Induced Calcium Influx Research Articles

α-Linolenic acid attenuates pseudo-allergic reactions by inhibiting Lyn kinase activity

BackgroundPseudo-allergic reactions are potentially fatal hypersensitivity responses caused by mast cell activation. α-linolenic acid (ALA) is known for its anti-allergic properties. However, its potential anti-pseudo-allergic effects were not much investigated. PurposeTo investigate the inhibitory effects of ALA on IgE-independent allergy in vitro, and in vivo, as well as the mechanism underlying its effects. Methods/study designsThe anti-anaphylactoid activity of ALA was evaluated in passive cutaneous anaphylaxis reaction (PCA) and systemic anaphylaxis models. Calcium imaging was used to assess intracellular Ca2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of Lyn-PLCγ-IP3R-Ca2+ and Lyn-p38/NF-κB signaling pathway. ResultsALA (0, 1.0, 2.0, and 4.0 mg/kg) dose-dependently reduced serum histamine, chemokine release, vasodilation, eosinophil infiltration, and the percentage of degranulated mast cells in C57BL/6 mice. In addition, ALA (0, 50, 100, and 200 μM) reduced Compound 48/80 (C48/80) (30 μg/ml)-or Substance P (SP) (4 μg/ml)-induced calcium influx, mast cell degranulation and cytokines and chemokine release in Laboratory of Allergic Disease 2 (LAD2) cells via Lyn-PLCγ-IP3R-Ca2+ and Lyn-p38/NF-κB signaling pathway. Moreover, ALA (0, 50, 100, and 200 μM) inhibited C48/80 (30 μg/ml)- and SP (4 μg/ml)-induced calcium influx in Mas-related G-protein coupled receptor member X2 (MrgX2)-HEK293 cells and in vitro kinase assays confirmed that ALA inhibited the activity of Lyn kinase. In response to 200 μM of ALA, the activity of Lyn kinase by (7.296 ± 0.03751) × 10−5 units/μl and decreased compared with C48/80 (30 μg/ml) by (8.572 ± 0.1365) ×10−5 units/μl. ConclusionOur results demonstrate that ALA might be a potential Lyn kinase inhibitor, which could be used to treat pseudo-allergic reaction-related diseases such as urticaria.

17β‐Estradiol via Orai1 activates calcium mobilization to induce cell proliferation in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most lethal estrogen-sensitive gynecological cancer. Studies have reported that estrogen induces rapid cellular calcium mobilization in cells and can determine the fate of a cell. We found that estrogen increased the calcium release-activated calcium channel modulator 1 (Orai1) protein expression levels in SK-OV-3 cells. However, to date, there has been no research on the functional relationship and molecular mechanism of estrogen-regulating Orai1 during EOC development. In our study, Orai1 had a high expression level in high-grade serous ovarian tumor tissues and SK-OV-3 cells. Estrogen promoted cell proliferation and migration while inhibiting cell apoptosis in SK-OV-3 cells. Orai1 silencing suppressed estrogen-induced cell migration and proliferation. Overexpression of Orai1, however, enhanced the ability of 17β-estradiol (E2) to exert its function. Estrogen induced rapid calcium influx in SK-OV-3 cells. Knockdown of Orai1 in SK-OV-3 cells blocked E2-induced stored-operated Ca2+ influx. The messenger RNA expression of caspase 3, matrix metallopeptidase 1, and cyclin-dependent kinase 6 were regulated via Orai1 under E2 treatment. Our results suggest that estrogen, by regulating Orai1, induced calcium influx to determine cell fate.

Regulatory role of receptor-like cytoplasmic kinases in early immune signaling events in plants.

Receptor-like cytoplasmic kinases (RLCKs) play crucial roles in regulating plant development and immunity. Conserved pathogen-associated molecular patterns (PAMPs) derived from microbes are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Microbial effectors, whose initial function is to promote virulence, are recognized by plant intracellular nucleotide-binding domain and leucine-rich repeat receptors (NLRs) to initiate effector-triggered immunity (ETI). Both PTI and ETI trigger early immune signaling events including the production of reactive oxygen species, induction of calcium influx and activation of mitogen-activated protein kinases. Research progress has revealed the important roles of RLCKs in the regulation of early PTI signaling. Accordingly, RLCKs are often targeted by microbial effectors that are evolved to evade PTI via diverse modulations. In some cases, modulation of RLCKs by microbial effectors triggers the activation of NLRs. This review covers the mechanisms by which RLCKs engage diverse substrates to regulate early PTI signaling and the regulatory roles of RLCKs in triggering NLR activation. Accumulating evidence suggests evolutionary links and close connections between PAMP- and effector-triggered early immune signaling that are mediated by RLCKs. As key immune regulators, RLCKs can be considered targets with broad prospects for the improvement of plant resistance via genetic engineering.

Engineering and Rewiring of a Calcium-Dependent Signaling Pathway.

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.

The role of Piper chaba Hunt. and its pure compound, piperine, on TRPV1 activation and adjuvant effect

BackgroundPiper chaba Hunt. is used as an ingredient in Thai traditional preparation for arthritis. Its isolated compound is piperine which shows anti-inflammatory activity. Piperine produces a burning sensation because it activates TRPV1 receptor. The TRPV1 activation involved with the analgesic and adjuvant effect. P. chaba Hunt. has not been reported about TRPV1 activation and adjuvant effect. The aim of this study was to investigate the effect of P. chaba extract and piperine on TRPV1 receptor, which is considered as a target for analgesic and their adjuvant effects to support the development of an analgesic drug from herbal medicine.MethodsThe effect of P. chaba extract and piperine on HEK cells expressing TRPV1 channel was examined by calcium imaging assay. Adjuvant effects of P. chaba extract and piperine were investigated by a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) model in mice.ResultsP. chaba extract induced calcium influx with EC50 value of 0.67 μg/ml. Piperine induced calcium influx with EC50 value of 0.31 μg/ml or 1.08 μM. For mouse CHS model, we found that 1% piperine, 5% piperine, 1% P. chaba extract and 5% P. chaba extract significantly enhanced sensitization to FITC as revealed by ear swelling responses.ConclusionP. chaba extract and piperine activated TRPV1 channel and enhanced contact sensitization to FITC.

Regulation of the SIRT1 signaling pathway in NMDA-induced Excitotoxicity

Silent Information Regulator 1 (SIRT1), an NAD+-dependent deacetylase, contributes to the neuroprotective effect. However, intracellular signaling pathways that affect SIRT1 function remain unknown. It is well known that N-methyl-D-aspartate (NMDA) receptor activation induces calcium influx which then activates PKC, and SIRT1 is a mRNA target for HuR protein. We hypothesize that Ca2+-PKC-HuR-SIRT1 pathway modulates SIRT1 function. The present study is to investigate the potential pathway of SIRT1 in the SH-SY5Y cell line as an in vitro model of NMDA-induced neurotoxicity. The results showed that: (1) SIRT1 levels were downregulated in NMDA model; (2) NMDA induced an increase in serine phosphorylation of HuR, while inhibition of serine phosphorylation of HuR increased SIRT1 levels, promoting cell survival; (3) PKC inhibitor (Gö 6976) reversed NMDA insults and also suppressed serine phosphorylation of HuR; (4) 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, fully reversed NMDA insults and also inhibited PKC activity evoked by NMDA. These results indicate that intracellular elevated Ca2+ activates PKC, which phosphorylates HuR and then promotes SIRT1 mRNA decay and subsequent neuronal death in NMDA model. Therefore, the study suggests that inhibition of Ca2+-PKC-HuR-SIRT1 pathway could be an effective strategy for preventing certain neurological diseases related to NMDA excitotoxicity.

Granisetron Alleviates Alzheimer's Disease Pathology in TgSwDI Mice Through Calmodulin-Dependent Protein Kinase II/cAMP-Response Element Binding Protein Pathway.

Alzheimer's disease (AD) is characterized by a compromised blood-brain barrier (BBB) and disrupted intracellular calcium homeostasis in the brain. Therefore, rectifying the BBB integrity and restoring calcium homeostasis could provide an effective strategy to treat AD. Recently, we developed a high throughput-screening assay to screen for compounds that enhance a cell-based BBB model integrity, which identified multiple hits among which is granisetron, a Food and Drug Administration approved drug. Here, we evaluated the therapeutic potential of granisetron against AD. Granisetron was tested in C57Bl/6J young and aged wild-type mice, and in a transgenic mouse model of AD namely TgSwDI for its effect on BBB intactness and amyloid-β (Aβ)-related pathology. Our study findings showed that granisetron enhanced BBB integrity in both aged and TgSwDI mice. This effect was associated with an overall reduction in Aβ load and neuroinflammation in TgSwDI mice brains. In addition, and supported by proteomics analysis, granisetron significantly reduced Aβ induced calcium influx in vitro, and rectified calcium dyshomeostasis in TgSwDI mice brains by restoring calmodulin-dependent protein kinase II/cAMP-response element binding protein pathway, which was associated with cognitive improvement. These results support granisetron repurposing as a potential drug to hold, slow, and/or treat AD.

Missiles of Mass Disruption: Composition and Glandular Origin of Venom Used as a Projectile Defensive Weapon by the Assassin Bug Platymeris rhadamanthus.

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug Platymeris rhadamanthus, a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators.

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100nM and 3μM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.

Quercetin as a Lyn kinase inhibitor inhibits IgE-mediated allergic conjunctivitis

BackgroundAllergic conjunctivitis (AC) resulting from conjunctival reactive inflammation is a common ocular surface disease. Quercetin is known for its anti-allergic properties but its effects on conjunctivitis are less well understood. PurposeIn this study, we evaluated the anti-allergic effects of quercetin in animal models of conjunctivitis, and explored its molecular mechanism(s) of action in cultured human mast cells (MCs). Key resultsQuercetin inhibited the ovalbumin (OVA) induced expression of IgE, HA, IL-4, TNF-α and substance-P in the peripheral blood of AC mouse models. Quercetin also attenuated OVA induced MC degranulation, eosinophil number, substance P concentrations, and mRNA IL-4/TNF-α expression in the conjunctival tissue of AC models. In vitro analysis showed that quercetin reduced DNP-HSA/IgE induced calcium (Ca2+) influx, and suppressed degranulation and chemokine release in LAD2 cells (human primary mast cell). Quercetin also inhibited DNP-HSA/IgE induced Lyn/PLCγ/IP3R–Ca2+ activation, Lyn/ERK1/2 signaling, and Lyn/NF-κB activation in LAD2 cells, all of which promote inflammation. When added alone, quercetin had no effect on PLCγ1 phosphorylation or expression, but potently inhibited Lyn and phosphorylation-Lyn. Quercetin (200 μM) and Lyn inhibitors (Bafetinib, 10 μM) inhibit the activity of Lyn kinase, and quercetin can reduce the activation of Lyn kinase by Lyn agonist (Tolimidone, 10 μM). These data can be preliminarily determined that quercetin can inhibit allergic conjunctivitis as a Lyn kinase inhibitor. Conclusions and implicationsThis study illustrated the use of quercetin for the treatment of allergic conjunctivitis, which might act through its ability to inhibit Lyn/PLCγ/IP3R–Ca2+, Lyn/ERK1/2, and Lyn/NF-κB signaling. The inhibition of Lyn likely represents a major mechanism by which quercetin dampens the inflammatory response in AC disease models.

Neurogenesis-on-Chip: Electric field modulated transdifferentiation of human mesenchymal stem cell and mouse muscle precursor cell coculture

A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding on the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of murine myoblasts (C2C12) with hMScs [hMSc:C2C12=1:10] in a custom designed polymethylmethacrylate (PMMA) based microfluidic device with in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysical stimulation in the range of the endogenous physiological electric field strength (8 ± 0.06 mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, the hMScs exhibited the neurosphere-like clusters with SOX2, nestin, βIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signalling among the differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly supports neural-like behaviour for the electric field stimulated cells in coculture. The intriguing results are explained in terms of the paracrine signalling among the transdifferentiated cells in the electric field stimulated cellular microenvironment. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro.

Broadening and Enhancing Functions of Antibodies by Self-Assembling Multimerization at Cell Surface.

Monoclonal antibody therapy has offered treatment benefits. Nonetheless, a lack of efficacy still exists, partially because monovalent binding of antibodies to specific receptors fails to translate into an active response. Here, we report a pretargeting–postassembly approach that exploits the selective Watson–Crick base pairing properties of oligonucleotides and multivalently tethers receptor-prebound antibodies to albumin at the cell surface. We demonstrate that this two-step self-assembling strategy allows sequential actions of receptor binding and clustering that broadens and strengthens the functions of antibodies. We show that anti-CD20 obinutuzumab (OBN) modified with one morpholino oligonucleotide (OBN-MORF1) maintains the feature of naked OBN antibody upon CD20 binding, and results in actin redistribution, homotypic adhesion, and lysosome-mediated cell death. Consecutive treatment with albumin grafted with multiple copies of a complementary morpholino oligonucleotide (HSA-(MORF2)x) hybridizes with surface-attached OBN-MORF1, manipulates CD20 clustering, and engages additional signals to induce calcium influx and caspase-related apoptosis. With the two types of different mechanisms collaborating in one system, the simple design exerted a notable survival extension of mice bearing disseminated B-cell lymphomas.

Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells.

ABSTRACTObjective:To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors.Methods:We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings.Results:Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM).Conclusion:These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.

TRPA1-expressing lamina propria mesenchymal cells regulate colonic motility.

The physiological process of defecation is directly controlled by colorectal motility. The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in small intestine enterochromaffin cells and is involved in gastrointestinal motility via serotonin release. In the colorectum, however, enterochromaffin cell localization is largely distinct from that in the small intestine. Here, we investigated the role of lower gastrointestinal tract TRPA1 in modulating colorectal motility. We found that in colonic tissue, TRPA1 is predominantly expressed in mesenchymal cells of the lamina propria, which are clearly distinct from those in the small intestine. These cells coexpressed COX1 and microsomal prostaglandin E synthase-1. Intracolonic administration of TRPA1 agonists induced colonic contraction, which was suppressed by a prostaglandin E2 (PGE2) receptor 1 antagonist. TRPA1 activation induced calcium influx and PGE2 release from cultured human fibroblastic cells. In dextran sulfate sodium-treated animals, both TRPA1 and its endogenous agonist were dramatically increased in the colonic lamina propria, accompanied by abnormal colorectal contractions. Abnormal colorectal contractions were significantly prevented by pharmacological and genetic inhibition of TRPA1. In conclusion, in the lower gastrointestinal tract, mesenchymal TRPA1 activation results in PGE2 release and consequently promotes colorectal contraction, representing what we believe is a novel physiological and inflammatory bowel disease-associated mechanism of gastrointestinal motility.

Trans-Anethole of Fennel Oil is a Selective and Nonelectrophilic Agonist of the TRPA1 Ion Channel.

Transient receptor potential (TRP) cation channels are molecular targets of various natural products. TRPA1, a member of TRP channel family, is specifically activated by natural products such as allyl isothiocyanate (mustard oil), cinnamaldehyde (cinnamon), and allicin (garlic). In this study, we demonstrated that TRPA1 is also a target of trans-anethole in fennel oil (FO) and fennel seed extract. Similar to FO, trans-anethole selectively elicited calcium influx in TRPA1-expressing mouse sensory neurons of the dorsal root and trigeminal ganglia. These FO- and anethole-induced calcium responses were blocked by a selective TRPA1 channel antagonist, HC-030031. Moreover, both FO and trans-anethole induced calcium influx and transmembrane currents in HEK293 cells stably overexpressing human TRPA1 channels, but not in regular HEK293 cells. Mutation of the amino acids S873 and T874 binding site of human TRPA1 significantly attenuated channel activation by trans-anethole, whereas pretreating with glutathione, a nucleophile, did not. Conversely, activation of TRPA1 by the electrophile allyl isothiocyanate was abolished by glutathione, but was ostensibly unaffected by mutation of the ST binding site. Finally, it was found that trans-anethole was capable of desensitizing TRPA1, and unlike allyl isothiocyanate, it failed to induce nocifensive behaviors in mice. We conclude that trans-anethole is a selective, nonelectrophilic, and seemingly less-irritating agonist of TRPA1.

Wnt5a Induces Association of ROR1 with Ca2+/Calmodulin-Dependent Protein Kinase II and ROR1-Dependent Calcium Influx in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could enhance CLL-cell proliferation (Yu J, et al, JCI 126:585, 2016; Yu J, et al, Leukemia 31:2608, 2017; Hasan MK, et al, Blood 132:170, 2018). We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified Ca2+/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine-specific protein kinase. Recent studies demonstrated that high levels of different isoforms of CaMK, especially CaMKII, were expressed in several cancers. CaMKII phosphorylates nearly 40 different proteins, including enzymes, ion channels, kinases, and transcription factors and can play a critical role in the regulation of proliferation and survival of various cancer cells (Wang YY, et al, Oncotarget 20:11725, 2015; Chi M, et al, Sci Rep 6:33132, 2016; Hoffman A, et al, Cell Signal 26:748, 2014). We validated our mass spectrometry finings in co-immunoprecipitation studies and immunoblot analyses. These studies showed that Wnt5a could induce the specific phosphorylation and recruitment of CaMKII to ROR1. Moreover, these studies demonstrated that Wnt5a-induced recruitment of CaMKII to ROR1 could be blocked by cirmtuzumab, a first-in-class humanized anti-ROR1 mAb undergoing clinical testing in patients with CLL (Choi MY, et al, Cell Stem Cell 22:951, 2018). CaMKs serve as a ubiquitous intracellular receptor for Ca2+ and their activity are regulated through Ca2+ signaling. Therefore, we examined calcium influx in CLL cells by flow cytometry. Treatment of CLL cells cultured overnight in serum-free medium induced calcium influx within seconds. Moreover, the relative capacity of Wnt5a to induce calcium influx was associated with the relative expression level of ROR1 on CLL cells of different patients. Furthermore, Wnt5a could enhance calcium influx induced by treatment with anti-µ in CLL cells that expressed ROR1, but not in CLL cells that lacked expression of ROR1. Knockdown of ROR1 using ROR1 siRNA or treatment with cirmtuzumab inhibited the capacity of Wnt5a to enhance calcium influx induced by treatment with anti-µ. Collectively, these data demonstrate that Wnt5a-induced ROR1-signaling in CLL can influence BCR-signaling and activation of the Ca2+/calmodulin-dependent protein kinase II signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

Histones Induce the Release of Extracellular Hemoglobin and Red Blood Cell-Derived Microvesicles with Procoagulant Activity

BackgroundHistones are nuclear proteins that can be released extracellularly following cell death or certain mechanisms of cell activation. Infusion of high doses of histones into mice causes lethality. Circulating levels of extracellular histones are elevated in several disease states associated with inflammation and thrombosis. The mechanisms by which extracellular histones induce morbidity and mortality are not fully understood. Histones may activate or be toxic to several cell types in vitro. Most of these activities have been shown to be dependent on histone signaling through toll-like receptors (TLRs). Red blood cells (RBC) do not express TLRs. However, previous studies have reported that histones promote RBC aggregation, increase shear-dependent RBC fragility, reduce deformability under flow and cause hemolytic anemia in mice. Histones also induce calcium influx and promote PS expression on RBC membranes thereby supporting the prothrombinase activity and shortening clotting times in plasma. Histones may insert into cell membranes (independently of TLRs), and form pore-like structures similar to ionophore. The role of this process in histone-induced RBC toxicity has not been fully investigated.ObjectiveWe assessed the ability of individual histones to promote the release of extracellular hemoglobin (Ex-Hb) and RBC-derived microvesicles (RBC-MVs), as well as RBC-MVs' ability to initiate or propagate thrombin generation (TG) in MV-free plasma.MethodsRBCs were isolated from citrated whole blood from healthy volunteers, and were re-suspended in buffer with or without 10uM A23187 (calcium ionophore), recombinant histones H2A, H2B, H3 or H4 for 1 hour at 37°C.To assess histone binding, RBCs were washed and dual-stained with fluorophore-conjugated anti-glycophorin A (GPA) and anti-panhistone antibodies. Stained RBCs were analyzed using imaging flow cytometry, and were considered positive for histone binding if the fluorescence intensity was > 95th percentile of RBCs processed in the absence of histones.RBCs were removed by centrifugation at 2,500g for 15 minutes x2. RBC-MVs present in the supernatant were pelleted by centrifugation at 20,000g for 30 min. Isolated RBC-MVs were then re-suspended in buffer to assess PS exposure using the microparticle activity assay (Hyphen Biomed). RBC-MVs were also re-suspended in re-calcified MV-free plasma supplemented with 4uM 41%PC/44%PE/15%PS lipid vesicles to assess their ability to initiate TG by monitoring the activity of thrombin on a fluorogenic substrate over time. To assess their ability to propagate TG, coagulation was initiated by 1pM tissue factor in MV-free plasma containing histone-induced RBC-MVs without addition of synthetic lipid vesicles. In this condition, phospholipids present on the surface of histone-induced RBC-MVs are the limiting factor for TG.The supernatant collected after pelleting RBC-MVs was used to measure Ex-Hb by absorbance at 540 nm.ResultsThe percentage of RBCs positive for histone binding was 21.6%, 2.3%, 76.9% and 98.1% for H2A, H2B, H3 and H4, respectively. Representative images of RBC staining for GPA and histone H4 is shown in Figure 1A.All histone types induced the release of Ex-Hb (Fig. 1B) and RBC-MVs that possess PS activity (Fig. 1C) in a dose-dependent manner, with the highest PS activity observed with H2A. RBC-MVs released by H2A or H2B clearly initiated TG in MV-free normal pool plasma (Fig.1D). They also significantly enhanced TF-initiated TG in the absence of additional lipid vesicles. The release of Ex-Hb and procoagulant RBC-MVs induced by histones were re-capitulated by ionophore to a greater degree, as expected (Fig.1B-E).ConclusionsAll 4 types of core histones bind to RBCs and induce the release of Ex-Hb. Our findings suggest that despite their lower binding to RBCs, histones H2A and H2B may have a greater ability to induce the release of procoagulant RBC-MVs that can initiate and propagate TG in plasma. The reason for the differential toxicity of histone types on RBCs and the discrepancy between the relative effect of histone-induced RBC-MVs on PS equivalents and TG are unknown. Histone-induced RBC toxicity appears to mimic calcium ionophore, supporting the idea that histones may insert and form functional pores in cell membranes. The contribution of Ex-Hb and procoagulant RBC-MVs to the morbidity and mortality of extracellular histones in vivo requires further studies. [Display omitted] DisclosuresMonroe:Novo Nordisk A/S: Honoraria, Research Funding.

Quercetin inhibits Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R related Ca2+ fluctuations

An allergic reaction is a potentially fatal hypersensitivity response caused by mast cell activation, particularly histamine and lipid mediators. Histamine release caused by reaction to drugs is considered a pseudo-allergic reaction. Quercetin is known for its anti-allergic immune effect. However, at present, its anti-pseudo-allergic effect and its mechanism are less investigated. Therefore, the purpose of this study was to evaluate the anti-pseudo-allergic effect of Quercetin in vivo and to explore the mechanism in vitro. The anti-pseudo-allergic activity of Quercetin was evaluated in vivo using a mouse model, while Quercetin mechanism of action was examined in vitro using HEK293 cells expressing Mrgprx2, a mast cell specific receptor, and LAD2 mast cell line. Our in vivo results showed that Quercetin could attenuate Evans blue leakage in the paws and hind paw thickness in C57BL/6 mice in a dose-dependent manner, and could significantly inhibit serum histamine and chemokines release. In addition, it suppressed calcium mobilization and attenuated the release of histamine and MCP-1 in peritoneal mast cells in a dose-dependent manner. Furthermore, it inhibited the vasodilation due to histamine, the release of eosinophils, and the percentage of degranulated mast cells, indicating that Quercetin antagonized mast cell mediators in vivo, histamine-induced vasodilation and eosinophil release.In vitro results showed that Quercetin reduced pseudo-allergic induced calcium influx, suppressed degranulation and chemokines release in a similar way as dexamethasone (100 μM) (mast cell stabilizer) in LAD2 mast cell line. In addition, Quercetin inhibited Mrgprx2-induced both calcium influx and pseudo-allergic reaction in HEK293 cells expressing Mrgprx2. C48/80, a histamine promoter, and Substance P (a neuropeptide) EC50 was higher when combined with Quercetin compared to the EC50 of these compounds alone, suggesting that Quercetin could inhibit Mrgprx2-induced pseudo-allergic reaction. Furthermore, Quercetin decreased PLCγ-IP3R signaling pathway activation induced by C48/80 in LAD2 mast cell line. In Mrgprx2 knockdown LAD2 cells, the effect of Quercetin (200 μM) reduced C48/80 induced calcium flux and the release of β‑hexosaminidase, histamine, MCP-1 and IL-8 compared with non-atopic control (NC) transfected LAD2 human mast cells, suggesting that Quercetin anti-pseudo-allergic effect was related to Mrgprx2. The docking results showed that Quercetin had a good binding affinity with Mrgprx2 similar to the one of Substance P and C48/80. Therefore, Quercetin inhibited Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations.Our results validated Quercetin as an effective small molecule inhibiting Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations, thus highlighting a potential candidate to suppress Mrgprx2 induced pseudo-allergic related diseases.

Microtubule dynamics modulate sensing during cold acclimation in grapevine suspension cells

Cold acclimation is of practical relevance, since it can avoid cold-induced damage in various crops. To efficiently activate cold acclimation requires that the chilling stress is perceived and processed efficiently. In the current work, we use a transgenic cell line of V. rupestris expressing a GFP-labelled tubulin to follow the effect of cold acclimation and the relation between microtubules and the expression of the transcription factor Cold Box Factor 4 (CBF4) as molecular readout for adaptive responses to cold stress. We find that chilling induced cold tolerance correlated with increased CBF4 expression. We show that cold acclimation can be achieved through stabilisation of microtubules by taxol, as well as through transient elimination of microtubules by pronamide in the absence of cold stress. Furthermore, results from inhibitor studies indicate that transcriptional activation of CBF4 appears to be under control of calcium influx. We screened a population of the ancestor of V. sylvestris and could identify different clades with strong induction of CBF4, indicative of genetic variation in cold adaptability that can be used for breeding. We summarize our findings into a working model where microtubule dynamics controls the sensitivity of cold induced calcium influx mediating the induction of CBF4 culminating in cold hardening.

Azelnidipine Attenuates the Oxidative and NFκB Pathways in Amyloid-β-Stimulated Cerebral Endothelial Cells.

Cerebral amyloid angiopathy (CAA), a condition depicting cerebrovascular accumulation of amyloid β-peptide (Aβ), is a common pathological manifestation in Alzheimer's disease (AD). In this study, we investigated the effects of Azelnidipine (ALP), a dihydropyridine calcium channel blocker known for its treatment of hypertension, on oligomeric Aβ (oAβ)-induced calcium influx and its downstream pathway in immortalized mouse cerebral endothelial cells (bEND3). We found that ALP attenuated oAβ-induced calcium influx, superoxide anion production, and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and calcium-dependent cytosolic phospholipase A2 (cPLA2). Both ALP and cPLA2 inhibitor, methylarachidonyl fluorophosphate (MAFP), suppressed oAβ-induced translocation of NFκB p65 subunitto nuclei, suggesting that cPLA2 activation and calcium influx are essential for oAβ-induced NFκB activation. In sum, our results suggest that calcium channel blocker could be a potential therapeutic strategy for suppressing oxidative stress and inflammatory responses in Aβ-stimulated microvasculature in AD.