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Cell-to-cell transmission of α-synuclein (α-syn) pathology underlies the spread of neurodegeneration in Parkinson's disease. α-Syn secretion is an important factor in the transmission of α-syn pathology. However, it is unclear how α-syn secretion is therapeutically modulated. Here, we investigated effects of monoamine oxidase (MAO)-B inhibitor selegiline on α-syn secretion. Treatment with selegiline promoted α-syn secretion in mouse primary cortical neuron cultures, and this increase was kept under glial cell-eliminated condition by Ara-C. Selegiline-induced α-syn secretion was blocked by cytosolic Ca 2+ chelator BAPTA-AM in primary neurons. Selegiline-induced α-syn secretion was retained in MAOA siRNA knockdown, whereas it was abrogated by ATG5 knockdown in SH-SY5Y cells. Selegiline increased LC3-II generation with a reduction in intracellular p62/SQSTM1 levels in primary neurons. The increase in LC3-II generation was blocked by co-treatment with BAPTA-AM in primary neurons. Additionally, fractionation experiments showed that selegiline-induced α-syn secretion occurred in non-extracellular vesicle fractions of primary neurons and SH-SY5Y cells. Collectively, these findings show that selegiline promotes neuronal autophagy involving secretion of non-exosomal α-syn via a change of cytosolic Ca 2+ levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here, in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput data-independent acquisition-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and SnRK2s, EGTA treatment also activates mitogen-activated protein kinase cascades, Calcium-dependent protein kinases, and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases and receptor-like protein kinases in the osmotic stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca 2+ signaling, offering insights into the (exocellular) Ca 2+ deprivation during early hyperosmolality sensing and signaling., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca 2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1 +/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca 2+ -dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Intracellular Ca 2+ signals play a vital role in a broad range of cell biological and physiological processes in all eukaryotic cell types. Dysregulation of Ca 2+ signaling has been implicated in numerous human diseases. Over the past four decades, the understanding of how cells use Ca 2+ as a messenger has flourished, largely because of the development of reporters that enable visualization of Ca 2+ signals in different cellular compartments, and tools that can modulate cellular Ca 2+ signaling. One such tool that is frequently used is BAPTA; a fast, high-affinity Ca 2+ -chelating molecule. By making use of a cell-permeable acetoxymethyl ester (AM) variant, BAPTA can be readily loaded into the cytosol of cells (referred to as BAPTAi), where it is trapped and able to buffer changes in cytosolic Ca 2+ . Due to the ease of loading of the AM version of BAPTA, this reagent has been used in hundreds of studies to probe the role of Ca 2+ signaling in specific processes. As such, for decades, researchers have almost universally attributed changes in biological processes caused by BAPTAi to the involvement of Ca 2+ signaling. However, BAPTAi has often been used without any form of control, and in many cases has neither been shown to be retained in cells for the duration of experiments nor to buffer any Ca 2+ signals. Moreover, increasing evidence points to off-target cellular effects of BAPTA that are clearly not related to Ca 2+ chelation. Here, we briefly introduce Ca 2+ signaling and the history of Ca 2+ chelators and fluorescent Ca 2+ indicators. We highlight Ca 2+ -independent effects of BAPTAi on a broad range of molecular targets and describe some of BAPTAi's impacts on cell functions that occur independently of its Ca 2+ -chelating properties. Finally, we propose strategies for determining whether Ca 2+ chelation, the binding of other metal ions, or off-target interactions with cell components are responsible for BAPTAi's effect on a particular process and suggest some future research directions., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest to report., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA expresses various effects on intracellular calcium signaling, which are not related to its ability to bind Ca 2+ . In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 μM Ca 2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off-target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M 1 muscarinic receptors coupled to the G q protein. Although PIP 2 depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (I M ), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of I M with Ca 2+ measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of I M was not blocked either by PIP 2 or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of I M uses multiple signaling pathways by blocking both PIP 2 depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of I M . Additionally, muscarinic inhibition of I M was not prevented either by sequestering of G-protein βγ subunits from G α -transducin or anti-G βγ antibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca 2+ signals in muscarinic inhibition of I M . Ca 2+ measurements showed that muscarinic stimulation increased intracellular Ca 2+ and was comparable to the Ca 2+ mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of I M . In contrast, muscarinic inhibition of I M was completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca 2+ signaling in muscarinic modulation of I M . The differential effects of EGTA and BAPTA imply that Ca 2+ microdomains or spatially local Ca 2+ signals contribute to inhibition of I M ., (© 2022. The Author(s).)

Calcium homeostasis plays a vital role in protecting against Alzheimer's disease (AD). In this study, amyloid- β (A β )-induced C. elegans models of AD were used to elucidate the mechanisms underlying calcium homeostasis in AD. Calcium acetate increased the intracellular calcium content, exacerbated A β 1-42 aggregation, which is closely associated with oxidative stress, aggravated neuronal degeneration and dysfunction, and shortened the lifespan of the C . elegans models. Ethylene glycol tetraacetic acid (EGTA) and nimodipine were used to decrease the intracellular calcium content. Both EGTA and nimodipine showed remarkable inhibitory effects on A β 1-42 aggregations by increasing oxidative stress resistance. Moreover, both compounds significantly delayed the onset of A β -induced paralysis, rescued memory deficits, ameliorated behavioral dysfunction, decreased the vulnerability of two major (GABAergic and dopaminergic) neurons and synapses, and extended the lifespan of the C. elegans AD models. Furthermore, RNA sequencing of nimodipine-treated worms revealed numerous downstream differentially expressed genes related to calcium signaling. Nimodipine-induced inhibition of selective voltage-gated calcium channels was shown to activate other calcium channels of the plasma membrane ( clhm-1 ) and endoplasmic reticulum ( unc-68 ), in addition to sodium-calcium exchanger channels ( ncx-1 ). These channels collaborated to activate downstream events to resist oxidative stress through glutathione S-transferase activity mediated by HPGD and skn-1 , as verified by RNA interference. These results may be applied for the treatment of Alzheimer's disease., Competing Interests: The authors declare that this research was conducted with no commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zihui Zheng et al.)

This study was designed to determine the inhibitory effect of astragaloside Ⅳ(AS-Ⅳ), a principal bioactive component extracted from the Chinese medicinal Astragali Radix, on the inflammatory response of vascular endothelial cells induced by angiotensin Ⅱ(Ang Ⅱ), the most major pathogenic factor for cardiovascular diseases, and to clarify the role of calcium(Ca~(2+))/phosphatidylinosi-tol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO) pathway in the process. To be specific, human umbilical vein endothelial cells(HUVECs) were cultured in the presence of AS-Ⅳ with or without the specific inhibitor of NO synthase(NG-monomethyl-L-arginine, L-NMMA), inhibitor of PI3K/Akt signaling pathway(LY294002), or Ca~(2+)-chelating agent(ethylene glycol tetraacetic acid, EGTA) prior to Ang Ⅱ stimulation. The inhibitory effect of AS-Ⅳ on Ang Ⅱ-induced inflammatory response and the involved mechanism was determined with enzyme-linked immunosorbent assay(ELISA), cell-based ELISA assay, Western blot, and monocyte adhesion assay which determined the fluorescently labeled human monocytic cell line(THP-1) adhered to Ang Ⅱ-stimulated endothelial cells. AS-Ⅳ increased the production of NO by HUVECs in a dose-and time-dependent manner(P<0.05) and raised the level of phosphorylated eNOS(P<0.05). The above AS-Ⅳ-induced changes were abolished by pretreatment with L-NMMA, LY294002, or EGTA. Compared with the control group, Ang Ⅱ obviously enhanced the production and release of cytokines(tumor necrosis factor-α, interleukin-6), chemokines(monocyte chemoattractant protein-1) and adhesion molecules(intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), and the number of monocytes adhered to HUVECs(P<0.05), which were accompanied by the enhanced levels of phosphorylated inhibitor of nuclear factor-κBα protein and activities of nuclear factor-κB(NF-κB)(P<0.05). This study also demonstrated that Ang Ⅱ-induced inflammatory response was inhibited by pretreatment with AS-Ⅳ(P<0.05). In addition, the inhibitory effect of AS-Ⅳ was abrogated by pretreatment with L-NMMA, LY294002, or EGTA(P<0.05). This study provides a direct link between AS-Ⅳ and Ca~(2+)/PI3K/Akt/eNOS/NO pathway in AS-Ⅳ-mediated anti-inflammatory actions in endothelial cells exposed to Ang Ⅱ. The results indicate that AS-Ⅳ attenuates endothelial cell-mediated inflammatory response induced by Ang Ⅱ via the activation of Ca~(2+)/PI3K/Akt/eNOS/NO signaling pathway.

To explore whether there is an interaction between melatonin (MT) and calcium (Ca 2+ ) in regulating heat tolerance of plants, we analyzed the response of endogenous MT and Ca 2+ to heat stress, and examined the effect of MT and Ca 2+ on the reactive oxygen (ROS) accumulation, antioxidant system, and transcripts of heat shock factor ( HSF ) and heat shock proteins ( HSPs ) of cucumber seedlings under high temperature stress. Seedlings were foliar sprayed with 100 μmol·L -1 MT, 10 mmol·L -1 CaCl 2 , 3 mmol·L -1 ethylene glycol tetraacetic acid (EGTA, Ca 2+ chelating agent) +100 μmol·L -1 MT, 0.05 mmol·L -1 chlorpromazine (calmodulin antagonist, CPZ) +100 μmol·L -1 MT, 100 μmol·L -1 p-chlorophenylalanine (p-CPA, inhibitor of MT) +10 mmol·L -1 CaCl 2 or deionized water (H 2 O), respectively. The results showed that both endogenous MT and Ca 2+ in cucumber seedlings were induced by high temperature stress. The seedlings treated with exogenous MT showed significant increases in the mRNA expression of calmodulin ( CaM ), calcium-dependent protein kinase ( CDPK5 ), calcineurin B-like protein ( CBL3 ) and CBL interacting protein kinase ( CIPK2 ) compared with the control at normal temperature. The mRNA levels of tryptophane decarboxylase ( TDC ), 5-hydroxytryptamine-N-acetyltransferase ( SNAT ) and N-acetyl-5-hydroxytryptamine methyltransferase ( ASMT ), key genes of MT biosynthesis and endogenous MT content were also induced by Ca 2+ in cucumber seedlings. Exogenous MT and CaCl 2 alleviated the heat-induced oxidative damage through increasing antioxidant ability, reducing the accumulation of reactive oxygen species (ROS), and upregulating the mRNA abundances of HSF7 , HSP70.1 and HSP70.11 , as evidenced by mild thermal damage symptoms, lower heat injury index and electrolyte leakage under heat stress. The positive effect of MT-induced antioxidant capacity and mRNA expression of HSPs was removed by adding EGTA and CPZ in stressed seedlings. Similarly, the mitigating role of Ca 2+ in the peroxidation damage to high temperature stress was reversed by p-CPA. These results suggested that both MT and Ca 2+ could induce heat tolerance of cucumber seedlings, which had crosstalk in the process of heat stress signal transduction.

Background: Mechanical stimuli induce the release of adenosine triphosphate into the extracellular environment by human periodontal ligament cells (hPDLCs). Extracellular adenosine triphosphate (eATP) plays the role in both inflammation and osteogenic differentiation. eATP involves in immunosuppressive action by increasing immunosuppressive molecules IDO and IFNγ expression on immune cells. However, the role of eATP on the immunomodulation of hPDLCs remains unclear. This study aimed to examine the effects of eATP on the IDO and IFNγ expression of hPDLCs and the participation of purinergic P 2 receptors in this phenomenon., Methods: hPDLCs were treated with eATP. The mRNA and protein expression of indoleamine-pyrrole 2,3-dioxygenase (IDO) and interferon-gamma (IFNγ) were determined. The role of the purinergic P 2 receptor was determined using calcium chelator (EGTA) and PKC inhibitor (PKCi). Chemical inhibitors (KN62 and BBG), small interfering RNA (siRNA), and P 2 X 7 receptor agonist (BzATP) were used to confirm the involvement of P 2 X 7 receptors on IDO and IFNγ induction by hPDLCs., Results: eATP significantly enhanced mRNA expression of IDO and IFNγ. Moreover, eATP increased kynurenine which is the active metabolite of tryptophan breakdown catalyzed by the IDO enzyme and significantly induced IFNγ protein expression. EGTA and PKCi reduced eATP-induced IDO and IFNγ expressions by hPDLCs, confirming the role of calcium signaling. Chemical P 2 X 7 inhibitors (KN62 and BBG) and siRNA targeting the P 2 X 7 receptor significantly inhibited the eATP-induced IDO and IFNγ production. Correspondingly, BzATP markedly increased IDO and IFNγ expression., Conclusion: eATP induced immunosuppressive function of hPDLCs by promoting IDO and IFNγ production via P 2 X 7 receptor signaling. eATP may become a promising target for periodontal regeneration by modulating immune response and further triggering tissue healing., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex., Methods: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 μmol/l) plus the GABA receptor blocker bicuculline (20 μmol/l)., Results: When the slow Ca2+ chelator EGTA was in the intracellular solution, RAP elicited significant enhancement of NMDAR-gated current at a 1 μmol/l concentration, and significantly reduced current at 10 μmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 μmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 μmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism., Conclusion: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Key Message: After cryopreservation, the Ca 2+ content increased, which affected the intracellular ROS content, then participated in the occurrence of programmed cell death in pollen. Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca 2+ ) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca 2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca 2+ content significantly increased. The regulation of Ca 2+ content had a significant effect on PCD indices, which showed that the Ca 2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca 2+ -chelating agent EGTA had the opposite effect. The addition of the Ca 2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca 2+ -chelating agent EGTA had the opposite effect, and Ca 2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca 2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Capsaicin and zinc have recently been highlighted as potential treatments for glucose metabolism disorders; however, the effect of these two natural compounds on signalling pathways involved in glucose metabolism is still uncertain. In this study, we assessed the capsaicin- or zinc- induced activation of signalling molecules including calcium/calmodulin-dependent protein kinase 2 (CAMKK2), cAMP-response element-binding protein (CREB), and target of rapamycin kinase complex 1 (TORC1). Moreover, the expression status of genes associated with the control of glucose metabolism was measured in treated cells. The activation of cell signalling proteins was then evaluated in capsaicin- or zinc treated cells in the presence or absence of cell-permeant calcium chelator (BAPTA-AM) and the CAMKK inhibitor (STO-609). Finally, capsaicin- and zinc-induced glucose uptake was measured in the cells pre-treated with or without BAPTA-AM. Our results indicate that calcium flux induced by capsaicin or zinc led to activation of calcium signalling molecules and promoting glucose uptake in skeletal muscle cells. Pharmacological inhibition of CAMKK diminished activation of signalling molecules. Moreover, we observed an increase in intracellular cAMP levels in the cells after treatment with capsaicin and zinc. Our data show that capsaicin and zinc mediate glucose uptake in C2C12 skeletal muscle cells through the activation of calcium signalling.

Osteoarthritis (OA) is a common joint disease that is characterized by cartilage degradation. Iron deposition in the joints is common during the pathogenic progression of OA and recent studies have indicated that iron overload is an important contributor to OA progression. Calcium chelators have been reported to inhibit iron influx via modulating transferrin receptor protein 1 internalization, and they have been identified as a potential approach to the treatment of iron overload‑induced diseases. The aim of the present study was to investigate the effect of calcium chelators on the progression of iron overload‑induced OA. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro , followed by co‑treatment with the calcium chelator BAPTA acetoxymethyl ester (BAPTA‑AM). Subsequently, intracellular iron levels, cell viability, reactive oxygen species (ROS) levels, mitochondrial function and morphological changes, as well as MMP levels, were detected using commercial kits. It was demonstrated that FAC treatment significantly promoted chondrocyte apoptosis and the expression of MMPs, and these effects were reversed by co‑treatment with BAPTA‑AM. Moreover, BAPTA‑AM suppressed iron influx into chondrocytes and inhibited iron overload‑induced ROS production and mitochondrial dysfunction. These results indicated that calcium chelators may be of value in the treatment of iron metabolism‑related diseases and iron overload‑induced OA progression.

Rationale: Disruption of intracellular calcium (Ca2+) homeostasis is implicated in inflammatory responses. Here we investigated endoplasmic reticulum (ER) Ca2+ efflux through the Inositol 1,4,5-trisphosphate receptor (IP3R) as a potential mechanism of inflammatory pathophysiology in a ventilator-induced lung injury (VILI) mouse model., Methods: C57BL/6 mice were exposed to mechanical ventilation using high tidal volume (HTV). Mice were pretreated with the IP3R agonist carbachol, IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) or the Ca2+ chelator BAPTA-AM. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected to measure Ca2+ concentrations, inflammatory responses and mRNA/protein expression associated with ER stress, NLRP3 inflammasome activation and inflammation. Analyses were conducted in concert with cultured murine lung cell lines., Results: Lungs from mice subjected to HTV displayed upregulated IP3R expression in ER and mitochondrial-associated-membranes (MAMs), with enhanced formation of MAMs. Moreover, HTV disrupted Ca2+ homeostasis, with increased flux from the ER to the cytoplasm and mitochondria. Administration of carbachol aggravated HTV-induced lung injury and inflammation while pretreatment with 2-APB or BAPTA-AM largely prevented these effects. HTV activated the IRE1α and PERK arms of the ER stress signaling response and induced mitochondrial dysfunction-NLRP3 inflammasome activation in an IP3R-dependent manner. Similarly, disruption of IP3R/Ca2+ in MLE12 and RAW264.7 cells using carbachol lead to inflammatory responses, and stimulated ER stress and mitochondrial dysfunction., Conclusion: Increase in IP3R-mediated Ca2+ release is involved in the inflammatory pathophysiology of VILI via ER stress and mitochondrial dysfunction. Antagonizing IP3R/Ca2+ and/or maintaining Ca2+ homeostasis in lung tissue represents a prospective treatment approach for VILI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ye, Zeng, Ling, Ma, Chen, Lin, Li and Pan.)

Aim: Despite animal evidence of a role of calcium in the pathogenesis of spinal cord injury, several studies conducted in the past found calcium blockade ineffective. However, those studies involved oral or parenteral administration of Ca++ antagonists. We hypothesized that Ca++ blockade might be effective with local/immediate application (LIA) at the time of neural injury., Methods: In this study, we assessed the effects of LIA of BAPTA (1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid), a cell-permeable highly selective Ca++ chelator, after spinal cord transection (SCT) in mice over 4 weeks. Effects of BAPTA were assessed behaviorally and with immunohistochemistry. Concurrently, BAPTA was submitted for the first time to multimodality assessment in an in vitro model of neural damage as a possible spinal neuroprotectant., Results: We demonstrate that BAPTA alleviates neuronal apoptosis caused by physical damage by inhibition of neuronal apoptosis and reactive oxygen species (ROS) generation. This translates to enhanced preservation of electrophysiological function and superior behavioral recovery., Conclusion: This study shows for the first time that local/immediate application of Ca++ chelator BAPTA is strongly neuroprotective after severe spinal cord injury., (© 2021 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Background: Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood., Objective: To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes., Methods: The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM., Results: The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM., Conclusion: Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

This study was carried out to investigate the effect of acibenzolar-S-methyl (ASM) and ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) treatments on calcium-dependent protein kinases (CDPKs) and reactive oxygen species (ROS) metabolism in apples. Postharvest ASM treatment increased H 2 O 2 content, reduced glutathione and ascorbic acid contents, and NADPH oxidase, peroxidase, ascorbate peroxidase, superoxide dismutase and glutathione reductase activities and retarded catalase activity and MdCAT expression in apples. ASM treatment enhanced MdSOD, MdPOD, MdAPX, MdGR, MdCDPK1, MdCDPK4, MdCDPK5, MdCDPK7, and MdCDPK21 expressions in apples. However, EGTA + ASM treatments suppressed H 2 O 2 , glutathione and ascorbic acid contents, NADPH oxidase, peroxidase, superoxide dismutase, ascorbate peroxidase and glutathione reductase activities. EGTA + ASM treatments suppressed the selected genes expressions in ROS metabolism and CDPKs, but up-regulated MdCAT expression in apples. These findings suggest that CDPKs play a vital role in regulating ROS metabolism and involve in inducing resistance in apples by ASM., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In 15% of cases, autosomal dominant polycystic kidney disease arises from defects in polycystin-2 (PC2). PC2 is a member of the polycystin transient receptor potential subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a procystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca 2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca 2+ have yielded widely varying results. Here, we report the mean macroscopic Ca 2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from the murine inner medullary collecting duct. Under conditions designed to isolate inward Ca 2+ currents, a small inward Ca 2+ current was detected in cilia with active PC2 channels but not in cilia lacking those channels. The current was activated by the addition of 10 µM internal Ca 2+ , which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was -1.8 pA at -190 mV; its conductance from -50 to -200 mV averaged 20 pS. Thus, native PC2 channels of renal primary cilia are able to conduct a small but detectable Ca 2+ influx under the conditions tested. The possible consequences of this influx are discussed. NEW & NOTEWORTHY In autosomal dominant polycystic kidney disease, it is proposed that Ca 2+ entering the primary cilium through polycystin-2 (PC2) channels may limit the formation of cysts. Recent studies predict that any macroscopic Ca 2+ influx through these channels should be small. We report that the native PC2 channels in primary cilia of cultured renal epithelial cells can allow a small macroscopic calcium influx. This may allow a significant accumulation of Ca 2+ in the cilium in vivo.

Hypaconitine, a neuromuscular blocker, is a diterpene alkaloid found in the root of Aconitum carmichaelii. Although hypaconitine was shown to affect various physiological responses in neurological models, the effect of hypaconitine on cell viability and the mechanism of its action of Ca 2+ handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca 2+ signalling in HCN-2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST-1). Cytosolic Ca 2+ concentrations [Ca 2+ ] i was measured by the Ca 2+ -sensitive fluorescent dye fura-2. In HCN-2 cells, hypaconitine (10-50 μmol/L) induced cytotoxicity and [Ca 2+ ] i rises in a concentration-dependent manner. Removal of extracellular Ca 2+ partially reduced the hypaconitine's effect on [Ca 2+ ] i rises. Furthermore, chelation of cytosolic Ca 2+ with BAPTA-AM reduced hypaconitine's cytotoxicity. In Ca 2+ -containing medium, hypaconitine-induced Ca 2+ entry was inhibited by modulators (2-APB and SKF96365) of store-operated Ca 2+ channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn 2+ influx indirectly suggesting that hypaconitine evoked Ca 2+ entry. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin abolished hypaconitine-induced [Ca 2+ ] i rises. Conversely, treatment with hypaconitine inhibited thapsigargin-induced [Ca 2+ ] i rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine-induced [Ca 2+ ] i rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca 2+ ] i rises by Ca 2+ influx via store-operated Ca 2+ entry involved PKC regulation and evoking PLC-independent Ca 2+ release from the endoplasmic reticulum. Because BAPTA-AM loading only partially reversed hypaconitine-induced cell death, it suggests that hypaconitine induced a second Ca 2+ -independent cytotoxicity in HCN-2 cells., (© 2021 John Wiley & Sons Australia, Ltd.)

Background & Aims: In upper airway cells, T helper 2 cytokines that signal through interleukin-4 (IL-4) receptor-α have been shown to stimulate eotaxin-3 secretion via a nongastric proton pump (ngH + ,K + ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH + ,K + ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL-4 receptor-α signaling., Methods: ngH + ,K + ATPase expression in EoE cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting. IL-4-stimulated eotaxin-3 secretion was measured by enzyme-linked immunosorbent assay after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester (calcium chelator), 2-aminoethoxydiphenyl borate (inhibitor of endoplasmic reticulum calcium release), verapamil, and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of patients with EoE and controls., Results: EoE cells expressed ngH + ,K + ATPase messenger RNA and protein. Omeprazole and SCH 28080 decreased IL-4-stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4-stimulated eotaxin-3 secretion was blocked by ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester, 2-aminoethoxydiphenyl borate, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4-stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls., Conclusions: EoE cells express a nongastric proton pump that mediates T helper 2 cytokine-stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block T helper 2 cytokine-stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca 2+ ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca 2+ chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), and recorded 19 F NMR spectra. The RBCs were suspended in gelatin gel in a special stretching/compression apparatus. The 5FBAPTA was loaded into the cells as the tetraacetoxymethyl ester; and 13 C NMR spectroscopy with [1,6- 13 C]D-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca 2+ influx is concluded to be via the mechanosensitive cation channel Piezo1. The increased rate of influx brought about by the activator of Piezo1, 2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1) supported this conclusion; while the specificity of the cation-sensing by 5FBAPTA was confirmed by using the Ca 2+ ionophore, A23187.

CaCl 2 , Ca 2+ chelator (EGTA) and Ca 2+ channel blocker (verapamil) were used to investigate mechanism of glucoraphanin metabolism in broccoli sprouts under ZnSO 4 stress. CaCl 2 treatment promoted sprout growth, reduced MDA (malonaldehyde) content and electrolyte leakage in sprouts under ZnSO 4 stress. The highest MDA content and electrolyte leakage were obtained in ZnSO 4 plus verapamil-treated sprouts. In addition, ZnSO 4 plus CaCl 2 treatment significantly enhanced glucoraphanin content and sulforaphane formation, while an opposite result was observed after ZnSO 4 plus EGTA treatment; which were further supported by expression of glucoraphanin biosynthetic and hydrolytic genes as well as myrosinase (MYR) and epithiospecifier protein (ESP) activities. These results indicated that exogenous and endogenous calcium promoted glucoraphanin biosynthesis and the conversion rate of glucoraphanin into sulforaphane. Verapamil treatment also stimulated glucoraphanin biosynthesis, but exerted an adverse influence on sulforaphane formation from the hydrolysis of glucoraphanin because of much higher ESP expression and ESP activity than ZnSO 4 treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin β3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss., Conclusions: Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53 . chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca 2+ /protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca 2+ /PKC axis as a potential therapeutic target in patients with chRCC. SIGNIFICANCE: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner., (©2020 American Association for Cancer Research.)

Acetylcholinesterase (AChE) hydrolyses acetylcholine to choline and acetate, playing an important role in terminating the neurotransmission in brain and muscle. Recently, the non-neuronal functions of AChE have been proposed in different tissues, in which there are various factors to regulate the expression of AChE. In mammalian skin, AChE was identified in melanocytes and keratinocytes. Our previous study has indicated that AChE in keratinocyte affects the process of solar light-induced skin pigmentation; however, the expression of AChE in keratinocytes in responding to sunlight remains unknown. Here, we provided several lines of evidence to support a notion that AChE could be upregulated at transcriptional and translational levels in keratinocytes when exposed to solar light. The light-mediated AChE expression was triggered by Ca 2+ , supported by an induction of Ca 2+ ionophore A23187 and a blockage by Ca 2+ chelator BAPTA-AM. In addition, this increase on AChE transcriptional expression was eliminated by mutagenesis on the activating protein 1 (AP1) site in ACHE gene. Hence, the solar light-induced AChE expression is mediated by Ca 2+ signalling through AP1 site. This finding supports the role of solar light in affecting the cholinergic system in skin cells, and which may further influence the dermatological function., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms., Methods: Fluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry., Results: Both norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [\(Ca_i^{2 + }\)]., Conclusions: Our results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of β-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.

Plants are under constant attack by a suite of insect herbivores. Over millions of years of coexistence, plants have evolved the ability to sense insect feeding via herbivore-associated elicitors in oral secretions, which can mobilize defense responses. However, herbivore-associated elicitors and the intrinsic downstream modulator of such interactions remain less understood. In this study, we show that tobacco hornworm caterpillar ( Manduca sexta ) oral secretion (OS) induces reactive oxygen species (ROS) in tomato ( Solanum lycopersicum ) protoplasts. By using a dye-based ROS imaging approach, our study shows that application of plant-fed (PF) M. sexta OS generates significantly higher ROS while artificial diet-fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. Elevation in ROS generation was saturated after ~140 s of PF OS application. ROS production was also suppressed in the presence of an antioxidant NAC ( N -acetyl- L -cysteine). Interestingly, PF OS-induced ROS increase was abolished in the presence of a Ca 2+ chelator, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid). These results indicate a potential signaling cascade involving herbivore-associated elicitors, Ca 2+ , and ROS in plants during insect feeding. In summary, our results demonstrate that plants incorporate a variety of independent signals connected with their herbivores to regulate and mount their defense responses.

Haloperidol, a typical antipsychotic medication, has been shown to possess various biological effects in different brain models. However, the impact of haloperidol on Ca 2+ signaling in astrocytes is elusive. This study explored the effect of haloperidol on cytosolic free Ca 2+ levels ([Ca 2+ ] i ) and viability, and established these two connections in Gibco® Human Astrocytes (GHAs) and DI TNC1 rat astrocytes. Haloperidol (5-20 μM) caused [Ca 2+ ] i rises in a concentration-dependent manner in GHAs but not in DI TNC1 cells. Furthermore, removal of extracellular Ca 2+ reduced haloperidol's effect by approximately 30% in GHAs. Haloperidol (20-40 μM) evoked concentration-dependent cytotoxicity in GHAs and DI TNC1 cells. However, chelating cytosolic Ca 2+ with the Ca 2+ chelator BAPTA/AM significantly reversed haloperidol's cytotoxicity only in GHAs. In GHAs, haloperidol-induced Ca 2+ entry was inhibited by store-operated Ca 2+ modulators (2-APB and SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. This Ca 2+ entry induced by haloperidol was confirmed by Mn 2+ entry-induced quench of fura-2 fluorescence. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished haloperidol-induced [Ca 2+ ] i rises. Conversely, treatment with haloperidol inhibited 45% of BHQ-evoked [Ca 2+ ] i rises. Moreover, haloperidol-induced Ca 2+ release from the endoplasmic reticulum was abolished by inhibition of phospholipase C (PLC) by U73122. Together, in GHAs but not in DI TNC1 cells, haloperidol caused Ca 2+ -associated cell death, induced Ca 2+ entry via PKC-sensitive store-operated Ca 2+ channels, and evoked PLC-dependent Ca 2+ release from the endoplasmic reticulum. The protective effect of Ca 2+ chelating on haloperidol-induced cytotoxicity in human astrocytes was also demonstrated.

Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans -Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription. IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1 , which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency., (Copyright © 2020 Madigan et al.)

Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca 2+ chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca 2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation., Competing Interests: Declaration of Competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca 2+ ] i . Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca 2+ -sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH 2 -terminal EF-hand pair (res. 9-76) is replaced by the COOH-terminal pair (res. 82-148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: "Ca-CaM-Cork" and "CaM-Cork". In the first, gating involves Ca 2+ -induced CaM-activation. In the second, gating takes place without [Ca 2+ ] i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca 2+ ] i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

Appropriate chelator may increase plant tolerance and accumulation for Cd in soil, but its molecular mechanism is unclear. In this experiment, the technology of isobaric tags for relative and absolute quantitation (iTRAQ) was used to compare the differential expression proteins (DEPs) and differential expression genes (DEGs) characteristics of poplar accumulating Cd combined with EDTA and/or EGTA. The results showed that the Cd concentrations, biomasses and activities of antioxidant enzymes of poplar were significantly increased in the treatments of chelator addition compared to the T Cd. The number of co-intersecting specific proteins of T Cd /CK, T Cd+EDTA /T Cd , T Cd+EGTA /T Cd and T Cd+EDTA+EGTA /T Cd was 49. Using the GO function and KEGG analysis, it was found that EDTA and EGTA might improve some main metabolic pathways of poplar leaves, which were involved in the enhancement of the expression of carbohydrate and energy metabolism-related proteins, regulation of cell energy metabolism, complementing and cooperating with each other in various ways, and dynamic regulation of energy metabolism. Particularly, chelator might induce the regulation of protein synthesis, folding and transport, and degradation of abnormal proteins in response to Cd toxicity. These results provided a theoretical basis for further elucidation of molecular mechanisms of poplar response to Cd stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Cryopreserved platelets are phenotypically and functionally different to conventionally stored platelets. Calcium may be released from internal stores during the freeze-thaw process, initiating signaling events which lead to these alterations. It was hypothesized that the addition of a calcium chelator prior to cryopreservation may mitigate some of these changes., Methods: Buffy coat-derived platelets that had been pooled and split were tested fresh and following cryopreservation (n = 8 per group). Platelets were cryopreserved using 5%-6% dimethylsulfoxide (DMSO) or were supplemented with increasing concentrations of the internal calcium chelator, BAPTA-AM (100 μM, 200 μM, or 400 μM), prior to storage at -80°C., Results: Supplementation of platelets with BAPTA-AM prior to freezing improved platelet recovery in a dose response manner (400 μM: 84 ± 2%) compared to standard DMSO cryopreserved platelets (70 ± 4%). There was a loss of GPIbα, GPVI, and GPIIb/IIIa receptors on platelets following cryopreservation, which was rescued when platelets were supplemented with BAPTA-AM (400 μM: p < 0.0001 for all). Platelet activation markers, such as phosphatidylserine and P-selectin, were externalized on platelets following cryopreservation. However, the addition of BAPTA-AM significantly reduced the increase of these activation markers on cryopreserved platelets (400 μM: p < 0.0001 for both). Both cryopreserved platelet groups exhibited similar functionality as assessed by thromboelastography, forming clots at a faster rate than fresh platelets., Conclusions: This study demonstrates that calcium plays a crucial role in mediating cryopreservation-induced damage to frozen platelets. The addition of the calcium chelator, BAPTA-AM, prior to cryopreservation reduces this damage., (© 2020 AABB.)

Extracellular calcium is required for intracellular Ca 2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca 2+ ] i rise. When egg activation was conducted in intracellular Ca 2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca 2+ ] i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca 2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII., (© 2020 Wiley Periodicals, Inc.)

Myrosinase (β-thioglucoside glucohydrolase, enzyme nomenclature, EC 3.2.1.147, TGG) is a highly abundant protein in Arabidopsis guard cells, of which TGG1 and TGG2 function redundantly in abscisic acid (ABA)- and methyl jasmonate-induced stomatal closure. Reactive carbonyl species (RCS) are α,β-unsaturated aldehydes and ketones, which function downstream of reactive oxygen species (ROS) production in the ABA signalling pathway in guard cells. Among the RCS, acrolein is the most highly reactive, which is significantly produced in ABA-treated guard cells. To clarify the ABA signal pathway downstream of ROS production, we investigated the responses of tgg mutants (tgg1-3, tgg2-1 and tgg1-3 tgg2-1) to acrolein. Acrolein induced stomatal closure and triggered cytosolic alkalization in wild type (WT), tgg1-3 single mutants and in tgg2-1 single mutants, but not in tgg1-3 tgg2-1 double mutants. Exogenous Ca2+ induced stomatal closure and cytosolic alkalization not only in WT but also in all of the mutants. Acrolein- and Ca2+-induced stomatal closures were inhibited by an intracellular acidifying agent, butyrate, a Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA) and a Ca2+ channel blocker, LaCl3. Acrolein induced cytosolic free calcium concentration ([Ca2+]cyt) elevation in guard cells of WT plants but not in the tgg1-3 tgg2-1 double mutants. Exogenous Ca2+ elicited [Ca2+]cyt elevation in guard cells of WT and tgg1-3 tgg2-1. Our results suggest that TGG1 and TGG2 function redundantly, not between ROS production and RCS production, but downstream of RCS production in the ABA signal pathway in Arabidopsis guard cells., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The Ca 2+ signal is essential in both hypoxia- and epidermal growth factor (EGF)-mediated epithelial to mesenchymal transition (EMT) in MDA-MB-468 breast cancer cells. This finding suggests that Ca 2+ -permeable ion channels participate in the induction of expression of some mesenchymal markers such as vimentin. However, the ion channels involved in vimentin expression induction have not been fully characterized. This work sought to define how differential modulation of the calcium signal effects the induction of vimentin and the Ca 2+ influx pathways involved. We identified that the intracellular Ca 2+ chelator EGTA-AM, cytochalasin D (a modulator of cytoskeletal dynamics and cell morphology), and the sarco/endoplasmic reticulum ATPase inhibitor thapsigargin are all inducers of vimentin in MDA-MB-468 breast cancer cells. EGTA-AM- and thapsigargin-mediated induction of vimentin expression in MDA-MB-468 cells involves store-operated Ca 2+ entry, as evidenced by sensitivity to silencing of the molecular components of this pathway, STIM1 and ORAI1. In stark contrast, cytochalasin D-mediated vimentin induction was insensitive to silencing of ORAI1, despite sensitivity to silencing of its canonical activator the endoplasmic reticulum Ca 2+ sensor STIM1. Cytochalasin D-mediated vimentin induction was, however, sensitive to silencing of another reported STIM1 target, TRPC1. Subsequent studies identified that EGTA-AM-induced vimentin expression also partially involved a TRPC1-dependent pathway. These studies define a complex interplay between vimentin expression in this model and the specific Ca 2+ -permeable ion channels involved. The complexity in the engagement of different Ca 2+ influx pathways that regulate vimentin induction are opportunities but also potential challenges in targeting Ca 2+ signaling to block EMT in cancer cells. Our findings further highlight the need to identify potential indispensable ion channels that can regulate induction of specific mesenchymal markers via different stimuli.

The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly understood. Here, we show that selective block of Panx1 in single postsynaptic hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, seconds long increases in the frequency of sEPSC following Schaffer collateral stimulation. The increase in sEPSC frequency occurred without an effect on evoked neurotransmission. Consistent with a presynaptic origin of the augmented glutamate release, the increased sEPSC frequency was prevented by bath-applied EGTA-AM or TTX. Manipulation of a previously described metabotropic NMDAR pathway (i.e., by preventing ligand binding to NMDARs with competitive antagonists or blocking downstream Src kinase) also increased sEPSC frequency similar to that seen when Panx1 was blocked. This facilitated glutamate release was absent in transient receptor potential vanilloid 1 (TRPV1) KO mice and prevented by the TRPV1 antagonist, capsazepine, suggesting it required presynaptic TRPV1. We show presynaptic expression of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block increases tissue levels of the endovanilloid, anandamide. Together, these findings demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in suppression of facilitated glutamate neurotransmission. SIGNIFICANCE STATEMENT The postsynaptic ion and metabolite channel, pannexin-1, is regulated by metabotropic NMDAR signaling through Src kinase. This pathway suppresses facilitated release of presynaptic glutamate during synaptic activity by regulating tissue levels of the transient receptor potential vanilloid 1 agonist anandamide., (Copyright © 2020 the authors.)

The transient receptor potential vanilloid 1 (TRPV1) protein is a pain receptor that elicits a hot sensation when an organism eats the capsaicin of red chili peppers. This calcium (Ca2+)-permeable cation channel is mostly expressed in the peripheral nervous system sensory neurons but also in the central nervous system (e.g. hippocampus and cortex). Preclinical studies found that TRPV1 mediates behaviors associated with anxiety and depression. Loss of TRPV1 functionality increases expression of genes related to synaptic plasticity and neurogenesis. Thus, we hypothesized that TRPV1 deficiency may modulate Alzheimer's disease (AD). We generated a triple-transgenic AD mouse model (3xTg-AD+/+) with wild-type (TRPV1+/+), hetero (TRPV1+/-) and knockout (TRPV1-/-) TRPV1 to investigate the role of TRPV1 in AD pathogenesis. We analyzed the animals' memory function, hippocampal Ca2+ levels and amyloid-β (Aβ) and tau pathologies when they were 12 months old. We found that compared with 3xTg-AD-/-/TRPV1+/+ mice, 3xTg-AD+/+/TRPV1+/+ mice had memory impairment and increased levels of hippocampal Ca2+, Aβ and total and phosphorylated tau. However, 3xTg-AD+/+/TRPV1-/- mice had better memory function and lower levels of hippocampal Ca2+, Aβ, tau and p-tau, compared with 3xTg-AD+/+/TRPV1+/+ mice. Examination of 3xTg-AD-derived primary neuronal cultures revealed that the intracellular Ca2+ chelator BAPTA/AM and the TRPV1 antagonist capsazepine decreased the production of Aβ, tau and p-tau. Taken together, these results suggested that TRPV1 deficiency had anti-AD effects and promoted resilience to memory loss. These findings suggest that drugs or food components that modulate TRPV1 could be exploited as therapeutics to prevent or treat AD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Inflammatory bowel disease, which typically manifests as Crohn's disease and ulcerative colitis, is caused by the abnormal production of cytokines such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β. These cytokines damage intestinal epithelial cells and trigger fibrosis, respectively, for which the current in vitro models have many limitations. Therefore, we tested whether human induced pluripotent stem cell-derived intestinal organoids (HiOs) can mimic inflammatory bowel disease (IBD), and whether such a model is suitable for drug screening. HiOs were treated with TNF-α and TGF-β to construct mucosal damage and fibrosis models. TNF-α diminished the mRNA expression of intestinal epithelial cell and goblet cell markers in HiOs. TNF-α also induced epithelial cell damage and degradation of tight junctions but not in the presence of infliximab, an antibody used in the clinic to deplete TNF-α. Furthermore, permeation of the non-absorbable marker FD-4 was observed in HiOs treated with TNF-α or ethylene glycol tetraacetic acid (EGTA), but not in the presence of infliximab. In contrast, TNF-α and TGF-β induced mRNA expression of mesenchymal and fibrosis markers, as well as epithelial-mesenchymal transition. SB431542, a TGF-β inhibitor, significantly reversed these events. The data indicate that HiOs mimic mucosal damage and fibrosis due to IBD and are thus suitable models for drug screening.

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.

Pleurotus ostreatus is a widely cultivated edible fungus around the world. At present, studies on the developmental process of the fruiting body are limited. In our study, we compared the differentially expressed proteins (DEPs) in the stipe and cap of the fruiting body by high-throughput proteomics. GO and pathway analysis revealed the great differences in the metabolic levels, including sucrose and starch metabolism, and sphingolipid signaling and metabolism, and the differences of 16 important DEPs were validated further by qPCR analysis in expression level. In order to control the cap and stipe development, several chemical inducers were applied to the primordium of the fruiting body according to the pathway enrichment results. We found that CaCl 2 can affect the primordium differentiation through inhibiting the stipe development. EGTA (ethyleneglycol bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) treatment confirmed the inhibitory role of Ca 2+ in the stipe development. Our study not only shows great metabolic differences during the cap and stipe development but also reveals the underlying mechanism directing the primordium differentiation in the early development of the fruiting body for the first time. Most importantly, we provide a reliable application strategy for the cultivation and improvement of the Pleurotus ostreatus , which can be an example and reference for a more edible fungus.

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing, and cancer invasion. Here, we report a simple optochemical method to induce cell contractions in vivo during Drosophila morphogenesis at single-cell resolution. We employed the photolabile Ca 2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca 2+ by laser photolysis in the epithelial tissue. Ca 2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, causing an approximately 50% drop in the cross-sectional area. Increased Ca 2+ levels are reversible, and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (ROCK) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin II accumulation in the apico-medial cortex, indicating that Ca 2+ bursts trigger non-muscle myosin II activation. Our approach can be, in principle, adapted to many experimental systems and species, as no specific genetic elements are required., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell-cell connections were essential for increased invasion on substrates with higher curvature, while cell-substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases-a cell-cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the past few decades, intracellular calcium overload has been shown to induce cell death through multiple signaling pathways. In this study, we used BAPTA-AM, a well-known membrane-permeable Ca 2+ chelator, to prevent cell injury by allaying the intracellular calcium overload. We explored the clinical potentials of BAPTA-AM-loaded liposome (BAL) in the treatment of the acute liver failure (ALF) mouse model, which is characterized by severe hepatic necrosis and apoptosis. We discovered that BAL can significantly inhibit D-GalN-induced LO2 cell damage as it increased cell viability by 60% and downregulated the LPS-stimulated inflammatory response in RAW 264.7 macrophages by reversing the morphological change and modulating TNF-α and NF-κB expressions. Through systemic administration, BAL can rapidly accumulate in damaged liver tissue and exhibit excellent treatment effects on the D-GalN/LPS-induced ALF mouse model, including elevation of the survival rate (from 10 to 80%), recovery of normal liver indexes and liver health indicators, improvement of liver blood microcirculation (increased the blood flow volume by 80% and flow rate by 60%), and blood coagulation. The underlying hepatoprotective effect of BAL is presumably based on the antinecrosis and antiapoptosis abilities attributed to its inhibition on oxidative stress, restriction on TNF-α receptor, and mitochondria-mediated apoptotic pathway by effectively clearing the overloaded intercellular calcium. BAL holds great potential as a new therapeutic strategy for ALF treatment, and its prominent cell rescue ability provides ample opportunities for the treatment of many other diseases that are characterized by rapid and massive cell damage.

β,β-Dimethylacrylshikonin (DMAS), an active ingredient of Lithospermum erythrorhizon and Arnebia euchroma, possess anti-neoplasm properties. Recently, DMAS was reported to stimulate autophagy in lung adenocarcinoma cells. However, the mechanisms by which DMAS modulates autophagy. have not yet been clearly elucidated. In this study, we found that DMAS significantly elevated intracellular free calcium accumulation. This activated the CaMKKβ-AMPK-mTOR pathway, subsequently inhibited mTOR and its substrate p70s6k and 4E-BP1, eventually leading to autophagy. In addition, we demonstrated that inhibition of autophagy by BAPTA-AM or STO-609 or compound C potently enhanced DMAS-induced lung adenocarcinoma cells apoptosis and growth inhibition. Overall, our results suggested that cytoprotective autophagy was triggered by DMAS via CaMKKβ-AMPK-mTOR signaling cascade in human lung adenocarcinoma cells, meaning that combining use of DMAS and autophagy inhibitors as a novel therapeutic option for lung adenocarcinoma will be very promising., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca 2+ , Mn 2+ and Zn 2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Prostate cancer (PCa) patients' mortality is mainly attributed to complications caused by metastasis of the tumor cells to organs critical for survival, such as bone. We hypothesized that PCa cell-bone interactions would promote paracrine signaling. A panel of PCa cell lines were cocultured with hydroxyapatite ([HA]; inorganic component of bone) of different densities. Conditioned media (CM) was collected and analyzed for calcium levels and effect on paracrine signaling, cell migration, and viability in vitro and in vivo. Our results showed that calcium levels were elevated in CM from cancer cell-bone cocultures, compared to media or cancer cells alone, and this could be antagonized by ethylene glycol-bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator, or knockdown of Snail protein. We also observed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation and paracrine cell proliferation and migration in LNCaP cells incubated with CM from various cell lines; this phosphorylation and cell migration could be antagonized by Snail knockdown or various inhibitors including EGTA, STAT3 inhibitor (WP1066) or cathepsin L inhibitor (Z-FY-CHO). In vivo, higher HA bone density increased tumorigenicity and migration of tumor cells to HA implant. Our study shows that cancer-bone microenvironment interactions lead to calcium-STAT3 signaling, which may present an area for therapeutic targeting of metastatic PCa., (© 2019 Wiley Periodicals, Inc.)

Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a gene-trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca 2+ levels, mitochondrial membrane potential measurements, and Ca 2+ -ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca 2+ -ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/grinaa mutants was tightly associated with disrupted intracellular Ca 2+ homeostasis, followed by mitochondrial Ca 2+ overload and cytochrome c release, leading to the activation of caspase 9- and caspase-3-mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N , N , N ', N '-tetraacetate-acetoxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP 3 R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9- and caspase 3-mediated apoptosis during zebrafish development., (© 2019 Chen et al.)