expressing CXCR1 but not of cells expressing CXCR2. CXCL8-Glu4AlaLeu5AlaArg6His was identified as the first specific CXCR1 antagonist. The binding of CXCL8-ELR6H to CXCR1 created a Zn2+ coordination site at the receptor activation domain responsible for calcium release, as ZnCl2 specifically blocked CXCL8-Arg6His-induced calcium release without affecting CXCL8-induced calcium release. This work provides the basis for further exploration of the activation mechanisms of chemokine receptors and will assist in the design of the next generation of modulators of CXCR1 and CXCR2.

Greater Protective Potent of s-Methyl Cysteine and Syringic Acid Combination for NGF-differentiated PC12 Cells against Kainic acid-induced Injury.

Objective: The effects of pre-treatments from s-methyl cysteine (SMC) alone, syringic acid (SA) alone and SMC plus SA against kainic acid (KA) induced injury in nerve growth factor (NGF) differentiated PC12 cells were investigated.Methods: NGF-differentiated PC12 cells were treated with 1 μM SMC, 1 μM SA or 0.5 μM SMC plus 0.5 μM SA for 2 days. Subsequently, cells were further treated by 150 μM KA.Results: KA suppressed Bcl-2 mRNA expression, enhanced Bax mRNA expression and casued cell death. SMC was greater than SA, and similar as SMC+SA in increasing Bcl-2 mRNA expression. SMC+SA led to greater increase in mitochondrial membrane potential and cell survival than SMC or SA alone. SMC+SA resulted in more reduction in reactive oxygen species and tumor necrosis factor-alpha generation, more increase in glutathione content and glutathione reductase activity than SMC or SA alone. KA up-regulated protein expression of nuclear factor kappa B (NF-κB) p65 and phosphorylated p38 (p-p38). SMC or SA pre-treatments alone limited protein expression of both factors. SMC+SA resulted in more suppression in NF-κB p65 and p-p38 expression. KA decreased glutamine level, increased glutamate level and stimulated calcium release. SMC pre-treatments alone reversed these alterations. SMC alone elevated glutamine synthetase (GS) activity and mRNA expression. SMC+SA led to greater GS activity and mRNA expression than SMC pre-treatments alone.Conclusion: These findings suggested that this combination, SMC+SA, might provide greater protective potent for neuronal cells.

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance.

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL.

ENMD-1068 inhibits liver fibrosis through attenuation of TGF-\u03b21/Smad2/3 signaling in mice

Protease-activated receptor 2 (PAR-2) plays an important role in the pathogenesis of liver fibrosis. We studied the effect of N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068), a PAR-2 antagonist, on the development of CCl4-induced liver fibrosis in mice and activation of hepatic stellate cells (HSCs) isolated from the mice. Before CCl4 injection, the mice were injected intraperitoneally with either 25 mg/kg or 50 mg/kg ENMD-1068 or with 200 μL of the vehicle control twice per week for 4 weeks. The isolated HSCs were stimulated by TGF-β1 with or without ENMD-1068 to evaluate the role of PAR-2 in TGF-β1 induced HSCs activation and collagen production. We showed that the levels of ALT/AST, collagen content, and α-smooth muscle actin (α-SMA) were significantly reduced by treatment with ENMD-1068 in CCl4-induced fibrotic mice. Interestingly, we found TGF-β1 signaling-related expression levels of α-SMA, type I and III collagen, and C-terminal phosphorylation of Smad2/3 were significantly decreased in the ENMD-1068 treated HSCs. Moreover, we showed ENMD-1068 treatment inhibited trypsin or SLIGRL-NH2 stimulated calcium release and TGF-β1 induced Smad transcriptional activity in HSCs. We demonstrated that ENMD-1068 reduces HSCs activation and collagen expression through the inhibiton of TGF-β1/Smad signal transduction.

The Sec14-like phosphatidylinositol transfer proteins Sec14l3/SEC14L2 act as GTPase proteins to mediate Wnt/Ca2+ signaling.

The non-canonical Wnt/Ca2+ signaling pathway plays important roles in embryonic development, tissue formation and diseases. However, it is unclear how the Wnt ligand-stimulated, G protein-coupled receptor Frizzled activates phospholipases for calcium release. Here, we report that the zebrafish/human phosphatidylinositol transfer protein Sec14l3/SEC14L2 act as GTPase proteins to transduce Wnt signals from Frizzled to phospholipase C (PLC). Depletion of sec14l3 attenuates Wnt/Ca2+ responsive activity and causes convergent and extension (CE) defects in zebrafish embryos. Biochemical analyses in mammalian cells indicate that Sec14l3-GDP forms complex with Frizzled and Dishevelled; Wnt ligand binding of Frizzled induces translocation of Sec14l3 to the plasma membrane; and then Sec14l3-GTP binds to and activates phospholipase Cδ4a (Plcδ4a); subsequently, Plcδ4a initiates phosphatidylinositol-4,5-bisphosphate (PIP2) signaling, ultimately stimulating calcium release. Furthermore, Plcδ4a can act as a GTPase-activating protein to accelerate the hydrolysis of Sec14l3-bound GTP to GDP. Our data provide a new insight into GTPase protein-coupled Wnt/Ca2+ signaling transduction.

High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

A new paradigm for IP 3 signaling

Cell Biology The second-messenger inositol trisphosphate (IP3) stimulates calcium release from the endoplasmic reticulum (ER). Dickinson et al. triggered the focal release of IP3 in animal cells and measured calcium “puffs”—intense, localized increases in calcium released from the ER (see the Focus by Leybaert). IP3 diffused much more slowly within cells than had been originally measured in vitro. Thus, rather than functioning as a global cellular signal, IP3 can produce local signals, increasing the complexity of information that can be encoded in responses to IP3-generating stimuli. Sci. Signal. 9 , ra108 and fs17 (2016).

HPM Method Applied to Solve the Model of Calcium Stimulated, Calcium Release Mechanism

In this paper, homotopy perturbation method (HPM) is employed to provide an approximate, but detailed, solution for the nonlinear differential equation that describes the calcium stimulated calcium release mechanism. Comparison to the exact solutions shows that the method is extremely efficient, if initial guess is suitably chosen.

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 μm) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 μm) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

The Regulation of Calcium Homeostasis in T Lymphocytes

The Regulation of Calcium Homeostasis in T Lymphocytes

Granule Calcium for Cell Killing

NAADP promotes exocytosis of cytolytic granules by stimulating calcium release from the granules.

Adenine Dinucleotide Second Messengers and T-lymphocyte Calcium Signaling

Calcium signaling is a universal signal transduction mechanism in animal and plant cells. In mammalian T-lymphocytes calcium signaling is essential for activation and re-activation and thus important for a functional immune response. Since many years it has been known that both calcium release from intracellular stores and calcium entry via plasma membrane calcium channels are involved in shaping spatio-temporal calcium signals. Second messengers derived from the adenine dinucleotides NAD and NADP have been implicated in T cell calcium signaling. Nicotinic acid adenine dinucleotide phosphate (NAADP) acts as a very early second messenger upon T cell receptor/CD3 engagement, while cyclic ADP-ribose (cADPR) is mainly involved in sustained partial depletion of the endoplasmic reticulum by stimulating calcium release via ryanodine receptors. Finally, adenosine diphosphoribose (ADPR) a breakdown product of both NAD and cADPR activates a plasma membrane cation channel termed TRPM2 thereby facilitating calcium (and sodium) entry into T cells. Receptor-mediated formation, metabolism, and mode of action of these novel second messengers in T-lymphocytes will be reviewed.

Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes.

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.