The basis to this transition is unclear. We investigate it using a biophysical Purkinje cell model under conditions of CF and PF input. The model can replicate these toggle and gain functions, dependent upon a novel account of intracellular calcium dynamics that we hypothesize to be applicable in real Purkinje cells.Without synaptic input, Purkinje neurons can spontaneously fire in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence. Climbing fiber input (CF) switches Purkinje neurons out of the trimodal firing pattern and  In contrast to the complexity of other brain regions, the cerebellar cortex has a single repeating connectivity motif Ito, . This moIn vitro, within cerebellar slices, Purkinje neurons can spontaneously fire action potentials in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence  input (1 Hz) can switch a Purkinje cell out of the trimodal firing pattern and into a tonic firing pattern interrupted, at the frequency of CF input, by a complex spike and its short evoked after-pause (~20 ms long)  and tonic firing periods of ~1 s alternate with quiescent periods of ~1 s i—added to the quiescent state's lower basal [Ca2+]i level—cannot confer sufficient hyperpolarising current flow (through Ca2+-gated SK K+ channels) to outweigh the depolarising effect of the Ca2+ entry; there is a net depolarisation and the cell is toggled to firing.During the firing state, the CF conferred rise in the intracellular Ca2+ levels, which remain elevated between 1 Hz CF stimuli , or with SK blocked by apamin, CF discharges are unable to block trimodal output  switches a Purkinje cell out of the trimodal firing pattern and into a tonic firing pattern interrupted by either quiescent periods (~1 s long) or short pauses (~20 ms)  that have the same characteristics as the long quiescent periods (>>1 s) in the experimental data. Principally, they are punctuated by CF driven spike events that fail to evoke a state transition into the firing state. Extrapolating from this similarity, we suggest that the long quiescent periods in the experimental data are, as in the model, the enduring quiescent mode of the trimodal firing pattern. Furthermore, we propose that they too are generated by the electrogenic action of Na+/K+ pumping.In the model, repetitive CF input (1 Hz) blocks the bursting mode of the model's trimodal firing pattern but not its quiescent mode, generated by electrogenic Na+/K+ pump generated silences can still occur under conditions of synaptic input and we tentatively suggest that Na+/K+ pump generated silences occur physiologically. Indeed, a sizable fraction of the quiescent periods observed in Purkinje cell firing, in vivo, have an onset and termination that is not co-incidental or driven by CF input events . This reduced model has 41 compartments as compared to 1089 in the full model. It runs significantly faster than the full model and yet faithfully reproduces its intrinsic electrical behavior  and maximal conductance values are multiplied by a correction factor, Cd = 3.80 . However, it is useful for studying the effect of multiple synaptic inputs that are diffuse over the dendritic tree. Indeed, the reduction algorithm employed (Ra conservation) has been shown to produce reduced, surrogate models that capture the synaptic integration properties of their full, parent models i.e., reduced models produced by this algorithm have been shown to perform the same non-linear integration of dendritic EPSPs and IPSPs (to have the same input-output function) as their parent models  is equivalent to Equation 55, except that it is without I+/K+ pump descriptions—one of which is highly simplified and that electrically counterbalances a highly simplified Na+/Ca2+ exchanger mechanism. Our model here only has one Na+/K+ pump description at the soma as it dispenses with the simplified Na+/K+ and Na+/Ca2+ exchanger mechanisms. It retains a realistic Na+/K+ pump description at the soma. The second disparity is that the model here, unlike that of Forrest et al.  description in the dendrites is modified to slow its inactivation, and the smooth dendrites' description of intracellular Ca2+ dynamics is reworked. A SK type Ca2+-activated K+ conductance is added to the smooth dendrites, sourced from Moczydlowski and Latorre . The dendrites do not have voltage-dependent Na+ conductances , which has been modified to inactivate very slowly (over seconds rather than milliseconds). This current is hyperpolarizing. It clamps dendritic excitability and prevents Ca2+ spike generation, which permits the tonic mode of firing. However, when this current inactivates, this excitability clamp is lost, it no longer prevents dendritic spiking and the model is switched from the tonic to the burst mode.So, in the model of Forrest et al. , the tonD is a simulacrum of the absent {Kv1.2, [K+]o accumulation, Na+/K+ pump, Na+/Ca2+ exchanger} system in the dendrites. There is some evidence that Kv1.2 is actually the channel correlate to the ID current  that slows its inactivation (refer to Equation 96 and 97). The rate of ID inactivation, modulated by this k parameter, sets the duration of the model's tonic mode in the trimodal pattern of firing.So, the model's It et al. , with an+/K+ pump at the soma  rises during the tonic and burst firing modes, as a function of voltage-gated Na+ entry. This increased intracellular Na+ enzymatically increases pump activity. Eventually, during the bursting mode, the pump generates such a hyperpolarizing current that firing stops and the model cell is driven to quiescence. In this case, without any spike associated Na+ entry, [Na+]i would be expected to stop rising. However, [Na+]i continues to increase as the Na+ influx is lagged by a parameter τ, which phenomenologically encodes the long duration of Na+ diffusion from the Na+ channel to Na+/K+ pump   inputs were introduced. CF inputs make 17 excitatory synaptic contacts upon 17 of the model's proximal smooth dendrites . The Purkinje cell is known to receive ~200,000 parallel fiber synaptic contacts  inputs were introduced. PF inputs make one excitatory synaptic contact upon each of the model's spiny dendrites , E-type (ICaE) and P-type (ICaP) voltage-gated Ca2+ currents. Parameter y is the set point Ca2+ concentration that the system strives to return the Ca2+ concentration to after a perturbation. The equation form and all of the aforementioned parameters are as in Forrest et al. (y specified as a constant (2.4 * 10−4 mM), in this model y is dictated by the following relationship:Here i accumulation on the scale of milliseconds, during Ca2+/[Ca2+]i spiking for instance, and the latter captures more gradual accumulation over seconds and minutes. So, with this layered system that uses an intermediary parameter y, it is possible to have [Ca2+]i accumulation over longer time scales without disrupting the fast spiking in [Ca2+]i, This fast dendritic spiking is crucial to the Purkinje cell model because it drives somatic bursting. Anyhow, we believe that a “creeping” Ca2+ set point is a realistic proposition, as Ca2+ efflux and buffering systems are sub-linearly dependent on [Ca2+]i because they rely on enzymes that have saturation kinetics  in the Ca2+ set point concentration (y). Their value is controlled by the value of a parameter w:The w is 0 and so the default values of g and τm are 1 * 105 and 100 respectively. However, if ICa2+ surpasses 0.06 mA/cm2, as it does when there is a climbing fiber (CF) input, w is set to 1 and the g and τm parameters are updated to 1 * 104 and 1000 respectively. The rational for these parameter changes are that the dramatic CF associated Ca2+ influx floods the nonlinear Ca2+ regulatory systems. They cannot increase their activity fast enough to match the increasing [Ca2+]i, leading to a rise in the rate of increase of [Ca2+]i. The parameter f sets the lifespan of the changed g and τm values by controlling the rate at which w attenuates. When w declines below 0.1, g and τm revert to their default small values. However, if there is another CF input event before w falls to below 0.1, w is reset to 1, which prolongs the lifespan of g and τm at their larger values for another cycle of w attenuation.The default value of 2+ spike event in the dendrites, which produces a burst event at the soma  at default but is assigned a higher value upon CF input. The value of gsk is controlled by the value of parameter r:A CF input produces a Car is 0 and so gsk at default is 1 * 10−7 S/cm2. However, if there is CF input, the synaptic current Isyn across the CF synapses exceeds 3 nA and r is set to 1, updating gsk to 0.72 S/cm2. The parameter s sets the lifespan of the changed gsk value by controlling the rate at which r attenuates. When r declines below 0.1, gsk reverts to its default value of 1 * 10−7 S/cm2. However, if there is another CF input event before r falls to below 0.1, r is reset to 1, which prolongs the lifespan of gsk at its higher value for another cycle of r attenuation.The default value of 2+ dynamics permits CF input to toggle the Purkinje cell model between a firing and a quiescent state (refer to Results). It is an extremely abstract description, but it is founded upon good rationale in the absence of conclusive experimental data—our present understanding of intracellular Ca2+ dynamics is extremely limited and so there is not much to guide its modeling. Available data does not constrain the free parameters of the model: g, τm, f, w, z, gsk, r, s. These were tuned manually , Ra is the specific axial resistivity (35.4 Ω cm), F is the Faraday constant, R is the gas constant and gmax is the maximal conductance. gmax values, for the different currents, are shown in Table m, h and z are Hodgkin-Huxley “particles”/gates  dependence of a Hodgkin–Huxley (H–H) current , either  OR . These entities are voltage (and/or intracellular Ca2+) dependent. The latter set can give the former set through the relations:The voltage , moderately TEA sensitive (IK_mid) and TEA insensitive (IK_slow) voltage-gated K+ currents, a BK voltage-and-Ca2+-gated K+ current (IBK), a resurgent Na+ current (INa−R), a P-type Ca2+ current (ICaP), a hyperpolarization activated cation current (IH), a leak current (IL), a Na+/K+ pump (ispump), an intracellular Ca2+ dynamics description and an account of intracellular Na+ accumulation. It receives a current from, or sends a current to, its connecting, adjacent dendrite compartment (Itransfer_DS). The membrane equation for the soma:The soma is a cylinder . K is the reversal potential for K+ (initiated at −88 mV), ENa is the reversal potential for Na+(initiated at +70 mV), ECa is the reversal potential for Ca2+, EL is the reversal potential for the Leak current (−70 mV), Eh is the reversal potential for the hyperpolarisation activated cation current (−30 mV), Intracellular Ca2+ concentration is initiated at the NEURON default of 5e−5 mM; Extracellular Ca2+ concentration is initiated at the NEURON default of 2 mM. The somatic membrane voltage (V) is initiated at the NEURON default of −65 mV.ED is the membrane voltage at the center of the dendrite compartment, VS is the membrane voltage at the center of the soma compartment and RDS is the axial Resistance between the two. Ra is the specific axial Resistivity, LS and rS are the length and radius of the soma respectively; LD and rD are the length and radius of the dendrite compartment respectively.VGoldman–Hodgkin–Katz (ghk) equation:2+Ca is 5 * 10−5 cm/sec, [Ca2+]i = 100 nM, [Ca2+]o = 2 mM, T = 295 K, F is the Faraday constant and R is the gas constant. [Ca2+]i and [Ca2+]o are fixed constants, as seen by this equation—it does not access the changing value of [Ca2+]i as set by the intracellular Ca2+ equations (given later).P2+] is calculated for the intracellular space within 100 nm of the membrane. [Ca2+] changes as I2+Ca  brings Ca2+ into this space and as Ca2+ leaves by diffusion to the bulk cytoplasm. The diffusion rate constant, β, is set to 1/ms.[Ca2+] at time step, t: was constrained to not fall below 100 nM by coding of the form:−1):This current is described by a Markov scheme, shown in Figure +/K+ pump  transports 3 Na+ out (ispump_Na) for every 2 K+ in (ispump_K). It has an exponential relation to intracellular Na+ concentration ([Na+]i).The somatic Na+]i is initiated at 10 mM and then changes in time,i is the intracellular Ca2+ concentration in a supra-membrane shell of depth = 0.1 μm, F is the Faraday constant, ICa2+ is the Ca2+ membrane current , kt = 4e−5 mM/ms, kd = 4e−5 mM, taur = 2 ms and y = 4e−5 mM.Where i—added to the quiescent state's lower basal [Ca2+]i level—cannot confer sufficient hyperpolarising current flow (through Ca2+-gated SK K+ channels) to outweigh the depolarising effect of the Ca2+ entry; there is a net depolarisation and the cell is toggled to firing.During the firing state, the CF conferred rise in ; (2) Na+/K+ pump conferred [>>1 s long]. The former is regulated by intracellular Ca2+ dynamics, the latter by intracellular Na+ dynamics. In addition, there can be short quiescent periods: short pauses after complex spikes (~20 ms long), which are a function of intracellular Ca2+ dynamics.So, we tentatively suggest that there are 2 different classes of In vitro, PF input increases tonic firing frequency. CF input decreases it. In fact, CF input decreases the mean frequency of firing to a range where PF input can greatly increase it, setting the gain of the PF response , the mean frequency of tonic firing shifts from 99 to 54 Hz—a 55% decrease. With CF input already introduced and then with the subsequent introduction of PF inputs, the mean frequency of tonic firing shifts from 54 to 80 Hz—a 68% increase. So, CF input confers a gain in the PF induced frequency change.h) as the critical conductance to Purkinje cell bistability. This model is heavily reduced, with no spiking dynamics and the bistability is between two different rest states (hyperpolarized and depolarized). The model cannot account for the fact that Ih block by ZD 7288, or Ih downregulation by serotonin, does not abolish bistability  then the CF toggled bistable pattern is “masked” by a pattern of continuous tonic firing, punctuated by complex spike events and their short after-pauses at the frequency of CF input  can rescue and reinstate the bistable pattern  . This Ca2+ memory has a lifespan and once it has expired the model cell responds by default. Unless there is another CF input to renew and perpetuate the memory setting.The model's intracellular CaV) is not the Purkinje cell's only coding variable. We hypothesize that [Ca2+]i is a coding variable as well. These two interact, with the Ca2+ memory being encoded and decoded by the membrane potential. Encoding is by way of CF input causing membrane depolarisation, which then opens voltage-gated Ca2+ channels to raise [Ca2+]i. Decoding is by way of [Ca2+]i modulating the membrane potential via Ca2+-activated K+ channels.So, we hypothesize that the membrane potential  in the Purkinje cell model. This system might be modulated in the physiological setting by signaling cascades, which have been shown to regulate intracellular Ca2+ dynamics . So a pause in its spiking would convey disinhibition Jaeger, . We specIn vitro, a physiological frequency of CF input (~1 Hz) switches Purkinje cells out of the trimodal firing pattern and into a nonbursting pattern of activity. On this basis it is probable that the trimodal firing pattern is not applicable in vivo. However, in these experiments, although CF input blocks the trimodal pattern's bursting mode, long (>>1 s) quiescent periods can still be observed. We hypothesize that these are the enduring quiescent mode of the trimodal pattern, generated by electrogenic Na+/K+ pumping. On this basis, we suggest that Na+/K+ pump generated silences occur physiologically. Furthermore, we propose that the intracellular Na+ concentration ([Na+]i) acts as a memory element in the Purkinje cell. It memorizes firing history and sets Na+/K+ pump activity to dictate the timing and duration of quiescent periods. This action could be externally and internally regulated: the Na+/K+ pump is a receptor for the endo-ouabain signaling molecule , which has symptoms to indicate that it is a pathology of cerebellar computation   of Na+/K+ conferred silences, via Na+ computation, we could propose that these two systems are specialized for, and operating on, different time scales.We propose that the cerebellar Purkinje cell has two memory stores in parallel: [Caup and down states in different Purkinje cells. These network states could store information and perform computations. So, these network computations sit upon the proposed intracellular ion computations  that dictate the activity state of individual Purkinje neurons.Our view is that these slow processes  are relevant and computationally advantageous because, by conferring an access to longer time scales, they permit storage and short-term processing of sensory information in the cerebellar cortex. To elaborate, they permit different dynamical states to be sustained in the cerebellar cortex for extended periods. Each of these states is associated with a specific configuration of Purkinje cells provide inhibitory input to the Deep Cerebellar Nuclei (DCN). Long quiescent periods may be important in cerebellar functioning because numerous Purkinje neurons converge upon—and inhibit—a single DCN neuron (~40:1). If all these Purkinje neurons are continually, simultaneously active then it might be that the DCN neuron is unable to fire in any meaningful way.With a proportion of the Purkinje cells quiescent, only a fraction of the population is active and relevant. The members making up this relevant sub-set can be switched and changed in a controlled fashion, which can be utilized as a computational feature.in vivo with anesthetized and awake animals. Toggling is observable in anesthetized animals  and, secondly, CF toggled quiescent periods (~1 s long); both a function of intracellular Cahttp://www.mrc.ac.uk/index.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The author declares that no competing interests exist. This work was reviewed by the author, and written up for journal publication, in 2012 (with no funding appropriated for this task). The author had previously presented his findings as a component of a poster presentation in 2009: Forrest MD, Wall MJ, Press DA. The Sodium-Potassium Pump Controls the Intrinsic Firing of the Cerebellar Purkinje Neuron. Poster session presented at: 16th International Conference on Neural Information Processing; 2009 Dec 1–5; Bangkok, Thailand.The author conducted this work in the period 2006–2007, whilst doing his Ph.D. degree at the University of Warwick. He was funded at this time by the Medical Research Council (MRC) of the UK, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, 45Ca uptake was measured, and changes in the intracellular Ca2+ concentration ([Ca2+]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca2+]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of 45Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca2+ and the NMDAR-mediated influx of Ca2+ into neurons participate in the mechanism of TBBPA-induced Ca2+ imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca Tetrabromobisphenol A (TBBPA) belongs to a large group of brominated flame retardants (BFRs) that are heat-resistant and decrease the chance of ignition of electronic equipment, textiles, and building materials. The economic profits of the use of BFRs are overshadowed by potential environmental and toxicological hazards (Alaee and Wenning 2+ ([Ca2+]i) , which suggested that glutamate receptors can mediate these processes. Using the TM4 Sertoli cell line model, Ogunbayo et al. (2+ stores via ryanodine receptors (RyRs) and proposed a role of this effect in TBBPA cytotoxicity. Other studies revealed that in addition to intracellular Ca2+ release, TBBPA strongly inhibits the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) and voltage-sensitive Ca2+ channels   -5-methyl-10,11-dihydro-5H-dibenzo·cyclohepten-5,10-imine hydrogen maleate (MK-801), dimethyl sulfoxide (DMSO), and propidium iodide, which were of analytical grade, as well as fetal bovine serum, cytosine β-d-arabinofuranoside, and the other materials for cell culturing were purchased from Sigma-Aldrich Chemical Poland . Bastadin 12, which was provided by Dr. Emmanuel N. Pitsinos , was synthesized there according to the method of Couladouros et al. . All the other chemicals were of analytical grade.Tetrabromobisphenol A propane, TBBPA) and 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95) were obtained from LCG Standards Poland . s et al. . Fluo-3APrimary neuronal cultures were prepared from 7-day-old Wistar rats. For these experiments, we used rat pups, which were obtained from the animal colony of the Mossakowski Medical Research Centre, Polish Academy of Sciences in Warsaw. Mothers and pups were fed and watered ad libitum and kept on a 12:12 h dark-light cycle, at room temperature with a constant humidity of approximately 60 %. The procedures were approved by the Fourth Local Ethical Committee in Warsaw and were performed in accordance with EC Directive 86/609/EEC of 24 November 1986. We made all efforts to reduce the number of animals used and to minimize their suffering.6 cells per well on 24-,12- or 6-well plates (Thermo Scientific™ Nunc™) coated with poly-l-lysine. After 48 h of culturing, 7.5 μM cytosine arabinofuranoside was applied to inhibit DNA replication and the growth of non-neuronal cells. The experiments were performed after 7 days in vitro.CGC were isolated and cultured according to a standard method , and 5 mM glucose and pre-incubated for 5 min in this medium. Then, 0.5 µM MK-801, and/or 2.5 µM bastadin 12 together with 200 µM ryanodine were added for an additional 5 min before application of TBBPA, DMSO, and positive controls glutamate or PCB 95, as presented in Figs. 6 cells per well) were preincubated with 4 μM fluo-3AM. After washing the cultures three times with Locke 5 buffer, fluorescence was measured using a microplate reader  set at 485 nm excitation and 538 nm emission wavelengths. Baseline fluorescence was measured, and then the changes in fluorescence after the addition of each of the tested compounds were recorded every 60 s.Changes in intracellular CaF/F0%). In addition, quantitative data characterizing the increase in fluo-3 fluorescence above the basal level, measured just after the rapid phase of the Ca2+ transient and at the steady-state level, i.e., 60 and 300 s after TBBPA or glutamate application, respectively, and the significance of the differences are presented in Tables p < 0.05), the corresponding data obtained in separate experiments were combined, analyzed as a single representative set for each treatment and presented as the mean ± SD of 15 repetitions. The data in Fig. There was no need to correct for autofluorescence in either of the two experimental settings, as it was stable and did not exceed 10 % of the basal level of fluorescence of plates with fluo-3-loaded CGC. The results in Figs. 6/well) were pre-incubated for 5 min in the Locke 5 medium, followed by a 5-min incubation without or with 0.5 µM MK-801 and/or 2.5 µM bastadin 12 together with 200 µM ryanodine. Then, 7.5, 10 or 25 µM TBBPA, 0.5 % DMSO (the vehicle control), or else the positive controls 100 µM glutamate or 25 µM PCB 95, were added for 10 min together with 45CaCl2 (1 μCi/well). The incubation was terminated by washing with ice-cold calcium-free medium containing 2 mM EGTA, and the cells were dissolved in 0.5 M NaOH. The content of radioactive 45Ca in the cultures was measured by liquid scintillation spectroscopy using a Wallac 1409 counter . The data in Figs. The procedure was basically performed as described previously  with Locke 25 buffer containing 134 mM NaCl, 25 mM KCl, 2.3 mM CaCl2, 4 mM NaHCO3, 5 mM HEPES (pH 7.4), and 5 mM glucose. As required, freshly prepared solutions of TBBPA or bastadin 12 (both in DMSO), vehicle (0.5 % DMSO), glutamate, or PCB 95 in DMSO (the positive controls), ryanodine, or MK-801 were also included. To evaluate TBBPA or glutamate toxicity, the cells were incubated with these substances at 37 °C for 30 min; then, the exposure was terminated by two washes with Locke 25 buffer, and the CGC were cultured in the original growth medium for an additional 24 h under standard conditions  was used.The results are presented as mean ± SD. Differences in corresponding data points between different groups were tested with one-way ANOVA followed by Dunn’s correction method. For all tests, 2+]i, in the primary CGC cultures are presented in Figs. 2+]i to the maximal levels of 292, 417, and 521 % relative to the basal level, respectively, whereas administration of the vehicle, 0.5 % DMSO, did not change basal fluo-3 fluorescence , did not interfere with the increases in i evoked by 7.5 µM TBBPA in CGC cultures using a fluorescence plate reader as a platform for measuring fluo-3 fluorescence. In contrast to the experiments using a fluorescence microscope, data from the fluorescence plate reader showed a steady upward trend of F/F0% . Notwithstanding the slight increase in [Ca2+]i evoked by 7.5 µM TBBPA, and a tendency to its partial inhibition by both MK-801 and bastadin 12 plus ryanodine, the application of these substances in combination resulted in a nearly complete inhibition of the Ca2+ transients . DMSO (0.5 %), the vehicle for TBBPA, bastadin 12, and PCB 95, had no effect on 45Ca uptake, whereas an antagonist of NMDARs, 0.5 µM MK-801, reduced basal 45Ca uptake by half  higher than the viability observed after separate application of these antagonists of NMDARs and RyRs  concentration-dependently increased 45Ca uptake, which was completely abolished by MK-801. Treatment with the RyR-inhibiting mixture composed of bastadin 12 with ryanodine did not reduce this effect, but instead produced MK-801-sensitive increases in 45Ca uptake . These data indicate that the mechanisms that trigger TBBPA cytotoxicity in CGC are complex, comprising both Ca2+-dependent, NMDAR- and RyR- mediated processes and Ca2+-independent processes.Our results Fig. a, b show2+-free buffer provided less extensive protection from this cytotoxicity, suggesting a role of Ca2+ influx via NMDARs in TBBPA cytotoxicity. Our present results, which were obtained under different conditions involving the acute exposure of CGC to TBBPA and using 0.5 µM MK-801, together with new data on Ca2+ transients, directly confirmed the role of NMDARs in TBBPA cytotoxicity. Moreover, we confirmed the cytoprotective potential of the well-tolerated RyR-inhibiting mixture composed of bastadin 12 with ryanodine. Although the efficacy of these substances in the inhibition of intracellular Ca2+ release in CGC challenged with thapsigargin or TBBPA was demonstrated previously , are involved in the acute cytotoxicity of TBBPA.In conclusion, the results of this in vitro study directly demonstrated that in CGC, TBBPA-induced increases in [Ca


Background. We hereby describe what we believe to be the first reported case of t   associated with acute myeloid leukemia (AML). Methods. PubMed, Embase, and OVID search engines were used to review the related literature and similar published cases. Case. A47-year-old female presented in December 2011 with AML (acute myelomonocytic leukemia) with normal cytogenetics; molecular testing revealed FLT-3 internal tandem duplication (ITD) mutation, while no mutations involving FLT3 D385/I836, NPM1 exon 12, or KIT exons 8 and 17 were detected. She was induced with 7 + 3 (cytarabine + idarubicin) and achieved complete remission after a second induction with high-dose cytarabine (HiDAC) followed by uneventful consolidation. She presented 19 months after diagnosis with relapsed disease. Of note, at relapse cytogenetic analysis revealed t  , while FLT-3 analysis showed a codon D835 mutation (no ITD mutation was detected). She proved refractory to the initial clofarabine-based regimen, so FLAG-idarubicin then was used. She continued to have persistent disease, and she was discharged on best supportive care. Conclusion. Based on this single case of AML with t  , this newly reported translocation may be associated with refractory disease. Genetic evaluation plays an integral role in the classification of AML –3. Appro FLT-3 D835/I836. FLT3-ITD mutations are an adverse prognostic factor. However, the significance of FLT3-TKD mutations is unclear; they are overrepresented in the PML-RARA and inv(16) AML subtypes and more generally in the intermediate-risk cytogenetic subgroup [Similarly, mutational analysis plays an important and expanding role in the diagnosis and management of AML. FLT3 is a member of the class III receptor tyrosine kinase family important for the normal development of hematopoietic stem cells and the immune system. FLT3 is the most commonly mutated gene in AML, with mutations occurring in about 30% of cases overall. There are several known activating mutations of FLT3 in AML, including internal tandem duplications (ITD) and mutations in the activation loop of the second tyrosine kinase domain (TKD) such assubgroup .We hereby describe what we believe to be the first reported case of t   associated with AML. Relapse in this case also is notable for alterations in the FLT3 status  in addition to the appearance of this newly described translocation.3 , a hemoglobin of 7.5 g/dL, and a platelet count of 43,000/mm3. She did not have a significant medical or surgical history, although she had a 27-pack-year smoking history. Her father died from pancreatic cancer. On exam her vital signs were within normal limits. No splenomegaly or lymphadenopathy was noted.The patient was a 47-year-old white female who initially presented with abdominal pain. She was noted to have leukocytosis with numerous blasts in her peripheral blood; her CBC included a WBC count of 16,900/mmBone marrow specimens were obtained for morphologic, immunophenotypic, and genetic analyses. Based on morphology and flow cytometric analysis, the patient was diagnosed with AML (acute myelomonocytic leukemia). Flow cytometry showed 25% blasts expressing CD45 (moderate intensity), CD34, CD13, CD117 , CD33 , HLA-DR , CD4 , and CD15 ; the blasts had no significant expression of CD14, CD64, CD10, CD19, CD20, surface immunoglobulin light chains, CD2, CD3, CD5, CD7, CD8, or CD56. It also noted 31% monocytes expressing CD45 (bright), CD13, CD33, CD4, CD64, HLA-DR, CD14 , and CD15  with possible slight expression of CD34 and CD2; the monocytes exhibited no expression of CD117 or the other analyzed lymphoid antigens.Routine cytogenetic analysis revealed a normal female karyotype . A multiplex, nested reverse transcription PCR assay for 16 of the more common recurrent chromosomal rearrangements associated with acute leukemia did not detect any abnormalities. Molecular assays for detection of mutations involving FLT3 ITD, FLT3 D835/I836, NPM1 exon 12, and KIT exons 8 and 17 were performed; only a FLT3 ITD mutation was detected.2/day continuous IV infusion on days 1–7 and idarubicin 12 mg/m2/d IV on days 1–3). The day 18 bone marrow evaluation was suspicious for residual AML, so a second induction was completed with HiDAC . A bone marrow evaluation after the second induction revealed no evidence of residual AML. She was consolidated with 4 additional cycles of HiDAC. She achieved complete remission with a normal CBC and transfusion independence. She was not a candidate for allogeneic stem cell transplantation due to the lack of an appropriate donor and concerning psychosocial assessment.The patient was started on induction chemotherapy with 7 + 3 , a hemoglobin of 11.0 g/dL, and a platelet count of 83,000/mm3.Fourteen months later, she presented with relapsed AML. Her CBC showed a WBC count of 4,000/mmA restaging bone marrow confirmed relapsed AML. Flow cytometry showed somewhat similar results to those at diagnosis. There were 36% blasts expressing CD45 (dim), CD13, CD117 , CD33, CD4 , and CD15 ; the blasts exhibited minimal expression of CD34 and HLA-DR and no significant expression of CD14, CD64, CD10, CD19, CD20, surface immunoglobulin light chains, CD2, CD3, CD5, CD7, CD8, or CD56. There also were 25% monocytes expressing CD45 (bright), CD13 , CD33, CD4, CD64, HLA-DR , CD14 , and CD15; the monocytes exhibited no expression of CD34, CD117, or the analyzed lymphoid antigens.Repeated routine cytogenetic analysis revealed an abnormal karyotype with t   in 16 of 20 analyzed cells . Molecul2/day IV on days 1–5, cytarabine 20 mg/m2/day SC on days 1–14, and sorafenib 400 mg PO BID on days 1–14). Unfortunately the postinduction day 22 bone marrow showed persistent AML, including the presence of t  . A second induction was performed with FLAG-idarubicin ; this treatment was complicated with prolonged neutropenia and a disseminated Fusarium infection involving sinus and cutaneous tissues. The day 40 bone marrow biopsy was consistent with persistent AML; it was hypocellular with left-shifted granulocytic maturation, decreased erythropoiesis, and dysmegakaryopoiesis. As a result, the patient was discharged on hospice care.Her relapse initially was treated with a clofarabine-based regimen  q32; q13). The genes activated or inactivated as a result of this translocation as well as the overall significance of this translocation in the disease process are unclear. Chromosome 14q32 encodes the IgH gene, which produces heavy chain immunoglobulin. Translocations involving the IgH gene/14q32 are well established in multiple hematologic malignancies including diffuse large B-cell lymphoma [2; q13. T; 15 q32;Additional sporadic cases of AML with translocations involving 14q32 have been reported. For example, Tecimer et al.  reportedIn our case the relapsed leukemia demonstrated refractoriness to multiagent chemotherapy regimens. This may represent either clonal evolution or an entirely new clone which can reflect a therapy related resistant disease after exposure to leukemia treatment on initial presentation. The lack of the FLT-3 ITD mutation, the presence of a FLT-3 D835 codon mutation, and the new onset of t   could support the second interpretation. In the setting of AML, this translocation is of unknown potential but may be linked to refractoriness to chemotherapy. However, potential unidentified mutation involving partner genes can lead also refractory disease. Collecting and reporting data of rare chromosomal abnormalities will potentially add more information concerning the pathogenesis and prognosis of AML. Additionally, it may translate to improved patient outcome and management in the future if identified earlier in the clinical course which may aid in selecting patients to undergo more aggressive chemotherapy regimens and allogeneic stem cell transplant.


This Frontiers issue on Ca2+ signaling and cardiac rhythm first focuses on the spontaneous membrane depolarization triggering action potential (AP) pacing by sino-atrial node (SAN) cells. These drive normal rhythmic atrial followed by ventricular depolarization initiating effective systolic contraction  increases produced the expected INCX changes . Furthermore, intracellular [Ca2+] proved instrumental in determining pacing rates: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) dose-dependently slowed, ultimately abolishing, AP firing in isolated guinea-pig SAN myocytes (Capel and Terrar). Involvement of ICa in both SAN pacing and atrioventricular conduction was indicated in mice homozygously lacking L-type, Cav1.3, or T-type, Cav3.1, channels normally expressed in mouse, rabbit and human pacemaker tissue . Volume and pressure overload-induced heart failure in rabbit SAN cells markedly influenced both Ca2+ transients and pacemaker activity . Finally, the hypothesis generated schemes amenable to quantitative modeling and reconstruction .The present articles first complete necessary conditions for such a Ca2+ entry are up-regulated in clinical and experimental atrial fibrillation (AF), and are implicated in SAN dysfunction and atrioventricular block . Similarly, intracellular Ca2+ store depletion increased Ca2+ entry in isolated firing mouse SAN pacemaker cells, findings reduced by store-operated Ca2+ entry (SOCE) blockers. SAN pacemaker cells further expressed the endoplasmic reticular, Ca2+-sensing, stromal interacting molecules (STIM) and surface membrane Orai1 channels likely involved in SOCE. Ca2+ store depletion redistributed STIM1 to the cell periphery increasing STIM1-Ora1 co-localization .The articles then explore further ion channel mechanisms possibly contributing to this regulation. TRPC3 channels mediating Ca2+ transients and diastolic SR Ca2+ release appear to be important AF triggers in murine hearts  .SAN and surrounding atrial tissue form a SAN-atrial pacemaker complex. SAN disorders accordingly can produce re-entrant substrate causing AF in addition to bradycardic, sinus node, dysfunction  deficiency, previously identified with hypertrophic ventricular remodeling in heart failure, through altered post-transcriptional activity of key Ca2+-handling proteins, particularly SR Ca2+-ATPase . Similarly, altered protein phosphatase 2A expression and activity, likely acting downstream of Pak1, may compromise responses to β-adrenergic stimulation with implications for arrhythmia and cardiac failure . Finally, membrane protein regulation, trafficking and recycling are fundamental to all cellular physiological processes including those involving Ca2+ homeostasis. This prompted review of a particular, endosome-based, trafficking process, involving endocytic C-terminal Eps15 homology domain-containing regulatory proteins .Further articles bear upon modulatory influences upon the complex of Ca2+-mediated modulatory effects on cardiac function as a whole must extend such molecular and cellular analysis from Ca2+ homeostatic to contractile function in entire cardiac chambers . Simulations of this kind offer openings into more detailed and quantitative studies of sino-atrial and atrial intricacies.Ultimately, quantitative analysis of CaExplorations of the kind described in this series of articles thus contribute to development of a systems basis for sinus node disorder (SND), atrial arrhythmia, and their translational consequences Nattel, . SND is CH, Drafting and planning of the editorial, response to editors report; RS, Re-reading and rewriting of the editorial; YK, Re-reading and checking of the editorial; and ML, Final corrections and rewordings, response to editors report.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chemical (acetylcholine) activation triggered spatiotemporally complex, propagating inositol trisphosphate (IP3)‐mediated Ca2+ waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3‐mediated Ca2+ signals in all activated cells. By computationally modelling Ca2+ release, endothelial shape changes were shown to alter the geometry of the Ca2+ diffusive environment near IP3 receptor microdomains to limit IP3‐mediated Ca2+ signals as pressure increased. Changes in cell shape produce a geometric microdomain regulation of IP3‐mediated Ca2+ signalling to explain macroscopic pressure‐dependent, endothelial mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3‐mediated Ca2+ signalling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that gives access to hundreds of endothelial cells in intact arteries in physiological configuration.The endothelium is an interconnected network upon which haemodynamic mechanical forces act to control vascular tone and remodelling in disease. Ca 1) how pressure is sensed by the endothelium and (2) how the vascular response is inhibited.Increased pressure suppresses endothelial control of vascular tone but it remains uncertain (This study used a novel imaging method to study large numbers of endothelial cells in arteries that were in a physiological configuration and held at normal blood pressures.3‐mediated Ca2+ signals.Increased pressure suppressed endothelial IPPressure modulated endothelial cell shape.2+ signals by modulating the diffusive environment for Ca2+ near IP3 receptors.The changes in cell shape may alter endothelial CaEndothelial pressure‐dependent mechanosensing may occur without a requirement for a conventional molecular mechanoreceptor. While Ca2+ release via IP3R is well established the contribution of the ryanodine receptor (RyR) to the control of endothelial Ca2+ (if any) is less clear . The sources of Ca2+ are not independent: Ca2+ influx regulates Ca2+ release and Ca2+ release regulates Ca2+ influx. For example, Ca2+ release from the endoplasmic reticulum may alter the activity of ion channels present on the plasma membrane to regulate Ca2+ entry either via Ca2+‐gated ion channels  is increased  of naturally connected endothelial cells to be imaged with subcellular resolution and has a high depth of field (141 μm) sufficient to maintain good focus across the highly curved, intact endothelial layer of a large artery. To handle the data from such a large number of cells a largely automated image processing routine was also developed. We show in native endothelial cells in their physiological configuration, that acetylcholine‐evoked Ca2+ rises originate as IP3‐mediated Ca2+ signals in particular regions of the endothelium from which they progress to other cells as Ca2+ waves. Detection of pressure‐dependent mechanical forces by the endothelium is integrated effortlessly into the same signalling pathway by geometric modulation of IP3‐evoked Ca2+ release brought about by changes in endothelial cell shape. We suggest the suppression of IP3‐mediated Ca2+ signals may underlie the inhibition of endothelial responses with increased pressure and, significantly, may not require a conventional mechanosensor for mechanotransduction to occur.To study the function of endothelial cells in intact arteries, some investigations have used either wide‐field or point‐scanning fluorescence microscopes to visualize the endothelium through the wall of the artery  Act 1986, UK), under UK Home Office Project and Personal Licence authority.All experiments employed tissue obtained from male Sprague–Dawley rats . Rats were humanely killed by overdose of pentobarbital sodium with the approval of the University of Strathclyde Local Ethical Review Panel (200 mg kg−1) before the arteries were pressurized to 60 mmHg, gently heated to 37°C and allowed to equilibrate at 37oC for 30 min. Flow was provided by a peristaltic pump  connected to the proximal cannula. The endothelium of mounted arteries was then selectively loaded with a fluorescent Ca2+ indicator by intraluminal perfusion of PSS containing the membrane‐permeant form of Oregon Green BAPTA‐1  and 0.04% Pluronic acid. Flow was stopped for 30 min to permit sufficient loading. Arteries were continuously superfused with PSS during this time. Following removal of excess dye, the distal cannula was removed from the artery and the artery was mounted on a gradient index (GRIN) microprobe (described below) and secured with suture thread. Transmural pressure was then incrementally increased to 160 mmHg, whilst stretching the artery to remove any buckle. Following this procedure, the pressure was decreased to 60 mmHg and the artery left for a further 30 min to equilibrate. During equilibration arteries were tested for leaks  by switching off the feedback on the pressure servo. Arteries that showed signs of leakage, identified as a reduction in pressure whilst isolated from the servo system, which could not be stopped by re‐tying the sutures were discarded at this point.The left and right common carotid arteries were exposed by blunt dissection. To prevent collapse of the artery upon removal, the rostral and caudal ends of the exposed carotid arteries were ligated with 8‐0 suture and the arteries were then rapidly excised. Arteries were then cleaned of connective tissue under a dissection microscope, and visually checked for the presence of side branches. Subsequently, arteries without side branches were mounted onto two blunted and deburred 22‐gauge cannula in a custom‐designed imaging bath using two lengths of suture thread. Blood was removed from the arteries by flushing the lumen with physiological saline solution (PSS) for 10 min (150 μl minA) and consisted of a 0.5 mm diameter, 30.2 mm long, single pitch GRIN relay lens  with a 0.5 mm × 0.5 mm × 0.5 mm aluminium‐coated micro‐prism  attached to the distal surface with ultraviolet curing optical epoxy . The lens was sheathed in a surgical stainless steel tube (0.71 mm outer diameter) for mechanical protection. In this single lens configuration, the GRIN rod acts to reconjugate the image plane of detection optics through the length of the cylinder . By focusing a conventional microscope objective to a sufficient depth inside the GRIN rod, the image plane can be extended beyond the front surface of the prism and into tissue . This property renders the probe suitable for the replacement of a cannula in a custom‐made pressure myograph, where the vessel must be tied to the probe to maintain pressure . The focal plane of the optical system can be varied in response to diameter variations in the vessel , without moving the probe, by focusing the microscope objective  further into the GRIN lens . Second, to maximize fluorescence detection, the NA of the collection lens should be higher than that of the GRIN rod. The solution to these opposing constraints is to illuminate the proximal end of the GRIN lens with collimated light, which is re‐collimated at the output   across the entire endothelial surface of curved artery. However, the use of a low NA GRIN relay lens imposes two opposing constraints on the transmission of light through the system. First, to maximize the delivery of excitation light through the GRIN rod, the NA of the excitation light should not be greater than the NA of the GRIN lens  that was mounted on the microscope stage of a standard inverted microscope . The fluorescence excitation and delivery system was constructed using 30 mm cage system components , and was also mounted onto the microscope stage via a three‐axis translation stage. Two axes of the translation stage permitted the external optical system to be coupled to the GRIN microprobe, whilst the third enabled the focus of the system to be altered. Mounting both the arteriograph and the external optical system on the stage ensured that movement of the microscope stage did not decouple the probe from the system. Fluorescence excitation was provided by a fibre‐coupled diode‐pumped solid‐state laser operating at 488 nm, which was collimated  before being focused by another lens and guided to the back of a 20 × 0.5 NA infinity‐corrected microscope objective  via a dichroic mirror. Optical excitation power density, measured at the output of the GRIN probe was 1 nW μm2. The microscope objective and a 65 mm focal length tube lens imaged fluorescence emission, returning through the probe, through an emission filter and onto a sCMOS camera  controlled by μManager , and of large 15.45 ± 0.04 μm  diameter fluorescent beads . To image fluorescein fluorescence, the bath chamber was filled with the fluorescent solution. Large‐diameter fluorescent beads were diluted in water (100× dilution in water) and left to settle on the microprobe prism surface. Figure A shows the normalized intensity across the centre of the circular field of view (raw image of fluorescein fluorescence inset). Due to vignetting of the illumination light and of the fluorescence excitation by the cylindrically shaped GRIN lens, the efficiency drops to 50% at a distance approximately 150 μm from the centre of the probe. Translating the focal plane 500 μm along the z‐axis, beyond the distal prism surface resulted in a slight increase in fluorescence signal, relative to maxima. Note that due to the curvature of pressurized arteries, this intensity profile does not necessarily reflect that obtained in imaging experiments performed in intact arteries. Average optical excitation power density (1 nW μm−2) was calculated across the full 500 μm field of view from power measurements made at the output of the GRIN microprobe using a photodiode power sensor and power meter .To demonstrate the fluorescence signal detected by the microendoscope camera, we recorded images of a diffuse fluorescein solution (1 μ6 dilution in water) was manually pipetted onto the distal prism surface. Fluorescent beads were left to settle on the distal prism surface before being brought into focus and imaged . Line intensity profiles of the fluorescence emitted from individual beads were taken . Gaussian fits of these line intensity profiles yielded a lateral resolution of 4.51 ± 0.04 μm (n = 6 beads). This measured value may be considered to be the actual resolution of our system because it is much larger than the actual size of the beads . The mean fluorescent microsphere diameter (as provided by the manufacturer) was 1.1 μm with a coefficient of variation (standard deviation/mean) of 4%. A small droplet of a fluorescent microsphere suspension  from ∼200 endothelial cells in each artery by GRIN microendoscopy. The plane of focus of the GRIN imaging system was set to the endothelial layer. Arteries at resting diameter were stimulated by application of ACh (100 μm), delivered by a handheld pipette to the outside of the pressurized artery to confirm endothelial viability. Preliminary experiments established that ∼80% of the cells in the field of view responded to ACh (100 μm). Arteries in which the majority of endothelial cells did not exhibit a Ca2+ response to ACh were discarded. To examine the effect of increases in transmural pressure, endothelial Ca2+ signalling was recorded at pressures within the physiological range for the artery . Following each change in pressure, the imaging system was refocused on the endothelium and arteries were left to equilibrate for 20 min. The Ca2+ responses evoked by ACh at various transmural pressures were studied in single experiments and expressed relative to the control response (60 mmHg). There was significant overlap in the cells imaged at each pressure, but matching individual cells was not possible. In other experiments, the extraluminal PSS was exchanged for PSS containing various pharmacological inhibitors or Ca2+‐free PSS and left for 20 min, before activation again with 100 μm extraluminally applied ACh. The Ca2+ responses evoked by ACh in arteries treated with pharmacological inhibitors or Ca2+‐free bath solution were studied (control and treatment) in the same artery and expressed relative to the control response. Following each acquisition period, the bath solution was immediately exchanged and the arteries were left for at least 20 min to re‐equilibrate. Smooth muscle cells were not loaded with OGB‐1/AM in any of our preparations, as indicated by an absence of fluorescence staining orthogonal to the longitudinal vessel axis.Endothelial Ca2+ signals has been achieved by assigning regions of interest (ROIs), of predetermined size and shape to image sequences based on subcellular activity  change. First, to facilitate visual inspection of endothelial Ca2+ signals, the active Ca2+ wavefronts themselves were examined by generating the forward difference of [Ca2+]c changes ; Bradley et al. et al. 2+ activity, were created by taking projections of the standard deviation (STDev) of intensity of SS image stacks. Unsharp masking of standard deviation projections was used to create sharpened, background‐corrected STDev images, where ROIs encompassing individual cells could be easily obtained by intensity thresholding. ROIs corresponding to individual cell outlines were verified for each image series and erroneous ROIs were corrected manually. Cell outlines were stored as polygon descriptions within text (.txt) files for subsequent processing and analysis. Except for the creation of standard deviation projections , calculated by dividing the raw signals by the average value of using a user‐defined period  preceding ACh‐evoked Ca2+ activity. There was a significant variation in the time taken for each cell to respond to ACh. To aid analysis, individual F/F0 traces were aligned with respect to their peak rate of change. The alignment provides a clear illustration of total Ca2+ activity. Baseline values of F/F0, peak amplitudes and the time of peak rate of change for each signal were calculated automatically and stored as data tables within .csv files. These .csv files were then imported into Origin 9.1 for calculation of peak changes in fluorescence intensity (ΔF/F0), and for plotting using custom analysis scripts. For presentation, an 6‐point (1.2 s), third‐order polynomial Savitzky–Golay filter was applied within Origin; all measurements were from unsmoothed traces.Individual fluorescence signals were extracted for each polygonal region from the raw image stacks using a custom program in the Python language. Fluorescence signals are expressed as baseline corrected values  mounted on the side‐port of the inverted microscope on which our GRIN imaging system was mounted. Images were captured from the CCD camera using Micromanager software (Edelstein Rat carotid arteries were fixed at pressure after length adjustment (to remove buckle) at 160 mmHg. Following length adjustment, lumenal PSS was replaced with Zenker's fixative. Once the lumen was filled with fixative, the artery was sealed and the pressure was immediately raised to 60 mmHg or 160 mmHg, and the extraluminal PSS was replaced with Zenker's solution. Arteries were left to fix for at least 2 h – a time determined, in preliminarily experiments to be required to prevent a reduction in arterial dimensions upon removal of pressure. Following fixation, arteries were removed from the myography chamber, washed overnight in tap water and stored in 70% ethanol at 4°C until use. Arteries were paired for analysis .Following fixation, arteries were dehydrated in alcohol series , cleared , overnight, 4 changes), infiltrated and embedded . Wax blocks were cut into 5 μm thick sections and mounted onto slides. Slides were rehydrated in Histo‐Clear , then alcohol series , before being washed in distilled water. Rehydrated slides were stained with Harris's haematoxylin solution (10 min), washed with warm running tap water (10 min) then Scotts tap water (1 min), differentiated in acid alcohol , stained with eosin (5 min), washed with warm running tap water (10 min), then dehydrated in alcohol series , cleared in Histo‐Clear , before being mounted with Histo‐Clear. Stained artery cross‐sections were imaged using a Leica DM LB2 microscope with a Leica DFC320 camera . The thickness of endothelial cell nuclei, used as an indication of height of the endothelial cell layer, were measured using Image Pro Plus.m): NaCl (145), KCl (4.7), Mops (2.0), NaH2PO4 (1.2) glucose (5.0), ethylenediamine‐tetraacetic acid , sodium pyruvate (2.0), MgCl2 (1.17), CaCl2 (2.0) (pH adjusted to 7.4 with NaOH). In Ca2+‐free PSS, no Ca2+ was added, the concentration of MgCl2 was increased to 3.17 mm, and ethylene glycol tetraacetic acid  was included.PSS consisted of  produced approximately equivalent responses (i.e. ∼80% cells responding) to ACh (100 μm) applied to the bath in intact artery. Secondly, the concentration of ACh was also estimated from the time course of diffusion of fluorescein across the vascular wall in the same experimental conditions as the pressurized artery. The fractional fluorescence change  at the time (10 s after addition) that ACh evoked Ca2+ responses was used to estimate the fraction of the ACh present in the lumen (∼1 μm). In other studies, the potency of extraluminally applied ACh was reported to be ∼1/50 of intraluminally applied ACh in the dog mesenteric artery . Zenker's fixative was obtained from Fisher Scientific.post hoc test as appropriate) was used for comparisons between groups, and biological replicate  was treated as a random factor. Statistical analyses were performed using Minitab 17 . A P‐value less that 0.05 was considered significant and n is number of animals.Summarized data are expressed as means ± standard error of the mean (SEM). One‐way ANOVA  microdomain was determined computationally in the time period encompassing ion channel opening. The cytosolic concentrations of ionic Ca2+, buffer and buffered Ca2+ are represented by CCa, cyt, CB, cyt and CCaB, cyt respectively. The partial differential equation governing the concentration, Cs, of a species, s, is given by:Cs is the concentration, Ds is the diffusion coefficient, ϕs is the source term derived from chemical reactions and Js is the source term resulting from trans‐membrane flux, which was taken as zero for all species except Ca2+, for which it comprises IP3R‐mediated Ca2+ currents.To understand how changes in cell geometry alter IPKon and Koff are the rate constants for the buffer.We employed the first order mass action reaction kinetic:2+ pumps (SERCAs) and membrane (plasma and ER) leakage currents are typically continuous, low magnitude processes that function to maintain specific concentrations of Ca2+ in the cytosol  and the ER  in the long term  to a molar concentration for the voxel to which the current is applied.CaCCa,cyt did not exceed a level 1% above baseline at the edges (excluding the edge containing the IP3Rs), except where we intentionally simulated compressed cells. The Courant–Freidcrichs–Lewy condition:dt is the simulation time step and dx is the linear size of a voxel. Simulation was conducted using bespoke C code employing Intel AVX‐2 vector extensions. The Python language was used to process and analyse simulation results.Equation 2+ and buffer concentrations as given in Table CCaB,cyt being initialized to equilibrium values. A zero flux boundary condition was applied to the edges of the cuboidal simulation volume.Our model was initialized with homogeneous Ca2+ source in a strongly buffered environment.Our simulation code was compared with analytical expressions for simplified test cases of 3D diffusion from a point source and for the microdomain profile of an isolated CaM released at position r = 0 and time t = 0, where r is scalar radius from the origin 0.5 is a space constant and τ = 1/(KonB) is the mean capture time of the buffer, of concentration B. Our simulation was configured to match, as well as possible, this analytical case. Specifically parameters from Table KOff was set to zero (forward binding only), ϕB,cyt from Eqn 2+ was rapidly depleted in this model, so ∞c was also set to zero to facilitate direct comparison. After running the model, the total quantity of free (unbuffered) cytosolic Ca2+ was examined . This level rose rapidly, stabilizing to equilibrium in ∼0.1 ms. A comparison of Eqn r and edge effects raising the concentration at high r.An analytical solution for the equilibrium concentration profile, at distance ned Fig. C. This Parameters for the simulation were chosen to be physiologically relevant, and are given in Table I) with subcellular resolution . In arteries pressurized to 60 mmHg, activation of the endothelium by extra‐luminal acetylcholine  evoked reproducible rises in cytoplasmic Ca2+ concentration ([Ca2+]c) in the majority of cells in the field . The response was composed of temporally distinct components. Initially, small spatial groupings of cells (macrodomain) activated  in small regions of the endothelium and from which waves expanded  to recruit the remaining majority of cells in the field of view  in various cells  which complicated summarizing and evaluating responses. Therefore, the temporal latency between individual Ca2+ traces (F/F0) was removed by aligning the signals to each other  . This procedure removed heterogeneity resulting from the time to onset of individual signals and provided a clearer illustration of total Ca2+ activity. Upon consecutive stimulation with ACh, the response, as measured from the number of cells activated and the average peak change in F/F0, was approximately reproducible .As a result of the wave progression there was a large spread of times to first Cable Fig. D, n = 52+ rise evoked by ACh was approximately reproducible on successive application of ACh . The ACh‐evoked Ca2+ rise persisted in a Ca2+‐free bathing solution (n = 4), but was blocked by the SERCA inhibitor cyclopiazonic acid  and the IP3R blocker 2‐aminoethoxydiphenyl borate  did not evoke a Ca2+ increase and the ACh‐evoked Ca2+ rise was unaltered by ryanodine . These results suggest that the Ca2+ rise originated from an IP3‐sensitive Ca2+ store and that RyRs play a minor role in Ca2+ signalling in the endothelium  did not decrease the ACh‐evoked endothelial response at 60 mmHg  – as expected from previous findings in larger arteries , occurs with variation in transmural pressure, and hence mechanical force. At 60 mmHg, ACh (100 μm) evoked asynchronous Ca2+ waves and oscillations that originated in macrodomains . As transmural pressure increased (60 to 110 to 160 mmHg), both the amplitude of the Ca2+ rise that occurred in each cell and the temporal spread of total activation decreased . The suppression of the amplitude of the Ca2+ signals reversed as pressure decreased (not shown). These results suggest that increased pressure in the intact artery suppressed IP3‐evoked Ca2+ signals. IP3R itself is not reported to be stretch sensitive, and mechanical activation of IP3‐generating processes is unlikely to explain our observations as transient increases in IP3 production and IP3‐evoked Ca2+ release occur typically . Increased transmural pressure may have also compressed the endothelial cells. The change in cell geometry is likely to alter Ca2+ dynamics in the endothelial cells at the microdomain level near IP3R. The effect of changes in cell geometry on IP3‐evoked Ca2+ signals was therefore considered further. To explore the effects of changes in the cells’ geometry on IP3‐mediated Ca2+ release at the relevant scale, which exists below functional optical microscopy, we computationally simulated events at an IP3R cluster.The question arises as to how increased pressure suppressed IPure Fig. . Increasure Fig. A and B.3Rs are arranged in clusters and the sensitivity of IP3Rs to Ca2+ is such that the [Ca2+]c in the microdomain of a single open IP3R (or cluster) may be high enough to activate nearby IP3Rs (or clusters) around the channel. The Ca2+ concentration decreased sharply with distance from the IP3R with a characteristic scale (the distance at which [Ca2+] remains elevated by at least one order of magnitude) on the order of 100 nm . The presence of many such point sources resulted in a greater [Ca2+]c in the vicinity of IP3R clusters than isolated receptors . The raised [Ca2+]c that occurred as a result of IP3R clustering reduced the local ion gradient between the store ([Ca2+]ER) and the cytosol, and so reduced the entropic force driving Ca2+ release through the channel. In addition, the elevated [Ca2+]c merged the microdomain of each IP3R into a unified, larger domain with scales approaching the 500 nm thickness typical of endothelial cells.The opening of a single IP nm Fig. B. The pors Fig. C. The r3Rs release Ca2+ into the cytosolic space between the ER and the cellular plasma membrane, where proximity of the IP3R to the cellular plasma membrane may create a restricted environment limiting the diffusion of Ca2+. The decreased cell height as a result of the change in cell shape . Thus, the decreased height reduced the total Ca2+ liberated from the ER by reducing the entropic force driving Ca2+ release . In this way, a mechanical force  can reduce Ca2+ release via IP3R, in the absence of any force‐ or stretch‐sensitive molecular machinery. Notably, the clustering of IP3Rs extends this effect, when compared to isolated receptors, from sub‐100 nm thick cells to those with a 100–500 nm thickness typical of the endothelium  IPape Fig.  reduces ors Fig. A and C.ase Fig. D. In thium Fig. .et al. et al. et al. b; Qian et al. 2+ activity from within an intact, pressurized artery to be observed. The microprobe bypasses the difficulties created by the artery wall in imaging blood vessels in a physiological configuration. Furthermore, the absence of biological tissue between the end of the probe and the endothelium minimized the light intensity required. The microprobe has a large depth‐of‐focus (141 μm) and delivers a 0.5 mm field of view to allow 200 cells to be viewed simultaneously.The endothelium's control over cardiovascular structure and function requires cooperation among endothelial cells to both sense and communicate stimuli efficiently with itself and other cell types  appear to be more sensitive to ACh. The path of the Ca2+ signal from the sensitive regions at times appeared poorly coordinated among endothelial cells – even chaotic. Nonetheless, the path and pattern were repeatable on successive application of ACh suggesting an encoded signal communicated among cells. The response to ACh was derived from an IP3‐mediated release of Ca2+ from the internal store. The Ca2+ rise persisted in a Ca2+‐free bath solution and was abolished by the SERCA pump inhibitor cyclopiazonic acid. The ACh‐evoked Ca2+ rise persisted in ryanodine and caffeine failed to evoke a Ca2+ rise. However, release was inhibited by the IP3R blocker 2‐APB. The transmission of the Ca2+ signal among endothelial cells may involve Ca2+ or IP3 or both.The detection of chemical stimuli appears to be distributed among endothelial cells. In response to ACh, Caet al. et al. et al. et al. et al. et al. et al. et al. et al. 2+ signalling is suppressed when pressure is elevated. The suppressed Ca2+ signalling will presumably result in reduced production of nitric oxide and reduced endothelium‐dependent dilatation  with increased ion channel and cell activity, the mechanical response to pressure differs and endothelial activity is suppressed. For example, transient and chronic increases in pressure each inhibit endothelial nitric oxide‐mediated vasodilatation  within the cell to modulate the overall response of the cell.The sensing system(s) responsible for detecting changes in pressure in the endothelium are poorly understood. Pressure‐evoked mechanical ced Fig. . Reduced3‐evoked Ca2+ signalling provides an additional level of complexity and subtly to mechanosensitive signalling, which may induce further signals to generate a functional response. For example, the changes in [Ca2+]c as a result of geometric modulation of IP3‐evoked Ca2+ release may modulate the activity of plasma membrane‐located Ca2+‐sensitive ion channels to explain, at least in part, some contradictory observations of apparent mechanical activation , and an IP3R blocker (xestospongin C) almost abolished Ca2+ events at 80 mmHg and significantly inhibited responses at 5 mmHg. These results suggest that IP3‐mediated Ca2+ release may contribute significantly to the spontaneous Ca2+ events. Geometric regulation of IP3‐evoked Ca2+ release may provide the pressure sensor for the change in TRPV4 activity.The pressure‐sensitive change in IP. et al.  found thet al. et al. et al. et al. et al. et al. Endothelial cells are acknowledged to be sensitive to their geometry and changes are known to regulate proliferation and cell death  and British Heart Foundation (PG/11/70/29086 and Research Excellence Award (Edinburgh/Durham University)) and EPSRC; their support is gratefully acknowledged.Movie S1. ACh‐evoked endothelial Ca2+ signals in a pressurised (60 mmHg) rat carotid artery. Activation of IP3‐evoked endothelial Ca2+ signals, by bath application of ACh (100 μM), causes a rise in cytoplasmic Ca2+ concentration in the majority of cells across the field of view. The movie is composed of raw data, to which a 5‐frame rolling average has been applied, that has been linearly contrast adjusted for visualisation. Data was acquired at 5 Hz and the scale bar is 100 μm.Click here for additional data file.Movie S2. ACh activated IP3‐evoked endothelial Ca2+ signals which were recorded from inside pressurized arteries. At 60 mmHg, ACh caused an initial burst of Ca2+ increases that propagate as waves. As ACh‐evoked Ca2+ waves expanded collision between adjacent cells occurred and wave annihilation occurred. As a result of multiple waves progressions and annihilation events, complex spatiotemporal patterns of Ca2+ signaling developed. The movie is composed of a sequential subtraction of the temporally smoothed Ca2+ activity (green) overlaid on standard deviation image . Data was acquired at 5 Hz and the scale bar is 100 μm.Click here for additional data file.Movie S3. ACh activated IP3‐evoked Ca2+ signals were significantly attenuated as the artery transmural pressure increased from 60 mmHg to 110 mmHg to 160 mmHg. After each application of ACh the bath solution was washed with >20 times the bath volume, and the artery was allowed to re‐equilibrate for 20 minutes before the next pressure change and ACh addition. The movie corresponds to Ca2+ traces and time‐series data shown in Figure 7A and Figure 7B respectively. The movie is a time series of Ca2+ wave activity (green) overlaid on standard deviation images (STDev) . Note that STDev images only show cells that exhibit Ca2+ activity. Data was acquired at 5 Hz and the scale bar corresponds to 100 μm.Click here for additional data file.


A previous study by our group has shown that increased levels of Ca2+ can promote hDPC-mediated mineralization in long-term cultures (21 days). However, the initiation of mineralization occurs in the early stage of osteogenic inductive culture, and the effects of Ca2+ on the mineralization of hDPCs in short-term cultures (five days) have not been studied in detail. Furthermore, the underlying mechanism by which Ca2+ stimulates the mineralization of hDPCs has remained controversial. A strong correlation between mineralization and cell apoptosis and/or death has been identified. Thus, the present study hypothesized that Ca2+ may promote the onset of hDPC-mediated mineralization through inducing their apoptosis and/or death. To verify this hypothesis, Ca2+ was added to the growth culture medium and osteogenic culture medium at various concentrations. Alizarin Red S staining and reverse transcription-polymerase chain reaction analysis were used to evaluate the onset of mineralization. Furthermore, the cell counting kit-8 and fluorescein isothiocyanate-Annexin V/propidium iodide double-staining method were adopted to detect the proliferation and apoptosis of hDPCs in the growth culture medium. An animal experiment and scanning electron microscopic observation of ceramic graft implants were applied to measure the mineralization in vivo. The results showed that 5.4 and 9.0 mM Ca2+ accelerated the onset of mineralized matrix nodule formation, promoted osteopontin mRNA expression and induced marked cell apoptosis and necrosis, but had no obvious effect on cell proliferation. These findings indicated a positive association between cell apoptosis and/or death and the timing of formation as well as the quantity of extracellular mineralization induced by Ca2+ in short-term cultured hDPCs.Calcium ions (Ca The main goal of restorative dentistry is to re-construct the tooth and maintain the vitality and function of human dental pulp tissue . Numerou2] and its derivatives have been used for direct pulp capping therapies  are major components of the pulp-capping materials currently used in clinical practice   to investigate cell mineralization, and in growth media to examine cell apoptosis and proliferation. Finally, an animal experiment was performed to test the performance of hDPCs on porous three-dimensional scaffolds cultured in the growth media in the presence of Ca2+.Calcium ions (Capractice . Ca2+ relization –14. A pr21 days) . Howeveror death . Thus, thDPCs were isolated from healthy impacted teeth extracted from five subjects <30 years of age, and informed consent was acquired from all subjects. Primary cultures of hDPCs were established as previously described using an explant culture method . All exp2+ supplementation was performed using CaCl2 as a sterile 1.8 M solution in double distilled water. The Ca2+ concentration in commercial α-MEM media is 1.8 mM. Based on this concentration in the culture medium, which was used as the control, various quantities of Ca2+ were added immediately prior to incubation of the cell cultures to achieve final concentrations of 5.4, 9.0, 12.6 and 16.2 mM. Accordingly, treatment groups are being referred to as Ca1.8, Ca5.4, Ca9.0, Ca12.6 and Ca16.2. To assess cell mineralization and apoptosis, Ca2+ was also added to the osteogenic differentiation medium  and basic growth medium [α-MEM containing 10% FBS (Invitrogen Life Technologies) and 2% penicillin/streptomycin].Ca2+ levels on cell mineralization, mineralized nodules were detected using an Alizarin Red S staining kit  according to the manufacturer’s instruction at days 3 and 5 of osteogenic inductive culture. The concentrations of Ca2+ tested in were as follows: 1.8 mM Ca2+, Ca5.4, Ca9.0, Ca12.6 and Ca16.2 mM. hDPCs were cultured at a density of 1×104/cm2 in 24-well microplates. A series of cell-free controls parallel to each group tested were used to check whether spontaneous precipitation occurred following Ca2+ supplementation under identical conditions to those in the cell culture. The stained cells in each well were observed using an inverted phase-contrast microscope , and microscopic images were captured at different magnification.To observe the effect of Ca2+ were defined as the experimental conditions for the subsequent assays. It is known that osteopontin (OPN) is required for the initiation of hard-tissue mineralization (2+ groups was extracted using TRIzol reagent (Invitrogen Life Technologies), and reverse transcribed by RevertAid™ Moloney Murine Leukemia Virus Reverse Transcriptase . Target gene expression was standardized relative to the housekeeping gene GAPDH. The 30 µl PCR mixture consisted of 3 µl 10X Taq Buffer with (NH4)2SO4, 3 µl 2 mM dNTP Mix, 2 µl 25 mM MgCl2, 1.5 U Taq DNA Polymerase, 0.6 µl (10 pg-1 µg) cDNA template, and 10 µM of each primer set. The reaction conditions for the PCR were as follows: 94°C (3 min), followed by 28 cycles of 94°C (30 sec), 60°C (30 sec) and 72°C (20 sec), with a final 3-min extension at 72°C. Forward and reverse primers were as follows: forward, GCCGAGGTGATAGTGTGGTT and reverse, TGAGGTGATGTCCTCGTCTG, with a product size of 101 bp. The primer sequence for GAPDH was as follows: forward, CATGTTCCAATATGATTCCACC; and reverse, GATGGGATTTCCATTGATGAC, with a product size of 88 bp. All primers were custom made by Invitrogen Life Technologies. The PCR product was separated with DYY-12 electrophoresis apparatus  by 2% agarose at 120V.Based on the results of the cell mineralization assay, 5.4 and 9.0 mM Calization . Hence, lization . The tot2+. Briefly, cells were seeded into the 96-well plate at a density of 2×103 cells/well, incubated for 24 h and starved in serum-free media for another 24 h. Subsequently, media in every well were replaced with the designated culture medium. After 1, 3, 5 and 7 days of culture, the medium was replaced with 15 µl CCK-8 solution. This solution was incubated with the cell cultures at 37°C for 4 h in a humidified atmosphere containing 5% CO2. Optical densities of the supernatants were measured at 490 nm with an ELISA spectrophotometer .The cell proliferation was measured using the Cell Counting kit-8  in growth media containing the abovementioned concentrations of Ca5 cells were harvested with 0.25% trypsin/EDTA (Invitrogen Life Technologies) and re-suspended in binding buffer . Apoptotic cells were identified by double supravital staining using the recombinant fluorescein isothiocyanate-conjugated Annexin-V and propidium iodide (Annexin V/PI) Apoptosis Detection kit (BD Biosciences) according to the manufacturer’s instructions. Apoptotic cell death was examined by FACSCalibur flow cytometry (BD Biosciences). Viable cells were Annexin V−/PI−, early apoptotic cells were Annexin-V+/PI−, late apoptotic cells were Annexin-V+/PI+ and necrotic cells were Annexin V−/PI+.hDPCs were seeded into six-well culture plates in the basic growth medium for 24 and 72 h, and 1×102+ group was selected for in vivo analysis. Six-week-old female severe combined immu nodeficient (SCID) mice  were used for the subcutaneous transplantation study. Animal protocols were approved by the Sun Yat-sen University Animal Care and Use Committee . In brief, 5×5×5-mm cube sizes of HA/TCP scaffolds were sterilized for 15 min  by autoclaving, and soaked in DMEM supplemented with 10% FBS and 2% penicillin/streptomycin at 37°C for 4 h. For a single transplant, 20 µl hDPC suspension at a density of 1×105 cells/ml was seeded using a pipettor  onto hydroxyapatite/tri-calcium phosphate (HA//TCP) scaffolds . After incubation in the growth culture medium containing 5.4 mM Ca2+ for three days, the mice were anaesthetised by an intraperitoneal injection of 4% chloral hydrate  (400 mg/kg dose), and then the hDPC-scaffold constructs were implanted into the dorsal surface of subcutaneous pockets. The constructs cultured in the classic α-MEM media served as the control, and HA/TCP only (without any cells) was implanted in the same animal as a blank control. Four transplants were placed into each animal. The incisions were closed with surgical staples. The mice were kept in a specific pathogen-free (SPF) environment at a constant temperature of 25±2°C with a constant humidity of 45–50%, and were fed inside the laminar air flow rack. After 1 week, the mice were sacrificed by an excessive injection of 4% chloral hydrate  and implants were harvested, fixed for 1 day in 4% formaldehyde in phosphate-buffered saline, and the cell growth and mineralization were examined by scanning electron microscopy .Based on the results of cell mineralization and apoptosis, the 5.4 mM CaValues are expressed as the mean ± standard deviation. Statistical analyses were performed by one-way analysis of variance with the least significant difference test using the SPSS 17.0 software package . P<0.05 was considered to indicate a statistically significant difference between values.2+ groups, while few nodules were observed in the 12.6 and 16.2 mM Ca2+ groups. After five days of osteogenic inductive culture, nodules appeared in all groups; the most obvious mineralization nodes were found in the 5.4 and 9.0 mM Ca2+groups. After three days of osteogenic differentiation culture, mineral matrix nodules first appeared in the cell cultures in the 5.4 and 9.0 mM Ca2+ levels displayed mRNA expression of OPN. Amongst all groups, 5.4 mM Ca2+ caused the most marked increase in OPN mRNA expression , the cell number of the hDPCs in all experimental groups analyzed was decreased at all time-points. However, there were no statistically significant differences in cell proliferation levels between these experimental groups and the control (P>0.05) .2+ for 24 or 72 h, hDPCs exhibited higher apoptotic and necrotic rates compared with those in the control group. As compared with the control group, following incubation with 9.0 mM Ca2+ for 24 h, the early apoptotic rate was increased (1.31 vs. 4.22%) and the necrotic rate was elevated (0.22 vs. 8.54%). Furthermore, as compared with the control group, following incubation with 9.0 mM Ca2+ for 72 h, the early apoptotic rate was increased (0.02 vs. 1.02%) and the necrotic rate was elevated (4.42 vs. 12.50%) (After incubation in media supplemented with 5.4 and 9.0 mM Ca 12.50%) .2+ into SCID mice (2+ displayed intercellular fusions and produced an extracellular matrix on the surface (At 7 days following implantation of HA/TCP scaffolds containing hDPCs incubated with 5.4 mM CaCID mice , the tra surface .Mineralization is an essential requirement for normal bone and tooth development, which is generally achieved through the function of two cell types, osteoblasts and chondrocytes . It is g2+ promoted the mineralization of hDPCs at day 3, but 12.6 and 16.2 mM Ca2+ did not stimulate the mineralization. This result was similar to that of a study by Maeno et al (2+, which significantly influenced the mineralization of osteoblasts at levels of 6–10 mM, whereas further increases beyond this range led to a decrease in the amount of mineral deposition. These findings suggested that there is no linear trend between the onset of mineralization and extracellular Ca2+ concentrations, and there appears to be a limit beyond which increases in the Ca2+ concentration do not lead to continued increases in hDPCs mineralization. This marked decrease in mineralization in the 12.6 and 16.2 mM Ca2+ groups is most likely a result of the cytotoxic effects outweighing any benefit in mineral production. The mineralization assay of the present study also showed that 5.4 and 9.0 mM Ca2+ induced the maximum amount of mineral nodules at days 3 and 5. Based on this result and that of a previous study by our group (2+ is an essential factor to trigger and maintain the hDPCs-mediated mineralization process.In the present study, 5.4 and 9.0 mM Cano et al , who repur group , it can 2+, indicating that the elevated calcium levels triggered hDPCs-mediated mineralization. By contrast, OPN mRNA was not detected in the control group. These findings suggested a positive association between the onset of hDPCs-mediated mineralization and OPN gene expression.OPN is a key marker of osteogenic differentiation and mineralization. High levels of OPN are associated with the initiation of tissue mineralization and ectopic calcification . However2+ concentration have remained controversial. A marked correlation between mineralization and cell death has been observed, and in particular, pathological mineralization has often been associated with apoptotic or necrotic processes (2+ and cell apoptosis, the present study determined the apoptotic and necrotic rates of hDPCs cultured in growth medium supplemented with 5.4 and 9.0 mM Ca2+ using Annexin V/PI staining followed by flow cytometric analysis. The results showed that elevated concentrations of Ca2+ increased the apoptotic rate, and the number of necrotic cells was also significantly increased. Hence, it was speculated that the mineralization of hDPCs induced by Ca2+ is associated with the induction of cell apoptosis or cell death.The biochemical mechanisms that initiate mineralization subsequent to the increase in the Carocesses . To inve2+ and the control cells. The hDPCs cultured in medium containing 5.4 mM Ca2+ displayed reduced proliferation compared with that of the control cells and had produced a larger amount of extracellular matrix. Apart from the effect of Ca2+ in the culture media, free Ca2+ released from the scaffolds is also an important factor that contributes to the mineralization (2+ promoted hDPC-mediated mineralization and contributed to the formation of a calcific barrier.Finally, SEM images of hDPCs engrafted onto HA/TCP scaffolds and incubated in SCID mice for 7 days showed differences in morphology between the hDPCs cultured in the presence of 5.4 mM Calization ,26–28. T2+ levels in stimulating and maintaining hDPCs-mediated mineralization. However, mineralization induced by elevated concentrations of Ca2+ was likely to be a trigger of apoptosis and/or necrosis of hDPCs. In conclusion, the present study provided a rationale for the design of novel pulp-capping materials, suggesting that an optimal extracellular Ca2+ concentration is able to improve clinical outcomes.In conclusion, the results of the present study and those of previous studies indicated the significance of optimal extracellular Ca