Increased Calcium Mobilization Research Articles

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

Loss of P2Y1 receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro

BackgroundThe hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors. After ADP exposure, the P2Y1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth. ObjectiveThe objective of this study was to examine the role of rapid P2Y1 receptor desensitization on platelet function and thrombus formation in vivo. MethodsWe analyzed a novel knock-in mouse strain expressing a P2Y1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y1340-0P), thereby preventing the desensitization of the receptor. ResultsP2Y1340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y1340-0P/340-0P and control mice. In vitro, P2Y1340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions. ConclusionOur studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.

Calcium signaling induced by 15-deoxy-prostamide-J2 promotes cell death by activating PERK, IP3R, and the mitochondrial permeability transition pore.

Melanoma is the deadliest form of skin cancer in the US. Although immunotherapeutic checkpoint inhibitors and small-molecule kinase inhibitors have dramatically increased the survival of patients with melanoma, new or optimized therapeutic approaches are still needed to improve outcomes. 15-deoxy-Δ12,14-prostamide J2 (15d-PMJ2) is an investigational small-molecule that induces ER stress-mediated apoptosis selectively in tumor cells. Additionally, 15d-PMJ2 reduces melanoma growth in vivo. To assess the chemotherapeutic potential of 15d-PMJ2, the current study sought to uncover molecular pathways by which 15d-PMJ2 exerts its antitumor activity. B16F10 melanoma and JWF2 squamous cell carcinoma cell lines were cultured in the presence of pharmacological agents that prevent ER or oxidative stress as well as Ca2+ channel blockers to identify mechanisms of 15d-PMJ2 cell death. Our data demonstrated the ER stress protein, PERK, was required for 15d-PMJ2-induced death. PERK activation triggered the release of ER-resident Ca2+ through an IP3R sensitive pathway. Increased calcium mobilization led to mitochondrial Ca2+ overload followed by mitochondrial permeability transition pore (mPTP) opening and the deterioration of mitochondrial respiration. Finally, we show the electrophilic double bond located within the cyclopentenone ring of 15d-PMJ2 was required for its activity. The present study identifies PERK/IP3R/mPTP signaling as a mechanism of 15d-PMJ2 antitumor activity.

In Arctic ground squirrels, diet and torpor alter skeletal muscle relaxation kinetics but not force development

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as ‐3°C), during which they are mostly physically inactive. Once Tb reaches ~15°C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~35°C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω‐6 and the ω‐6:ω‐3 PUFA ratio correlates with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. Muscular activity is characterized by contraction and relaxation mediated by calcium ions cycling released from SR stores. Ryanodine receptors (RyR1) on SR membranes release calcium ions into the cytosol, facilitating muscle contraction via actin‐myosin cross‐bridging. Whereas SERCA catalyzes Ca2+ reuptake back into the SR, releasing Ca2+ from actin/myosin filaments, completing the calcium cycle, and causing muscle relaxation. We hypothesized that diet (increased ω‐6:ω‐3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex‐vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. 1) Standard rodent chow with an ω‐6:ω‐3 ratio of 5:1, and 2) a balanced diet with an ω‐6:ω‐3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4°C, 15°C, 25°C, and 37°C to determine functional resilience. Our data show that torpid animals fed standard rodent chow appear to have an increase in SERCA activity, resulting in a significant increase in calcium mobilization from the sarcoplasm to the sarcoplasmic reticulum by the SERCA pump, allowing faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω‐6:ω‐3 PUFA ratio improves skeletal muscle performance during hypothermic stress in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Dimerized Translationally Controlled Tumor Protein-Binding Peptide 2 Attenuates Systemic Anaphylactic Reactions Through Direct Suppression of Mast Cell Degranulation.

Dimerized translationally controlled tumor protein (dTCTP) amplifies allergic responses through activation of several types of immune cells and release of inflammatory mediators. In particular, dTCTP plays an important role in histamine release by triggering mast cells and has been proposed as a target in the treatment of allergic diseases. dTCTP-binding peptide 2 (dTBP2) is known to attenuate severe allergic rhinitis and asthma through inhibition of dTCTP activity on airway epithelial cells and T cells; however, it is unclear whether dTBP2 affects mast cell function and mast cell disease. In this study, we explored the effects of dTBP2 on mast cell degranulation and allergen-induced anaphylactic reactions. We found that bacterial product lipopolysaccharide increased the expression of dTCTP in mast cells and rapidly released dTCTP by the mast cell stimulator compound 48/80. Interestingly, the released dTCTP further promoted mast cell degranulation in an autocrine activation manner and increased calcium mobilization in mast cells, which is essential for degranulation. Furthermore, dTBP2 directly and dose-dependently inhibited in vitro mast cell degranulation enhanced by compound 48/80, suggesting a direct and potent anti-anaphylactic activity of dTBP2. dTBP2 also significantly suppressed the dTCTP-induced degranulation and histamine release through inhibition of the p38 MAPK signaling pathway and suppression of lysosomal expansion and calcium mobilization in mast cells. More importantly, in vivo administration of dTBP2 decreased mortality and significantly attenuated histamine release and inflammatory cytokine production in compound 48/80-induced systemic anaphylactic reactions. These results suggest that dTBP2 is beneficial for the control of anaphylaxis with increased dTCTP.

Histamine-1 Receptors Expression in Primary Pterygium Tissue is Higher than Normal Conjunctival Tissue

BACKGROUND: Pterygium is an eye disease with multifactorial etiopathogenesis. Molecular factors such as cell proliferation and inflammatory mediators are associated with increased calcium mobilization and activation of nuclear factor kβ mediated by histamine-1 receptors (H1R).
 AIM: This study aims to determine whether the expression of H1R primary pterygium tissue is higher than normal conjunctival tissue and the expression of H1R based on pterygium grades.
 METHODS: This study was an analytic observational study with a case-control study approach at Sanglah General Hospital, Bali Mandara Eye Hospital, and Mangusada Hospital. The study was conducted from November 2017 to April 2018. The pterygium and conjunctival tissues obtained from 28 subjects in the same eye and examined for H1R expression by immunohistochemistry.
 RESULT: The results of this study obtained 64.3% of women with a mean age of 54.2 ± 7.8 years. There was no difference in mean H1R expression between pterygium grades in the final score (P = 0.759). There was a mean difference of H1R between primary pterygium (42.50) and normal conjunctival tissue (14.50) with P <0.001. Only tissue types affected the expression of H1R in the final score (B = 4.893; 95% CI 4.363-5.423; P <0.001).
 CONCLUSION: It was concluded that the expression of H1R primary pterygium tissue was higher in primary pterygium than normal conjunctival tissue.

Dextromethorphan - A widely-used cough suppressant - Induces local anaphylaxis via MRGPRX2 on mast cells

Dextromethorphan (DM) is a cough suppressant available in many prescribed and over-the-counter medications. Adverse reactions induced by DM have been regularly reported, including allergic skin reactions in some cases. However, the underlying mechanisms of local anaphylaxis induced by DM have not been elucidated. In this study, we found that DM could activate mast cells to increase calcium mobilization and release β-hexosaminidase, histamine, tumor necrosis factor-α, MCP-1, and IL-8 in a dose-dependent manner. The allergic reactions were confirmed by hind paw swelling and extravasation assay in vivo. Furthermore, DM was revealed to induce local anaphylaxis via MRGPRX2 by the mast cell-deficient kitW-sh/W−sh mice and MRGPRX2 knockdown mast cells. And the MRGPRX2-HEK293/CMC analysis and frontal analysis also showed that DM has a considerable affinity with MRGPRX2. Together, our findings suggest that close monitoring should be drawn on patients with DM for its potential anaphylaxis via MRGPRX2.

Toxicity and antimicrobial effect of silver nanoparticles in swine sperms

ABSTRACT Bacterial contamination in swine semen affects the quality and longevity of sperm and consequently fertility is reduced. Antibiotics have been used to prevent bacterial growth, but the frequency of bacterial resistance to various antibiotics are increasing. Silver nanoparticles (AgNPs) of 10–20 nm in size have shown a biocide effect in bacteria and fungi microorganisms without toxicity to certain mammalian cells. The goal of this study was to analyze both, antimicrobial activity against Staphylococcus aureus and toxicity in swine sperms after 10–20 nm AgNPs treatment. S. aureus proliferation decreased when concentrations from 0.4 to 10 mM AgNPs were assayed. Also, sperm viability measured by mitochondrial metabolism after AgNPs treatment up to a concentration of 10 mM, was viable. In addition, viability determined by membrane integrity of sperms showed that AgNPs treatment up to a concentration of 10 mM was safe. Sperm morphology was evaluated by automated quantification of proximal and distal drops and whiptails. Data indicated that AgNPs treatment up to a concentration of 4 mM were harmless. Finally, sperm capacitation and acrosome reactions were determined by (chlortetracycline) CTC assay. Data showed that no changes in sperm capacitation were observed when sperms were treated with 2 mM of AgNPs, but data showed increased calcium mobilization when treated with 10 mM AgNPs, which suggested sperm capacitation. Finally, there were no significant changes encountered on sperm acrosome reaction for any of the treatments after AgNPs treatment. Taken together, these results show the potential of AgNPs as an alternative to conventional antimicrobial agents that are currently used in extenders to preserve semen required for storage. Abbreviations AgNPs: silver nanoparticles; AMK: amikacin; AMP: adenosine monophosphate; AR: acrosome reaction; C: capacitation; CF: cefallotin; CFU: colony-forming unit; CTC: chlortetracycline; CXM: cefuroxime; DMSO: dimethyl sulfoxide; NC: non-capacitation; NOM: Norma Oficial Mexicana; PBS: phosphate buffered saline; RLUs: relative light units; ROS: reactive oxygen species; SQS: Seminal Quality System

Ca2+ mediates extracellular vesicle biogenesis through alternate pathways in malignancy

ABSTRACT Extracellular vesicles (EVs) are small membrane vesicles that serve as important intercellular signalling intermediaries in both malignant and non-malignant cells. For EVs formed by the plasma membrane, their biogenesis is characterized by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. EV-production is significantly higher in malignant cells relative to non-malignant cells and previous work suggests this is dependent on increased calcium mobilization and activity of calpain. However, calcium-signalling pathways involved in malignant and non-malignant EV biogenesis remain unexplored. Here we demonstrate; malignant cells have high basal production of plasma membrane EVs compared to non-malignant cells and this is driven by a calcium–calpain dependent pathway. Resting vesiculation in malignant cells occurs via mobilization of calcium from endoplasmic reticulum (ER) stores rather than from the activity of plasma membrane calcium channels. In the event of ER store depletion however, the store-operated calcium entry (SOCE) pathway is activated to restore ER calcium stores. Depleting both ER calcium stores and blocking SOCE, inhibits EV biogenesis. In contrast, calcium signalling pathways are not activated in resting non-malignant cells. Consequently, these cells are relatively low vesiculators in the resting state. Following cellular activation however, an increase in cytosolic calcium and activation of calpain increase in EV biogenesis. These findings contribute to furthering our understanding of extracellular vesicle biogenesis. As EVs are key mediators in the intercellular transfer of deleterious cancer traits such as cancer multidrug resistance (MDR), understanding the molecular mechanisms governing their biogenesis in cancer is the crucial first step in finding novel therapeutic targets that circumvent EV-mediated MDR.

Histamine-1 Receptors Expression in Primary Pterygium Tissue is Higher than Normal Conjunctival Tissue

Pterygium is an eye disease with multifactorial etiopathogenesis. Molecular factors such as cell proliferation and inflammatory mediators are associated with increased calcium mobilization and activation of nuclear factor kβ mediated by histamine-1 receptors (H1R). This study aims to determine whether the expression of H1R primary pterygium tissue is higher than normal conjunctival tissue and the expression of H1R based on pterygium grades. This study was an analytic observational study with a case-control study approach at Sanglah General Hospital, Bali Mandara Eye Hospital, and Mangusada Hospital. The study was conducted from November 2017 to April 2018. The pterygium and conjunctival tissues obtained from 28 subjects in the same eye and examined for H1R expression by immunohistochemistry. The results of this study obtained 64.3% of women with a mean age of 54.2 ± 7.8 years. There was no difference in mean H1R expression between pterygium grades in the final score (P = 0.759). There was a mean difference of H1R between primary pterygium (42.50) and normal conjunctival tissue (14.50) with P <0.001. Only tissue types affected the expression of H1R in the final score (B = 4.893; 95% CI 4.363-5.423; P <0.001). It was concluded that the expression of H1R primary pterygium tissue was higher in primary pterygium than normal conjunctival tissue.

Phenothiazine antipsychotics exhibit dual properties in pseudo-allergic reactions: Activating MRGPRX2 and inhibiting the H1 receptor

Phenothiazines are a class of antipsychotics that share the same tricyclic structure and are widely used in clinical settings. Adverse reactions from these drugs, however, have been regularly reported, with allergic skin reactions noted in some cases. Nevertheless, the mechanisms underlying anaphylaxis by these drugs have not been described. In the present study, we found that phenothiazine antipsychotics increased calcium mobilization and activated mast cells to release β-hexosaminidase, histamine, and tumor necrosis factor-α via Mas-related G-protein-coupled receptor member X2 (MRGPRX2) in vitro. In addition, they induced histamine release in serum via Mrgprb2 in C57BL/6 mice without Evans blue extravasation or paw swell. Further experiments indicated these drugs had good interaction with the histamine H1 receptor (H1R) and show an anti-calcium mobilization effect on H1R-HEK293 cells, which confirmed a potential antagonist effect of these drugs on the H1R. The molecular docking and activity experiments indicated that the N-methyl substitution on the side chain of these drugs played a significant role in activating MRGPRX2, while the phenothiazine tricyclic ring was associated with the inhibiting effect on the H1R. Therefore, due to their dual properties of increasing histamine levels without obvious allergic symptoms, clinicians should be highly vigilant for damage from histamine accumulation and long-term inflammatory reactions during the clinical use of phenothiazine antipsychotics.

TAK-442, a Direct Factor Xa Inhibitor, Inhibits Monocyte Chemoattractant Protein 1 Production in Endothelial Cells via Involvement of Protease-Activated Receptor 1.

Oral blood coagulation inhibitors and their receptors, such as factor Xa (FXa), thrombin, and the thrombin receptor protease-activated receptor 1 (PAR1), are entered into clinical trials for acute coronary syndrome therapy; however, the results obtained so far are different for each drug. The underlying mechanisms of the results have not been fully investigated. We studied the in vitro anti-inflammatory effects of the selective FXa inhibitor TAK-442 on human endothelial cells, with comparing those of the selective thrombin inhibitor melagatran and the PAR1 antagonist vorapaxar. In human umbilical vein endothelial cells, FXa-increased production of monocyte chemoattractant protein 1 (MCP-1), a key inflammatory mediator, was inhibited by TAK-442 but not melagatran, and was also remarkably suppressed by vorapaxar. As thrombin did, FXa increased calcium mobilization in PAR1-overexpressed Chinese hamster ovary cells, which was selectively inhibited by TAK-442 and vorapaxar. We therefore confirmed the inhibitory effect of TAK-442 in endothelial MCP-1 production and the PAR1 intervention in the response. Our results suggest that TAK-442 may have anti-inflammatory potential in addition to its anti-thrombotic effects.

The Human Cytomegalovirus US27 Gene Product Constitutively Activates Antioxidant Response Element-Mediated Transcription through Gβγ, Phosphoinositide 3-Kinase, and Nuclear Respiratory Factor 1.

Human cytomegalovirus (HCMV) is a widespread pathogen that modulates host chemokine signaling during persistent infection in the host. HCMV encodes four proteins with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. Each of the four receptors modulates host CXCR4 signaling. US28, UL33, and UL78 impair CXCR4 signaling outcomes, while US27 enhances signaling, as evidenced by increased calcium mobilization and cell migration to CXCL12. To investigate the effects of US27 on CXCR4 during virus infection, fibroblasts were infected with bacterial artificial chromosome-derived clinical strain HCMV TB40/E-mCherry (wild type [WT]), mutants lacking US27 (TB40/E-mCherry-US27Δ [US27Δ]) or all four GPCRs (TB40 E-mCherry-allΔ), or mutants expressing only US27 but not US28, UL33, or UL78 (TB40/E-mCherry-US27wt [US27wt]). CXCR4 gene expression was significantly higher in WT- and US27wt-infected fibroblasts. This effect was evident at 3 h postinfection, suggesting that US27 derived from the parental virion enhanced CXCR4 expression. Reporter gene assays demonstrated that US27 increased transcriptional activity regulated by the antioxidant response element (ARE), and small interfering RNA treatment indicated that this effect was mediated by NRF-1, the primary transcription factor for CXCR4. Increased translocation of NRF-1 into the nucleus of WT-infected cells compared to mock- or US27Δ-infected cells was confirmed by immunofluorescence microscopy. Chemical inhibitors targeting Gβγ and phosphoinositide 3-kinase (PI3K) ablated the increase in ARE-driven transcription, implicating these proteins as mediators of US27-stimulated gene transcription. This work identifies the first signaling pathway activated by HCMV US27 and may reveal a novel regulatory function for this orphan viral receptor in stimulating stress response genes during infection.IMPORTANCE Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, causing deafness, blindness, and other serious birth defects. CXCR4 is a human chemokine receptor that is crucial for both fetal development and immune responses. We found that the HCMV protein US27 stimulates increased expression of CXCR4 through activation of the transcription factor nuclear respiratory factor 1 (NRF-1). NRF-1 regulates stress response genes that contain the antioxidant response element (ARE), and HCMV infection is associated with increased expression of many stress response genes when US27 is present. Our results show that the US27 protein activates the NRF-1/ARE pathway, stimulating higher expression of CXCR4 and other stress response genes, which is likely to be beneficial for virus replication and/or immune evasion.

Manipulation of CXCR4 signaling by human cytomegalovirus (HCMV) interleukin-10

Abstract Herpesviruses have developed numerous strategies for manipulating host immune responses to avoid clearance and establish lifelong latent infection. Human cytomegalovirus (HCMV) encodes cmvIL-10, an ortholog of human interleukin-10 that binds to the cellular IL-10 receptor (IL-10R) with high affinity to elicit potent immunosuppressive effects, including inhibition of inflammatory cytokine production. In direct contrast, we found that cmvIL-10 promotes heightened chemokine signaling. CXCR4 is an essential cellular chemokine receptor that guides cell movement toward its natural ligand, CXCL12. We found that monocytes and epithelial cells exhibited significantly increased calcium mobilization and migration to CXCL12 in the presence of cmvIL-10. Treatment with a Stat3 inhibitor or siRNA blocked these effects, indicating that Stat3 activation is required for potentiation of CXCR4 signaling activity by cmvIL-10. Using immunofluorescence microscopy, we found evidence of association between IL-10R and CXCR4 in cell membranes and observed that cmvIL-10 treatment led to dramatic changes in the cellular distribution of Stat3. Since intracellular calcium release is a rapid response, Stat3 is likely acting here via a non-canonical pathway, rather than in its classic role as a transcription factor. These findings demonstrate that cmvIL-10 triggers a complex series of events that augment the signaling activity of a cellular chemokine receptor. Given that monocytes are a primary reservoir of latent virus, manipulation of CXCR4 signaling may enable HCMV to modify cell trafficking patterns to promote virus dissemination.

N-arachidonoyl glycine, another endogenous agonist of GPR55

Interest in lipoamino acids as endogenous modulators of G-protein coupled receptors has escalated due to their involvement in a variety of physiologic processes. In particular, a role for these amino acid conjugates has emerged in the endocannabinoid system. The study presented herein investigated the effects of N-arachidonoyl glycine (NAGly) on a candidate endocannabinoid receptor, GPR55. Our novel findings reveal that NAGly induces concentration dependent increases in calcium mobilization and mitogen-activated protein kinase activities in HAGPR55/CHO cells. These increases were attenuated by the selective GPR55 antagonist ML193 (N-[4-[[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl]phenyl]-6,8-dimethyl-2-(2-pyridinyl)-4-quinolinecarboxamide), supporting receptor mediated signaling. To our knowledge this is the first report identifying GPR55 as a target of the endogenous lipoamino acid, NAGly.

The MARCKS protein amount is differently regulated by calpain during toxic effects of methylmercury between SH-SY5Y and EA.hy926 cells.

Methylmercury (MeHg) is an environmental pollutant that shows severe toxicity to humans and animals. However, the molecular mechanisms mediating MeHg toxicity are not completely understood. We have previously reported that the MARCKS protein is involved in the MeHg toxicity to SH-SY5Y neuroblastoma and EA.hy926 vascular endothelial cell lines. In addition, calpain, a Ca2+-dependent protease, is suggested to be associated with the MeHg toxicity. Because MARCKS is known as a substrate of calpain, we studied the relation between calpain activation and cleavage of MARCKS and its role in MeHg toxicity. In SH-SY5Y cells, MeHg decreased cell viability along with increased calcium mobilization, calpain activation and a decrease in MARCKS amounts. However, pretreatment with calpain inhibitors attenuated the decrease in cell viability and MARCKS amount induced only by 1 µM but not by 3 µM MeHg. In cells with a MARCKS knockdown, calpain inhibitors failed to attenuate the decrease in cell viability caused by MeHg. In EA.hy926 cells, although MeHg caused calcium mobilization and a decrease in MARCKS levels, calpain activation was not observed. These results indicate that the participation of calpain in the regulation of MARCKS amounts is dependent on the cell type and concentration of MeHg. In SH-SY5Y cells, calpain-mediated proteolysis of MARCKS is involved in cytotoxicity induced by a low concentration of MeHg.

Hypochlorhydria-induced calcium malabsorption does not affect fracture healing but increases post-traumatic bone loss in the intact skeleton.

ABSTRACTEfficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr−/− mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr−/− and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr−/− mice. However, Cckbr−/− mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr−/− mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016.

Microenvironment involved in FPR1 expression by human glioblastomas.

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-015-1777-2) contains supplementary material, which is available to authorized users.

Cloning, identification and functional characterization of bovine free fatty acid receptor-1 (FFAR1/GPR40) in neutrophils.

Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid.

Mechanisms by which calcium receptor stimulation modifies electromechanical coupling in isolated ventricular cardiomyocytes

The calcium-sensing receptor (CaR) is widely expressed throughout the entire cardiovascular system and is capable of activating signaling pathways in different cells. Alongside calcium, the CaR also responds to physiological polycations such as putrescine underlining a participation in physiological and pathophysiological processes. Here, we aimed to determine mechanisms as to how CaR activation affects the contractile responsiveness of ventricular cardiomyocytes under basal and stimulated conditions. For that purpose, cardiac myocytes from 3-month-old male Wistar rats were isolated, and the acute effects of an antagonist (NPS2390), agonists (putrescine and gadolinium), or of downregulation of the CaR by siRNA on cell shortening were recorded in a cell-edge-detection system. In addition, experiments were performed on muscle stripes and Langendorff preparations. Mechanistic insights were taken from calcium transients of beating fura-2 AM-loaded cardiomyocytes and western blots. Isolated ventricular cardiomyocytes constitutively express CaR. The expression in the atria is less pronounced. Acute inhibition of CaR reduced basal cell shortening of ventricular myocytes at nearly physiological levels of extracellular calcium. Inhibition of CaR strongly reduced contractility of ventricular muscle stripes but not of atria. Activation of CaR by putrescine and gadolinium influences the contractile responsiveness of isolated cardiomyocytes. Increased calcium mobilization from the sarcoplasmic reticulum via an IP3-dependent mechanism was responsible for amplified systolic calcium transients and a subsequent improvement in cell shortening. Alongside with these effects, activation of CaR increased relaxation velocity of the cells. In conclusion, ventricular CaR expression affects contractile parameters of ventricular heart muscle cells and modifies electromechanical coupling of cardiomyocytes.