Trigger Ca2 Research Articles

Extracellular Matrix-Mimetic Intrinsic Versatile Coating Derived from Marine Adhesive Protein Promotes Diabetic Wound Healing through Regulating the Microenvironment.

The management of diabetic wound healing remains a severe clinical challenge due to the complicated wound microenvironments, including abnormal immune regulation, excessive reactive oxygen species (ROS), and repeated bacterial infections. Herein, we report an extracellular matrix (ECM)-mimetic coating derived from scallop byssal protein (Sbp9Δ), which can be assembled in situ within 30 min under the trigger of Ca2+ driven by strong coordination interaction. The biocompatible Sbp9Δ coating and genetically programmable LL37-fused coating exhibit outstanding antioxidant, antibacterial, and immune regulatory properties in vitro. Proof-of-concept applications demonstrate that the coating can reliably promote wound healing in animal models, including diabetic mice and rabbits, ex vivo human skins, and Staphylococcus aureus-infected diabetic mice. In-depth mechanism investigation indicates that improved wound microenvironments accelerated wound repair, including alleviated bacterial infection, lessened inflammation, appearance of abundant M2-type macrophages, removal of ROS, promoted angiogenesis, and re-epithelialization. Collectively, our investigation provides an in situ, convenient, and effective approach for diabetic wound repair.

Studies on Membrane Potential and Ca2+ Oscillations in Rat Carotid Body Type I Cells

Carotid body (CB) glomus cells sense changes in arterial O2 pressure (PO2) and pH, and adjust the secretory and carotid sinus nerve activity and ventilation to help maintain normal levels of blood PO2 and pH. Our recent studies showed that isolated CB cell clusters perfused with physiological buffer solution generate spontaneous Ca2+ oscillations at low frequency in normoxia. Mild and moderate levels of hypoxia (2-5%O2) and acidosis (pHo7.2-7.3) increased the frequency and amplitude of Ca2+ oscillations. Inhibitors of voltage-dependent Ca2+ channels and removal of external Ca2+ abolished Ca2+ oscillations, indicating that Ca2+ influx was critical for generating Ca2+ oscillations. To better understand the phenomenon of Ca2+oscillations in glomus cells, we examined the potential role of oscillations in cell membrane potential (Em) in CB cells in triggering Ca2+ influx and Ca2+ oscillations. Using the cell-attached patch configuration, we assessed cell Em by recording TASK single channels, because a linear relationship exists between TASK amplitude and cell Em. Recording of TASK in CB cells in normoxia showed that many CB cells exhibit oscillations in TASK amplitude, indicating the presence of spontaneous oscillations in cell Em. The oscillation frequency of cell Em was similar to that of Ca2+ oscillations. Oscillations in TASK single channel amplitude were blocked by nifedipine (Ca2+ channel antagonist), by removal of extracellular Ca2+, and by 2-APB (an inhibitor of ER Ca2+ channel and store-operated Ca2+ entry). Mild hypoxia increased the frequency of oscillations of TASK amplitude, indicating that mild hypoxia increased the frequency of cell Em oscillations. These findings suggest that cell Em oscillations (that open voltage-dependent Ca2+ channels and increase Ca2+ influx) are an integral component of the cellular signaling mechanism by which Ca2+ oscillations are produced in CB cells. This work was funded by National Institutes of Health (NIH) grants to D.K. (HL111497) and C.W. (HL142906), and an award from Rosalind Franklin University of Medicine and Science. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The context-dependent role of the Na+/Ca2+-exchanger (NCX) in pancreatic stellate cell migration

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and − 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.

Abstract P2035: Rad Reduction Improves Heart Failure

Background: Heart failure with reduced ejection fraction (HFrEF) entails depressed systolic function. Targeting calcium handling provides a direct, restorative treatment for the loss of systolic function. We recently showed that induced, cardiomyocyte-restricted Rad knockout mice (cRadKO) show increased trigger Ca 2+ with improved contractility and no signs of pathological remodeling. Hypothesis: In the present study we tested the hypothesis that cRadKO provides inotropic support to hearts subjected to chronic pressure overload. Results: We first show that Rad-reduction in human myocardial slices provides an inotropic boost. We then performed transverse aortic constriction (TAC) in mice. cRadKO imposed either before TAC or 1-month after TAC promoted survival and attenuated cardiac remodeling. cRadKO induced after TAC reflects an interventional proof-of-principle that Rad reduction can attenuate heart failure progression. The intervention timeline group was further probed using bulk RNA sequencing. cRadKO attenuated key fetal gene program transcripts including NPPA, NPPB, and MYH7. RCAN1 levels were decreased with cRadKO consistent with reduced calcineurin-NFAT signaling, and BIN1 levels were restored in cRadKO, suggesting improved cardiac remodeling despite ongoing pressure overload. Conclusions: The prevention (cRadKO before TAC) and intervention (cRadKO after TAC) studies demonstrate that reduced Rad levels can provide an inotropic boost in HFrEF and stimulate transcriptional effects consistent with reparative remodeling.

Arrhythmogenic mechanism of a novel ryanodine receptor mutation underlying sudden cardiac death.

The ryanodine receptor 2 (RyR2) is essential for cardiac muscle excitation-contraction coupling; dysfunctional RyR2 participates in the development of inherited arrhythmogenic cardiac disease. In this study, a novel RyR2 mutation A690E is identified from a patient with family inheritance of sudden cardiac death, and we aimed to investigate the pathogenic basis of the mutation. We generated a mouse model that carried the A690E mutation. Mice were characterized by adrenergic-induced ventricular arrhythmias similar to clinical manifestation of the patient. Optical mapping studies revealed that isolated A690E hearts were prone to arrhythmogenesis and displayed frequency-dependence calcium transient alternans. Upon β-adrenoceptor challenge, the concordant alternans was shifted towards discordant alternans that favour triggering ectopic beats and Ca2+ re-entry; similar phenomenon was also found in the A690E cardiomyocytes. In addition, we found that A690E cardiomyocytes manifested abnormal Ca2+ release and electrophysiological disorders, including an increased sensitivity to cytosolic Ca2+, an elevated diastolic RyR2-mediated Ca2+ leak, and an imbalance between Ca2+ leak and reuptake. Structural analyses reveal that the mutation directly impacts RyR2-FK506 binding protein interaction. In this study, we have identified a novel mutation in RyR2 that is associated with sudden cardiac death. By characterizing the function defects of mutant RyR2 in animal, whole heat, and cardiomyocytes, we demonstrated the pathogenic basis of the disease-causing mutation and provided a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

Cadmium induced mouse spermatogonia apoptosis via mitochondrial calcium overload mediated by IP3R-MCU signal pathway

Cadmium (Cd) is a toxic metal and also a well-known reproductive toxicant. Cd could induce germ cells apoptosis in mouse testes, however, the mechanism remains unclear. This study designed in vitro using GC-1 spermatogonial (spg) cells to explore the cytotoxicity and the molecular mechanisms induced by cadmium chloride(CdCl2). As expected, CdCl2 elevated the levels of reactive oxygen species (ROS) and induced the release of AIF and Cyt-c from the mitochondria to the cytosol in spermatogonia. Correspondingly, CdCl2 apparently increased the apoptotic rate in spermatogonia. Further researches found that CdCl2 could activate IP3R-MCU pathway, trigger Ca2+ transfer from endoplasmic reticulum to mitochondria, and cause mitochondrial Ca2+ overload. BAPTA acetoxymethyl ester (BAPTA-AM), a calcium chelator, almost completely attenuated IP3R phosphorylation, inhibited the mRNA and protein expression levels of VDAC1, MCU and MCUR1 upregulated by CdCl2, reduced the calcium ion content in the mitochondria. Moreover, BAPTA-AM could decrease the level of ROS, antagonize CdCl2-induced release of AIF and Cyt-c from the mitochondria to the cytosol and alleviate CdCl2-induced apoptosis in spermatogonia. As above, these results provided the evidence that CdCl2 might induce apoptosis of spermatogonia via mitochondrial Ca2+ overload mediated by IP3R-MCU signal pathway.

TRPV4 channel is involved in HSV-2 infection in human vaginal epithelial cells through triggering Ca2+ oscillation.

Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 μM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.

Unravelling neuron-astrocyte communication in the dorsal raphe nucleus.

Introduction: Serotonin is a neuromodulator widely spread throughout the central nervous system and involved in a vast variety of behaviors, such as cognitive functions and emotional states. On this basis, serotonergic dysfunction has been related to several psychiatric disorders such as Major Depressive Disorder, schizophrenia, or autism. Despite serotonin’s widespread distribution, the location of serotonergic neurons is restricted to the midbrain raphe nuclei. In this context, astrocytes are known to participate in synaptic transmission modulating neural circuits; however, their role in the serotonergic system remains largely unknown. My study aims to elucidate the neuron-astrocyte signaling at serotonergic nuclei, looking at the dorsal raphe nucleus (DRN). Materials and methods: For the purpose of this project, we performed intracranial injections of viral constructs in adult mice to express the Ca2+ indicator GCaMP6 specifically in DRN astrocytes, Ca2+ imaging experiments to record astrocytic activity, electrophysiological recordings of neuronal activity using patch-clamp and immunohistochemistry techniques. Results and conclusions: Preliminary data show that astrocytes from DRN respond to serotonin with an increase in the amplitude and frequency of intracellular Ca2+ events, which is partly mediated by serotonin type 2 receptors. As a consequence of triggering Ca2+ signaling, we found that serotonin induces gliotransmission, shown by an increased frequency of Slow Inward Currents (SICs) and modulation of excitatory synaptic transmission in dorsal raphe neurons. In conclusion, these data suggest that astrocytes might play a role in synaptic transmission and neuronal excitability in the DRN and, therefore, in the serotonergic-mediated actions of DRN.

Reversing thromboxane A2 receptor activity from calcium to cAMP signaling by shifting Gαq to Gαs covalently linked to the receptor

The conversion of a G-protein coupling receptor (GPCR) signaling from one G-protein to another could be a novel concept for drug designs. Thromboxane A2 (TXA2) receptor (TP) plays a key role in mediating platelet aggregation and thrombosis through its binding to TXA2 and coupling with Gαq-calcium signaling. To alter TP coupling from Gαq to Gαs, which mediates the opposite anti-platelet aggregation and vasodilation activities, we first created a recombinant single-chain (SC) TP-Gαq complex (SC-TP-Gαq) in which the Gαq N-terminus is covalently linked to the TP C-terminus. The SC-TP-Gαq expressed on HEK293 cells could perform identical functions of wild-type TP binding to agonist and activating Gαq triggering Ca2+ signal. The results were further confirmed by expressing the SC-TP-Gαq on CRISPR-edited Gαq-knocked-out HEK293 cells. A step further, by replacing Gαq using Gαs for SC-TP-Gαq, a second recombinant SC-TP-Gαs was created and expressed on wild type and CRISPR-edited Gαs-knocked-out HEK293 cells. Upon the binding of the same TXA2 agonist to the cells expressing SC-TP-Gαs, cAMP signaling was observed. The results have provided strong evidence that covalently linking TP with different Gα could control cell signaling. This study has opened a door to developing a method to redirect TP signaling from thrombotic calcium to anti-thrombotic cAMP, and apply it to that of other GPCRs.

PO-614-03 ALTERED SUBCELLULAR CALCIUM RELEASE IN THE HEART FAILURE ATRIA

Transverse (t)-tubules enable close coupling between L-type calcium (Ca2+) channels and ryanodine receptors (RyR) to facilitate triggered Ca2+ release throughout the cell. In heart failure (HF) there is disruption of the t-tubule network that contributes to dyssynchronous Ca2+ release. Despite the importance of t-tubules in triggering Ca2+ release in the atria of large mammals, little is known about Ca2+ release sites and how they are altered in HF.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down).Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot.DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone.Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

Meningitic Escherichia coli \u03b1-hemolysin aggravates blood\u2013brain barrier disruption via targeting TGF\u03b21-triggered hedgehog signaling

Bacterial meningitis is a life-threatening infectious disease with severe neurological sequelae and a high mortality rate, in which Escherichia coli is one of the primary Gram-negative etiological bacteria. Meningitic E. coli infection is often accompanied by an elevated blood–brain barrier (BBB) permeability. BBB is the structural and functional barrier composed of brain microvascular endothelial cells (BMECs), astrocytes, and pericytes, and we have previously shown that astrocytes-derived TGFβ1 physiologically maintained the BBB permeability by triggering a non-canonical hedgehog signaling in brain microvascular endothelial cells (BMECs). Here, we subsequently demonstrated that meningitic E. coli infection could subvert this intercellular communication within BBB by attenuating TGFBRII/Gli2-mediated such signaling. By high-throughput screening, we identified E. coli α-hemolysin as the critical determinant responsible for this attenuation through Sp1-dependent TGFBRII reduction and triggering Ca2+ influx and protein kinase A activation, thus leading to Gli2 suppression. Additionally, the exogenous hedgehog agonist SAG exhibited promising protection against the infection-caused BBB dysfunction. Our work revealed a hedgehog-targeted pathogenic mechanism during meningitic E. coli-caused BBB disruption and suggested that activating hedgehog signaling within BBB could be a potential protective strategy for future therapy of bacterial meningitis.

A short isoform of STIM1 confers frequency-dependent synaptic enhancement

Store-operated Ca2+-entry (SOCE) regulates basal and receptor-triggered Ca2+ signaling with STIM proteins sensing the endoplasmic reticulum (ER) Ca2+ content and triggering Ca2+ entry by gating Orai channels. Although crucial for immune cells, STIM1's role in neuronal Ca2+ homeostasis is controversial. Here, we characterize a splice variant, STIM1B, which shows exclusive neuronal expression and protein content surpassing conventional STIM1 in cerebellum and of significant abundance in other brain regions. STIM1B expression results in a truncated protein with slower kinetics of ER-plasma membrane (PM) cluster formation and ICRAC, as well as reduced inactivation. In primary wild-type neurons, STIM1B is targeted by its spliced-in domain B to presynaptic sites where it converts classic synaptic depression into Ca2+- and Orai-dependent short-term synaptic enhancement (STE) at high-frequency stimulation (HFS). In conjunction with altered STIM1 splicing in human Alzheimer disease, our findings highlight STIM1 splicing as an important regulator of neuronal calcium homeostasis and of synaptic plasticity.

Can Foliar Pulverization with CaCl2 and Ca(NO3)2 Trigger Ca Enrichment in Solanum tuberosum L. Tubers?

This study aimed to assess the efficiency of Ca enrichment in tubers of three genotypes of Solanum tuberosum L., through foliar spraying with CaCl2 and Ca(NO3)2 solutions. In this context, soil heterogeneity of three potato-growing fields, as well as the implications of Ca accumulation among tissues and some quality parameters were assessed. Three potato varieties (Agria, Picasso and Rossi) were grown in three production fields and during the life cycle, four pulverizations with calcium chloride (3 and 6 kg ha−1) or calcium nitrate (0.5, 2 and 4 kg ha−1) were applied. For screening the potential phytotoxicity, using Agria as a test system, the potential synthesis of photoassimilates was determined, and it was found that after the 3rd Ca application, leaf gas exchanges were moderately (net photosynthesis), to strongly (stomatal conductance) affected, although without impact on Ca accumulation in tubers. At harvest, the average Ca biofortification index varied between 5–40%, 40–35% and 4.3–13% in Agria, Picasso and Rossi, respectively. Moreover, the equatorial region of the tubers in general showed that Ca accumulation prevailed in the epidermis and, in some cases, in inner areas of the potato tubers. Biofortified tubers with Ca also showed some significant changes in total soluble solids and colorimetric parameters. It is concluded that Ca enrichment of potato tubers through foliar spraying complemented the xylem mass flow of Ca from roots, through phloem redistribution. Both fertilizers showed similar efficiency, but Rossi revealed a lower index of Ca accumulation, eventually due to different metabolic characteristics. Although affected by Ca enrichment, potato tubers maintained a high quality for industrial processing.

Glucose-induced cAMP elevation in β-cells involves amplification of constitutive and glucagon-activated GLP-1 receptor signalling.

AimcAMP typically signals downstream of Gs‐coupled receptors and regulates numerous cell functions. In β‐cells, cAMP amplifies Ca2+‐triggered exocytosis of insulin granules. Glucose‐induced insulin secretion is associated with Ca2+‐ and metabolism‐dependent increases of the sub‐plasma‐membrane cAMP concentration ([cAMP]pm) in β‐cells, but potential links to canonical receptor signalling are unclear. The aim of this study was to clarify the role of glucagon‐like peptide‐1 receptors (GLP1Rs) for glucose‐induced cAMP signalling in β‐cells.MethodsTotal internal reflection microscopy and fluorescent reporters were used to monitor changes in cAMP, Ca2+ and ATP concentrations as well as insulin secretion in MIN6 cells and mouse and human β‐cells. Insulin release from mouse and human islets was also measured with ELISA.ResultsThe GLP1R antagonist exendin‐(9‐39) (ex‐9) prevented both GLP1‐ and glucagon‐induced elevations of [cAMP]pm, consistent with GLP1Rs being involved in the action of glucagon. This conclusion was supported by lack of unspecific effects of the antagonist in a reporter cell‐line. Ex‐9 also suppressed IBMX‐ and glucose‐induced [cAMP]pm elevations. Depolarization with K+ triggered Ca2+‐dependent [cAMP]pm elevation, an effect that was amplified by high glucose. Ex‐9 inhibited both the Ca2+ and glucose‐metabolism‐dependent actions on [cAMP]pm. The drug remained effective after minimizing paracrine signalling by dispersing the islets and it reduced basal [cAMP]pm in a cell‐line heterologously expressing GLP1Rs, indicating that there is constitutive GLP1R signalling. The ex‐9‐induced reduction of [cAMP]pm in glucose‐stimulated β‐cells was paralleled by suppression of insulin secretion.ConclusionAgonist‐independent and glucagon‐stimulated GLP1R signalling in β‐cells contributes to basal and glucose‐induced cAMP production and insulin secretion.

Excitation-contraction coupling and calcium release in atrial muscle.

In cardiac muscle, the process of excitation-contraction coupling (ECC) describes the chain of events that links action potential induced myocyte membrane depolarization, surface membrane ion channel activation, triggering of Ca2+ induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store to activation of the contractile machinery that is ultimately responsible for the pump function of the heart. Here we review similarities and differences of structural and functional attributes of ECC between atrial and ventricular tissue. We explore a novel "fire-diffuse-uptake-fire" paradigm of atrial ECC and Ca2+ release that assigns a novel role to the SR SERCA pump and involves a concerted "tandem" activation of the ryanodine receptor Ca2+ release channel by cytosolic and luminal Ca2+. We discuss the contribution of the inositol 1,4,5-trisphosphate (IP3) receptor Ca2+ release channel as an auxiliary pathway to Ca2+ signaling, and we review IP3 receptor-induced Ca2+ release involvement in beat-to-beat ECC, nuclear Ca2+ signaling, and arrhythmogenesis. Finally, we explore the topic of electromechanical and Ca2+ alternans and its ramifications for atrial arrhythmia.

Short Novel STIM1B Uncovers a Mechanism of Synaptic Enhancement

Store-operated Ca2+-entry (SOCE) regulates basal and receptor-triggered Ca2+ signaling with STIM proteins sensing the endoplasmic reticulum (ER) Ca2+ content and triggering Ca2+ entry by gating Orai channels. Although crucial for immune cells, STIM1’s role in neuronal Ca2+ homeostasis is controversial. Here, we characterize a novel splice variant, STIM1B, with exclusive neuronal expression, protein content surpassing conventional STIM1 in cerebellum and significant abundance in hippocampus. STIM1B results in a truncated protein with slower kinetics of ER-PM cluster formation and ICRAC as well as reduced inactivation. In primary wild-type neurons, Stim1B is targeted by its spliced-in domain B to presynaptic sites where it converts classic synaptic depression into Ca2+- and Orai-dependent short-term synaptic enhancement (STE) at high frequency stimulation (HFS). In conjunction with altered STIM1 splicing in human Alzheimer disease, our findings highlight STIM1 splicing as an important regulator of neuronal calcium homeostasis and synaptic plasticity.

Biodistribution and immunomodulatory activities of a proteoglycan isolated from Ganoderma lucidum

A proteoglycan, namely FYGL extracted from Ganoderma lucidum, was proved previously efficient for anti-diabetes in vivo. However, many medicine functions of FYGL have not been revealed, such as immunoregulation, an important function of Ganoderma in the traditional Chinese medicine. In this work, we firstly investigated the distribution of FYGL orally administrated by mice and found that FYGL was available in small intestine and viscera organs and enriched mostly in liver. Consequently, we studied the immunoregulation mechanism of FYGL in RAW 264.7 macrophages. The results showed that FYGL could induce initially ROS and trigger Ca2+ ions influx into cells, leading to the activation of NF-κB and MAPK signaling to secret NO and cytokines for immune responses, and the promotion of phagocytosis. Meanwhile, FYGL absorbed in cells could reduce ROS, therefore preventing mitochondrial membrane from damage for cells safety. The results provided the immunoregulation basis of FYGL potentially applicable in clinics.

Epigallocatechin-3-gallate mobilizes intracellular Ca2+ in prostate cancer cells through combined Ca2+ entry and Ca2+-induced Ca2+ release

AimsTo elucidate the mechanism by which (−)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. Main methodsFollowing exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. Key findingsEGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 μM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. SignificanceEGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.

Anti-CD20 rituximab IgG1, IgG3, and IgG4 but not IgG2 subclass trigger Ca2+ mobilization and cytotoxicity in human NK cells.

NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc-gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca2+ signaling and NK cell-mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low-affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high-affinity (VV) and VF SNP variants was two- to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low-affinity FF variation. In conclusion, this is the first study correlating Ca2+ signaling and NK cell-mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy.