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Background: Mag-Fluo-4 is increasingly employed for studying Ca 2+ signaling in skeletal muscle; however, the lack of information on the Ca 2+ -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 K d of the Ca 2+ -Mag-Fluo-4 reaction. Rate constants (k on , k off ), fluorescence maximum (F max ), minimum (F min ), 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 Ca 2+ /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 K d was 1.652 × 10 5 μM 2 (n = 58 fibres, R 2  = 0.99). With an F max of 150.9 (8.8) A.U. (n = 8), F min of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca 2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca 2+ ] 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 Ca 2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca 2+ transients., General Significance: Our results may be relevant to the quantitative study of Ca 2+ kinetics in many other cell types., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium-independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium-transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura-2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes., (© 2020 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

An increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) plays a key role in controlling endothelial functions; however, it is still unclear whether endothelial Ca 2+ handling is altered by type 2 diabetes mellitus, which results in severe endothelial dysfunction. Herein, we analyzed for the first time the Ca 2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca 2+ -sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca 2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The "Ca 2+ add-back" protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca 2+ pool, while store-operated Ca 2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca 2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca 2+ toolkit.

The fluorescent dye fura-2 AM was employed to record activation of Ca 2+ entry in response to a decrease in Ca 2+ concentration in the endoplasmic reticulum. Using whole-cell voltage clamp technique, we revealed Ca 2+ currents with an amplitude of 0.46±0.13 pA/pF that passed through selective channels with current-voltage characteristics similar to those of classical store-operated CRAC channels. These currents were sensitive to 2-APB (50 μM), an inhibitor of store-operated channels. The data suggest that store-operated calcium entry is a characteristic feature of mature ventricular cardiomyocytes. Pathological alterations in store-operated Ca 2+ entry can be implicated in the development of heart diseases.

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 [Ca 2+ ] ([Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] i . RvD1-, but not the RvE1-, stimulated increase in [Ca 2+ ] i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca 2+ ] 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 [Ca 2+ ] i ., Conclusions: We conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca 2+ ] i , activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca 2+ ] I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Atrial tachyarrhythmia (AT) is a common complication in patients who have undergone a Fontan operation. In this study, we investigated whether abnormal Ca 2+ handling contributes to the Fontan operation-related atrial arrhythmogenic substrate., Methods: Mongrel dogs were randomly assigned to sham and Fontan groups. The Fontan operation model was developed by performing an atriopulmonary anastomosis. After 14 days, an electrophysiological study was performed to evaluate the AT vulnerability. Ca 2+ handling properties were measured by loading atrial cardiomyocytes (CMs) with fura-2 AM. The L-type Ca 2+ (I Ca-L ) and Na + -Ca 2+  exchanger (I NCX ) currents of the CMs were recorded by the whole-cell patch-clamp technique. The key Ca 2+ handling proteins expression was assessed by Western blotting., Results: The AT inducibility was higher in the Fontan group than in the sham group (85.71 vs. 14.29%, P < 0.05). The Fontan operation resulted in decreased Ca 2+ transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca 2+ content, but in enhanced diastolic intracellular Ca 2+ concentration and SR Ca 2+ leak in the atrial CMs. The spontaneous CaT events, triggered ectopic activity and I NCX density were increased, but I Ca-L density was reduced in CMs from the Fontan atria (all P < 0.05). Additionally, the Fontan operation resulted in decreased SR Ca 2+ ATPase expression and Cav1.2 expression, but in increased NCX1 and Ser2814-phosphorylated ryanodine receptor 2. The calmodulin-dependent protein kinase II expression and function were markedly enhanced in the Fontan atria., Conclusion: The Fontan operation caused atrial CM Ca 2+ handling abnormalities that produced arrhythmogenic-triggered activity and increased vulnerability to AT in experimental Fontan dogs.

Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca 2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca 2+ release was investigated. The changes in the concentration of intracellular Ca 2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca 2+ entry into the cytoplasm and fluorescence emission decreased. The IC 50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways. Graphical abstract ᅟ.

Glycoconjugate mucin secretion from conjunctival goblet cells is tightly regulated by nerves and specialized pro-resolving mediators (SPMs) to maintain ocular surface health. Here we investigated the actions of the SPM resolvin E1 (RvE1) on cultured rat conjunctival goblet cell glycoconjugate secretion and intracellular [Ca 2+ ] ([Ca 2+ ] i ) and the signaling pathways used by RvE1. Goblet cells were cultured from rat conjunctiva in RPMI medium. The amount of RvE1-stimulated glycoconjugate mucin secretion was determined using an enzyme-linked lectin assay with Ulex Europaeus Agglutinin 1 lectin. Cultured goblet cells were also incubated with the Ca 2+ indicator dye fura 2/AM and [Ca 2+ ] i was measured. Cultured goblet cells were incubated with inhibitors to phospholipase (PL-) C, D, and A2 signaling pathways. RvE1 stimulated glycoconjugate secretion in a concentration dependent manner and was inhibited with the Ca 2+ chelator BAPTA. The Ca 2+ i response was also increased in a concentration manner when stimulated by RvE1. Inhibition of PLC, PLD, and PLA2, but not Ca 2+ /calmodulin-dependent kinase blocked RvE1-stimulated increase in [Ca 2+ ] i and glycoconjugate secretion. We conclude that under normal, physiological conditions RvE1 stimulates multiple pathways to increase glycoconjugate secretion and [Ca 2+ ] i . RvE1 could be an important regulator of goblet cell glycoconjugate mucin secretion to maintain ocular surface health., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to determine if histamine receptors interact with the epidermal growth factor receptor (EGFR) in cultured rat conjunctival goblet cells., Methods: Goblet cells from rat conjunctiva were grown in organ culture. First-passage goblet cells were used in all experiments. Phosphorylated (active) and total EGFR, AKT, and extracellular signal-regulated kinase (ERK)1/2 were measured by Western blot analysis. Cells were preincubated with the EGFR antagonist AG1478 for 30 minutes or small interfering RNA specific to the EGFR for 3 days prior to stimulation with histamine or agonists specific for histamine receptor subtypes for 2 hours. Goblet cell secretion was measured using an enzyme-linked lectin assay. Goblet cells were incubated for 1 hour with the calcium indicator molecule fura-2/AM, and intracellular [Ca2+] ([Ca2+]i) was determined. Data were collected in real time and presented as the actual [Ca2+]i with time and as the change in peak [Ca2+]i., Results: Histamine increased the phosphorylation of the EGFR. Mucin secretion and increase in [Ca2+]i stimulated by histamine, and agonists specific for each histamine receptor subtype were blocked by inhibition of the EGFR. Increase in [Ca2+]i stimulated by histamine and specific agonists for each histamine receptor was also inhibited by TAPI-1, a matrix metalloproteinase (MMP) inhibitor. The histamine-stimulated increase in activation of AKT, but not ERK1/2, was blocked by AG1478., Conclusions: In conjunctival goblet cells, histamine, using all four receptor subtypes, transactivates the EGFR via an MMP. This in turn phosphorylates AKT to increase [Ca2+]i and stimulate mucin secretion.

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% O 2 /92%N 2 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.

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-β1 [TGFβ1], 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 (TGFβ1, 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.

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 Ca 2+ entry induced by endoplasmic Ca 2+ -ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest calsequestrin involvement in the regulation of the store-dependent Ca 2+ entry in macrophages.

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.

Objective: To establish a method of isolation and primary culture of mice distal pulmonary artery smooth muscle cells (PASMCs) and identify the functional properties. Methods: PASMCs were harvested from the distal pulmonary artery (PA) tissue of mice by enzymatic digestion of collagenaseⅠand papain; and the growth characteristics were observed under inverted microscope and identified by Immunofluorescence technique. Effects on the intracellular calcium ion concentration of distal PASMCs were detected by Fura-2-AM fluorescent probe tracer under a fluorescence microscope in Krebs solution containing clopiazonic acid (CPA) and nifedipin (Nif). Results: PASMCs density reached approximately to 80% in a typical valley-peak-like shape after 6 days. Cell α-smooth muscle actin (α-SMA) immunofluorescence identified that 95% of the cultured cells were PASMCs. More than 95% PASMCs responded well to calcium-potassium Krebs solution (potassium ion concentration of 60 mmol/L) and showed a rapid increase in basal [Ca(2+) ](i) after 1 minute's perfusion (Δ[Ca(2+) ](i)>50), which demonstrated that the voltage-dependent calcium channels (VDCC) of distal PASMCs were in good function; after the perfusion of calcium Krebs, calcium-free/calcium-Krebs containing CPA and Nif, distal PASMCs showed two typical peaks, indicated the full function of store-operated calcium channel (SOCC) in distal PASMCs. Conclusion: This experiment successfully established a stable and reliable mice distal PASMCs model and the study of pulmonary vascular diseases could benefit from its higher purity and better functional condition.

Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division. The depletion of chromosome scaffold proteins does not prevent chromosome condensation despite structural defects. This suggests that other factors contribute to condensation. Here we investigated the contribution of divalent cations, particularly Ca 2+ , to chromosome condensation in vitro and in vivo. Ca 2+ depletion caused defects in proper mitotic progression, particularly in chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca 2+ . Chromosomes had compact globular structures when exposed to Ca 2+ and expanded fibrous structures without Ca 2+ . Therefore, we have clearly demonstrated a role for Ca 2+ in the compaction of chromatin fibres.

Using Fura-2AM microfluorimetry, we have shown for the first time that methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, significantly inhibits glutoxim- and molixan-induced Ca 2+ -responses in rat peritoneal macrophages. The results suggest that intact rafts are necessary for signaling cascade induced by glutoxim or molixan and leading to intracellular Ca 2+ concentration increase in macrophages.

We propose a tool for a rapid high-resolution detection of calcium ions which can be used in parallel with other techniques. We have applied a new approach by  photo-oxidation of diaminobenzidine in presence of the emission of an excited fluorochrome specific for calcium detection. This method combines the selectivity of available fluorophores to the high spatial resolution offered by transmission electron microscopy to detect even fluorescing molecules even when present in low amounts in membrane-bounded organelles. We show in this paper that Mag-Fura 2 photoconversion via diaminobenzidine oxidation is an efficient way for localizing Ca2+ ions at EM level, is easily carried out and reproducible, and can be obtained on a good amount of cells, since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp. The end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary.

Using Fura-2AM microfluorimetry, we have shown for the first time that 5-lipoxygenase specific inhibitor antiasthmatic agent zileuton significantly inhibits Ca(2+)-responses induced by glutoxim and molixan in macrophages. The results support 5-lipoxygenase involvement in the effect of glutoxim and molixan on intracellular Ca(2+) concentration in macrophages and indicate the inadvisability of a combined use of drugs glutoxim and molixan and antiasthmatic agent zileuton.

Magnesium is an important micronutrient essential for various biological processes and its deficiency has been linked to several inflammatory disorders in humans. Topical magnesium delivery is one of the oldest forms of therapy for skin diseases, for example Dead Sea therapy and Epsom salt baths. Some anecdotal evidence and a few published reports have attributed amelioration of inflammatory skin conditions to the topical application of magnesium. On the other hand, transport of magnesium ions across the protective barrier of skin, the stratum corneum, is contentious. Our primary aim in this study was to estimate the extent of magnesium ion permeation through human skin and the role of hair follicles in facilitating the permeation. Upon topical application of magnesium solution, we found that magnesium penetrates through human stratum corneum and it depends on concentration and time of exposure. We also found that hair follicles make a significant contribution to magnesium penetration.

Background: ITC is a powerful technique that can reliably assess the thermodynamic underpinnings of a wide range of binding events. When metal ions are involved, complications arise in evaluating the data due to unavoidable solution chemistry that includes metal speciation and a variety of linked equilibria., Scope of Review: This paper identifies these concerns, provides recommendations to avoid common mistakes, and guides the reader through the mathematical treatment of ITC data to arrive at a set of thermodynamic state functions that describe identical chemical events and, ideally, are independent of solution conditions. Further, common metal chromophores used in biological metal sensing studies are proposed as a robust system to determine unknown solution competition., Major Conclusions: Metal ions present several complications in ITC experiments. This review presents strategies to avoid these pitfalls and proposes and experimentally validates mathematical approaches to deconvolute complex equilibria that exist in these systems., General Significance: This review discusses the wide range of complications that exists in metal-based ITC experiments. It provides a starting point for scientists new to this field and articulates concerns that will help experienced researchers troubleshoot experiments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The activity of TRPA1 and TRPV1 channels, their sensitivity to selective activators - allyl isothiocyanate (AITC) and capsaicin (Caps), especially their interaction were studied. The method of microfluorescent microscopy and Ca2+ sensitive dye fura- 2AM. Registration of changes in the concentration of intracellular Ca2+ was performed by using the ratio of fluorescence signals measured at two wavelengths (R = F1/ F2). Researches were conducted on cultured neurons of rat dorsal ganglia (DRG neurons). Application of AITC and Caps on soma of DRG neurons resulted in an increase in intracellular Ca2+. Consistent repeated Caps applications resulted in a significant reduction in the amplitude of Ca2+ transients ( desensitization of TRPV1 channels), which accounted 20,7% of initial value. Further application of selective TRPA1 channel agonist (AITC) resulted in restoration of sensitivity to capsaicin TRPV1 channels ( resensitization TRPV1 channels). Thus, we have established the presence of regulation of TRPV1 channel activity by TRPA1 channels.

In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy., (© 2015 International Society for Neurochemistry.)

Intestinal nutrient transport and sensing are of emerging interest in research on obesity and diabetes and as drug targets. Appropriate in vitro models are lacking that allow both, studies on transport processes as well as sensing and subsequent incretin hormone secretion including intracellular signaling. We here demonstrate that murine small-intestinal organoids are the first in vitro model system enabling concurrent investigations of nutrient and drug transport, sensing and incretin hormone secretion as well as fluorescent live-cell imaging of intracellular signaling processes. By generating organoid cultures from wild type mice and animals lacking different nutrient transporters, we show that organoids preserve the main phenotypic features and functional characteristics of the intestine. This turns them into the best in vitro model currently available and opens new avenues for basic as well as medical research.

Purpose: This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells., Methods: Changes in LC cell intracellular calcium [Ca(2+)]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR., Results: Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i (p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05)., Conclusions: This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aims: Vibrio vulnificusis an opportunistic pathogen that causes primary septicemia and wound infection with high mortality rate. This pathogen produces an RTX toxin (RtxA1) which can cause host cell rounding, cell death and interference with internalization by host phagocytes. However, the mechanism of RtxA1-induced phagocyte paralysis is not clear., Main Methods: Using the murine macrophage cell line RAW264.7, we measured cytotoxicity and phagocytosis of V. vulnificusin normal and calcium-depleted media. To deplete extracellular and cytosolic Ca(2+), cells were exposed to the calcium chelators ethylene glycol tetraacetic acid (EGTA) and 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl esteris (BAPTA-AM), respectively. The cytotoxicity was examined by measuring the activity of lactate dehydrogenase (LDH) released from the damaged cells. The gentamicin protection assay was conducted to determine the number of internalized bacteria, while acridine orange staining was applied to visualize the intracellular bacteria. The fluorescent indicator fura-2-acetoxymethyl ester (fura 2-AM) was used to measure the Ca(2+)signal post-infection., Key Findings: We revealed that extracellular Ca(2+)was essential for phagocytes to internalize V. vulnificus. Meanwhile, cytosolic Ca(2+)flux in RAW264.7 cells induced by an RtxA1 isogenic mutant was repressed by the parent strain. Furthermore, depletion of extracellular Ca(2+)level by EGTA significantly reduced the cytotoxicity but did not affect the antiphagocytic activity of RtxA1 toxin., Significance: Our results indicated that RtxA1 may interfere with cytosolic Ca(2+)flux of phagocyte to promote bacteria colonization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

Aims: Lysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. LPC could be deacylated to form glycerophosphocholine by neuropathy target esterase (NTE)/Swiss cheese protein (SWS). Although NTE/SWS is important in maintaining Ca(2+) homeostasis, the role of LPC in regulating the intracellular calcium concentration ([Ca(2+)]i) in Drosophila remains poorly understood. We aimed to study the mechanism of LPC-induced [Ca(2+)]i changes in Drosophila S2 cells., Main Methods: The [Ca(2+)]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM., Key Findings: Our results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca(2+)]i in the presence of external calcium ([Ca(2+)]e). The LPC-induced [Ca(2+)]i increase was reduced by 60.7% in the absence of [Ca(2+)]e. Furthermore, the Ca(2+) influx was inhibited by 37.3% after the cells were preincubated with an L-type Ca(2+) channel blocker. In the Ca(2+)-free medium, the LPC-induced [Ca(2+)]i increase was completely blocked using an inositol triphosphate receptor (IP3R) inhibitor. However, a ryanodine receptor (RyR) inhibitor had no effect on the LPC-induced [Ca(2+)]i increase., Significance: The LPC-induced [Ca(2+)]i increase in S2 cells was dependent on both the release of Ca(2+) stored in the endoplasmic reticulum and [Ca(2+)]e influx. Both L-type Ca(2+) channels and IP3R might be involved in this process. The LPC-induced [Ca(2+)]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.

Ghrelin, a gut and brain peptide, has recently been shown to be involved in motivated behavior and regulation of the sleep and wakefulness cycle. The laterodorsal tegmental nucleus (LDT) is involved in appetitive behavior and control of the arousal state of an organism, and accordingly, behavioral actions of ghrelin could be mediated by direct cellular actions within this nucleus. Consistent with this interpretation, postsynaptically mediated depolarizing membrane actions of ghrelin on LDT neurons have been reported. Direct actions were ascribed solely to closure of a potassium conductance however this peptide has been shown in other cell types to lead to rises in calcium via release of calcium from intracellular stores. To determine whether ghrelin induced intracellular calcium rises in mouse LDT neurons, we conducted calcium imaging studies in LDT brain slices loaded with the calcium binding dye, Fura-2AM. Ghrelin elicited TTX-insensitive changes in dF/F indicative of rises in calcium, and a portion of these rises were independent of membrane depolarization, as they persisted in conditions of high extracellular potassium solutions and were found to involve SERCA-pump mediated intracellular calcium stores. Involvement of the ghrelin receptor (GHR-S) in these actions was confirmed. Taken together with other studies, our data suggest that ghrelin has multiple cellular actions on LDT cells. Ghrelin's induction of calcium via intracellular release in the LDT could play a role in behavioral actions of this peptide as the LDT governs processes involved in stimulation of motivated behavior and control of cortical arousal., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Deoxycholic acid (DOA) is one of the secondary bile acids used as a mild detergent for the isolation of membrane associated proteins. This study examined whether the secondary bile acid, DOA, altered Ca(2+) movement, cell viability and apoptosis in SCM1 human gastric cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i. DOA-evoked [Ca(2+)]i rises concentration dependently. The response was reduced by removing extracellular Ca(2+). DOA-evoked Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (nifedipine, econazole and SKF96365), the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA) and the PKC inhibitor GF109203X. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished DOA-evoked [Ca(2+)]i rises. Conversely, treatment with DOA abolished TG-evoked [Ca(2+)]i rises. Inhibition of phospholipase C with U73122 abolished DOA-evoked [Ca(2+)]i rises. At 100-500 μM, DOA decreased cell viability, which was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). DOA between 100 and 300 μM also induced apoptosis. Collectively, in SCM1 cells, DOA-induced [Ca(2+)]i rises by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. DOA also caused Ca(2+)-independent apoptosis.

The Fura-2AM fluorescent Ca(2+) probe was used to study the possibility that the Arp2/3 complex and WASP proteins are involved in the effects of glutoxim and molixan on the intracellular Ca(2+) concentration in macrophages. It has been demonstrated that preincubation of macrophages with inhibitors of the Arp2/3 complex or WASP proteins (CK-0944666 or wiskostatin, respectively) results in a significant suppression of Ca(2+)-responses induced by glutoxim or molixan. This suggests that polymerization of actin filaments is a process involved in the effect of glutoxim or molixan on intracellular Ca(2+) concentration in macrophages.

Intracellular calcium transient ([Ca(2+)]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca(2+)]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca(2+)]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2AM were respectively used to detect membrane potential and [Ca(2+)]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca(2+)]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca(2+)]i transients. Replacing extracellular Na(+) with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca(2+)]i transients. However, voltage-activated K(+) channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca(2+) or blocking of L-type voltage-sensitive Ca(2+) channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca(2+)]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was -43.3mV and the depolarization induced by FSS was about 44mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca(2+) mobilization wherein FSS activated SACs to promote Na(+) entry to depolarize membrane that, in turn, activated L-VSCCs and Ca(2+) influx though L-VSCCs switched on [Ca(2+)]i response in osteoblasts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cytoplasmic free Mg(2+) concentration ([Mg(2+)]i) was measured in rat ventricular myocytes with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K(+) (Ca(2+)- and Mg(2+)-free) solution, [Mg(2+)]i decreased from ? 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg(2+)]i was recovered by perfusion with Ca(2+)-free Tyrode's solution containing 1 mM Mg(2+). The time course of the [Mg(2+)]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as being proportional to the initial Mg(2+) influx rate. The Mg(2+) influx rate was inversely related to [Mg(2+)]i, being higher at low [Mg(2+)]i. The Mg(2+) influx rate was augmented by the high extracellular Mg(2+) concentration (5 mM), whereas it was greatly reduced by cell membrane depolarization caused by high K(+). Known inhibitors of TRPM7 channels, 2-aminoethoxydiphenyl borate (2-APB), NS8593, and spermine reduced the Mg(2+) influx rate with half inhibitory concentrations (IC50) of, respectively, 17 ?M, 2.0 ?M, and 22 ?M. We also studied Ni(2+) influx by fluorescence quenching of intracellular furaptra by Ni(2+). The Ni(2+) influx was activated by lowering intra- and extracellular Mg(2+) concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg(2+) influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg(2+) influx. Under the whole-cell patch-clamp configuration, the removal of intracellular and extracellular divalent cations induced large inward and outward currents, MIC (Mg-inhibited cation) currents or IMIC, carried by monovalent cations likely via TRPM7 channels. IMIC measured at -120 mV was diminished to ? 50% by 100 ?M 2-APB or 10 ?M NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg(2+) influx in rat ventricular myocytes.

Strong β-adrenergic stimulation induced spontaneous diastolic Ca(2+) transients (SCTs) in electrically paced murine cardiac myocytes [28]. To obtain further insights into the underlying mechanism, we developed a method for a simultaneous analysis, in which the free luminal Ca(2+) concentration in the sarcoplasmic reticulum (SR) ([Ca(2+)]SR) and the free cytosolic Ca(2+) concentration ([Ca(2+)]i) were measured in parallel in the same cell. Each spontaneous diastolic Ca(2+) transient was exactly mirrored by a decrease of [Ca(2+)]SR. Since antagonism of the Ca(2+) mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was shown to block SCTs in single cardiac myocytes [28], we analyzed the effect of the novel ADP-ribosyl cyclase inhibitor SAN4825 on both cytosolic and intra-luminal Ca(2+) transients upon strong β-adrenergic stimulation. A strong antagonist effect of SAN4825 on SCTs at low micromolar concentrations was observed. Our results suggest that the underlying mechanism of spontaneous diastolic Ca(2+) transients observed upon strong β-adrenergic stimulation is sensitization of type 2 ryanodine receptor by the Ca(2+) releasing activity of the products of ADP-ribosyl cyclase activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aims: Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca., Methods and Results: Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s(-1). Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased., Conclusions: This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

I(Ca)-gated Ca(2+) release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca(2+), but the extent to which mitochondria contribute to the overall Ca(2+) signaling remains controversial. To examine the possible role of mitochondria in Ca(2+) signaling, we developed a low affinity mitochondrial Ca(2+) probe, mitycam-E31Q (300-500 MOI, 48-72h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca(2+) in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca(2+) while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca(2+)) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na(+) increased global cytosolic and mitochondrial Ca(2+) signals in one population of mitochondria, but unexpectedly decreased it (release of Ca(2+)) in another mitochondrial population. Such mitochondrial Ca(2+) release signals were seen not only during long lasting Na(+) withdrawal, but also when Ca(2+) loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca(2+) transients appear to activate with a delay following the cytosolic rise of Ca(2+) and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca(2+) signaling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Organophosphates (OPs) and carbamates are widely used insecticides that exert their neurotoxicity via inhibition of acetylcholine esterase (AChE) and subsequent overexcitation. OPs can induce additional neurotoxic effects at concentrations below those for inhibition of AChE, indicating other mechanisms of action are also involved. Since tight regulation of the intracellular calcium concentration ([Ca(2+)]i) is essential for proper neuronal development and function, effects of one carbamate (carbaryl) and two OPs (chlorpyrifos, parathion-ethyl) as well as their -oxon metabolites on [Ca(2+)]i were investigated. Effects of acute (20min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were measured in fura-2-loaded PC12 cells using single-cell fluorescence microscopy. Acute exposure to chlorpyrifos and its metabolite chlorpyrifos-oxon (10μM) induced a modest increase in basal [Ca(2+)]i. More importantly, the tested OPs concentration-dependently inhibited depolarization-evoked [Ca(2+)]i. Chlorpyrifos already induced a ∼30% inhibition at 0.1μM and a 100% inhibition at 10μM (IC50=0.43μM), whereas parathion-ethyl inhibited the depolarization-evoked [Ca(2+)]i increase with ∼70% at 10μM. Interestingly, -oxon metabolites were more potent inhibitors of AChE, but were less potent inhibitors of depolarization-evoked [Ca(2+)]i compared to their parent compound (chlorpyrifos-oxon) or were even without effect (paraoxon-ethyl and -methyl). Similarly, acute exposure to carbaryl had no effect on [Ca(2+)]i. Exposure to mixtures of chlorpyrifos with its oxon-analog or with parathion-ethyl did not increase the degree of inhibition, indicating additivity does not apply. These data demonstrate that concentration-dependent inhibition of depolarization-evoked [Ca(2+)]i is a novel mechanism of action of (sub)micromolar concentrations of OPs that could partly underlie OP-induced neurotoxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Kenyon cells, the intrinsic neurons of the insect mushroom body, have the intriguing property of responding in a sparse way to odorants. Sparse neuronal codes are often invariant to changes in stimulus intensity and duration, and sparse coding often depends on global inhibition. We tested if this is the case for honeybees' Kenyon cells, too, and used in vivo Ca²⁺ imaging to record their responses to different odorant concentrations. Kenyon cells responded not only to the onset of odorant stimuli (ON responses), but also to their termination (OFF responses). Both, ON and OFF responses increased with increasing odorant concentration. ON responses were phasic and invariant to the duration of odorant stimuli, while OFF responses increased with increasing odorant duration. Pharmacological blocking of GABA receptors in the brain revealed that ionotropic GABA(A) and metabotropic GABA(B) receptors attenuate Kenyon cells' ON responses without changing their OFF responses. Ionotropic GABA(A) receptors attenuated Kenyon cell ON responses more strongly than metabotropic GABA(B) receptors. However, the response dynamic, temporal resolution and paired-pulse depression did not depend on GABA(A) transmission. These data are discussed in the context of mechanisms leading to sparse coding in Kenyon cells.

Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca(2+) homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca(2+) concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca(2+) rise. Here we report that the channel responsible for Ca(2+) entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H2O2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H2O2. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.

Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca(2+)]i) handling in Raw 264.7 macrophages. In the present study, [Ca(2+)]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+) channel blockers had no effect on the resting [Ca(2+)]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca(2+)]i transient elevation independent of extracellular Ca(2+). The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca(2+)]i transient elevation, but pinacidil and other unselective K(+) channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+)]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+)]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+)]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

Neuronal plasticity allows an animal to respond to environmental changes by modulating its response to stimuli. In the honey bee (Apis mellifera), the biogenic amine octopamine plays a crucial role in appetitive odor learning, but little is known about how octopamine affects the brain. We investigated its effect in the antennal lobe, the first olfactory center in the brain, using calcium imaging to record background activity and odor responses before and after octopamine application. We show that octopamine increases background activity in olfactory output neurons, while reducing average calcium levels. Odor responses were modulated both upwards and downwards, with more odor response increases in glomeruli with negative or weak odor responses. Importantly, the octopamine effect was variable across glomeruli, odorants, odorant concentrations and animals, suggesting that the octopaminergic network is shaped by plasticity depending on an individual animal's history and possibly other factors. Using RNA interference, we show that the octopamine receptor AmOA1 (homolog of the Drosophila OAMB receptor) is involved in the octopamine effect. We propose a network model in which octopamine receptors are plastic in their density and located on a subpopulation of inhibitory neurons in a disinhibitory pathway. This would improve odor-coding of behaviorally relevant, previously experienced odors.

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca(2+) concentration ([Ca(2+)]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 μM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 μM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca(2+)]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca(2+)]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca(2+)]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

Background: Fluid secretion by sweat glands in response to heat and exercise is underpinned by increases in intracellular calcium. In horses, this is primarily via β2-adrenoceptors, but studies in equine sweat gland cell lines have indicated a possible role for purinergic agonists. Knowledge of equine sweating stimulus-secretion mechanisms in intact glands from healthy animals would allow future comparison to determine whether these mechanisms are affected in equine anhidrosis., Hypothesis/objectives: To determine whether purinergic agonists can induce changes in intracellular calcium in intact, freshly isolated equine sweat glands., Animals: Eleven healthy thoroughbred horses from the Hong Kong Jockey Club were used in this study., Methods: Freshly isolated equine sweat glands were loaded with the calcium-sensitive fluorescent dye fura-2 AM, and changes in intracellular calcium were recorded before, during and after stimulation by purinergic agonists., Results: Purinergic agonists ATP and UTP generated significant increases in intracellular calcium., Conclusions and Clinical Importance: The results show that it is possible to investigate stimulus-secretion coupling mechanisms by fluorescence imaging in equine sweat glands that have been isolated from fresh skin samples. Such isolated glands retain functional β2-adrenoceptors and P2Y purinergic receptors that couple to a calcium-signalling pathway. Using isolated, intact sweat glands therefore offers a very useful model for the further study of secretory processes in equine sweat glands, and using this experimental approach could facilitate a better understanding of how these mechanisms are affected in equine anhidrosis., (© 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.)

A microfluidic oxygenator is used to deliver constant oxygen to rodent brain slices, enabling the loading of the cell-permeant calcium indicator Fura-2/AM into cells of adult brain slices. When compared to traditional methods, our microfluidic oxygenator improves loading efficiency, measured by the number of loaded cells per unit area, for all tested age groups. Loading in slices from 1-year-old mice was achieved, which has not been possible with current bulk loading methods. This technique significantly expands the age range for which calcium studies are possible without cellular injection. This technique will facilitate opportunities for the study of calcium signaling of aging and long term stress related diseases. Moreover, it should be applicable to other membrane-permeant physiological indicator varieties., (Published by Elsevier B.V.)

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca(2+) concentration ([Ca(2+)](i)) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca(2+)-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca(2+) levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca(2+)](i). Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca(2+) transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca(2+)](i), in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Intracerebral hemorrhage (ICH) can lead to tragic disability and mortality. Accumulating evidence has shown that sodium calcium exchanger (NCX) may contribute to the secondary injury of a stroke. Recently, a novel member of NCX, SLC24A6, was discovered with knowledge of its abundant distribution in brain. In the present study, we examined the time course of expression of SLC24A6 and its mediated intracellular calcium concentration ([Ca(2+)]i) to investigate its potential roles in brain damage after ICH. An ICH model was established as previously reported. Real-time PCR and Western blotting were used to test the mRNA and protein levels of SLC24A6 on the hemorrhagic side and on the contralateral side caudate nucleus tissues at 6 h, and on days 1, 3, 5, and 7 after ICH. Immunohistochemistry was used to analyze the morphological changes. Fura-2/AM loaded, dual wavelength spectrophotofluorometry was used to test [Ca(2+)]i. The data presented a remarkable decrease in SLC24A6 early after ICH, along with a comparable increase in [Ca(2+)]i. Our results indicated that SLC24A6 presents specific and remarkable alterations in both mRNA and protein levels after ICH. Decreases in SLC24A6 level were correlated with [Ca(2+)]i elevation. These data suggest that SLC24A6-mediated calcium overload plays an important role in brain damage after ICH.

Although elevated levels of H(2)O(2) have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H(2)O(2) on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H(2)O(2)-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca(2+)](i) was measured on fura-2-loaded vessels. Perfusion of H(2)O(2) (10 μM) caused a delayed and progressively increased endothelial [Ca(2+)](i) and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H(2)O(2) induced two phases of NO production followed by caspase activation, intracellular Ca(2+) accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser(1177) phosphorylation in the absence of elevated endothelial [Ca(2+)](i), whereas the second phase of NO depended on increased [Ca(2+)](i) and was associated with Thr(495) dephosphorylation without increased Ser(1177) phosphorylation. Inhibition of NOS prevented H(2)O(2)-induced caspase activation, cell apoptosis, and increases in endothelial [Ca(2+)](i) and Lp. Our results indicate that H(2)O(2) at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca(2+) accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases.

Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000-different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand-receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the "odor space" of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.