Modulation Of Calcium Signaling Research Articles

Interplay Between Calcium and AMPK Signaling in Human Cytomegalovirus Infection.

Calcium signaling and the AMP-activated protein kinase (AMPK) signaling networks broadly regulate numerous aspects of cell biology. Human Cytomegalovirus (HCMV) infection has been found to actively manipulate the calcium-AMPK signaling axis to support infection. Many HCMV genes have been linked to modulating calcium signaling, and HCMV infection has been found to be reliant on calcium signaling and AMPK activation. Here, we focus on the cell biology of calcium and AMPK signaling and what is currently known about how HCMV modulates these pathways to support HCMV infection and potentially contribute to oncomodulation.

Modulators of calcium signalling at fertilization.

Calcium (Ca2+) signals initiate egg activation across the animal kingdom and in at least some plants. These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca2+ is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca2+ influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca2+. Following the Ca2+ rise, the reuptake of Ca2+ into intracellular stores or efflux of Ca2+ out of the egg drive the return of cytoplasmic Ca2+ back to baseline levels. The molecular mediators of these Ca2+ fluxes in different organisms include Ca2+ release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca2+ signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling.

Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pellicle-compartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites.

TRPM5 Negatively Regulates Calcium-Dependent Responses in Lipopolysaccharide-Stimulated B Lymphocytes

B cells produce high amounts of cytokines and immunoglobulins in response to lipopolysaccharide (LPS) stimulation. Calcium signaling cascades are critically involved in cytokine production of Tcells, and the cytosolic calcium concentration is regulated by calcium-activated monovalent cation channels (CAMs). Calcium signaling is also implicated in B cell activation; however, its involvement in the cytokine production of LPS-stimulated B cells remains less well characterized. Here, we show that the transient receptor potential melastatin 5 channel (TRPM5), which is one of the CAMs, negatively modulates calcium signaling, thereby regulating LPS-induced proliferative and inflammatory responses by B cells. LPS-stimulated B cells of Trpm5-deficient mice exhibit an increased cytosolic calcium concentration, leading to enhanced proliferation and the production of the inflammatory cytokines interleukin-6 and CXCL10. Furthermore, Trpm5-deficient mice show an exacerbation of endotoxic shock with high mortality. Our findings demonstrate the importance of TRPM5-dependent regulatory mechanisms in LPS-induced calcium signaling of splenic B cells.

The Role of Ca2+-NFATc1 Signaling and Its Modulation on Osteoclastogenesis.

The increasing of intracellular calcium concentration is a fundamental process for mediating osteoclastogenesis, which is involved in osteoclastic bone resorption. Cytosolic calcium binds to calmodulin and subsequently activates calcineurin, leading to NFATc1 activation, a master transcription factor required for osteoclast differentiation. Targeting the various activation processes in osteoclastogenesis provides various therapeutic strategies for bone loss. Diverse compounds that modulate calcium signaling have been applied to regulate osteoclast differentiation and, subsequently, attenuate bone loss. Thus, in this review, we summarized the modulation of the NFATc1 pathway through various compounds that regulate calcium signaling and the calcium influx machinery. Furthermore, we addressed the involvement of transient receptor potential channels in osteoclastogenesis.

Role of Calcium Signaling in Prostate Cancer Progression: Effects on Cancer Hallmarks and Bone Metastatic Mechanisms.

Advanced prostate cancers that progress to tumor metastases are often considered incurable or difficult to treat. The etiology of prostate cancers is multi-factorial. Among other factors, de-regulation of calcium signals in prostate tumor cells mediates several pathological dysfunctions associated with tumor progression. Calcium plays a relevant role on tumor cell death, proliferation, motility-invasion and tumor metastasis. Calcium controls molecular factors and signaling pathways involved in the development of prostate cancer and its progression. Such factors and pathways include calcium channels and calcium-binding proteins. Nevertheless, the involvement of calcium signaling on prostate cancer predisposition for bone tropism has been relatively unexplored. In this regard, a diversity of mechanisms triggers transient accumulation of intracellular calcium in prostate cancer cells, potentially favoring bone metastases development. New therapies for the treatment of prostate cancer include compounds characterized by potent and specific actions that target calcium channels/transporters or pumps. These novel drugs for prostate cancer treatment encompass calcium-ATPase inhibitors, voltage-gated calcium channel inhibitors, transient receptor potential (TRP) channel regulators or Orai inhibitors. This review details the latest results that have evaluated the relationship between calcium signaling and progression of prostate cancer, as well as potential therapies aiming to modulate calcium signaling in prostate tumor progression.

Glucocorticoids rapidly stimulate cAMP production in a Gαs‐dependent manner

Glucocorticoids have wide therapeutic applications in inflammation and immune disorders and have traditionally been used to treat diseases such as asthma and COPD. They exert their anti‐inflammatory effects by binding to the cytosolic glucocorticoid receptor (GRα), a nuclear receptor that translocates into the nucleus to regulate gene transcription. Several studies have shown that glucocorticoids can have rapid, non‐genomic actions. For instance, short duration studies on several cell lines revealed that glucocorticoids can rapidly modulate calcium signaling, reactive oxygen species and arachidonic acid release. Moreover, studies show that a membrane GR (mGR) may interact directly with a GPCR coupled to Gαs and/or Gq/11.ObjectiveTo better understand the underlying mechanism of the non‐genomic effects of glucocorticoids, we tested the hypothesis that glucocorticoids can directly stimulate cAMP production via a Gαs coupled GPCR.ResultsThe glucocorticoids prednisone, fluticasone and budesonide stimulate cAMP rapidly in the absence of phosphodiesterase (PDE) inhibitors as measured in real time via the cADDis assay. These effects were seen in human airway smooth muscle (HASM), HEK‐293, HFL‐1, MCF‐7 and A375 cells. Glucocorticoids retained cAMP stimulating activity in HASM cells preincubated with 10 μM IBMX, a broad spectrum PDE inhibitor, implying that they do not increase cAMP via inhibition of PDEs. Cortisol‐albumin conjugate, a cell membrane impermeable glucocorticoid, stimulated cAMP suggesting that an outward facing membrane‐bound receptor mediates glucocorticoid non‐genomic signaling. GRα knockdown had no effect on glucocorticoid‐stimulation of cAMP, implying a GRα‐independent mechanism. We tested whether glucocorticoid‐stimulated cAMP depends upon the G protein Gαs by transfecting HASM with siRNA for GNAS. Immunoblot analysis confirmed a reduction in expression of both long and short forms of Gαs in the knockdown condition. Gαs knockdown lead to diminished cAMP production by fluticasone, budesonide and formoterol but not by forskolin, indicating that Gαs is an essential part of the non‐genomic signaling mechanism of glucocorticoids. The G protein estrogen receptor (GPER) is a G‐protein coupled receptor (GPCR) that couples to Gαs and binds to specific steroid ligands and is expressed in various cell types including HASM, HFL‐1 and HEK‐293. In the presence of either G15 or G36, GPER‐specific antagonists, both budesonide‐ and fluticasone‐stimulated cAMP was unaltered, suggesting that GPER does not mediate cAMP signaling by glucocorticoids.ConclusionTaken together these results reveal that glucocorticoids induce rapid, non‐genomic signaling via the Gαs‐cAMP pathway. The identity of the cell surface receptor for glucocorticoids is unknown, but given the rapid, GRα‐independent, Gαs‐dependent nature of the response we hypothesize that a GPCR or family of GPCRs is involved.Support or Funding InformationThis work was supported by NIH grant GM107094

Cyclophilins A and B oppositely regulate renal tubular epithelial cell phenotype

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial–mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor β (TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGFβ-induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium-buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.

Ca2+ signaling in T lymphocytes: the interplay of the endoplasmic reticulum, mitochondria, membrane potential, and CRAC channels on transcription factor activation

T cell receptor stimulation initiates a cascade of reactions that cause an increase in intracellular calcium (Ca2+) concentration mediated through inositol 1,4,5-trisphosphate (IP3). To understand the basic mechanisms by which the immune response in T cells is activated, it is useful to understand the signaling pathways that contain important targets for drugs in a quantitative fashion. A computational model helps us to understand how the selected elements in the pathways interact with each other, and which component plays the crucial role in systems. We have developed a mathematical model to explore the mechanism for controlling transcription factor activity, which regulates gene expression, by the modulation of calcium signaling triggered during T cell activation. The model simulates the activation and modulation of Ca2+ release-activated Ca2+ (CRAC) channels by mitochondrial dynamics and depletion of endoplasmic reticulum (ER) store, and also includes membrane potential in T-cells. The model simulates the experimental finding that increases in Ca2+ current enhances the activation of transcription factors and the Ca2+ influx through CRAC is also essential for the NFAT and NFκB activation. The model also suggests that plasma membrane Ca2+-ATPase (PMCA) controls a majority of the extrusion of Ca2+ and modulates the activation of CRAC channels. Furthermore, the model simulations explain how the complex interaction of the endoplasmic reticulum, membrane potential, mitochondria, and ion channels such as CRAC channels control T cell activation.

The Proposed Multimodal Mechanism of Action of Cannabidiol (CBD) in Epilepsy: Modulation of Intracellular Calcium and Adenosine-mediated Signalling

The Proposed Multimodal Mechanism of Action of Cannabidiol (CBD) in Epilepsy: Modulation of Intracellular Calcium and Adenosine-mediated Signalling

Dual function of miR-1248 links interferon induction and calcium signaling defects in Sjögren's syndrome.

BackgroundSjögren's syndrome (SS) is one of the most common autoimmune disorders leading to exocrine gland dysfunction. Both immune-dependent processes – like Type I Interferon (IFN) signaling and immune-independent processes – such as calcium signaling in epithelial cells – contribute to disease pathophysiology. However, a mechanistic link between these processes has not been demonstrated.MethodsPrimary human salivary gland cells were used to evaluate the differential expression of miRNAs with smRNA-seq in primary epithelial cells culture and digital PCR was conducted in SS human salivary glands (SG) biopsies to verify the results. With siRNA screening and pull-down assays to establish the role of miRNA in IFN activation.FindingsActivation of IFN-β by miR-1248 is through the direct association with both RIG-I and AGO2. Further functional studies establish a unique dual functional role of miR-1248 in phSG cells: i) activation of the RIG-I pathway by acting as ligand of this sensor leading to IFN production and ii) regulation of the expression of mRNAs through the canonical microRNA function. Importantly, ITPR3, a key component of calcium signaling in epithelial cells, that has previously shown to be downregulated in SS SG, was directly targeted and downregulated by miR-1248, inducing the same functional calcium signaling changes as observed in SS SGs.InterpretationIdentification of the first endogenous mammalian microRNA that binds to RIG-I inducing IFN production but also demonstrate a novel pathophysiological underlying mechanism in which miR-1248 overexpression links two major pathways associated with SS, namely activation of IFN production with modulation of calcium signaling. Together, these findings suggest a unifying hypothesis for the immune-independent and -dependent processes contributing to the pathogenesis of SS.FundThis research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Dental and Craniofacial Research (NIDCR).

Magnetic field regulates plant functions, growth and enhances tolerance against environmental stresses.

Global climatic fluctuations and the increasing population have been responsible for the decline in the crop productivity. The chemical fertilizers, pesticides, and suitable genetic resources are commonly used for improving the crop yield. Magnetic field (MF) therapy for plants and animals has been found to be an effective and emerging tool to control diseases and increase tolerance against the adverse environment. Very limited studies have been attempted to determine the role of MF on plant tolerance against various stress conditions. This review aims to highlight the mitigating effect of MF on plants against abiotic and biotic stresses. MF interacts with seeds and plants and accelerates metabolism, which leads to an improved germination. The primary and secondary metabolites, enzyme activities, uptake of nutrient and water are reprogrammed to stimulate the plant growth and yield under favorable conditions. During adverse conditions of abiotic stress such as drought, salt, heavy metal contamination in soil, MF mitigates the stress effects by increasing antioxidants and reducing oxidative stress in plants. The stunted plant growth under different light and temperature conditions can be overcome by the exposure to MF. An MF treatment lowers the disease index of plants due to the modulation of calcium signaling, and proline and polyamines pathways. This review explores the basic and recent information about the impact of MF on plant survival against the adverse environment and emphasizes that thorough research is required to elucidate the mechanism of its interaction to protect the plants from biotic and abiotic stresses.

Resveratrol derivatives increase cytosolic calcium by inhibiting plasma membrane ATPase and inducing calcium release from the endoplasmic reticulum in prostate cancer cells

Resveratrol (RES) is a putative chemotherapeutic naturally found in grapes, peanuts, and Japanese knotweed. Previous studies demonstrate that RES modulates calcium signaling as part of its chemotherapeutic activity. In this study, we determined the chemotherapeutic activity of three RES esters that have been modified at the 4’ hydroxyl by the addition of pivalate, butyrate, and isobutyrate. All of the RES derivatives disrupted the calcium signaling in prostate cancer cells more than the parent compound, RES. Further, we demonstrate that the RES derivatives may disrupt the calcium homeostasis by activating calcium release from the endoplasmic reticulum and inhibiting plasma membrane Ca2+-ATPase. The pivalated and butyrated RES derivatives decreased cell viability significantly more than RES. Because pivalated and butyrated RES are more effective than RES at targeting calcium signaling pathways, pivalated and butyrated RES may serve as more effective chemotherapeutics.

Vascular defects of DYRK1A knockouts are ameliorated by modulating calcium signaling in zebrafish.

ABSTRACTDYRK1A is a major causative gene in Down syndrome (DS). Reduced incidence of solid tumors such as neuroblastoma in DS patients and increased vascular anomalies in DS fetuses suggest a potential role of DYRK1A in angiogenic processes, but in vivo evidence is still scarce. Here, we used zebrafish dyrk1aa mutant embryos to understand DYRK1A function in cerebral vasculature formation. Zebrafish dyrk1aa mutants exhibited cerebral hemorrhage and defects in angiogenesis of central arteries in the developing hindbrain. Such phenotypes were rescued by wild-type dyrk1aa mRNA, but not by a kinase-dead form, indicating the importance of DYRK1A kinase activity. Chemical screening using a bioactive small molecule library identified a calcium chelator, EGTA, as one of the hits that most robustly rescued the hemorrhage. Vascular defects of mutants were also rescued by independent modulation of calcium signaling by FK506. Furthermore, the transcriptomic analyses supported the alterations of calcium signaling networks in dyrk1aa mutants. Together, our results suggest that DYRK1A plays an essential role in angiogenesis and in maintenance of the developing cerebral vasculature via regulation of calcium signaling, which may have therapeutic potential for DYRK1A-related vascular diseases.

The proposed multimodal mechanism of action of cannabidiol (CBD) in epilepsy: modulation of intracellular calcium and adenosine-mediated signaling (P5.5-007)

The proposed multimodal mechanism of action of cannabidiol (CBD) in epilepsy: modulation of intracellular calcium and adenosine-mediated signaling (P5.5-007)

Cardiac sigma receptors - an update.

More than four decades passed since sigma receptors were first mentioned. Since then, existence of at least two receptor subtypes and their tissue distributions have been proposed. Nowadays, it is clear, that sigma receptors are unique ubiquitous proteins with pluripotent function, which can interact with so many different classes of proteins. As the endoplasmic resident proteins, they work as molecular chaperones - accompany various proteins during their folding, ensure trafficking of the maturated proteins between cellular organelles and regulate their functions. In the heart, sigma receptor type 1 is more dominant. Cardiac sigma 1 receptors regulate response to endoplasmic reticulum stress, modulates calcium signaling in cardiomyocyte and can affect function of voltage-gated ion channels. They contributed in pathophysiology of cardiac hypertrophy, heart failure and many other cardiovascular disorders. Therefore, sigma receptors are potential novel targets for specific treatment of cardiovascular diseases.

Dietetic ingredients of small animals suffering from obesity and their biological role

Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have a negative effect on health. The daily ration of an adult cat must contain 40–45% of proteins, 20–25% of fats, 25–30% of carbohydrates. Low- fat diets are recommended to cats with overweight. Necessary amino acids are in the diet of cats with excess weight. Taurine is a sulfonic acid, which synthesizes in the body of animals and humans from the amino acid of cysteine. It plays an essential role in the digestion and assimilation of fats and lipids. The need for cats in taurine is due to their limited ability to synthesize taurine from amino acids that contain sulfur, as well as the fact that it holds bile acids. The latter is very important because cats do not produce bile acid salts associated with glycine, even in the case of taurine deficiency. Taurine has many fundamental biological roles, such as conjugation of bile acids, antioxidation, osmoregulation, membrane stabilization, and modulation of calcium signaling. It is essential for cardiovascular function, and development and function of skeletal muscle, the retina, and the central nervous system. The deficiency of taurine leads to degeneration of the retina and blindness, deafness, cardiomyopathy, disorders in the functioning of the immune and reproductive systems, suppression of neonatal growth, and the occurrence of birth defects. The recommended amount for a cat per day is 100–200 mg. L-Tryptophan is an α-amino acid that is used in the biosynthesis of proteins. Together with vitamin B6, magnesium and niacin, tryptophan is responsible for the serotonin production in the brain (a mediator that regulates the activity of nerve cells and transmits signals between them). Also, tryptophan is involved in the production of hemoglobin and affects the reproductive function of animals. The recommended amount for a cat per day is 0.3 g/1000 kcal of energy value (EV). DL-methionine is a synthetic analog of natural methionine. Methionine is an essential amino acid for animals. As the substrate for other amino acids such as cysteine and taurine, and the important antioxidant glutathione, methionine plays a critical role in the metabolism and health of many species, including humans. Methionine is a source of sulfur that forms the keratin protein. Keratin is simply necessary for the health of the hair, skin, and claws of the animal. The recommended amount for a cat per day (methionine + cystine) is 1.5 g/1000 kcal EV. Nowadays the problem of overweight in small domestic animals, in particular in cats and dogs, is becoming more widespread. In order to prevent the spread of obesity, therapeutic rations should be balanced by all indicators, such as proteins, fats, carbohydrates, amino acids, minerals, and vitamins.

190 Effects of endocrine-disrupting chemical on calcium signalling in cardiomyocyte differentiation from mouse embryonic stem cells

Endocrine-disrupting chemicals (EDC) are substances similar to steroid hormones that can disturb normal physiological functions of male and female reproductive organs. Endocrine-disrupting chemicals tend to bind to steroid hormone receptors. Sex steroid hormones modulate calcium signalling in the cardiac muscle in early embryo development. Among the steroid hormones, progesterone (P4) has been reported to affect blood pressure and other aspects of the cardiovascular system. The EDC that have similar structure to P4, such as octyl-phenol (OP) and bisphenol A (BPA), are potentially harmful to development of the heart. To confirm the effect of OP and BPA on early differentiation of mouse embryonic stem cells (mES) into cardiomyocytes, the hanging-drop method with mESC cell line (ES-E14TG2a) was used for forming embryoid bodies. Pluripotent mESC were cultured in basal medium with leukemia inhibitory factor and grown on mitomycin C-treated mouse embryonic fibroblasts in a 60-mm plate at 37°C in a 5% CO2 humidified tissue culture incubator with basal medium consisting of DMEM/F-12 supplemented with nonessential amino acids, 10% heat-inactivated and certified fetal bovine serum (FBS), 2-mercaptoethanol, penicillin, and streptomycin. The mouse embryoid bodies (mEB) were suspended onto 6-well plates and cultured with differentiation medium containing steroid-free FBS without leukemia inhibitory factor. Progesterone, OP, and BPA were added on Day 2 from mEB attachment. In addition, mifepristone (RU486), an antagonist for progesterone receptor (PR), was used to confirm the effect of P4 through PR. To determine whether RU486 is capable of attenuating the inhibition effect of P4, RU486 was added for 1 day starting on Day 11. Assessment of cardiomyocyte differentiation was determined by checking the number of beating cell populations divided by the total number of attached mEB. Total RNA was extracted using Trizol reagent and synthesised to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase. Messenger RNA level was assessed using quantitative real-time PCR. To investigate calcium signalling, the mRNA levels of calcium channel gene Trpv2 and contraction-related genes Ryr2, Cam2, and Mylk3 were analysed. Beating ratio was decreased in P4, OP, and BPA treatments. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison tests, and P<0.05 was considered statistically significant in least 3 different replicates. The mRNA level of Pgr was significantly increased in P4, OP, and BPA-treated group. However, the mRNA level of calcium channel gene Trpv2 was significantly decreased in the P4, OP, and BPA-treated group. Expression of contraction-related genes such as Ryr2, Cam2, and Mlck3 were significantly decreased in the P4, OP, and BPA-treated group. In addition, treatment with RU486 rescues altered calcium channel gene and contraction-related genes. Taken together, these results suggest that OP and BPA may affect the differentiation of mESC into cardiomyocyte and disrupts differentiation of cardiomyocytes.

Systems Biology Analyses to Delineate Mechanisms of Anti-CD20 Antibody Resistance in Non-Hodgkin Lymphoma (NHL): Influence of BCR Signaling and the Critical Importance of Calcium Polarization

Introduction:CD20 a cell surface protein selectively expressed in mature B cells is involved in regulation of B cell differentiation and is a well-established target for anti-CD20 therapy in the treatment of B cell malignancies. However, therapeutic resistance to anti-CD20 antibodies, including rituximab and obinutuzumab, continues to remain a major obstacle in the treatment for NHL patients. Known mechanisms of anti-CD20 resistance include loss of CD20 expression, apoptotic dysfunction and/or poor immunoreactivity. Further mechanisms, including potential biologic etiologies, which engender resistance to anti-CD20 antibodies remain largely unclear. We sought to delineate the immuno-biological landscape and mechanisms associated with anti-CD20 resistance via systems biology analyses and immune functional studies with rituximab and obinutuzumab resistant (RR & OR) NHL models in a novel single-cell microfluidics platform.Methods:RR or OR cells (SUDHL4 and SUDHL10) were developed by repeated culturing with chronic low dose exposure to antibody (Ab) drugs. Anti-CD20 resistance was biologically characterized by CD20 immunostaining-flow cytometry, gene expression profiling and systems biology analysis (with Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Immunological characterization was performed using NK mediated ADCC activity (AcellaTox-Glo), real-time analysis of NK cell-NHL interaction kinetics by microfluidics and cytokine/immune factor secretion using proximity extension assay (Olink immuno-oncology panel). Ionomycin induced release of Calcium assay was performed using Fluo-4 cell-based assay and plate reader.Results: Flow cytometric studies and ADCC assays using anti-CD20 Abs and primary NK cells, exhibited prominent downregulation of CD20 cell surface expression and markedly decreased NK mediated ADCC activity in RR (11%, P=0.0002) and OR (17%, P=0.001) compared with 10mg/ml of rituximab (51%) or obinutuzumab (56%), respectively, in the parental SUDHL4 cells. The basal NK mediated Furthermore, microfluidic analyses revealed decreased cell-cell interactions among NK cells and anti-CD20 resistant NHL cells in the presence of either antibody. Systems biology analysis of the transcriptome with these RR and OR cells showed downregulation of immune signaling mechanisms closely related MAPK, NFkB, mTOR and JAK/STAT pathways, with partial upregulation of PLCγ, B-cell receptor (BCR) signaling (via western blot). Using the Olink 92-plex cytokine assay, we observed hyposecretion of the majority of cytokines including IL-2, IL-6, IL-8, IL-10, TNFα, IFNγ and FASL all CD20 resistant cells (Fig. 1A). Based in part on these biological changes, we hypothesized that loss of CD20 associated with decreased calcium signaling could be the master regulator observed with anti-CD20 resistance. Assessment of ionomycin-induced releasable calcium showed lack of ionomycin releasable calcium in anti-CD20 resistant cells (Fig. 1B). Treatment with the depolarizing agent, veratridine, restored ionomycin releasable calcium levels, reversed BCR signaling, and it was accompanied with significantly decreased cell viability (60%, P<0.0001) in anti-CD20 resistant cells compared with 100% in sensitive cells. In contrast, use of the hyperpolarizing ivermectin resulted in mimicking anti-CD20 resistant upregulation of BTK and anti-CD20 sensitive cells intolerant to decreased calcium levels.Conclusions: RR and OR NHL cells were associated with downregulation of immune signaling pathways, cytokine hyposecretion, and upregulation of BCR signaling. Furthermore, these observed biological changes were associated with decreased intracellular calcium suggesting its role as a master regulator in anti-CD20 resistance. Utilizing depolarizing and hyperpolarizing pharmacologic agents, we were able to restore favorable signaling changes associated with anti-CD20 antibody sensitivity as well as mimic a resistant phenotype in sensitive cells. Additional investigation of the modulation of calcium signaling as a novel mechanism and target for the treatment of anti-CD20 resistant NHL is warranted. [Display omitted] DisclosuresEvens:Pharmacyclics International DMC: Membership on an entity's Board of Directors or advisory committees; Affimed: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Tesaro: Research Funding; Novartis: Consultancy.

Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons.

Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca2+ ]i ) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca2+ ]i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca2+ ]i signaling in a dose-dependent manner when tested at 10 and 100 μM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca2+ ]i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 μM U73122, and 10 μM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca2+ ]i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.