SERCA1 Overexpression in Skeletal Muscle Attenuates Muscle Atrophy and Improves Motor Function in a Mouse Model of ALS.

Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of muscle mass and muscle function. Previous work from our lab demonstrated that skeletal muscles from a mouse model of ALS show elevated intracellular calcium (Ca2+) levels and heightened endoplasmic reticulum (ER) stress. Objective: To investigate whether overexpression of sarcoplasmic reticulum (SR) Ca2+ ATPase 1 (SERCA1) in skeletal muscle would improve intracellular Ca2+ handling, attenuate ER stress, and improve motor function ALS transgenic mice. Methods: B6SJL-Tg (SOD1*G93A)1Gur/J (ALS-Tg) mice were bred with skeletal muscle α-actinin SERCA1 overexpressing mice to generate wild type (WT), SERCA1 overexpression (WT/+SERCA1), ALS-Tg, and SERCA1 overexpressing ALS-Tg (ALS-Tg/+SERCA1) mice. Motor function (grip test) was assessed weekly and skeletal muscles were harvested at 16 weeks of age to evaluate muscle mass, SR-Ca2+ ATPase activity, levels of SERCA1 and ER stress proteins - protein disulfide isomerase (PDI), Grp78/BiP, and C/EBP homologous protein (CHOP). Single muscle fibers were also isolated from the flexor digitorum brevis muscle to assess changes in resting and peak Fura-2 ratios. Results: ALS-Tg/+SERCA1 mice showed improved motor function, delayed onset of disease, and improved muscle mass compared to ALS-Tg. Further, ALS-Tg/+SERCA1 mice returned levels of SERCA1 protein and SR-Ca2+ ATPase activity back to levels in WT mice. Unexpectedly, SERCA-1 overexpression increased levels of the ER stress maker Grp78/BiP in both WT and ALS-Tg mice, while not altering protein levels of PDI or CHOP. Lastly, single muscle fibers from ALS-Tg/+SERCA1 had similar resting but lower peak Fura-2 levels (at 30 Hz and 100 Hz) compared to ALS-Tg mice. Conclusions: These data indicate that SERCA1 overexpression attenuates the progressive loss of muscle mass and maintains motor function in ALS-Tg mice while not lowering resting Ca2+ levels or ER stress.

Point mutations in RyR2 Ca2+-binding residues of human cardiomyocytes cause cellular remodelling of cardiac excitation contraction-coupling.

AIMS: CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences. METHODS AND RESULTS: Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5-20 mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; (iv) and 20 mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red. CONCLUSION: Mutating RyR2 Ca2+-binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+- and caffeine-binding sites. Enhanced transmembrane calcium influx and remodelling of EC-coupling pathways may underlie the persistence of spontaneous beating in Ca2+-induced Ca2+ release-suppressed mutant myocytes.

Mechanosensitivity and mechanotransductive properties of osteoclasts.

Mechanical loading is essential for maintaining bone health. Here, we aimed to investigate the role of ATP and ADP in the mechanotransduction of bone-resorptive osteoclasts. Single osteoclast in primary cultures from 10 to 12-wk-old mice was mechanically stimulated by a gentle touch with a micropipette. Changes in cytosolic free calcium [Ca2+]i were analyzed in Fura-2 loaded osteoclasts. The cell injury was assessed by analyzing the cellular Fura-2 loss and classified as severe or mild using k-means. Osteoclasts responded to mechanical stimuli with transient calcium elevation (primary responders) and transduced these signals to neighboring cells, which responded with delayed calcium elevations (secondary responders). Severely injured osteoclasts had higher calcium transients than mildly injured cells. Fluid shear stress similarly induced reversible cell injury in osteoclasts. Secondary responses were abolished by treatment with A-804598, a specific inhibitor of P2X7, but not suramin, a broad P2 receptor blocker. Osteoclasts responded to ATP and ADP with concentration-dependent changes in [Ca2+]i. We performed osteoclast micropipette stimulation in the presence of phosphoenolpyruvate and pyruvate kinase which converted all ADP in solution to ATP, or with hexokinase converting all ATP to ADP. Osteoclasts with mild membrane injury demonstrated similar calcium responses in ATP and ADP-rich environments. However, when the mechanotransductive signal to severe osteoclast injury was converted to ADP, the fraction of secondary responders and their [Ca2+]i amplitude was higher. This study suggests the importance of osteoclast mechanobiology and the role of ADP-mediated signaling in conditions of altered mechanical loading associated with bone loss.NEW & NOTEWORTHY Osteoclasts are rarely considered as cells that participate in mechanical signaling in bone. We show that osteoclasts are capable of sensing and transmitting mechanical signals to neighboring cells. Mechanical stimulation commonly induces minor repairable membrane injury in osteoclasts. ATP and especially ADP were found to play important roles in the mechanoresponsiveness of osteoclasts. This study highlights the importance of osteoclast mechanobiology especially in conditions of altered mechanical loading associated with bone loss, such as in microgravity.

Calibration of mammalian skeletal muscle Ca2+ transients recorded with the fast Ca2+ dye Mag-Fluo-4.

BACKGROUND: Mag-Fluo-4 is increasingly employed for studying Ca2+ signaling in skeletal muscle; however, the lack of information on the Ca2+-Mag-Fluo-4 reaction limits its wider usage. METHODS: Fluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 µM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C. RESULTS: The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca2+/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ Kd was 1.652 × 105µM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca2+]peak reached 22.5 (7.8) µM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10). CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca2+ transients. GENERAL SIGNIFICANCE: Our results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types.

Impaired Ca2+ signaling due to hepatic steatosis mediates hepatic insulin resistance in Alström syndrome mice that is reversed by GLP-1 analog treatment.

Ca2+ signaling plays a critical role in the regulation of hepatic metabolism by hormones including insulin. Changes in cytoplasmic Ca2+ regulate synthesis and posttranslational modification of key signaling proteins in the insulin pathways. Emerging evidence suggests that hepatocyte intracellular Ca2+ signaling is altered in lipid-loaded liver cells isolated from obese rodent models. The mechanisms of altered Ca2+-insulin and insulin-Ca2+ signaling pathways in obesity remain poorly understood. Here, we show that the kinetics of insulin-initiated intracellular (initial) Ca2+ release from endoplasmic reticulum is significantly impaired in steatotic hepatocytes from obese Alström syndrome mice. Furthermore, exenatide, a glucagon-like peptide-1 (GLP-1) analog, reversed lipid-induced inhibition of intracellular Ca2+ release kinetics in steatotic hepatocytes, without affecting the total content of intracellular Ca2+ released. Exenatide reversed the lipid-induced inhibition of intracellular Ca2+ release, at least partially, via lipid reduction in hepatocytes, which then restored hormone-regulated cytoplasmic Ca2+ signaling and insulin sensitivity. This data provides additional evidence for the important role of Ca2+ signaling pathways in obesity-associated impaired hepatic lipid homeostasis and insulin signaling. It also highlights a potential advantage of GLP-1 analogs when used to treat type 2 diabetes associated with hepatic steatosis.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.

Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs) and spontaneously differentiated neurons derived from a pluripotent human embryonic stem cell (hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.

Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Under physiologic conditions, conjunctival goblet cells (CGCs) secrete mucins into the tear film to preserve ocular surface homeostasis. Specialized proresolving mediators (SPMs), like resolvins (Rvs), regulate secretion from CGCs and actively terminate inflammation. The purpose of this study was to determine if RvD2 stimulated mucin secretion and to investigate the cellular signaling components. Goblet cells were cultured from rat conjunctiva. Secretion was measured by an enzyme-linked lectin assay, change in intracellular [Ca2+] ([Ca2+]i) using Fura-2, and cellular cAMP levels by ELISA. RvD2 (10-11-10-8 M) stimulated secretion, increased cellular cAMP levels and the [Ca2+]i. RvD2-stimulated increase in [Ca2+]i and secretion was blocked by Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride but not by the cAMP exchange protein inhibitor α-[2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile. Forskolin, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP (8-Br-cAMP) increased [Ca2+]i. Increasing cAMP with 8-Br-cAMP inhibited the increase in [Ca2+]i stimulated by the cAMP-independent agonist cholinergic agonist carbachol. In conclusion, RvD2 uses both cellular cAMP and [Ca2+]i to stimulate glycoconjugate secretion from CGCs, but the interaction of cAMP and [Ca2+]i is context dependent. Thus RvD2 likely assists in the maintenance of the mucous layer of the tear film to sustain ocular surface homeostasis and has potential as a novel treatment for dry eye disease.-Botten, N., Hodges, R. R., Li, D., Bair, J. A., Shatos, M. A., Utheim, T. P., Serhan, C. N., Dartt, D. A. Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Methods to Measure Intracellular Ca2+ Concentration Using Ca2+-Sensitive Dyes.

Ca2+ ion is universally considered the most versatile second messenger responsible for decoding and regulating the majority of the signaling pathways within the cell. The study of intracellular Ca2+ concentration ([Ca2+]i) dynamics is consequently of primary importance for the interpretation of cellular biology. This chapter will present a relatively simple, largely diffused, and nevertheless robust method to measure variations of [Ca2+]i by the use of the Ca2+-sensitive chemical dye Fura-2. A general protocol for the assessment of [Ca2+]i in adherent cells, applicable to a variety of cell systems, will be first presented. Then, the implementation of Fura-2 to detect [Ca2+]i in two specific cell types, namely, human adrenocortical cells and primary skin fibroblasts, will be discussed in more particulars. Finally, the procedure to monitor Ca2+ influx through the plasma membrane using Fura-2 will be described.

Resolvin D1, but not resolvin E1, transactivates the epidermal growth factor receptor to increase intracellular calcium and glycoconjugate secretion in rat and human conjunctival goblet cells.

PURPOSE: To identify interactions of the epidermal growth factor receptor (EGFR) with the pro-resolving mediator receptors for RvD1 and RvE1 to stimulate an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion from cultured human and rat conjunctival goblet cells. METHODS: Goblet cells from human and rat conjunctiva were grown in culture using RPMI media. Cultured goblet cells were pre-incubated with inhibitors, and then stimulated with RvD1, RvE1, EGF or the cholinergic agonist carbachol (Cch). Increase in [Ca2+]i was measured using fura-2/AM. Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-1. Western blot analysis was performed with antibodies against AKT and ERK 1/2. RESULTS: In cultured human conjunctival goblet cells RvE1 -stimulated an increase in [Ca2+]i. RvD1-, but not the RvE1-, stimulated increase in [Ca2+]i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca2+]i that was also inhibited by TAPI-1, an inhibitor of the matrix metalloprotease ADAM 17. Inhibition of the EGFR also blocked RvD1-stimulated increase in AKT activity and both RvD1-and RvE1-stimulated increase in ERK 1/2 activity. Pretreatment with either RvD1 or RvE1 did not block the EGFR-stimulated increase in [Ca2+]i. CONCLUSIONS: We conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca2+]i, activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca2+]I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity.

Trifluoperazine Attenuates Store-Dependent Ca2+ Entry in Macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with the calsequestrin inhibitor neuroleptic trifluoperazine leads to a significant inhibition of the store-dependent Ca2+ entry induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest calsequestrin involvement in the regulation of the store-dependent Ca2+ entry in macrophages.

Activation of the NFAT-Calcium Signaling Pathway in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2+]i and ECM gene expression in normal and glaucoma LC cells. Methods: [Ca2+]i was measured with dual-wavelength Ca2+ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-ß1 [TGFß1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. Results: Basal and hypotonic cell membrane stretch-induced [Ca2+]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2+]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2-induced elevation in profibrotic ECM genes (TGFß1, Col1A1, and periostin), both in normal and in glaucoma LC cells. Conclusions: Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

Poly-arginine R18 and R18D (D-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat.

We examined the neuroprotective efficacy of the poly-arginine peptide R18 and its D-enantiomer R18D in a perinatal hypoxic-ischaemic (HI) model in P7 Sprague-Dawley rats. R18 and R18D peptides were administered intraperitoneally at doses of 30, 100, 300 or 1000 nmol/kg immediately after HI (8% O2/92%N2 for 2.5 h). The previously characterised neuroprotective JNKI-1-TATD peptide at a dose of 1000 nmol/kg was used as a control. Infarct volume and behavioural outcomes were measured 48 h after HI. For the R18 and R18D doses examined, total infarct volume was reduced by 25.93% to 43.80% (P = 0.038 to < 0.001). By comparison, the JNKI-1-TATD reduced lesion volume by 25.27% (P = 0.073). Moreover, R18 and R18D treatment resulted in significant improvements in behavioural outcomes, while with JNKI-1-TATD there was a trend towards improvement. As an insight into the likely mechanism underlying the effects of R18, R18D and JNKI-1-TATD, the peptides were added to cortical neuronal cultures exposed to glutamic acid excitotoxicity, resulting in up to 89, 100 and 71% neuroprotection, respectively, and a dose dependent inhibition of neuronal calcium influx. The study further confirms the neuroprotective properties of poly-arginine peptides, and suggests a potential therapeutic role for R18 and R18D in the treatment of HIE.

Resolvin D1 Increases Mucin Secretion in Cultured Rat Conjunctival Goblet Cells via Multiple Signaling Pathways.

Purpose: Goblet cells in the conjunctiva secrete mucin into the tear film protecting the ocular surface. The proresolution mediator resolvin D1 (RvD1) regulates mucin secretion to maintain homeostasis during physiological conditions and in addition, actively terminates inflammation. We determined the signaling mechanisms used by RvD1 in cultured rat conjunctival goblet cells to increase intracellular [Ca2+] ([Ca2+]i) and induce glycoconjugate secretion. Methods: Increase in [Ca2+]i were measured using fura 2/AM and glycoconjugate secretion determined using an enzyme-linked lectin assay with the lectin Ulex Europaeus Agglutinin 1. Signaling pathways activated by RvD1 were studied after goblet cells were pretreated with signaling pathway inhibitors before stimulation with RvD1. The results were compared with results when goblet cells were stimulated with RvD1 alone and percent inhibition calculated. Results: The increase in [Ca2+]i stimulated by RvD1 was blocked by inhibitors to phospholipases (PL-) -D, -C, -A2, protein kinase C (PKC), extracellular signal-regulated kinases (ERK)1/2 and Ca2+/calmodulin-dependent kinase (Ca2+/CamK). Glycoconjugate secretion was significantly inhibited by PLD, -C, -A2, ERK1/2 and Ca2+/CamK, but not PKC. Conclusions: We conclude that RvD1 increases glycoconjugate secretion from goblet cells via multiple signaling pathways including PLC, PLD, and PLA2, as well as their signaling components ERK1/2 and Ca2+/CamK to preserve the mucous layer and maintain homeostasis by protecting the eye from desiccating stress, allergens, and pathogens.

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells.

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca2+ oscillations in bEND5 cells, 300 µM being the most effective dose to generate a prolonged Ca2+ burst. Pharmacological manipulation revealed that Ach-evoked Ca2+ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca2+ release via inositol-1,4,5-trisphosphate receptors (InsP3Rs) and store-operated Ca2+ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP3R1 and InsP3R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

C1q/tumor necrosis factor-related protein-3 enhances the contractility of cardiomyocyte by increasing calcium sensitivity.

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that protects against myocardial infarction-induced cardiac dysfunction through its pro-angiogenic, anti-apoptotic, and anti-fibrotic effects. However, whether CTRP3 can directly affect the systolic and diastolic function of cardiomyocytes remains unknown. Adult rat cardiomyocytes were isolated and loaded with Fura-2AM. The contraction and Ca2+ transient data was collected and analyzed by IonOptix system. 1 and 2µg/ml CTRP3 significantly increased the contraction of cardiomyocytes. However, CTRP3 did not alter the diastolic Ca2+ content, systolic Ca2+ content, Ca2+ transient amplitude, and L-type Ca2+ channel current. To reveal whether CTRP3 affects the Ca2+ sensitivity of cardiomyocytes, the typical phase-plane diagrams of sarcomere length vs. Fura-2 ratio was performed. We observed a left-ward shifting of the late relaxation trajectory after CTRP3 perfusion, as quantified by decreased Ca2+ content at 50% sarcomere relaxation, and increased mean gradient (µm/Fura-2 ratio) during 500-600ms (-0.163 vs. -0.279), 500-700ms (-0.159 vs. -0.248), and 500-800ms (-0.148 vs. -0.243). Consistently, the phosphorylation level of cardiac troponin I at Ser23/24 was reduced by CTRP3, which could be eliminated by preincubation of okadaic acid, a type 2A protein phosphatase inhibitor. In summary, CTRP3 increases the contraction of cardiomyocytes by increasing the myofilament Ca2+ sensitivity. CTRP3 might be a potential endogenous Ca2+ sensitizer that modulates the contractility of cardiomyocytes.

Effect of Methoxsalen on Ca²âº Homeostasis and Viability in Human Osteosarcoma Cells.

Methoxsalen is a natural compound found in many seed plants. The effect of methoxsalen on Ca²âº- related physiology in human osteosarcoma is unclear. This study investigated the effect of methoxsalen on cytosolic free Ca²âº concentrations ([Ca²âº]i) in MG63 human osteosarcoma cells. Methoxsalen induced [Ca²âº]i rises concentration-dependently. Methoxsalen-induced Ca²âº entry was confirmed by Mn²âº-induced quench of fura-2 fluorescence. This Ca²âº entry was suppressed by nifedipine, econazole, and SKF96365. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) inhibited methoxsalen-evoked [Ca²âº]i rises by 96%. In contrast, incubation with methoxsalen abolished BHQ-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished methoxsalen-induced [Ca²âº]i rises. Methoxsalen was cytotoxic at 300-700 µM in a concentration-dependent fashion. Chelating cytosolic Ca²âº with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) did not prevent methoxsalen-induced cytotoxicity. Collectively, our data suggest that in MG63 cells, methoxsalen induced [Ca²âº]i rises by evoking PLC-dependent Ca²âº release from the endoplasmic reticulum, and Ca²âº entry via store-operated Ca²âº entry. Methoxsalen also induced Ca²âº- disassociated cell death.

Effect of oleic acid on store-operated calcium entry in immune-competent cells.

PURPOSE: To study the mechanism by which oleic acid (OA) (C18:1) exerts its beneficial effects on immune-competent cells. Since store-operated Ca2+ entry (SOCE) is a Ca2+ influx pathway involved in the control of multiple physiological processes including cell proliferation, we studied the effect of OA in Ca2+ signals of Jurkat T cells and THP-1 monocytes, paying particular attention to SOCE. METHODS: Changes in [Ca2+]i were measured using the Fura-2 fluorescence dye. Mn2+ uptake was monitored as a rate of quenching of Fura-2 fluorescence measured at the Ca2+-insensitive wavelengths. Thapsigargin was used to induce SOCE in Fura-2-loaded cells. RESULTS: We showed a clear dose-dependent SOCE-inhibitory effect of OA in both cell lines. Such an inhibitory effect was PKC independent and totally restored by albumin, suggesting that OA exerts its effect somewhere in the membrane. We also demonstrated that OA induces increases in [Ca2+]i partly mediated by an extracellular Ca2+ influx through econazole-insensitive channels. Finally, we compared the effect of OA with stearic acid (C18:0), assuming the emerged evidence concerning the link between saturated fats and inflammation disorders. Stearic acid failed to inhibit SOCE, independently on the concentration tested, thus intensifying the physiological relevance of our findings. CONCLUSION: We suggest a physiological pathway for the beneficial effects of OA in inflammation.

Chorein Sensitive Orai1 Expression and Store Operated Ca2+ Entry in Rhabdomyosarcoma Cells.

BACKGROUND: Chorein, a protein encoded by VPS13A (vacuolar protein sorting-associated protein 13A), is defective in chorea acanthocytosis, a rare disease characterized by acanthocytosis of red blood cells and neuronal cell death with progressive hyperkinetic movement disorder, cognitive dysfunction, behavioral abnormalities and chronic hyperkalemia. Chorein is highly expressed in ZF rhabdomyosarcoma cells and counteracts apoptosis of those cells. Chorein is effective in part by interacting with and fostering stimulation of phosphoinositide-3-kinase (PI3K)-p85-subunit. PI3K dependent signaling includes the serum and glucocorticoid inducible kinase SGK1. The kinase activates NFκB with subsequent up-regulation of the Ca2+ channel subunit Orai1, which accomplishes store operated Ca2+ entry (SOCE). Orai1 and SOCE have been shown to confer survival of tumor cells. The present study thus explored whether chorein impacts on Orai1 expression and SOCE. METHODS: In rhabdomyosarcoma cells chorein, Orai1, NFκB and SGK1 transcript levels were quantified by RT-PCR, Orai1 protein abundance by Western blotting, FACS analysis and confocal laser microscopy, [Ca2+]i utilizing Fura-2 fluorescence, and SOCE from the increase of [Ca2+]i following store depletion with extracellular Ca2+ removal and inhibition of the sarcoendoplasmatic reticular Ca2+ ATPase with thapsigargin. RESULTS: The mRNA coding for chorein was most abundant in drug resistant, poorly differentiated human ZF rhabdomyosarcoma cells. Chorein silencing significantly decreased Orai1 transcript levels and Orai1 protein expression, as well as SGK1 and NFκB transcript levels. SOCE in ZF rhabdomyosarcoma cells was significantly blunted by chorein silencing, Orai1 inhibitor 2-APB (50 µM), SGK1 inhibitor EMD638683 (50 µM, 10 h) and NFκB inhibitor wogonin (50 µM, 24 h). CONCLUSION: Chorein is a stimulator of Orai1 expression and thus of store operated Ca2+ entry. The effect may involve SGK1 and NFκB.