Each transient Ca2+ concentration change was accompanied by a small coordinated movement of the cytoplasm that could be detected using PIV analysis.Microinjection of PLCζ cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca2+ oscillations, a critical parameter in activating human zygotes, can be monitored by PIV analysis of cytoplasmic movements. This simple method provides a novel, noninvasive approach to determine in real time the occurrence and frequency of Ca2+ oscillations in human zygotes.The occurrence and frequency of cytoplasmic Ca The combined application of fluorescent dyes and high-intensity excitation light is invasive and, when used at frequencies needed to assess Ca2+ oscillations, can cause damage to the oocyte, which then would interfere with later embryo development 2+ oscillations with dyes cannot be readily used in combination with longer term studies of embryo development. The invasive and predicted harmful effect of loading oocytes with fluorescent dyes also excludes their use in monitoring the success, or progress, of oocyte activation in any clinical IVF or ICSI treatments. However, it was previously reported that the first few Ca2+ transients during fertilization in mouse oocytes are also simultaneously accompanied by abrupt, diminutive movements in the ooplasm 2+ oscillations in the absence of fluorescent dyes 2+ transient in human oocytes that had failed to fertilize after ICSI. The method we describe may have potential as a non-invasive method for monitoring Ca2+ oscillation patterns in human oocytes.The Ca2+-sensitive dye, 0.5 mM Oregon Green BAPTA dextran, . This mixture was then microinjected using a micropipette (∼1-μM tip diameter). The micropipette was inserted into the oocyte using a brief pulse of electrical oscillation from an amplifier that was connected in line to the micropipette needle. A pressure pulse (∼1 second of 20 psi) was then applied to the back of the micropipette to push a bolus of the injection mixture into the oocyte. PLCζ cRNA was prepared as described elsewhere Human oocytes were donated by patients in the IVF Wales clinic at the University Hospital of Wales, Cardiff. The current project and all associated procedures have been approved by the South East Wales Research Ethics Committee and the Human Fertilisation and Embryology Authority (R0161 held by K.S. and N.A.). Aged “failed ICSI” oocytes and fresh oocytes derived from follicle reduction procedures were used. ICSI was performed under standard conditions, and the oocytes were cultured for a further 16–18 hours before they were judged to be fertilized or not. Fresh oocytes were derived from follicle reduction and were used within 5 hours of collection. Only those oocytes that were found to be not activated were used in experiments. During the subsequent 1–4 hours, these oocytes were transferred from the clinic to the research laboratory where they were microinjected with ∼0–20 pL of human PLCζ cRNA, as described elsewhere 2 CCD camera (Photometrics). The Lambda-10, image collection, and initial analysis were controlled using InVivo software (Media Cybernetics) and images stored as tiff stacks.The oocytes or zygotes were imaged for several hours after microinjection using a Nikon TiU epifluroescence microscope with a ×20 0.75 NA objective. Fluorescence excitation from a halogen lamp was passed through a 490-nm bandpass filter, reflected via a 505-nm dichroic, and collected with a 530-nm bandpass filter. White light from another halogen lamp was used to visualize the oocytes with differential interference phase contrast (DIC) microscopy. Shutters were placed in the path of these light sources, and filter wheels were in the path of both the fluorescence excitation and emission light such that oocytes were only briefly exposed to light during image acquisition. The shutters and filter wheels were controlled by a Lambda-10 controller (Sutter Instruments). Images were taken with 100- to 200-ms exposures in immediate succession every 10 seconds with a Coolsnap HQhttp://rsbweb.nih.gov/ij/), and the fluorescence intensity of the dye from the whole oocyte was plotted against time. The movements in the cytoplasm were analyzed with cross-correlation methods that had been developed for studying similar movements in mouse zygotes http://users.ox.ac.uk/∼zool0847/code.html.The fluorescence images were analyzed using ImageJ . It is noteworthy that the movements detected in the oocyte cytoplasm are relatively small in scale: the mean magnitude of all vectors at a speed peak never exceeded 40 nm/s, and within these speed peaks, local regions never moved faster than 120 nm/s. Even though in most cases there was a sudden increase in movement associated with a Ca2+ transient, it was also notable that the higher level of Ca2+ achieved in the first spike of some recordings was often accompanied by reduced movement in the cytoplasm  and are not related directly to activation events 2+ increases that occur repetitively in response to injection of sperm PLCζ cRNA.In this study we have shown that Ca2+ oscillations were also induced by the injection of PLCζ. This could be explained by the greater postovulatory age of the oocytes that had failed to fertilize after ICSI. Alternatively, although we did not verify sperm incorporation in our oocytes, the potential presence of a sperm after ICSI could also be important for effective observation of cytoplasmic movements in human oocytes.Previous work in mouse zygotes has demonstrated the role of myosin light chain kinase and the actin cytoskeleton in promoting the cytoplasmic movements 2+ concentration previously described in human oocytes after IVF or ICSI occur about once every 10–30 minutes 2+ spike frequencies that we observed in the initial period after injection in our previous and current experiments involving microinjecting PLCζ cRNA 2+ increases that are triggered by PLCζ being identical to those seen at fertilization in mouse, bovine, and pig oocytes 2+ oscillations that we have induced with PLCζ injection should be a close mimic of the corresponding pattern detected in normal fertilization.The repetitive spikes in Ca2+ spikes, will prove to be that which predicts the success rate of development of the zygote to the blastocyst stage in humans. In the mouse and rabbit, the number of Ca2+ spikes can influence the timing and degree of oocyte activation and later development 2+ oscillations in human embryo development. In possibly the only relevant study, oocytes that failed to fertilize after ICSI were subjected to one or three electroporation pulses to generate one or three Ca2+ transients 2+ pulses showed greater development to the blastocyst stage than those receiving one single Ca2+ pulse. This type of study has not been followed up, in part because of the technical constraints imposed by the requirement to measure Ca2+ in the same oocyte that is then assessed for embryo development. Our present approach of monitoring cytoplasmic movements with PIV offers a potential method that could be used in future for studies of such oocytes to relate specific changes  in the cytoplasmic Ca2+ profile to the success rate of human preimplantation embryo development.It remains unclear at present whether a single parameter, such as the total number of Ca2+ oscillations in mouse oocytes A recent study found that frequent imaging of the developing human embryo can be used to predict the ability of the zygote to develop to the blastocyst 2+ transients occurring in aged human oocytes that failed to fertilize after ICSI. This has the potential to be a simple and minimally invasive clinical method for assessing the occurrence of Ca2+ oscillations in such oocytes. Studies of mouse fertilization have suggested that the analysis of such cytoplasmic movements  may provide an early and effective indication of zygote viability after IVF The data in this study show that the temporal analysis of oocyte cytoplasmic movements can provide a directly correlative measure of the number and timing of Ca


BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD−ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated  Hairy cell leukaemia (HCL) is a rare B cell leukaemia with characteristic hair-like cytoplasmic projections on tumor cells, displaying distinctive activation markers FMC7, CD103, CD25 and CD11c Seminal studies have established that a functional BCR is essential for survival of normal B-cells, enabling response to cognate antigen or tonic, antigen-independent stimuli IGV) region genes The response of normal B-cells to antigen is stage and context dependent Whether the BCR plays a comparable role in sustaining survival in malignant B-cells is less well characterized and remains a central question in understanding origins and progression of mature B-cell neoplasms. A role for antigen recognition by the BCR in selection of the tumor clone or driving persistence via the BCR has been investigated in diverse B-cell tumors: it is clear that signalling via the BCR is implicated, as recent data from genome sequencing highlights abnormalities in key downstream pathways such as CARD11 in DLBCL, and constitutive activation of the PI3K pathway in lymphoma, or that the BCR itself is selectively modified to promote tumor: niche interactions, as in follicular lymphoma to potentially bypass a requirement for antigen Review, .AID expressed, implicating initial contact with antigen via the BCR BRAF V(600)E as almost universal in this tumor type IGHV4-34 genes, in which pathogenesis may differ BRAF V(600)E, ERK1/2 is constitutively activated and levels of phosphorylated ERK (p-ERK) are raised in HCL In mult-HCL, the functional role of BCR in primary tumor cells has not been examined to date. Most mult-HCL tumors exhibit SHM with a low level of on-going mutations and BRAF V(600)E and mutated IGHV genes. This study extends our preliminary observations on BCR function in HCL Here, we report on BCR function in mult-HCL, focusing on the role of individual isotypes and their relevance to tumor persistence, in cases uniformly displaying mutant Ethical approval was obtained from institutional bodies. The HCL samples were stored and provided by Professor H. Kluin-Nelemans, and have been approved for use by Ethical Review by the NHS Health Research Authority UK, NRES Committee South Central under REC M228/02/t. The HCL samples comprise an existing holding collected in 1980–1990 and for which consent is waived by the Human Tissue Authority.Hi/CD19+/CD22+/CD25+) 2. Unselected cases were analysed. Frozen splenocyte samples were thawed, washed, resuspended and allowed to stabilise prior to use  subpopulations in individual tumors Multiparameter flow cytometry (FC) with fluorochrome conjugated F(ab’)IGHV genes and the BRAF V(600)E mutation were identified as reported BRAF V(600)E mutations Tumor-derived 2+ sensor dye Fluo-3AM and labelled with anti-CD19/CD11c antibodies for FC, and stimulated with isotype specific or sIgL antibodies for tracing Ca2+ flux 2 anti-goat F(ab’)2 and a secondary antibody to detect remaining goat F(ab’)2 on the cell surface, or stained with rabbit F(ab’)2 anti- human Ig antibodies, as for immunophenotyping, to allow analysis of changes in paired IgH and IgL chains (detailed protocol is described in Two methods were employed: either cells were stained with a FITC-labeled rabbit F(ab’)2 anti-IgH or IgL, then incubated at 37°C, washed and loaded with fluo3-AM, followed by staining with α-CD19-V450 and α-CD11c-APC 2 α-human IgA, IgD, IgG, IgM, kappa or lambda antibody respectively or with immunobead-conjugated anti-human IgA, IgG or IgM coated immunobeads in medium for 2/3 hours at 37°C. Induction of apoptosis was measured using Apostat or Apo2.7-PE as detailed in Splenocytes were stimulated with soluble goat F(ab)2 α-human IgA, IgD, IgG, IgM, kappa or lambda at 37°C and proliferation assessed using a metabolite capture Cell Proliferation Kit II (XTT) (detailed protocol is described in HCL cells were stimulated with F(ab)+CD103+CD11cHi expression to gate specifically on HCs, and F(ab’)2 antibodies to exclude non-specific FcR binding. CD103 expression correlated tightly with CD11cHi expression (+CD11cHi FC gates to delineate tumor cells and exclude non-tumor B-cells (n = 10 mult-HCL cases). To confirm this, tumor cells obtained in 2/2 cases by flow sorting using CD19+CD103+CD11cHIgates achieved purities of 91.1–99.8%, and in these the tumor-derived IGHV marker gene was found in >98% of all clones examined in each case (data not shown). Splenic tumor burden ranged from 52–90% using these markers , IgG (95%), IgM (89%) and kappa (88%), and similarly, in IgD+ve Case 6 all cells expressed IgD/lambda with subpopulations expressing IgA (34%), IgG (62%), IgM (20%) and kappa (72%).In IgD−ve mult-HCL, tumor cells displayed IgG/M or IgG/M/A isotypes but clearly lacked surface IgD expression . In IgD−ve Case 5, 100% of cells expressed IgA/IgG/lambda, with subpopulations of cells co-expressing IgM (48%) or kappa (86%) respectively. IgD−ve Case 10 expressed IgG/lambda on 100% of cells, with subpopulations of cells co-expressing IgA (72%) and IgM (18%), respectively E mutation was verified in 10/10 cases , with clonally-derived multiple isotype switched transcripts in the tumor population  that fluoresce in the presence of free Ca2+. BCR mediated Ca2+ flux was examined in HCs gated by CD19+CD11cHi (7/10) or CD19+CD103+CD11cHi (3/10) expression. Initial studies indicated that anti-CD19/CD103/CD11c antibodies respectively did not induce Ca2+ flux (data not shown). All cases (10/10) revealed functional BCRs that transduced Ca2+ flux , invariably more than one sIgH isotype triggered Ca2+ flux , only signals via a single switched isotype (IgG/A) induced flux but not the co-expressed IgM  or IgG (case 5) or IgA (cases 10 and 11) , this does not account for lack of function in more highly co-expressed isotypes . These non-functional isotypes in IgD−ve cases are co-expressed at comparable levels to functional isotypes in IgD+ve cases. It may instead point to a selected feature that distinguishes the two mult-HCL subsets.BCR stimulation triggers tyrosine kinase phosphorylation events and activation of phospholipase C to release intracellular Caa2+ flux . Howevera2+ flux . In thes and 11) . While, 2+ flux observed. When IgD was co-expressed, it tended to trigger persistent responses accompanied by transient responses from the less prominent isotype(s)  , whereasent type . In norm2+ flux in mult-HCL were carried out in 6/6 IgD−ve and 4/4 IgD+ve cases, confirming observations in ‘biological’ replicates. Whether these patterns are consistently observed in each IgD subset awaits further study when larger cohorts are examined.Overall, measurements of Ca+ve mult-HCL Case 13 a low level of Ca2+ flux was observed via the second light chain κ in addition to the predominant λ response; in Case 5 and 6, this cationic flux was only induced by the λ chain 2 anti-goat F(ab’)2 secondary antibody  where, due to limited availability of material, a reduced analysis was performed. Endocytosis by anti-IgD stimulation resulted in a reduction in sIgD/sIgλ, and a loss of λ-induced Ca2+ flux, whereas the corresponding anti-λ mediated endocytosis resulted in reduction of sIgλ/sIgD expression and reduction in IgD induced Ca2+ flux (data not shown).Comparable patterns of BCR response were observed in IgD−ve mult-HCL Cases 2, 3 and 4 , endocytosis of sIgG and sIgκ followed BCR engagement with anti-sIgG, and endocytosis triggered via sIgκ reduced sIgG and sIgκ expression, but had no effect on levels of sIgM expression in 3/3 cases , stimulation via functional sIgG or light chain with soluble goat F(ab’)2 anti-human antibody induced significant levels of apoptosis as compared to dysfunctional sIg isotype, which were used as controls , an additional XTT based assay for proliferation showed no such effects when stimulated with functional anti-sIg antibodies -HCL case. Again, anti-sIgD stimuli did not trigger apoptosis E, that constitutively activates a critical component of the signaling cascade normally activated by a functional BCR. Such a study is a pre-requisite to address whether the BCR sustains tumor survival in-vivo in mult-HCL, and what the nature of the antigen might be that triggers BCR signals.This study addressed the intriguing question of a functional role for individual variant sIg isotypes in mult-HCL, in a background of mutant + tumor cells by CD103 and/or CD11cHi co-expression. Two functionally distinct mult-HCL sets emerged by immunophenotype differing by IgD co-expression (IgD+ve and IgD−ve), in which marked differences were apparent in isotype function. In IgD+ve cases, more than one sIgH isotype transduced Ca2+ flux with a persistent more robust pattern, whereas in IgD−ve cases only a single switched isotype signaled Ca2+ flux of a fast transient nature, despite the presence of other sIgH isotypes. Functional Ca2+ flux via specific functional sIgH isotype or sIgL associated tightly with the ability to endocytose BCR, substantiating the linkage of endocytic internalization of the BCR as dependent on functional signaling Variant isotype function on HCs was analyzed by a rigorous strategy that delineated CD19+ flux also associated closely with amplification of ERK1/2 phosphorylation in mult-HCL. These cases uniformly harbored a monoallelic BRAF V600E mutation, which associates with constitutive activation of ERK1/2 reported in HCL 599VKS602) induces kinase activity by conformational changes BRAF V(600)E, that either the wild type BRAF allele is being activated or that possibly alternative pathways are being recruited by BCR transduced signals to increase constitutive levels of ERK1/2 phosphorylation, as observed in this study. HCL therefore presents a model disease in which to dissect the molecular interplay between mutant B-RAF and wild type A-RAF and C-RAF in transducing BCR-mediated signals to the MAPK pathway. It also suggests that other receptor-ligand interactions have potential to augment phospho-ERK levels in HCs, as signalling conduits for specific tumor requirements.Functional sIg-mediated Ca2−ve mult-HCL dysfunctional sIgH isotypes were apparent, notably with sIgM which failed to transduce measurable intracellular signals and remained immobilized on the cell surface. It is possible that sIgM or other isotypes in the IgD−ve subset may not be expressed in a fully functional form. However, unless sIgM is fully assembled, it fails to reach the cell surface −ve cases, it would tend to argue that in the IgD−ve subset there appears to be a successive loss of function of co-expressed isotype. Whether IgD−ve cases result from loss of expression of IgD, with subsequent successive loss of function of individual isotypes, remains to be determined. We have previously suggested that individual variant isotypes in mult-HCL arise by a mechanism of differential RNA processing IgD co-expression correlated with a functional assembly of each variant isotype BCR on single HCs, suggesting that there are no intrinsic steric constraints to multiple sIgH isotypes packaging in the endoplasmic reticulum prior to surface expression. In contrast, in IgDBRAF are isotype specific, using both soluble and immobilised anti-sIgH stimuli, although due to sample availability of this rare leukemia the latter observations with immobilised ‘antigen’ were restricted, but nevertheless experimentally replicated. These data suggest that in IgD−ve mult-HCL, BCR transduced signals appear not to be relevant to tumor survival. The data also suggest that signals downstream of ERK1/2 phosphorylation bifurcate when initiated via the non-IgD BCR complex (apoptosis) or via BRAF V(600)E . They raise the intriguing possibility that acquisition of the BRAF V(600)E mutation may be a mechanism to by-pass any BCR signalling constraints in mult-HCL. Constitutively active BRAF V(600)E in HCL would also abrogate a requirement for ligand independent tonic signals via the BCR, as these signals are transduced via ERK1/2 phosphorylation BRAF V(600)E in HCL clearly mediates pro-survival tumor signals, as targeting the mutant protein with Vemurafenib (VEM) leads to clinical resolution of disease Notably, our data reveal that BCR mediated signals that lead to apoptosis in HCs carrying mutant −ve mult-HCL that lead to apoptosis may then be recapitulating AICD observed in B-cells activated via the BCR in the absence of T-cell help, and tend to support the contention that mult-HCL origins implicate a T-cell independent pathway. Whether co-stimuli such as TLR ligands can rescue the sIgM/G/A signalling cascade to apoptosis in mult-HCL remains to be established.BCR signaling in response to antigen in the absence of co-stimuli  is known to lead to activation-induced cell death (AICD) 2+ influx and activated tyrosine kinase signals in both, only anti-IgM antibodies induce cell death, indicative of differing downstream effector pathways in-vivo. Whether mult-HCL has entirely by-passed any requirements for BCR, possibly due to mutant BRAF constitutively activating the ERK1/2 pathway, remains at present speculative.In HCL, an opposing role of IgD as compared with other isotypes is clearly apparent as sIgD transduced stimuli abrogate pro-apoptotic signals from the BCR, revealing differences in signals initiated by sIgD as compared to other isotypes. They appear to parallel known divergence in IgD and IgM signals in normal B-cells, with the same antigenic specificity. IgD elicits a faster, stronger and more durable signal than IgM whereas only the latter is under control of a negative feedback loop in-vivo, although as with normal B-cells, it is as yet not clear whether specific sIgD signals are recruited for mult-HCL survival. In non-IgD expressing mult-HCL, the possibility exists that BCR signals that lead to apoptosis can be exploited to synergise with VEM activity to maximize potency in refractory disease. Targeting specific components of BCR signaling to ablate tumors is already at the clinical application stage in several B-cell malignancies Mult-HCL presents with unique features of multiple variant sIg isotypes, and in this first study of the individual isotypes in the BCR in this disease these isotypes emerge as differing functionally. Of these, sIgD appears to have a potential role in tumor survival and progression Figure S1Multiparameter flow cytometry to define immunophenotype of single tumor cells in mult-HCL. Normal PBMNCs and isolated spleen cells from HCL cases were immunophenotyped by gating on live cells and staining for CD19/CD11c/CD103 and sIgH/L surface expression. (A) Following FSC/SSC gating, CD19hiCD11chi cells (for 7/10 cases) or CD19hiCD11chiCD103+ cells (for 3/10 cases) were examined for sIg expression using rabbit F(ab’)2 anti-human Ig antibodies. Normal PBMNC are virtually devoid of B-cells that are dual CD11chiCD103+. (B) Representative example of immunophenotype of a mult-HCL tumor. IgD−ve mult-HCL Case 10 CD19hiCD11chiCD103+ cells display co-expression of sIgG and λ in 100% of HCL cells, with subsets of cells also sIgA (72%) and sIgM (18%) positive.(TIF)Click here for additional data file.Figure S2+ mult-HCL.Anti-sIg stimulation results in endocytosis of functional BCR in IgD Cells were stimulated with goat F(ab’)2 anti-Ig antibodies for 1 hour either at 37°C to allow endocytosis or at 4°C to prevent it. Changes in stimulated sIg expression levels were then measured by secondary staining using rabbit F(ab’)2 anti-goat F(ab)2 . Changes in expression of paired sIg were measured using rabbit F(ab’)2 anti-Ig . Loss of functional sIg was further confirmed by measuring induction of calcium flux following endocytosis (B). Using the anti-goat secondary antibody method  stimulation via sIgD, λ and sIgM in Case 13 as a representative example resulted in reduction of surface expression of these specific isotypes. Stimulation of non-functional sIgA in this tumor did not result in endocytosis . Using the rabbit F(ab’)2 staining method  stimulation of sIgD (left panels) in Case 13 resulted in loss of l, but not k light chain expression. Lambda stimulation, second column, resulted in loss of both surface IgD and IgM expression. IgM stimulation, third column, resulted in marginal loss of l but not κ surface expression. Inset of a Table of the sIg phenotype and functional sIg isotypes is included for reference purposes to evaluate data . The ability to induce Ca2+ flux (B) via IgD was markedly reduced following endocytosis by λ stimulation . Ability to induce Ca2+ flux via λ was ablated by IgD endocytosis and also greatly reduced by IgM endocytosis . Similarly, IgM induced flux was completely ablated following endocytosis following anti-λ stimulation .(TIF)Click here for additional data file.Figure S3Stimulation of BCR in mult-HCL triggers apoptosis not proliferation. Cells were stimulated with goat F(ab`)2 anti-Ig antibodies for 4 hours at 37°C and early apoptosis was measured in CD19+CD11c+CD103+ HCL cells using Apostat (left panels). Alternatively, XTT, a tetrazolium salt that is cleaved to formazan in metabolically active cells only was used to assess viability and proliferation following anti-BCR stimuli and OD assayed at 490 nm (right panels). In 2/2 HCL cases  ApoStat assays revealed significant increases in apoptosis in response to both functional heavy (IgG) and light /λ;HCL 10) chain stimulation. No apparent increase in numbers of metabolically active tumor cells was observed in response to BCR stimulation in either HCL case by XTT assay (right panels).(TIF)Click here for additional data file.File S1Contains supplementary materials and methods and Table S1.(DOCX)Click here for additional data file.


Mitochondria regulate cytosolic [Ca2+] over an exceptional breath of concentrations (∼200 nM to >10 μM) to provide a wide dynamic range in the control of Ca2+ signals. Ca2+ uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca2+ signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca2+ signaling in native smooth muscle.Mitochondrial Ca To enable the ion to modulate such a diversity of activities, there are a wealth of different types of Ca2+ signals . Ca2+ enters the cell from the extracellular fluid via channels on the plasma membrane such as the voltage-dependent and store-operated Ca2+ channels. The other main Ca2+ source is the SR store from which release proceeds via two receptor-controlled channels—the IP3R and the RyR 2+ are not independent; Ca2+ influx regulates Ca2+ release and Ca2+ release regulates Ca2+ influx. For example, Ca2+ release from the SR may alter plasma membrane ion channel activity to regulate the membrane potential and Ca2+ entry, whereas depletion of the SR of Ca2+ activates influx via store-operated Ca2+ channels 2+]c arising from the activity of channels in the plasma membrane or the SR will itself regulate ion channel activity to provide feedback control of Ca2+ signals.The two main sources of Ca2+ to act as either a highly localized signal or to evoke more widespread effects through the cell. For example, in sympathetic neurons, while muscarinic and bradykinin receptors each stimulate PLC to produce IP3, only bradykinin receptors co-immunoprecipitate with, and activate, IP3R to evoke Ca2+ release The strategic positioning of channels, receptors, and organelles is important in facilitating the operation of feedback processes. Various channels, receptors, and organelles combine to become functional units and enable Ca3R are positioned near the plasma membrane providing another mechanism for agonists, acting via IP3, to target specific cellular responses by generating Ca2+ rises in specific regions of the cell 3-mediated Ca2+ release events (referred to as “pulsars”) activate intermediate conductance, Ca2+-sensitive potassium channels, which co-localize to the same region, to hyperpolarize the endothelial membrane. The resultant membrane potential change is transmitted from the endothelium to the smooth muscle cells by coupling of the membrane via the projections. In this way localized IP3-mediated Ca2+ release in endothelial cells triggers relaxation in smooth muscle cells 2+ release events from RyR (Ca2+ sparks), may activate either Ca2+-activated K+ channels or Ca2+-activated Cl− channels or both to generate spontaneous transient outward (hyperpolarizing) or inward (depolarizing) currents on the plasma membrane 2+ release via RyR to activate or inhibit smooth muscle function. Another structural element to the organization of Ca2+ signals lies in the clustering surface receptors in certain regions on the plasma membrane. The clustering of surface receptors provides areas with increased sensitivity to extracellular stimuli 2+ signaling.Active IP2+ rise (a “Ca2+ wave”) occurs by positive feedback control of the Ca2+ rise and is another example of interaction among components, in this case to increase the reach of a local Ca2+ signal. In smooth muscle, local Ca2+ release from the SR may activate neighboring closely positioned ion channels on the SR to propagate the signal from site to site through the cell 2+ rise begins at precisely the same small site on each activation despite the cell being stimulated uniformly across the plasma membrane, indicating that is there is a preferred site of wave initiation 3R1 that are structurally and functionally coupled  and the SR membrane (IP3R) is one element of feedback control in the Ca2+ toolkit enabling receptors to generate differences in the signaling pathways subsequently activated Regenerative propagation of the Ca coupled  60. mACh2+ signals to contribute to the Ca2+ toolkit. Mitochondria may take up and sequester a large amount of Ca2+ from the cytoplasm and modulate the time course and amplitude of Ca2+ signals and shape the resulting message. The mitochondrial capacity to sequester Ca2+ is enormous and the buffer power c increases in aortic and arterial myocytes 2+ uptake did not alter caffeine-evoked SR Ca2+ release in cardiac or various smooth muscles 2+ transient arising from voltage-dependent Ca2+ entry has an accelerated rate of decline as a consequence of mitochondrial Ca2+ uptake and when the uptake is inhibited, the rate of Ca2+ decline is substantially slowed  2+]c range 200–600 nM, which demonstrates that mitochondria have a high affinity for Ca2+  near active voltage-dependent Ca2+ channels , Ca2+ puffs are inhibited 2+ (as Ca2+ puffs are >10 μM) and that Ca2+ uptake is fast enough to modulate concentrations near ion channels.In controlling Cachannels  or the a2+ from the cytoplasm , generate a larger [Ca2+]c rise? IP3R is regulated by Ca2+-dependent positive and negative feedback mechanisms. Mitochondrial Ca2+ uptake limits a negative feedback inhibition of Ca2+ on IP3R. There are at least two types of Ca2+-dependent negative feedback mechanisms, which may deactivate smooth muscle IP3R. In the first, a Ca2+-dependent deactivation of IP3R occurs at [Ca2+]c which exceed ∼300 nM 2+]c increase ends in permeabilized vascular smooth muscle 2+-dependent deactivation of IP3R, once initiated by an increased [Ca2+]c, persisted long (tens of seconds) after [Ca2+]c had regained resting values, that is became, at least partially, refractory 2+ release via IP3R. Mitochondrial Ca2+ uptake by buffering the Ca2+ rise at IP3R presumably prevents a persistent deactivation of IP3R to increase the overall release of Ca2+ . When mitochondria are prevented from taking up Ca2+, waves and oscillations halt  occur at all.The control that mitochondria exert on Caons halt , that is2+ signal from reaching certain regions of the cell. In pancreatic acinar cells, mitochondria are arranged as a “belt” and prevent Ca2+ signals from entering the basal part of the cell 2+ wave from progressing through the cell, but rather the organelle performs a decision-making role to determine whether or not the signal is permitted to progress. If the ΔΨM is normal and polarized, the Ca2+ signal progresses. On the other hand, if ΔΨM is depolarized  the mitochondria exerts control by inhibiting the local IP3-mediated Ca2+ signal so that the wave does not progress. Thus, as the Ca2+ signal regeneratively propagates from site to site through the cell (like a “fire-beacon” network), mitochondria act as “beacon-wardens” and determine whether or not the Ca2+ signal is transmitted to the next point of the cell 3R, but not voltage-dependent Ca2+ channel activity because the organelle is held close to the former, but not to the latter.In some cell types, mitochondria act as a “firewall” and prevent the Ca2+] range over which mitochondria are effective in regulating Ca2+ signaling prompted widespread interest into precisely how this is achieved. One mechanism is the MCU, a ruthenium red-sensitive, inwardly rectifying, and highly Ca2+ selective voltage-dependent channel of acknowledged importance in Ca2+ uptake by the organelle. In voltage-clamp experiments Ca2+ flux via MCU did not saturate until [Ca2+] exceeded 100 mM and the K1/2 was 19 mM 2+ increases near Ca2+ channels . Another proposed Ca2+ transporter present on the inner mitochondrial membrane is LetM1 2+/H+ exchanger that transports Ca2+ into mitochondria during low (<1 μM) global [Ca2+]c increases when the free mitochondrial matrix [Ca2+] is also low (∼5 μM) 2+ uptake by mitochondria. However, LetM1 was suggested to be a K+/H+ exchanger 2+ transporter. The Ca2+ transport facility attributed to LetM1 is not universally accepted The large  gradient. The [H+] gradient is significant; cytoplasmic pH is ∼7.2 while the pH within the mitochondrial matrix is ∼7.8. Indeed the effectiveness of complexes I, III, and IV in exporting H+ is emphasized by considering the average number of free protons within the matrix . A mitochondrial pH of 7.8 corresponds to a [H+] of ∼16 nM (1.58 E−8M). Taking the mitochondrion to be a prolate spheroid with dimensions of 2 μm (major axis) by 0.5 μm (minor axis) the volume (a is the minor axis radius and b the major axis radius) is 0.26 fL. 1 g-H+/L = 6.023 E23 ions/L so that a [H+] concentration of 1.58 E−8 M = 9.5 E15 ions/L (1.58 E−8 × 6.023 E23) and the number of H+ per mitochondrion = 9.5 E15 × 0.26 E−15= 2.5. Thus, on average there are only ∼2.5 H+ free within the mitochondrial matrix.Mitochondrial Ca+] gradient and ΔΨM provide the protomotive force (approximately −180 mV) to drive ADP phosphorylation . ATP production approximately doubles with each 10 mV increase in protomotive force 2+ ions is driven by ΔΨM. Unsurprisingly, a major method of determining the contribution of mitochondria to various cell activities  is to collapse the proton gradient using drugs such as protonophores and electron transport chain inhibitors. Protonophores  are mildly acidic lipophilic compounds that are deprotonated in the mitochondrial matrix to form lipophilic anions. The deprotonated form crosses the inner mitochondrial membrane from the matrix, picks up a proton on the cytoplasmic side, and returns. In this way protonophores collapse the proton gradient and ΔΨM and, as a result, inhibit ATP synthesis and mitochondrial Ca2+ uptake. For example, protonophores slow the rate of [Ca2+]c decline in smooth muscle  while cytoplasmic pH is ∼7.2 . The [H+] is thus highest in cytoplasm and lower in the extracellular space. However, the resting plasma membrane potential  may remain unaltered in the presence of protonophores. Because of its magnitude, the plasma membrane potential will determine the net flux of H+ and the concentration of H+ in the cytoplasm will increase via protonophore activity . A 60 mV (inside negative) membrane potential difference will result in ∼10-fold increase in cytoplasmic [H+] to 400 nM . Therefore, cytoplasmic pH will decrease from 7.2 to 6.4 when a protonophore is applied. Such a substantial decrease in pH is likely to exert several physiological changes and could result in a false-positive misinterpretation of the effects of protonophores on mitochondrial activity. A way around the pH change is to control cytoplasmic pH (in patch clamp experiments) using high concentrations of H+ buffers for example, 30 mM HEPES However, protonophores may have significant off target effects and care is required in interpreting data from these experiments. Protonophores incorporate into the plasma membrane as well as the inner mitochondrial membrane and by facilitating the flux of protons may substantially alter the cytoplasmic pH. The effect of protonophores may be substantial. Extracellular pH is ∼7.4  are fluorescent lipophilic cations that pass readily through lipid bilayers because their charge is dispersed over a large surface area. The mitochondria's negative membrane potential drives the fluorescent lipophilic cations into the matrix decrease the fluorescent signal. Conversely, when mitochondria are depolarized, fluorophore concentration will decrease and fluorescence emission will now increase because the lower fluorophore concentration results in unquenching  so that fluorescence emission is reduced. Under these conditions, when mitochondria are hyperpolarized more fluorophore will enter the organelle, but this will cause additional quenching to uenching B. Under uenching B.M of a few millivolts may be detected and imaging ΔΨM of single mitochondria is possible. The signal may also be quantified in unquench mode. The relationship between fluorescence and ΔΨM is described from the ratio of the free mitochondrial fluorophore concentration to free cytosolic fluorophore concentration and is an exponential function of ΔΨMMM.The response from the rhodamine fluorophores therefore depends on the concentration at which they are used, which are referred to as either “quenching” B or “unqM depolarization. Some consideration must also be given to the sampling frequency; transient changes in whole cell fluorescence in response to ΔΨM alterations may be missed with low frequency measurements.“Quench” mode has the advantage of producing larger signals (when compared with those in unquench mode) which are detected readily at a whole cell  level resulting in a more straightforward imaging procedure. The response of the fluorophore to membrane potential changes may be highly nonlinear in quench mode, however, and will show both increases and decreases in response to ΔΨAs with all agents that are introduced into cells, fluorophores may have some toxicity to mitochondria and cells. Problems can arise from “phototoxicity” when fluorescent molecules react with molecular oxygen to produce free radicals that may damage subcellular components. The lowest possible excitation light intensity should be used. The fluorophores themselves at high concentrations may also inhibit the electron transport chain so the lowest possible fluorophore concentration should be also used.2+] in native cells 2+ indicator, which bears a net positive charge. This net positive charge is suggested to promote the uptake of rhod-2 AM into the mitochondrial matrix because of the strongly negative ΔΨM. Once inside the mitochondrial matrix, esterases hydrolyse the AM group leaving the Ca2+-sensitive membrane impermeable form of rhod-2 trapped. However, while rhod-2 bears one net positive charge in the AM form, the de-esterfied rhod-2 bears three net negative charges and  is unlikely to accumulate in mitochondria. The balance of esterase activity in the cytoplasm and mitochondria will therefore determine the major source of the signal from rhod-2 and is likely to contribute to variations in results among studies.Rhod-2 have also been used to measure mitochondrial [CaM. In some studies JC-1 has been used successfully to measure ΔΨMM. Moreover, while the monomeric (green) form of JC-1 has been reported to equilibrate on a time scale similar to that of TMRM/TMRE, the aggregate (red) form of the dye takes six times longer to equilibrate M when none exist, perhaps because of the dye's sensitivity to H2O2. Notwithstanding, JC-1 has been used successfully in some studies to image change in ΔΨM.The other major class of fluorophore used commonly to measure the mitochondrial membrane potential is represented by JC-1. JC-1 is fluorescent and, like the rhodamine type fluorophores, accumulates in the mitochondria in a membrane potential dependent way. JC-1 has the useful feature of being a ratiometric indicator. In the cytoplasm JC-1 is usually “non-aggregated” (monomeric) and shows green fluorescence emission. When the concentration increases (in mitochondria) JC-1 aggregates and the fluorescence emission shifts from green to red. It is this fluorescence shift that permits ratiometric imaging of ΔΨM indicator, is the contribution that the plasma membrane potential makes to the signal measured from mitochondria, that is the fluorescence signal in the mitochondria is not independent of changes in plasma membrane potential. This situation arises because the concentration of fluorophore in mitochondria results from an equilibrium established by the concentration in the cytoplasm and the driving force for dye entry to mitochondria provided by ΔΨM. Significantly, the concentration of fluorophore in the cytoplasm is itself an equilibrium involving the extracellular dye concentration and the plasma membrane potential. The contribution of the plasma membrane potential is significant and there is a 10-fold increase in cytoplasmic dye concentration for a 60 mV inside negative membrane potential. Drugs or physiological activators that change the plasma membrane potential will alter the concentration of fluorophore in the cytoplasm. As a result, there will be a re-equilibration of fluorophore concentration in mitochondria with the new cytoplasmic concentration and a change in mitochondrial fluorescence even in the absence of a ΔΨM change. This change in fluorescence could result in a misinterpretation of an effect of a drug or activator as altering ΔΨM when no such change has occurred. Solutions are either to clamp the plasma membrane potential or to correct the mitochondrial signals after measuring alterations in the plasma membrane potential One additional issue that requires consideration, regardless of the ΔΨ2+ signals.Another significant consideration in the use of these drugs is an absence of spatial control of the organelles affected. One approach to overcome the lack of spatial control has been the development of photoactivatable forms of uncouplers M drive Ca2+ uptake into the organelle. In studying the role of mitochondria in Ca2+ signaling, the proton gradient and ΔΨM are both measured and manipulated experimentally. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the mitochondrial electrochemical gradient and are altered by changes in plasma membrane potential. The solution to the latter problem is either to clamp the plasma membrane voltage or to determine the contribution of plasma membrane voltage changes to the mitochondrial signal. In determining the contribution of mitochondria to the cytosolic Ca2+ signal a large number of drugs offer a potent mechanism to alter the organelle's ability to take up Ca2+. These drugs alter mitochondrial Ca2+ uptake by changing the proton gradient across the inner mitochondrial membrane but may have significant off target effects. The off target effects include cytosolic pH changes, ATP depletion and alterations in free radical production. However, each off target effect may be controlled experimentally so the drugs offer a powerful method to study the role of mitochondria in Ca2+ signaling.The mitochondrial proton gradient and ΔΨ


Arachidonic acid (AA) and its metabolites, prostaglandins (PG) are known to be involved in regulation of vascular homeostasis including vascular tone and vessel wall tension, but their potential role in Hypoxic pulmonary vasoconstriction (HPV) remains unclear. In this study, we examined the effects of AA and PGE2 on the hypoxic response in isolated rat intrapulmonary arteries (IPAs).We carried out the investigation on IPAs by vessel tension measurement. Isotetrandrine (20 µM) significantly inhibited phase I, phase IIb and phase IIc of hypoxic vasoconstriction. Both indomethacin (100 µM) and NS398 attenuated KPSS-induced vessel contraction and phase I, phase IIb and phase IIc of HPV, implying that COX-2 plays a primary role in the hypoxic response of rat IPAs. PGE2 alone caused a significant vasoconstriction in isolated rat IPAs. This constriction is mediated by EP4. Blockage of EP4 by L-161982 (1 µM) significantly inhibited phase I, phase IIb and phase IIc of hypoxic vasoconstriction. However, AH6809 (3 µM), an antagonist of EP1, EP2, EP3 and DP1 receptors, exerted no effect on KPSS or hypoxia induced vessel contraction. Increase of cellular cAMP by forskolin could significantly reduce KPSS-induced vessel contraction and abolish phase I, phase II b and phase II c of HPV.2+ channels.Our results demonstrated a vasoconstrictive effect of PGE2 on rat IPAs and this effect is via activation of EP4. Furthermore, our results suggest that intracellular cAMP plays dual roles in regulation of vascular tone, depending on the spatial distribution of cAMP and its coupling with EP receptor and Ca HPV is restricted to the segments of the vasculature perfusing the poorly ventilated (or hypoxic) part of the lung, thereby maintaining an appropriate ventilation/perfusion ratio. HPV has been demonstrated in isolated resistance pulmonary artery rings The normal pulmonary circulation is a low pressure and low resistance system with little or no resting vascular tone. Oxygen tension is a major mediator in determining pulmonary vascular tone. Unlike the systemic arterials which dilate in response to hypoxia, the pulmonary artery constricts when oxygen tension is lowered, a phenomenon known as hypoxic pulmonary vasoconstriction (HPV) 2+]i is a primary event in the contraction of PASMC. Ca2+ entry via voltage-gated Ca2+ channels (VGCC) 2+ channels 2+ release from the store via Ryanodine receptors in HPV remains controversial despite several previous reports suggesting that Ca2+ release may be essential to HPV Although the mechanism responsible for HPV has still not been fully elucidated, an increase of i2+]i elevation was composed of Ca2+ release from store and Ca2+ entry via store operated Ca2+ entry (SOC). Higher concentrations (10 µM) of PGF2α led to Ca2+ entry via L-type VGCCs and receptor operated Ca2+ channels as a result of TP receptor activation. Noticeably, the presence of varying concentrations of PGF2α may have influence on the hypoxic responses and thus may bias interpretation of our data, when experiments were performed to investigate effects of EPs. We therefore have performed experiments by using varying concentrations of KPSS (30–40 mM KCl) to adjust the baseline tone. In addition, vessel contraction elicited by 80 mM KPSS prior to and after application of chemicals was compared. The significant difference in KPSS response with or without tested chemicals indicates the modulation of VGCCs by applied chemicals.In order to examine the contributions of Ca2+-free bath medium abolished hypoxic vasoconstriction in the rat IPAs, confirming the key role of Ca2+ entry in the hypoxic response. 2-APB and SKF96365 were employed to identify the role of Ca2+ entry via voltage-independent channels in hypoxic vasoconstriction. They both exert an inhibitory effect on voltage independent Ca2+ influx. SKF-96365 not only inhibits voltage-independent Ca2+ entry e.g. TRP channel, but also attenuates voltage dependent Ca2+ entry, which is interpreted by its effect on decrease of 80 KPSS induced vasoconstriction, as vearapmil almost abolishes the 80 mM KPSS induced vasoconstriction. 2 APB as a membrane permeable SOCE inhibitor exerts no effect on KPSS induced contraction but attenuates hypoxic phase I and phase II constriction. In conclusion, SKF96365 works on both voltage dependent and voltage independent Ca2+ entry. 2APB in dosage (50 µM) has more specificity on SOCE. In addition to VGCC, voltage independent Ca2+ entry contributes to HPV. It is noticeable that SKF96365 exerted a similar inhibition effect in the phase I of HPV as 2-APB performed, but had less inhibition in the phase II of HPV than 2-APB did. It is well documented that phase II of HPV is endothelium dependent and voltage-independent Ca2+ channel is predominant in endothelium, suggesting that SKF96365 in compare with 2APB is less favour on voltage-independent Ca2+entry.Consistent with previous reports 2+ elevation in cultured human pulmonary artery smooth muscle cells via inhibition on voltage-independent Ca2+ entry A variety of lipids including AA and its metabolites have been demonstrated to activate and/or modulate diverse channels including K channels Two major isoforms of COX have been identified in rat pulmonary artery, namely COX-1 and COX-2. COX1 is constitutively expressed and is associated with the basal level of prostanoid. Its product PGH2 is converted to PGE2 by cytosolic PGEs (cPGEs). COX-2 is induced in response to inflammatory cytokines and mediators, resulting in increased and sustained prostanoid release. PGH2 produced by COX-2 is converted to PGE2 via microsomal PGEs (mPGEs). We found that the non-selective COX inhibitor indomethacin significantly inhibited hypoxic vasoconstriction in rat IPAs and this effect was mimicked by the COX-2 selective inhibitor NS398. However, DuP697 only inhibit phase I responses, suggesting that COX-2-mPGEs-PGE2 pathway plays a primary role in regulating rat pulmonary arterial tone during hypoxia at least in phase I. The difference derived from the two cox-2 inhibitors is likely due to NS398 binding within the cyclooxygenase channel of COX-2 in a similar fashion to indomethacin, but in a different position for other COX-2-selective inhibitors We further explored the prostanoid receptor subtypes that might be involved in hypoxic vasoconstriction in rat IPAs. We found that prostanoid receptor antagonists with efficacy on EP4 inhibited hypoxic vasoconstriction, whereas inhibitors of EP1-3 exerted no effect. AH6809 inhibited phase II in denuded IPAs, and it may be contributed by the lower pretone in the presence of AH6809. These results implicated that endogenous prostanoids, presumably PGE2 regulate vessel tone during hypoxia via interacting with EP4 receptor.2+ channel Activation of EP4 is reported to be coupled with an increase of cellular cAMP via Gs


Background: Pancreatic cancer cells exhibit up-regulated glycolysis.Results: Inhibition of glycolysis, but not mitochondrial metabolism, induced ATP depletion, plasma membrane calcium pump (PMCA) inhibition, Ca2+ overload, and cell death.Conclusion: Glycolytic ATP fuels the PMCA in pancreatic cancer.Significance: Glycolytic regulation of the PMCA may represent a novel chemotherapeutic target for pancreatic cancer. 2+ ([Ca2+]i), as the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) is critical for maintaining low [Ca2+]i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca2+]i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca2+]i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca Pancreatic ductal adenocarcinoma (PDAC)A hallmark of cancer cells, including PDAC, is a shift from predominantly mitochondrial metabolism toward glycolysis, even when oxygen is abundant  concentration  is an ATP-dependent process that is critical for cell survival. Of particular importance is the ubiquitously expressed plasma membrane Ca2+ ATPase (PMCA), an ATP-dependent pump with a high affinity for Ca2+ that extrudes cytosolic Ca2+ (+/Ca2+ exchanger (NCX) is either lacking or expressed with low abundance , inhibition of glycolysis induced severe ATP depletion, cytosolic Ca2 (95%:5%) at 37 °C. Cells were used up to passage 30 and then discarded.MIA PaCa-2 and PANC1 cells (ATCC) were grown in DMEM  in a humidified atmosphere of air/COm NaCl, 4.7 mm KCl, 1.28 mm CaCl2, 0.56 mm MgCl2, 5.5 mm glucose, 10 mm HEPES, pH 7.4). Rinse buffer was replaced with 4 μm fura-2 AM in 1 ml HEPES-PSS and incubated for 40 min at room temperature. Cells were then rinsed with HEPES-PSS followed by a further 20 min in dye-free HEPES-PSS to allow uncleaved dye to re-equilibrate. Fura-2-loaded cells were mounted onto imaging systems, and [Ca2+]i was measured as previously described  and a CoolSNAP HQ interline progressive-scan CCD camera . Both systems used a monochromator illumination system  and were controlled by MetaFluor image acquisition and analysis software . Cells were continually perfused with HEPES-PSS using a gravity-fed perfusion system (Harvard apparatus) and were excited at 340 and 380 nm (50-ms exposure). Emitted light was separated from excitation using a 400-nm dichroic with 505LP filter. Background-subtracted images of a field of view of cells were acquired every 5 s for both excitation wavelengths (340 and 380 nm). For all experiments, [Ca2+]i was measured as fura-2 340/380 nm fluorescence ratio. [Ca2+]i clearance was measured using an in situ [Ca2+]i clearance assay as previously described (2+ HEPES-PSS contained 1 mm EGTA. Experiments (between 5 and 32 cells) were performed at room temperature.Cells were seeded onto glass coverslips in a 6-well culture plate and grown to >30% confluency. To load cells with fura-2 dye, seeded coverslips were rinsed with HEPES-buffered physiological saline solution . Stocks of La3+, 3-bromopyruvate (BrPy), sodium iodoacetate (IAA), and ATP  were prepared in MilliQ water. Stocks of oligomycin (OM), carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and cyclopiazonic acid (CPA) were prepared in DMSO. Fura-2 AM  was prepared in 50:50 DMSO (Sigma), and 0.1% Pluronic® F-127 . Working solutions in HEPES-PSS were prepared from frozen stocks immediately before an experiment. Reagents were prepared in media for the ATP and cell death experiments.Nam), BrPy (100–1000 μm), or a combination  for 0.5–6 h. Cells were then stained with propidium iodide  and Hoechst 33342 . PI-positive cell count was normalized to Hoechst 33342-positive cell count (%). Assays were run in duplicate on a Thermo Fisher Scientific Cellomics® ArrayScan® VTI HCS Reader fitted with Hamamatsu ORCAR-ER camera and both ×10 and ×20 objectives. Imaging was performed by Imagen Biotech Ltd. Image analysis was assessed using the Compartmental Analysis Bioapplication (Thermo Fisher Scientific) whereby a threshold gate was set (∼500 levels of gray) along an intensity histogram plot to distinguish positive PI fluorescent cells (∼5000 levels of gray) from background noise (∼10 levels of gray).PANC1 cells were seeded into black-walled, clear-bottom 96-well plates at 70% confluency and allowed to adhere overnight. Cells were then incubated in culture conditions with blank media or ascending concentrations of CCCP (1–10 μ5 cells/ml) and allowed to adhere overnight. Cells were then treated with varying concentrations of metabolic inhibitors and incubated for 15 min in culture conditions. Each experiment included untreated cells (control), which represented total ATP, and a positive control with cells treated with an ATP depletion mixture  to achieve maximal ATP depletion. After treatment, cells were lysed, and ATP was determined using the luciferase-based ViaLight® Plus kit . Luminescence was measured using a Synergy HT multiwell reader (BioTEK). Each experiment was run in duplicate; the luminescence counts of each duplicate pair were averaged. To correct for background luminescence under conditions of maximal ATP depletion, the averaged luminescence count from the ATP depletion mixture duplicates was subtracted from all other values. These values were normalized to the corresponding control (%).Cultured MIA PaCa-2 and PANC1 cells were seeded into white-walled, clear-bottom 96-well plates , and MIA PaCa-2 cells (n = 25 cells). Once [Ca2+]i reached a minimum (Rmin), cells were perfused with 20 mm Ca2+ to induce a maximum increase in [Ca2+]i (Rmax). Fura-2 ratios were then calibrated to determine [Ca2+]i as previously described , and the second rate was normalized to the first. Values were averaged for all cells in an experiment, and the resulting experimental means for each condition were averaged to give the presented group means ± S.E.Repeated measurements of [Ca2+]i were quantified by measuring the area under the curve (AUC) and the maximum increase in [Ca2+]i over 30 min and compared using an unpaired Student's t test. Cell viability was quantified as the percentage of cells responding to 100 μm ATP and compared using a Mann-Whitney U test. Changes in [Ca2+]i clearance rate were compared using a Mann-Whitney U test. ATP depletion was assessed using a one-sample t test against the hypothetical control value of 100%. Presented data are the means ± S.E. of the indicated number (n) of independent experiments.Cell death was statistically assessed using a two-way analysis of variance with a post hoc Bonferroni correction. Changes in resting i responses, thereby testing for reversibility of responses and thus viability.We next tested the effects of glycolytic and mitochondrial inhibitors on resting i overload, with an average maximum increase in [Ca2+]i of 531 ± 32 nm in MIA PaCa-2 cells (n = 5) and 425 ± 134 nm in PANC1 cells . Similarly, treatment with 2 mm IAA induced an average maximum increase in [Ca2+]i of 261 ± 29 nm in MIA PaCa-2 cells (n = 7) and 160 ± 26 nm in PANC1 cells . These responses were significant compared with corresponding time-matched control cells for both MIA PaCa-2  and PANC1 cells .Treatment with BrPy  increased the AUC to 485 ± 104 μm·s in MIA PaCa-2 cells and 399 ± 86 μm·s in PANC1 cells, whereas IAA (2 mm) elevated the AUC to 421 ± 44 μm·s in MIA PaCa-2 cells and 213 ± 28 μm·s in PANC1 cells (E). These responses were significantly elevated compared with control MIA PaCa-2  and PANC1 cells . AUC represents a measure of not only the magnitude of [Ca2+]i increase but also the recovery of response.Similarly, BrPy  and CCCP  had no effect on the maximum increase in [Ca2+]i observed in either MIA PaCa-2 cells  or PANC1 cells . Interestingly, CCCP elicited a small but significant increase in AUC to 128 ± 16 μm·s (n = 5) in MIA PaCa-2 cells  but not in PANC1 cells . Despite this, OM had no effect on AUC in either MIA PaCa-2 or PANC1 cells .In contrast, OM 10 μm, A and CCCCP 4 μm, C had no 2+]i, BrPy (500 μm) and IAA (2 mm) abolished the ability of both cell lines to elicit Ca2+ responses to 100 μm ATP . In healthy cells, ATP evoked robust spike-like increases in [Ca2+]i, indicating that these cells were viable. In contrast to the glycolytic inhibitors, ATP-evoked Ca2+ responses were observed in the majority of cells treated with the mitochondrial inhibitors OM  or CCCP . Together these results suggest that glycolysis, but not mitochondrial metabolism, is critically important for maintaining a low resting [Ca2+]i in human PDAC cells.In addition to their effects on i clearance assay in pancreatic acinar cells in which PMCA activity is pharmacologically and functionally isolated (2+ ATPase (SERCA) activity was blocked by continuous application of CPA in the absence of extracellular Ca2+ and the presence of 1 mm EGTA, leading to endoplasmic reticulum Ca2+ store depletion. Rapid store-operated Ca2+ entry was then induced upon perfusion with 20 mm Ca2+. The subsequent removal of extracellular Ca2+ resulted in clearance of [Ca2+]i, presumably via the PMCA. This influx-clearance phase was repeated, and test reagents or maneuvers were applied during the second influx-clearance phase. Thus, the resulting paired experimental design controlled for cell-to-cell and temporal variability.We previously developed an isolated , 18. Bri2+]i clearance can be quantified either by fitting the falling phase to a single exponential decay or by measuring the initial linear clearance rate from a standardized [Ca2+]i value. However, fitting to an exponential decay is only valid if the [Ca2+]i clears to approximately the same base-line [Ca2+]i (asymptote) and is not accurate if clearance approaches a slow linear phase. This was almost always the case when clearance was severely inhibited. We, therefore, normalized the linear clearance rate over 60 s during the second clearance phase to that of the first (%), both measured from the same [Ca2+]i value. Before assessing the effects of metabolic inhibitors on [Ca2+]i clearance during our in situ [Ca2+]i clearance assay, control experiments revealed that clearance rate was maintained between sequential influx-clearance phases in untreated PANC1  and MIA PaCa-2 cells .[Ca2+]i clearance in pancreatic acinar cells . NMDG is not transported by NCX yet maintains the osmotic balance, thereby removing the Na+ gradient required for NCX-mediated Ca2+ efflux.Although the NCX is not thought to contribute to i clearance in PANC1  or MIA PaCa-2 cells  compared with control cells . These data indicate that NCX does not contribute to [Ca2+]i clearance in these cell lines under the current experimental conditions.Na2+]i clearance in PANC1 and MIA PaCa-2 cells during the in situ [Ca2+]i clearance assay, 1 mm La3+ was applied during the second clearance phase. La3+ inhibits Ca2+ efflux (PMCA) at millimolar concentrations  and 18 ± 5% in MIA PaCa-2 cells  compared with 100 ± 3% (n = 18) and 98 ± 5% (n = 17) in corresponding time-matched control cells, respectively .Furthermore, to test whether the PMCA is responsible for i clearance in these cells under these conditions. Any inhibition of [Ca2+]i clearance observed using this experimental design can, therefore, be interpreted as an effect on PMCA activity, consistent with our previous studies  and BrPy (500 μm)) or mitochondrial inhibitors (OM (10 μm) and CCCP (4 μm)) during the second Ca2+ clearance phase of our in situ [Ca2+]i clearance assay. In PANC1 cells, both IAA and BrPy markedly decreased PMCA activity to 57 ± 4%  and 63 ± 5% , respectively, compared with time-matched control cells . In contrast, the mitochondrial inhibitors CCCP and OM had no effect .After establishing that inhibition of glycolysis results in [Cam) and BrPy (500 μm) also inhibited PMCA activity. We first attempted to repeat the [Ca2+]i clearance experiments in MIA PaCa-2 cells using an identical protocol to that used for PANC1 cells. BrPy  and IAA  both significantly decreased PMCA activity to 10 ± 10% (n = 4) and 23 ± 10% , respectively, compared with the corresponding time-matched control cells . Interestingly, CCCP decreased PMCA activity to 58 ± 6% ; OM on the other hand had no effect . However, application of IAA and BrPy before Ca2+ influx resulted in inhibition of Ca2+ entry and dramatically reduced the plateau from which [Ca2+]i clearance was initiated and subsequently measured (B and D). As a result, [Ca2+]i clearance could only be measured over a very narrow range of Ca2+ concentrations. Furthermore, the slowed Ca2+ influx meant it took longer to achieve a sufficient increase in [Ca2+]i from which to measure [Ca2+]i clearance rate. Using the current protocol where metabolic inhibitors were applied before Ca2+influx, this resulted in prolonged exposure (>15 min) to the glycolytic inhibitors before initiation of [Ca2+]i clearance. These confounding factors cast doubt on the validity of the [Ca2+]i clearance data obtained from MIA PaCa-2 cells with the current experimental design.Similarly, in MIA PaCa-2 cells, IAA . Similar to PANC1 cells, BrPy  and IAA  both significantly decreased PMCA activity to 25 ± 8%  and 40 ± 6% , respectively, compared with corresponding time-matched control cells . In contrast, 10 μm OM had no effect on [Ca2+]i clearance . Interestingly, however, CCCP caused a significant decrease in [Ca2+]i clearance .Similar to previous experiments, i after each treatment. To measure recovery, the difference was calculated between the fura-2 ratio upon removal of La3+ during the second influx-clearance phase and the minimum value the steady state plateau subsequently reached. This was then normalized to the difference between the fura-2 ratio upon removal of La3+ during the second influx-clearance phase and the lowest fura-2 ratio value observed before the first influx-clearance phase. BrPy  and IAA  both significantly inhibited recovery to 45 ± 8% (n = 6) and 73 ± 3% (n = 6), respectively, compared with 107 ± 4% for the corresponding time-matched control cells . In contrast, after treatment with the mitochondrial inhibitors, cells were able to fully recover [Ca2+]i to base-line values; neither OM  nor CCCP  had any effect on recovery compared with corresponding control cells. Taken together, these data suggest that glycolysis, but not mitochondrial metabolism, is critically important for both maintaining PMCA [Ca2+]i clearance rate and for ensuring full recovery to a low resting [Ca2+]i.We, therefore, also assessed the degree of recovery to base-line i clearance experiments. Moreover, 15-min incubation was chosen because it was sufficient to induce [Ca2+]i overload and inhibition of the PMCA during our in situ [Ca2+]i clearance assays. Cells were also treated with a combination of all four metabolic inhibitors to induce maximum ATP depletion . Raw luminescence counts obtained from those cells treated with the ATP depletion mixture were subtracted from each individual treatment before normalization to the luminescence counts of untreated control cells (%). On average, the raw luminescence count in control cells was 37,994 ± 5,976 in PANC-1 (n = 7) and 56,974 ± 10,865 in MIA PaCa-2 cells (n = 8) and in those treated with the ATP depletion mixture was 2,923 ± 519 in PANC-1 and 4,036 ± 1,032 in MIA PaCa-2 cells. Thus, the ATP depletion mixture reduced global ATP to 8 ± 1% in PANC-1 cells and 9 ± 3% in MIA PaCa-2 cells over 15 min. All statistical comparisons were made using a one-sample t test against the hypothetical control value of 100%.To assess the effects of glycolytic and mitochondrial inhibitors on ATP depletion, cells were treated with varying concentrations of BrPy, CCCP, IAA, or OM for 15 min before the addition of ViaLight® Plus ATP kit assay reagents . These concentrations were chosen because they were below, equal to, and higher than those used in the i clearance experiments; 10 μm CCCP caused a significant decrease in ATP in both MIA PaCa-2  and PANC1 cells , whereas 30 μm OM only caused a significant decrease in ATP in PANC1 cells .The glycolytic inhibitors BrPy and IAA caused a profound decrease in ATP in both MIA PaCa-2 and PANC1 cells. IAA caused a significant decrease in ATP at all three concentrations in both MIA PaCa-2  within minutes and ultimately caused cell death within 3–6 h. Inhibition of mitochondrial metabolism, on the other hand, had almost no effect on resting [Ca2+]i, PMCA activity, ATP depletion, or cell death. This is at variance with studies in non-cancerous pancreatic duct cells and the related pancreatic acinar cells, in which separate inhibition of either glycolysis or mitochondrial metabolism induced ATP depletion , [Ca2+]i will recover so long as the PMCA remains active. On the other hand, an inability to extrude [Ca2+]i via the PMCA would render [Ca2+]i overload an inevitability, as the endoplasmic reticulum capacity is finite. This leaves the PMCA as the “final gatekeeper” for the control of low resting [Ca2+]i in PDAC. Given that non-cancerous cells have a greater reliance on mitochondrial ATP production, targeting the glycolytic regulation of the PMCA may be an effective strategy to selectively kill PDAC cells while leaving non-cancerous cells unharmed. Furthermore, this may be relevant to other similar cancers where the PMCA is the major Ca2+ efflux pathway.The present study is the first to show that in human PDAC cell lines (PANC1 and MIA PaCa-2) both i , 39, andIt has previously been described that PDAC cells exhibit the highly glycolytic phenotype characteristic of the Warburg effect , 40. IndATP depletion is well known to drive necrosis , and inh2+]i overload. A sustained increase in [Ca2+]i is catastrophic for cells as it leads to the inappropriate activation of cytosolic enzymes such as proteases, phospholipases, and nucleases; it has long been known that a prolonged elevation in [Ca2+]i has cytotoxic and pathological effects , we are presented with a conundrum with respect to the sequence of events, as either could be expected to promote cell death. Furthermore, in addition to PMCA inhibition, ATP depletion would also be expected to inhibit the Na+/K+ ATPase. The Na+/K+ ATPase consumes a large fraction (>20%) of intracellular ATP to maintain the resting membrane potential of the cell  affinity for ATP  suggests that ATP depletion-induced inhibition of the PMCA impairs the maintenance of resting [Cafunction . This haion rate . [Ca2+]i pipette . Collect2+]i homeostasis in PDAC selectively.In light of these findings, it is tempting to speculate that glycolytic regulation of the PMCA may be an important pro-survival mechanism in PDAC tumors. Previous experiments using human erythrocyte membranes suggest that glycolytic enzymes associate with the plasma membrane  and that2+]i in human PDAC cell lines. Furthermore, these findings are translational and provide insights into a potentially new therapeutic avenue for the treatment of PDAC. Although targeting the glycolytic regulation of the PMCA in PDAC alone may be insufficient to eradicate the cancer unless part of a combination chemotherapy regimen, the link between the Warburg effect and [Ca2+]i homeostasis is an avenue previously unexplored. Moreover, glycolytic regulation of the PMCA might represent a critical pro-survival phenotype in other cancer types, particularly those derived from cells that rely on the PMCA as the major Ca2+ efflux pathway. Targeting key glycolytic enzymes that are up-regulated or uniquely expressed in highly glycolytic tumors such as PDAC could result in selective PMCA inhibition, thereby inducing [Ca2+]i overload-induced cell death in the tumor while sparing adjacent non-cancerous cells.Collectively the present study suggests that glycolytic ATP synthesis is critically important for maintaining PMCA activity and a low resting [Ca


We characterized the activation of the TMEM16A-encoded CaCC  by Ca2+, Sr2+, and Ba2+, and discovered that Mg2+ competes with Ca2+ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca CaCCs can be reversibly inhibited by inhibitors/blockers such as niflumic acid (NFA) The CaCC channel molecules encoded by TMEM16A and TMEM16B are also called anoctamin 1 (ANO1) and anoctomin 2 (ANO2), respectively 2+ activation, the degree of current rectification, or even the calculated permeability ratios of various anions. From the literature it can be found that the apparent affinity of Ca2+ from dose-dependent activation curves of CaCCs varies significantly, and this large variation cannot be fully explained by alternative splicing of the TMEM16A protein 2+ sensitivities among various alternatively spliced variants of TMEM16A differed by ∼4–6 fold 2+  in the literature ranged from <100 nM While CaCCs consist of well-defined functional properties, a technical complication is frequently encountered in studying these channels–the “rundown” or “desensitization” of CaCCs. The rundown of CaCCs could affect the apparent affinity of Ca2+ solution to a solution containing specified Ca2+ concentrations ([Ca2+]). We circumvented the problem of channel rundown by normalizing the Ca2+-induced current to the maximally-activated current obtained within a time period in which the channel rundown is negligible. We found that two other divalent cations, Sr2+ and Ba2+, can activate ANO1 to the same level as that activated by the saturating [Ca2+]. On the other hand, Mg2+ cannot induce ANO1 current, but it appears that Mg2+ can bind to the divalent cation binding site(s) to antagonize channel activation by Ca2+. We also discovered that the apparent affinity of a well-known CaCC blocker, NFA, was inversely related to the occupancy of anions in the pore of ANO1. On the other hand, the NFA block was not affected by the degree of channel activation. The mechanism of the NFA block of ANO1 is not well understood. Our experiments revealed functional properties that may help to unravel the mechanism for the NFA inhibition on the ANO1 channel.In this study we employed a fast solution exchange method to induce ANO1 current upon switching the intracellular solution from a zero-Ca2+ activation was changed in the process of rundown. 2+] (I666 nM) bracketed by maximal currents (Is) induced by 20 µM [Ca2+], a saturating [Ca2+]. A repeated trace recorded 8 minutes later is shown in s’s within the same recording sweep did not show a significant difference, but the amplitude of Is was reduced between sweeps separated by minutes because of the current rundown. s of each sweep to that of the initial current sweep (Is(0)). The ratio I666 nM/Is from each sweep was also calculated as a measure of the sensitivity of ANO1 to Ca2+ activation. The results showed that ANO1 revealed a prominent current rundown, with ∼50% current attenuation after 8 minutes. However, the sensitivity of ANO1 to Ca2+ activation remained the same throughout the 8-min recording period as indicated by the unchanged ratio of I666 nM/Is  of 466 nM and 588 nM at +40 mV and −40 mV, respectively, and a Hill coefficient (nCa) of ∼4. We further performed the experiments using Ba2+  of the channel current under bi-ionic conditions. Two different approaches were employed to determine the Erev. First, the Erev of I-V curves from voltage-clamp recordings were measured (rev of the membrane current) when the intracellular Cl− was replaced by other anions . After junction potential correction, the calculated permeability ratios (PX/PCl) were ∼11, ∼4.1, ∼3.5, and ∼2.1 for SCN−, I−, NO3−, or Br−, respectively. On the other hand, Erev = −27.6±2.4 mV  when HCO3− was the major intracellular anion  recording approach under bi-ionic conditions . Because the measurement of Erev could be imprecise if the amplitude of the current varies from patch to patch, we further compared the Erev’s of the Ca2+-activated and the Sr2+-activated current from the same patch, using SCN− as the intracellular anion  or at ±20 mV (right three panels). The averaged dose-dependent NFA inhibitions at these four different voltages are plotted in 1/2) of NFA were in the range of 18–20 µM and were not voltage dependent between −40 mV and +40 mV  while the pipette solution contained 140 mM [Cl−]. At +20 mV (red traces), an inward current was observed in the I– or SCN–containing solution because the Erev values in these two intracellular solutions were ∼+35 mV and ∼+60 mV, respectively : 17.9±1.1, 39.2±1.1, 54.8±3.4, 85.5±3.6, and 189.4±7.4, respectively  in ANO1. This finding may be physiologically pertinent because K1/2,Ca is affected by millimolar [Mg2+], a physiological relevant concentration. It should also be pointed out that many experiments in the literature included millimolar [Mg2+] in the intracellular solution, and these studies usually reported values of K1/2,Ca in the µM range 2+ competes with Ca2+ for binding to the activation sites could explain the higher values of K1/2,Ca reported in these previous experiments.In contrast to Ca2+ cannot induce ANO1 current is probably due to a failure of coupling the Mg2+-binding to the opening of the channel. This situation is similar to the major divalent activation sites (with µM Ca2+ affinity) in the large conductance Ca2+-activated K+ channel (or BK channel) 2+ and Ba2+ can bind and activate the BK channel while Mg2+ binds and prevents the activation of the channel by other divalent cations 2+ can bind and activate the channel 2+ activation of the BK channel was thought to be due to a modulation of the voltage-dependent BK channel activation 2+ in the intracellular side. The recorded current in the absence of Ca2+ at ±40 mV was small and was not proportional to the amplitude of the saturated Ca2+-activated current  and the half-activating concentrations of the intracellular blockers were much higher than those of the extracellular blockers, it was concluded that A9C and DPC blocked the Xenopus oocyte CaCC from the extracellular side. On the other hand, it was more difficult to determine the sideness of the NFA block in this early study because NFA blocked CaCC of Xenopus oocytes from intracellular and extracellular sides with similar affinities 2+ application  encoding ANO1 CaCC was a generous gift from Dr. Lily Jan. We subcloned the coding region of the cDNA into pEGFP-N3 vector , resulting in a construct in which an enhanced green fluorescent protein (eGFP) was attached to the C-terminus of the ANO1 protein. The sequence was confirmed by commercially-available DNA sequencing service. This eGFP-attached ANO1 cDNA construct was used for channel expression throughout the study. The cDNA was transfected into human embryonic kidney (HEK) 293 cells using lipofectamine transfection methods 2, SrCl2, BaCl2, MgCl2 or sodium salts of various anions) were purchased from Fisher Chemicals , J. T. Baker  or Sigma Chemicals Co. . EGTA, HEPES, and NFA were from Sigma Chemicals Co. Excised inside-out patch-clamp recordings were performed throughout the study. The extracellular (pipette) solution in all experiments contained: 140 mM NaCl, 10 mM HEPES, 0.1 mM EGTA, pH = 7.4. The regular intracellular solution contained 10 mM HEPES and 140 mM sodium salt of Cl− or other anions. Various concentrations of divalent cations were made by adding certain total concentrations of divalent cations  into the solution containing a defined concentration of EGTA. We defined the pipette solution as the “zero-Ca2+” solution. A solution containing a saturating [Ca2+] was made by adding 0.12 mM total [Ca2+] into the aforementioned zero-Ca2+ solution, resulting in a solution containing ∼20 µM free [Ca2+]. For making non-saturating [Ca2+] solutions, the solutions included 1 mM EGTA. To make solutions containing Sr2+ and Ba2+, only 0.1 mM EGTA was used. The pH of all solutions was adjusted to 7.4.All chemicals were reagent grade. Regular salts  kindly provided by Chris Patton from the Stanford University. This program was developed based on the binding constants available in the National Institute of Standard and Technology (NIST) between EGTA and these three divalent cations. 2+], [Sr2+], and [Ba2+] used in this study. All added divalent cations were Cl− salts. When the added salts resulted in an increase of [Cl−] larger than 2 mM, [Cl−] was adjusted by reducing the added amount of NaCl. For example, in a solution containing 5 mM, 10 mM or 30 mM MgCl2, only 130 mM, 120 mM or 80 mM of NaCl, respectively, were added to the solution.Because of weak MgThe patch-clamp recording pipettes were fabricated from borosilicate glass capillaries (World Precision Instruments) using PP830 electrode puller . When filled with the pipette solution, the electrode resistance was ∼1.5–3 MΩ. The experiments were carried out at room temperature (20–23°C) using the Axopatch 200B amplifier controlled by the Digidata I/O converter and the pClamp9 software (Axon Instruments/Molecular Devices).2+ solution. The membrane potential was then clamped to ±40 mV (or ±20 mV in some experiments) followed by a switch of the solution to a different solution containing certain free [Ca2+]. 2+-containing solution three times: two with a solution containing the saturating [Ca2+] (20 µM free Ca2+) and one with a solution containing a specific [Ca2+] (0.666 µM). The current rundown within one recording sweep of <20 s was normally negligible (see the time course of the current rundown in s) obtained in every recording sweep was normalized to the Is of the initial recording sweep. To evaluate the sensitivity of the channel to divalent cations, the amplitude of the current induced by the activation ligand was measured and normalized to the averaged value of the two Is’s in the same sweep (see Solution exchange was achieved using the SF-77 solution exchanger (Warner Instruments). After the excised inside-out patch mode was established, the tip of the patch pipette was positioned at the exit of the solution-delivery pipes of the SF-77 solution exchanger. The switch of solution pipes was controlled by a digital signal delivered from the Digidata 1320 digitizing board. The time required for the motor moving the solution-pipes to respond, the time for the solution junction passing the patch, and the time for the ligand to diffuse into the Ω-shaped inside-out patch added up to tens of ms (sometimes 100–200 ms) as the dead time before the ligand effect could be observed . The recweep see . The nor2+ solution). The intracellular solution contained 140 mM sodium salts of various anions with the exception of the HCO3− experiment in which the solution contained 130 mM NaHCO3 and 10 mM NaCl. In all intracellular solutions for the bi-ionic experiments, 0.1 mM EGTA and 0.12 mM total Ca2+ or Sr2+ were added to the solution to activate the ANO1 channel (Free [Ca2+] or [Sr2+] were saturating concentrations). Erev of the open channel was measured in two ways. First, under voltage-clamp configuration, a 3-sec voltage ramp from −80 mV to +80 mV was applied and the Erev of the leak-subtracted I-V curve was then determined  was normally close to 0 mV. When the intracellular solution was replaced by the one containing other anions, the value of EI = 0 changed to a new value corresponding to the Erev of the new ionic condition. It is important to point out that the measurement of Erev can be imprecise if the amplitude of the ANO1 current is not within a proper range. Therefore, in permeability ratio measurements we only used the data obtained from patches with a maximal current in the range of 100 pA–1000 pA at +40 mV. All the presented Erev values (and the recording traces for measuring Erev) are not corrected for junction potential difference between solutions because the junction potentials are relatively small. However, the reported permeability ratios were calculated based on the junction potential-corrected Erev. The correction for the junction potential results in very small changes in the calculated permeability ratios.The anion permeability of the ANO1 pore was examined under bi-ionic conditions in which the extracellular solution contained 140 mM NaCl (the zero-Cained see . The secined see . When th2+] followed by the application of various [NFA] in the solution containing the same [Ca2+]. The steady-state current (I) at the end of NFA application was divided by the current in the absence of NFA (I0) before and after the drug application to obtain the percentage of current remained in the presence of NFA.The inhibition of ANO1 by intracellular NFA was achieved by directly applying NFA to the intracellular side of the membrane patch using fast-solution exchange. Except where indicated, the ANO1 current was induced by saturating [Ca2+] in the same recording sweep. The percentage current activation (Inorm) was plotted against the divalent cation concentration ([D]), and the dose-response activation curve was fitted to a Hill equation (eq. 1), Inorm = 1/{1+ n}, whereas K1/2,D is the half-activation concentration of the divalent cation, D, and n is the Hill coefficient.Data analyses were conducted using the software of pClamp10 (Axon Instrument/Molecular Devices) and Origin 8 (OriginLab). All the averaged data were presented as mean ± S.E.M. For the analysis of ANO1 activation, the current induced by non-saturating concentrations of divalent cations were normalized to the maximally-activated current induced by 20 µM [Ca0). The ratio I/I0 was then plotted against [NFA], and the concentration-dependent inhibition curve was fitted to a Langmuir function (eq. 2), I/I0 = 1/(1+ [NFA]/K1/2), where K1/2 is the half-inhibition [NFA].For dose-response inhibition by NFA, the current (I) measured at the end of the NFA application was normalized to the current amplitude in the absence of NFA  and in the presence of Mg2+ (KMg) were compared. In Mg/K0)−1] was plotted as a function of [Mg2+] , and in such a Schild plot the X-axis crossing point of the linear regression line of data points denoted the [Mg2+] that increases K1/2,Ca by 2-fold.The apparent binding affinity of Mg−  were calculated using the Erev obtained from bi-ionic experiments according to the following equation (eq. 3), PX/PCl = exp[Erev/(RT/F)], where R, T, and F are the gas constant, absolute temperature, and Faraday constant, respectively. Comparing the difference between Erev’s of the Ca2+- and Sr2+-activated current was performed using the pair t test at the significant level of 0.05.The permeability ratios of various anions versus ClFigure S12+ and NFA.The dead time of observing the effects upon switching solutions was identical for applying Ca (A) Comparison between the delay of the Ca2+-induced current increase (upper panel) and that of the NFA-induced current reduction (lower panel) in two different patches. Digital signals indicating the start of moving the solution pipes are shown at the bottom of each recording. (B) Averaged delay time for the Ca2+ activation and the NFA inhibition. Because this dead time varied from patch to patch, most likely due to different Ω-shape of the patch, we compared the delay of the Ca2+-induced current increase and that of the NFA-induced current decrease in the same patch. The difference is not statistically different (pair t-test).(TIF)Click here for additional data file.Table S12+] in the conditions of 1 mM EGTA, pH = 7.4, 0.14 mM salt solution, temperature = 22°C, with various [Mg2+] (in mM).Calculated free [Ca(DOC)Click here for additional data file.Table S22+] in the conditions of 0.1 mM EGTA, pH = 7.4, 0.14 mM salt solution, temperature = 22°C.Calculated free [Sr(DOC)Click here for additional data file.Table S32+] in the conditions of 0.1 mM EGTA, pH = 7.4, 0.14 mM salt solution, temperature = 22°C.Calculated free [Ba(DOC)Click here for additional data file.


After years of initial aggressive surgical intervention and a subsequent shift to damage control surgery (DCS), non operative management (NOM) has been shown to be safe and effective.In fact trauma surgeons realized that in liver trauma, it was safer to pack livers  than do Damage control was not the paradigm shift for spleen and liver, but rather to address coagulopathy that was more commonly associated with penetrating major abdominal vascular injuries .The shift to nonoperative care was largely motivated by intraoperative observations that many minor liver  and spleThen CT arrived in the early 1980s and confirmed that many moderate liver and spleen injuries did not require OR intervention. Pediatric surgeons first lead the shift to nonoperative management for splenic trauma ,7.In the 90's it became the gold standard for liver injuries in hemodynamically stable patients, regardless of injury grade and degree of hemoperitoneum , allowinAlthough recent reports  showed tMore recently several authors have highlighted an excessive use of NOM, which for some high grade liver injuries is pushed far beyond the reasonable limits, carrying increased morbidity at short and long term, such as bilomas, biliary fistulae, early or late haemorrhage, false aneurysm, arteriovenous fistulae, haemobilia, liver abscess, and liver necrosis . IncidenLiver resection in hepatic trauma should be considered when (1) massive bleeding related to a hepatic venous injury, (2) massive destruction and devitalized hepatic tissue is present, often partially resected by the injury itself, or (3) a major bile leak coming from a proximal, main intrahepatic biliary duct are found.NOM of liver injuries grade > = 3, especially when treated with combined AngioEmbolization (AE), is not without risks  and may lead to significant morbidity and possible mortality in up to 11% of cases due to liver-related complications .Although AE has been defined the logical augmentation of damage control techniques for controlling hemorrhage, the overall liver-related complication rate can be as high as 60.6% with 42.2% incidence of Major Hepatic Necrosis . Early lFurther concerns for both liver and spleen NOM, arise when associated hepatic and splenic injuries coexist and/or potentially missed injuries can be suspected. Patients with associated liver and spleen injuries are twice as likely to fail non-operative therapy as those with only a single organ injured . MissingNOM is actually used blunt splenic as the initial standard of care for blunt splenic injuries, not only in children (rates above 90-95%) but also in adults (60-77% ). Even ip = .07) [A significantly higher failure rate (38%) has been observed in grade IV-V Blunt Splenic injury(BSI) patients and above all, mortality of patients for whom NOM failed was almost 7-fold higher than those with successful NOM in this series  . Furtherp = .07) . That mep = 0.002). Higher rates of septic complications including splenic abscess, septicemia, and pneumonia have also been recorded, and lastly (4) a non significant trend to higher amount of PRBC (packed red blood cell) transfusions, higher mortality and longer Length Of Stay [In the most recent years a liberal and more aggressive use of angiography has often been observed and is associated with higher rates of NOM (80%) and lower rates of failure (2-5%); nonetheless several concerns raise because it is labour intensive and there have been several reports reporting a surprisingly high rate of complications . In WTA  Of Stay .Splenic preservation can also have deleterious side effects in otherwise salvageable patients. A review of 78 patients who failed NOM revealed a mortality rate of 12.6%. The authors concluded that the majority of their deaths were a result of delayed treatment of intra-abdominal injuries, and suggested that 70% of deaths after failing NOM were potentially preventable . When exThus we surgeons must keep in our minds that post-splenectomy sepsis is rare and can be minimized with polyvalent vaccines of encapsulated bacteria, whilst operative mortality of splenectomy in the otherwise normal patient is < 1% .Whereas Non Operative Management of Liver Injury (NOMLI) has not been shown to increase mortality rates for those that fail, the same cannot be said for the NOMSI and the balance between concerns with bleeding and infection has in the most recent years shifted illogically to favour infection. As Richardson highlighted, it should be made clear that these delayed bleeding and late failures of NOM are not harmful. "Anecdotally, I have been impressed in private discussions about deaths or "near misses" from bleeding occurring in NOM failures. These are rarely reported in the literature. Additionally, many reports list multiple organ failure as a leading cause of death. Does unrecognized shock play a role in these deaths?" .st century, when NOM for liver and spleen injuries is often advocated beyond the limits of a reasonable safety and the need for surgery is considered as a defeat or "failure". We should not forget in making the best treatment choice, to keep in mind not only the predictors of NOM failure, such as the injury grade, the presence of associated intra-abdominal injuries and the risk of missing injuries with the subsequent sequelae, of a failed NOM and of delayed surgical treatment, but we must also consider the potential drawbacks of angioembolization, the environmental setting and factors, i.e. the level of the hospital (trauma center), availability of Angio Suite and ICU for continuous monitoring, the initiation of NOM during night shift, the need of an eventual time consuming spine surgery in a prone position for a concomitant vertebral fracture, and last but not least, the time needed for complete and safe resumption of normal life .In conclusion, at the beginning of the 21