However, the patterns of expression and physiological/pathological functions of B1 and B2 receptors in trigeminal ganglion (TG) neurons remain to be fully elucidated. We investigated the functional expression of BK receptors in rat TG neurons. We observed intense immunoreactivity of B2 receptors in TG neurons, while B1 receptors showed weak immunoreactivity. Expression of the B2 receptor colocalized with immunoreactivities against the pan-neuronal marker, neurofilament H, substance P, isolectin B4, and tropomyosin receptor kinase A antibodies. Both in the presence and absence of extracellular Ca2+ ([Ca2+]o), BK application increased the concentration of intracellular free Ca2+ ([Ca2+]i). The amplitudes of BK-induced [Ca2+]i increase in the absence of [Ca2+]o were significantly smaller than those in the presence of Ca2+. In the absence of [Ca2+]o, BK-induced [Ca2+]i increases were sensitive to B2 receptor antagonists, but not to a B1 receptor antagonist. However, B1 receptor agonist, Lys-[Des-Arg9]BK, transiently increased [Ca2+]i in primary cultured TG neurons, and these increases were sensitive to a B1 receptor antagonist in the presence of [Ca2+]o. These results indicated that B2 receptors were constitutively expressed and their activation induced the mobilization of [Ca2+]i from intracellular stores with partial Ca2+ influx by BK. Although constitutive B1 receptor expression could not be clearly observed immunohistochemically in the TG cryosection, cultured TG neurons functionally expressed B1 receptors, suggesting that both B1 and B2 receptors involve pathological and physiological nociceptive functions.Bradykinin (BK) and its receptors (B The existence of B1 and B2 receptors has been confirmed by pharmacological and radioligand-binding studies, as well as by mRNA expression analyses, in a wide variety of cells , which are released by nociceptive afferents and/or non-neural cells in the injured area of the tissue  regarding the care and use of animals for experimental procedures. This study was approved by the Animal Research Ethics Committee of Tokyo Dental College .2). The primary cells were cultured in Leibovitz’s L-15 medium (Life Technologies) containing 10% fetal bovine serum, 1% penicillin-streptomycin (Life Technologies), 1% fungizone (Life Technologies), 26 mM NaHCO3, and 30 mM glucose (pH 7.4). For the immunocytochemistry, TG cells were subjected to primary culture on poly-L-lysine-coated cover glasses .TG cells were isolated from neonatal Wistar rats (7 days old) , including mouse anti-Neuronal nuclei (NeuN), anti-microtubule-associated protein 2 (MAP2), anti-βIII tubulin, and anti-neurofilament H (NF-H) antibodies, was used as a neuronal marker. TG cells were also incubated with either mouse anti-NF-H  as an A-neuron marker, mouse anti-substance P  as a peptidergic C-neuron marker, FITC-conjugated anti-isolectin B4  as a non-peptidergic C-neuron marker, goat anti-high-affinity nerve growth factor (NGF) receptor (a tropomyosin receptor kinase A (TrkA); R&D Systems; 1.5 μg/100 μl dilution) as an NGF-responsive nociceptor marker  and rabbit anti-B2 receptor    were fixed in optimal cutting temperature compound and rapidly frozen in liquid nitrogen. Frozen tissues were cut at a thickness of 10 μm and placed on slides. After fixation by 50% ethanol and 50% acetone at −20°C for 30 min, primary cultured TG cells and cryosections were treated with 10% donkey serum at room temperature for 20 min and then incubated overnight at 4°C with primary antibodies  Figure . The cel2, 0.5 MgCl2, 0.44 KH2PO4, 0.34 Na2HPO4, 4.17 NaHCO3, and 5.55 glucose (pH 7.4) was used as an extracellular solution. A high-K+ solution containing (in mM) 91 NaCl, 50 KCl, 2.0 CaCl2, 0.5 MgCl2, 0.44 KH2PO4, 0.34 Na2HPO4, 4.17 NaHCO3, and 5.55 glucose (pH 7.4) was used to discern TG neurons from glial cells by activation of depolarization-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in neurons. BK, a selective B2 receptor antagonist (HOE140), a selective B1 receptor antagonist (R715) and a highly selective B1 receptor agonist (Lys-[Des-Arg9]BK) were obtained from Tocris Bioscience . All the other reagents were purchased from Sigma-Aldrich Co. LLC , except where indicated.A standard solution containing (in mM) 137 NaCl, 5.0 KCl, 2.0 CaClF340) and 380 nm (F380) with an Aquacosmos system and software , which controls the excitation wavelength selector and intensified charge-coupled device camera system (Hamamatsu Photonics K.K.). [Ca2+]i was measured as the fluorescence ratio of F340 and F380 (RF340/F380) and expressed as F/F0 units. The RF340/F380 value (F) was normalized to the resting value (F0).Primary cultured TG cells were loaded for 90 min at 37°C in Hank’s solution containing 10 μM of fura-2 acetoxymethyl ester  and 0.1% (w/v) pluronic acid F-127 (Life Technologies). Cultured TG cells were then rinsed with fresh Hank’s solution and mounted on a microscope stage . Fura-2 fluorescence emission was measured at 510 nm in response to alternating excitation wavelengths of 340 nm .The data were expressed as the mean ± standard error (S.E.) or standard deviation of the mean of 2+]i on each pharmacological agent was determined by fitting the data to the following function with Origin 8.5 :The dependence of the changes in o indicates the applied concentration of the pharmacological agents, and F/F0int and F/F0fin are the initial and final F/F0 responses, respectively.where TM pan-neuronal marker), which contained mouse anti-NeuN, anti-MAP2, and anti-βIII tubulin antibodies  in the presence of external Ca2+  of 1.0 nM.We observed rapid and transient [Ca2+]i responses in both the presence and absence of external Ca2+. The application of BK (1.0 nM) rapidly increased [Ca2+]i to a peak F/F0 value of 1.7 ± 0.03 F/F0 units in the presence (2.0 mM) of external Ca2+ and 1.4 ± 0.03 F/F0 units in the absence (0 mM) of external Ca2+ -induced [Ca2+]i increases were significantly inhibited by a B2 receptor antagonist (100 nM of HOE140)   in the presence (2.0 mM) of external Ca2+  increases in [Ca Figures .2+]i increases during the administration of Lys-[Des-Arg9]BK, which is an endogenous, potent, and highly selective B1 receptor agonist  in the presence of extracellular Ca2+ (2.0 mM)   Figure . A semil) Figure  illustra Figures .1 and B2) in TG neurons. B2 receptors were present on axons and dendrites in A-neurons, non-peptidergic C-neurons, peptidergic C-neurons, and NGF-responsive nociceptors. While the localization pattern of the B1 receptor was not clear in the TG cryosections, weak immunoreactivity for B1 receptors was observed in the primary cultured TG neurons. The application of BK activated B2 receptors and Lys-[Des-Arg9]BK activated the B1 receptors. B2 receptor activation mobilized [Ca2+]i by releasing Ca2+ from internal Ca2+ stores with partial Ca2+ influx from the extracellular medium.The present study demonstrated the functional expression of BK receptors  analyses  neurons have been inconsistent. In DRG neurons, some immunohistochemical studies have reported constitutive B1 receptor expression  than those in the presence of Ca2+  was mainly composed of Ca2+ release from internal stores with partial Ca2+ influx from the extracellular medium. Notably, BK has been reported to activate voltage-dependent Ca2+ channels in rat submucosal plexus neurons (Avemary and Diener, 2+ currents could not be recorded in TG neurons (Kitakoga and Kuba, 2+ influx pathways contribute to the BK-induced Ca2+ influx in TG neurons, the present results clearly indicate that BK mobilizes [Ca2+]i through both intracellular Ca2+ release and Ca2+ influx.BK-induced i by releasing Ca2+ from intracellular stores with partial Ca2+ influx. In contrast, B1 receptor expression was faint in cultured TG neurons and absent in neurons in TG cryosections, although a metabolite of endogenous BK elicited [Ca2+]i increases. These results indicated that both BK and its metabolites activated [Ca2+]i mobilization in TG neurons through B2 and B1 receptor activation, respectively.In conclusion, BThe 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.


Films formed through the drying of nanoparticle suspensions release the build-up of strain through a variety of different mechanisms including shear banding, crack formation and delamination. Here we show that important connections exist between these different phenomena: delamination depends on the dynamics of crack hopping, which in turn is influenced by the presence of shear bands. We also show that delamination does not occur uniformly across the film. As cracks hop they locally initiate the delamination of the film which warps with a timescale much longer than that associated with the hopping of cracks. The motion of a small region of the delamination front, where the shear component of interfacial crack propagation is believed to be enhanced, results in the deposition of a complex zig-zag pattern on the supporting substrate. The drying of colloidal dispersions has strong relevance to many industrial coating processes such as those involved in the deposition of paints, inks and specialty coatings24687101112As nanoparticle suspensions dry, a film is formed as the result of the evaporation of the suspending fluid. The details of the processes involved in the deposition of particles are subtle and complex, leading to a number of distinct regions in the drying film which are shown in When the nanoparticle suspension is spread on a substrate, a film begins to form at the edges of the drying fluid. Evaporation of the suspending fluid then results in a quasi-steady state being set up in which distinct regions can be clearly observed behind the retreating front of liquid  and arrest (σstop) fracture. Whilst a critical stress for crack propagation may be estimated from the Griffith’s criterion, this can only explain the origin of the crack initiating stress and leaves a description of the origin of the arresting stress lackinget al. were able to produce a model which predicted a regular and consistent pattern of hops. However, this differs from the real situation where there is an apparent randomness in the final positions of cracks, despite the average behaviour obeying the predicted pattern. It was proposed that the stochastic nature of propagation could be explained in terms of changes in the shape of crack tips or as a result of inhomogeneities in the films.Dufresne et al.In all of the experiments reported here, cracks were observed to hop discretely from one position to another as observed by Dufresne Strong candidates for these inhomogeneities are the shear bands that form behind the compaction front. These shear bands result in the formation of diagonal lines at an angle of 45° to the drying direction and occupy approximately 10% of the volume and free surface area of the filmTo test the generality of the idea that shear bands influence the final positions of crack tips, crack trajectories were also studied in films containing 47 nm diameter polystyrene (PS) colloids. We chose to use polymer particles because they display the same qualitative crack hopping characteristics as drying silica nanoparticles, but the size of the gaps between shear bands is much larger which makes the dynamics easier to study.It is important to emphasise that other inhomogeneities in the film may also arrest the crack. Similar experiments performed with larger polymer particles and also for samples with added salt, where shear bands do not exist, also display sporadic crack arrest and hops. Since cracks do not stop at every shear band see  it is al719Crack hopping is particularly important since it is this that controls the delamination dynamics of the adjacent film. For the image shown in the main panel, the motion of the drying front proceeded from right to left. Two types of structure can be observed in this image. Firstly, regions of film/particles left behind by the DP and secondly regions of film/particles left behind by the edges of each crack. Line profiles across these features show that in both cases they have thicknesses ranging from 50–140 nm.The process by which pieces of film are left behind at each crack edge can be observed in 212324The motion of the apex of the delamination front results in the deposition of a zig-zag pattern on the surface. This process, like the crack widening, is also slow compared to the initial delamination of the edge of the film. et al.In a detailed finite element study of the delamination front in “telephone cord” blisters, Faou This type of behaviour has also been observed experimentally during the delamination of polymeric strips, with a similar geometry to the one described in this workc) exceeds the energy gained from the elastic energy recovered from the film (G0)27c larger than G0 near the apex would also explain why the delamination front becomes pinned at the apex, as it is energetically unfavourable to propagate the interfacial crack.These studies report many of the same phenomena observed in the experiments described here. Near the curved apex, the component of the stresses arising due to shear is expected to increase (ie mode II). During the crack widening process it was observed that shear resulted in the deposition of a thin film and it is reasonable to assume that shear could produce the same effect in the apex region of the delaminating films. Away from the apex, delamination has a stronger peeling component and the crossover between these two behaviours (shear and peeling) occurs when the crack mode dependent work of adhesion  for a delaminating film of thickness, h, can be related to the Young’s modulus, E, and the compressive stress, σ0, by the equationAs discussed above, the apex of the delamination front is located where the two sides of the delamination front meet. Where this occurs, the film is placed under a mixture of compression and shear and exhibits excess curvature see . The exc0, can be obtained from the capillary stress such that σ0  ~ 5.3 γ/a = 34 MPaet al. have also measured the Young’s Modulus of films of the same type of silica particles that were used in this study and found it to be ~100 MPad, relates to the size of the folded region of the film not the cracked region which would give rise to the DP. However it is reasonable to assume that the size of the spalled section of the film that would result in the formation of the DP scales with the length of the folded region. Such an assumption provides an estimate of the scaling of the width of the delamination pattern with film thickness and predicts a linear scaling between the width of the DP and h according to et al.An upper bound for the stress, σThe fact that the delamination pattern is deposited only at the apex of the delamination front means that motion of the apex is controlled by the asymmetry of crack hops. When a crack hops, the DP is usually situated relatively close to the crack. The film that is adjacent to the crack then peels relatively quickly but only as far across as the delamination pattern. At this point, the motion of the delamination front becomes pinned and peeling forces then act on the region near the DP resulting in the lateral motion of the pattern towards the trailing crack tip. As the DP moves laterally, a larger area of the film is then allowed to delaminate, releasing stored strain energy in the film. As the cracks hop, alternate peeling from each corner causes the DP to track this motion and results in the observed zig zag pattern.In conclusion, our experiments demonstrate the important links between shear banding, crack propagation and delamination in drying colloidal films. Shear bands play a role in influencing the positions to which cracks hop. The alternating nature of crack propagation results in delamination of each edge of the film in response to each crack hop with a timescale that is controlled by plastic deformations within the film. Over longer timescales widening of the crack and a slowing of the motion of the apex of the delamination front are observed. This results in the deposition of a thin film of material on the substrate which is believed to be the result of an increased shear component in compressed regions of the film near the apexLudox silica suspensions  with concentrations in the range 2.5–10 wt% were prepared by diluting the concentrated suspension using deionised water. These suspensions were deposited on glass coverslips to produce films with thickness values in the 20–60 μm range. Experiments were also conducted on 2.5 wt% solutions of PS colloids of diameter 47 nm. The suspensions were then spread on pieces of glass with the edge of a clean glass slide, resulting in a planar drying front. Prior to deposition all the pieces of glass were cleaned with detergent, ethanol, acetone and deionised water before being dried with nitrogen.Images of the drying films were obtained using a Bruker inverted microscope equipped with a colour camera . All image analysis was performed using software written in Matlab. The thickness of deposited films was measured using a DektakTo measure the width of the delamination pattern, films of different thicknesses were dried on glass coverslips. The films which delaminated from the substrate, were easily removed with a gentle shake, leaving the delamination pattern and film near the crack edges behind on the surface. Microscope images were taken using a 10x objective. At particular locations the width of the delamination pattern was measured 5x to capture the variations, together with the crack spacing. The measured relationship between crack spacing and film thickness (λ ~ 5.5 h) was then used to estimate the film thickness.How to cite this article: Yang, B. et al. The interplay of crack hopping, delamination and interface failure in drying nanoparticle films. Sci. Rep.6, 32296; doi: 10.1038/srep32296 (2016).


Macadamia seed coats, known to exhibit astoundingly high strength and toughness. We performed an extensive materials science characterization of the complex hierarchical structure of these coats, using light and scanning electron microscopy in 2D as well as microCT for 3D characterization. We differentiate nine hierarchical levels that characterize the structure ranging from the whole fruit on the macroscopic scale down to the molecular scale. From a biological viewpoint, understanding the hierarchical structure may elucidate why it is advantageous for these seed coats to be so difficult to break. From an engineering viewpoint, microstructure characterization is important for identifying features that contribute to the high strength and cracking resistance of these objects. This is essential for revealing the underlying structure-function-relationships. Such information will help us develop engineering materials and lightweight-structures with improved fracture and puncture resistance.The shells/coats of nuts and seeds are often very hard to crack. This is particularly the case with  This concept forms the basis of biomimetic inventions and products, for example light-weight constructions with high bending resistance based on the structure of plant stems e.g.. “BiomimMacadamia nuts exhibit particularly high strength values. For biologists, this poses questions about the evolutionary advantages of investing efforts in producing such high strength materials. For engineers, this makes the shell interesting as a source of inspiration for the development of impact- and puncture-resistant materials. A detailed understanding of the microstructure is the basis for identifying the structural features that are most important for bringing about the exceptional strength of these shells, and for understanding the role of their interactions during mechanical loading.It is well known that the shells of nuts and the coats of seeds are hard to crack despite their relatively thin walls. They have to protect the seed against deleterious environmental influences, for example UV radiation, water loss, mechanical damage when they fall or inadvertent crushing by animals seeking food Macadamia nuts come from trees that have their origin in the rainforests of North-East Australia belonging to the family of Proteaceae. Of the ten known species, only Macadamia integrifolia and Macadamia tetraphylla nuts are edible and therefore economically important e.g.Macadamia seed coats, forces in the range of 1800 to 4000 N are needed: our own measurements  & Macadamia seed coat material was found to exhibit the same specific tensile yield and ultimate strength  that are found in commercially pure annealed aluminium To break Macadamia and other “nuts” have been described by several authors  structure of  biology fig. 2. Macadamia integrifolia seeds were supplied by Mac Nuts WA, Australia. The average moisture content was 10.1 wt% (standard deviation: 0.7 wt%), determined gravimetrically based on 10 fracture pieces of 5 seeds by weighing them before and after treatment at 105°C for at least 24 h, according to the method described by Braga and co-workers Dry For microstructural investigations, coats of several seeds were cut normal and parallel to the suture, a line clearly visible on the outer surface  CamScan CS24  on several polished sections as well as on fracture surfaces and outer surfaces of the seed coat. For these investigations, the specimens were coated with a thin gold-layer of approximately 10 to 20 nm thickness. The outer surfaces were investigated in the natural state and after dewaxing the samples by submerging whole seeds for 12 hours in a dishwashing detergent solution, followed by drying in ambient air for 2 hours as described by Kaupp & Kaupp Macadamia seed coat was quantitatively analysed on a total of 35 micrographs of full-thickness fracture surfaces of eight different nuts. To determine whether the sclerenchymatous fibre bundles have a preferred orientation, two sections cut normal and four sections cut parallel to the suture were investigated in greater detail. The procedure of this analysis is schematically shown in The thickness of the different layers of the To assess the three-dimensional organisation of internal structural elements, whole nuts were scanned by microCT  with a spatial resolution of 16 µm. Following reconstruction, the 3D volume data was observed using ImageJ  and Amira 5.1 .Macadamia seed coat exhibits a hierarchically organised microstructure. Nine main levels exist, ranging from the level of the entire fruit down to the molecular scale. An overview of the hierarchical levels that we differentiated and the correlation to the scheme usually applied in biology is shown in The Macadamia fruit in the mature state has a round shape with outer diameters of 25 to 40 mm A whole Macadamia integrifolia seeds have a spherical shape with outer diameters between 22 and 27 mm in the case of the seeds we investigated. The diffusely arranged light-brown, round or elongated speckles with diameters between 1 and 10 mm form an individual pattern for each seed coat , and the inner seed. The seed is a hollow shell-structure enclosing the seedling fig. 3e.Sutures appear also on the inside of the testa, as one or two notch-like depressions, joining the hilum and the micropyle  consists of one cell-layer of flat pancake-like cells fig. 6a.The outer sclereid layer (L2) is about 800 µm thick, and therefore makes up about one third of the testa table 2.The sclerenchymatous fibre layer (L3) has an average thickness of about 1300 µm, which corresponds to about 50% of the seed coat thickness table 2.SEM investigations of different fracture surfaces show that shells, fractured parallel to the outer suture, exhibited more fibre bundles running parallel to the suture (hilum-micropyle direction) than shells fractured normal to the outer suture fig. 9c.The cream-coloured layer on one half of the inside of the shell (L5.1) is composed of non-sclerenchymatous tissue, as can be seen in the micrographs in Other features on this hierarchical level are the vascular bundles, visible as white structures that may be observed in photographs of cut seed coats , which vary in shape, dimension and composition. The cell dimensions were estimated by evaluating the light and SEM micrographs  have a thickness of 5 to 10 µm and diameters of approximately 20 to 40 µm. The cells of the cream-coloured layer (L5.1) have a polyhedral shape with approximately 10 to 14 faces. Their diameter spans 20 to 30 µm and their length is about 30 to 40 µm fig. 10dThe vascular bundles are composed of entwined hollow tubes fig. 12.This level relates to the structure of the biological cells in the single layers of the sandwich. The sclereids and the sclerenchymatous fibres show a similar composition with strong thickened and lignified cell walls  that could be identified as “elastic stiffeners”. Kaupp & Naimi-Jamal On the level of the whole seed coat (H3), we found a sandwich arrangement of five, or locally six concentric layers. Three of these layers (L2 to L4) belong to the so-called sclerenchymatous tissue and consist of different amounts of two cell-types: isodiametric sclereid cells, and elongated fibre cells. The sclereid layers  and the intermediate sclerenchymatous fibre layer (L3) make up more than 95% of the shell. The results correspond well with previous investigations Macadamia seeds. These were the sclerenchymatous fibres, their arrangements in bundles, vascular bundles and the single spiral vessels and tracheids. We showed a highly anisotropic arrangement of these elongated structures on different hierarchical levels. This is in agreement with the anisotropic arrangement of fibrous structures described by Kaupp & Naimi-Jamal Macadamia seed coats as an isotropic arrangement of elongated fibres The observed morphology and dimensions of the sclerenchymatous cells (sclereids and sclerenchymatous fibres) and their arrangement correspond well with the features described in former work Macadamia ternifolia, only two groups Macadamia integrifolia as we did. However, confusion exists in the literature as to what extent these species are different: several authors point out that Macadamia integrifolia is a variety of Macadamia ternifoliaIt may be that some of the differences that we see between the results of others and our description may come from “inter-species” differences. While most groups Macadamia integrifolia hierarchical levels and their main building blocks interact and result in astounding mechanical properties of the seed coat. The strength of these seed shells is much higher than the strength of comparable shells  seem to be the most important animal vectors in orchards in Australia Macadamia seeds found in rat burrows are damaged Macadamia. As to the importance of other animals  as vectors in the natural habitat, only little information exists Macadamia seed coat is primarily necessary as protection against abrasive transport over ground, and that the structure of Macadamia integrifolia is well suited to fulfil this function.Finally, we discuss a question relevant from a biological viewpoint: why do the seeds need to be so strong? Detailed knowledge of the microstructure may help to identify the types of loading and or abrasion that the seed coat faces when protecting the seed, although the intricate structure may have formed due to other reasons. In order to elucidate the ecological significance of the extraordinarily hard and tough seed coats of


We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death.Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca Summary: Monomeric and oligomeric α-synuclein induce Ca2+ signal in neurons and astrocytes by incorporating into the membrane. We utilised two different methods for the preparation of different α-synuclein species. In method 1, we add Alexa Fluor 594 (AF594)- or Alexa Fluor 647 (AF647)-labelled monomeric α-synuclein to Alexa Fluor 488 (AF488)-labelled monomeric α-synuclein in a 1:1 ratio. As the aggregation proceeds, oligomers consisting of both the AF488–α-synuclein and the AF594–α-synuclein will form, and, as the AF488–α-synuclein acts as a FRET donor, and the AF594–α-synuclein as a FRET acceptor, we can use single-molecule confocal FRET analysis to differentiate the oligomeric α-synuclein from the rest of the monomeric protein. Moreover, we can further utilize the FRET efficiency analysis to determine the formation of the compact β-sheet-containing oligomers  to generate an enriched oligomer preparation, which allowed us to determine oligomer-specific effects on cells. The enriched oligomers generated in this method have been extensively characterised and reported previously . Using t2+ signal in both neurons and astrocytes. In these experiments, cells loaded with the Ca2+ indicator were initially observed at basal levels for 2 min prior to application of α-synuclein. Cells that exhibited spontaneous oscillations prior to addition of α-synuclein were excluded from the analysis. The α-synuclein species was then added to the cells and recordings are taken for 30–40 min. Two patterns of α-synuclein-induced Ca2+ signal were observed. Oligomeric α-synuclein induces a transient Ca2+ signal, consisting of Ca2+ spikes, with and without complete recovery to baseline, at a concentration range of 40–250 nM, after 2–6 min (n=189). Monomeric α-synuclein induces transient Ca2+ spikes superimposed on a gradual increase in the basal Ca2+ signal (n=568). We report that the amplitude and shape of the α-synuclein-induced Ca2+ signal was consistently one of these patterns, and appeared to be independent of the concentration of α-synuclein applied. 2+ response in a dose–response experiment performed on cells of the same batch at the same number of days in vitro (DIV), using the same preparation of aggregated α-synuclein. At concentrations of 100 nM (oligomer 1 nM), 11.5±6.3% cells responded; at 250 nM (oligomer 2.5 nM), 13.0±4.1% cells responded; at 500 nM (oligomer 5 nM), 26.8±17.6% cells responded; at 750 nM (oligomer 7.5 nM), 25.7±21.9% cells responded; at 1.75 µM (oligomer 17.5 nM); 75.6±5.6% cells responded (mean±s.e.m.). In summary, the amplitude of the α-synuclein-induced Ca2+ signal did not vary with dose. However, the number of cells in the culture that responded to extracellular α-synuclein did vary, increasing with higher concentrations of α-synuclein. 2+ signalling induced by α-synuclein in the representative dose–response experiment shown in 2+ , or transient spikes , or a mixed pattern . Oligomers induced a sustained rise in basal Ca2+ , or transient spikes  or a mixed pattern . Thus overall, we show that the majority of cells will exhibit both transient Ca2+ spikes as well as a sustained rise in basal Ca2+. A small proportion of cells will demonstrate exclusively a sustained rise in basal Ca2+ particularly on exposure to monomers.It is well established that α-synuclein is present extracellularly, and we, and others, have shown that it is rapidly taken up by neurons and astrocytes in its monomeric, oligomeric and fibrillar forms . Applica 2–6 min D; n=189.+ signal C; n=568.2+ signals as low as 10 nM (for monomers) and 100 pM (for oligomers). However, at these concentrations <5% of the cells in the culture responded. Therefore, for all further experiments, concentrations of α-synuclein were employed that ensured a Ca2+ signal in at least 30% of cells. Of note, the α-synuclein-induced Ca2+ signal was independent of cell type and was observed in both neurons and astrocytes, and was also independent of brain region as it was observed in primary co-cultures from midbrain and cortex, but also from human cortical neurons derived from iPSCs .The physiological concentrations of extracellular α-synuclein are estimated to be in the low nanomolar range for monomer , and, as only 1% of the monomeric form aggregates, it is estimated to be in the picomolar range for oligomers. We were able to detect α-synuclein-induced Caom iPSCs G. Furthe2+ signal in neurons, we also applied the enriched oligomer preparation (from method 2) to primary co-cultures to determine whether oligomeric α-synuclein alone induced a Ca2+ signal. We observed a similar α-synuclein Ca2+ signal  and demonstrated that α-synuclein no longer induced any Ca2+ signal  [Ca2+]c release induced by inositol 1,4,5-trisphosphate (IP3). External Ca2+ can activate phospholipase C (PLC), which could in turn generate IP3 and thus mobilise ER Ca2+. To assess this hypothesis, we employed U73122 (5 µM), an inhibitor of phospholipase C . Similarly, Xestospongin C (10 µM), an inhibitor of IP3-dependent Ca2+ release, failed to reduce the oligomeric-α-synuclein-induced [Ca2+]c signals in astrocytes and neurons  to primary co-cultures in order to deplete Ca2+ from the ER. To ensure that the concentration of thapsigargin was adequate for complete ER depletion of Ca2+, ATP was then applied to the culture, and this did not induce a cytosolic Ca2+ response. This was followed by the addition of monomeric (n=199) or oligomeric (n=218) α-synuclein, which still induced a similar [Ca2+]c response as before. Taken together, these data strongly suggest that ER Ca2+ does not contribute to the α-synuclein-induced [Ca2+]c responses.We applied 0.5 µM thapsigargin -induced quenching technique whereby fura-indicators irreversibly bind Mn2+ and this quenches their fluorescence. The Mn2+ quench process can be quantified by measuring the fluorescence of fura-2 at its isobestic wavelength of ∼360 nm. Mn2+, when applied extracellularly, will enter cells through Ca2+ permeable channels, and thus the Mn2+ entry through Ca2+ channels can be quantified using the emission of fura-2 at 360 nM . As a control, in the presence of monomeric (n=62 cells) α-synuclein but in the absence of Mn2+, the fura-2 signal excited at 360 nm was unaltered showing that this method reliably reports a Ca2+-independent signal . However, in the presence of 40 µM Mn2+ and monomeric α-synuclein (n=81 cells), the fura-2 signal at 360 nm showed step-like irreversible decreases (quench) confirming the entry of Mn2+ through Ca2+ permeant channels. Each quench directly corresponds to the α-synuclein-induced transient increase in [Ca2+]c . These results suggest that the α-synuclein-induced transient [Ca2+]c signals are caused by pulses of Ca2+ influx from the extracellular medium into astrocytes and neurons across the plasmalemmal membrane through Ca2+-permeant channels.Having excluded intracellular Caynuclein A or olig2+ influx across the plasmalemmal membrane occurred through the known voltage-dependent Ca2+ channels or glutamate receptor mechanisms. We first incubated primary cultures with a variety of inhibitors of channels and receptors for 10 min, and then applied α-synuclein in order to determine their effect on the Ca2+ signal induced by monomers and oligomers. The concentration of these inhibitors has been previously tested and confirmed to induce inhibition of their channels or receptors. Nifedipine (1 µM) or Verapamil (20 µM), inhibitors of L-type VDCCs, did not alter the shape or amplitude of the monomeric (data not shown) or oligomeric  or neurons . Inhibitors of either ionotropic or metabotropic glutamate receptors, including 20 µM CNQX (n=156 cells), 10 µM MK-801 (n=286 cells) or 50 µM (S)-MCPG (n=215 cells), did not have any effect on the α-synuclein-induced Ca2+ signals, as shown on 2+ signals are not reflective of glutamate release in the culture, and are not dependent on known voltage-dependent Ca2+ channels.We then investigated whether the α-synuclein Caigomeric C α-synuc2+ signal caused by Ca2+ influx, we investigated whether the presence of α-synuclein was able to affect Ca2+ responses to physiological stimuli, such as depolarising stimuli: glutamate and KCl in neurons, and non-depolarising stimuli such as ATP in astrocytes. We therefore pre-incubated astrocytes and neurons with monomeric α-synuclein for 10 min. Subsequent stimulation of neurons with 5 µM glutamate resulted in a significantly slower [Ca2+]c efflux in neurons when compared to control . Both of these channel modes were cation selective. In the presence of a 10:1 NaCl concentration gradient across the lipid membrane, the reversal potential  was 57 mV, which is close to the theoretical Nernst potential for Na+ selectivity under these conditions (58 mV) . In our experiments we used the planar lipid bilayer (BLM) approach. This approach allows use of artificial membranes formed by pure lipids without participation of protein components, which are normally present in biological membranes. We detected that addition of oligomeric α-synuclein to the recording causes an increase in membrane permeability. We observed two distinct conductance modes consistent with channel-like behaviour of this protein. The first mode is characterized by a stable open state with no transitions to closed or sub-conductance states ( (58 mV) C. These  bilayer D. This aOverall these data suggest that oligomers might disrupt lipid membranes through different mechanisms that might depend on the lipid composition, the curvature of the lipid bilayer and the protein-to-lipid ratio.2+ dysregulation is known to be a key player in cell death induction, we investigated whether the α-synuclein-induced Ca2+ signal leads to neurotoxicity and ultimately cell death. We treated primary co-cultures with monomeric or oligomeric α-synuclein, assessed caspase-3 activation using the fluorescent-labelled caspase-3 substrate NucView and recorded the number of cells undergoing apoptosis over a 30-min period. We found that within 15 min, a significant percentage of cells exposed to oligomeric, but not monomeric α-synuclein, started to undergo apoptosis , containing monomeric or oligomeric α-synuclein for 6 h, and counted the number of Hoechst-33342- or propidium-iodide-positive cells  a gradual increase in the basal cytosolic Ca2+ level and (2) transient Ca2+ spikes. Patterns 1 and 2 could occur distinctly or in combination in the same cell. However, the monomer- and oligomer-induced Ca2+ signal was entirely dependent on the presence of extracellular Ca2+. Thus, both monomeric and oligomeric α-synuclein induce a cytosolic Ca2+ signal in cells by inducing pulses of Ca2+ influx across the plasmalemmal membrane through Ca2+-permeable channels.In this study, we sought to unravel the link between misfolded α-synuclein protein and aberrant Ca2+ influx seen with aggregated α-synuclein. First is the amyloid pore hypothesis in which oligomeric α-synuclein is able to form pore-like structures with non-selective leakage of compounds  binds membranes with higher affinity than monomers and retains a β-sheet structure. There are currently two major biophysical hypotheses that explain the Caompounds . Atomic ompounds . The secompounds .Monomers of α-synuclein have also been reported to affect membranes through differing mechanisms, that is, altering permeability or inducing channel formation. High concentrations of monomers have been shown to permeabilise membranes through detergent-like mechanisms . One stu2+ signal in cells, our bilayer membrane data suggest that the presence of both monomeric and oligomeric α-synuclein causes an increase in lipid membrane permeability. In the presence of monomeric α-synuclein, membrane conductance was highly irregular with frequent ‘spikes’ of ionic currents. Conversely, in the presence of oligomeric α-synuclein, we detected ion currents with distinct stable conductance levels, which are typical for ion channels and pores. For both monomers and oligomers, their effect on cell membrane conductance and Ca2+ influx can therefore be explained by their direct interaction between cell lipid membranes and the protein. Overall, our data is in agreement with both hypotheses of the interaction of monomers and oligomers with membranes: lipid thinning and resultant increase in permeability leads to ionic currents and Ca2+ spiking with both monomers and oligomers. However, in the case of oligomers, membrane permeabilisation produces a stronger effect likely due to the formation of stable pores resulting in rare, but regularly occurring, ion channel formation. Thus the α-synuclein-induced Ca2+ signal seen in cells is due to the interaction of monomeric and oligomeric α-synuclein with the neuronal membrane affecting its permeability and allowing different types of Ca2+ influx to occur.In order to understand the effect of our monomeric and oligomeric species of α-synuclein on membranes, we applied them to artificial bilayer membranes and recorded changes in conductance. In agreement with the monomeric and oligomeric α-synuclein-induced Ca2+ signals, impaired Ca2+ homeostasis and cell death in Parkinson's disease. We report that although monomeric and oligomeric forms of α-synuclein both induce Ca2+ signals, it is only the oligomeric species that induces apoptosis and cell death when applied to cells. Furthermore, the toxicity of the oligomeric α-synuclein can be abolished by preventing the Ca2+ influx through removal of extracellular Ca2+. This finding supports the fact that β-sheet-rich oligomeric species are the most toxic species in cells, and that this toxicity is crucially dependent on Ca2+ dysregulation. We have previously shown that the β-sheet-rich oligomeric species (and not the monomeric species) generates reactive oxygen species (ROS) resulting in oxidative stress  to induce cell death in this timeframe. Interestingly it has been reported that α-synuclein cytotoxicity in a yeast model is mediated by the ability of α-synuclein to increase the basal intracellular Ca2+ levels (through a PMR1-mediated mechanism) and this results in an oxidative burst that ultimately kills cells. Abolishing the raised intracellular Ca2+ prevents cell death in this model  at 1 mg/ml to prevent α-synuclein species from adsorbing to the surface, as we have recently shown  and cardiolipin were obtained from Avanti Polar Lipids . Planar BLMs were formed from a 20 mg/ml lipid solution of either DOPC or DOPC:Cardiolipin (3:1) in n-decane (Aldrich) as has been shown previously  150 mM NaCl, 2 mM CaCl2, 10 mM Tris-HCl pH 7.4. The liquid junction potential offset was compensated for prior to the membrane formation.For the measurements of the selectivity of the induced conductance, reversal potential  was measured in a presence of 10:1 (trans-to-cis) transmembrane ion gradient. In these experiments, membranes were formed between compartments containing: (cis) 15 mM NaCl, 2 mM CaClCurrents across the bilayer were recorded using a Planar Lipid Bilayer Workstation (Warner Instruments). The cis compartment was connected to the head stage input and the trans compartment was held at virtual ground by a pair of matched Ag–AgCl electrodes. Signals from voltage-clamped BLMs were high-pass-filtered at 2.1 kHz using an eight-pole Bessel filter LPF-8 (Warner Instruments), digitized using Data Translation Digitizer and recorded on a computer using software developed by Elena Pavlova (available on request).2+- and Mg2+-free, Invitrogen, Paisley, UK). The tissue was minced and trypsinised (0.25% for 5 min at 37°C), triturated and plated on poly-D-lysine-coated coverslips and cultured in Neurobasal A medium  supplemented with B-27  and 2 mM L-glutamine. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air, fed twice a week and maintained for a minimum of 12 days before experimental use to ensure expression of glutamate and other receptors. Neurons were easily distinguishable from glia: they appeared phase bright, had smooth rounded somata and distinct processes, and lay just above the focal plane of the glial layer. Cells were used at 12–15 days in vitro (DIV).Mixed cultures of hippocampal or cortical neurons and glial cells were prepared as described previously  with mod2PO4, 1.25; glucose, 10; MgSO4, 1.8; CaCl2, 1.6; KCl, 3; NaHCO3, 26, pH 7.4) continuously bubbled with 95% O2 and 5% CO2. Slices were imaged in HBSS for up to 3 h after preparation.Horizontal hippocampus or entorhinal cortex slices (100 µm) were prepared from P9 Wistar rats, using standard procedures. Tissue was sliced at ∼4°C in artificial cerebrospinal fluid . Ca2+-free medium contained 0.5 mM EGTA.Primary neurons and astrocytes were loaded for 30 min at room temperature with 5 µM fura-2 AM or fluo-4 AM and 0.005% pluronic in a HEPES-buffered salt solution (HBSS) . Emitted fluorescence light was reflected through a 515-nm long-pass filter to a cooled CCD camera  and digitised to a 12-bit resolution. All imaging data were collected and analysed using software from Andor . Fura-2 is a ratiometric dye with a high affinity for Ca2+ and this allows an accurate measurement of the cytosolic Ca2+ independent of loading variations. The experimental setup used for measuring fura-2 also enables signals with transient rapid kinetics and decay times to be measured. The measurement of this dye is a ratio between the emitted light at 340 nm (high Ca2+) and the emitted light at 380 nm (low Ca2+). The fura-2 data have not been calibrated in terms of [Ca2+]c because of the uncertainty arising from the use of different calibration techniques.Fluorescence measurements were obtained on an epifluorescence inverted microscope equipped with a 20× fluorite objective. [Ca2+ sensor where the emission intensity depends on the levels of bound Ca2+. It has a lower Kd and is useful for visualising Ca2+ variations around the basal concentration. As fluo-4 intensity is measured on the confocal microscope, the units used for these experiments are (arbitrary) fluorescence units.Confocal images were obtained using a Zeiss 710 CLSM equipped with a 40× oil immersion objective. The 488-nm argon laser line was used to excite fluo-4 which was measured between 510–550 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ∼2 µm. All data presented were obtained from at least five coverslips and two or three different cell preparations. Fluo-4, in contrast is a single-wave CaFor toxicity assays, we loaded cells simultaneously with 20 µM propidium iodide, which is excluded from viable cells but exhibits a red fluorescence following a loss of membrane integrity, and 4.5 µM Hoechst 33342 , which gives a blue staining to chromatin, to count the total number of cells. Using phase-contrast optics, a bright-field image allowed identification of neurons, which look quite different to the flatter glial component and also lie in a different focal plane, above the glial layer. A total number of 600–800 neurons or glial cells were counted in 10 fields of each coverslip. Each experiment was repeated five or more times using separate cultures.For measurements of caspase-3 activation, cells were loaded for 15 min at room temperature with 10 μM NucView 488 caspase-3 substrate  in HBSS. NucView 488 is a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. The substrate can rapidly cross the cell membrane to enter the cell cytoplasm, where it is cleaved by caspase-3 to release the high-affinity DNA dye. The released DNA dye migrates to the cell nucleus to stain the nucleus brightly green.Confocal images were obtained using Zeiss 710 confocal laser scanning microscope and a 40× oil immersion objective. The 488-nm argon laser was used to excite NucView 488 fluorescence, which was measured using a bandpass filter from 510 and 560 nm.t-tests were performed where appropriate and P-values provided in results section.Statistical analysis and exponential curve fitting were performed using Origin 9  software. Results are expressed as mean±s.e.m. Student's


Eryptosis is triggered by a wide variety of xenobiotics. It is inhibited by several xenobiotics and endogenous molecules including NO and erythropoietin. The susceptibility of erythrocytes to eryptosis increases with erythrocyte age. Phosphatidylserine exposing erythrocytes adhere to the vascular wall by binding to endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor for phosphatidylserine and oxidized low density lipoprotein (CXCL16). Phosphatidylserine exposing erythrocytes are further engulfed by phagocytosing cells and are thus rapidly cleared from circulating blood. Eryptosis eliminates infected or defective erythrocytes thus counteracting parasitemia in malaria and preventing detrimental hemolysis of defective cells. Excessive eryptosis, however, may lead to anemia and may interfere with microcirculation. Enhanced eryptosis contributes to the pathophysiology of several clinical disorders including metabolic syndrome and diabetes, malignancy, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson's disease. Facilitating or inhibiting eryptosis may be a therapeutic option in those disorders.Suicidal erythrocyte death or eryptosis is characterized by erythrocyte shrinkage, cell membrane blebbing, and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca The lifespan of circulating erythrocytes is limited by senescence to 100–120 days . In seneThe present paper lists triggers and inhibitors or eryptosis, the mechanisms involved in the regulation of eryptosis, and the (patho-) physiological significance of eryptosis. The reader is encouraged to study earlier reviews on further aspects of eryptosis –12.As listed in The susceptibility to stimulation of eryptosis increases with erythrocyte age . The enhErythrocytes from newborns are relatively resistant to several triggers of eryptosis but are highly susceptible to eryptosis following oxidative stress –24. The 2+ activity ([Ca2+]i) [2+]i mainly results from Ca2+ entry through Ca2+-permeable unselective cation channels i and eryptosis following Cl− removal were thus blunted in erythrocytes from gene-targeted mice lacking TRPC6 [A major trigger of eryptosis is the increase of cytosolic Ca[Ca2+]i) . The incchannels , 27, whiand Ca2+ . The cha2 (PGE2) , 30. Phaic shock . The Ca2sorbitol  and by s or SCN− . They ar or SCN− –34, whicryptosis , 28. Actryptosis . Accordiryptosis –37 glutaryptosis , salidroryptosis , 7-monohryptosis , trolox ryptosis , or N-acryptosis , 20. Theryptosis . The incng TRPC6 .2+]i following activation of the cation channels is followed by cell shrinkage due to activation of Ca2+-sensitive K+ channels i further stimulates cell membrane scrambling with breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane and translocation of phosphatidylserine to the erythrocyte surface i and subsequent stimulation of eryptosis upon exposure of erythrocytes to oxidative stress or following energy depletion [α opens cation channels and thus triggers Ca2+ influx into erythrocytes [Pharmacological evidence points to a role of casein kinase 1epletion . Pharmachrocytes . Osmotichrocytes  and pharhrocytes . Eryptoshrocytes  and sunihrocytes  sensitiv2+]i [2+ as it protects against eryptosis induced by the Ca2+ ionophore ionomycin without appreciably affecting the ionomycin-induced increase of [Ca2+]i. NO blunts apoptosis of nucleated cells in part by caspase inhibition [2+]i [Eryptosis is further inhibited by cGMP-dependent protein kinase (cGKI) . cGKI de2+]i . The kin2+]i –233, a p2+]i . NO is s2+]i –236. Ery2+]i  at conce2+]i , 238. NOhibition , 240. Hoon [2+]i –245 NO ion [2+]i . Converson [2+]i , 242. Ason [2+]i . The effon [2+]i . In conton [2+]i , excession [2+]i –248. NO on [2+]i , 250. Inon [2+]i  and pres2+]i, ceramide, oxidative stress, caspases, nitroxide, and a variety of kinases. Most triggers of eryptosis are mainly effective by increasing [Ca2+]i and/or enhancing ceramide abundance in the cell membrane. Unlike in apoptosis of nucleated cells, caspases do not play a dominant role in the triggering of eryptosis. Survival of erythrocytes does require the activity of several kinases including AMPK and cGKI. Activation of other kinases, such as CK1α and JAK3, triggers eryptosis. The phosphorylation targets of the kinases required for the stimulation or inhibition of eryptosis are still ill-defined. Clearly, tremendous additional experimental effort is required for full understanding of the eryptotic machinery.Collectively erythrocyte survival and eryptosis are regulated by an amazingly complex cellular machinery involving i results from activation of the Ca2+ permeable unselective cation channels i i . The pat [Ca2+]i . Plasmodylserine , 299 andylserine , 301. Thylserine . Adherenhrocytes . P. falciparum, however, the formation of PGE2, Ca2+ permeability, phosphatidylserine exposure at the cell surface, and removal by macrophages are all augmented in HbA/S carriers [Upon infection, eryptosis is accelerated in erythrocytes from heterozygous carriers of the sickle-cell trait (HbA/S), beta-thalassemia-trait, homozygous Hb-C, and G6PD-deficiency thus leading to early clearance of infected erythrocytes, decreased parasitemia, and a relatively mild course of the disease , 300. Ascarriers . The acccarriers . Moreovecarriers , chlorprcarriers , and inhcarriers . Importa2+-permeable unselective cation channels, ceramide, caspases, and a variety of kinases including Janus-activated kinase 3, AMP-activated kinase, cGMP-dependent protein kinase, casein kinase 1α, p38 kinase, protein kinase C, and p21-activated kinase 2. The sensitivity to eryptosis is enhanced in aged erythrocytes. Fetal erythrocytes are particularly sensitive to oxidative stress. Eryptosis is triggered by a wide variety of xenobiotics and enhanced eryptosis is observed in several clinical conditions including dehydration, diabetes, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson's disease. Drugs and nutrients inhibiting eryptosis may open novel therapeutic options in the treatment of anemia and deranged microcirculation. Eryptosis stimulating xenobiotics may at least in theory accelerate removal of Plasmodium infected erythrocytes and thus favourably influence the clinical course of malaria.Similar to apoptosis of nucleated cells, eryptosis is a physiological mechanism eliminating defective erythrocytes in order to prevent hemolysis and subsequent release of hemoglobin into circulating blood. Excessive eryptosis may, however, cause anemia and impede microcirculation. Orchestration of eryptosis involves Ca


Resveratrol has been reported to prevent the development of hypertrophy and contractile dysfunction induced by PO. However, other studies found that resveratrol treatment for a longer period of time failed to regress cardiac hypertrophy. The aim of this study is to determine the timing of resveratrol treatment to achieve antihypertrophic effect and investigate whether resveratrol prevents the development of HF through preservation of myocardium structure and modulation of Ca2+ handling proteins were measured by western blot analysis.To generate rats with cardiac hypertrophy, male Sprague–Dawley rats were subjected to PO (aortic banding procedure) for 4 weeks. Sham-operated animals served as controls. Rats with cardiac hypertrophy were given resveratrol (4 mg/kg/day) for 4, 6, and 8 weeks, respectively. Histological and echocardiographic analysis and transmission electron microscopy were performed to assess cardiac structure and function. The levels of Ca2+ handling proteins induced by aortic banding, including downregulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and ryanodine receptor 2 (RyR2), hypophosphorylated phospholamban (PLB), upregulation of Na+/Ca2+-exchangers (NCX1) and increased expression and phosphorylation of Ca2+/calmodulin -dependent protein kinase II (CaMKII). Moreover, resveratrol alleviated the decreased SERCA activity induced by aortic banding.Histological analysis showed that resveratrol treatment regressed developed cardiac hypertrophy at 8 and 10 weeks postsurgery, but not at 12 weeks. However, resveratrol strongly and continuously prevented the development of cardiac dysfunction and dilation of cardiac chamber as evaluated by echocardiography and H&E staining of heart cross-sections. In addition, PO-induced cardiac fibrosis was completely inhibited by resveratrol treatment. Resveratrol markedly prevented the disrupted myocardium but partially rescued mitochondrial abnormality in banded rats. Moreover, resveratrol prevented the alteration of Ca2+ handling proteins.Resveratrol effectively prevented the transition from compensatory to decompensatory stage of cardiac hypertrophy induced by PO, but this effect is dependent on the timing of treatment. We suggest that resveratrol may exert beneficial effects on cardiac hypertrophy through protection of cardiac structure and modulation of CaThe online version of this article (doi:10.1186/s12967-014-0323-x) contains supplementary material, which is available to authorized users. Heart failure (HF) is a major cause of hospitalization and mortality worldwide . IncreasPrevious studies including The Dietary Approaches to Stop Hypertension (DASH) trial and Lyon Diet Heart Study have demonstrated that increased intake of fruits and vegetables lowered blood pressure in patients with hypertension, reduced the incidence of cardiovascular diseases, and improved survival after myocardial infarction . FurtherAdditionally, it is well demonstrated that resveratrol protects against the development of HF by improving contractile functions -5. Howev2+ cycling, a critical process referring to the mobilization of intracellular Ca2+ in excitation-contraction (EC) coupling, determines cardiac contractility and relaxation. It is now generally accepted that defective Ca2+ handling proteins in the cycling play an important role in HF pathophysiology. The key defects in Ca2+ cycling occur at the level of the sarcoplasmic reticulum (SR), a Ca2+ storage bulk in muscle. The SR Ca2+ release channel  is oxidized, nitrosylated, and hyperphosphorylated, resulting in “leaky” channels and depletion of Ca2+ from the SR in failing hearts. Downregulated SR Ca2+ ATPase 2a (SERCA2a) and hypophosphorylated phospholamban (PLB) contribute to impaired SR Ca2+ uptake that conspires with leaky RyR2 to deplete SR Ca2+ in failing hearts [2+ handling proteins in SR to preserve contractile function in rats subjected to PO.Cag hearts . AccordiThe investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Male Sprague–Dawley rats weighing 80-100 g were obtained from Central Animal Care Services at Southern Medical University and subjected to the abdominal aortic banding procedure for induction of PO. Briefly, rats were kept in a room with constant temperature of 26°C, humidity of 55%, and a 12 h light: 12 h dark cycle throughout the study. Animals were given standard rat chow and tap water ad libitum. All rats were anesthetized for surgery with 10% chloral hydrate (3 ml/kg). A midline laparotomy was performed, and the suprarenal abdominal aorta was exposed. The aorta between the branches of celiac artery and anterior mesenteric artery was tied off by a 4–0 silk suture with a blunt 24-gauge needle as a guide. Sham-operated rats were served as controls and subjected to the same surgeries except for the creation of the aortic band. Resveratrol treatment started from 4 weeks postsurgery when LV hypertrophy ocurred, as indicated by the results of echocardiography, and aortic banded rats were divided into treated and untreated subgroups. Resveratrol  was suspended in 0.5% carboxymethylcellulose  dissolved in 0.9% saline. This solution was administered to treated rats by oral gavages for 4, 6 and 8 weeks, respectively. 0.9% saline was used as a vehicle to treat sham and banded animals.At 4, 8, 10 and 12 weeks postsurgery, rats from all groups were weighed and anesthetized with chloral hydrate. Transthoracic two-dimensionally guided M-mode echocardiography was performed using an IE33 echocardiographic system  equipped with a 15-MHz (s12) transducer. The following parameters were measured: percentage of left ventricular (LV) fractional shortening (FS), LV ejection fraction (EF), maximal velocity through left ventricular outflow tract (Vmax), Cardiac output, LV pressure half-time, LV internal dimensions at both diastole and systole , LV posterior wall dimensions at both diastole and systole , and interventricular septal dimensions at both diastole and systole .Body weight, heart and LV with septum were weighed, and the LV mass-to-body weight ratio (LVm/BW) was calculated. LV samples were fixed in 10% neutral buffered formalin, embedded with liquid paraffin, and sectioned into 6 μm in thickness. Myocyte cross-sectional area was used for the evaluation of the degree of LV hypertrophy. Briefly, sections were stained with hematoxylin and eosin (HE) and examined under a light microscope (Nikon ECLIPSE Ti-U). Five random fields from each of 4 sections per animal were analyzed, and 10 ~ 15 myocytes per section were measured. The quantification of diameter and area of myocytes were determined with Image Pro Plus 6.0 . To assess fibrosis, sections were stained with a Masson trichrome kit  according to manufacturer’s instructions and stained sections were examined under a light microscope. Interstitial and perivascular fibrosis content were quantified in tissue sections using Image Pro Plus 6.0 as the percentage of connective tissue per cross-sectional surface area. The cross sectional area of LV chamber was also measured.Hearts were perfused and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer followed by post-fixation with 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature. The tissue cubes were then dehydrated in an ascending series of ethanol solutions, followed by acetone dehydration. The tissue cubes were embedded with Embed 812 resin overnight at 37°C. 40 ~ 60 nm ultrathin sections were cut on an ultramicrotome, picked up on grids, and stained with a solution of lead citrate and uranyl acetate. The sections were then observed, and photographs were taken under a Hitachi 7650 electron microscope.2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin (20 ml/g tissue) and 1 mM phenylmethylsulfonyl fluoride. Protein samples (20 ~ 25 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were probed with the following primary antibodies: anti-Ca2+/calmodulin-dependent protein kinase II (CaMKII) antibody , anti-phospho-CaMKII (Thr286) antibody , anti-PLB antibody , anti-phospho-PLB (S16) antibody , anti-SERCA2 antibody , anti-RyR2 antibody  or anti-Na+/Ca2+-exchangers (NCX1) antibody . Appropriate secondary antibodies were used to detect the corresponding primary antibodies, and the antibody-antigen complexes in all membranes were detected by the ECL PLUS kit (Bio-Rad). The protein bands were quantified by Image Pro Plus 6.0.LV tissues frozen in liquid nitrogen were pulverized and homogenized in a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM NaSamples of the left ventricle were prepared as the method previously described by Lizotte E . The ATPP <0.05 was considered significant.All statistical analyses were performed using the SPSS Statistical software (version 17.0). Values are expressed as mean ± standard error (SEM). One-way analysis of variance was used to analyze the differences between the means of groups, followed by Tukey post hoc test. A value of Cardiac structure and function in rats were assessed by echocardiography at 4 weeks postsurgery  in banded rats were decreased continuously compared to sham rats at 8, 10, and 12 weeks postsurgery. Of note, resveratrol treatment significantly increased EF by 14%, 15%, 26%, FS by 30%, 32%, 26%, and Vmax by 30%, 26%, 25% at 8, 10, 12 weeks postsurgery respectively, compared with banded rats. FS and Vmax in resveratrol-treated rats were even remarkably higher than sham rats at 12 weeks postsurgery  inhibition of cardiac fibrosis, (2) prevention of myocardium ultrastructural alterations, (3) modulation of activation and expression of CaAntihypertrophic effect of resveratrol and its analogue has been documented in different experimental settings of PO ,3,4,8-124-week aortic banding caused compensatory centric hypertrophy characterized by high contractile function of LV, increased thickness of LV wall and decreased LVIDs. However, with long-standing untreated PO, LV systolic performance decreased and LV dilatation occurred, which are the key features of decompensatory phase, namely HF. Resveratrol treatment effectively preserved cardiac systolic function and LV chamber dimension. Previous studies demonstrated that resveratrol improved contractile function with abolishing cardiac hypertrophy caused by PO ,3. InterThe development of HF is associated with marked myocardial fibrosis which is characterized by excessive extracellular matrix (ECM) deposition and myocardial stiffness . LimitinSecond, we found that resveratrol protected against myocardial ultrastructural abnormalities induced by PO. The most obvious detriment to myocardial ultrastructure induced by PO was decrease of myofibrils as a result of rupture and degradation of myofilaments. Notably, resveratrol treatment normalized myocardial ultrastructure as evidenced by intact myofibrils and organized sarcomeres, which may be attributed to multiple mechanisms beneficial to cell survival including prevention of cardiomyocyte apoptosis, regulation of autophagy, and reduction of oxidative stress ,18-20. T2+ cycling which refers to the release and reuptake of intracellular Ca2+ is highly regulated in cardiomyocytes and determines the process of cardiac muscle contraction and relaxation. Defects in the regulation of Ca2+ handling proteins contribute to HF. To our knowledge, the effects of resveratrol on Ca2+ handling proteins in HF remain unclear. Impaired SERCA2 function and enhanced NCX activity have been proposed as causes of reduced SR Ca2+ load in HF. Moreover, PLB, a regulator of the affinity of SERCA2 for Ca2+, balances SR Ca2+ uptake through inhibiting the affinity of SERCA2 for Ca2+ by unphosphorylated PLB and relieving this inhibition by phosphorylation. As a negative regulator of SERCA2, hypophosphorylated PLB is an important cause of deficient SR Ca2+ uptake in failing hearts. Accordingly, both SERCA2 overexpression and PLB inhibition have been designed as therapeutic strategies for HF [2 expression and ATPase activity, elevated NCX1, increased PLB and decreased p-PLB, indicating that SR Ca2+ load in this animal model is deficient, consequently leading to impaired contractile function. Resveratrol effectively upregulated SERCA2 levels, ATPase activity and the ratio of phosphorylated to unphosphorylated PLB, accompanied by a significant downregulation of NCX1, indicative of improved SR Ca2+ load in hearts. Consistent with our results, a previous study showed that increased SERCA2a expression by resveratrol improved contractile function in chronic type 1 diabetes [Cas for HF . In thisdiabetes .2 accounts for excessive diastolic SR Ca2+ leak in non-ischemic (aortic banding) cardiomyopathy, but not in ischemia (post-MI) in which PKA phosphorylation of RyR2 is involved [2 open probability and a diastolic SR Ca2+ leak because of a higher sensitivity to Ca2+-induced Ca2+ release at low cytoplasmic Ca2+ concentrations. In our experiment, resveratrol attenuated the increase in CaMKII level and completely inhibited hyperphosphorylation of CaMKII. It has been demonstrated that RyR2 leak was inhibited through inhibition of CaMKII phosphorylation. Knock-in mice with an inactivated CaMKII phosphorylation site on RyR2 had lower SR Ca2+ leak and improved SR Ca2+ load, and were relatively protected from HF development after transverse aortic constriction [2 phosphorylation was significantly increased, accompanied by increased SR Ca2+ leak, reduced systolic Ca2+ transients, depletion of SR Ca2+ storage and elevated diastolic Ca2+ levels. Moreover, CaMKII inhibition, but not inhibition of PKA yielded a reduction of the SR Ca2+ leak [2 leak.Additionally, we detected a significant increase in expression and phosphorylation of CaMKII due to PO. It has been reported that CaMKII hyperphosphorylation of RyRinvolved . This letriction . In patia2+ leak . Therefo2 channel in HF was investigated in many studies, very little is known about expression of RyR2 and its significance in failing heart. We found that RyR2 protein expression was significantly decreased in myocardium of HF and resveratrol effectively enhanced the expression of RyR2. We speculate that downregulation of RyR2 may decrease systolic Ca2+ release and impair cardiac contractility. Similarly, Kubalova et al. found that RyR2 content in failing hearts was decreased to approximately half of control values, but the levels of other proteins of the Ca2+ release channel complex such as triadin and junctin were not changed. The altered stoichiometry of triadin and junctin to RyR2 may increase activity of RyR2, thus leading to the abnormal Ca2+ handling in HF [Although the activity of RyRng in HF .2+ spark-mediated SR leak by reducing oxidative stress in RyR2 in burn-traumatized heart, leading to protection of cardiac function against burn injury [2 and other Ca2+ handling proteins in aortic banded rats.A prevailing theory is that one of the major mechanisms proposed to underlie resveratrol mediated cardioprotection is reduction of oxidative stress ,27,28. Pn injury . Further2+ handling proteins.These data demonstrated that resveratrol treatment prevented cardiac hypertrophy induced by PO with time-limitation and inhibited the development of HF via improving cardiac structure and function. The novel findings on mechanisms underlying cardioprotection of resveratrol include protection of myocardium ultrastructure and regulation of global Ca


Methods: RT-PCR, Q-PCR, western blotting and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca2+ channels in leukemia cell lines. The function of T-type Ca2+ channels in leukemia cell growth and the possible mechanism of the effect of T-type Ca2+ channel antagonists on cell proliferation and apoptosis were examined in T-lymphoma cell lines.T-type Ca2+ channel inhibitors, mibefradil and NNC-55-0396 in a concentration-dependent manner. Mechanistically, these inhibitors played a dual role on cell viability: (i) blunting proliferation, through a halt in the progression to the G1-S phase; and (ii) promoting cell apoptosis, partially dependent on the endoplasmic reticulum Ca2+ release. In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.We show that leukemia cell lines exhibited reduced cell growth when treated with T-type Ca2+ channel expressing leukemia cell lines and suggest a potential therapeutic target for leukemia.These results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type CaThe online version of this article (doi:10.1186/s13046-015-0171-4) contains supplementary material, which is available to authorized users. Cancer cells are able to pass these phases with much lower extracellular Ca2+ levels than normal cells [2+ influx.Intracellular Capoptosis –3. Intraferation , otherwill death . In normal cells , indicat2+ influx, T-type Ca2+ channel expression and relationship to proliferation and apoptosis have been demonstrated in many cancer types, including leukemic [2+ channels can be detected in tumor samples collected from patients. In addition, these reports also show that pharmacological inhibition by small molecule antagonists or RNAi-mediated downregulation of T-type Ca2+ channels leads to inhibition of cancer cell proliferation and inducing cancer cell apoptosis. Therefore, T-type Ca2+ channels pose an attractive potential target for cancer therapy. T-type Ca2+ channels have unique electrophysiological characteristics: low voltage-activated Ca2+ current, fast (transient) inactivation, slow deactivation and low unitary conductance [2+ channel subunits, the α1G (Cav3.1), α1H (Cav3.2) and α1I (Cav3.3) has been revealed [2+ channels are known to mediate a “window current” [2+ current carried by the portion of channels that are not completely inactivated. Hence, T-type Ca2+ channels are well suited to regulate Ca2+ oscillations under non-stimulated or resting membrane conditions. This regulation of Ca2+ homeostasis allows T-type Ca2+ channels to control cell proliferation and apoptosis, or death. There are increasing data suggest that the expression of T-type Ca2+ channels is cell cycle-dependent [Among the routes for Caleukemic , ovarianleukemic , 9, glioleukemic , 11, breleukemic , esophagleukemic , hepatomleukemic , melanomleukemic , and colleukemic . Moreoveductance . To daterevealed . At low current” , i.e. a ependent –22.2+ currents with 10 to 20 times higher selectivity for T-type over L-type Ca2+ channels [2+ channels, which exerts no effect against high voltage Ca2+ channels at 100 μM, but inhibits T-type Ca2+ channels in HEK293 cells with a potency comparable to that of mibefradil  [2+ channels [Mibefradil is a potent inhibitor of T-type Cachannels . NNC-55-10.1 μM) . A growichannels , 23, 24.2+ channels in leukemic cell lines. We showed that inhibition of T-type Ca2+ channels with antagonists, mibefradil and NNC-55-0396, led to a decrease in proliferation, and an increase in apoptosis of leukemia cells in vitro, which was preceded by disrupting endoplasmic reticulum (ER) Ca2+ homeostasis. We also demonstrated down-regulating ERK signaling in MOLT-4 cells following the application of T-type Ca2+ channel antagonists. Since human normal blood cells do not express T-type Ca2+ channels, our results suggest that T-type Ca2+ channel inhibitors may be useful in the treatment of acute lymphocytic leukemia (ALL).In this study, we examined the function of T-type Ca2 with 95 % humidity.Human leukemic cell lines MOLT-4, Jurkat, Ball, HL-60, NB4, HEL, K-562, and U937 were purchased from the American Type Culture Collection  and were cultured in RPMI 1640 medium containing 10 % heat-inactivated fetal bovine serum , 1 % pen/strep  and 2 mM L-glutamine at 37 °C in 95 % air/5 % COn = 8 each), according to The Code of Ethics of the World Medical Association. Mononuclear cells were isolated with human lymphocyte separation medium  according to manufacturer’s instructions. Briefly, PBMCs were separated by centrifugation at 900 × g for 30 min at 18–20 °C over a Ficoll-Paque PLUS gradient. The resulting PBMC layer was washed twice with nuclease-free 0.9 % NaCl solution and prepared for RNA isolation.Whole blood (5–10 ml) was collected from healthy human male and female donors . The cDNA product was used as a template for subsequent PCR amplifications for α1G, α1H, and α1I subunit, using sequence-specific primers. Primer sequences, product sizes and PCR conditions are summarized in Table Total cellular RNA was isolated from exponentially growing cells and human PBMCs using RNAsimple Total RNA Kit . Messenger RNA was reverse-transcribed (RT) to cDNA using oligo(dT)t method. Samples were assayed in triplicate for each gene, and the mean expression was used during subsequent analysis. Q-RT-PCR was carried out under the following reaction conditions: stage 1, 95 °C for 30 s (Rep 1); stage 2, 95 °C for 5 s then 60 °C for 1 min (Reps 40).Total RNA 1 μg was used to generate cDNA with GoScript reverse transcriptase as above. A 1-μl aliquot of each synthesized cDNA was analyzed by Quantitative Real-Time PCR  using SYBR Green PCR Master Mix  according to manufacturer’s protocols and message level was determined using the △CWestern blotting was performed as described previously , 26. Imm2, 10 glucose, pH 7.4 adjusted with TEA-OH. The resistance of pipettes ranged 3–5 MΩ when filled with internal solution containing (in mmol/L): 10 HEPES, 120 CsCl, 1 MgCl2, 10 TEA-Cl, 10 EGTA, 5 Na2ATP, 1.2 Creatine phosphase, pH 7.2 adjusted with CsOH. Liquid junction potential was not compensated. Following whole cell access, the cells were held at- 80 mV with test pulses ranging from −60 mV to +60 mV with 10 mV increments.Whole-cell voltage-clamp recordings were performed by following the procedures as described in our previous studies . For T-c4 cells/well in 100 μL of cell culture media. Cells were treated with different concentrations of mibefradil or NNC-55-0396 . After drug exposure, 20 μL of CellTiter 96 AQ One Solution Reagent was added to each well and allowed to incubate for 2 h at 37 °C. The quantity of formazan product formed, which is directly proportional to the number of viable cells, was measured on a Multi-Mode Microplate Reader  at 490 nm wavelength using a reference filter at 650 nm wavelength. Viability assays were performed at least three times in independent experiments, using triplicate measurements in each.To determine cell survival and proliferation, cell growth was quantified using the CellTiter 96 AQ One Solution Cell Proliferation Assay Kit . Cells were plated in 96-well culture plates at a density of 1–2 × 10v3.1 and Cav3.2 T-type Ca2+ channels was designed according to a previous report [v3.1/3.2, 5′- GCCATCTTCCAGGTCATCACA -3′; negative control scramble sequence, 5′-TTCTCCGAACGTGTCACGT-3′) were synthesized by Integrated DNA Technologies and cloned into the lentiviral small interference RNA (siRNA) vector GV115 . Transduction of shRNA into the MOLT-4 cells was achieved by a lentiviral infection method. The positive transfected cells were sorted using the flow cell sorter and subjected to the CellTiter 96 AQ One Solution Cell Proliferation Assay. Q-RT-PCR was used to verify that shRNA decreased T-type Ca2+ channel genes expression.The target sequence against human both Cas report . RNAi ol6 cells were fixed in 70 % ethanol for 2 h on ice. The cell pellets were washed with PBS and incubated with 100 μL RNase A solution for 30 min at 37 °C. PI (400 μL) was then added and allowed to incubate for an additional 30 min at 4 °C in dark. DNA content was measured by exciting PI at 488 nm and measuring the emission at 620 nm, using a flow cytometer . Data analysis was carried out using FlowJo software. Each experiment is representative of at least three independent experiments.Analysis of cell cycle distribution was determined by propidium iodide (PI) staining and flow cytometry according to manufacturer’s instructions . Briefly, following treatment, approximately 1 × 10Apoptosis of ALL cells was detected using an annexin V apoptosis assay, followed by flow cytometry analysis. In brief, cells were harvested following treatment, washed in PBS, and subjected to Annexin V/PI staining according to the manufacturer’s protocol . The percentage of apoptotic cells was evaluated using flow cytometer (BD Accuri C6).Briefly, cells were loaded with 1 μM Fluo-4/AM (Invitrogen) for 60 min at 37 °C in 1640 medium, washed 3 times with PBS and resuspended in 1640 or calcium-free medium. The loaded cells were measured by flow cytometry in a FACScan (BD Accuri C6) at an excitation wavelength of 488 nm and an emission wavelength of 520 nm as described below.m, was assessed with 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide fluorescent probe (JC-1) . The treated and control cells were harvested and incubated with JC-1 for 20 min at 37 °C in the dark. The cells were washed and resuspended in 100 μL of cold PBS and then analyzed with flow cytometer (BD Accuri C6).Mitochondrial membrane potential, ψPlots were produced using Origin 7.0 . Results were compared using unpaired t-tests (for comparing two groups) or one factor ANOVA analysis followed, where appropriate, by Student-Newman-Keuls (for multiple comparisons) post-test. A p-value of less than 0.05 indicated statistically significant differences between observed effects. The results are expressed as mean ± SEM.2+ channels in human leukemia cell lines  using standard reverse transcriptase PCR (RT-PCR). As shown in Fig. 2+ channels. We also examined the expression of T-type Ca2+ channels in MOLT-4 and Jurkat T cells employing western blot analysis. Both Cav3.1 (very weak) and Cav3.2 subunits were stained in a size of ~260 kD from the cellular extract of MOLT-4 cells, whereas only Cav3.2 subunit (very weak) was detected in Jurkat cells , and the mean T-current density was 0.69 ± 0.15 pA/pF. Figure 2+ current was elicited by a depolarizing pulse at 0 mV when held at −80 mV (control). After perfusion of 2 μM of NNC-55-0396, the T-type Ca2+ current was inhibited by ~70 %. In addition, MOLT-4 cells showed a mean resting potential of −30.5 ± 1.8 mV (n = 12) and membrane capacitance of 14.5 ± 0.7 pF (n = 15). Treatment of T lymphocytes with mibefradil, a selective inhibitor against T-type Ca2+ channels, blocked Ca2+ influx  to knocked down T-type Ca2+ channels. As shown in Fig. 2+ channels contribute to the growth of human T cell leukemia lines.To further demonstrate that T-type Ca2+ channel antagonists induced a remarkable increase in the number of cells at the sub-G1 phase, a hallmark of cell apoptosis. While in Jurkat cells, mibefradil and NNC-55-0396 mainly induced cell apoptosis confirmed by a significant increase in the percentage of cells at the sub-G1 phase  diminished or abolished the inhibiting effect of intracellular baseline Ca2+ levels in the present of extracelluar Ca2+  endoplasmic reticulum Ca2+-ATPase (SERCA) and depletes ER Ca2+ stores) could be altered by NNC-55-0396. As shown in Fig. 2+ from the ER.Because endoplasmic reticulum (ER) is the major Ca storage , we aske2+ signals arising from the ER is the mitochondrial network [2+ flux in stimulating apoptosis [2+ release had a putative effect on depolarization of the mitochondrial membrane potential, resulting in cell apoptosis. To this end, we first measured the effect of NNC-55-0396 on mitochondrial membrane potential in Jurkat cells. As shown in Fig. A proximal target of Ca network . Severalpoptosis . Therefo2+ release, which disrupts mitochondrial Ca2+ homeostasis, Ru360  or cyclosporine A  inhibitor) was preincubated with Jurkat cells for 1 h before NNC-55-0396 treatment. Unexpectedly, neither of the compounds had significantly protective effect on mitochondrial membrane potential  may play a role in remodelling Ca2+ homeostasis. Abnormal up-regulation of the gene encoding T-type Ca2+ channel was detected in various tumour cells [2+ channels play a role in cancer development.Cancer cells have been reported to be relatively insensitive to reductions in extracellular calcium concentration . Ca2+-deur cells , suggest2+ channels, they were used for patch-clamp recording analysis. The patch-clamp recording results demonstrate that the current in MOLT-4 cells activated at −30 mV, with peak current at 0 mV, inconsistent with other reports of recording T-current [2+ influx which can be reduced by T-type Ca2+ channel blockers. Together, these results are consistent with the occurrence of T-type Ca2+ channel window currents, providing the pattern of Ca2+ signaling required for cell cycle progression.In the present study, because MOLT-4 cells expressed high level of T-type Ca-current , 13, 39.2+ channels reduce cancer cell proliferation and viability [2+ channels with mibefradil had been shown to induce apoptosis in breast cancer cells [2+ channels function as regulators of survival and/or apoptosis signaling. In this study, blocking the functional T-type Ca2+ channels significantly decreased the growth of Jurkat and MOLT-4 cells, while mibefradil and NNC-55-0396 had no effect on the growth in U937 and HEL cells, which didn’t express T-type Ca2+ channels. These results demonstrate a strong correlation between T- type Ca2+ channels expression and growth inhibition. Interestingly, we found that the lower-expression cell line (Jurkat) showed a larger growth inhibition than the higher-expressing cell line (MOLT-4), especially for NNC-55-0396 treatment. The phenomenon may attribute to NNC-55-0396-induced Ca2+ release in Jurkat cells, resulting in a larger cell death. In addition, the high percentage of sub-G1 phase upon NNC-55-0396-treatment also indicates that the Jurkat cell death is due to its inherent strong cytotoxicity as well as T-type Ca2+ channel blockade. Cell cycle analysis data demonstrated that mibefradil and NNC-55-0396 had a dual effect on cell viability: (a) decreasing proliferation rate; (b) inducing cell apoptosis. As shown in Fig. Several studies with in vitro systems have demonstrated that antagonists of T-type Caiability . In addier cells  and glioer cells . This ob2+ is an essential regulator of the cell cycle and is indispensable for cell proliferation. For example, the transition from the G1/S interphase (initiation of DNA synthesis) and the G2/M interphase (initiation of mitosis), is dependent upon Ca2+/calmodulin-dependent kinase II (CaM-kinase II) [2+ signals are often organized in oscillatory patterns involving entry of external Ca2+ and release of Ca2+ from internal stores. T-type Ca2+ channels are particular well suited to participate in such oscillations due to their low voltage activation ranges, transient kinetics of inactivation and “window current”. Indeed, many proliferating cells exhibit T-type Ca2+ current, including a variety of tumour cells [2+ channels with pharmacological blockers reduced intracellular calcium concentration, confirming the role of these channels in calcium concentration maintenance.Canase II) . In prolur cells , 40. As 2+ channel blocker and has been used in many studies to establish this putative causal link between T-type Ca2+ channels and cell proliferation. However, mibefradil has also been reported to inhibit cell proliferation through an association with cell swelling and the inhibition of volume-sensitive Cl− channels [et al. reported that NNC-55-0396 inhibited voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells [2+ channel blockers should be carefully attributed to T-type Ca2+ channel blockage.Mibefradil was originally presented as a T-type Cachannels , 44 or schannels –47. Son le cells . Thus, t2+ homeostasis have long been associated with apoptotic cell death [2+ changes (Ca2+ surge or Ca2+ overload) could trigger cell death. Therefore, the question arises, why blocking T-type Ca2+ calcium channels, which should inhibit calcium influx from the external environment, paradoxically induces an extensive apoptotic response in ALL cells? One possible explanation lies on the fact that cytosolic Ca2+ can be increased not only through influx from outside, but also via release of calcium ions from the internal stores. As shown in Fig. 2+ level from inducing ER Ca2+ release. In addition, mibefradil at high concentration (≥10 μM) also induced intracellular Ca2+ overload  and, possibly, also of the protein kinase RNA-like ER kinase (PERK) or ATF6 pathways of the UPR (inducing GADD153) [2+ has close association with MAPK signaling pathway, we next investigated whether mibefradil and NNC-55-0396 can modulate MAP kinase activity. MAP kinase signaling pathway plays an important role in regulating cell cycle progression, and T-type Ca2+ channel inhibitors blunted cell proliferation—through a halt in the progression to the G1-S phase in MOLT-4 cells, so MOLT-4 cells were used as a model to study ERK signaling pathway. We report here that both inhibitors down-regulated ERK signaling pathway in MOLT-4 cells, in agreement with Kotturi report that inhibition of Ca2+ influx decreased the phosphorylation of ERK1/2 [The ER and mitochondria are crucial nodes at which intracellular Canecrosis . In our GADD153) . Thus ERf ERK1/2 . Since E2+ channel in leukemia cell lines. Mibefradil and NNC-55-0396 had a dual role on cell viability: (a) inhibiting cell proliferation; (b) promoting cell apoptosis. Mechanistically, both T-type Ca2+ channel inhibitors induced ER Ca2+ release and disrupted ERK1/2 signaling pathway. Based on these observations and in vivo results reported elsewhere, we propose that T-type Ca2+ channel blockers may be utilized as future therapies for neoplasm expressing T-type channels.We have shown both molecular and extensive pharmacological evidence for the presence of a T-type Ca