Intracellular Calcium Mobilization Research Articles

C5aR2 Activation Broadly Modulates the Signaling and Function of Primary Human Macrophages.

The complement activation fragment C5a is a potent proinflammatory mediator that is increasingly recognized as an immune modulator. C5a acts through two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), to powerfully modify multiple aspects of immune cell function. Although C5aR1 is generally acknowledged to be proinflammatory and immune-activating, the potential roles played by C5aR2 remain poorly defined. Despite studies demonstrating C5aR2 can modulate C5aR1 in human cells, it is not yet known whether C5aR2 functionality is limited to, or requires, C5aR1 activation or influences immune cells more broadly. The present study, therefore, aimed to characterize the roles of C5aR2 on the signaling and function of primary human monocyte-derived macrophages, using a C5aR2 agonist (Ac-RHYPYWR-OH; P32) to selectively activate the receptor. We found that although C5aR2 activation with P32 by itself was devoid of any detectable MAPK signaling activities, C5aR2 agonism significantly dampened C5aR1-, C3aR-, and chemokine-like receptor 1 (CMKLR1)-mediated ERK signaling and altered intracellular calcium mobilization mediated by these receptors. Functionally, selective C5aR2 activation also downregulated cytokine production triggered by various TLRs (TLR2, TLR3, TLR4, and TLR7), C-type lectin receptors (Dectin-1, Dectin-2, and Mincle), and the cytosolic DNA sensor stimulator of IFN genes (STING). Surprisingly, activity at the C-type lectin receptors was particularly powerful, with C5aR2 activation reducing Mincle-mediated IL-6 and TNF-α generation by 80-90%. In sum, this study demonstrates that C5aR2 possesses pleiotropic functions in primary human macrophages, highlighting the role of C5aR2 as a powerful regulator of innate immune function.

Structure-Activity Relationship Analysis of Psychedelics in a Rat Model of Asthma Reveals the Anti-Inflammatory Pharmacophore.

Psychedelic drugs can exert potent anti-inflammatory effects. However, anti-inflammatory effects do not appear to correlate with behavioral activity, suggesting different underlying mechanisms. We hypothesized that the distinct structural features of psychedelics underlie functionally selective mechanisms at the target 5-HT2A receptor to elicit maximal anti-inflammatory effects. In order to test this hypothesis, we developed a new rat-based screening platform for allergic asthma. Next, we investigated 21 agonists at the 5-HT2A receptor from the three primary chemotypes (phenylalkylamine, ergoline, and tryptamine) for their ability to prevent airways hyperresponsiveness as a measure of pulmonary inflammation. Furthermore, we assessed each drug for in vitro activation of the canonical signaling pathway, calcium mobilization, from the 5-HT2A receptor. We find that the drug 2,5-dimethoxyphenethylamine (2C-H) represents the pharmacophore for anti-inflammatory activity and identify structural modifications that are either permissive or detrimental to anti-inflammatory activity. Additionally, there is no correlation between the ability of a particular psychedelic to activate intracellular calcium mobilization and to prevent the symptoms of asthma or with behavioral potencies. Our results support the notions that specific structural features mediate functional selectivity underlying anti-inflammatory activity and that relevant receptor activated pathways necessary for anti-inflammatory activity are different from canonical signaling pathways. Our results inform on the nature of interactions between ligands at the 5-HT2A receptor as they relate to anti-inflammatory activity and are crucial for the development of new 5-HT2A receptor agonists for anti-inflammatory therapeutics in the clinic that may be devoid of behavioral activity.

3121 – ERYTHROPOIETIN DEPENDENT SIGNALING IN ERYTHROBLASTIC ISLAND MACROPHAGES INDUCES PGE2 DEPENDENT DIFFERENTIATION OF STRESS ERYTHROID PROGENITORS

Inflammation caused by infection or tissue damage has a profound effect on hematopoiesis. Pro-inflammatory cytokines skew hematopoiesis to increase the production of myeloid effector cells, but this change comes with a cost as erythropoiesis is inhibited by these signals. In order to compensate for the decrease in steady state erythropoiesis, stress erythropoiesis is induced. This process is distinct from steady sate erythropoiesis and shares many properties with other stem cell mediated tissue regenerative responses. It is conserved between mouse and human. Unlike steady state erythropoiesis which constantly produces new erythrocytes, the strategy of stress erythropoiesis is to produce a bolus of erythrocytes that will maintain homeostasis until steady state erythropoiesis can resume. Previous work showed that immature stress erythroid progenitors rapidly proliferate but do not differentiate, which generates a transient amplifying population of progenitors. These progenitors develop in close contact with macrophages in structures known as erythroblastic islands (EBIs). Erythropoietin (Epo) dependent signaling in EBI macrophages changes the signals made by the macrophages from those promoting proliferation (Wnt and Hedgehog) to those promoting differentiation (PGJ2 and PGE2). Here we report that PGE2 dependent mobilization of intracellular calcium activates the Perk kinase (Eif2ak3) mediated integrated stress pathway in stress erythroid progenitors. This pathway acts to increase the expression of amino acid transporters and tRNA synthases resulting in the activation of the mTorC1 complex. These two pathways drive the commitment to erythroid differentiation and are required for the generation of new erythrocytes that will maintain homeostasis until steady state erythropoiesis can resume.

Oleuropein promotes hippocampal LTP via intracellular calcium mobilization and Ca2+-permeable AMPA receptor surface recruitment

Oleuropein (OLE), a major phenolic compound in olive oil, has been demonstrated to possess several pharmacological properties, including neuroprotection. However, the cognitive effects of OLE and its action mechanism have remained unclear. Here, we examined the effect of OLE on long-term potentiation (LTP) using field excitatory postsynaptic potential recorded in the CA1 region of both wild-type and 5XFAD mouse hippocampal slice preparations. In initial experiments with wild-type mice, 100 μM/1 h of OLE produced significant enhancements in the LTPs of Schaffer collateral synapses in the CA1 regions of treated mice, as compared to the vehicle-treated controls. As assessed by surface biotinylation and Western blot analysis, OLE caused a significant increase in protein kinase A (PKA)-mediated phosphorylation and the surface expression of GluA1 containing calcium permeable- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) in the hippocampus. Furthermore, we found that OLE enhanced LTP induction, while GluA1 phosphorylation occurred in an N-methyl-d-aspartate receptors (NMDARs)-independent manner. The OLE-induced CP-AMPAR trafficking resulted from elevated intracellular Ca2+ levels via regulation of phospholipase C (PLC). Consistently, we also found involvement of NMDAR-independent LTP and GluA1 phosphorylation in 5XFAD transgenic mice hippocampal slices treated with OLE. Together, our findings indicate that OLE may regulate beneficial effects on memory through the facilitation of CP-AMPAR trafficking and synaptic transmission.

P2X7 Receptor is Involved in Mitochondrial Dysfunction Induced by Extracellular Alpha Synuclein in Neuroblastoma SH-SY5Y Cells.

The purinergic P2X7 receptor (P2X7R) belongs to a family of trimeric ion channels that are gated by extracellular adenosine 5′-triphosphate (ATP). Several studies have pointed to a role of P2X7R-dependent signalling in Parkinson's disease (PD)-related neurodegeneration. The pathology of (PD) is characterized by the formation of insoluble alpha-synuclein (α-Syn) aggregates—Lewy bodies, but the mechanisms underlying α-Syn-induced dopaminergic cell death are still partially unclear. Our previous studies indicate that extracellular α-Syn directly interact with neuronal P2X7R and induces intracellular free calcium mobilization in neuronal cells. The main objective of this study was to examine the involvement of P2X7R receptor in α-Syn-induced mitochondrial dysfunction and cell death. We found that P2X7R stimulation is responsible for α-Syn-induced oxidative stress and activation of the molecular pathways of programmed cell death. Exogenous α-Syn treatment led to P2X7R-dependent decrease in mitochondrial membrane potential as well as elevation of mitochondrial ROS production resulting in breakdown of cellular energy production. Moreover, P2X7R-dependent deregulation of AMP-activated protein kinase as well as decrease in parkin protein level could be responsible for α-Syn-induced mitophagy impairment and accumulation of dysfunctional mitochondria. P2X7R might be putative pharmacological targets in molecular mechanism of extracellular α-Syn toxicity.

Ulmus parvifolia Modulates Platelet Functions and Inhibits Thrombus Formation by Regulating Integrin αIIbβ3 and cAMP Signaling.

BackgroundThe prevalence of cardiovascular diseases (CVDs) is increasing at a high rate, and the available treatment options, sometimes, have complications which necessitates the need to develop safer and efficacious approaches. Ethnomedicinal applications reportedly reduce CVD risk. Ulmus parvifolia Jacq. (Ulmaceae) commonly known as Chinese Elm or Lacebark Elm, is native to China, Japan, and Korea. It exhibits anti-inflammatory, antiviral, and anticancer properties, but its anti-platelet properties have not yet been elucidated.PurposeTo investigate the pharmacological anti-platelet and anti-thrombotic effects of U. parvifolia bark extract.Study Design and MethodsHuman and rat washed platelets were prepared; light transmission aggregometry and scanning electron microscopy was performed to assess platelet aggregation and the change in platelet shape, respectively. Intracellular calcium mobilization, ATP release, and thromboxane-B2 production were also measured. Integrin αIIbβ3 activation was analyzed in terms of fibrinogen binding, fibronectin adhesion, and clot retraction. The expression of MAPK, Src, and PI3K/Akt pathway proteins was examined. Cyclic nucleotide signaling pathway was evaluated via cAMP elevation and VASP phosphorylation. Anti-thrombotic activity of the extract was evaluated in vivo using an arteriovenous shunt rat model, whereas its effect on hemostasis in mice was assessed via bleeding time assay.Results U. parvifolia extract significantly inhibited human and rat platelet aggregation in a dose-dependent manner along with inhibition of calcium mobilization, dense granule secretion, and TxB2 production. Integrin αIIbβ3 mediated inside-out and outside-in signaling events, as evidenced by the inhibition of fibrinogen binding, fibronectin adhesion, and clot retraction. The extract significantly reduced phosphorylation of Src, MAPK (ERK, JNK, and p38MAPK), and PI3K/Akt pathway proteins. Cyclic-AMP levels were elevated in U. parvifolia-treated platelets, while PKAαβγ and VASPser157 phosphorylation was enhanced. U. parvifolia reduced thrombus weight in rats and moderately increased bleeding time in mice.Conclusion U. parvifolia modulates platelet responses and inhibit thrombus formation by regulating integrin αIIbβ3 mediated inside-out and outside-in signaling events and cAMP signaling pathway.

SAT-404 Neonatal Hypocalcemic Seizures in Offspring of a Mother with Familial Hypocalciuric Hypercalcemia Type 1 (FHH1)

Background: Familial hypocalciuric hypercalcemia type 1 (FHH1) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR), and considered to be a benign condition associated with mild-to-moderate hypercalcemia (1). However, the children of parents with FHH1 can develop a variety of disorders of calcium homeostasis in infancy. Objective: To further characterise the range of calcitropic phenotypes in the children of a mother with FHH1. Methods: We assessed a three generation FHH kindred by clinical, biochemical and mutational analysis following informed consent. Results: The kindred comprised a hypercalcemic male, his daughter who had hypercalcemia and hypocalciuria, and her four children, of whom two had asymptomatic hypercalcemia, one was normocalcemic, and one suffered from transient hypocalcemic seizures during infancy. The hypocalcemic infant had a serum calcium of 1.57 mmol/L (normal, 2.0-2.8) and PTH of 2.2 pmol/L (normal, 1.0-9.3) as a consequence of maternal hypercalcemia, and required treatment with I-V calcium gluconate infusions. Mutational analysis identified a novel heterozygous p.Ser448Pro CaSR variant in the hypercalcemic family members, but not in the children with hypocalcemia or normocalcemia. Three-dimensional modelling using a reported crystal structure of the dimeric CaSR showed the mutated Ser448 residue to be located in the CaSR extracellular domain, and predicted the p.Ser448Pro variant to disrupt a hydrogen bond interaction across the extracellular CaSR dimer interface. The variant Pro448 CaSR, when expressed in HEK293 cells, was shown to significantly impair CaSR-mediated intracellular calcium mobilisation and mitogen-activated protein kinase (MAPK) responses following stimulation with extracellular calcium, thereby demonstrating it to represent a loss-of-function mutation. Conclusion: These studies have identified a novel loss-of-function CaSR mutation which caused asymptomatic hypercalcemia in a mother and her children who had inherited the mutation. However, one child who did not inherit the mutation developed transient neonatal hypocalcemic seizures as a consequence of maternal hypercalcemia. These findings highlight the importance of assessing serum calcium and undertaking CaSR mutational analysis in the newborn offspring of a mother with FHH1. Reference: (1) Hannan FM, Kallay E, Chang W, Brandi ML, Thakker RV. The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases. Nat Rev Endocrinol. 2018; 15(1): 33-51.

Protein tyrosine phosphatase SHP-1 modulates microtubule organization in later stages of mast cell activation

Abstract Antigen-mediated activation of mast cells initiates signaling events leading to degranulation and release of inflammatory mediators. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Important role in microtubule nucleation from centrosomes play γ-tubulin complexes. Here we report on the regulation of microtubule organization in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1). Reciprocal immunoprecipitation experiments revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Pull-down experiments with truncated forms of γ-tubulin and SHP-1 unveiled corresponding interaction domains. When compared to controls, activated BMMCs without SHP-1 had more protrusions containing microtubules, higher level of tyrosine phosphorylation, enhanced activity of Syk kinase, as well as increased expression of cytokines and prostaglandins. Intracellular calcium mobilization and degranulation also increased. Microtubule regrowth experiments in cells lacking SHP-1 revealed stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes of γ-tubulin accumulation in centrosomes. Our data suggest a novel SHP-1 dependent mechanism for the suppression of microtubule formation in later stages of mast cell activation.

Lysophosphatidylcholine Containing Anisic Acid Is Able to Stimulate Insulin Secretion Targeting G Protein Coupled Receptors.

Diabetes mellitus is a worldwide health problem with high rates of mortality and morbidity. Management of diabetes mellitus by dietary components is achievable especially at the initial stage of the disease. Several studies confirmed the antidiabetic activities of simple phenolic acids and lysophosphatidylcholine (LPC). The main goal of this study was to identify new potential insulin secretion modulators obtained by combining the structures of two natural compounds, namely O-methyl derivatives of phenolic acids and phospholipids. LPC and phosphatidylcholine bearing methoxylated aromatic carboxylic acids were tested as potential agents able to improve glucose-stimulated insulin secretion (GSIS) and intracellular calcium mobilization in MIN6 β pancreatic cell line. Our results show that LPC with covalently bonded molecule of p-anisic acid at the sn-1 position was able to induce GSIS and intracellular calcium flux. Notably, 1-anisoyl-2-hydroxy-sn-glycero-3-phosphocholine did not affect the viability of MIN6 cells, suggesting its potential safe use. Furthermore, we have shown that three G protein coupled receptors, namely GPR40, GPR55, and GPR119, are targeted by this LPC derivative.

Adipose mesenchymal stromal/stem cells expanded by a GMP compatible protocol displayed improved adhesion on cancer cells in flow conditions

BackgroundAdipose tissue derived mesenchymal stromal/stem cells (ASC) can be expanded using supernatant rich in growth factors (SRGF) as Good Manufacturing Practice compatible additive, instead of fetal bovine serum (FBS). After transendothelial migration, ASC can migrate to cancer masses where they can release active substances. Due to their homing and secretion properties ASC can be used as targeted drug delivery vehicles. Nevertheless, the fraction of ASC actually reaching the tumor target is limited. The impact of culture conditions on ASC homing potential on cancer cells is unknown.MethodsIn dynamic in vitro conditions, we perfused FBS or SRGF ASC in flow chambers coated with collagen type I and fibronectin or seeded with endothelial cells or with HT1080, T98G and Huh7 cancer cells. Expression of selected adhesion molecules was evaluated by standard cytofluorimetry. Dynamic intracellular calcium concentration changes were evaluated in microfluidic and static conditions.ResultsWhen compared to FBS ASC, not specific adhesion of SRGF ASC on collagen type I and fibronectin was lower (−33.9%±12.2% and −45.3%±16.9%), while on-target binding on HT1080 and T98G was enhanced (+147%±8% and 120.5%±5.2%). Adhesion of both FBS and SRGF ASC on Huh7 cells was negligible. As confirmed by citofluorimetry and by function-blocking antibody, SRGF mediated decrease of CD49a expression accounted for lower SRGF-ASC avidity for matrix proteins. Upon stimulation with calcium ionophore in static conditions, mobilization of intracellular calcium in SRGF ASC was greater than in FBS ASC. In dynamic conditions, upon adhesion on matrix proteins and HT1080 cells, SRGF ASC showed marked oscillatory calcium concentration changes.ConclusionsSRGF can enhance specific ASC binding capacity on selected cancer cells as HT1080 (fibrosarcoma) and T98G (glioblastoma) cells. Upon cell-cell adhesion, SRGF ASC activate intracellular responses potentially improving cell secretion functions. SRGF ASC could be considered as suitable drug delivery vehicle for cancer therapy.

Efforts Toward GPR88 Deorphanization: Challenges and Alternative Approaches to Troublesome Cell‐Based Second Messenger Assays

G protein‐coupled receptors (GPCR) comprise a family of over 800 integral membrane proteins involved in signal transduction and serve as the primary cellular sensors for chemical stimuli. About 35% of current drugs act on GPCR targets. In addition, of the 300 agents in current clinical trials, greater than 20% target novel GPCRs for which there are no approved drugs (orphan GPCRs). Ligand discovery and deorphanization efforts seeking to assign function to understudied GPCRs are not trivial. Notoriously complex in nature, GPCR‐dependent signaling centers around receptor‐ligand interactions that serve to stabilize conformational access to intracellular signaling partners, G proteins. In GPCR signaling, receptors and heterotrimeric G proteins (made of the Gα and Gβγ subunits) work together to transmit signals via downstream effectors and distinct pathways. The measurement of GPCR mediated second messengers such as cyclic AMP (cAMP), intracellular calcium (Ca2+) mobilization, and ERK activation (MAP kinase) are commonly used as tools to detect ligand induced GPCR activation. However, experimental pathway elucidation is complicated by the tendencies for pathway crosstalk, receptor desensitization, and receptor‐G protein interaction promiscuity. Cell‐based assays that monitor second messenger responses are often used as screening tools for potential GPCR agonists and antagonists, however, commonly used cell‐lines such as HEK‐293, express many endogenous GPCRs that can complicate data analysis. Many classes of Gα subunits exist: Gαs and Gα i/o G‐proteins modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylate cyclase while Gαq subunits activate phospholipase C and a subsequent rapid release of intracellular Ca2+. Specifically, the orphan receptor, GPR88, has been linked with psychiatric diseases including schizophrenia and bipolar disorder. Lacking a validated endogenous agonist, the GPR88 signaling pathway remains poorly understood, however, in vivo knockout experiments and in vitro preclinical studies with small molecule synthetic agonists indicate that GPR88 couples to Gαi/o G‐proteins to inhibit adenylate cyclase activity and reduce intracellular cAMP in cell‐based assays. In this work, we describe our challenges in the study of the GPR88 signaling pathway using second messenger cell‐based assays and explain our decision to move to a real‐time NanoLuc‐based GPCR/G protein complementation assay.Support or Funding InformationNIH grant 1 R15 MH109034

Pleiotropic Activation of Endothelial Function by Angiotensin II Receptor Blockers is Crucial to Their Protective Anti‐vascular Remodeling Effects

RationaleEndothelial function is strongly associated with vascular health and homeostasis. However, therapeutically relevant approaches capable of stimulating the protective properties of the endothelium remain elusive.ObjectiveTo determine whether modulation of the Angiotensin II (AngII) system with Angiotensin receptor blockers (ARBs) can improve endothelial function and promote downstream vascular homeostasis.Methods and ResultsWe show that all 4 ARBs tested (losartan, telmisartan, olmesartan, and valsartan) can promote robust chronic activation of endothelial nitric oxide (NO) in mice. With telmisartan, a well‐known pleiotropic ARB, in vivo treatment lead to a 58% inhibition of vascular contractility after two weeks of treatment and up to 77% at 6 months, in a fully L‐NAME sensitive fashion. Endothelial NO synthase (eNOS) knockout (KO) mice showed complete resistance against the endothelial function effects of telmisartan, whereas the Angiotensin converting enzyme inhibitor (ACEi) captopril showed little to no endothelial function activity at a similar blood pressure (BP) lowering dose. In a myograph chamber, direct stimulation of aortic vessels with telmisartan in the absence of AngII stimulated endothelial function, confirming pleiotropism. Moreover, in vitro stimulation of cultured endothelial cells caused rapid intracellular Calcium ([Ca2+]i) mobilization, which is a known upstream event of eNOS activation. In vivo, telmisartan completely abolished aortic wall remodeling in aging animals as well as a well‐established mouse model of Marfan syndrome (MFS), which causes accelerated and pre‐mature vascular aging and remodeling of the aortic root. In addition, in vivo inhibition of NOS activity with L‐NAME rendered telmisartan inactive in a BP‐independent manner.ConclusionARBs have unique and robust endothelial function activation properties independent of their inhibitory effects on the AngII system. Direct stimulation of the endothelial NO system with ARBs results in vascular protection and homeostasis independent of BP lowering effects, which supports a broader prophylactic use of this class of medication.Support or Funding InformationSupported by CIHR, HSFC, BCKDF, Marfan Foundation, SPH Foundation and Rare Disease Foundation.

Rational development of a high-affinity secretin receptor antagonist

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu22 with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with receptor residues Leu31, Val34, and Phe92 that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y10,c[E16,K20],I17,Cha22,R25)sec(6–27), exhibited high binding affinity (4 nM), similar to natural secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or β-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr5, exhibited partial agonist activity, while further truncation of even a single residue (Phe6) reduced binding affinity. This sec(6–27)-based peptide will be an important new tool for pharmacological and structural studies.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

Multiple calcium sources are required for intracellular calcium mobilization during gastric organoid epithelial repair.

Calcium (Ca2+) is a known accelerator for gastric wound repair. We have demonstrated in vivo and in vitro that intracellular Ca2+ increases in the gastric epithelial cells directly adjacent to a damaged cell, and that this Ca2+ rise is essential for the cellular migration that rapidly repairs the epithelium (restitution). While intracellular Ca2+ has been shown to be an important signaling factor during epithelial restitution, the source from which this intracellular Ca2+ originates remains unclear. Using gastric organoids derived from mice transgenic for a genetically encoded Ca2+ indicator, we sought to investigate the potential sources of intracellular Ca2+ mobilization. During confocal imaging, photodamage (PD) was induced to 1–2 gastric organoid epithelial cells and epithelial restitution measured simultaneously with changes in intracellular Ca2+ (measured as FRET/CFP ratio in migrating cells adjacent to the damaged area). Inhibition of voltage‐gated Ca2+ channels (verapamil, 10 µM) or store‐operated calcium entry (YM58483, 20 µM) resulted in delayed repair and dampened intracellular Ca2+ response. Furthermore, inhibition of phospholipase C (U73122, 10 µM) or inositol trisphosphate receptor (2‐APB, 50 µM) likewise resulted in delayed repair and dampened Ca2+ response. Results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ mobilization that stimulates repair.

Modulation of Drosophila suzukii type 1 tyramine receptor (DsTAR1) by monoterpenes: a potential new target for next generation biopesticides

This study proposes a biochemical and molecular model for the interaction between the Drosophila suzukii type 1 tyramine receptor (DsTAR1) and monoterpenes.A preliminary molecular and functional characterization of DsTAR1 cDNA revealed that a 1.8 kb long ORF codes for a 600 amino acid polypeptide featuring seven transmembrane domains, as expected for a GPCR. A stable HEK 293 cell line expressing DsTAR1 was tested for responsiveness to tyramine (TA) and octopamine (OA). In intracellular calcium mobilization studies, TA led to a concentration-dependent increase in [Ca2+]i (pEC50 ~ 6.40), completely abolished by pre-incubation with the antagonist yohimbine 1 μM. Besides, in dynamic mass redistribution (DMR) studies, TA evoked a positive DMR signal in a concentration-dependent manner (pEC50 ~ 6.80).The recombinant cell line was then used to test three monoterpenes (thymol, carvacrol and α-terpineol) as putative ligands for DsTAR1. The terpenoids showed no agonist effects in both DMR and calcium mobilization assays, but they increased the potency of the endogenous ligand, TA, acting as positive allosteric modulators.Moreover, expression analysis on adults D. suzukii, exposed for 24, 72 or 120 h to a sublethal concentration of the three monoterpenes, showed a downregulation of DsTAR1.This evidence has led to hypothesize that the downregulation of DsTAR1 might be a compensatory mechanism in response to the positive allosteric modulation of the receptor induced by monoterpenes.Therefore, these findings might be useful for the development of a new generation of biopesticides against Drosophila suzukii, targeting TAR1.

Design and synthesis of potent PAR-1 antagonists based on vorapaxar

A series of novel vorapaxar analogues with different amino substitutes at the C-7, C-9a and aromatic substitutes at the C-4 position were designed, synthesized, and evaluated for their inhibitory activity to PAR-1. Several compounds showed good potency in antagonist activity based on the intracellular calcium mobilization assay and excellent pharmacokinetics profile in rats. Among these analogues, 3d exhibited excellent PAR-1 inhibitory activity (IC50 = 0.18 μM) and the lower ability to cross the blood-brain barrier compared with vorapaxar (IC50 = 0.25 μM). Compound 3d has the potential to be developed as a new generation of PAR-1 antagonists with a better therapeutic window.

TRPM2, linking oxidative stress and Ca2+ permeation to NLRP3 inflammasome activation.

The NLRP3 inflammasome is an innate immune platform that senses various pathogens and sterile insults. NLRP3 stimulation leads to activation of caspase-1, the secretion of pro-inflammatory cytokines and an inflammatory cell death called pyroptosis. Effectors of the NLRP3 inflammasome efficiently drive an immune response, not only providing protection in physiological settings but also promoting pathology when over activated. Generation of reactive oxygen species (ROS) and intracellular calcium mobilization can activate the NLRP3 inflammasome. Recent studies suggest that TRPM2 is a calcium-permeable cation channel mediating ROS-dependent NLRP3 activation. Here, we review the role of TRPM2 in NLRP3 inflammasome activation and provide an update on new functional and structural discoveries. Understanding the molecular mechanism of TRPM2 dependent NLRP3 inflammasome activation will shed lights on this complex pathway and help the developing of therapeutic strategies.

The anti-anaphylactoid effects of Piperine through regulating MAS-related G protein-coupled receptor X2 activation.

Mast cells play an important role in inflammatory and allergic diseases. MAS-related G protein-coupled receptor X2 (MRGPRX2) is a novel G protein-coupled receptor in mast cells that mediates drug-induced anaphylactoid reactions. Piperine has been reported to have anti-inflammatory and anti-allergic pharmacological activities. However, whether the pharmacological effects are regulated by MRGPRX2 has not yet been reported. The purpose of this study was to assess the anti-anaphylactoid effect of Piperine and to explore its potential mechanism. The anti-anaphylactoid effect of Piperine was assessed by an in vivo mouse hindpaw extravasation model. Mast cell intracellular calcium mobilization was measured by a calcium imaging assay. An enzyme immunoassay was used to evaluate the release of pro-inflammatory factors from stimulated mast cells. Activated mast cell related signals were assessed by western blot. A cell membrane chromatography assay was used to determine the binding characteristics of Piperine and MRGPRX2. The results showed that Piperine suppressed mast cell intracellular Ca2+ mobilization, inhibited cytokines and chemokines release, and down-regulated the phosphorylation level of phospholipase Cγ1, protein kinase C, inositol 1,4,5-triphate receptor, P38, protein kinase B, and ERK. Meanwhile, Piperine can bind to MRGPRX2 as a specific antagonist. Hence, Piperine can be served as a novel therapeutic drug candidate for MRGPRX2-mediated anaphylactoid reactions.

Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells.

Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H37Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.