The pattern of Ca2+ influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity, including Ca2+/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bind up to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime, supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+ with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning.During the acquisition of memories, influx of Ca 2+ ions that flow across the membrane at the postsynaptic site, and by the pattern of influx over time. The mechanisms of activation of regulatory enzymes that decode this Ca2+ signal have been extensively studied under laboratory conditions which are different from the conditions encountered inside a neuron. Therefore, we created a dynamic model of activation of one enzyme that is critical for learning by Ca2+. The model allows us to simulate activation of the enzyme within a biochemical milieu similar to what it will encounter at the postsynaptic site. It predicts unexpected behaviors of the enzyme in vivo and provides a framework for quantitative exploration of complex mechanisms of synaptic plasticity.Networks of neurons in the brain are connected together by specialized signaling devices called synapses. One way an active neuron relays its activity to other neurons is by releasing small amounts of chemical transmitters from its presynaptic terminals which induce electrical activity in postsynaptic neurons connected to it. Memories are formed when synapses in the network encoding the memory change their strength in order to stabilize the network. The decision whether or not a synapse becomes potentiated is controlled by delicate variations in the amount of Ca For example, it has long been recognized that Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors initiates changes at synapses that enable us to form memories and to learn. Transient influx of Ca2+ through NMDA receptors triggers activation of complex protein signaling networks that regulate changes in synaptic efficacy including long-term potentiation (LTP) and long-term depression (LDP) 2+ binding sites, is a molecular detector of influxes of Ca2+ across the synaptic membrane. It is ubiquitous in all cells including neurons 2+ enters the spine, it binds to CaM and to other Ca2+-binding proteins. As Ca2+ binds to CaM, the Ca2+/CaM complex can then bind to and regulate its enzyme targets, many of which are immobilized in the “postsynaptic density” (PSD), a scaffold for signaling molecules attached to the postsynaptic membrane 2+/CaM are believed to determine their level of activity in a sensitive and selective fashion. Among the prominent CaM targets in the spine is Ca2+/CaM-dependent protein kinase II (CaMKII) 2+, CaM, and CaMKII can provide important insight into the initiation of mechanisms of synaptic plasticity.Calcium  strongly influences the overall dissociation rate of the peptide from CaM. The kinetics of Ca2+ binding to CaM are likely to be particularly important in determining the outcome of Ca2+ signaling in neuronal spines because Ca2+ triggers biochemical events in the spine during a period when the Ca2+ concentration is fluctuating rapidly 2+, including basal Ca2+ or during relatively low amplitude Ca2+ transients, very few of the free CaM molecules will have four bound Ca2+ ions. Yet physiological responses to small increases in Ca2+ are observed 2+ ions participates in initiating these responses.The structure and Car region . The pai2+/CaM to this domain releases inhibition of the active site and stimulates catalytic activity 2+-independent catalytic activity 2+ ions bound to its C-terminal sites, binds to CaMKII and activates autophosphorylation, though at a ten-fold lower catalytic rate than fully loaded CaM (CaM with 4 Ca2+ bound) 2+ binding to each of the individual CaM termini, as well as binding of Ca2+ to the CaM/CaMKII complex is important for a complete description of the activation dynamics of CaMKII in spines. Furthermore, a model that accounts for the activity of CaM/CaMKII with less than 4 bound Ca2+, is necessary to understand the extent of activation of CaMKII at relatively low and/or fluctuating Ca2+ concentrations such as occur in the spine cytosol.CaMKII is a large holoenzyme comprising 12 homologous catalytic subunits held together by association of their carboxyl terminal domains 2+ to free CaM and to CaM bound to individual (ie. monomeric) CaMKII subunits. The model of monomeric CaMKII (mCaMKII) allows us to examine the significance of the dynamics of Ca2+ and CaM binding independent of the effects of cooperativity of binding of CaM between subunits within the holoenzyme 2+/CaM binding at the N and C termini, and the thermodynamic stabilization of Ca2+-binding when CaM is bound to a target protein 2+ to the two CaM termini, including 9 Ca2+/CaM states and their interactions with mCaMKII. It differs from a recently published allosteric model 2+ binding rates depend explicitly on whether CaM is in one of two abstracted ensemble conformational states, R or T. Most of the required kinetic rates in our model are well constrained by previous experimental studies; however, a few have not been measured directly. In these cases, we used the principle of microscopic reversibility and fitting of existing experimental data to derive reasonable ranges of values for the kinetic rates. The second model is a coarse-grained model that is motivated by experiments showing high cooperativity of binding between Ca2+ ions at each terminus 2+ to each terminus of CaM is assumed to be rapid; thus, binding of pairs of Ca2+ to each terminus is treated as a single event. The resulting model includes 4 Ca2+/CaM states and their interactions with mCaMKII.Here we present two kinetic models of activation of monomeric subunits of CaMKII which include binding of Ca2+, CaM, and mCaMKII, and under conditions that are closer to those believed to exist in synaptic spines. We determined a range of initial conditions under which the results of the coarse grained, pair-binding model are indistinguishable from those of the complete model, and a range under which the two deviate significantly. We show that Ca2+/CaM species with fewer than four bound Ca2+ predominate under many conditions that are believed to prevail in spines, and can sometimes completely determine the level of autophosphorylation. We find that activation of mCaMKII is highest at a particular frequency of Ca2+ fluctuations. The frequency that gives highest activation depends on the ratio of the time interval between Ca2+ transients and the rates of Ca2+ binding to the N and C termini of CaM, as well as on the the width of the Ca2+ transients. Finally, we performed global variation and sensitivity analyses to determine which parameters most affect the levels of autophosphorylation at particular times and under various conditions. We use these analyses to help infer the kinetic pathways through which autophosphorylation of CaMKII is likely to occur and to identify parameters whose refinement by direct measurement will be most important for the accuracy of predictions from our models.We created computer simulations based on each of these two models and explored their behavior under commonly used experimental concentrations of Ca2+ fluctuations in postsynaptic spines 2+/CaM dependent enzymes, including the CaM-dependent protein kinases , phosphatases  and others . Detailed kinetic analysis of these interactions are critical for understanding the molecular mechanisms that underlie synaptic plasticity because the events that determine whether a synapse undergoes LTP or LTD are determined under non-equilibrium conditions, when the Ca2+ concentration is fluctuating. Such analyses may also be useful for understanding Ca2+/CaM signaling in other tissues such as cardiac myocytes and cells of the immune system.The models presented here are a first step in a larger project to build kinetic simulations of activation of the CaMKII holoenzyme in the context of physiologically realistic models of Ca2+ ions to free CaM and to the resulting intermediate Ca2+/CaM complexes. The models also describe reversible binding of Ca2+ to the Ca2+/CaM complexes after they have bound to individual subunits of CaMKII (mCaMKII). Finally, they describe the kinetics of irreversible autophosphorylation of mCaMKII, which is triggered by binding of Ca2+/CaM.We constructed a detailed model (Model 1) and a coarse-grained model (Model 2), both of which describe the kinetics of reversible binding of Ca2+ ions to the CaM termini, resulting in nine distinct Ca2+/CaM species or “states”, characterized by the number of Ca2+ ions bound at each terminus , which represent four independent variables, are constrained by fitting to three sets of experimental data , similar to those thought to occur in neurons. We also examine autophosphorylation at steady-state concentrations of Ca2+ ranging from 0.5 to 250 µM, which mimic experimental conditions. In all simulations, the concentration of Mg2+-ATP is assumed to be saturating, as it would be in the cell.The concentration of CaReaction networks were entered into Mathematica 2+ spikes were simulated as exponential functions t is time. This function was used as a fixed boundary condition representing the free Ca2+ concentration. Thus, total Ca2+ was not conserved over the sum of the driving function and the Ca2+ bound to various molecular species. This algorithm simulates a neuronal environment in which Ca2+ enters the cytosol through voltage and ligand-gated channels and is then rapidly sequestered or removed. Mathematica packages implementing the models are available from the authors.Ca2+, CaM, and mCaMKII (above). We then calculated output of the model for each set of randomized parameters every 0.05 s for a 2 s simulation. In one set of calculations, we used a series of fixed concentrations of Ca2+ to examine how the importance of individual parameters varies at different Ca2+ concentrations. The contribution of variations in each input parameter to variation in the output was quantified by calculating the partial rank correlation coefficient (PRCC) We used sensitivity analyses to determine which parameters of Model 1  produce 2+ to CaM and of Ca2+/CaM to mCaMKII after a rapid increase in concentration of Ca2+. In particular, we examined the time evolution when the concentrations of Ca2+ or CaM are not high enough to saturate binding to mCaMKII; conditions that are likely to prevail in postsynaptic spines during activation of NMDARs 2+ ions) when 10 µM Ca2+ was introduced at time zero into a system containing 30 µM CaM and 80 µM mCaMKII. We picked these values because they are within the likely physiological ranges of concentrations in a spine during activation of NMDARs. Changes in the species of free CaM , but free CaM1C (blue) reached a relatively high equilibrium concentration because Ca2+ has a higher affinity for the C-terminal sites. The equilibrium concentration of free CaM2C remained low because this species (purple) binds very rapidly to mCaMKII . This result means that Model 1 is required to obtain the most acccurate estimates of binding of Ca2+ to CaM and CaMKII under the conditions that prevail in a spine; the simpler Model 2 can be used when accuracy within a factor of 2 is adequate. In contrast, the differences between the empirical model and Model 1 in the same physiological range of Ca2+ are much greater. For example, at 10 µM Ca2+, the empirical model predicts ∼100 fold higher autophosphorylation after 1 sec than does Model 1; whereas, Model 2 predicts ∼1.12 fold higher autophosphorylation than does Model 1. This result means that the empirical model, and by inference other 2 state models, do not accurately predict Ca2+/CaM dynamics at concentrations of Ca2+, CaM, and CaMKII present in vivo. Thus, in order to achieve the highest accuracy in predictions of CaMKII activity in a spine, it is necessary to include the kinetic details of binding among Ca2+, CaM, and CaMKII.We compared predictions of Model 1 to two other models of interactions of Ca Model 1  and the o 60 sec . The out2+ transients such as occur inside a cell during signaling. One experimental study demonstrated a frequency dependence by applying brief pulses (80 ms to 1 s at 0.1 to 10 Hz) of fully saturated Ca2+/CaM  to immobilized CaMKII and then measuring the resulting Ca2+-independent catalytic activity 2+/CaM from a single subunit is significantly slower than the inter-stimulus interval of the Ca2+ transients, some Ca2+/CaM will remain bound individual subunits and contribute to activation of autophosphorylation during the next transient stimulus. The theoretical model of Kubota and Bower, which included the empirical model described in 2+, CaM, and CaMKII, also supported this same mechanism 2+ binding to the C terminus of CaM in the K•CaM complex give rise to frequency dependence of autophosphorylation in the 1 to 8 Hz range.Previous investigators have studied the dependence of activation of CaMKII on the frequency of rapid series of Ca2+ pulses, as a function of frequency of the pulses. To determine whether this form of frequency dependence requires that two CaM's must bind to two kinase monomers to initiate autophosphorylation, Model 1 was altered to permit zero-order autophosphorylation (that is autophosphorylation without the requirement for monomer-monomer interactions). The modified model showed similar frequency dependence (data not shown), indicating that the requirement for two CaM's binding to two monomers does not play a large role in this mechanism of frequency dependence.2+ pulses affects the accumulation of CaM species during each pulse. Ten and 1 sec of 20 ms pulses of 10 µM Ca2+ were simulated at 0.5 and 7 Hz, respectively. At 0.5 Hz , medium (10–50 µM), and high (50 to 250 µM). The low regime encompasses the range believed to arise in and near the PSD during low frequency stimulation of NMDA-receptors 2+ regimes. We used PRCC values  to that for −1), a range of 16-fold. In contrast, the narrower range of values  of CaM2C and CaM2N for CaMKII . The lowest estimate of the dissociation constant (highest affinity) was given by Hanson and Schulman (1994) as 1.3 µM. However, a number of other studies suggest that the affinity is considerably lower (KD≈20–40 µM) We have used global sensitivity analysis to identify parameters that have a strong impact on autophosphorylation of mCaMKII in particular concentration regimes. Thus, we have identified which of the relatively uncertain parameters will be most important to measure experimentally in the future. One of the least well defined parameters is the affinity of CaMKII monomers for each other in the autophosphorylation reaction  for CaMKII.In general, the parameters that describe binding of CaM to CaMKII do not have a strong influence on autophosphorylation. One exception is binding of CaM0 to CaMKII at low Cantration . At low 2+, CaM, and monomeric subunits of CaMKII that can be used to understand the dynamics of activation of CaMKII by Ca2+ in the environment of a postsynaptic glutamatergic spine. Activation of CaMKII by Ca2+ flowing through NMDA-type glutamate receptors is a critical early step in synaptic changes that underlie learning and memory. We constructed this model to represent binding of Ca2+ and CaM to monomeric subunits, rather than to the dodecameric holoenzyme, so that it can be used to experimentally test and verify parameters for activation of CaMKII in the absence of cooperativity of CaM binding caused by the structure of the holoenzyme We have constructed a kinetic model of interactions of Ca2+ concentrations lower than ∼50 µM, with concentrations of CaM and CaMKII set to approximate those present in the spine. We conclude that these coarse-grained models are inadequate to predict the timing and extent of activation of CaMKII under physiological conditions because they do not capture critical aspects of the dynamics in the physiological regime. For example, a model that treats the binding of two Ca2+ ions at the amino- or carboxyl-termini of CaM as simultaneous (Model 2), overestimates the rate of autophosphorylation by 30 to 60% under these conditions, compared to Model 1. An Empirical model which is similar to those often used in previous models of activation of CaMKII An important finding is that two types of models, which are more coarse-grained than our Model 1, produced significantly different predictions for rates of autophosphorylation in a physiological regime; in particular, when simulations were run for times shorter than ∼5 sec, and at Ca2+ ions are major activators of CaMKII at Ca2+ concentrations as high as ∼30 µM, a concentration that falls in the middle of the physiological regime. Simulations of formation of CaM species such as those shown in 2+ binds to CaM, and Ca2+/CaM binds to and activates CaMKII, differ during the first sec of Ca2+ influx, depending on the peak Ca2+ concentration Click here for additional data file.Figure S1Models of calcium binding to calmodulin. A) Sequential binding model. In this model a state of calmodulin is characterized by the number of calcium ions bound. The dissociation constants are called macroscopic constants. B) Terminal binding model. Here, a state of calmodulin is characterized by the number of calcium ions bound to each of the calmodulin termini. The dissociation constants are called microscopic constants.(0.62 MB PDF)Click here for additional data file.Table S1Parameters for Model S1. Most values are taken from the literature or derived from values in the literature. In a few instances values were derived by fitting to published experimental data as described in (0.76 MB PDF)Click here for additional data file.Table S2Fitted cooperativity coefficients with their on and off components. The parameters were fit as described in (0.04 MB PDF)Click here for additional data file.Table S32+ concentration ranges. Model parameters are ranked by their PRCC values at 0.1, 1 and 2 seconds after Ca2+ addition. Parameters with PRCC values lower than 0.3 are not shown.Sensitivity of autophosphorylation of CaMKII to variations in input parameters. Parameters were varied over a 2.5 fold range from mean of data in (0.68 MB PDF)Click here for additional data file.


The present experiments went on to correlate acute initiation of atrial arrhythmogenesis in Langendorff-perfused mouse hearts with changes in Ca2+ homeostasis in isolated atrial myocytes following pharmacological procedures that modified the storage or release of sarcoplasmic reticular (SR) Ca2+ or inhibited entry of extracellular Ca2+.Intracellular Ca2+ waves in regularly stimulated atrial myocytes immediately following addition. This was followed by a decline in the amplitude of the evoked transients and the disappearance of such diastolic events, suggesting partial SR Ca2+ depletion.Caffeine (1mmol/L) elicited diastolic Ca2+ transients and abolished diastolic Ca2+ events produced by the further addition of caffeine.Cyclopiazonic acid  produced more gradual reductions in evoked Ca2+ transients. Further addition of caffeine produced an immediate increase followed by a decline in the amplitude of the evoked Ca2+ transients, without eliciting diastolic Ca2+ events.Nifedipine (0.5µmol/L) produced immediate reductions in evoked CaThese findings correlated with changes in spontaneous and provoked atrial arrhythmogenecity in mouse isolated Langendorf-perfused hearts. Thus, caffeine was pro-arrhythmogenic immediately following but not >5min after application and both CPA and nifedipine pretreatment inhibited such arrhythmogenesis.2+ events in atrial myocytes that, in turn, depend upon a finite SR Ca2+ store and diastolic Ca2+ release following Ca2+-induced Ca2+ release initiated by the entry of extracellular Ca2+.Together, these findings relate acute atrial arrhythmogenesis in intact hearts to diastolic Ca However, the precise cause-and-effect relationships between atrial arrhythmogenecity, any accompanying anatomical or functional remodelling and changes in SR Ca2+ release remain unclear. Consequently, the mechanisms involved in the initiation or termination, particularly of acute, as opposed to established, AF remain poorly understood. This acute situation is exemplified by the condition of catecholaminergic polymorphic ventricular tachycardia (CPVT), associated with cardiac ryanodine receptor (RyR2) mutations that result in episodic atrial arrhythmias, including sinus bradycardia, junctional rhythms and AF, in addition to ventricular tachyarrhythmias. These are initiated by stress and adrenergic stimulation, despite the absence of anatomical abnormalities.7Atrial arrhythmias constitute the most common sustained disorders of cardiac rhythm encountered in clinical practice. For example, atrial fibrillation (AF) is associated with substantial mortality and morbidity from stroke, thromboembolism, heart failure and impaired quality of life.2+ homeostasis in the acute initiation of atrial arrhythmogenecity in otherwise functionally and structurally normal hearts. Previous studies on atrial systems have not made such correlations.2+ imaging and a separation of atrial from ventricular electrophysiological activity in intact Langendorf-perfused mouse hearts through a range of pacing conditions.Rather than considering established atrial arrhythmogenesis, the present paper is concerned with the possible roles of pharmacological changes to cellular CaInbred 129/Sv wild-type mice  were kept in an animal house at room temperature under a 12h light–dark cycle and fed sterile rodent chow with constant access to water. All procedures conformed to the UK Animals (Scientific Procedures) Act (1986).3 25; KCl 4.0; KH2PO4 1.2; MgCl2 1.0; CaCl2 1.8; glucose 10; sodium pyruvate 2.0) maintained at pH7.4 by bubbling with 95% O2–5% CO2 . Solution B was the basic solution from which other solutions used in the myocyte isolation procedure were derived (composition (in mmol/L): NaCl 125; KCl 4.75; MgSO4 1.2; KH2PO4 1.2; HEPES 30; glucose 10; taurine 50, titrated to pH7.4 with NaOH). The solution was filtered through a 0.2µm filter  to remove microbes and small particles. This provided the stock solution for the production of Solutions C–H used during myocyte isolation.The following solutions were used during the course of the preparation and experimental procedures. Solution A for electrophysiological experiments on Langendorff-perfused hearts consisted of normal bicarbonate-buffered Krebs’–Henseleit (KH) solution (composition (in mmol/L): NaCl 119; NaHCO2+-containing perfusion solution was prepared by adding 750µmol/L CaCl2 to Solution B.Solution C: this 750µmol/L Ca2+-free nitrilotriacetic acid (NTA)-containing solution was prepared by adding 5mmol/L NTA to Solution B and titrating the resulting solution to pH6.95 with NaOH.Solution D: this Ca2 to Solution B.Solution E: an enzyme solution prepared by adding 1.5mg/mL collagenase (Worthington Type II), 2mg/mL hyaluronidase  and 100µmol/L CaClSolution F: this was a further digestion buffer made by adding 1mg/mL collagenase and 1mg/mL bovine servum albumin (BSA) to Solution B.2 to Solution B.Solution G: this was an enzyme washout solution made by adding 1mg/mL BSA and 250µmol/L CaCl2+-containing solution was made by adding 1.2mmol/L CaCl2 to Solution B.Solution H: this CaAfter preparation, Solutions D–G were always filtered using a 0.2µm filter to remove microbes and small particles.2+ signals were isolated using an enzymatic digestion protocol substantially modified and combined from previously established procedures.g for 3min. The resulting isolated cells were then washed using Solution G, followed after 5min by centrifugation at 30g for 2min. The cells were then resuspended in Solution H and, after a 5min interval, centrifuged again at 30g for 2min. The cells were then maintained at room temperature in Solution H for the experiments that followed, in common with previous studies in atrial myocytes.Atrial myocytes were obtained from mice aged 3 months. Single mouse atrial myocytes used for the imaging of Ca2) for 10–20min in the dark before washout of the Fluo-3-containing solution. Cells were then transferred onto the stage of a Zeiss LSM-510 laser scanning confocal system  with a ×20 air objective on a Zeiss Axiovert 100M inverted microscope. Fluo-3 fluorescence emission was excited with a 488nm argon laser and measured at wavelengths between 505 and 550nm. Images were then analysed using an in-house custom-made software program. Series of 500 frames (128×64pixels/frame) were collected at a scanning frequency of 25msec/frame to monitor fluorescence changes over time. Fluorescence measurements, corrected for background signal in regions outside the cells, were made within defined regions of interest (F) and were normalized to their resting fluorescence (F0) values. For each of the myocytes studied, peak F/F0 values were calculated throughout each time series acquired and a mean peak F/F0 was calculated for that series. Where indicated, cells were paced at 1Hz (5V above excitation threshold of 30–60V for 2msec) with two field electrodes. All fluorescence studies were performed at room temperature. Ca2+ transients were measured both from regions of interest (ROIs) covering entire cells and from sets of three  ROIs placed at the cell margin, the cell centre and regions between these, approximately 2–3µm from the surface.Cells were then placed on a Grade 1 circular laminin-coated coverslip  that formed the floor of a 1.5mL perfusion chamber, to which it was fixed with vacuum grease. Cells were then loaded with the acetoxymethyl (AM) ester of Fluo-3  by incubation with 5µmol/L Fluo-3 AM in Solution H . The cannulated heart was perfused with Solution A at room temperature before being mounted onto a Langendorff systemThe whole-heart experiments used modifications of previously established procedures to set up a Langendorff perfusion system for the murine heart.in situ cannulation procedure gave preparations with higher intrinsic rates corresponding to baseline cycle lengths of 155.0±58.4msec (n=33 hearts) and more consistent 1:1 AV conduction  than hearts cannulated following prior separation and immersion in ice-cold buffer .The perfusate was first filtered through 200 and 5µm membranes  and warmed to 37°C by a water-jacketed heat-exchange coil  before entering the coronary arterial network. The aortic valve was shut by the pressure of the perfusate that ultimately drained through the vena cava. Viable hearts regained a pink appearance and spontaneous rhythmic contractions upon warming. Hearts were perfused retrogradely for not less than 10min in the absence of stimulation. Experiments were only performed in intact Langendorff preparations showing clearcut 1:1 atrioventricular (AV) conduction during the intrinsic activity following cannulation. The plus; Cambridge Electronic Design, Cambridge, UK).The electrophysiological studies performed in isolated perfused hearts were designed to distinguish atrial from ventricular bipolar electrogram (BEG) waveforms and involved comparisons of records from simultaneous recordings made at two sites. Thus, in addition to the paired platinum stimulating electrodes placed on the right atrium, two bipolar recording electrodes of 1mm interpole spacing were placed on the left atrium and left ventricle. At the beginning of each experiment, the ventricular recording electrode was placed at a series of positions at successively greater distances from the atria until there was no demonstrable atrial far-field deflection in the ventricular traces while ensuring persistent far-field deflections in the atrial traces. Hearts were initially paced for not less than 5min at 10Hz to permit them to regain their physiological steady state. Three types of pacing protocols were used: (i) hearts were studied at their intrinsic rates in the absence of stimulation; (ii) hearts were subject to regular pacing at 10Hz using 2msec square-wave stimuli set at 2× the excitation threshold ; and (iii) hearts were studied using a programmed electrical stimulation (PES) procedure adopted from clinical techniques used previously in ventricular studiesn=33) than the AV node .The present protocols differed from previous studies on ventricular arrhythmogenesisanova to compare data using spsssoftware . Results from individual hearts acquired during pharmacological intervention were compared with their respective untreated controls using one-way anova for correlated samples (spsssoftware). P<0.05 was considered significant. Cross-tabulations with Chi-squared or Fisher's exact test were used as appropriate for categorical variables.Statistical analysis was performed using a repeated-measures one-way n denotes the number of peaks from which F/F0 values were obtained; the numbers of cells involved are given separately. For experiments in whole hearts, n denotes the number of whole hearts studied.Data are expressed as the mean ± SEM. For experiments with single cells, All drugs and other chemical agents were purchased from Sigma-Aldrich , unless indicated otherwise. Nifedipine was dissolved in 96% ethanol to make a 1mmol/L stock solution, kept wrapped in foil to prevent light degradation and was kept refrigerated at 4°C. Cyclopiazonic acid (CPA) was prepared in 96% ethanol to make a 10mmol/L stock solution and was stored at –20°C. Final drug concentrations were achieved by dilution with Solution A for electrophysiological experiments and Solution H for experiments in single cells. Caffeine was dissolved directly in Solution A or H, as appropriate, and kept at room temperature.n=15 cells) with well-defined striations. We studied a total of 90 cells from seven hearts. Of these cells, we examined the resting unstimulated properties in approximately 20 cells. The remaining cells were subjected to stimulation protocols in the absence of (n=15 cells) or following the addition of caffeine (n=16 cells), CPA (n=10 cells), nifedipine (n=8 cells), CPA+ caffeine (n=14 cells) or nifedipine+caffeine (n=5 cells). Even the approximately 20 resting, unstimulated, Fluo-3 loaded cells exhibited two forms of spontaneous Ca2+ activity, with some cells showing evidence of both . This was demonstrated by comparing signals from regularly spaced ROIs  positioned at 2–3µm intervals along the cell lengths ; width 13.1±2.0µm (range 10.1–17.2µm); 2+ transients in regularly stimulated (1Hz) Fluo-3-loaded atrial myocytes exposed to agents that sought to increase the release of SR Ca2+ through CICR or to inhibit SR uptake of cytosolic Ca2+ or extracellular Ca2+ entry. In each cell, Ca2+ transients were measured from both ROIs covering entire cells and sets of three  ROIs placed at the cell margin, the cell centre and regions between these, approximately 2–3µm from the surface. Before the addition of pharmacological agents, myocytes showed regular successions of entrained Ca2+ transients with stable amplitudes of mean F/F0 5.49±0.97 (from n=176 peaks), determined from ROIs covering the entire cell area, that decayed to a stable baseline , consistent with a partial depletion of SR Ca2+ stores  from 119±8 to 126±16 and 302±42msec <5min and >10min after its addition, respectively  exposures to caffeine. Such a hypothesis was corroborated by the results of independent pharmacological changes in such SR Ca2+ stores and extracellular Ca2+ entry using CPA and nifedipine, respectively. Following the addition of CPA, atrial cells showed only nine diastolic Ca2+ transients over 130 evoked peaks and then in only three of 10 cells at <5min following the addition of CPA  at 10–15min and 3.29±0.73 (n=39 peaks) at >15min, respectively  and CPA alone (>15min) were statistically similar at 2.61±0.13 (n=52 peaks), 2.37±0.08 (n=88 peaks) and 3.29±0.73 (n=39 peaks), respectively (P<0.01), consistent with effects of caffeine in prolonging Ca2+ release , it did not result in the appearance of diastolic Ca2+ transients  at approximately 5min and to 173±24msec (P<0.01) at 10min  ROIs placed at the cell margin, the cell centre and regions between these, approximately 2–3µm from the surface. The present findings are compatible with an effect of caffeine in changing the characteristics of such CICR; such a hypothesis would predict that caffeine, but not nifedipine or CPA, would enhance its propagation with distance from the cell surface. 2+ transients obtained from localized 4×1 pixel (2.4× 0.6µm)ROIs ROIs  and nife6µm)ROIs . However6µm)ROIs  or in co6µm)ROIs , consist2+ homeostasis correlated closely with atrial arrhythmogenic tendency in intact Langendorff-perfused hearts. Control experiments (n=34 hearts) assessed whether atrial arrhythmogenesis was initiated during 10–15min of intrinsic activity, 5min of regular pacing at 10Hz and following application of the PES protocol, with stimulation applied at the right atrium. These control experiments recorded episodes of either atrial tachycardia (AT), in the form of a normal sequence of electrical waveforms at increased frequency, or AF, in the form of irregularly irregular electrical deflections, extending for more than five deflections during intrinsic or regular pacing and for >1s following PES. Similar protocols were applied in experiments that then involved 20min pretreatment with CPA or nifedipine. Experiments that investigated the effect of caffeine used PES and regular pacing both immediately after and >5min after application; this was in parallel with observations in isolated myocytes in which diastolic Ca2+ events were observed immediately following, but not 5min after, the addition of caffeine.The changes in cellular Can=11 hearts), but not after >5min exposure to caffeine  increases in the incidence of atrial arrhythmias only in the case immediately following the addition of caffeine and not with prolonged caffeine exposure or manoeuvres involving CPA (n=6 hearts) or nifedipine (n=6 hearts) pretreatment. These results in intact hearts precisely parallel the occurrence of diastolic Ca2+ transients in isolated atrial myocytes.2+ homeostasis in the acute initiation of atrial arrhythmogenesis in mouse intact hearts. The findings demonstrated that such arrhythmogenic phenomena depend on both a definite SR Ca2+ store and the diastolic release of Ca2+. Accordingly, atrial arrhythmogenecity was increased by pharmacological manipulations that increased SR Ca2+ release and this effect was abolished by either inhibiting this release or depleting SR Ca2+. Thus, in atrial myocytes, caffeine produced diastolic Ca2+ events immediately following, but not >5min after, its application, consistent with an initially increased CICR subsequently resulting in the partial depletion of a finite SR Ca2+ store. Both CPA and nifedipine pretreatment abolished these effects. In whole hearts, caffeine correspondingly produced pro-arrhythmogenic effects immediately following, but not >5min after, application that were abolished by CPA or nifedipine pretreatment.The present study investigated the possible roles of altered Ca2+ homeostatic processes. Atrial myocytes do not possess extensive T-tubular systems2+ channel (LTCC) clusters are confined to the cell peripheries.2+, either by sensitizing RyR2 to cytosolic Ca2+ or inhibiting phosphodiesterase activity, thereby increasing cellular cAMP2+ release that would subsequently cease with the resulting reduction in SR Ca2+, as reported previously for ventricular cells.37Atrial myocytes show important differences from ventricular myocytes, particularly in their tubular and SR membrane systems, which may reflect functional differences in their Ca2+ release. However, there was a subsequent disappearance of these events that was accompanied by a progressive reduction of amplitude, but a prolongation in time-course, of the evoked Ca2+ transients with time. The former observation was consistent with a reduction in store Ca2+ levels available for release by electrical stimulation consistent with a delayed reduction of SR Ca2+ and consistent with earlier studies that have actually used caffeine as a means of depleting these stores.2+ transients consistent with actions on the CICR process.2+ release events (sparks) and Ca2+ waves in atrial cardiomyocytes from AF patients.2+ release channels resulting in increased open probabilities and diastolic leak of Ca2+ relates to an increased triggered activity in heart failure.39In regularly stimulated single murine atrial myocytes, application of caffeine resulted in the early appearance of diastolic Ca2+-ATPase activity and ultimately reduce SR Ca2+.2+ signals without altering their kinetics or spatial heterogeneities and inhibited the diastolic Ca2+ events caused by the subsequent addition of caffeine. These findings similarly suggest a depletion by CPA of SR Ca2+ releasable by electrical stimulation that also results in a reduction in caffeine-induced diastolic events. Nifedipine is a known competitive dihydropyridine LTCC blocker in ventricular cells with a KD of 40 nmol/L;2+ entry.2+ transients while preserving their kinetics and spatial heterogeneity. Subsequent addition of caffeine similarly restored the amplitudes of the evoked Ca2+ transients, again without the expected induction of diastolic Ca2+ transients, consistent with CICR reduced by inhibited entry of extracellular Ca2+. These findings at the cellular level correlate with the presence or absence of atrial arrhythmogenicity in whole hearts. The presence or absence of spontaneous and provoked atrial arrhythmogenesis were examined under conditions of intrinsic and regular pacing from the right atrium and using programmed electrical stimulation, respectively. Control hearts showed no evidence of arrhythmogenicity, whether during intrinsic or regular pacing, and a low incidence of AT during programmed electrical stimulation, recapitulating previous clinical observations of brief periods of AT that follow the imposition of extra stimuli.27These findings were corroborated by observations from experiments using the complementary agents CPA and nifedipine. Cyclopiazonic acid is thought to inhibit Ca2+ release attributable to an enhanced CICR, but their subsequent disappearance is due to the consequent SR Ca2+ reduction. This would be consistent with the correlations between the observed abolition of atrial arrhythmogenicity in the whole hearts by CPA, which appeared to reduce myocyte SR Ca2+ even in the presence of caffeine. Finally, the effects of nifedipine in inhibiting caffeine-induced arrhythmogenesis were clearly correlated with its actions in reducing extracellular Ca2+ entry, which would similarly reduce CICR, albeit through a different mechanism.In contrast, the addition of caffeine initially resulted in episodes of both AT and AF during regular pacing and an increased incidence of arrhythmogenic phenomena during PES in intact hearts, but this effect disappeared over the next 5min. These findings are directly comparable with the early appearance of diastolic Ca2+ transient kinetics at the cellular level, as evidenced in our observations of increased FWHM values despite the reduced Ca2+ peaks. Nevertheless, such changes in FWHM indicate changes in the kinetics as opposed to the quantity of Ca2+ release. They confirm actions of caffeine in atrial cells not shared by the other agents tested. Thus, CPA and nifedipine alone, thought to act primarily on SR Ca2+ reuptake and voltage-dependent Ca2+ entry, respectively, produced no observable changes in FWHM. Thus, the results are consistent with the action of caffeine on CICR that has been established previously for ventricular cells.2+ signals also implicate changes in CICR: caffeine abolished such heterogeneities, whereas CPA and nifedipine did not.The presence of caffeine continued to result in prolonged Ca2+, itself dependent upon a finite SR Ca2+ store and initiation of Ca2+ entry, both of which may then offer possible therapeutic targets in the clinical management of acute AF. We have demonstrated that caffeine produces early arrhythmogenesis in intact hearts. This correlates with the early appearance of diastolic Ca2+ transients in single cells, suggesting an immediate cause for this arrhythmogenesis. However, caffeine also produces a progressive decline in Ca2+ release, consistent with a depletion of cellular SR Ca2+ stores. It is then possible to demonstrate increased FWHM, not seen either when SR Ca2+ is depleted by CPA or in the presence of nifedipine. This is consistent with an action of caffeine on atrial CICR and possible roles of this CICR in atrial arrhythmogenesis. Therefore, the findings of the present study directly complement earlier studies that explored the effects of pharmacological changes of Ca2+ homeostasis.36Taken together, the findings from single cells and intact hearts suggest acute atrial arrhythmogenic phenomena that are dependent on a diastolic release of SR Ca


Chronic urticaria, a major health problem causing patient's distress, induces often physicians' dilemma while dealing with its etiology, investigations and management. Clinical approach of such cases should include apart from clinical history and physical examination laboratory investigations like routine blood test, thyroid profile, etc. as well as sometimes special test like autologous serum skin test. Management includes reassurance, avoidance of precipitating factors, treatment of underlying disorders, and non-pharmacological approach along with pharmacotherapy. First line drug therapy comprises non-sedative and sedative antihistamines, second line doxepin, nifedipine, leukotriene-inhibitors, sulfasalazine, etc. and third line cyclosporine, dapsone, colchicin, etc. Ordinary chronic urticaria is appropriately termed as a “Cinderella” disease with a capricious course and a demoralizing response to treatments. There are no guaranteed means of controlling attacks. It can be difficult to determine the appropriate medication, as the condition is intermittent and outbreaks also typically clear up without any treatment.There are several recognizable clinical patterns of urticaria. It is characterized by the rapidity of its fluctuation. Urticaria becomes chronic when wheals fluctuate daily or almost daily for six weeks or more. It may occur continuously for a long duration or daily for a few days and then not for a week or two, just to reappear again. When continuous urticarial activity is punctuated by gaps of several weeks or months or, in other words, if the episodes are of shorter duration than the symptom-free period, it is better to name it episodic or recurrent. Recognizing this pattern may be helpful in identifying an external trigger factor.Chronic urticaria remains a major problem in terms of etiology, investigation, and management. It may cause considerable distress and may last for years, but it can often be alleviated by appropriate management. The remainder of this article will review the management of chronic urticaria excluding autoimmune urticaria, contact urticaria, and delayed pressure urticaria, as these topics are discussed elsewhere.We still do not know the etiology in a majority of urticaria patients. Establishing the cause of chronic urticaria is difficult and at times almost impossible. A “Practice Parameter” for the diagnosis and management of chronic urticaria was published in 2000, emphasizing the conditions that need to be considered in the differential diagnosis. The mostRoutine laboratory screening does not contribute substantially to the diagnosis and etiology of chronic urticaria. It has bAutologous serum skin test (ASST) should be performed in all antihistamine-nonresponders. The ASST has a sensitivity of 70% and a specificity of 80%. A positive test is suggestive, but not diagnostic of an autoimmune basis for the patients' chronic urticaria. Skin bioChronic idiopathic urticaria is a frequent problem where response to treatment is often disappointing. Treatment of patients with chronic idiopathic urticaria involves reducing symptoms with least invasive therapy and carefully balancing risk and benefit. The goal of the therapy is to reduce symptoms until spontaneous resolution occurs. Management of a patient with chronic idiopathic urticaria can be both frustrating and rewarding. The initial management of chronic urticaria should be directed according to the results of a full clinical assessment. A step by step approach should be taken.Many pharmacological and nonpharmacological interventions are available, but none is accepted universally. For this reason, the treatment regime should be tailored to individual patients. The key features of all regimes should be removal of any identifiable cause, avoidance of aggravating factors, advice, explanation, information, and reassurance.Treatment of chronic urticaria presents a challenge to both physicians as well as patients. Advice on general measures and information can be helpful to most patients with chronic urticaria. Reassurance is another important aspect of therapy, as appearance of urticarial rash is often more frightening than the generally favorable prognosis warrants. Also some patients become quite distraught because of their own inability to figure out what is bringing on their hives and many get more discouraged by the physician's apparent inability to delineate a specific cause.Patients should be informed that: (1) although the diagnosis is clear, in most cases the cause may not be identifiable; (2) the condition is not contagious, serious, or a sign of cancer; (3) unless it is related to underlying systemic disease, the condition will resolve eventually. A clear explanation that chronic ordinary urticaria is not an allergic problem is often a useful point to address and failure to do this often leads to dissatisfaction, with the fact that allergy tests are not offered immediately.Most treatment plans for urticaria involve being aware of one's own triggers, but this can be difficult or impossible. Obviously, the preferred form of treatment is avoidance of the causative agent in the exceptional case where it can be identified and avoidance is feasible. In addition to avoidance of the possible causative factor, patients with chronic urticaria should be appropriately advised to avoid, to the extent feasible, common potentiating factors, such as, alcohol overuse, excessive tiredness, emotional stress, overheated surroundings, aspirin, and other nonsteroidal anti-inflammatory drugs (NSAIDs).Avoidance of aspirin and other NSAIDs should usually be recommended, because these drugs aggravate chronic urticaria in 30% of the patients.–8 RegulaAvoidance of dietary pseudoallergens including food additives , natural salicylates , and unidentified aromatic substances in tomatoes, herbs, and white wine have not been found beneficial in treating chronic urticaria.–11 A strHelicobacter pylori might be associated with chronic urticaria in some patients, and eradication of H. pylori is associated with remission of chronic urticaria.[Chronic urticaria may be a manifestation of an underlying disease, and in these cases, treatment of the underlying condition may be warranted. The best example of a systemic condition that is commonly associated with chronic urticaria is autoimmune thyroid disease, that is, Hashimoto's thyroiditis. Treatment with thyroxine has been reported to alleviate the urticaria. A few rerticaria.–14 Accorrticaria. Use of nrticaria. A few carticaria.Similar too many other therapeutically challenging disorders, chronic idiopathic urticaria has seen an abundance of fad therapies, including ayurvedic and homeopathic medications and naturopathy come and go.Frequent tepid showers and application of soothing lotions can be prescribed as cooling agents when wheals erupt and are most pruritic. These inA practical approach to chronic idiopathic urticaria bases its treatment on the severity of the disease and the patient's previous response to therapy. The mainstay of this therapy is the use of antihistamines that are considered as first-line drugs for treating chronic idiopathic urticaria. Second-line drugs include systemic corticosteroids, leukotriene antagonists, mast cell stabilizer, doxepin, thyroxine, nifedipine, terbutaline, sulfasalazine, and so on.–24Histamine is the main mediator of urticaria and antihistamines are reasonably effective for treating patients with chronic urticaria. Various studies on the role of antihistamines in chronic urticaria showed a 44 to 90% response rate. To get cClassic (sedating) H-1 antihistamine—chlorpheniramine, hydroxyzine, diphenhydramine.Second/Newer generation (nonsedating) H-1 antihistamine—loratadine, cetirizine, terfenadine, mizolastine.Second generation H-1 antihistamine derivatives—desloratadine, levocetirizine, fexofenadine.H-2 antihistamines—cimetidine, ranitidine.The newer generation H-1 antihistamines with less sedating and less anticholinergic effects are preferred over the older generation H-1 antihistamines, as the initial choice of therapy. Combining drugs from the different groups and with nonhistaminic drugs can lead to a better control of urticaria. Treatment should be started with a nonsedating antihistamine at the licensed dosage, taken at the same time each day. Some of the workers prefer to combine newer antihistamine with classic sedating antihistamine at night and also with H-2 antihistamine. Addition of H-2 antihistamine may sometimes provide a better control of urticaria than an H-1 antihistamine taken alone. Although in practice, it may be more helpful for the dyspepsia that may accompany severe urticaria. H-2 antihistamines alone have no effect on histamine-induced pruritus, hence should not be used on their own. It has bIt is best to avoid all antihistamines in pregnancy, especially during the first trimester, although none has been shown to be teratogenic in humans. Hydroxyzine is the only antihistamine to be specifically contraindicated during the early stages of pregnancy. Chlorpheniramine is often chosen for pregnant women when antihistamines are essential, because it has a long safety record. None of the currently licensed anti-histamines is contraindicated in children 12 years and older. Short acting classic antihistamines still have a place in the primary management of urticaria in children between the ages of 1 and 6 years.–27Cetirizine and levocetirizine should be avoided in severe renal impairment (creatinine clearance <10ml/min), whereas, loratadine and desloratadine should be used with caution. Mizolastine is contraindicated by significant hepatic impairment. Clinically important drug interactions are infrequent, but coadministration of mizolastine with systemic erythromycin and ketoconazole is contraindicated, because of a concern about the possible occurrence of cardiac arrhythmia, as noted with terfenadine and astemizole. Caution is also advised with use of other hepatic cytochrome P450 3A4 inhibitors or substrates, including cimetidine, cyclosporine, and nifedipine, all of which may be used for urticaria in special situations.[Sixty percent of the patients with chronic urticaria respond well or reasonably well to antihistamines, but 40% derive little or no relief. There are many possible second-line drugs to consider when urticaria does not respond to antihistamines. Helping these patients is challenging and is influenced considerably by a full clinical assessment, experience, and thorough knowledge of the literature of the clinician. It is unusual for children to require second-line drugs, except for the occasional use of systemic corticosteroids for very severe, acute exacerbations of chronic urticaria. Second-line drugs should be used with considerable care in pregnancy.Doxepin is a tricyclic antidepressant with very potent H-1 and H-2 antihistaminic properties. Doxepin is more effective than diphenhydramine at a dosage of 10 mg thrice daily. Doxepin should not be advised after a recent myocardial infarction or for patients with severe liver disease. It is best to start at a low dose of 10 mg daily at night, which can be increased to 20 or 30 mg a day.–29 Mirtaβ-agonists like terbutaline and epinephrine also have been combined with H-1 antihistamine in a small number of patients, althoughNifedipine is a calcium channel blocking drug that possesses mast cell stabilizing properties as well. Nifedipine should be started at a dosage of 5 mg thrice daily and can be increased up to 20 mg thrice daily.The use of prednisolone in the treatment of urticaria is a controversial issue. It is effective for nearly all presentations of chronic urticaria, but often needs to be used at high doses to achieve a good control of urticaria. A short course of systemic steroids (30-40 mg of prednisolone/day) can be given in resistant cases of chronic urticaria that have not responded to antihistamines. Patients must understand that although systemic steroids may be the only treatment that completely eliminates their lesions, long-term use is unacceptable because of their known adverse effects, such as, diabetes mellitus, hypertension, osteoporosis, and gastrointestinal bleeding.–34Leukotriene receptor antagonists zafirlukast and montelukast have been seen to have a beneficial effect in the treatment of a few cases of chronic urticaria. The daily dosage of montelukast is 10 mg once a day at bed time and that of zafirlukast is 20 mg twice a day. However, most studies do not support their role in treating chronic urticaria.–37 ZileuSulfasalazine has been reported for the treatment of steroid-dependent chronic urticaria, ordinary urticaria. The usual starting dosage is 1 gm twice daily, leading to a maximum regular dosage of 4 gm daily.Thyroxine supplementation has been reported to suppress chronic ordinary urticaria in biochemically euthyroid patients with antithyroid autoantibody. The dosage of thyroxine used in the studies varied from 50 mg to 250 mg daily. Thyroid function tests should be done before starting the treatment and rechecked after four to six weeks of therapy.[Rofecoxib, a newer cyclo-oxygenase-2 (COX-2) inhibitor, has shown promise in treating patients with refractory chronic idiopathic urticaria.Third-line therapy typically involves the use of immunomodulatory drugs for treating refractory chronic urticaria. These agCyclosporine has been shown to be effective in severe unremitting urticaria that has had a poor response to conventional treatment with antihistamines. Cyclosporine 3-5mg/kg/day appears to benefit about two-thirds of patients with chronic urticaria who do not respond to antihistamine.High dose of intravenous immunoglobulin (IVIg) and plasmapheresis have been found to be associated with some apparent benefits in the treatment of chronic urticaria.–42Dapsone, colchicine, hydroxychloroquine, oral tacrolimus, cyclophosphamide, and low dose methotrexate, which have immunomodulatory properties, are effective in the treatment of chronic urticaria.3343–4533et al., documented a significant improvement in nine patients with chronic urticaria after they had taken mycophenolate mofetil for 12 weeks.[A recent report by Shahar 12 weeks.A few studies have found that warfarin appears to have beneficial effects in some patients with chronic urticaria.Omalizumab, a humanized monoclonal antibody, has recently been reported to have been effective in three patients with chronic urticaria.In a recent placebo controlled study, autohemotherapy has shown a beneficial role in ASST positive chronic urticaria patients. Autologo


Here, we identify the first component in a presynaptic calcium signaling pathway.Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca2+]o than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca2+]o sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca2+]o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca2+]o following its entry via voltage-activated Ca2+ channels. CaSR may detects such falls in [Ca2+]o and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca2+]o on release probability. CaSR is positioned to detect the dynamic changes of [Ca2+]o and provide presynaptic feedback that will alter brain excitability.Our experiments identify CaSR as the first component in the [Ca Recent studies have underlined the central role in regulation of neurotransmission of a number of calcium signaling pathways 2+2+ entry via voltage-activated Ca2+ channels (VACC) and less attention has been focused on the presynaptic role of other Ca2+ signaling pathways, such as surface charge screening 2+-dependent ion channels 2+]o employed in most studies ensured maximal receptor activation and decreased the impact of physiological decreases in external [Ca2+].Neurotransmitter release from nerve terminals underlies synaptic communication in the brain. Invasion of the nerve terminal by an axonal spike activates voltage-gated channels, triggering calcium entry and exocytosis of transmitter-containing vesicles 2+]o receptors include the extracellular calcium-sensing receptor (CaSR), metabotropic glutamate receptor (mGluR) and γ-aminobutyric acid B receptor (GABABR). All of these receptors have been identified as sensitive to [Ca2+]oBR In this study we identify the receptor activating a novel calcium signaling pathway using direct patch clamp recordings from nerve terminals. Candidate [Ca2+]o sensor-NSCC pathway in nerve terminals, show that the calcium-sensing receptor (CaSR), the mainstay in serum calcium homeostasis, is the extracellular calcium sensor regulating NSCC activity in neocortical nerve terminals. In contrast, nerve terminals were insensitive to glutamate and GABA arguing strongly against the mGluR and the GABABR mediating these effects. This approach provides insight into a novel pathway through which [Ca2+]o influences nerve terminal excitability.Our experiments, studying the impact of CaSR agonists and a CaSR mutation on the [Ca2+]o and indirectly modulate a NSCC current , which has an elevated serum [Ca2+] reflecting lower affinity for [Ca2+]o but normal growth and survival 2+]o. In cell-attached recordings, 71 of 76 mouse neocortical nerve terminals (93%) possessed the characteristic [Ca2+]o-modulated outward current. Outward NSCC currents were activated by depolarization from −40 mV to 110 mV  while [Ca2+]o was changed between 6 µM and 60 mM . We measured the NSCC current amplitudes elicited by 6 µM and 60 mM bath Ca2+ to test if the maximum and minimum currents respectively were also dependent on genotype. However, CaSR+/+ and CaSR+/− terminals had similar maximum  and minimum currents  indicating the IC50, but not NSCC current amplitude was dependent on the CaSR genotype. The reduction in affinity for [Ca2+]o in CaSR+/− terminals was consistent with CaSR involvement in modulation of the NSCC currents. The relatively modest shift in affinity is similar to the changes observed following heterologous co-expression of normal and other mutant CaSR Small, acutely isolated neocortical nerve terminals sense o-modulated NSCC. Allosteric CaSR agonists bind to a transmembrane pocket of CaSR 2+]o-modulated NSCC currents in rat synaptosomes were sensitive to Calindol, an allosteric CaSR agonist 2+]o-modulated outward current. In the cell-attached configuration, NSCC currents activated by depolarization with a [Ca2+]o of 60 µM were substantially and reversibly inhibited by bath application of 10 µM Calindol  from 6 mM to 60 µM  resulted in increases in NSCC current amplitude at a fixed bath [Ca2+] (blue line). Like the initial decrease in response to Calindol, the other changes in NSCC current amplitude were well described by exponential time courses. The average IC50 for Calindol was 6.3±1.1 µM at 60 µM bath [Ca2+] . Increasing bath [Ca2+] from 1 to 5 mM (at t = 0) resulted in an average increase in F/F0 of 0.400±0.017 . After a five minute wash in 1 mM Ca, application of Calindol (10 µM) resulted in a F/F0 rise of 0.269±0.017 in the same 12 cells (2+. Measurement of the latency (by deflection >4 S.D. above baseline noise) showed a substantial increase with Calindol compared to 5 mM Ca2+  in heterologously expressed CaSR consistent with our observation at isolated small terminals.Calindol has not previously been reported to exhibit any delay or latency of action. If Calindol's action on the o-modulated NSCC signaling pathway in nerve terminals involves CaSR. In addition, the distinct kinetics of action for Calindol and Ca2+ confirm different mechanisms of action for these two classes of CaSR agonist.These data strongly support the proposal that the [CaBR were all possible candidates for the neocortical [Ca2+]o sensor as they are sensitive to [Ca2+]oBR were mediating part of the response of the terminals to changes in [Ca2+]o. We addressed this issue by testing whether glutamate or GABA modulate the NSCC currents in isolated rat neocortical nerve terminals. In cell-attached recordings, depolarization from −40 to 110 mV activated outward currents through NSCCs that increased as bath [Ca2+] was reduced  in a parallel set of experiments (n = 3). GABA was equally ineffective at altering current amplitude, activation or responsiveness to [Ca2+]o . The abs2+]o will depress subsequent synaptic transmission due to the exquisite sensitivity of transmitter release to [Ca2+]o2+]o and indirectly activates a voltage-dependent NSCC was identified in nerve terminals in the neocortex 2+]o-sensor in the [Ca2+]o-sensor-NSCC signaling pathway in neocortical terminals. Consequently presynaptic CaSR is positioned to alter presynaptic excitability and attenuate the impact of falls in [Ca2+]o on synaptic transmission 2+]o suggesting the previously isolated CaSR-mGluR and CaSR-GABABR heterodimers may operate in alternative subcellular compartments or in other brain regions.Sustained decreases in [Ca2+]o has reduced potency at terminals from heterozygous mutant CaSR- containing mice, and that Calindol modulates terminal NSCC currents. These data strongly support the hypothesis that the mechanism by which changes in [Ca2+]o are transduced in neocortical nerve terminals involves CaSR. Moreover, the lack of any action of glutamate or GABA on the NSCC current indicate that neither mGluR nor GABABR act as neocortical nerve terminal [Ca2+]o-sensors that regulate the NSCC current.We demonstrated that CaSR is present in nerve terminals by immunoblot and immunofluorescence, that [Ca2+]o-sensor-NSCC signaling pathway was present in the vast majority of neocortical nerve terminals from mouse (93%) and rat (79%) consistent with a pervasive role in neuronal signaling. The IC50 of Ca2+ inhibition of mouse neocortical terminal NSCC current (1.1 mM) was lower than the EC50 for Ca2+ activation of heterologously expressed CaSR (1.75–4.1 mM) 2+. Second, desensitization has also been shown to alter the CaSR concentration-effect relationship and reduce the apparent affinity of Ca2+2+]i in CaSR expressing HEK cells . This modest shift in affinity is in line with the changes reported following heterologous co-expression of normal and mutant CaSR +/+ and CaSR+/− mice are 1.2±0.1 and 1.4±0.0 mM, respectively The  ([Ca2+]i) imaging experiments, ∼105 HEK cells were plated in 500 µl medium onto poly-D-lysine-coated glass coverslips in 24-well plate. Cells were transfected with pEGFP/CaSR plasmid DNA using Lipofectamine one day after plating. Briefly, 0.8 µg pEGFP/CaSR plasmid DNA was mixed in 50 µl Opti-MEM® I Reduced Serum Medium and 10 µl Lipofectamine 2000 (Invitrogen) mixed in 50 µl Opti-MEM® I Reduced Serum Medium. The mixtures were incubated for 20 minutes and then added to each well containing cells and medium. The transfection medium was replaced with DMEM containing 5% FBS, six hours later. Cells were kept in culture for another 18 to 24 hours before use. Untransfected cells were used in control experiments 42–48 hours after plating. Stable lines of HEK cells containing mGluR1 were used once confluent.HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (FBS). For intracellular  was greater than 0.6 mM, NaCl was decreased isotonically for CaCl2. Voltage-clamp recordings were obtained using Pulse software and an EPC- amplifier 9 . Leak currents were subtracted with a p/-4 protocol. Currents were prefiltered with a 2 or 5 kHz Bessel filter and digitized at 20–100 µs per point. Calindol stock solution was made up at 10 mM in ethanol and stored as aliquots at −20°C.Recordings were made from acutely isolated single nerve terminals (synaptosomes) visualized using an inverted microscope  as previously described 2+]i was measured in HEK cells transfected with CaSR-EGFP or mGluR1 using the fluorescent indicators, X-rhod-1 or Fluo-4 respectively. Briefly, cells were loaded for 30 min at 37°C with 2 µM AM X-rhod-1 or 5 µM AM Fluo-4 in Tyrode solution, and then placed on the stage of an inverted microscope . Images were acquired using a 1.2 N.A. 63x water immersion or PlanApo 1.42 N.A. 60x oil-immersion objective and cooled CCD camera  with computer controlled shutter . Fluorophores were excited with a halogen lamp. Fluo-4 was excited at 470–490 nm and fluorescence emission measured via a LP filter 510 nm while X-rhod-1 was excited at 542–582 nm and emission measured at 604–644 nm . Images were captured and processed using Wasabi image software. The changes in [Ca2+]i are reported as fluorescence ratios (F/F0) where F0 represents the baseline fluorescence. About 10–20 cells with similar cell surface GFP fluorescence from a single field were analyzed simultaneously using Wasabi software.o were decreased by co-application of 10 µM NPS-467. Each trace represents an average of 10–20 currents at steady state solution conditions from the same synaptosome-attached recording. Timecourse of NSCC current amplitude from the same recording at three [Ca2+]o (upper trace) revealed that NPS reversibly inhibited the NSCC current amplitude.NPS-467 reversibly inhibits NSCC currents in nerve terminal recordings. NSCC currents activated by step depolarizations  in the presence of 60 µM [Ca(0.28 MB EPS)Click here for additional data file.Figure S32+]o-modulated NSCC current in rat nerve terminals was unaffected by 2 mM glutamate. NSCC currents activated by step depolarizations  over a range of bath [Ca2+]o  superimposed with those recorded in the presence of 2 mM glutamate (red traces). Each trace represents average of 10–20 currents at steady state solution conditions from the same synaptosome-attached recording.The [Ca(0.44 MB EPS)Click here for additional data file.


Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP3) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca2+ and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca2+ wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca2+ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca2+ waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.Our results suggest that PIP2 plays comprehensive roles in shaping Ca When exposed to the maturation hormone , the oocytes reenter the cell cycle and proceed with meiosis to become mature eggs. The eggs of starfish and nearly all animal species display intense mobilization of intracellular Ca2+ release in fertilized eggs in part facilitates exocytosis of cortical granules. The initial rise of Ca2+ induced by the sperm occurs at the egg cortex , and is followed by the propagation of Ca2+ waves starting from the site of sperm interaction 2+ from internal stores is mediated by several second messengers, i.e., InsP3, cyclic ADP-ribose (cADPr), and nicotinic acid adenine dinucleotide phosphate (NAADP), which bind to the cognate cytoplasmic receptors functioning as ligand-gated Ca2+ channels 3 may play distinct roles in priming (NAADP) and propagating (InsP3) the Ca2+ signals The massive Ca2+ plays a role in remodeling the actin cytoskeleton through the actin-binding proteins whose activity is regulated by Ca2+, e.g. gelsolin, but conversely the actin cytoskeleton itself may modulate the efficacy of the intracellular Ca2+-releasing mechanisms 2+ signaling in several different experimental paradigms 2+ release at fertilization while abolishing the cortical flash 2+ signaling and in exocytosis In recent studies, however, it has been shown that fine regulation of the subplasmalemmal actin cytoskeleton is also required for exocytosis in neuroendocrine cells and fertilized eggs, as well as in non-excitable cells 33 at fertilization in echinoderm eggs has been found to take place much after the peak of Ca2+ release 2+ signaling and the local PIP2 levels would provide invaluable first insights.A growing body of evidence has suggested that PIP2, a phospholipid enriched at the plasma membrane, serves not only as a metabolic precursor of InsP2+ signaling inside the egg.The PH domain of PLC-δ1 binds to PIP2 with high affinity and specificity, and has thus been widely used as a marker to visualize this phospholipid 2+  and F-actin , the PH domain of PLC-δ1 was tagged with either RFP or GFP. Within 5–10 minutes after microinjection into Astropecten aranciacus oocytes, both PH-GFP and RFP-PH were predominantly localized at the plasma membrane /Ft−1]), it was evident that the initial increase of PIP2 took place at the sperm interaction site 9 sec after the sperm-induced Ca2+ release (see the images at 0:27 and 0:36 in rel  = [Fp-Fc]/Fc) increased after fertilization, but it took place nearly 10 sec after the onset to the Ca2+ signaling (2+ over a certain level (>0.3 R.F. unit), the relative level of PIP2 at the plasma membrane  began to decrease at 40 sec  was slightly lower than that of the R40A-injected eggs  (P<0.05), which was expected from the role of PIP2 as the substrate of PLC. When Ca2+ was released by photoactivation of the caged InsP3, sequestration of PIP2 at the plasma membrane with RFP-PH failed to influence the magnitude of the Ca2+ response, as the values for the eggs microinjected with RFP-PH  and R40A  were virtually the same  than in R40A-microinjected eggs  (P<0.01)  than in the R40A-injected eggs  (P<0.05). To see if the delayed kinetics of the Ca2+ rise is due to the specific effect of RFP-PH on PIP2 binding, we have performed control experiments using neomycin that is known to bind PIP2 and inhibit PLC. It has been reported that rather a high concentration of neomycin is required to block Ca2+ signalling or PIP2 hydrolysis at fertilization, 2+ peak at fertilization was not significantly changed in neomycin-microinjected eggs  in comparison with the control eggs . On the other hand, the neomycin-injected eggs required significantly longer time  to reach the Ca2+ peaks in comparison to the control eggs  (P<0.05), which was in line with the results obtained with RFP-PH.PIP2 is coupled with InsPthe same . The maj(P<0.01) . Similar2+ response during fertilization and InsP3 uncaging  frequency of hemispheric impairment of vitelline layer elevation  and sea urchin (Paracentrotus lividus) were briefly stained prior to fertilization, by use of a lipophilic dye that selectively delineates the plasma membrane. As shown in To test if the spike formation in the perivitelline space is limited to a certain species of starfish or to a detection method (RFP-PH) , we visu3 and diacylglycerol. Furthermore, PIP2 by itself has other roles in regulating cytoskeletal structures and ion channel activities 2+ mobilization and dynamic restructuration of the actin cytoskeleton are the two major initial events at fertilization, it was of great interest to see how the local level of PIP2 changes at the site of fertilization. However, the issue of PIP2 metabolism at fertilization has been somewhat controversial. Upon fertilization, mouse eggs displayed an increase of PIP2 at the plasma membrane 2+ signaling P2 with high specificity and affinity (Kd = 1–5 µM) 3 uncaging , we have observed that the RFP-PH protein inhibits the sperm-induced Camodestly . It is cagnitude  and 7. T2+-evoked exocytosis of cortical granules deposits their contents into the perivitelline space with the assistance of protease activities 2+ signaling According to the prevailing view, the Ca2+A. pectinifera) at fertilization displayed no evident increase in lipophilic labeling at the baseline of the plasma membrane  were captured in the Gulf of Naples during the breeding season (January to June) and maintained in circulating cold seawater (16°C). The gonads containing oocytes were dissected from the central dorsal area near the arms and transferred to the filter-sterilized cold seawater. Free oocytes were isolated as single cells by sieving through gauze several times followed by repeated rinsing and low speed  sedimentation in cold filtered seawater. For fertilization experiment, immature oocytes were stimulated with 10 µM of 1-methyladenine (1-MA) for 1 h before being exposed to the spermatozoa in the filtered seawater.The Mediterranean species of starfish (The PH domain of rat PLC-δ1 (amino acids 1-140) was prepared from RNA of Wister rat brains by RT-PCR and ligated into pCRII-TOPO vector (Invitrogen). To facilitate ligation and to remain in frame, restriction site sequences and a few extra nucleotides were added to the primers whenever necessary. After DNA sequence verification, the cDNA were ligated to the pET-28b vector (Novagen), which was already ligated to the GFP cDNA. The histidine-tagged protein expressed from the resulting vector contained the PH domain at the N-terminus side of GFP, and was accordingly named PH-GFP. To make RFP-PH, the cDNA for RFP was amplified from pCDNA3.1-HSP-RFP  in a similar procedure, and ligated to the pET28b vector, which already contained the PH domain. Between the PH domain and fluorescent tag, we inserted a tether of 5 glycine residues by adding the coding sequences to the PCR primers. The intermediate plasmid constructed in this procedure expressed either the fusion protein or the PH domain alone. The plasmid producing R40A mutant version of the PH fusion proteins were prepared from RFP-PH and PH-GFP plasmid by use of the QuickChange site-directed mutagenesis kit (Strategen) and the targeting primer pairs: R40f (5′-gtggcgtagggaagccttctacaagctac-3′) and R40r (5′- gtagcttgtagaaggcttccctacgccac-3′).E.coli strain BL12 was transformed with the plasmid through the chemical method (heat shock at 42°C for 2 min). After overnight growth on LB/kanamycin plates and recovery at 4°C for >6 h, green or red colonies were selected for mass production. The overnight inoculates containing the transformed E. coli were diluted 10 fold, and further grown in 250 ml LB/kanamycine. After 90 min incubation at 30°C, the transformed E. coli were stimulated with 1 mM IPTG at 20°C for 18 h. The bacterial pellet was resuspended and sonicated in the lysis buffer containing 50 mM Tris, pH 7.9, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF and the protease inhibitors CØmplete (Roche). Supplemented with 1% Triton X-100 and agitated on ice for 15 min, the lysate was centrifuged 20,000× g at 4°C for 30 min to collect soluble fraction. The supernatant containing the fusion proteins was applied to nickel-affinity chromatography , and the imidazole-eluted 6H fusion protein was dialyzed against 10 mM Hepes buffer, pH 7.0 at 4°C for 4 h and supplemented with 100 mM L-Asp (injection buffer). The dialyzed protein was concentrated in Micron columns (Millipore) and adjusted to the desired concentration. The final protein was typically >95% pure, as judged by the coomassie-stained SDS-PAGE analysis.2+ signaling or other physiological traits of the eggs (data not shown). Fluorescent calcium dye  conjugated with 10 kDa dextran (Molecular Probes) was used in 500 µM (pipette concentration). Caged InsP3 (Molecular Probes) and the bacterially expressed proteins were prepared in the injection buffer . To activate the caged InsP3 (Molecular Probes), microinjected oocytes were irradiated with 330 nm UV light for 25 seconds by using the computer-controlled shutter system Lambda 10-2 . Alexa Fluor 488-phalloidin (Molecular Probes) was injected as described previously 2+ changes were detected using a cooled CCD camera  mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective. The quantified Ca2+ signal at a given time point was normalized to the baseline fluorescence (F0) following the formula Frel  =  [F-F0]/F0, where F represents the average fluorescence level of the entire oocyte. The incremental changes of the Ca2+ rise was analyzed by applying the formula Finst = [(Ft-Ft-1)/Ft-1] in order to visualize the site of instantaneous Ca2+ release. Likewise, the instantaneous changes of PIP2 were assessed by the same formula. PIP2 labeling in the plasma membrane was compared with that in cortical cytoplasm by applying the formula Frel = [Fpm-Fct]/Fct, where Fpm and Fct refer to the levels of fluorescence in the plasma membrane and the cortical cytoplasm, respectively. Fluorescence of Ca2+ and PIP2 images were analyzed with the MetaMorph Imaging System software . In the simultaneous monitoring of the intracellular Ca2+ response and the changes in the egg morphology and PIP2 levels at the sperm entry site (2+ dye (50 ms), transmission photomicrograph (50 ms) were captured by the cooled CCD camera, and the data were transmitted to the computer for recording.Microinjection of the oocytes was performed with an air-pressure Transjector (Eppendorf). For calibration of the injected volume for each substance, a predefined volume of the sample was delivered into the egg by use of Narishige IM16 readout type microinjector  and used for the standard. The amount of injected material was targeted to be 1% of oocyte volume, and the final concentration of the injected material was thus estimated 100-fold lower than the concentration in the injection pipette. In the case of RFP-PH, the intensity of the plasma membrane labeling was proportionally increased with the dose of the injected protein up to 330 µM (concentration in pipette). Beyond this concentration, the labeling in the plasma membrane was saturated, and the surplus probes were remained in the cytoplasm. The dose of RFP-PH used to delineate the plasma membrane (150 µM in pipette) was more than two-fold lower than the saturation point, and did not have an effect on Catry site , the timP<0.05 was considered as statistically significant.The numerical MetaMorph data were compiled and analyzed with Excel of Microsoft Office 2003. The average and variation of the data were reported as ‘mean ± standard deviation (SD)’ in all cases in this manuscript. The paired t-test was performed by use of Prism 3.0 , and Astropecten aranciacus) were microinjected with of RFP-PH or its R40A (330 µM pipette concentration) and stimulated with 1-MA (10 µM) incubation for 1 h. The resulting oocytes at metaphase I (before the extrusion of the first polar body) were fixed in filtered seawater containing 1% glutaraldehyde (pH 8.0) for 1 h at room temperature and then treated with 1% osmium tetroxide for 1 h. Specimens were dehydrated in increasing concentrations of alcohol and embedded in EPON 812. Sections were stained with 2% uranyl acetate and 0.2% lead citrate and examined with a LEO 912 AB energy filter transmission electron microscope.Immature oocytes at prophase I (Asterina pectinifera) and sea urchin (Paracentrotus lividus) eggs were treated with 1M citric acid for 30 sec and washed with FSW. To delineate the plasma membrane, the eggs were then stained by incubating with 4 or 1 µM of FM 1-43 (Molecular Probes), respectively, in FSW for 2 min. After rinse with FSW, sperm were added and the fertilized eggs were imaged with confocal microscopy for real-time monitoring.Starfish Click here for additional data file.Data S2A movie clip of video animation showing the formation of fertilization envelope. The microfilament-filled spikes were visualized with RFP-PH, and superimposed with bright field view images to delineate the perivitelline space.(3.01 MB AVI)Click here for additional data file.Data S3A short movie clip of video animation showing the fluctuating microvilli on the surface of a starfish oocyte. Following microinjection with PH-GFP, a serial of 10 sequential images were taken by confocal microscopy  and composited in MetaMorph.(1.88 MB AVI)Click here for additional data file.


Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis. All these changes occurred at lower concentrations of the drug than those required to induce a reduction in viability of melanoma cells. We also found that low concentrations of Ca2+-ionophore induced an increase in adenosine 5′-triphosphate (ATP), which most probably resulted from the increase in mitochondrial-bound hexokinase, which reflects a defence mechanism. This mechanism can no longer operate at high concentrations of the Ca2+-ionophore, which causes a decrease in mitochondrial and cytosolic hexokinase, leading to a drastic fall in ATP and melanoma cell death. The present results suggest that drugs which are capable of inducing accumulation of intracellular-free Ca2+ in melanoma cells would cause a reduction in energy-producing systems, leading to melanoma cell death. © 1999 Cancer Research CampaignCancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca